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Posts Tagged ‘propagation’


Alzheimer Disease Developments – Spring 2015

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

 

Cognitive Stimulation Modulates Platelet Total Phospholipases A2 Activity in Subjects with Mild Cognitive Impairment

 

JNK: A Putative Link Between Insulin Signaling and VGLUT1 in Alzheimer’s Disease

Omega-3 Fatty Acid Status Enhances the Prevention of Cognitive Decline by B Vitamins in Mild Cognitive ImpairmentOpenly Available
Oulhaj, Abderrahim | Jernerén, Fredrik | Refsum, Helga | Smith, A. David | de Jager, Celeste A.

Preliminary Study of Plasma Exosomal Tau as a Potential Biomarker for Chronic Traumatic EncephalopathyOpenly Available
Stern, Robert A. | Tripodis, Yorghos | Baugh, Christine M. | Fritts, Nathan G. | Martin, Brett M. | Chaisson, Christine | Cantu, Robert C. | Joyce, James A. | Shah, Sahil | Ikezu, Tsuneya | Zhang, Jing | Gercel-Taylor, Cicek | Taylor, Douglas D

AZD3293: A Novel, Orally Active BACE1 Inhibitor with High Potency and Permeability and Markedly Slow Off-Rate KineticsOpenly Available
Eketjäll, Susanna | Janson, Juliette | Kaspersson, Karin | Bogstedt, Anna | Jeppsson, Fredrik | Fälting, Johanna | Haeberlein, Samantha Budd | Kugler, Alan R. | Alexander, Robert C. | Cebers, Gvido

Predictive Value of Cerebrospinal Fluid Visinin-Like Protein-1 Levels for Alzheimer’s Disease Early Detection and Differential Diagnosis in Patients with Mild Cognitive Impairment
Babić Leko, Mirjana | Borovečki, Fran | Dejanović, Nenad | Hof, Patrick R. | Šimić, Goran

Plasma Phospholipid and Sphingolipid Alterations in Presenilin1 Mutation Carriers: A Pilot Study
Chatterjee, Pratishtha | Lim, Wei L.F. | Shui, Guanghou | Gupta, Veer B. | James, Ian | …… | Wenk, Marcus R. | Bateman, Randall J. | Morris, John C. | Martins, Ralph N.

Cognitive reserve in ageing and Alzheimer’s disease / Stern Y / Lancet Neurol. 2012 Nov; 11(11):1006-12. PMID: 23079557.

A mutation in APP protects against Alzheimer’s disease and age-related cognitive decline/ Jonsson T, Atwal JK, Steinberg S, Snaedal J, Jonsson PV, Bjornsson S, Stefansson H, Sulem P, Gudbjartsson D, Maloney J, et al. / Nature. 2012 Aug 2; 488(7409):96-9. PMID: 22801501.

 Propagation of tau pathology in a model of early Alzheimer’s disease / de Calignon A, Polydoro M, Suárez-Calvet M, William C, Adamowicz DH, Kopeikina KJ, Pitstick R, Sahara N, Ashe KH, Carlson GA, et al. / Neuron. 2012 Feb 23; 73(4):685-97. PMID: 22365544.

Stages of the pathologic process in Alzheimer disease: age categories from 1 to 100 years/ Braak H, Thal DR, Ghebremedhin E, Del Tredici K / J Neuropathol Exp Neurol. 2011 Nov; 70(11):960-9. PMID: 22002422.

Neuroinflammation in Alzheimer’s disease and mild cognitive impairment: a field in its infancy / McGeer EG, McGeer PL / J Alzheimers Dis. 2010; 19(1):355-61. PMID: 20061650.

Metallothioneins in Prion- and Amyloid-Related Diseases

MICROGLIA

Microglia are the immune cells of the CNS and account for approximately 10% of the CNS cellpopulation, with regional variation in density [27, 28]. During embryonic development, microglia originate from yolk sac progenitor cells that migrate into the developing CNS during early embryogenesis [29,30].Following construction of the blood-brain barrier (BBB), microglia are renewed by local turnover [31]. In the healthy brain, microglia actively support neurons through the release of insulin-like growth factor 1, nerve growth factor, ciliary neurotrophic factor, and brain-derived neurotrophic factor (BDNF) [32–34]. Microglia also provide indirect support to neurons by clearance of debris to maintain the extracellular environment, and phagocytosis of apoptotic cells to facilitate neurogenesis [35, 36]. In the adult brain, microglia coordinate much of their activity with astrocytes and activate in response to similar stimuli [37, 38]. Dysfunctional signaling between microglia and astrocytes often results in chronic inflammation, a characteristic of many neurodegenerative diseases [39, 40].

