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Reversing Heart Disease: Combination of PCSK9 Inhibitors and Statins – Opinion by Steven Nissen, MD, Chairman of Cardiovascular Medicine at Cleveland Clinic

Reporter: Aviva Lev-Ari, PhD, RN

UPDATED on 2/25/2019
https://www.medpagetoday.com/cardiology/prevention/78202?xid=nl_mpt_SRCardiology_2019-02-25&eun=g99985d0r&utm_source=Sailthru&utm_medium=email&utm_campaign=CardioUpdate_022519&utm_term=NL_Spec_Cardiology_Update_Active

While nearly 10% of middle-age adults in China have high risk for cardiovascular disease, only 0.6% of these high-risk individuals use statins and 2.4% take aspirin, a national screening project reported in the Annals of Internal Medicine.

UPDATED on 5/5/2017

Europeans Mull PCSK9i Post-FOURIER Fallout on Clinical Practice

Patrice Wendling, May 04, 2017

But it was panelist Dr Stephen Nicholls (University of Adelaide, Australia) who took aim at the elephant in the packed auditorium. At an annual cost of about $14,100 for evolocumab and $14,600 for alirocumab (Praluent, Sanofi/Regeneron), the important question facing cardiologists is who will be eligible for these drugs “in a world where we can’t just write a scrip for every FOURIER-type patient; we won’t be allowed to.”

He suggested initially this will include patients with familial hypercholesterolemia and only those with established atherosclerotic CVD whose LDL-C remains unacceptably high despite therapy. Future FOURIER subanalyses may define other eligible high-risk groups.

http://www.medscape.com/viewarticle/879523?nlid=114642_3802&src=WNL_mdplsnews_170505_mscpedit_card&uac=93761AJ&spon=2&impID=1342003&faf=1#vp_2

 

 

UPDATED on 3/14/2017

PCSK9 Inhibitor Access Snarled in Red Tape, Rejections

Patrice Wendling, March 21, 2017

To determine whether this experience is happening nationwide, Navar and colleagues examined first PCSK9 prescriptions in 45,029 patients (median age 66 years; 51% female) between August 1, 2015 and July 31, 2016 in the Symphony Health Solutions database, which covers 90% of retail, 70% of specialty, and 60% of mail-order pharmacies in the US.

Nearly half (48%) of prescribers were cardiologists, and 37% were general practitioners. Most patients (52%) had government insurance, typically Medicare, and 40% had commercial insurance.

In the first 24 hours after being submitted to the pharmacy, 79.2% of prescriptions were rejected. Ultimately, 52.8% of all PCSK9 prescriptions were rejected.

Of special note, 34.7% of prescriptions for the pricy lipid-lowering drugs were abandoned at the pharmacy.

http://www.medscape.com/viewarticle/877515?nlid=113592_3802&src=WNL_mdplsnews_170324_mscpedit_card&uac=93761AJ&spon=2&impID=1314983&faf=1

 

How 2 Drugs Lower Cholesterol Remarkably — and Reverse Heart Disease

Study shows promise for combination of newer drug and statins

How 2 Drugs Lower Cholesterol Remarkably --- and Reverse Heart Disease

Newer cholesterol-lowering drugs combined with more conventional medicine reduces bad cholesterol to incredibly low levels, a new study shows. Perhaps even more important, the combination also reduces the heart-attack-inducing plaque that forms inside the arteries, the study says.

The study was led by cardiologist Steven Nissen, MD, Chairman of Cardiovascular Medicine at Cleveland Clinic. Results appeared recently in the Journal of the American Medical Association (JAMA).

The study looked at the use of a drug called evolocumab by people who took statins to lower the amount of LDL, or bad, cholesterol in their blood. Evolocumab is a drug called a PCSK9 inhibitor. This is a newer kind of medicine that can make LDL cholesterol levels plummet.

The people who took statins and evolocumab had greater reductions in atherosclerosis compared with people who took statins and a placebo.  Atherosclerosis is  a disease in which plaque builds up inside your arteries.  The condition can lead to serious problems, including heart attack, stroke, or even death.

The results are an intriguing indicator — rather than definite proof — that evolocumab may have benefit for patients taking statins, Dr. Nissen says. Researchers are still awaiting the results of large clinical trials investigating whether evolocumab is safe and will prevent heart attack, stroke or death. The first results of these studies are expected in April 2017.

