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Posts Tagged ‘platelet-leukocyte aggregate’


Platelets in Translational Research – Part 1

 

Reviewer and Curator: Larry H Bernstein, MD, FCAP 

 

Introduction

This article is one of a 2 part presentation posted as an example of a central role of platelet biology in translational medicine investigations leading to the prevention and control of hemolytic and coagulopathic conditions, and to an understanding of atherosclerotic cardiovascular disease. The study of coagulation traces back to the early work on Warfarin in bleeding, and even earlier than that to the geneological evidence of inherited hemophilia in the Royal family of 18th Century Victoria.  The amount of work has been voluminous, and the conceptual framework has been difficult to put into practice over generations of postgraduate physicians.  No wonder, considering the clotting proteins and the amazing platelet.

Part I of Platelets in Translaional Research is a comprehensive coberage of the signaling and control involved in platelet-endothelial reactions, platelet-platelet reactions, and platelet transciptomics, all of which have a significant bearing on atherosclerotic plaque buildup, plaque rupture, and acute coronary syndrome as well as chronic ischemic heart disease.

Part II will cover a range of studies pointing to anti-platelet therapeutic targets.

Related work in Pharmaceutical Intelligence

Advanced Topics in Sepsis and the Cardiovascular System at its End Stage

Larry H Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2013/08/18/advanced-topics-in-sepsis-and-the-cardiovascular-system-at-its-end-stage/

‎ Special Considerations in Blood Lipoproteins, Viscosity, Assessment and Treatment

Larry H. Bernstein, MD, FCAP and Curator: Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2012/11/28/special-considerations-in-blood-lipoproteins_ viscosity_assessment-and-treatment/

What is the role of plasma viscosity in hemostasis and vascular disease risk?

Larry H Bernstein, MD  and Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2012/11/28/What-is-the-role-of-plasma-viscosity-in-hemostasis-and-vascular-disease-risk?

Biochemistry of the Coagulation Cascade and Platelet Aggregation – Part I

Larry H. Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2012/11/26/Biochemistry-of-the-Coagulation-Cascade-and-Platelet-Aggregation-Part_I

Nitric Oxide Function in Coagulation

Larry H. Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2012/11/26/nitric-oxide-function-in-coagulation/

Coagulation: Transition from a familiar model tied to laboratory testing, and the new cellular-driven model

Larry H. Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2012/11/10/coagulation-transition-from-a familiar-model-tied-to-laboratory-testing-and-the-new-cellular-driven-model/

Nitric Oxide, Platelets, Endothelium and Hemostasis

Larry H. Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2012/11/08/nitric-oxide-platelets-endothelium-and-hemostasis/

Interaction of Nitric Oxide and Prostacyclin in Vascular Endothelium

Larry H. Bernstein, MD

https://pharmaceuticalintelligence.com/2012/09/14/interaction-of-nitric-oxide-and-prostacyclin-in-vascular-endothelium

The Effects of Aprotinin on Endothelial Cell Coagulant Biology

Demet Sag, PhD*†, Kamran Baig, MBBS, MRCS; James Jaggers, MD, Jeffrey H. Lawson, MD, PhD

https://pharmaceuticalintelligence.com/2013/07/20/the-effects-of-aprotinin-on-endothelial-cell-coagulant-biology/

Platelet Role in Atheroscleosis

Platelets and Cardiovascular Disease

David Gregg, MD; Pascal J. Goldschmidt-Clermont, MD
Duke University Medical Center, Durham, NC.

Platelets are specialized disk-shaped cells in the blood stream that are involved in the formation of blood clots that play an important role in heart attacks, strokes, and peripheral vascular disease. In most people, the more than 200 million platelets in a milliliter of blood act as tiny building blocks to form the basis of a clot to stop bleeding from cuts or injuries. Platelets can detect a disruption in the lining of a blood vessel and react to build a wall to stop bleeding.

F1.large  platelr forms plug

Figure 1. Platelets form a platelet plug to stop bleeding from an injured blood vessel.

In cardiovascular disease, abnormal clotting occurs that can result in heart attacks or stroke. Blood vessels injured by smoking, cholesterol, or high blood pressure develop cholesterol-rich build-ups (plaques) that line the blood vessel; these plaques can rupture and cause the platelets to form a clot. Even though no bleeding is occurring, platelets sense the plaque rupture and are confused, thinking that an injury has taken place that will cause bleeding. Instead of sealing the vessel to prevent bleeding as would occur with a cut, a clot forms in an intact blood vessel, causing a blockage of blood flow (Figure 2). Without blood, a portion of the heart muscle can die, leading to a heart attack.

F2.large   clot formation blocks flow

Figure 2. Plaque rupture results in clot formation to block blood flow, which may result in a heart attack or stroke

Platelet Disorders

Platelets may be abnormal either quantitatively (too many or too few) or qualitatively (the right number but they do not work correctly). The number of platelets is routinely tested as part of the complete blood count (CBC). Normal counts range from 150 000 to 450 000. A decrease in the number of platelets indicates a condition known as thrombocytopenia and may result in increased bleeding, the first signs of which may include gum bleeding, nose bleeds, and increased bruising. In cardiology, the most frequent cause of a low platelet count is an abnormal immune response caused by drug therapy, particularly with the intravenous blood thinner heparin (heparin-induced thrombocytopenia), and rarely with other drugs to control high blood pressure or symptoms of congestive heart failure (diuretics), to control diabetes (antidiabetic medications), or to regulate your blood clotting (antiplatelet drugs). Elevated platelet counts can also occur, usually in association with diseases in the elderly, and can result in either excess clotting or even abnormal bleeding.

Because platelets are so important in stopping bleeding from everyday injuries such as cuts or bruises, severe inherited disorders of platelets are quite rare. Researchers, however, have discovered more subtle genetic variations in platelets called polymorphisms that may alter platelets in subtle ways to raise the risk of cardiovascular disease when combined with other risk factors, but which on their own do not result in overt disease. These polymorphisms may also be important in understanding who may gain the greatest benefit from anti-platelet drugs.

The most commonly used antiplatelet agent is aspirin, although you may also be prescribed other oral agents, such as ticlopidine, clopidogrel, or dipyridamole, or intravenous antiplatelet drugs such as abciximab or eptifibatide while you are in the hospital or undergoing angioplasty procedures. Each agent affects platelets in slightly different ways and may have unique side effects, but all either cause the platelets to stick together or induce them to clot less well. Your doctor will choose the drug that best suits your situation. Table 1 shows some of the unique features of commonly used oral antiplatelet drugs

TABLE 1. Commonly Used Oral Antiplatelet Drugs: Uses and Side Effects

Aspirin Clopidogrel Ticlopidine Dipyridamole + Aspirin
Uses Heart disease and stroke; inexpensive Heart disease and stroke, particularly after stenting Mostly in stroke; requires blood count monitoring Stroke; may not be suitable for patients with heart disease
Side effects Gastrointestinal (GI) intolerance; GI bleeding Diarrhea (much less common than with ticlopidine); rash and itching Diarrhea and GI upset (usually resolves in 2 weeks); may decrease blood counts, particularly white blood cells Headache; GI bleeding; GI intolerance

Antiplatelet drugs are different than blood thinners or anticoagulants such as warfarin (Coumadin, Bristol-Myers Squibb) or heparin. Anticoagulants block a second step in clotting known as coagulation but do not directly affect the platelets.

Eur J Cardiovasc Nurs. 2002 Dec;1(4):273-88.

Platelets and cardiovascular disease

Willoughby S, Holmes A, Loscalzo J.
Queen Elizabeth Hospital, Adelaide University, South Australia, Adelaide, Au

Platelets play an important, but often under-recognized role in cardiovascular disease. For example, the normal response of the platelet can be altered, either by increased pro-aggregatory stimuli or by diminished anti-aggregatory substances to produce conditions of increased platelet activation/aggregation and occur in active cardiovascular disease states both on a chronic (e.g. stable angina pectoris) and acute basis (e.g. acute myocardial infarction). In addition, platelet hyperaggregability is also associated with the risk factors for coronary artery disease (e.g. smoking, hypertension, and hypercholesterolaemia). Finally, the utility of an increasing range of anti-platelet therapies in the management of the above disease states further emphasizes the pivotal role platelets play in the pathogenesis of cardiovascular disease. This paper provides a comprehensive overview of the normal physiologic role of platelets in maintain homeostasis, the pathophysiologic processes that contribute to platelet dysfunction in cardiovascular disease and the associated role and benefits of anti-platelet therapies.   PMID: 14622657

Triggering of Plaque Disruption and Arterial Thrombosis in an Atherosclerotic Rabbit Model

GS Abela, PD Picon, SE Friedl, OC Gebara, A Miyamoto, et al.
Deaconess Hospital, Harvard Medical School, Boston, Mass; Federal Univ and Univ Passo Fundo (P.D.P.), Rio Grande de Sul, Brazil;  Heart Institute, Univ São Paulo (O.C.G.), São Paulo, Brazil; National Defense Medical College (A.K.), Saitama, Jp.

It is now recognized that plaque disruption and thrombosis, a process often triggered by activities of the patient, is generally the cause of the onset of acute coronary syndromes. Plaque disruption and subsequent arterial thrombosis are now recognized as critical to the onset of acute coronary ischemic syndromes. It is hypothesized that occurrence of thrombotic coronary occlusion has three components. First, a plaque that is vulnerable to disruption must be present. Second, acute physiological events are required to induce plaque disruption and thrombosis. Third, a relatively hypercoagulable state and heightened vasomotor tone increase the likelihood that arterial thrombosis will produce complete lumen occlusion.

In human patients, the opportunity to study factors responsible for acute onset of myocardial infarction is limited because coronary angiography performed before the event cannot prospectively identify plaques vulnerable to disruption. After the event, angiography cannot distinguish the features of the plaque responsible for the disruption from those resulting from the disruption. Moreover, plaque disruptions producing total vascular occlusion and death may be more severe than those occurring in asymptomatic individuals or in patients with unstable angina or nonfatal myocardial infarction. These difficulties, inherent in the study of plaque disruption and thrombosis in human patients, create a great need for an animal model of the process.  An atherosclerotic rabbit model of triggering of arterial thrombosis that was introduced by Constantinides and Chakravarti more than 30 years ago but not subsequently used. Aortic plaques were induced by a high-cholesterol diet, by mechanical balloon injury of the artery, or by a combination of the two. Triggering was attempted by injection of Russell’s viper venom (RVV), which is a proteolytic procoagulant, and histamine. A recent review of the animal models of thrombosis currently in use noted that “thus far, it has not been possible to duplicate in a model the most common clinical cause of thrombosis—an ulcerated atherosclerotic plaque.” The advantage of the Constantinides model over other animal models used to study thrombosis is that it uses a biological intervention to trigger localized atherosclerotic plaque disruption and formation of platelet-rich arterial thrombi.  Disadvantages of the Constantinides model are (1) the low yield of triggering (only about one third of the rabbits developed thrombosis) and (2) the long (8-month) preparatory period. In addition, there is a need to replicate the findings of Constantinides and Chakravarti13 from 30 years ago because of the biological variability of rabbit strains and RVV. It cannot be assumed that the rabbits and RVV currently available will produce the results obtained in the 1960s.

A total of 53 New Zealand White rabbits were exposed to one of four preparatory regimens: rabbits in group I (n=9) were fed a regular diet for 8 months; rabbits in group II (n=13) were fed a diet of 1% cholesterol for 2 months alternated with 2 months of a regular diet for a total of 8 months; rabbits in group III (n=5) underwent balloon-induced arterial wall injury, then were given a regular diet for 8 months; and rabbits in group IV (n=14) underwent balloon-induced arterial wall injury, then were given a diet of 1% cholesterol for 2 months followed by a regular diet for 2 months for a total of 4 months. After completion of the preparatory regimen, triggering of plaque disruption and thrombosis was attempted by injection of RVV (0.15 mg/kg IP) and histamine (0.02 mg/kg IV). In group I, normal control rabbits without atherosclerosis, only one small thrombus was noted in 1 of 9 rabbits. In group II, cholesterol-fed rabbits, thrombosis occurred in 3 of 13 rabbits. Thrombus occurred in all rabbits in group III (5 of 5) and in 10 of 14 rabbits in group IV. Although the frequency of thrombosis was not significantly different between groups I and II, possibly due to a small sample size, it was significantly different among all four groups (P<.001). Also, the frequency and amount of thrombus formation were significantly different among all four groups (P<.001; P<.0001) but not between groups I and II. Rabbits with atherosclerosis (those in groups II and IV) demonstrated plaque disruption and overlying platelet-rich thrombus formation similar to that observed in patients with acute coronary syndromes. The surface area covered by thrombus was 2 mm2 in group I, 15.3±19.2 mm2 in group II, 223±119 mm2 in group III, and 263±222 mm2 in group IV. Rabbits in groups III and IV had the greatest amount of thrombus, and this amount was significantly greater than in rabbits in groups I and II (P<.001 and P<.03, respectively).