Historically, it has been thought that microglia ‘rest’ when not responding to inflammatory stimuli or damage [41, 42]. However, this notion is being increasingly recognized as inaccurate [43]. When not involved in active inflammatory signaling, microglia constantly patrol the neuropil by extension and retraction of their finely branched processes [44]. Microglial activation is often broadly classified into two states; pro-inflammatory (M1) or anti-inflammatory (M2) [36, 45], based on similar phenotypes in peripheral macrophages [46]. M1 activated microglia are characterized by increased expression of pro-inflammatory mediators and cytokines, including inducible nitric oxide synthase, tumor necrosis factor-α, and interleukin-1β, often under the control of the transcription factor nuclear factor-κB [45]. Pro-inflammatory microglia rapidly retract their processes and adopt an amoeboid morphology and often migrate closer to the site of injury [47]. Anti-inflammatory M2 activation of microglia, often referred to as alternative activation, represents the other side of microglial behavior. Anti-inflammatory activation is characterized by increased expression of cytokines including arginase 1 and interleukin-10, and is associated with increased ramification of processes [45]. The polarization of microglia into M1 or M2 throughout the brain is well characterized, especially in neurodegenerative diseases [48]. In the AD brain, microglia expressing markers of M1 activation are typically localized to brain regions such as the hippocampus that are most heavily affected in the disease [49]. However, it is important to note that M1 and M2 classifications of microglia may over-simplify microglial phenotypes and may only represent the extremes of microglial activation [50]. It has been more recently proposed that microglia likely occupy a continuum between these phenotypes [39, 51].

Do microglia have multiple roles in AD?

Classical pro-inflammatory activation of microglia has long been associated with AD [39, 49]. Samples taken from late-stage AD brains contain characteristic signs of inflammation, including amoeboid morphology of microglia, high levels of pro-inflammatory cytokines in the cerebrospinal fluid, and evidence of neuronal damage due to chronic exposure to pro-inflammatory cytokines and oxidative stress [52, 53]. The cause of this inflammation may be in response to direct toxicity of Aβ to neurons resulting in activation of nearby microglia and astrocytes [53, 54]. However, Aβ may also induce inflammatory activation of microglia and astrocytes. Activated immune cells are typically present surrounding amyloid plaques [55–57], with such peri-plaque cells exhibiting strong evidence of pro-inflammatory activation [56, 58–60]. The presence of undigested Aβ particles within these activated microglia may suggest that the Aβ peptide itself is a pro-inflammatory signal for microglia [61–64]. In vitro experiments provide supporting evidence for the in vivo studies, with Aβ promoting pro-inflammatory microglial activation [65, 66], and also acting as a potent chemotactic signal [67].

However, it is important to note that although widespread inflammation is characteristic of late-stage AD, it remains unclear what role inflammation could play in early stages of the disease. Some evidence suggests that reducing inflammation through the long-term use of some non-steroidal anti-inflammatory drugs (NSAIDs) can reduce the risk of AD [68]. However, these findings have not yet been verified in clinical trials [69, 70]. Little is understood about how NSAIDs and related compounds affect the delicate balance of pro- versus anti-inflammatory microglial activity within the brain. Although there is considerable evidence to suggest that chronic inflammation may contribute to pathology in the later stages of AD, it is important to note that inflammation normally only represents a small aspect of microglial function. The non-inflammatory functions of microglia may play a more important role in early disease; specifically, microglial functions relating to maintenance of the CNS.

Phagocytosis: A vital role of microglia that may be lost in AD    

SYNAPTIC PRUNING: MICROGLIA CAN REGULATE NETWORK ACTIVITY

Recently, a new function has been proposed for microglia. A number of studies have provided evidence that microglia prune synapses throughout life. Microglia are known to remove extraneous synapses during development to ensure that only meaningful connections remain [43]. It was, however, thought that differentiated astrocytes performed the majority of synaptic pruning in the adult brain [91]. The discovery that microglial processes are constantly active within the brain and are often positioned near synapses raised the question of whether microglial synaptic pruning continued throughout life [44, 47, 92–94]. This question was answered in 2014 in a study that demonstrated that microglia do prune synapses into adulthood, and that this activity is important for normal brain function [95]. These findings supported those found a year earlier in a study reporting that ablation of microglia from brain slices increases synapse density and results in abnormal firing of hippocampalneurons [96].

Altered microglial behavior may underlie altered neuronal firing in AD  

Altered neuronal activity is an early phenomenon in AD

The cause of DMN hypoactivity in AD is not yet clear; however studies performed in cohorts that are genetically predisposed to AD suggest that DMN hypoactivity is preceded by a period of hyperactivity and increased functional connectivity [123, 136], often manifesting as an absence of normal DMN deactivation during external tasks [137–140]. DMN hyperactivity may interfere with hippocampal memory encoding, leading to the memory deficits that are present in mild cognitive impairment [141, 142]. It has been proposed that hippocampal hyperexcitability in AD may develop as a protective mechanism against increased input from the DMN [142–144]. As AD progresses, the initial hyperexcitability of the DMN and hippocampus may result in hypoactivity due to exhaustion of compensatory mechanisms [123, 136]. Evidence from both transgenic AD mice and longitudinal human studies supports an exhaustion model of hyperactivation leading to later hypoactivation [143, 145–147]. Interestingly, a number of studies report a lower incidence of AD among those who regularly practice meditation which specifically ‘calms’ the DMN [148].