Special ultrasound

In the study, researchers treated for 18 months 968 high-risk people who had extremely high levels of blood cholesterol.

Participants were randomly assigned to take either a statin and a placebo, or a statin and evolocumab.

Researchers monitored the participants’ cholesterol levels. They also used a special ultrasound probe to measure the amount of plaque in their arteries at the beginning and the end of the study. 

“We were able to show that getting the bad cholesterol levels down to really low levels, down to the 20s and 30s, can actually remove plaque from the coronary arteries,” Dr. Nissen says. “This going to levels that we’ve never been able to achieve before.”           

Low cholesterol, less plaque

Results show the group treated with statins and a placebo reduced their LDL cholesterol levels to 93 on average. At the same time, the group treated with the combination of the statin plus evolocumab got down to an average bad cholesterol level of 36.6.

“No one’s ever reached levels that low in a clinical trial,” Dr. Nissen says.

Participants who took evolocumab also had less plaque in their arteries at the end of the study — essentially reversing their heart disease.

“We, for the first time now, have shown that this new class of drugs, the PCSK9 inhibitors, has a favorable effect on the development of plaques on the coronary artery and can actually regress those plaques,” Dr. Nissen says. “And it turns out about two-thirds of patients actually had less plaque at the end of 18 months than they started with.” 

PCSK9 inhibitors, which are expensive, are not for everybody, Dr. Nissen says. Currently, the drug is used in addition to statins for the highest-risk patients with particularly high cholesterol.

SOURCE

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Praluent FDA Approved

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

PRALUENT® (alirocumab) is now approved for additional LDL-C lowering on top of maximally tolerated statin therapy in patients with HeFH or clinical ASCVD1
INDICATIONS AND USAGE
PRALUENT is a PCSK9 (Proprotein Convertase Subtilisin/Kexin Type 9) inhibitor antibody indicated as adjunct to diet and maximally tolerated statin therapy for the treatment of adults with heterozygous familial hypercholesterolemia or clinical atherosclerotic cardiovascular disease, who require additional lowering of LDL-C.

The effect of PRALUENT on cardiovascular morbidity and mortality has not been determined.

DOSING INFORMATION
The recommended starting dose of PRALUENT is 75 mg administered subcutaneously once every 2 weeks, since the majority of patients achieve sufficient LDL-C reduction with this dosage. If the LDL-C response is inadequate, the dosage may be increased to the maximum dosage of 150 mg administered every 2 weeks.

Measure LDL-C levels within 4 to 8 weeks of initiating or titrating PRALUENT to assess response and adjust the dose, if needed.

PRALUENT is a human monoclonal antibody that binds to PCSK91
PRALUENT efficacy was investigated in 5 double-blind, placebo-controlled trials with 3499 patients enrolled: 36% with HeFH and 54% non-FH with clinical ASCVD.
All patients were receiving a maximally tolerated dose of statin with or without other lipid-modifying therapies
3 studies used an initial dose of 75 mg Q2W as part of an up-titration regimen with criteria-based up-titration to 150 mg Q2W at week 12 for patients who did not achieve their prespecified target LDL-C at week 8
2 studies with 150 mg Q2W dose only
Clinical ASCVD is defined in the ACC/AHA guidelines2 as acute coronary syndromes or a history of any of the following: myocardial infarction, stable or unstable angina, coronary or other arterial revascularization, transient ischemic attack or stroke, or peripheral arterial disease presumed to be of atherosclerotic origin.
All studies met their primary efficacy endpoint measured at week 241
All trials were at least 52 weeks in duration with the primary efficacy endpoint measured at week 24 (mean percent change in LDL-C from baseline)
The first and only FDA-approved PCSK9 inhibitor with 2 doses that allows you to adjust the dose based on your patients’ LDL-C lowering needs1
MyPRALUENT™: Comprehensive support for you and your patients
MyPRALUENT is designed to help meet your needs and your patients’ needs
Speak with a MyPRALUENT Care Specialist at 1-844-PRALUENT (1-844-772-5836), option 1
IMPORTANT SAFETY INFORMATION
PRALUENT is contraindicated in patients with a history of a serious hypersensitivity reaction to PRALUENT. Reactions have included hypersensitivity vasculitis and hypersensitivity reactions requiring hospitalization.