The intima in group I rabbits appeared normal by gross inspection. In group II rabbits, white-yellow plaque was widely distributed over the arterial surface, with focal punctate ulceration occasionally noted under a dissecting microscope. In group III rabbits, the intima was smooth and widely covered with white plaque. Group IV rabbits had extensive sheets of elevated white-yellow plaque. By gross visualization, ulceration of the surface was present without superimposed thrombus in two rabbits in group IV.  In sections from groups II and IV, some areas of plaque directly adjacent to the thrombi had marked thinning of the connective tissue cap and areas of dehiscent foam cells. These observations were rare and were noted in <0.5% of the examined lesions. In most cases, the arterial thrombus was not located at a site of obvious plaque rupture. Foam cell infiltration was also noted adjacent to sites of thrombosis. Scanning electron microscopy demonstrated fissures of various lengths below areas from which overlying thrombi were removed. Endothelial cells could be seen lining the intimal surface of the aorta in the rabbits that had undergone balloon-induced arterial wall injury 8 months earlier. Surface blebs and focal endothelial breakdown with ulcer formation, without grossly visible thrombosis, were occasionally seen in samples from groups II and IV. The base of these ulcers was layered with platelets, fibrin, and red blood cells. Transmission electron microscopy of areas with thrombosis confirms that the thrombi were platelet rich.

In the two groups that received cholesterol feeding, the total cholesterol content in tissue samples pooled from the thoracic and abdominal aorta was significantly higher in group IV (16±7.2 mg/g) than in group II (2.8±1.6 mg/g) (P<.0001). Rabbits that were maintained on a regular diet (groups I and III) had equally low levels of tissue cholesterol (0.05±0.04 versus 0.06±0.02 mg/g, P=NS). The average fibrinogen level before triggering in the 27 rabbits in which fibrinogen was measured was 210±119 mg/dL; it rose to 403±168 mg/dL 48 hours after triggering (P<.001). Plasma fibrinolytic activity did not change after triggering (85.5±37.8 versus 94.8±33.5 arbitrary units). Platelet counts (measured in only 19 rabbits in groups II and IV) decreased from 350±84×103 to 215±116×103 per cubic millimeter after triggering (P<.001).

Conclusions

The results demonstrate that vulnerable plaques can be produced and that plaque disruption and platelet-rich arterial thrombus formation may be triggered pharmacologically in an animal model of arterial plaque. This finding documents that the New Zealand White rabbit strains and the RVV currently available can be used to obtain the same results observed by Constantinides and Chakravarti13 more than 30 years ago. This animal model is suitable for the study of plaque disruption and arterial thrombosis. Hypercholesterolemia and mechanical arterial wall injury seemed to produce plaques vulnerable to triggering of disruption and thrombosis, whereas normal arteries were relatively resistant to triggering. The model provides a method to evaluate agents that might decrease the occurrence of vulnerable plaques or the amount of thrombus formed after triggering. Most important, the model can be used to identify the features of vulnerable plaques and the pharmacological stressors that trigger plaque disruption and thrombus formation.

Certain features of the lesions seen in this model are similar to those of human lesions seen at autopsy of patients with fatal myocardial infarction, ie, a lesion with a fissured collagen cap overlying a lipid mass of amorphous and crystalline lipid. However, most of the lesions in the model did not have these features and were more consistent with a recent pathological study of fatal coronary thrombosis, which revealed that in approximately half the cases, the plaque was relatively intact but an inflammatory infiltrate was present. Perhaps the incidence of plaque rupture causing thrombus may be even lower in patients with nonfatal coronary thrombosis, as suggested from angioscopic studies of coronary arteries that have shown plaque ulceration of various severities.  Analyses of human plaques have demonstrated that disrupted plaques have significantly less collagen, glycosaminoglycans, and smooth muscle cells and more extracellular lipid and macrophages  than do nondisrupted plaques. This is consistent with findings in our study that rabbits in group II had more connective tissue and a lower rate of disruption and thrombosis than those in groups III and IV.

references

Herrick JB. Clinical features of sudden obstruction of the coronary arteries. JAMA. 1912;59:2015-2020.
Chapman I. Morphogenesis of occluding coronary artery thrombosis. Arch Pathol. 1965;80:256-261.
Friedman M, van den Bovenkamp GJ. The pathogenesis of a coronary thrombus. Am J Pathol. 1966;80:19-44.

This reader sees a validation in this study of the noted cardiologist, Alan Jaffe, at Mayo Clinic, in referring to Type I and Type II myocardial infarcts, which accounts for differences in troponin elevations in patients.

The Platelet in Cardiovascular Disease

1. microthrombi adhering to foam cells
2. Platelets secrete

–  Platelet-derived growth factor (PDGF) that promotes smooth muscle cell migration and collagen production

– Plasminogen activator inhibitor-1 (PAI-1) that suppresses fibrinolysis

Davies MJ, Woolf N, Rowles PM, et al. Morphology of the endothelium over atherosclerotic plaques in human coronary arteries. Br Heart J 1988;60:459-64.

– Microhemorrhages attract and activate neighboring platelets, support fibrin generation

Inoue M, Itoh H, Ueda M, et al. Vascular endothelial growth factor (VEGF) expression in human coronary atherosclerotic lesions: Possible pathophysiological significance in progression of atherosclerosis. Circulation 1998;98:2108-16.

Systems biology of platelet-vessel wall interactions

Scott L. Diamond*, Jeremy Purvis, Manash Chatterjee and Matthew H. Flamm
Institute for Medicine and Engineering, University of Pennsylvania, Philadelphia, PA
Front Phys 26 August 2013    http://dx.doi.org/10.3389/fphys.2013.00229

Blood systems biology seeks to quantify outside-in signaling as platelets respond to numerous external stimuli, typically under flow conditions. Platelets can activate via GPVI collagen receptor and numerous G-protein coupled receptors (GPCRs) responsive to ADP, thromboxane, thrombin, and prostacyclin. A bottom-up ODE approach allowed prediction of platelet calcium and phosphoinositides following P2Y1 activation with ADP, either for a population average or single cell stochastic behavior. The homeostasis assumption (i.e., a resting platelet stays resting until activated) was particularly useful in finding global steady states for these large metabolic networks. Alternatively, a top-down approach involving Pairwise Agonist Scanning (PAS) allowed large data sets of measured calcium mobilization to predict an individual’s platelet responses. The data was used to train neural network (NN) models of signaling to predict patient-specific responses to combinatorial stimulation. A kinetic description of platelet signaling then allows prediction of inside-out activation of platelets as they experience the complex biochemical milieu at the site of thrombosis. Multiscale lattice kinetic Monte Carlo (LKMC) utilizes these detailed descriptions of platelet signaling under flow conditions where released soluble species are solved by finite element method and the flow field around the growing thrombus is updated using computational fluid dynamics or lattice Boltzmann method. Since hemodynamic effects are included in a multiscale approach, thrombosis can then be predicted under arterial and venous thrombotic conditions for various anatomical geometries. Such systems biology approaches accommodate the effect of anti-platelet pharmacological intervention where COX1 pathways or ADP signaling are modulated in a patient-specific manner.

CLOTTING UNDER FLOW CONDITIONS

Collagen is sufficient to capture and activate platelets under venous wall shear rates (ãw  100–200s_1). In the arterial circulation (ãw 1000–2000 s_1), collagen adsorbed von Willebrand factor (vWF) facilitates platelet capture, allowing col­lagen induced GPVI signaling and subsequent á2â1 and á2bâ3 activation. Under flow conditions, red blood cells help enrich the platelet concentration by 3–8x in the plasma layer near the wall. At pathological high shear exposures (>5000 s_1) encountered in severe stenosis, mechanical heart valves, and continuous LVAD pumps, the plasma vWF may undergo structural changes, such as a transition from a globular to an extended state (Schneider et al., 2007), likely increasing the availability of A1 domains in the vWF polymer for multivalent contacting with platelet GPIb receptors.

GROWTH OF THE PLATELET AGGREGATE VIA AUTOCATALYTIC SIGNALING

Collagen triggers GPVI clustering, leading to rapid phosphorylation of the GPVI-associated Fc receptor by Src family tyrosine kinases. Such phosphotyrosine residues are recognized by Syk, and the binding and activation of Syk activates PLCã2. PLCã2 converts phosphatidylinositol (PI)-4,5-P2 (PIP2) to inositol 1,4,5-trisphosphate (1,4,5-IP3 or IP3) and diacyclglycerol (DAG). IP3 opens Ca2+ channels in the platelet dense tubular system (DTS). Depletion of DTS Ca2+ results in STIM1 activation and bind­ing to Orai1, leading to store operated calcium entry (SOCE). DAG/Ca2+ activates protein kinase C (PKC) in platelets, which in turn governs several serine/threonine phosphorylation events.

Beyond the first monolayer of platelets adherent to colla-gen/VWF, the addition of subsequent layers of platelets to the growing thrombus is strongly potentiated by locally released ADP and thromboxane (TXA2) as well as locally generated thrombin. ADP activates P2Y1 and P2Y12 while TXA2 activates the TP receptor and thrombin cleaves PAR1 and PAR4. Activation of a GPCR causes an exchange of GTP for GDP on the α subunit of the G protein and dissociation of the α and γ subunits. Both these units in turn interact with secondary effectors such as PLC and adenylate cyclase. Human platelets express at least 10 forms of Gα (including members of the Gq, Gi, G12, and Gs fami­lies) (Brass et al., 2006; Offermanns, 2006). Thrombin, ADP, and TXA2 activate PLC via Gq. PLC generates IP3 from membrane PIP2. Rising Ca2+ levels activate the Ras family member, Rap1B via Cal-DAG GEF. Rap1B activation is a precursor to αIIb 3 acti­vation and allows the platelets to form aggregates with other platelets through fibrinogen cross-bridging. Ca2+-dependent signaling drives myosin light chain kinase and activation of GTP binding proteins of the Rho family. Rho acti­vation in turn activates kinases like p160ROCK and 5 LIM-kinase that can phosphorylate myosin light chain kinase and cofilin to regulate actin-dependent cytoskeletal shape changes. Endothelial derived prostacyclin (PGI2) binds the IP recep­tor and causes Gs mediated increase in adenyl cyclase activity. Also, NO from the endothelium and platelets can activate guany-late cyclase resulting in elevated cGMP levels that subsequently inhibit the hydrolysis of cAMP by intracellular phosphodi-esterases. Taken together these mechanisms elevate intracellular cAMP levels, which strongly downregulate platelet signaling. Agonists coupled to Gi family members inhibit cAMP production in platelets, thus allowing activation to proceed unhindered. Additionally the âã subunits of these receptors can activate PLCâ and the ã isoform of PI3K. The effectors for PI3K include Rap1b and Akt.

Fig 1 reaction schemes for platelet signaling

FIGURE 1 | Detailed reaction schemes for platelet signaling modules. Four interconnected models were defined: (A) Ca2+ module: cytosolic and DTS compartments are separated by the DTS membrane, which contains the IP3R and SERCA. (B) Phosphoinositide (PI) module: Membrane-bound PIs are cleaved by PLC-â to form diffusible inositol phosphates and DAG, which are substrates for resynthesis of PIs. (C) PKC module: Ca2+i and DAG activate PKC, which migrates to the plasmamembrane where it phosphorylates PLC-â. (D) P2Y1 module: extracellular ADP binds to and activates P2Y1. Active P2Y1 accelerates guanine nucleotide exchange on bound Gq. The Gq·GTP binds and activates PLC-â, which increases the GTPase activity of Gq·GTP.

ADP is stored in platelet dense granules and is released upon activation. P2Y1 and P2Y12 are the primary receptors for this agonist. P2Y1 is Gq coupled and signaling through this receptor causes Ca2+ mobilization, shape change, and thromboxane generation. P2Y12 is the target of the commonly used anti-platelet drug Plavix, and is a Gi2 coupled receptor that inhibits cAMP production in platelets. Thrombin is a potent platelet agonist that causes fast mobi­lization of intracellular Ca2+, and activation of phospholipase A2 and subsequent thromboxane generation (Offermanns et al., 1997). Also, thrombin can trigger Rho dependent signaling pathways in platelets (Moers et al., 2003), that contribute to actin modeling and shape change. Thrombin signals through the protease-activated receptor (PAR) family of GPCRs. PAR1 and PAR4 are expressed on human platelets, while PAR3 and PAR4 are expressed on mouse platelets. Thrombin cleaves the N-terminus of these receptors, exposing a new N-terminus that serves as a tethered ligand for these receptors. Synthetic pep­tides are able to selectively activate these receptors and mimic the actions of thrombin (for example, SFLLRN for PAR1, and AYPGKF for PAR4). Kinetic studies have shown that the human platelet response to thrombin is biphasic and involves first signal­ing through PAR1 and subsequent signaling through PAR4 (Covic et al., 2000). In mouse platelets signaling occurs primarily via PAR4, and is facilitated by PAR3. In addition to the PAR recep­tors, GP1bá has high affinity for thrombin. Absence of GP1bá reduces responses to low doses of thrombin and diminishes PAR1 signaling, suggesting that this receptor facilitates signaling through the PARs (Dormann et al., 2000). Ca2+ mobilization also activates phospholipase A2 (PLA2), which in turn converts mem­brane phospholipids to arachidonic Acid. TXA2 is produced from membrane arachidonate by the aspirin sensitive cyclooxygenase (COX-1) enzyme. TXA2 causes Ca2+ mobilization, aggregation, secretion, phosphoinositide hydrolysis, and protein phosphoryla-tion. TXA2 can diffuse across the membrane and activate nearby platelets, but its activity is limited by the molecule’s short half life (∼30 s).