Our understanding of AD as a disease is changing. Historically considered to be primarily a disease of neuronal degeneration, this neurocentric view has widened to encompass non-neuronal cells such as astrocytes into our understanding of the disease process and pathogenesis. A proposed model for microglia in AD is shown in Fig. 2. Microglia perform a wide range of functions in the CNS and although this includes induction of an inflammatory reaction in response to damage, they also have critical roles for maintaining normal function in the brain. Recent evidence shows that microglia regulate neuronal activity through synaptic pruning throughout life as an extension on their normal phagocytosis behavior. The discovery of a large number of AD risk genes associated with reduced immune cell function suggests that perturbed microglial phagocytosis could lead to AD. In our model, altered microglial phagocytosis of synapses results in network dysfunction and onset of AD, occurring downstream of Aβ.

The immune system and microglia represent a novel target for intervention in AD. Importantly, a large number of anti-inflammatory drugs are already in use for other conditions. What is important to know at this stage is exactly how to best target immune cell function. The studies outlined here provide evidence that an indiscriminate dampening down of all microglial activity may result in a worse outcome for individuals by suppressing normal microglial regulatory functions. We currently do not know whether future microglial-based therapies should focus on reducing chronic inflammation or conversely, whether they should be aimed at boosting microglial phagocytosis. It is also likely that future treatment strategies may use a combination of approaches to target Aβ, immune cell phagocytosis and network activity. An increasing view in the AD field is that any drug or therapy needs to be provided very early in the disease process to maximize its beneficial effects. Although we are currently unable to effectively target those at risk of AD at such an early stage, advances in neuroimaging for subtle changes in network activity, or in assays for immune cell function, may provide new avenues for identification of early damage and risk of disease.

REFERENCES

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Selkoe DJ ((2011) ) Alzheimer’s disease. Cold Spring Harb Perspect Biol 3: , pii: a004457.

[2]

Masters CL , Simms G , Weinman NA , Multhaup G , McDonald BL , Beyreuther K ((1985) ) Amyloid plaque core protein in Alzheimer disease and Down syndrome. Proc Natl Acad Sci U S A 82: , 4245–4249.

[3]

Glenner GG , Wong CW ((1984) ) Alzheimer’s disease: Initial report of the purification and characterization of a novel cerebrovascular amyloid protein. Biochem Biophys Res Commun 120: , 885–890.

[4]

Goldgaber D , Lerman MI , McBride OW , Saffiotti U , Gajdusek DC ((1987) ) Characterization and chromosomal localization of a cDNA encoding brain amyloid of Alzheimer’s disease. Science 235: , 877–880.

[5]

Kang J , Lemaire HG , Unterbeck A , Salbaum JM , Masters CL , Grzeschik KH , Multhaup G , Beyreuther K , Muller-Hill B ((1987) ) The precursor of Alzheimer’s disease amyloid A4 protein resembles a cell-surface receptor. Nature 325: , 733–736.

[6]

Robakis NK , Ramakrishna N , Wolfe G , Wisniewski HM ((1987) ) Molecular cloning and characterization of a cDNA encoding the cerebrovascular and the neuritic plaque amyloid peptides. Proc Natl Acad Sci U S A 84: , 4190–4194.

[7]

Levy E , Carman MD , Fernandez-Madrid IJ , Power MD , Lieberburg I , van Duinen SG , Bots GT , Luyendijk W , Frangione B ((1990) ) Mutation of the Alzheimer’s disease amyloid gene in hereditary cerebral hemorrhage, Dutch type. Science 248: , 1124–1126.

[8]

Levy-Lahad E , Wasco W , Poorkaj P , Romano DM , Oshima J , Pettingell WH , Yu CE , Jondro PD , Schmidt SD , Wang K , et al ((1995) ) Candidate gene for the chromosome 1 familial Alzheimer’s disease locus. Science 269: , 973–977.

[9]

Rogaev EI , Sherrington R , Rogaeva EA , Levesque G , Ikeda M , Liang Y , Chi H , Lin C , Holman K , Tsuda T , et al ((1995) ) Familial Alzheimer’s disease in kindreds with missense mutations in a gene on chromosome 1 related to the Alzheimer’s disease type 3 gene. Nature 376: , 775–778.

[10]

Sherrington R , Rogaev EI , Liang Y , Rogaeva EA , Levesque G , Ikeda M , Chi H , Lin C , Li G , Holman K , Tsuda T , Mar L , Foncin JF , Bruni AC , Montesi MP , Sorbi S , Rainero I , Pinessi L , Nee L , Chumakov I , Pollen D , Brookes A , Sanseau P , Polinsky RJ , Wasco W , Da Silva HA , Haines JL , Perkicak-Vance MA , Tanzi RE , Roses AD , Fraser PE , Rommens JM , St George-Hyslop PH ((1995) ) Cloning of a gene bearing missense mutations in early-onset familial Alzheimer’s disease. Nature 375: , 754–760.

 

Late-Onset Metachromatic Leukodystrophy with Early Onset Dementia Associated with a Novel Missense Mutation in the Arylsulfatase A Gene

Microbes and Alzheimer’s DiseaseOpenly Available
Itzhaki, Ruth F. | Lathe, Richard | Balin, Brian J. | Ball, Melvyn J. | Bearer, Elaine L. | Braak, Heiko | Bullido, Maria J. | Carter, Chris | Clerici, Mario | Cosby, S. Louise | Del Tredici, Kelly | Field, Hugh | Fulop, Tamas | Grassi, Claudio | Griffin, W. Sue T. | Haas, Jürgen | Hudson, Alan P. | Kamer, Angela R. | Kell, Douglas B. | Licastro, Federico | Letenneur, Luc | Lövheim, Hugo | Mancuso, Roberta | Miklossy, Judith | Otth, Carola | Palamara, Anna Teresa | Perry, George | Preston, Christopher | Pretorius, Etheresia | Strandberg, Timo | Tabet, Naji | Taylor-Robinson, Simon D. | Whittum-Hudson, Judith A.