Hypersensitivity reactions (e.g., pruritus, rash, urticaria), including some serious events (e.g., hypersensitivity vasculitis and hypersensitivity reactions requiring hospitalization), have been reported with PRALUENT treatment. If signs or symptoms of serious allergic reactions occur, discontinue treatment with PRALUENT, treat according to the standard of care, and monitor until signs and symptoms resolve.

The most commonly occurring adverse reactions (≥5% of patients treated with PRALUENT and occurring more frequently than with placebo) are nasopharyngitis, injection site reactions, and influenza.

Local injection site reactions including erythema/redness, itching, swelling, and pain/tenderness were reported more frequently in patients treated with PRALUENT (7.2% versus 5.1% for PRALUENT and placebo, respectively). Few patients discontinued treatment because of these reactions (0.2% versus 0.4% for PRALUENT and placebo, respectively), but patients receiving PRALUENT had a greater number of injection site reactions, had more reports of associated symptoms, and had reactions of longer average duration than patients receiving placebo.

Neurocognitive events were reported in 0.8% of patients treated with PRALUENT and 0.7% of patients treated with placebo. Confusion or memory impairment were reported more frequently by those treated with PRALUENT (0.2% for each) than in those treated with placebo (<0.1% for each).

Liver-related disorders (primarily related to abnormalities in liver enzymes) were reported in 2.5% of patients treated with PRALUENT and 1.8% of patients treated with placebo, leading to treatment discontinuation in 0.4% and 0.2% of patients, respectively. Increases in serum transaminases to greater than 3 times the upper limit of normal occurred in 1.7% of patients treated with PRALUENT and 1.4% of patients treated with placebo.

The most common adverse reactions leading to treatment discontinuation in patients treated with PRALUENT were allergic reactions (0.6% versus 0.2% for PRALUENT and placebo, respectively) and elevated liver enzymes (0.3% versus <0.1%).

PRALUENT is a human monoclonal antibody. As with all therapeutic proteins, there is a potential for immunogenicity with PRALUENT.

Please see full Prescribing Information.

 

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Pathophysiological Effects of Diabetes on Ischemic-Cardiovascular Disease and on Chronic Obstructive Pulmonary Disease (COPD)


Pathophysiological Effects of Diabetes on Ischemic-Cardiovascular Disease and on Chronic Obstructive Pulmonary Disease (COPD)

Curator:  Larry H. Bernstein, MD, FCAP

This is a multipart article that develops the pathological effects of type-2 diabetes in the progression of a systemic inflammatory disease with a development of neuropathy, and fully developing into cardiovascular disease.  It also identifies a systemic relationship to the development of chronic obstructive pulmonary disease (COPD).

The more we learn about diabetes, we learn about its generalized systemic effects.

This article has the following SIX Parts:

Part 1. Role of Autonomic Cardiovascular Neuropathy in Pathogenesis of ischemic heart disease in patients with diabetes mellitus

Part 2. A Longitudinal Cohort Study of the Cardiovascular Experience of Individuals at High Risk for Diabetes

Part 3.  Clinical significance of cardiovascular dysmetabolic syndrome

Part 4.   Waist circumference a good indicator of future risk for type 2 diabetes and cardiovascular disease

Part 5.   How to use C-reactive protein in acute coronary care

Part 6.  Chronic obstructive pulmonary disease and glucose metabolism: a bitter sweet symphony

INTRODUCTION

Type 2 diabetes mellitus is a common chronic disease which develops insidiously over time, and is associated with obesity, nutritional imbalance (high fructose beverages, high starch and processed foods, carbohydrate excess intake, and an imbalance of proinflammatory to anti-inflammatory polyunsaturated  fatty acids), which makes it an acquired and manageable disease.  The long term effects of T2DM is played out on cardiovascular disease and stroke-risk, obstructive sleep apnea, progressive renal insufficiency, development of neuropathy, congestive heart failure and chronic obstructive pulmonary disease, all of which are occuring related to an systemic inflammatory condition that proceeds for some time prior to the identification of overt diabetes.
A detailed story of a significant part of these associations continues in the SIX Part series.