These modules use previously validated or data-consistent kinetic networks for SERCA, IP3-Receptor, PKC translocation, and GPCR signaling (Figures 1E–H). Assembling the four modules together results in a global ODE model that has 77 reactions, 132 fixed kinetic rate constants, and 70 species. Since the reaction network (Figure 1) and the kinetic parameters are fixed, the reaction topology of the model is also fixed. Such a model takes the general form: dc/dt = F(c) and c(t = 0) = co where c is a vector of all species concentrations and co is a specified initial condition vector at t = 0. To determine appropriate sets of co that are suitable for use in modeling platelets, a challenge exists that the copy number of each species in a resting platelet is not known. Imposing a homeostasis assumption results in powerful tool to define a set of acceptable co vectors. The homeostasis assumption states that a resting platelet remains resting until activated. This means that an acceptable ini­tial condition co also represents a steady state for the system and will satisfy the equation dc/dt = 0. Finding a global co involves assembling the steady state solutions of each module (Figure 2).

Fig 2 Assembly of full model from steady-state modules

FIGURE 2 | Homeostasis requirement: Assembly of full model from steady-state modules using principle component analysis (PCA). The full model is assembled by combining PCA-reduced, steady-state solution spaces from each module into a combined steady state solution space. This global space is searched for full-length, steady-state solution vectors that satisfy both the steady state requirements of each module and the desired time-dependent properties when the steady-state is perturbed. A simple linear constraint is imposed for every pair of modules that share a common molecule ci to ensure that steady state solutions are Keywords: platelet, thrombosis, hemodynamic, ADP, thromboxane consistent. To assemble the platelet signaling model, a set of 16 PC vectors representing all 72 unknown variables in the model were used as search directions in a global optimization routine. The global solution space was searched for models with accurate dynamic behavior using experimental time-series data for ADP-stimulated Ca2+ release. Species are grouped according to compartment. Color values correspond to molar concentrations (mol/L or mol/m2) or as indicated: DTS species (mol L1). †Extracellular species (mol L1). DTS volume (L). §PM leak conductance/area (S m−2).

The first phase of the method involves generating a com­pact representation of the steady-state solutions for each module. First, conservative bounds are chosen for c based on physiological and practical considerations. Also, because molecular concentra­tions can span several orders of magnitude, it is most efficient to delineate this range of values on a logarithmic scale rather than a linear scale. Once the sampling distribution for c has been defined, steady-state solutions (co = c55) for each module are cal­culated using fixed kinetic parameters for each reaction in the module. For non-oscillating systems, steady-state solutions may be obtained by simulating the system until equilibrium is reached (i.e., until dc/dt = 0). In the third step, a large collection of steady-state solutions for each module is subjected to principal component analysis (PCA) (Purvis et al., 2009). PCA is then used to transform these points to a new coordinate set that optimally covers the space of steady-state solutions using the fewest num­ber of dimensions. For example, if two molecule concentrations in the steady-state space are highly correlated due to participation in the same reaction, PCA will locate a single dimension to rep­resent each pair of points in the transformed space. Ultimately, these new dimensions will be combined across all modules to search for global solutions that lie in the steady-state space for the fully combined network. Since PCA is a linear method, a steady-state solution space that is highly nonlinear may require more principal component vectors to accurately estimate the solutions. The reduction procedure is shown for the human platelet model comprising 4 interlinked signaling modules (Figure 2). For this step, we generated more than 109 sets of initial guesses (co) for each module, computed the initial value problem for each co until a steady state was reached (dc/dt ≈ 0), and selected only those steady states (c55) that were consistent with known con­centrations (i.e., [Ca2+]o ∼100 nM).  Interestingly, only a small fraction of initial guesses produce steady-state solutions that are also consistent with known concentration values. For example, it was shown that only 50,000 of 109 initial guesses (0.005%) in the Ca2+ balance module (Figure 1A) met both requirements and were suitable for further analysis. This observation shows that the kinetic topology of these molecular networks places very strong constraints on the range of concentrations that can exist at steady state. In biological terms, this suggests that fixed kinetic proper­ties at the molecular level (e.g., IP3R and SERCA kinetics) can affect not only the dynamical features of a biochemical system but can also determine the abundance of chemical species and the compartmental structures that contain them. A fully assem­bled initial condition vector results (bottom, Figure 2) results in new hypotheses about allowable concentrations and ratios of con­centrations (i.e., IP3/SERCA ratio is very small). The allowed co = css is consistent with the known resting levels of Ca2+, IP3, P2Y1, DAG, PA, PI, PIP2, and PIP (bottom, Figure 2) as well as the stimulated response of platelets to increasing amounts of ADP (right, Figure 2). With a global simulation of P2Y1 signaling, it is possible to simulate the ADP dose-response of calcium mobiliza­tion and IP3 generation in platelets as well as the mobilization of intracellular calcium in a single platelet due to stochastic fluctuations (Figure 3).

Fig 3. P2Y1 signalink model

FIGURE 3 | Tests of P2Y1 signaling model. ADP dose response for the full platelet model from 100 nM to 10 ìM ADP for calcium mobilization (A) or IP3 generation (B). Stochastic simulation of a single platelet (C). A single, fura-2-loaded platelet was immobilized on a fibrinogen-coated coverslip and activated with 40 ìM ADP at t = 90 [Ca2+ trace from Heemskerk et al. (2001)]. After 90 s of simulated rest, the platelet model was activated by setting extracellular [ADP] to 40ìM. Simulated interval times were binned in 2s increments for direct comparison with experiment (inset).

Since many initial condition vectors can be found to allow a resting platelet to remain resting and then respond appropriately to stimulation, investigation of these multiple steady states and associated cell responses can allow an ad-hoc sensitivity analysis. Some species (flexible nodes) may vary widely in the allowed ini­tial condition vectors but have little effect on system response. In contrast, other species (rigid nodes) may be forced to take on val­ues in a very narrow range due to the kinetic constraints of the problem.

To examine the changes in steady-state properties caused by kinetic perturbations in the P2Y1 model, we altered the rates of important regulatory reactions and observed the system response to each perturbation. Each perturbation cause a brief adjustment phase lasting ∼200 s followed by a more gradual phase char­acterized by a new steady-state profile. After 1 h of simulated time, steady-state concentrations and reaction fluxes were quan­tified relative to their original steady-state levels (Figure 4). In a computational perturbation, the inhibition of phospholipase C-β (PLC-β) activity by PKC was reduced 10-fold. Since PKC has a negative-feedback role in suppressing the platelet-stimulating activity of PLC-β, this perturbation caused a 2-fold increase

in steady-state PIP2 hydrolysis, elevated IP3 concentration, and accelerated Ca2+ release. This was a compensatory effect caused by the negative feedback loop involving Ca2+-regulated activity of PKC, a resulting new hypothesis that can be probed experi­mentally. In another example, increasing the hydrolytic activity of PLC-â for the substrate PIP2 by 10-fold caused an expected stimulatory effect, raising intracellular calcium and steady-state levels of cytosolic inositol phosphates (IP3, IP2, and IP) between 2- and 3-fold. Interestingly, reaction fluxes for phosphoinositide hydrolysis were diminished, possibly due to substrate depletion. Taken together, these examples illustrate the system-wide effects of perturbations in the kinetic rate processes. The procedure could easily be extended to examine multiple simultaneous per­turbations in both reaction rates and steady-state concentrations. In future applications of this approach, genomic or proteomic information of multiple perturbations could be used to help predict platelet signaling phenotypes.

Fig 4 Shifts in steady-state profiles caused by kinetic perturbations

FIGURE 4 | Shifts in steady-state profiles caused by kinetic perturbations. The steady-state platelet model was perturbed by changing selected kinetic parameters (±10-fold) and simulating for 1 h. After approaching a new steady state, the model concentrations and fluxes were determined relative to their original steady-state values and colored according to fold-change. Green indicates no change (NC) relative to initial flux/concentration. Red indicates a relative increase and blue indicates a relative decrease. Note that the color scale in each panel is normalized separately to maximize distinctions in fold change. New steady states were achieved after (top) 10-fold decrease in PKC-mediated inhibition of PLC-β, and (bottom) 10-fold increase in PIP2 hydrolysis (10-fold increase in kcat of hydrolysis). ∗, active state.

Fig 5. predicting global calcium response

FIGURE 5 | Pairwise agonist scanning to predict global calcium response in human platelets. (A) Simplified schematic of signaling pathways examined in this study that converge on intracellular calcium release in human platelets. (B) Dynamic NN model used to train platelet response to combinatorial agonist activation. A sequence of input signals representing agonist concentrations is introduced to the network at each time point. Processing layers integrate input values with feedback signals to predict the next time point. (C) A total of 154 calcium traces were measured for single and pairwise activation using 6 different agonists (“Experiment”) and used for neural network training. The NN training accurately predicted (“NN Prediction”) the training data.

Fig 6. Multiscale modeling with 4 components

FIGURE 6 | Multiscale modeling. The multiscale model has four main components (A) fluid flow, transport of soluble species, motion and binding of platelets, and the activation state of each platelet. The fluid flow is perturbed by the growing clot and is determined using the lattice Boltzmann method. The released soluble agonists form a boundary layer in the flow, and this process is determined using the finite element method. Platelet motion and bonding are simulated with lattice kinetic Monte Carlo. Platelet activation state is estimated from the history of intracellular calcium concentration, which is determined by a neural network model. (B) Multiscale simulation of patient-specific platelet deposition under flow for a specific donor and PAS-trained neural network of calcium signaling. Platelet activation (black, unactivated; white, activated) and deposition at 500 s (inlet wall shear rate, 200 s−1) showing released ADP (top) and TXA2 (middle) and perturbation of the flow field (bottom). Flow: left to right (streamlines, black lines); surface collagen (250 ìm long): red bar.  

PLATELET INTERACTIONS WITH THE VESSEL WALL

The multiscale systems biology model accommodates platelet sig­naling, platelet adhesion to collagen and other activated platelets, release of soluble agonists, thrombus growth, and distortion of the prevailing flow field (Figure 6A). The lattice Boltzmann (LB) method is used to solve for the velocity field of the fluid. Platelets in the growing aggregate release ADP and TXA2 into the fluid, and a boundary layer is formed with the flow. The dynamics of this process are determined with a finite element method solution of the convection-diffusion-reaction equation for each of the soluble species, ADP and TXA2. Platelets move in the fluid by convection and RBC-augmented dispersion. They also bind to the collagen surface as well as previously bound platelets. The motion and binding of platelets is simulated using the convective lattice kinetic Monte Carlo (LKMC) algorithm validated for stochastic convective-diffusive particle transport (Flamm et al., 2009, 2011, 2012). The level of integrin activation and associated adhesiveness for each platelet is related to the cumulative intracellular calcium concentration. The intracellu­lar calcium concentration is determined using a NN trained on a specifc patient’s platelet PAS phenotyping experiment. Using this multiscale approach, Multiscale simulations predicted the density of platelets adherent to the surface, platelet activation states, as well as the spatiotemporal dynamics of ADP and TXA2 release, morphology of the growing aggregate, and the distribu­tion of shear along the solid-fluid boundary (Figure 6B). Platelets stick to the collagen surface and release ADP and TXA2 which forms a boundary layer extending up to 10 pm from the throm­bus. Boundary layer concentrations of up to 10 pM ADP and 0.1 pM TXA2 were found by simulation. TXA2 concentrations were found to be sub-physiological (<0.0067 pM or <0.1 xEC50) until a sufficient platelet mass accumulated at the surface after ∼250 s. Boundary layer ADP concentrations were within the effective dynamic range (0.1–10 pM) throughout the simulation. The strong temporal and spatial fluctuations in the concentration of ADP were predominately driven by the short release time (5 s), whereas the longer release time of TXA2 (100 s) smoothed fluc­tuations. The shear rate along the solid-fluid boundary became nonuniform during the simulation (5–10-fold increase above 200 s−1) due to surface roughness. At 500 s, the platelet deposit was characterized by platelet clusters 20–30 pm in length, fully consistent with microfluidic measurements of platelet cluster size on collagen at this shear rate.

Developing tools to define platelet variations between patients and the relationship of platelet phenotype to prothrombotic or bleeding traits will have significant impact in stratifying patients according to risk. This multiscale approach also makes feasible patient-specific prediction of platelet deposi­tion and drug response in more complex in vivo geometries such as stenosis, aneurysms, stented vessels, valves, bifurcations, or ves­sel rupture (for prediction of bleeding risks) or in geometries encountered in mechanical biomedical devices.

 Platelet–Leukocyte–Endothelial Cell Interactions After Middle Cerebral Artery Occlusion

Mami Ishikawa, *Dianne Cooper, *Thiruma V. Arumugam, †John H. Zhang, †Anil Nanda, and *D. Neil Granger
Departments of *Molecular and Cellular Physiology, and †Neurosurgery, Louisiana State University Health Sciences Center, Shreveport, LA
Journal of Cerebral Blood Flow & Metabolism 24:907–915 © 2004 

Summary: The adhesion of both leukocytes and platelets to microvascular endothelial cells has been implicated in the pathogenesis of ischemia/reperfusion (I/R) injury in several vascular beds. The objectives of this study were to (1) assess the platelet–leukocyte–endothelial cell interactions induced in the cerebral microvasculature by middle cerebral artery occlu­sion (MCAO)/reperfusion, and (2) define the molecular deter­minants of the prothrombogenic and inflammatory responses in this model of focal I/R. MCAO was induced for 1 hour in wild-type (WT) mice, WT mice treated with a monoclonal antibody (mAb) to either P-selectin or GPIIb/IIIa, and in P-selectin−/−(P-sel−/−) chimeras. Isolated platelets labeled with carboxyfluorescein diacetate succinimidyl ester (CFDASE) were administered intravenously and observed with intravital fluorescence microscopy. Leukocytes were observed after in­travenous injection of rhodamine 6G. One hour of MCAO fol­lowed by 1 hour of reperfusion resulted in the rolling and adhesion of leukocytes in venules, and after 4 hours of reperfusion, the adhesion of both leukocytes and platelets was de­tected. Although both the P-selectin and GPIIb/IIIa mAbs sig­nificantly reduced the adhesion of leukocytes and platelets at 4 hours of reperfusion, the antiadhesive effects of the P-selectin mAb were much greater. The leukocyte and platelet adhesion responses were significantly attenuated in both P-sel−/−-WT and WT-P-sel−/− bone marrow chimeras, compared with WT-WT chimeras. Neutropenia, induced by antineutrophil serum treatment, also reduced the recruitment of leukocytes and platelets after cerebral I/R. These findings implicate a ma­jor role for both platelet-associated and endothelial cell– associated P-selectin, as well as neutrophils in the inflamma­tory and prothrombogenic responses in the microcirculation after focal cerebral I/R.
Key Words: Platelet—Leukocyte—P-selectin—GPIIb/IIIa—Cerebral ischemia—Reperfusion.