Longitudinal Relationships between Caloric Expenditure and Gray Matter in the Cardiovascular Health StudyOpenly Available
Raji, Cyrus A. | Merrill, David A. | Eyre, Harris | Mallam, Sravya | Torosyan, Nare | Erickson, Kirk I. | Lopez, Oscar L. | Becker, James T. | Carmichael, Owen T. | Gach, H. Michael | Thompson, Paul M. | Longstreth Jr., W.T. | Kuller, Lewis H.

Preliminary Study of Plasma Exosomal Tau as a Potential Biomarker for Chronic Traumatic EncephalopathyOpenly Available
Stern, Robert A. | Tripodis, Yorghos | Baugh, Christine M. | Fritts, Nathan G. | Martin, Brett M. | Chaisson, Christine | Cantu, Robert C. | Joyce, James A. | Shah, Sahil | Ikezu, Tsuneya | Zhang, Jing | Gercel-Taylor, Cicek | Taylor, Douglas D.

Unraveling Alzheimer’s: Making Sense of the Relationship between Diabetes and Alzheimer’s Disease1Openly Available
Schilling, Melissa A.

Pain Assessment in Elderly with Behavioral and Psychological Symptoms of DementiaOpenly Available
Malara, Alba | De Biase, Giuseppe Andrea | Bettarini, Francesco | Ceravolo, Francesco | Di Cello, Serena | Garo, Michele | Praino, Francesco | Settembrini, Vincenzo | Sgrò, Giovanni | Spadea, Fausto | Rispoli, Vincenzo

Editor’s Choice from Volume 50, Number 4 / 2016

Post Hoc Analyses of ApoE Genotype-Defined Subgroups in Clinical Trials
Kennedy, Richard E. | Cutter, Gary R. | Wang, Guoqiao | Schneider, Lon S.

Protective Effect of Amyloid-β Peptides Against Herpes Simplex Virus-1 Infection in a Neuronal Cell Culture Model
Bourgade, Karine | Le Page, Aurélie | Bocti, Christian | Witkowski, Jacek M. | Dupuis, Gilles | Frost, Eric H. | Fülöp, Tamás

Association Between Serum Ceruloplasmin Specific Activity and Risk of Alzheimer’s Disease
Siotto, Mariacristina | Simonelli, Ilaria | Pasqualetti, Patrizio | Mariani, Stefania | Caprara, Deborah | Bucossi, Serena | Ventriglia, Mariacarla | Molinario, Rossana | Antenucci, Mirca | Rongioletti, Mauro | Rossini, Paolo Maria | Squitti, Rosanna

Effects of Hypertension and Anti-Hypertensive Treatment on Amyloid-β (Aβ) Plaque Load and Aβ-Synthesizing and Aβ-Degrading Enzymes in Frontal Cortex
Ashby, Emma L. | Miners, James S. | Kehoe , Patrick G. | Love, Seth

AZD3293: A Novel, Orally Active BACE1 Inhibitor with High Potency and Permeability and Markedly Slow Off-Rate KineticsOpenly Available
Eketjäll, Susanna | Janson, Juliette | Kaspersson, Karin | Bogstedt, Anna | Jeppsson, Fredrik | Fälting, Johannad | Haeberlein, Samantha Budd | Kugler, Alan R. | Alexander, Robert C. | Cebers, Gvido

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Coagulation: Transition from a familiar model tied to Laboratory Testing, and the New Cellular-driven Model


Coagulation: Transition from a familiar model tied to Laboratory Testing, and the New Cellular-driven Model

 

Curator: Larry H. Bernstein, MD, FCAP 

Short Title: Coagulation viewed from Y to cellular biology.

PART I.

Summary: This portion of the series on PharmaceuticalIntelligence(wordpress.com) isthe first of a three part treatment of the diverse effects on platelets, the coagulation cascade, and protein-membrane interactions.  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.  The second part will be directed toward low flow states, local and systemic inflammatory disease, oxidative stress, and hematologic disorders, bringing NO and the role of NO synthase in the process.   A third part will be focused on management of these states.

Coagulation Pathway

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).