Part 1. Role of Autonomic Cardiovascular Neuropathy in Pathogenesis of ischemic heart disease in patients with diabetes mellitus

This article is an abstract only of a related publication of the pathogenesis of autonomic neuropathy in diabetics leading to ischemic heart disease.

Subjects: Medicine (General), Medicine, Medicine (General),
Health Sciences Authors: Popović-Pejičić Snježana, Todorović-Đilas Ljiljana, Pantelinac Pavle
Publisher: Društvo lekara Vojvodine Srpskog lekarskog društva
Publication: Medicinski Pregled 2006; 59(3-4): Pp 118-123 (2006) ISSN(s): 0025-8105  Added to DOAJ: 2010-11-11
http://dx.doi.org/10.2298/MPNS0604118P  http://www.doiserbia.nb.rs/img/doi/0025-8105/2006/0025-81050604118P.pdf

Keywords: diabetes mellitus, autonomic nervous system diseases, heart diseases, myocardial ischemia, comorbidity

Introduction.

Diabetes is strongly associated with macrovascular complications, among which

  • ischemic heart disease is the major cause of mortality.

Autonomic neuropathy increases the risk of complications, which calls for an early diagnosis. The aim of this study was to determine

  • both presence and extent of cardiac autonomic neuropathy,

in regard to the type of diabetes mellitus, as well as

  • its correlation with coronary disease and
  • major cardiovascular risk factors.

Material and methods. We have examined 90 subjects, classified into three groups, with 30 patients each: those with type 1 diabetes, type 2 diabetes and control group of healthy subjects. All patients underwent

  • cardiovascular tests (Valsalva maneuver, deep breathing test, response to standing, blood pressure response to standing sustained, handgrip test),
  • electrocardiogram,
  • treadmill exercise test and
  • filled out a questionnaire referring to major cardiovascular risk factors: smoking, obesity, hypertension, and dyslipidemia.

Results. Our results showed that cardiovascular autonomic neuropathy was

  • more frequent in type 2 diabetes,
  • manifesting as autonomic neuropathy.

In patients with autonomic neuropathy, regardless of the type of diabetes,

  • the treadmill test was positive, i.e. strongly correlating with coronary disease.

In regard to coronary disease risk factors,

  • the most frequent correlation was found for obesity and hypertension.

Discussion

Cardiovascular autonomic neuropathy is considered to be the principal cause of arteriosclerosis and coronary disease. Our results showed that the occurrence of cardiovascular autonomic neuropathy increases the risk of coronary disease due to dysfunction of autonomic nervous system.

Conclusions

Cardiovascular autonomic neuropathy is a common complication of diabetes that significantly correlates with coronary disease. Early diagnosis of cardiovascular autonomic neuropathy points to increased cardiovascular risk, providing a basis for preventive and therapeutic measures.

Part 2. A Longitudinal Cohort Study of the Cardiovascular Experience of Individuals at High Risk for Diabetes

This second part is a description of a longitudinal cohort study of individuals at high-risk for diabetes.  Unlike the SSA study, the study is not focused on protein-energy malnutrition.

Protocol for ADDITION-PRO: a longitudinal cohort study of the cardiovascular experience of individuals at high risk for diabetes recruited from Danish primary care

Subjects: Public aspects of medicine, Medicine, Public Health, Health Sciences
Authors: Johansen NB, Hansen Anne-Louise S, Jensen TM, Philipsen A, Rasmussen SS, Jørgensen ME, Simmons RK, Lauritzen T, Sandbæk A, Witte DR
Publisher: BioMed Central    Date of publication: 2012 Dec Published in: BMC Public Health 2012; 12(1): 1078    ISSN(s): 1471-2458   Added to DOAJ: 2013-03-12 http://dx.doi.org/10.1186/1471-2458-12-1078       http://www.biomedcentral.com/1471-2458/12/1078

Keywords: Diabetes, Cardiovascular disease, Primary care, Complications, Microvascular, Impaired fasting glucose, Impaired glucose intolerance, Aortic stiffness, Physical activity, Body composition

Background

Screening programmes for type 2 diabetes inevitably find more individuals at high risk for diabetes than people with undiagnosed prevalent disease. While well established guidelines for the treatment of diabetes exist, less is known about treatment or prevention strategies for individuals found at high risk following screening. In order to make better use of the opportunities for primary prevention of diabetes and its complications among this high risk group, it is important to

  • quantify diabetes progression rates and to examine
  • the development of early markers of cardiovascular disease and
  • microvascular diabetic complications.