Adhesion of leukocytes and platelets after treatment with mAb against P-selectin or GPIIIb/IIIa

The I/R-induced recruitment of rolling and adherent leuko­cytes was significantly attenuated in P-selectin mAb-treated mice, compared with the responses noted in untreated mice exposed to 1-hour MCAO and 4-hour reperfusion (Figs. 3A and 3B). However, the number of adherent leukocytes after P-selectin mAb treatment remained elevated above the level de­tected in sham experiments. Both the rolling and firm adhesion of platelets was reduced to sham levels in the P-selectin mAb-treated mice. Although treatment with a GPIIb/IIIa mAb sig­nificantly reduced the adhesion of both platelets and leukocytes after I/R, the reductions noted were relatively small compared with the responses seen with the P-selectin mAb.

Leukocyte and platelet adhesion in P-selectin–deficient bone marrow chimeras

Our findings related to the role of platelet-associated and endothelial cell–associated P-selectin in mediating the I/R-induced rolling and adhesion of leukocytes and platelets are summarized in Fig. 4.  In P-sel / -WT chimeras, the number of rolling and adherent leukocytes were significantly but not completely reduced compared with WT—WT chimeras. However, compared with WT—WT chimeras, the rolling and firm adhesion of platelets was virtually abolished after I/R. In WT—P-sel−/− chimeras, the number of rolling and adherent leu­kocytes and platelets also decreased significantly com­pare with WT—WT chimeras; however, some adhesion of leukocytes and platelets was still detected after I/R, similar to the responses noted in the group treated with the P-selectin blocking mAb.

Plateletleukocyte interaction

Platelets were noted to adhere directly onto adherent leukocytes and platelet-bearing leukocytes were occa­sionally observed rolling in postischemic venules. Some free-flowing platelets were seen to suddenly bind (with-out rolling) on adherent leukocytes. Some of these plate­lets detached from the adherent leukocyte whereas others adhered firmly on the leukocyte. Other platelets were seen to roll and adhere directly on venular endothelium. To quantify the contribution of leukocytes to I/R-induced platelet recruitment, some mice were rendered neutropenic with antineutrophil serum. Although leuko­cyte rolling and adherence were still observed in cerebral venules of serum-treated mice after I/R, the responses were dramatically reduced. The cerebral venules of neutropenic mice also exhibited large and significant reduc­tions in rolling and adherent platelets after I/R (Fig. 5).

Fig  platelet and endothelial cell–associated P-selectin in mediating rolling and adhesion of leukocytes

FIG. 4. Role of platelet-associated and endothelial cell–associated P-selectin in mediating I/R-induced rolling and adhesion of leuko­cytes (A) and platelets (B). Four or five animals were studied in each group. Mice in all groups were exposed to 1 hour of MCAO followed by 4 hours of reperfusion. WT—*WT and WT—*P-sel−/− chi­meras received CFDASE-labeled platelets from WT mice. P-sel−/−—*WT chimeras received CFDASE-labeled platelets from P-sel−/− mice. *P < 0.05 relative to the WT*WT (control) chimeras.

Signal-Dependent Protein Synthesis by Activated Platelets: New Pathways to Altered Phenotype and Function

Guy A. Zimmerman and Andrew S. Weyrich
Arterioscler Thromb Vasc Biol. 2008;28:s17-s24       http://dx.do.org/10.1161/ATVBAHA.107.160218      http://atvb.ahajournals.org/content/28/3/s17         Online ISSN: 1524-4636

New biologic activities of platelets continue to be discovered, indicating that concepts of platelet function in hemostasis, thrombosis, and inflammation require reconsideration as new paradigms evolve. Studies done over 3 decades ago demonstrated that mature circulating platelets have protein synthetic capacity, but it was thought to be low level and inconsequential. In contrast, recent discoveries demonstrate that platelets synthesize protein products with important biologic activities in a rapid and sustained fashion in response to cellular activation. This process, termed signal-dependent translation, uses a constitutive transcriptome and specialized pathways, and can alter platelet phenotype and functions in a fashion that can have clinical relevance. Signal-dependent translation and consequent protein synthesis are examples of a diverse group of posttranscriptural mechanisms in activated platelets that are now being revealed. (Arterioscler Thromb Vasc Biol. 2008;28:s17-s24)
Key Words: platelets . translation . protein synthesis . transcriptome . proteome . thrombosis

This article is part of a multi-part CME-certified activity titled Translational Therapeutics at the Platelet Vascular Interface. 

New Paradigms at the Vascular Interface

The acute hemostatic functions of platelets are well known, have dominated the attention of the field for decades, and have been the founda­tion for discoveries that generated new molecular therapies. Rapid, immediate activation responses mediate platelet-dependent thrombosis in a variety of pathologic conditions, and pharmacological antiplatelet strategies are largely aimed at these events. Nevertheless, the focus on adhesion, aggre­gation, and secretion, and the view that platelets have a repertoire of activities primarily restricted to these acute processes, have also generated a central dogma that may inappropriately limit our view of their actions at the vascular interface and in other settings in health and disease. Clearly, our understanding of the molecular mechanisms by which platelets influence hemostasis, thrombosis, regulated and dysregulated inflammation, and neoplasia remains incom­plete and continues to evolve. New paradigms are emerging as previously unrecognized pathways in platelets are identi­fied, and unanticipated activities are characterized. In this regard, the current state of the field of platelet biology may be akin to that of endothelial cells several decades ago, when endothelium was thought by most investigators and physi­cians to have a limited range of responses; on the contrary, however, when this dogma was reexamined using new approaches that included primary culture of human endothe-lium, active participation of these cells in interactions with leukocytes and a variety of other previously unrecognized functions were discovered. If the comparison is accurate, new paradigms relevant to activities of platelets at the vascular interface are likely to be reported with some frequency.

Alternative and traditional views of selected features of platelet biology are listed in the Table. There is already considerable evidence for some of the alternative themes, such as inflammatory and immune activities of platelets,10–16 whereas others are less well explored and more speculative. The remainder of this review summarizes evidence for one such functional capability not generally recognized in plate­lets until recent discoveries revealed it: synthesis of new protein products in response to cellular activation (reviewed in references5,17).

Table. New Biology of Platelets: Traditional Paradigms May Be Insufficient to Understand Platelet Activities at the Vascular Interface

Traditional View                                                                                                                                              Alternate View

Platelets are biologically simple because they are anucleate                   Platelets have specializations and biologic activities that are novel and complex. Some

and have a limited repertoire of responses.                                                                                     activities are yet to be discovered.

Platelets do not express new gene products.                                             Platelets have diverse posttranscriptional mechanisms and use a transcriptome and

specialized pathways to modify their proteome, phenotype, and functions.

Platelets are short-acting cells in clots and damaged tissue.                      Platelets can be relatively long-lived and can mediate cell-cell interactions for many

hours after initial adhesion, aggregation, and secretion.

Platelets operate exclusively in the intravascular                                               Platelets can influence critical events in the extravascular milieu in direct

compartment.                                                                                                                                            and indirect fashions.

Observations from a number of laboratories now demonstrate that physiologically relevant activation signals induce translation of proteins with impor­tant functions from constitutive or posttranscriptionally pro­cessed messenger RNAs (mRNAs) in human and murine platelets, a process that we have termed signal-dependent translation. These and other studies indicate that the platelet has intricate posttranscriptional mechanisms that allow it to alter its proteome, phenotype, and functions by accomplish­ing new protein synthesis in response to cellular activation. This capacity may allow platelets to modify the complex milieu of the vascular interface in ways that were previously unrecognized.

Essentially, all of the platelets isolated from normal subjects incorporated radiolabeled amino acids into new protein, demonstrating that this function is not a property of a subset of immature cells. Platelets from splenectomized subjects with idiopathic throm-bocytopenic purpura had increased levels of amino acid incorporation into protein, indicating that the physiological state of the subject or the age and maturity of the platelets influence protein synthesis. Extracellular factors were re­ported to alter protein synthesis by human platelets under some conditions. This provided evidence suggesting that the synthetic mechanisms involved are regulated.

The Platelet Transcriptome

Circulating human platelets have a substantial and diverse transcriptome, in addition to protein synthetic machinery. RNA-selective fluorescent dyes stain the entire population of platelets isolated from normal subjects, indicating the presence of RNA species transcribed by parent megakaryocytes. Messenger RNAs with 5′-methylguanosyl (m7G) caps and 3′ untranslated region polyadenylated tails are present, as are 18S and 28S ribosomal proteins, which are integral to the structure of ribo-somes. Early experiments with intact platelets from nor­mal subjects indicated that some of the mRNA transcripts are competent to serve as templates for proteins and have relatively long functional half lives that correlate with the lifespan of platelets in the circulation. This observation then lay fallow, for the most part, until the advent of reverse transcriptase polymer-ase chain reaction (RT-PCR) analysis and cDNA cloning meth-odologies. This infusion of new technology resulted in construction of cDNA libraries from platelet transcripts. Most recently, transcript profiling by microarray analysis and serial analysis of gene expression (SAGE) have been applied to platelets, identifying 1500 to 3000 unique transcripts in platelets from normal subjects, depending on the approach. Both cytoplasmic and mitochondrial transcripts are represented.35 There is substantial consistency between data generated by microarray analysis and SAGE, and in platelets isolated from different normal donors.

Multiple Proteins Are Synthesized by Activated Human Platelets

Although early studies indicated that platelets have protein synthetic capacity, the general concept in the field has been that it is low level, vestigial, and likely inconsequential. Several texts of hemostasis and platelet biology do not mention this function, and some commentaries conclude that platelets are simply incapable of any new protein synthesis. Consistent with the notion that platelets have low basal protein synthesis, little incorporation of the radiolabeled amino acid is detectable when freshly isolated human plate­lets are incubated with [35S] methionine under resting condi­tions in the absence of activation. However, when an activat­ing signal is delivered to platelets incubated in parallel, multiple labeled proteins are synthesized when lysates and soluble fractions are analyzed by 1-dimensional or 2-dimensional gel electrophoresis (Lindemann S, Weyrich AS, Zimmerman GA, 2001). Some of these newly synthe­sized proteins have been identified and mechanisms of their signal-dependent translation determined.

Recent findings provided clear evidence for signal-dependent (that is, induced by activating signals) translation of Bcl-3 from mRNA that is transcribed in parent megakaryocytes but is repressed, or “silenced,” in circulating platelets under resting, basal conditions. Immunocytochemical de­tection of Bcl-3 in platelets in inflamed and thrombosed human vessels in surgical specimens (Figure 1D) provided in situ evidence that the experimental observations have physi­ological and clinical relevance. We subsequently found that collagen, platelet-activating factor, ADP, and epinephrine are also agonists for signal-dependent translation in plate-lets. Collagen was recently reported to induce Bcl-3 synthesis by platelets in experiments by other investigators. The time course of Bcl-3 synthesis in response to thrombin yielded additional important insights: newly synthesized Bcl-3 could be detected in activated platelets within 15 to 30 minutes in some experiments, consistent with translation of constitutively present mRNA without a requirement for new transcription. This feature is also consistent with the biology of platelets as rapid response cells. Nevertheless, synthesis of Bcl-3 is also prolonged over many hours, indicating that platelets may have important functions in thrombi and injured vessels well beyond the first few minutes of acute activation.