According to WH Seegers [Seegers WH,  Postclotting fates of thrombin.  Semin Thromb Hemost 1986;12(3):181-3], prothrombin is virtually all converted to thrombin in clotting, but Factor X is not. Large quantities of thrombin are inhibited by plasma and platelet AT III (heparin cofactor I), by heparin cofactor II, and by fibrin.  Antithrombin III, a serine protease, is a main inhibitor of thrombin and factor Xa in blood coagulation. The inhibitory function of antithrombin III is accelerated by heparin, but at the same time antithrombin III activity is also reduced. Heparin retards the thrombin-fibrinogen reaction, but otherwise the effectiveness of heparin as an anticoagulant depends on antithrombin III in laboratory experiments, as well as in therapeutics. The activation of prothrombin is inhibited, thereby inactivating  any thrombin or other vulnerable protease that might otherwise be generated. [Seegers WH, Antithrombin III. Theory and clinical applications. H. P. Smith Memorial Lecture. Am J Clin Pathol. 1978;69(4):299-359)].  With respect to platelet aggregation, platelets aggregate with thrombin-free autoprothrombin II-A. Aggregation is dependent on an intact release mechanism since inhibition of aggregation occurred with adenosine, colchicine, or EDTA. Autoprothrombin II-A reduces the sensitivity of platelets to aggregate with thrombin, but enhances epinephrine-mediated aggregation. [Herman GE, Seegers WH, Henry RL. Autoprothrombin ii-a, thrombin, and epinephrine: interrelated effects on platelet aggregation. Bibl Haematol 1977;44:21-7.]

A tetrapeptide, residues 6 to 9 in normal prothrombin, was isolated from the NH(2)-terminal, Ca(2+)-binding part of normal prothrombin. The peptide contained two residues of modified glutamic acid, gamma-carboxyglutamic acid. This amino acid gives normal prothrombin the Ca(2+)-binding ability that is necessary for its activation.

Abnormal prothrombin, induced by the vitamin K antagonist, dicoumarol, lacks these modified glutamic acid residues and that this is the reason why abnormal prothrombin does not bind Ca(2+) and is nonfunctioning in blood coagulation. [Stenflo J, Fernlund P, Egan W, Roepstorff P. Vitamin K dependent modifications of glutamic acid residues in prothrombin.  Proc Natl Acad Sci U S A. 1974;71(7):2730-3.]

Interestingly, a murine monoclonal antibody (H-11) binds a conserved epitope found at the amino terminal of the vitamin K-dependent blood proteins prothrombin, factors VII and X, and protein C. The sequence of polypeptide recognized contains 2 residues of gamma-carboxyglutamic acid, and binding of the antibody is inhibited by divalent metal ions.  The antibody bound specifically to a synthetic peptide corresponding to residues 1-12 of human prothrombin that was synthesized as the gamma-carboxyglutamic acid-containing derivative, but binding to the peptide was not inhibited by calcium ion. This suggested that binding by divalent metal ions is not due simply to neutralization of negative charge by Ca2+. [Church WR, Boulanger LL, Messier TL, Mann KG. Evidence for a common metal ion-dependent transition in the 4-carboxyglutamic acid domains of several vitamin K-dependent proteins. J Biol Chem. 1989;264(30):17882-7.]

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.

 

Coagulation cascade

Coagulation cascade (Photo credit: Wikipedia)

Coagulation Cascade

The procoagulant plasma coagulation cascade has traditionally been divided into the intrinsic and extrinsic pathways. The Waterfall/Cascade model consists of two separate initiations,

  • intrinsic (contact)and
    • The intrinsic pathway is initiated by a complex activation process of the so-called contact phase components,
      • prekallikrein,
      • high-molecular weight kininogen (HMWK) and
      • factor XII

Activation of the intrinsic pathway is promoted by non-biological surfaces, such as glass in a test tube, and is probably not of physiological importance, at least not in coagulation induced by trauma.

Instead, the physiological activation of coagulation is mediated exclusively via the extrinsic pathway, also known as the tissue factor pathway.

  • extrinsic pathways

Tissue factor (TF) is a membrane protein which is normally found in tissues. TF forms a procoagulant complex with factor VII, which activates factor IX and factor X.

  • common pathway which ultimately merge at the level of Factor Xa

Regulation of thrombin generation. Coagulation is triggered (initiation) by circulating trace amounts of fVIIa and locally exposed tissue factor (TF). Subsequent formations of fXa and thrombin are regulated by a tissue factor pathway inhibitor (TFPI) and antithrombin (AT). When the threshold level of thrombin is exceeded, thrombin activates platelets, fV, fVIII, and fXI to augment its own generation (propagation).

Activated factors IX and X (IXa and Xa) will activate prothrombin to thrombin and finally the formation of fibrin. Several of these reactions are much more efficient in the presence of phospholipids and protein cofactors factors V and VIII, which thrombin activates to Va and VIIIa by positive feedback reactions.

We depict the plasma coagulation emphasizing the importance of membrane surfaces for the coagulation processes. Coagulation is initiated when tissue factor (TF), an integral membrane protein, is exposed to plasma. TF is expressed on subendothelial cells (e.g. smooth muscle cells and fibroblasts), which are exposed after endothelium damage. Activated monocytes are also capable of exposing TF.

A small amount, approximately 1%, of activated factor VII (VIIa) is present in circulating blood and binds to TF. Free factor VIIa has poor enzymatic activity and the initiation is limited by the availability of its cofactor TF. The first steps in the formation of a blood clot is the specific activation of factor IX and X by the TF-VIIa complex. (Initiation of coagulation: Factor VIIa binds to tissue factor and activates factors IX and X). Coagulation is propagated by procoagulant enzymatic complexes that assemble on the negatively charged membrane surfaces of activated platelets. (Propagation of coagulation: Activation of factor X and prothrombin).  Once thrombin has been formed it will activate the procofactors, factor V and factor VIII, and these will then assemble in enzyme complexes. Factor IXa forms the tenase complex together with its cofactor factor VIIIa, and factor Xa is the enzymatic component of the prothrombinase complex with factor Va as cofactor.