We also require a better understanding of the

  • mechanisms that underlie and drive early changes in cardiometabolic physiology.

The ADDITION-PRO study was designed to address these issues among individuals at different levels of diabetes risk recruited from Danish primary care.

Methods/Design

ADDITION-PRO is a population-based, longitudinal cohort study of individuals at high risk for diabetes. 16,136 eligible individuals were identified at high risk following participation in a stepwise screening programme in Danish general practice between 2001 and 2006.

  • All individuals with impaired glucose regulation at screening,
  • those who developed diabetes following screening, and
  • a random sub-sample of those at lower levels of diabetes risk

were invited to attend a follow-up health assessment in 2009–2011 (n = 4,188), of whom 2,082 (50%) attended. The health assessment included

  • detailed measurement of anthropometry,
  • body composition,
  • biochemistry,
  • physical activity and
  • cardiovascular risk factors including aortic stiffness and central blood pressure.

All ADDITION-PRO participants are being followed for incident cardiovascular disease and death.

Discussion

The ADDITION-PRO study is designed to increase

  • understanding of cardiovascular risk and
  • its underlying mechanisms among individuals at high risk of diabetes.

Key features of this study include

  • (i) a carefully characterised cohort at different levels of diabetes risk;
  • (ii) detailed measurement of cardiovascular and metabolic risk factors;
  • (iii) objective measurement of physical activity behaviour; and
  • (iv) long-term follow-up of hard clinical outcomes including mortality and cardiovascular disease.

Results will inform policy recommendations concerning cardiovascular risk reduction and treatment among individuals at high risk for diabetes. The detailed phenotyping of this cohort will also allow a number of research questions concerning early changes in cardiometabolic physiology to be addressed.

Part 3.  Clinical significance of cardiovascular dysmetabolic syndrome

This study also addresses the issue of diabetes insulin resistance leading to cardiovascular dysmetabolic syndrome.

Subjects: Diseases of the circulatory (Cardiovascular) system,
Specialties of internal medicine, Internal medicine, Medicine, Cardiovascular, Medicine (General), Health Sciences
Authors: Deedwania Prakash C Publisher: BioMed Central            Date of publication: 2002 Jan
Published in: Trials 2002; 3: 1(2)   ISSN(s): 1468-6708  Added to DOAJ: 2004-06-03
http://dx.doi.org/10.1186/1468-6708-3-2   http://cvm.controlled-trials.com/content/3/1/2

Keywords: cardiovascular dysmetabolic syndrome, coronary heart disease, diabetes mellitus, hyperinsulinemia, insulin resistance

Although diabetes mellitus is predominantly a metabolic disorder,

  • recent data suggest that it is as much a vascular disorder.
  • Cardiovascular complications are the leading cause
    • of death and disability in patients with diabetes mellitus.

A number of recent reports have emphasized that

  • many patients already have atherosclerosis in progression
  • at the time they are diagnosed with clinical evidence of diabetes mellitus.

The increased risk of atherosclerosis and cardiovascular complications in diabetic patients is related to

  • the frequently associated dyslipidemia, hypertension, hyperglycemia, hyperinsulinemia, and endothelial dysfunction.

The evolving knowledge regarding the variety of

  • metabolic,
  • hormonal, and
  • hemodynamic abnormalities in patients with diabetes mellitus

has led to efforts designed for early identification of individuals at risk of subsequent disease. It has been suggested that

  • insulin resistance, the key abnormality in type II diabetes,
  • often precedes clinical features of diabetes by 5–6 years.

Careful attention to the criteria described for the cardiovascular dysmetabolic syndrome

  • should help identify those at risk at an early stage.

The application of nonpharmacologic as well as newer emerging pharmacologic therapies can have beneficial effects

  • in individuals with cardiovascular dysmetabolic syndrome and/or diabetes mellitus
  • by improving insulin sensitivity and related abnormalities.