We examined the effect of rapamycin and found that it completely and selectively inhibited Bcl-3 synthesis in thrombin-stimulated platelets, and also inhibited phosphorylation of 4E-BP1 assayed as a marker of mTOR activation in parallel. Pharmacological inhibition of phosphatidylinositol-3-kinase, which lies upstream from

mTOR in signaling cascades linking surface receptors to mTOR activation,49 also blocked both 4E-BP1 phosphoryla-tion and Bcl-3 synthesis.52 Together, these studies demon­strated that synthesis of Bcl-3 is controlled by mTOR and provided evidence for a new and previously unrecognized activity of mTOR as a regulator of expression of specific protein products and phenotypic changes in terminally differ­entiated cells in response to signals delivered via G protein– coupled receptors and integrins.52 This observation in platelets contributed to a parallel set of discoveries demonstrating that mTOR has similar roles in myeloid leukocytes.69–71 The find­ings also suggest that inhibition of mTOR by rapamycin may have novel therapeutic effects on gene expression by platelets and leukocytes independent of inhibition of proliferation of other cell types when this agent is applied in antiangiogenic strategies and in “drug-eluting” vascular stents in the clinic.72

Although Bcl-3 provided an index example of specialized, signal-dependent translation of a protein product in activated platelets the functional relevance of this event was not immediately obvious and was initially perplexing because the activity assigned to Bcl-3 at that time was as a transcriptional regulator. A clue lay in the domain structure of Bcl-3, which includes ankyrin repeats and proline-rich N and C termini, suggesting the possibility of multiple protein-protein interactions. Based on this information, we designed experi­ments to determine whether newly synthesized Bcl-3 interacts with other intracellular proteins. We found that Bcl-3 specifi­cally binds to the tyrosine kinase Fyn via the Fyn SH2 domain in activated platelets and transfected COS cells. Bcl-3 also associates with the actin cytoskeleton in platelets.53

Because Fyn and related intracellular tyrosine kinases influence contractile responses of activated platelets, we examined the contributions of Bcl-3 and mTOR to fibrin clot retraction. Clot retraction is proposed to stabilize thrombi and to modify thrombus remodeling and resolution. It can be modeled in vitro, where activated platelets retract and condense fibrin strands in a fashion that can be examined macroscopically and microscopically (Figure 1E). In paral­lel loss-of-function and gain-of-function strategies, inhibition of mTOR activity in human platelets using rapamycin under conditions that block Bcl-3 synthesis inhibited clot retraction,

Common and Specialized Elements in Platelet Translational Pathways and Transcripts

Biologic Advantages of Signal-Dependent Translation, and Potential Roles in Disease

Novel Pathways to Signal-Dependent Translation in Activated Human Platelets

Activated Platelets Synthesize Additional Proteins Under Signal-Dependent Control

Translation in Activated Human Platelets

Discovery of synthesis of Bcl-3 by activated platelets sparked a search for the identities of other protein products, yielding IL-1J3 and TF. It also led to the unexpected discovery that their synthesis is preceded by signal-dependent cytoplasmic splicing of IL-1J3 and TF pre-mRNAs, yielding mature transcripts that are translated into precursor (IL-1J3) and active (TF) proteins.24,43,44 This identified a novel mechanism not previously recognized in activated mammalian cells. The splicing capacities of activated platelets are intricate and will be reviewed separately. Signal-dependent splicing, to­gether with the mTOR-dependent translational control mech­anism and other regulatory pathways discussed here, indicate that platelets have unexpected diversity in posttranscriptional control. Previous and ongoing studies add to this conclusion and suggest that platelets may also use ribosomal “stalling” or polypeptide termination, participation of micro RNAs (Denis MM, Trask B, Schwertz H, Weyrich AS, Zimmerman GA, 2004) and, potentially, other modes of control.

References

  1. Lindemann S, McIntyre TM, Prescott SM, Zimmerman GA, Weyrich AS. Platelet signal-dependent protein synthesis. In: Quinn M, Fitzgerald D, eds. Platelet Function: Assessment, Diagnosis, and Treatment. Totowa, NJ: Humana Press Inc.;2005:149-74.
  2. Weyrich AS, Lindemann S, Tolley ND, Kraiss LW, Dixon DA, Mahoney TM, Prescott SP, McIntyre TM, Zimmerman GA. Change in protein phenotype without a nucleus: translational control in platelets. Semin Thromb Hemost. 2004;30:491–498.

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Interaction of Nitric Oxide and Prostacyclin in Vascular Endothelium https://pharmaceuticalintelligence.com/2012/09/14/interaction-of-nitric-oxide-and-prostacyclin-in-vascular-endothelium/

Cardiovascular Disease (CVD) and the Role of Agent Alternatives in endothelial Nitric Oxide Synthase (eNOS) Activation and Nitric Oxide Production Aviva Lev-Ari, PhD, RN
https://pharmaceuticalintelligence.com/2012/07/19/cardiovascular-disease-cvd-and-the-role-of-agent-alternatives-in-endothelial-nitric-oxide-synthase-enos-activation-and-nitric-oxide-production/

 

nihms-292073-f0002  platelet and vessel

Protein_Slide_2  proteome

nihms-292073-f0001  platelets support integrity and barrier function

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Nitric Oxide, Platelets, Endothelium and Hemostasis (Coagulation Part II)

Curator: Larry H. Bernstein, MD, FCAP 

Subtitle: Nitric oxide and hemostatic mechanisms.  Part II.

Summary: This is the second of a coagulation series on

http://pharmaceuticalIntelligence.com

Treating the diverse effects of NO on platelets, the coagulation cascade, and protein-membrane interactions with low flow states, local and systemic inflammatory disease, oxidative stress, and hematologic disorders.  It is highly complex as the distinction between intrinsic and extrinsic pathways become blurred as a result of  endothelial shear stress, distinctly different than penetrating or traumatic injury.  In addition, other factors that come into play are also considered.

Please refer to Part I. Coagulation Pathway

https://pharmaceuticalintelligence.com/2012/11/26/biochemistry-of-the-coagulation-cascade-and-platelet-aggregation/

The workhorse tests of the modern coagulation laboratory, the prothrombin time (PT) and the activated partial thromboplastin time (aPTT), are the basis for the published extrinsic and intrinsic coagulation pathways.  This is, however, a much simpler model than one encounters delving into the mechanism and interactions involved in hemostasis and thrombosis, or in hemorrhagic disorders.

We first note that there are three components of the hemostatic system in all vertebrates:

  • Platelets,
  • vascular endothelium, and
  • plasma proteins.

The liver is the largest synthetic organ, which synthesizes

  • albumin,
  • acute phase proteins,
  • hormonal and metal binding proteins,
  • albumin,
  • IGF-1, and
  • prothrombin, mainly responsible for the distinction between plasma and serum (defibrinated plasma).

Role of vascular endothelium.

I have identified the importance of prothrombin, thrombin, and the divalent cation Ca 2+ (1% of the total body pool), mention of heparin action, and of vitamin K (inhibited by warfarin).  Endothelial functions are inherently related to procoagulation and anticoagulation. The subendothelial matrix is a complex of many materials, most important related to coagulation being collagen and von Willebrand factor.

What about extrinsic and intrinsic pathways?  Tissue factor, when bound to factor VIIa, is the major activator of the extrinsic pathway of coagulation. Classically, tissue factor is not present in the plasma but only presented on cell surfaces at a wound site, which is “extrinsic” to the circulation.  Or is it that simple?

Endothelium is the major synthetic and storage site for von Willebrand factor (vWF).  vWF is…

  • secreted from the endothelial cell both into the plasma and also
  • abluminally into the subendothelial matrix, and
  • acts as the intercellular glue binding platelets to one another and also to the subendothelial matrix at an injury site.
  • acts as a carrier protein for factor VIII (antihemophilic factor).
  • It  binds to the platelet glycoprotein Ib/IX/V receptor and
  • mediates platelet adhesion to the vascular wall under shear. [Lefkowitz JB. Coagulation Pathway and Physiology. Chapter I. in Hemostasis Physiology. In ( ???), pp1-12].

Ca++ and phospholipids are necessary for all of the reactions that result in the activation of prothrombin to thrombin. Coagulation is initiated by an extrinsic mechanism that

  • generates small amounts of factor Xa, which in turn
  • activates small amounts of thrombin.

The tissue factor/factorVIIa proteolysis of factor X is quickly inhibited by tissue factor pathway inhibitor (TFPI).The small amounts of thrombin generated from the initial activation feedback

  • to create activated cofactors, factors Va and VIIIa, which in turn help to
  • generate more thrombin.
  • Tissue factor/factor VIIa is also capable of indirectly activating factor X through the activation of factor IX to factor IXa.
  • Finally, as more thrombin is created, it activates factor XI to factor XIa, thereby enhancing the ability to ultimately make more thrombin.

The reconceptualization of hemostasis 

The common theme in activation and regulation of plasma coagulation is the reduction in dimensionality. Most reactions take place in a 2D world that will increase the efficiency of the reactions dramatically. The localization and timing of the coagulation processes are also dependent on the formation of protein complexes on the surface of membranes. The coagulation processes can also be controlled by certain drugs that destroy the membrane binding ability of some coagulation proteins – these proteins will be lost in the 3D world and not able to form procoagulant complexes on surfaces.

Assembly of proteins on membranes – making a 3D world flat

• The timing and efficiency of coagulation processes are handled by reduction in dimensionality

– Make 3 dimensions to 2 dimensions

• Coagulation proteins have membrane binding capacity

• Membranes provide non-coagulant and procoagulant surfaces

– Intact cells/activated cells

• Membrane binding is a target for anticoagulant drugs

– Anti-vitamin K (e.g. warfarin)

Modern View

It can be divided into the phases of initiation, amplification and propagation.

  • In the initiation phase, small amounts of thrombin can be formed after exposure of tissue factor to blood.
  • In the amplification phase, the traces of thrombin will be inactivated or used for amplification of the coagulation process.

At this stage there is not enough thrombin to form insoluble fibrin. In order to proceed further thrombin  activates platelets, which provide a procoagulant surface for the coagulation factors. Thrombin will also activate the vital cofactors V and VIII that will assemble on the surface of activated platelets. Thrombin can also activate factor XI, which is important in a feedback mechanism.

In the final step, the propagation phase, the highly efficient tenase and prothrombinase complexes have been assembled on the membrane surface. This yields large amounts of thrombin at the site of injury that can cleave fibrinogen to insoluble fibrin. Factor XI activation by thrombin then activates factor IX, which leads to the formation of more tenase complexes. This ensures enough thrombin is formed, despite regulation of the initiating TF-FVIIa complex, thus ensuring formation of a stable fibrin clot. Factor XIII stabilizes the fibrin clot through crosslinking when activated by thrombin.

Platelet Aggregation

The activities of adenylate and guanylate cyclase and cyclic nucleotide 3′:5′-phosphodiesterase were determined during the aggregation of human blood platelets with

  • thrombin, ADP,
  • arachidonic acid and
  • epinephrine.

[Aggregation is dependent on an intact release mechanism since inhibition of aggregation occurred with adenosine, colchicine, or EDTA.  (Herman GE, Seegers WH, Henry RL. Autoprothrombin ii-a, thrombin, and epinephrine: interrelated effects on platelet aggregation. Bibl Haematol 1977;44:21-7)].

  1. The platelet guanylate cyclase activity during aggregation depends on the nature and mode of action of the inducing agent.
  2. The membrane adenylate cyclase activity during aggregation is independent of the aggregating agent and is associated with a reduction of activity and
  3. Cyclic nucleotide phosphodiesterase remains unchanged during the process of platelet aggregation and release.

The role of platelets in arterial thrombosis

Formation of a thrombus on a ruptured plaque is the product of a complex interaction between coagulation factors in the plasma and platelets.

  • Tissue factor (TF) released from the subendothelial tissue after endothelial damage induces a cascade of activation of coagulation factors ultimately leading to the formation of thrombin.
  • Thrombin cleaves fibrinogen to fibrin, which assembles into a mesh that supports the platelet aggregates.

The Platelet

The platelets are …

  • anucleated,
  • discoid shaped cell fragments
  • originating from megakaryocytes
  •  fragmented as they are released from the bone marrow

Whether they can in circumstances be developed at extramedullary sites (liver sinusoid) is another matter. They have a lifespan of 7-10 days.  Of special interest is:

  • They have a network of internal membranes forming a dense tubular system and the open canalicular system (OCS).
  • The plasma membrane is an extension of the OCS, thereby greatly increasing the surface area of the platelet.
  • The dense tubular system is comparable to the endoplasmatic reticulum in other cell types and is the main storage place of the majority of the platelet’s Ca2+.

Three types of secretory granules exist in platelets:

  • the dense granules
    •  In the dense granules serotonin
    • adenosine diphosphate (ADP) and
    • Ca2+ are stored.
    • a-granules contain
      • P-selectin,
      • fibrinogen,
      •  thrombospondin,
      • Von Willebrand Factor,
      • platelet factor 4 and
      • platelet derived growth factor
      • lysosomes.

Circulating platelets are kept in a resting state by endothelial cell derived

  • prostacyclin (PGI2) and
  • nitric oxide (NO).

PGI2 increases cyclic adenosine monophosphate (cAMP), the most potent platelet inhibitor.

Contact activation

The major regulator of the activation of the contact system is the plasma protease inhibitor, C1-INH, which inhibits activated fXII, kallikrein and fXIa. In addition, α2-macroglobulin is an important inhibitor of kallikrein and α1-antitrypsin for fXIa. Factor XII also converts the fXI to an active enzyme, fXIa, which, in turn, converts fIX to fIXa, thereby activating the intrinsic pathway of coagulation.

Activation

Several agonists can activate platelets;

  • ADP,
  • collagen,
  • thromboxane A2 (TxA2),
  • epinephrin,
  • serotonine and
  • thrombin,

which lead to activation previously referred to:

  • platelet shape change is
  • followed by aggregation and
  • granule secretion.

Upon activation the discoid shape changes into a spherical form.

Activation of platelets is increased by two positive feedback loops

  1. arachidonic acid is cleaved from phospholipids and transformed by cyclooxygenase

(COX) to prostaglandin G2 and H2,

  • followed by the formation of TxA2, a potent platelet agonist.

2.   the secretion of ADP by the dense granules,

  • resulting in activation of the ADP receptor P2Y12.

This causes inhibition of cyclic AMP and sustained aggregation.

Aggregation

The integrin receptor αIIbβ3 plays a vital role in platelet aggregation. The platelet agonists

  • induce a conformational change of the αIIbβ3 receptor and
  • exposition of binding domains for fibrinogen and von Willebrand Factor.