Activation of protein C takes place on the surface of intact endothelial cells. When thrombin (IIa) reaches intact endothelium it binds with high affinity to a specific receptor called thrombomodulin. This shifts the specific activity of thrombin from being a procoagulant enzyme to an anticoagulant enzyme that activates protein C to activated protein C (APC).  The localization of protein C to the thrombin-thrombomodulin complex can be enhanced by the endothelial protein C receptor (EPCR), which is a transmembrane protein with high affinity for protein C.  Activated protein C (APC) binds to procoagulant surfaces such as the membrane of activated platelets where it finds and degrades the procoagulant cofactors Va and VIIIa, thereby shutting down the plasma coagulation.  Protein S (PS) is an important nonenzymatic  cofactor to APC in these reactions. (Degradation of factors Va and VIIIa).

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

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

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.

Activated protein C resistance

Activated protein C resistance (Photo credit: Wikipedia)

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)].

The activity of guanylate cyclase is altered to a much larger degree than adenylate cyclase, while cyclic nucleotide phosphodiesterase activity remains unchanged. During the early phases of thrombin- and ADP-induced platelet aggregation a marked activation of the guanylate cyclase occurs whereas aggregation induced by arachidonic acid or epinephrine results in a rapid diminution of this activity. In all four cases, the adenylate cyclase activity is only slightly decreased when examined under identical conditions.

Platelet aggregation induced by a wide variety of aggregating agents including collagen and platelet isoantibodies results in the “release” of only small amounts (1–3%) of guanylate cyclase and cyclic nucleotide phosphodiesterase and no adenylate cyclase. The guanylate cyclase and cyclic nucleotide phosphodiesterase activities are associated almost entirely with the soluble cytoplasmic fraction of the platelet, while the adenylate cyclase is found exclusively in a membrane bound form. ADP and epinephrine moderately inhibit guanylate and adenylate cyclase in subcellular preparations, while arachidonic and other unsaturated fatty acids moderately stimulate (2–4-fold) the former.

  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.

Furthermore, these observations suggest a role for unsaturated fatty acids in the control of intracellular cyclic GMP levels. Arachidonic acid, once deemed essential, is a derivative of linoleic acid. (Barbera AJ. Cyclic nucleotides and platelet aggregation effect of aggregating agents on the activity of cyclic nucleotide-metabolizing enzymes. Biochimica et Biophysica Acta (BBA) 1976; 444 (2): 579–595. http://dx.doi.org/10.1016/0304-4165(76)90402-5).

Leukocyte and platelet adhesion under flow

The basic principles concerning mechanical stress demonstrated by Robert Hooke (1635-1703) proved to be essential for the understanding of pathophysiological mechanisms in the vascular bed.

In physics, stress is the internal distribution of forces within a body that balance and react to the external loads applied to it. Stress is a 2nd order tensor. The hemodynamic conditions inside blood vessels lead to the development of superficial stresses near the vessel walls, which can be divided into two categories:

a) circumferential stress due to pulse pressure variation inside the vessel;

b) shear stress due to blood flow.

The direction of the shear stress vector is determined by the direction of the blood flow velocity vector very close to the vessel wall. Shear stress is applied by the blood against the vessel wall. Friction is the force applied by the wall to the blood and has a direction opposite to the blood flow. The tensions acting against the vessel wall are likely to be determined by blood flow conditions. Shear stresses are most complicated during turbulent flow, regions of flow recirculation or flow separation.

The notions of shear rate and fluid viscosity should be first clearly apprehended, since they are crucial for the assessment and development of shear stress. Shear rate is defined as the rate at which adjacent layers of fluid move with respect to each other, usually expressed as reciprocal seconds. The size of the shear rate gives an indication of the shape of the velocity profile for a given situation.  The determination of shear stresses on a surface is based on the fundamental assumption of fluid mechanics, according to which the velocity of fluid upon the surface is zero (no-slip condition). Assuming that the blood is an ideal Newtonian fluid with constant viscosity, the flow is steady and laminar and the vessel is straight, cylindrical and inelastic, which is not the case. Under ideal conditions a parabolic velocity profile could be assumed.

The following assumptions have been made:

  1. The blood is considered as a Newtonian fluid.
  2. The vessel cross sectional area is cylindrical.
  3. The vessel is straight with inelastic walls.
  4. The blood flow is steady and laminar.

The Haagen-Poisseuille equation indicates that shear stress is directly proportional to blood flow rate and inversely proportional to vessel diameter.

Viscosity is a property of a fluid that offers resistance to flow, and it is a measure of the combined effects of adhesion and cohesion. It increases as temperature decreases. Blood viscosity (non-Newtonian fluid) depends on shear rate, which is determined by blood platelets, red cells, etc. Moreover, it is slightly affected by shear rate changes at low levels of hematocrit. In contrast, as hematocrit increases, the effect of shear rate changes on blood viscosity becomes greater. Blood viscosity measurement is required for the accurate calculation of shear stress in veins or microcirculation.