Early identification and implementation of appropriate therapeutic strategies would be necessary

  • to contain the emerging new epidemic of cardiovascular disease related to diabetes.

Part 4.   Waist circumference a good indicator of future risk for type 2 diabetes and cardiovascular disease

Subjects: Public aspects of medicine, Medicine, Public Health, Health Sciences
Authors: Siren Reijo, Eriksson Johan G, Vanhanen Hannu
Publisher: BioMed Central      Date of publication: 2012 Aug
Published in: BMC Public Health 2012; 12: 1(631)    ISSN(s): 1471-2458   Added to DOAJ: 2013-03-12
http://dx.doi.org/10.1186/1471-2458-12-631    http://www.biomedcentral.com/1471-2458/12/631

Keywords: Waist circumference, Type 2 diabetes, Cardiovascular disease, Middle-aged men

Background

Abdominal obesity is a more important risk factor than overall obesity in

  • predicting the development of type 2 diabetes and cardiovascular disease.

From a preventive and public health point of view it is crucial that

  • risk factors are identified at an early stage,
  • in order to change and modify behaviour and lifestyle in high risk individuals.

Methods

Data from a community based study was used to assess

  • the risk for type 2 diabetes,
  • cardiovascular disease and
  • prevalence of metabolic syndrome in middle-aged men.

In order to identify those with increased risk for type 2 diabetes and/or cardiovascular disease

  • sensitivity and specificity analysis were performed, including
  • calculation of positive and negative predictive values, and
  • corresponding 95% CI for eleven different cut-off points,
    • with 1 cm intervals (92 to 102 cm), for waist circumference.

Results

A waist circumference ≥94 cm in middle-aged men,

  • identified those with increased risk for type 2 diabetes
  • and/or for cardiovascular disease

with a sensitivity of 84.4% (95% CI 76.4% to 90.0%), and a specificity of 78.2% (95% CI 68.4% to 85.5%). The positive predictive value was 82.9% (95% CI 74.8% to 88.8%), and negative predictive value 80.0% (95% CI 70.3% to 87.1%), respectively .

Conclusions

Measurement of waist circumference in middle-aged men

  • is a reliable test to identify individuals at increased risk for type 2 diabetes and cardiovascular disease.

This measurement should be used more frequently in daily practice in primary care

  • in order to identify individuals at risk and when planning health counselling and interventions.

Part 5.  How to use C-reactive protein in acute coronary care

Luigi M. Biasucci, Wolfgang Koenig, Johannes Mair, Christian Mueller, Mario Plebani, Bertil Lindahl, Nader Rifai,Per Venge,Christian Hamm, and the Study Group on Biomarkers in Cardiology of the Acute Cardiovascular Care Association of the European Society of Cardiology
Department of Cardiology B, Aarhus University Hospital, Tage Hansens Gade2, Aarhus DK-8000,Denmark; Germany, U.K., U.S., Italy
European Heart Journal Advance Access published Nov 7, 2013.  Current Opinion.  http://dx.doi.org/10.1093/eurheartj/eht435

Introduction

 C-reactive protein (CRP) is an acute phase protein and an established marker for detection, risk stratification, and monitoring of infections, and inflammatory and necrotic processes.. Because C-reactive protein is sensitive but not specific, its values must be nterpreted  in the clinical context. Inpatients with acute myocardial infarction (AMI), CRP increases within 4–6h of symptoms, peaks 2–4 days later,and returns to baseline after 7–10 days.

CRP has gained interest recently as a marker for risk stratification in acute coronary syndrome (ACS) when measured by high-sensitivity CRP assays. These assays have greater analytical sensitivity and reliably measure CRP concentrations within the reference range with low imprecision (5–10%). Because of evidence that atherosclerosis is an inflammatory disease, high-sensitivity CRP can be used as a biomarker of risk
in primary prevention and in patients with known cardiovascular disease. The aim of this review is to evaluate the use of CRP in patients with acute coronary disease.

The in-vitro stability of high-sensitivity C-reactive protein is excellent. Specific blood sampling conditions aren’t necessary.  However, retesting may be necessary with some assays if there is marked lipaemia.  Baseline and subsequent measures are in good for agreement for risk stratification despite biological variability of 30–60%.