This allows cross-linking of platelets and the formation of aggregates.

In addition to shape change and aggregation, the membranes of the α- and dense granules fuse with the membranes of the OCS. This causes the release of their contents and the transportation of proteins embedded in their membrane to the plasma membrane.

This complex interaction between

  • endothelial cells
  • clotting factors
  • platelets and
  • other factors and cells

can be studied in both in vitro and in vivo model systems. The disadvantage of in vitro assays is that it studies the role of a certain protein or cell in isolation. Given the large number of participants and the complex interactions of thrombus formation there is need to study thrombosis and hemostasis in intact living animals, with all the components important for thrombus formation – a vessel wall and flowing blood – present.

Endothelial Damage and Role as “Primer”

  • Endothelial injury changes the permeability of the arterial wall.
  • This is followed by an influx of low-density lipoprotein (LDL).
  • This elicits an inflammatory response in the vascular wall.
  • Monocytes and T-cells bind to the endothelial cells promoting increased migration of the cells into the intima layer
  • The monocytes differentiate into macrophages, which take up modified lipoproteins and transform them into foam cells.
  • Concurrent with this process macrophages produce cytokines and proteases.

This is a vicious circle of lipid driven inflammation that leads to narrowing of the vessel’s lumen without early clinical consequences. Clinical manifestations of more advanced atherosclerotic disease are caused by destabilization of an atherosclerotic plaque formed as described.

  • The first recognizable lesion of the stable atherosclerotic plaque is the fatty streak, which consists of the above described foam cells and T-lymphocytes in the intima.
  • Further development of the lesion leads to the intermediate lesion, composed
  • of layers of macrophages and smooth muscle cells.
  • A more advanced stage is called the vulnerable plaque.
    • It has a large lipid core that is covered by a thin fibrous cap.
    • This cap separates the lipid contents of the plaque from the circulating blood.
    • The vulnerable plaque is prone to rupture, resulting in the formation of a thrombus on the site of disruption or the thrombus can be superimposed on plaque erosion without signs of plaque rupture.

The formation of a superimposed thrombus on a disrupted atherosclerotic plaque in the lumen of the artery leads to

  • an acute occlusion of the vessel
  • hypoxia of the downstream tissue.

Depending on the location of the atherosclerotic plaque this will cause a myocardial infarction, stroke or peripheral vascular disease.

Endothelial regulation of coagulation

The endothelium attenuates platelet activity by releasing

  • nitric oxide and
  • prostacyclin.

Several coagulation inhibitors are produced by endothelial cells.

Endothelium-derived TFPI (on its surface) is rapidly released into circulation after heparin administration, reducing the pro-coagulant activities of TF-fVIIa. Endothelial cells also secrete heparin-sulphate, a glycosaminoglycan which catalyzes anti-coagulant activity of AT. Plasma AT binds to heparin-sulphate located on the luminal surface and in the basement membrane of the endothelium. Thrombomodulin is another endothelium-bound protein with anti-coagulant and anti-inflammatory functions. In response to systemic pro-coagulant stimuli, tissue-type plasminogen activator (tPA) is transiently released from the Weibel-Palade bodies of endothelial cells to promote fibrinolysis. Downstream of the vascular injury, the complex of TF-fVIIa/fXa is inhibited by TFPI. Plasma (free) fXa and thrombin are rapidly neutralized by heparan-bound AT. Thrombin is also taken up by endothelial surface-bound thrombomodulin.

The protein C pathway works in hemostasis to control thrombin formation in the area surrounding the clot. Thrombin, generated via the coagulation pathway, is localized to the endothelium by binding to the integral membrane protein, thrombomodulin (TM). TM by occupying exosite I on thrombin, which is required for fibrinogen binding and cleavage, reduces thrombin’s pro-coagulant activities. TM bound thrombin  on the endothelial cell surface is able to cleave PC producing activated protein C (APC), a serine protease.  In the presence of protein S, APC inactivates FVa and FVIIIa. The proteolytic activity of APC is regulated predominantly by a protein C inhibitor.

Fibrinolytic pathway

Fibrinolysis is the physiological breakdown of fibrin to limit and resolve blood clots. Fibrin is degraded primarily by the serine protease, plasmin, which circulates as plasminogen. In an auto-regulatory manner, fibrin serves as both the co-factor for the activation of plasminogen and the substrate for plasmin. In the presence of fibrin, tissue plasminogen activator (tPA) cleaves plasminogen producing plasmin, which proteolyzes the fibrin. This reaction produces the protein fragment D-dimer, which is a useful marker of fibrinolysis, and a marker of thrombin activity because fibrin is cleaved from fibrinogen to fibrin.

Nitric Oxide and Platelet Energy Production

Nitric oxide (NO) has been increasingly recognized as an important intra- and intercellular messenger molecule with a physiological role in

  • vascular relaxation
  • platelet physiology
  • neurotransmission and
  • immune responses.

In vitro NO is a strong inhibitor of platelet adhesion and aggregation. In the blood stream, platelets remain in contact with NO that is permanently released from the endothelial cells and from activated macrophages. It  has been suggested that the activated platelet itself is able to produce NO. It has been proposed that the main intracellular target for NO in platelets is soluble cytosolic guanylate cyclase. NO activates the enzyme. When activated, intracellular cGMP elevation inhibits platelet activation. Further, elevated cGMP may not be the sole factor directly involved in the inhibition of platelet activation.

The reaction mechanism of Nitric oxide synthase

The reaction mechanism of Nitric oxide synthase (Photo credit: Wikipedia)

Platelets are fairly active metabolically and have a total ATP turnover rate of about 3–8 times that of resting mammalian muscle. Platelets contain mitochondria which enable these cells to produce energy both in the oxidative and anaerobic pathways.

  • Under aerobic conditions, ATP is produced by aerobic glycolysis which can account for 30–50% of total ATP production,
  • by oxidative metabolism using glucose and glycogen (6–11%), amino-acids (7%) or free fatty acids (20–40%).

The inhibition of mitochondrial respiration by removing oxygen or by respiratory chain blockers (antimycin A, cyanide, rotenone) results in the stimulation of glycolytic flux. This phenomenon indicates that in platelets glycolysis and mitochondrial respiration are tightly functionally connected. It has been reported that the activation of human platelets by high concentration of thrombin is accompanied by an acceleration of lactate production and an increase in oxygen consumption.

The results (in porcine platelets) indicate that:

  • NO is able to diminish mitochondrial energy production through the inhibition of cytochrome oxidase
  • The inhibitory effect of NO on platelet secretion (but not aggregation) can be attributed to the reduction of mitochondrial energy production.

Porcine blood platelets stimulated by collagen produce more lactate. This indicates that both glycolytic and oxidative ATP production supports platelet responses, and blocking of energy production in platelets may decrease their responses. It is well established that platelet responses have different metabolic energy (ATP) requirements increasing in the order:

  • Aggregation
  • < dense and alfa granule secretion
  • < acid hydrolase secretion.

In addition, exogenously added NO (in the form of NO donors) stimulates glycolysis in intact porcine platelets. Since in platelets glycolysis and mitochondrial respiration are tightly functionally connected, this indicates the stimulatory effect of NO on glycolysis in intact platelets may be produced by non-functional mitochondria.

Can this be the case?

  • NO donors are able to inhibit both mitochondrial respiration and platelet cytochrome oxidase.
  • Interestingly, the concentrations of NO donors inhibiting mitochondrial respiration and cytochrome oxidase were similar to those stimulating glycolysis in intact platelets.

Studies have shown that mitochondrial complex I is inhibited only after a prolonged (6–18 h) exposure to NO and

  • This inhibition appears to result from S-nitrosylation of critical thiols in the enzyme complex.
  • Further studies are needed to establish whether long term exposure of platelets to NO affects Mitochondrial complexes I and II.

Comparison of the concentrations of SNAP and SNP affecting cytochrome oxidase activity and mitochondrial respiration with those reducing the platelet responses indicates that NO does not reduce platelet aggregation through the inhibition of oxidative energy production. The concentrations of the NO donors inhibiting platelet secretion, mitochondrial respiration and cytochrome oxidase were similar. Thus, the platelet release reaction strongly depends on the oxidative energy production, and  in porcine platelets NO inhibits mitochondrial energy production at the step of cytochrome oxidase.

Taking into account that platelets may contain NO synthase and are able to produce significant amounts of NO it seems possible that nitric oxide can function in these cells as a physiological regulator of mitochondrial energy production.

Key words: glycolysis, mitochondrial energy production, nitric oxide, porcine platelets.
Abbreviations: NO, nitric oxide; SNAP, S-nitroso-N-acetylpenicyllamine; SNP, sodium nitroprusside.

[M Tomasiak, H Stelmach, T Rusak and J Wysocka.  Nitric oxide and platelet energy metabolism.  Acta Biochimica Polonica 2004; 51(3):789–803.]

Nitric Oxide and Platelet Adhesion

The adhesion of human platelets to monolayers of bovine endothelial cells in culture was studied to determine the role of endothelium-derived nitric oxide in the regulation of platelet adhesion. The adhesion of unstimulated and thrombin-stimulated platelets, washed and labelled with indium-111, was lower in the presence than in the absence of bradykinin or exogenous nitric oxide. The inhibitory action of both bradykinin and nitric oxide was abolished by hemoglobin, but not by aspirin, and was potentiated by superoxide dismutase to a similar degree. It appears that the effect of bradykinin is mediated by the release of nitric oxide from the endothelial cells, and that nitric oxide release contributes to the non-adhesive properties of vascular endothelium.

(Radomski MW, Palmer RMJ, Moncada S.   Endogenous Nitric Oxide Inhibits Human Platelet Adhesion to Vascular Endothelium. The Lancet  1987 330; 8567(2): 1057–1058.
http://dx.doi.org/10.1016/S0140-6736(87)91481-4)

1 The interactions between endothelium-derived nitric oxide (NO) and prostacyclin as inhibitors of platelet aggregation were examined to determine whether release of NO accounts for the inhibition of platelet aggregation attributed to EDRF.

2 Porcine aortic endothelial cells treated with indomethacin and stimulated with bradykinin (10-100 nM) released NO in quantities sufficient to account for the inhibition of platelet aggregation attributed to endothelium-derived relaxing factor (EDRF).

3 In the absence of indomethacin, stimulation of the cells with bradykinin (1-3 nM) released small amounts of prostacyclin and EDRF which synergistically inhibited platelet aggregation.

4 EDRF and authentic NO also caused disaggregation of platelets aggregated either with collagen or with U46619.

5 A reciprocal potentiation of both the anti- and the disaggregating activity was also observed between low concentrations of prostacyclin and authentic NO or EDRF released from endothelial cells.

6 It is likely that interactions between prostacyclin and NO released by the endothelium play a role in the homeostatic regulation of platelet-vessel wall interactions.

(Radomski MW, Palmer RMJ & Moncada S. The anti-aggregating properties of vascular endothelium: interactions between prostacyclin and nitric oxide. Br J Pharmac 1987; 92: 639-646.

 

Factor Xa–Nitric Oxide Signaling

Although primarily recognized for maintaining the hemostatic balance, blood proteases of the coagulation and fibrinolytic cascades elicit rapid cellular responses in

  • vascular
  • mesenchymal
  • inflammatory cell types.

Considerable effort has been devoted to elucidate the molecular interface between protease-dependent signaling and pleiotropic cellular responses. This led to the identification of several membrane protease receptors, initiating intracellular signal transduction and effector functions in vascular cells. In this context, thrombin receptor activation

  • generated second messengers in endothelium and smooth muscle cells,
  • released inflammatory cytokines from monocytes, fibroblasts, and endothelium, and
  • increased the expression of leukocyte-endothelial cell adhesion molecules.

Similarly, binding of factor Xa to effector cell protease receptor-1 (EPR-1) participated in

  • in vivo acute inflammatory responses,
  • platelet and brain pericyte prothrombinase activity, and
  • endothelial cell and smooth muscle cell signaling and proliferation.

Factor Xa stimulated a 5- to 10-fold increased release of nitric oxide (NO) in a dose-dependent reaction (0.1–2.5 mgyml) unaffected by the thrombin inhibitor hirudin but abolished by active site inhibitors, tick anticoagulant peptide, or Glu-Gly-Arg-chloromethyl ketone. In contrast, the homologous clotting protease factor IXa or another endothelial cell ligand, fibrinogen, was ineffective.

A factor Xa inter-epidermal growth factor synthetic peptide L83FTRKL88(G) blocking ligand binding to effector cell protease receptor-1 inhibited NO release by factor Xa in a dose-dependent manner, whereas a control scrambled peptide KFTGRLL was ineffective.

Catalytically active factor Xa induced hypotension in rats and vasorelaxation in the isolated rat mesentery, which was blocked by the NO synthase inhibitor L-NG-nitroarginine methyl ester (LNAME) but not by D-NAME. Factor Xa/NO signaling also produced a dose-dependent endothelial cell release of interleukin 6 (range 0.55–3.1 ngyml) in a reaction

  • inhibited by L-NAME and by the
  • inter-epidermal growth factor peptide Leu83–Leu88 but
  • unaffected by hirudin.
We observe that incubation of HUVEC monolayers with factor Xa which resulted in a concentration-dependent release of NO, as determined by cGMP accumulation in these cells, was inhibited by the nitric oxide synthase antagonist L-NAME.

Catalytically inactive DEGR-factor Xa or TAP-treated factor Xa failed to stimulate NO release by HUVEC.