It has to be emphasised that the dependence of blood viscosity on hematocrit is more pronounced in the microcirculation than in larger vessels, due to hematocrit variations observed in small vessels (lumen diameter <100 Ìm). The significant change of hematocrit in relation to vessel diameter is associated with the tendencyof red blood cells to travel closer to the centre of the vessels. Thus, the greater the decrease in vessel lumen, the smaller the number of red blood cells that pass through, resulting in a decrease in blood viscosity.

Shear stress and vascular endothelium

Endothelium responds to shear stress through various pathophysiological mechanisms depending on the kind and the magnitude of shear stresses. More specifically, the exposure of vascular endothelium to shear forces in the normal value range stimulates endothelial cells to release agents with direct or indirect antithrombotic properties, such as prostacyclin, nitric oxide (NO), calcium, thrombomodulin, etc.  The possible existence of so-called “mechanoreceptors” has provoked a number of research groups to propose receptors which “translate” mechanical forces into biological signals.

Under normal shear conditions, endothelial as well as smooth muscle cells have a rather low rate of proliferation. Changes in shear stress magnitude activate cellular proliferation mechanisms as well as vascular remodeling processes. More specifically, a high grade of shear stress increases wall thickness and expands the vessel’s diameter, so that shear stress values return to their normal values. In contrast, low shear stress induces a reduction in vessel diameter. Shear stresses stimulate vasoregulatory mechanisms which, together with alterations of arterial diameter, serves to maintain a mean shear stress level of about 15 dynes/cm2. The presence of low shear stresses is frequently accompanied by unstable flow conditions (e.g. turbulence flow, regions of blood recirculation, “stagnant” blood areas).

(Papaioannou TG, Stefanadis C. Vascular Wall Shear Stress: Basic Principles and Methods. Hellenic J Cardiol 2005; 46: 9-15.)

Leukocyte adhesion under flow in the microvasculature is mediated by binding between cell surface receptors and complementary ligands expressed on the surface of the endothelium. Leukocytes adhere to endothelium in a two-step mechanism: rolling (primarily mediated by selectins) followed by firm adhesion (primarily mediated by integrins). These investigators simulated the adhesion of a cell to a surface in flow, and elucidated the relationship between receptor–ligand functional properties and the dynamics of adhesion using a computational method called ‘‘Adhesive Dynamics.’’ This relationship was expressed in a one-to-one map between the biophysical properties of adhesion molecules and various adhesive behaviors.

Behaviors that are observed in simulations include firm adhesion, transient adhesion (rolling), and no adhesion. They varied the dissociative properties, association rate, bond elasticity, and shear rate and found that the unstressed dissociation rate, kro, and the bond interaction length, γ, are the most important molecular properties controlling the dynamics of adhesion.

(Chang KC, Tees DFJ and Hammer DA. The state diagram for cell adhesion under flow: Leukocyte rolling and firm adhesion. PNAS 2000; 97(21):11262-11267.)

The study of the effect of leukocyte adhesion on blood flow in small vessels is of primary interest to understand the resistance changes in venular microcirculation when blood is considered as a homogeneous Newtonian fluid. When studying the effect of leukocyte adhesion on the non-Newtonian Casson fluid flow of blood in small venules; the Casson model represents the effect of red blood cell aggregation. In this model the blood vessel is considered as a circular cylinder and the leukocyte is considered as a truncated spherical protrusion in the inner side of the blood vessel. Numerical simulations demonstrated that for a Casson fluid with hematocrit of 0.4 and flow rate Q = 0:072 nl/s, a single leukocyte increases flow resistance by 5% in a 32 m diameter and 100 m long vessel. For a smaller vessel of 18 m, the flow resistance increases by 15%.

(Das B, Johnson PC, and Popel AS. Computational fluid dynamic studies of leukocyte adhesion effects on non-Newtonian blood flow through microvessels. Biorheology  2000; 37:239–258.)

Biologists have identified many of the molecular constituents that mediate adhesive interactions between white blood cells, the cell layer that lines blood vessels, blood components, and foreign bodies. However, the mechanics of how blood cells interact with one another and with biological or synthetic surfaces is quite complex: owing to the deformability of cells, the variation in vessel geometry, and the large number of competing chemistries present (Lipowski et al., 1991, 1996).

Adhesive interactions between white blood cells and the interior surface of the blood vessels they contact is important in inflammation and in the progression of heart disease. Parallel-plate microchannels have been useful in characterizing the strength of these interactions, in conditions that are much simplified over the complex environment these cells experience in the body. Recent computational and experimental work by several laboratories have attempted to bridge this gap between behavior observed in flow chamber experiments, and cell surface interactions observed in the microvessels of anesthetized animals.

We have developed a computational simulation of specific adhesive interactions between cells and surfaces under flow. In the adhesive dynamics formulation, adhesion molecules are modeled as compliant springs. One well-known model used to describe the kinetics of single biomolecular bond failure is due to Bell, which relates the rate of dissociation kr to the magnitude of the force on the bond F. The rate of formation directly follows from the Boltzmann distribution for affinity. The expression for the binding rate must also incorporate the effect of the relative motion of the two surfaces. Unless firmly adhered to a surface, white blood cells can be effectively modeled as rigid spherical particles, as evidenced by the good agreement between bead versus cell in vitro experiments (Chang and Hammer, 2000).