The upper reference limit is method-dependent but usually 8mg/L for standard assays. The distribution of high-sensitivity CRP concentrations is skewed in both genders with a 50th percentile of_1.5mg/L (excluding women on hormone replacement therapy). Race differences have been reported. Most studies have reported no relationship with age,  but to circadian and seasonal variation. CRP concentrations are increased by smoking, obesity, and hormone replacement therapy and reduced by exercise, moderate alcohol drinking, and statin use. Correction for these factors is essential in reference range studies. CRP assays are not standardized. We recommend  the use of third-generation high-sensitivity CRP assays that combine features of standard and high-sensitivity CRP assays.  Required assay precision should be < 10% in the range of 3 and 10 mg/L.

Biochemical and analytical issues

Critical clinical concepts

(1) CRP concentrations are reported in mg/L
(2) CRP test results are method-dependent

  •  classification of patients into risk categories is usually comparable
(3) Third generation CRP assay are recommended
(4) No specific patient preparation before blood sampling is necessary
(5) The in-vitro stability of CRP is high

This is only a portion of the published concensus document. What is relevant to this discussion is that the hs-CRP is an extremely valuable marker for inflammatory disease.  It is not ordered often enough because of the broad range of values that we have become accustomed to for years, and it is elevated in rheumatologic conditions, but even then, it is widely used in pediatrics because children may present with rapidly emergent sepsis with very minimal sympoms.
The hs-CRP has opened a window to subliminal inflammatory disease that is diabetes, with accompanied arteriolar endothelial inflammation.

Part 6.  Chronic obstructive pulmonary disease and glucose metabolism: a bitter sweet symphony

Subjects: Diseases of the circulatory (Cardiovascular) system,
Specialties of internal medicine, Internal medicine, Medicine, Cardiovascular, Medicine (General), Health Sciences
Authors: Mirrakhimov Aibek E
Publisher: BioMed Central      Date of publication: Oct 2012   ISSN(s): 1475-2840
Published in: Cardiovascular Diabetology 2012; 11(1):132   Added to DOAJ: 2013-03-12
http://dx.doi.org/10.1186/1475-2840-11-132      http://www.cardiab.com/content/11/1/132

Keywords: COPD, Dysglycemia, Insulin resistance, Obesity, Metabolic syndrome, Diabetes mellitus endothelial dysfunction, Vasculopathy

Chronic obstructive pulmonary disease, metabolic syndrome and diabetes mellitus

  • are common and underdiagnosed medical conditions.

It was predicted that chronic obstructive pulmonary disease

  • will be the third leading cause of death worldwide by 2020.

The healthcare burden of this disease is even greater

  • if we consider the significant impact of chronic obstructive pulmonary disease on
    • the cardiovascular morbidity and mortality.

Chronic obstructive pulmonary disease

  • may be considered as a novel risk factor for new onset type 2 diabetes mellitus via

multiple pathophysiological alterations such as:

  1. inflammation and oxidative stress,
  2. insulin resistance,
  3. weight gain and
  4. alterations in metabolism of adipokines.

On the other hand, diabetes may act as an independent factor,

  • negatively affecting pulmonary structure and function.

Diabetes is associated with an increased risk of

  1. pulmonary infections,
  2. disease exacerbations and
  3. worsened COPD outcomes.

On the top of that, coexistent OSA

  • may increase the risk for type 2 DM in some individuals.

The current scientific data necessitate a greater outlook on chronic obstructive pulmonary disease and

  • chronic obstructive pulmonary disease may be viewed as a risk factor for
  • the new onset type 2 diabetes mellitus.

Conversely, both types of diabetes mellitus should be viewed as

  • strong contributing factors for the development of obstructive lung disease.

Such approach can potentially improve the outcomes and medical control for both conditions,

  • and, thus, decrease the healthcare burden of these major medical problems.

CONCLUSIONS

This discussion  presents a spectrum of cardiovascular risk associated with type 2 diabetes mellitus, with high risk for CVD, stroke, endothelial dysfunction, and an association with obesity, measured by waist circumference, and an underlying proinflammatory state that can be measured by CRP.

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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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nihms-292073-f0002  platelet and vessel

Protein_Slide_2  proteome

nihms-292073-f0001  platelets support integrity and barrier function

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