To determine whether factor Xa-induced NO release could also modulate acute phase/inflammatory cytokine gene expression we examined potential changes in IL-6 release following HUVEC stimulation with factor Xa. HUVEC stimulation with factor Xa resulted in a concentration-dependent release of IL-6.

The specificity of factor Xa-induced cytokine release was investigated. Factor Xa-induced IL-6 release from HUVEC was quantitatively indistinguishable from that obtained with tumor necrosis factor-a or thrombin stimulation. This response was abolished by heat denaturation of factor Xa.

Maximal induction of interleukin 6 mRNA required a brief, 30-min stimulation with factor Xa, and was unaffected by subsequent addition of tissue factor pathway inhibitor (TFPI). These data suggest that factor Xa-induced NO release modulates endothelial cell-dependent vasorelaxation and IL-6 cytokine gene expression.

Here, we find that factor Xa induces the release of endothelial cell NO

  • regulating vasorelaxation in vivo and acute response cytokine gene expression in vitro.

This pathway requires a dual step cascade, involving

  • binding of factor Xa to EPR-1 and
  • a secondary as yet unidentified protease activated mechanism.

This pathway requiring factor Xa binding to effector cell protease receptor-1 and a secondary step of ligand-dependent proteolysis may preserve an anti-thrombotic phenotype of endothelium but also trigger acute phase responses during activation of coagulation in vivo.

In summary, these investigators have identified a signaling pathway centered on the ability of factor Xa to rapidly stimulate endothelial cell NO release. This involves a two-step cascade initiated by catalytic active site-independent binding of factor Xa to its receptor, EPR-1, followed by a second step of ligand dependent proteolysis.

(Papapetropoulos A, Piccardoni P, Cirino G, Bucci M, et al. Hypotension and inflammatory cytokine gene expression triggered by factor Xa–nitric oxide signaling. Proc. Natl. Acad. Sci. USA. Pharmacology. 1998; 95:4738–4742.)

Platelets and liver disease

Thrombocytopenia is a marked feature of chronic liver disease and cirrhosis. Traditionally, this thrombocytopenia was attributed to passive platelet sequestration in the spleen. More recent insights suggest an increased platelet breakdown and to a lesser extent decreased platelet production plays a more important role. Besides the reduction in number, other studies suggest functional platelet defects. This platelet dysfunction is probably both intrinsic to the platelets and secondary to soluble plasma factors. It reflects not only a decrease in aggregability, but also an activation of the intrinsic inhibitory pathways. (Witters P, Freson K, Verslype C, Peerlinck K, et al. Review article: blood platelet number and function in chronic liver disease and cirrhosis. Aliment Pharmacol Ther 2008; 27: 1017–1029).

The shortcomings of the old Y-shaped model of normal coagulation are nowhere more apparent than in its clinical application to the complex coagulation disorders of acute and chronic liver disease. In this condition, the clotting cascade is heavily influenced by numerous currents and counter-currents resulting in a mixture of pro- and anticoagulant forces that are themselves further subject to change with altered physiological stress such as super-imposed infection or renal failure.

Multiple mechanisms exist for thrombocytopenia common in patients with cirrhosis besides hypersplenism and expected altered thrombopoietin metabolism. Increased production of two important endothelial derived platelet inhibitors

  • nitric oxide and
  • prostacyclin

may contribute to defective platelet activation in vivo. On the other hand, high plasma levels of vWF in cirrhosis appear to support platelet adhesion.

Reduced levels of coagulation factors V, VII, IX, X, XI, and prothrombin are also commonly observed in liver failure. Vitamin K–dependent clotting factors (II, VII, IX, X) may be defective in function as a result of decreased  y-carboxylation (from vitamin K deficiency or intrinsically impaired carboxylase activity). Fibrinogen levels are decreased with advanced cirrhosis and in patients with acute liver failure.

A hyperfibrinolytic state may develop when plasminogen activation by tPA is accelerated on the fibrin surface. Physiologic stress including infection may be key in tipping this process off through increased release of tPA.  Not uncommonly, laboratory abnormalities in decompensated cirrhosis come to resemble disseminated intravascular coagulation (DIC). Relatively stable platelet levels and characteristically high factor VIII levels distinguish this process from DIC as does the absence of uncompensated thrombin generation. The features of both hyperfibrinolysis and DIC are often evident in the decompensated liver disease patient, and the term “accelerated intravascular coagulation and fibrinolysis” (AICF) has been proposed as a way to encapsulate the process under a single heading. The essence of AICF can be postulated to be the result of formation of a fibrin clot that is more susceptible to plasmin degradation due to elevated levels of tPA coupled with inadequate release of PAI to control tPA and lack of a-2 plasmin inhibitor to quench plasmin activity and the maintenance of high local concentrations of plasminogen on clot surfaces despite lower total plasminogen production. These normally balanced processes become pronounced when disturbed by additional stress such as infection.

Normal hemostasis and coagulation is now viewed as primarily a cell-based process wherein key steps in the classical clotting cascade

  • occur on the phospholipid membrane surface of cells (especially platelets)
  • beginning with activation of tissue factor and factor VII at the site of vascular breach
    •  which produces an initial “priming” amount of thrombin and a
    • subsequent thrombin burst.

Coagulation and hemostasis in the liver failure patient is influenced by multiple, often opposing, and sometimes changing variables. A bleeding diathesis is usually predominant, but the assessment of bleeding risk based on conventional laboratory tests is inherently deficient.

(Caldwell SH, Hoffman M, Lisman T, Gail Macik B, et al. Coagulation Disorders and Hemostasis in Liver Disease: Pathophysiology and Critical Assessment of Current Management. Hepatology 2006;44:1039-1046.)

Bleeding after Coronary Artery bypass Graft

Cardiac surgery with concomitant CPB can profoundly alter haemostasis, predisposing patients to major haemorrhagic complications and possibly early bypass conduit-related thrombotic events as well. Five to seven percent of patients lose more than 2 litres of blood within the first 24 hours after surgery, between 1% and 5% require re-operation for bleeding. Re-operation for bleeding increases hospital mortality 3 to 4 fold, substantially increases post-operative hospital stay and has a sizeable effect on health care costs. Nevertheless, re-exploration is a strong risk factor associated with increased operative mortality and morbidity, including sepsis, renal failure, respiratory failure and arrhythmias.

(Gábor Veres. New Drug Therapies Reduce Bleeding in Cardiac Surgery. Ph.D. Doctoral Dissertation. 2010. Semmelweis University)

Hypercoagulable State in Thalassemia

As the life expectancy of β-thalassemia patients has increased in the last decade, several new complications are being recognized. The presence of a high incidence of thromboembolic events, mainly in thalassemia intermedia patients, has led to the identification of a hypercoagulable state in thalassemia. Patients with thalassemia intermedia (TI) have, in general, a milder clinical phenotype than those with TM and remain largely transfusion independent. The pathophysiology of TI is characterized by extravascular hemolysis, with the release into the peripheral circulation of damaged red blood cells (RBCs) and erythroid precursors because of a high degree of ineffective erythropoiesis. This has also been recently attributed to severe complications such as pulmonary hypertension (PHT) and thromboembolic phenomena.

Many investigators have reported changes in the levels of coagulation factors and inhibitors in thalassemic patients. Prothrombin fragment 1.2 (F1.2), a marker of thrombin generation, is elevated in TI patients. The status of protein C and protein S was investigated in thalassemia in many studies and generally they were found to be decreased; this might be responsible for the occurrence of thromboembolic events in thalassemic patients.

The pathophysiological roles of hemolysis and the dysregulation of nitric oxide homeostasis are correlated with pulmonary hypertension in sickle cell disease and in thalassemia. Nitric oxide binds soluble guanylate cyclase, which converts GTP to cGMP, relaxing vascular smooth muscle and causing vasodilatation. When plasma hemoglobin liberated from intravascularly hemolyzed sickle erythrocytes consumes nitric oxide, the balance is shifted toward vasoconstriction. Pulmonary hypertension is aggravated and in sickle cell disease, it is linked to the intensity of hemolysis. Whether the same mechanism contributes to hypercoagulability in thalassemia is not yet known.

While there are diverse factors contributing to the hypercoagulable state observed in patients with thalassemia. In most cases, a combination of these abnormalities leads to clinical thrombosis. An argument has been made for the a higher incidence of thrombotic events in TI compared to TM patients  attributed to transfusion for TM. The higher rate of thrombosis in transfusion-independent TI compared to polytransused TM patients suggests a potential role for transfusions in decreasing the rate of thromboembolic events (TEE). The reduction of TEE in adequately transfused patients may be the result of decreased numbers of pathological RBCs.

(Cappellini MD, Musallam KM,  Marcon A, and Taher AT. Coagulopathy in Beta-Thalassemia: Current Understanding and Future Prospects. Medit J Hemat Infect Dis 2009; 1(1):22009029.
DOI 10.4084/MJHID.2009.0292.0), www.mjhid.org/article/view/5250.  ISSN 2035-3006.)

Microvascular Endothelial Dysfunction

Severe sepsis, defined as sepsis associated with acute organ dysfunction, results from a generalized inflammatory and procoagulant host response to infection. Coagulopathy in severe sepsis is commonly associated with multiple organ dysfunction, and often results in death. The molecule that is central to these effects is thrombin, although it may also have anticoagulant and antithrombotic effects through the activation of Protein C and induction of prostacyclin. In recent years, it has been recognized that chemicals produced by endothelial cells play a key role in the pathogenesis of sepsis. Thrombomodulin on endothelial cells coverts Protein C to Activated Protein C, which has important antithrombotic, profibrinolytic and anti-inflammatory properties. A number of studies have shown that Protein C levels are reduced in patients with severe infection, or even in inflammatory states without infection. Because coagulopathy is associated with high mortality rates, and animal studies have indicated that therapeutic intervention may result in improved outcomes, it was rational to initiate clinical studies.

Considering the coagulation cascade as a whole, it is the extrinsic pathway (via TF and thrombin activation) rather than the intrinsic pathway that is of primary importance in sepsis. Once coagulation has been triggered by TF activation, leading to thrombin formation, this can have further procoagulant effects, because thrombin itself can activate factors VIII, IX and X. This is normally balanced by the production of anticoagulant factors, such as TF pathway inhibitor, antithrombin and Activated Protein C.

It has been recognized that endothelial cells play a key role in the pathogenesis of sepsis, and that they produce important regulators of both coagulation and inflammation. They can express or release a number of substances, such as TF, endothelin-1 and PAI-1, which promote the coagulation process, as well as other substances, such as antithrombin, thrombomodulin, nitric oxide and prostacyclin, which inhibit it.

Protein C is the source of Activated Protein C. Although Protein C is a biomarker or indicator of sepsis, it has no known specific biological activity. Protein C is converted to Activated Protein C in the presence of normal endothelium. In patients with severe sepsis, the vascular endothelium becomes damaged. The level of thrombomodulin is significantly decreased, and the body’s ability to convert Protein C to Activated Protein C diminishes. Only when activated does Protein C have antithrombotic, profibrinolytic and anti-inflammatory properties.

Blood Coagulation (Thrombin) and Protein C Pat...

Blood Coagulation (Thrombin) and Protein C Pathways (Blood_Coagulation_and_Protein_C_Pathways.jpg) (Photo credit: Wikipedia)

Coagulation abnormalities can occur in all types of infection, including both Gram-positive and Gram-negative bacterial infections, or even in the absence of infection, such as in inflammatory states secondary to trauma or neurosurgery. Interestingly, they can also occur in patients with localized disease, such as those with respiratory infection. In a study by Günther et al., procoagulant activity in bronchial lavage fluid from patients with pneumonia or acute respiratory distress syndrome was found to be increased compared with that from control individuals, with a correlation between the severity of respiratory failure and level of coagulant activity.

Severe sepsis, defined as sepsis associated with acute organ dysfunction, results from a generalized inflammatory and procoagulant host response to infection.  Once the endothelium becomes damaged, levels of endothelial thrombomodulin significantly decrease, and the body’s ability to convert Protein C to Activated Protein C diminishes. The ultimate cause of acute organ dysfunction in sepsis is injury to the vascular endothelium, which can result in microvascular coagulopathy.

(Vincent JL. Microvascular endothelial dysfunction: a renewed appreciation of sepsis pathophysiology.
Critical Care 2001; 5:S1–S5. http://ccforum.com/content/5/S2/S1)

Endothelial Cell Dysfunction in Severe Sepsis

During the past decade a unifying hypothesis has been developed to explain the vascular changes that occur in septic shock on the basis of the effect of inflammatory mediators on the vascular endothelium. The vascular endothelium plays a central role in the control of microvascular flow, and it has been proposed that widespread vascular endothelial activation, dysfunction and eventually injury occurs in septic shock, ultimately resulting in multiorgan failure. This has been characterized in various models of experimental septic shock. Now, direct and indirect evidence for endothelial cell alteration in humans during septic shock is emerging.

The vascular endothelium regulates the flow of nutrient substances, diverse biologically active molecules and the blood cells themselves. This role of endothelium is achieved through the presence of membrane-bound receptors for numerous molecules, including proteins, lipid transporting particles, metabolites and hormones, as well as through specific junction proteins and receptors that govern cell–cell and cell–matrix interactions. Endothelial dysfunction and/or injury with subendothelium exposure facilitates leucocyte and platelet aggregation, and aggravation of coagulopathy. Therefore, endothelial dysfunction and/or injury should favour impaired perfusion, tissue hypoxia and subsequent organ dysfunction.