Various in vitro, in vivo, and computational methods have been developed to understand the complex range of transient interactions between cells, neighboring cells, and bounding surfaces under flow. Knowledge gained from studying physiologically realistic flow systems may prove useful in microfluidic applications where the transport of blood cells and solubilized, bioactive molecules is needed, or in miniaturized diagnostic devices where cell mechanics or binding affinities can be correlated with clinical pathologies.

(King MR. Cell-Surface Adhesive Interactions in Microchannels and Microvessels.   First International Conference on Microchannels and Minichannels. 2003, Rochester, NY. Pp 1-6. ICMM2003-1012.

P-selectin role in adhesion of leukocytes and sickle cells blocked by heparin

Vascular occlusion is responsible for much of the morbidity associated with sickle cell disease. Although the underlying cause of sickle cell disease is a single nucleotide mutation that directs the production of an easily polymerized hemoglobin protein, both the erythrocyte sickling caused by hemoglobin polymerization and the interactions between a proadhesive population of sickle cells and the vascular endothelium are essential to vascular occlusion.

Interactions between sickle cells and the endothelium use several cell adhesion molecules. Sickle red cells express adhesion molecules including integrin, CD36, band 3 protein, sulfated glycolipid, Lutheran protein, phosphatidylserine, and integrin-associated protein. The proadhesive sickle cells may bind to endothelial cell P-selectin, E-selectin, vascular cell adhesion molecule-1 (VCAM-1), CD36, and integrins. Activation of endothelial cells by specific agonists enhances adhesion by inducing the expression of cellular adhesion molecules and by causing cell contraction, which exposes extracellular matrix proteins, such as thrombospondin (TSP), laminin, and fibronectin. Initial events likely involve the adhesion of sickle erythrocytes to activated endothelial cells under laminar flow. The resultant adhesion of cells to the vascular wall creates nonlaminar and arrested flow, which propagates vascular occlusion by both static and flow adhesion mechanisms. It is likely too that the distinct mechanisms of adhesion and of regulation of endothelial cell adhesivity pertain under dissimilar types of flow.

The expression of adhesion molecules by endothelial cells is affected by cell agonists such as thrombin, histamine, tumor necrosis factor  (TNF-), interleukin 1 (IL-1), platelet activating factor (PAF), erythropoietin, and vascular endothelial growth factor (VEGF), and by local environmental factors such as hypoxia, reperfusion, flow, as well as by sickle erythrocytes themselves. An important effector in sickle cell vascular occlusion is thrombin. Increased thrombin activity correlates with sickle cell disease pain episodes. In addition to generating fibrin clot, thrombin also acts on specific thrombin receptors on endothelial cells and platelets. Work from our laboratory has demonstrated that thrombin treatment causes a rapid increase of endothelial cell adhesivity for sickle erythrocytes under static conditions

We have also reported that sickle cell adhesion to endothelial cells under static conditions involves P-selectin. Although P-selectin plays a major role in the tethering, rolling, and firm adhesion of leukocytes to activated endothelial cells, its contribution to the initial steps is singular and essential to the overall adhesion process. Upon stimulation of endothelial cells by thrombin, P-selectin rapidly translocates from Weibel-Palade bodies to the luminal surface of the cells. Others have shown that sickle cell adhesion is decreased by unfractionated heparin, but the molecular target of this inhibition has not been defined. We postulated that the adhesion of sickle cells to P-selectin might be the pathway blocked by unfractionated heparin. Heparin is known to block certain types of tumor cell adherence, TSP-independent sickle cell adherence, and coagulation processes that are active in sickle cell disease. In one uncontrolled study, prophylactic administration of heparin reduced the frequency of sickle cell pain crises. The role of P-selectin in the endothelial adhesion of sickle red blood cells, the capacity of heparin to block selected P-selectin–mediated adhesive events, and the effect of heparin on sickle cell adhesion suggest an association among these findings.

We postulate that, in a manner similar to that seen for neutrophil adhesion, P-selectin may play a role in the tethering and rolling adhesion of sickle cells. As with neutrophils, integrins may then mediate the firm adhesion of rolling sickle erythrocytes. The integrin  is expressed on sickle reticulocytes and can mediate adhesion to endothelial cells, possibly via endothelial VCAM-4. The endothelial integrin, V3, also mediates sickle cell adhesion to endothelial cells. Other 1 and 3 integrins may also fulfill this role.

In this report we demonstrate that the flow adherence of sickle cells to thrombin-treated human vascular endothelial cells also uses P-selectin and that this component of adhesion is inhibited by unfractionated heparin. We also demonstrate that sickle cells adhere to immobilized recombinant P-selectin under flow conditions. This adhesion too was inhibited by unfractionated heparin, in a concentration range that is clinically attainable. These findings and the general role of P-selectin in initiating adhesion of blood cells to the endothelium suggest that unfractionated heparin may be useful in preventing painful vascular occlusion. A clinical trial to test this hypothesis is indicated.

(Matsui NM, Varki A, and Embury SH.  Heparin inhibits the flow adhesion of sickle red blood cells to P-selectin  Blood. 2002; 100:3790-3796)

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