Anatomical damage to the endothelium during septic shock has been assessed in several studies. A single injection of bacterial lipopolysaccharide (LPS) has long been demonstrated to be a nonmechanical technique for removing endothelium. In endotoxic rabbits, observations tend to demonstrate that EC surface modification occurs easily and rapidly, with ECs being detached from the internal elastic lamina with an indication of subendothelial oedema.  Proinflammatory cytokines increase permeability of the ECs, and this is manifested approximately 6 hours after inflammation is triggered and becomes maximal over 12–24 hours as the combination of cytokines exert potentiating effects. Endothelial physical disruption allows inflammatory fluid and cells to shift from the blood into the interstitial space.

In sepsis

  • ECs become injured, prothrombotic and antifibrinolytic
  • They promote platelet adhesion
  • They promote leucocyte adhesion and inhibit vasodilation

An important point is that EC injury is sustained over time. In an endotoxic rabbit model, we demonstrated that endothelium denudation is present at the level of the abdominal aorta as early as after several hours following injury and persisted for at least 5 days afterward. After 21 days we observed that the endothelial surface had recovered. The de-endothelialized surface accounted for approximately 25% of the total surface.

Thrombomodulin and protein C activation at the microcirculatory level.

The endothelial cell surface thrombin (Th)-binding protein thrombomodulin (TM) is responsible for inhibition of thrombin activity. TM, when bound to Th, forms a potent protein C activator complex. Loss of TM and/or internalization results in Th–thrombin receptor (TR) interaction. Loss of TM and associated protein C activation represents the key event of decreased endothelial coagulation modulation ability and increased inflammation pathways.
( Iba T, Kidokoro A, Yagi Y: The role of the endothelium in changes in procoagulant activity in sepsis. J Am Coll Surg 1998; 187:321-329. Keywords: ATIII, antithrombin III; NF-κ, nuclear factor-κB; PAI,plasminogen activator inhibitor).

In order to test the role of the endothelial-derived relaxing factors NO and PGI2, we investigated, in dogs, the influence of a combination of NG-nitro-L-arginine methyl ester (an inhibitor of NO synthesis) and indomethacin (an inhibitor of PGI2 synthesis). In these dogs treated with indomethacin plus NG-nitro-L-arginine methyl ester, the severity of the oxygen extraction defect was lower than that observed in the deoxycholate-treated dogs, suggesting that other mediators and/or mechanisms may be involved in microcirculatory control during hypoxia. One of these mediators or mechanisms could be related to hyperpolarization. Membrane potential is an important determinant of vascular smooth muscle tone through its influence on calcium influx via voltage-gated calcium channels. Hyperpolarization (as well as depolarization) has been shown to be a means of cell–cell communication in upstream vasodilatation and microcirculatory coordination. It is important to emphasize that intercell coupling exclusively involves ECs.

Interestingly, it was recently shown that sepsis, a situation that is characterized by impaired tissue perfusion and abnormal oxygen extraction, is associated with abnormal inter-EC coupling and reduction in the arteriolar conducted response.  An intra-organ defect in blood flow related to abnormal vascular reactivity, cell adhesion and coagulopathy may account for impaired organ oxygen regulation and function. If specific classes of microvessels must or must not be perfused to achieve efficient oxygen extraction during limitation in oxygen delivery, then impaired vascular reactivity and vessel injury might produce a pathological limitation in supply. In sepsis, the inflammatory response profoundly alters circulatory homeostasis, and this has been referred to as a ‘malignant intravascular inflammation’ that alters vasomotor tone and the distribution of blood flow among and within organs. These mechanisms might coexist with other types of sepsis associated cell dysfunction. For example, data suggest that endotoxin directly impairs oxygen uptake in ECs and indicate the importance of endothelium respiration in maintaining vascular homeostasis under conditions of sepsis.

Consistent with the hypothesis that alteration in endothelium plays a major in the pathophysiology of sepsis, it was observed that chronic ecNOS overexpression in the endothelium of mice resulted in resistance to LPS-induced hypotension, lung injury and death . This observation was confirmed by another group of investigators, who used transgenic mice overexpressing adrenomedullin  – a vasodilating peptide that acts at least in part via an NO-dependent pathway. They demonstrated resistance of these animals to LPS-induced shock, and lesser declines in blood pressure and less severe organ damage than occurred in the control animals. It might therefore be of importance to favour ecNOS expression and function during sepsis. The recent negative results obtained with therapeutic strategies aimed at blocking inducible NOS with the nonselective NOS inhibitor NG-monomethyl-L-arginine in human septic shock further confirm the overall importance of favoring vessel dilatation.

(Vallet B. Bench-to-bedside review: Endothelial cell dysfunction in severe sepsis: a role in organ dysfunction?  Critical Care 2003; 7(2):130-138 (DOI 10.1186/cc1864). (Print ISSN 1364-8535; Online ISSN 1466-609X). http://ccforum.com/content/7/2/130

Thrombosis in Inflammatory Bowel Disease

An association between IBD and thrombosis has been recognized for more than 60 years. Not only are patients with IBD more likely to have thromboembolic complications, but it has also been suggested that thrombosis might be pathogenic in IBD.

Coagulation Described.  See Part I. (Cascade)

Endothelial injury exposes TF, which forms a complex with factor VII.  This complex activates factors X and, to a lesser extent, IX. TFPI prevents this activation progressing  further; for coagulation to progress, factor Xa must be produced via factors IX and VIII. Thrombin, generated by the initial production of factor Xa, activates factor VIII and, through factor XI, factor IX, resulting in further activation of factor X. This positive feedback loop allows coagulation to proceed. Fibrin polymers are stabilized by factor XIIIa. Activated proteins CS (APCS) together inhibit factors VIIIa and Va, whereas antithrombin (AT) inhibits factors VIIa, IXa, Xa, and XIa. Fibrinolysis balances this system through the action of plasmin on fibrin. Plasminogen activator inhibitor controls the plasminogen activator-induced conversion of plasminogen to plasmin.

Inflammation and Thrombotic Processes Linked

Although interest has recently moved away from the proposal that ischemia is a primary cause of IBD, it has become increasingly clear that inflammatory and thrombotic processes are linked.  A vascular component to the pathogenesis of CD was first proposed only a year after Crohn et al. described the condition.  Subsequently, in 1989, a series of changes comprising vascular injury, focal arteritis, fibrin deposition, arterial occlusion, and then microinfarction or neovascularization was proposed as a possible pathogenetic sequence in CD.  In this study, resin casts of the intestinal vasculature showed changes ranging from intravascular fibrin deposition to complete thrombotic occlusion. Furthermore, the early vascular changes appeared to precede mucosal changes, suggesting that they were more likely to cause rather than result from the pathologic features of CD. Subsequent studies showed that intravascular fibrin deposition occurred at the site of granulomatous destruction of mesenteric blood vessels, and positive immunostaining for platelet glycoprotein IIIa occurred in fibrinoid plugs of mucosal capillaries in CD. In addition, intracapillary thrombus has been identified in biopsies from inflamed rectal mucosa from patients with CD. When combined with evidence of ongoing intravascular coagulation in both active and quiescent CD, the above data point toward a thrombotic element contributing to the pathogenesis of CD.

Not only are many different prothrombotic changes described in association with IBD, but they can also have multiple causes. Hyperhomocysteinemia, for example, is known to predispose to thrombosis, and patients with IBD are more likely to have hyperhomocysteinemia than control subjects. Hyperhomocysteinemia in IBD might be due to multiple possible causes, such as deficiencies of vitamin B12 as a result of terminal ileal disease or resection; B6, which is commonly reduced in IBD.  A vegan diet can’t be discarded either because of seriously deficient methyl donors (S-adenosyl methionine).

The realization that platelets are not only prothrombotic but also proinflammatory has stimulated interest in their role in both the pathogenesis and complications of IBD. The association between thrombocytosis and active IBD was first described more than 30 years ago. More recent observations link decreased or normal platelet survival to IBD-related thrombocytosis, possibly due to increased thrombopoiesis. This in turn could be driven by an interleukin-6 –induced increase in thrombopoietin synthesis in the liver. Spontaneous in vitro platelet aggregation occurs in platelets isolated from 30% of patients with IBD but not in platelets from control subjects. Moreover, collagen, arachidonic acid, ristocetin, and ADP-induced platelet activation are more marked in platelets from patients with active IBD than in those from healthy volunteers.

The roles of activated platelets and PLAs in mucosal inflammation. Activated platelets can interact with other cells involved in the inflammatory response either through direct contact or through the release of soluble mediators. Activated platelets interact directly with activated vascular endothelium, causing the latter to express adhesion molecules and release inflammatory and chemotactic cytokines.

Platelet activation might be pathogenic in IBD in several ways. Platelet activation might increase platelet aggregation, hence increasing the likelihood of thrombus formation at sites of vascular injury, for example, within the mesenteric circulation. P-selectin is the major ligand for leukocyte-endothelial interaction and is responsible for the rolling of platelets, leukocytes, and PLAs on vascular endothelium. Moreover, platelets adherent to injured vascular endothelium support leukocyte adhesion via P-selectin, an effect that could contribute to leukocyte emigration from the vasculature into the lamina propria in patients with IBD. In addition, P-selectin is the major platelet ligand for platelet-leukocyte interaction, which in turn causes both leukocyte activation and further platelet activation.

Platelet-Leukocyte Aggregation

Recently, studies showing that platelets and leukocytes that circulate together in aggregates (PLA) are more activated than those that circulate alone have generated interest in the role of PLA in various inflammatory and thrombotic conditions. PLA numbers are increased in patients with ischemic heart disease, systemic lupus erythematosus and rheumatoid arthritis, myeloproliferative disorders, and sepsis and are increased by smoking.

We have recently shown that patients with IBD have more PLAs than both healthy and inflammatory control subjects (patients with inflammatory arthritides).  As with platelet activation, there was no correlation with disease activity, suggesting that increased PLA formation might be an underlying abnormality. PLAs could contribute to the pathogenesis of IBD in a number of ways. As previously mentioned, TF is key to the initiation of thrombus formation. TF has recently been demonstrated on the surface of activated platelets and in platelet-derived microvesicles. Interaction between neutrophils and activated platelets or microvesicles vastly increases the activity of “intravascular” TF.

Conclusion        

It is becoming increasingly apparent that thrombosis and inflammation are intrinsically linked. Hence the involvement of thrombotic processes in the pathogenesis of IBD, although perhaps not as the primary event, seems likely. Indeed, with the recently mounting evidence of the role of activated platelets and of their interaction with leukocytes in the pathogenesis of IBD, it seems even more probable that thrombosis plays some role in the pathogenic process.

(Irving PM, Pasi KJ, and Rampton DS. Thrombosis and Inflammatory Bowel Disease. Clinical Gastroenterology and Hepatology 2005;3:617–628. PII: 10.1053/S1542-3565(05)00154-0.)

Bleeding in Patients with Renal Insufficiency

Approximately 20–40% of critically ill patients will have renal insufficiency at the time of admission or will develop it during their ICU stay, depending on the definition of renal insufficiency and the case mix of the ICU. Such patients are also predisposed to bleeding because of uremic platelet dysfunction, typically multiple comorbidities, coagulopathies and frequent concomitant treatment with antiplatelet or anticoagulant agents.

The impairment in hemostasis in uremic patients is multifactorial and includes physiological defects in platelet hemostasis, an imbalance of mediators of normal endothelial function and frequent comorbidities such as vascular disease, anemia and the frequent need for medical interventions required to treat such comorbidities. Physiologic alterations in uremia include:

  • decreased platelet glycoprotein IIb–IIIa binding to both von Willebrand factor (vWf) and fibrinogen, causing an impairment in platelet aggregation;
  • increased prostacyclin and nitric oxide production, both potent inhibitors of platelet activation and vasoconstriction; and
  • decreased levels of platelet adenosine diphosphate (ADP) and serotonin, causing an impairment in platelet secretion.

In addition to other factors, small peptides containing the RGD (Arg-Gly-Asp) sequence of amino acids have been shown to be inhibitors of platelet aggregation that act by competing with vWf and fibrinogen for binding to the glycoprotein IIb–IIIa receptor.

Conclusion

ICU patients have dynamic risks of thrombosis and bleeding. Invasive procedures may require temporary interruption of anticoagulants. Consequently, approaches to thromboprophylaxis require daily reevaluation.

(Cook DJ, Douketis J, Arnold D, and Crowther MA. Bleeding and venous thromboembolism in the critically ill with emphasis on patients with renal insufficiency. Curr Opin Pulm Med 2009;15:455–462.)

Epicrisis

I have covered a large amount of material on one of the most complex systems in medicine, and still not comprehensive, with a sufficient dash of repetition.  The task is to have some grasp of the cell-mediated imbalances inherent if coagulation and bleeding disorders.  The key points are:

  • inflammation and oxidative stress invariably lurk in the background
  • the Y-shaped model with an extrinsic, intrinsic, and common pathway has no basis in understanding
  • the current model is based on a cell-mediated concept of endothelial damage and platelet-endothelial interaction
  • the model has 3 components: Initiation, Amplification, Propagation
  • NO and prostacyclin have key roles in the process
  • The plasma proteins involved are in the serine-protease class of enzymes
  • The conversion of Protein C to APC has a central role as anti-coagulant

Part II goes into organ aystem abnormalities that are all related to impairment of the Nitric Oxide balance and dual platelet-endothelial roles.

Part III will explore therapeutic targets and opportunities.

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