Effect of cocoa powder on the modulation of inflammatory biomarkers in patients at high risk of cardiovascular disease

Background: Epidemiologic studies have suggested that flavonoid intake plays a critical role in the prevention of coronary heart disease. Because atherosclerosis is considered a low-grade inflammatory disease, some feeding trials have analyzed the effects of cocoa (an important source of flavonoids) on inflammatory biomarkers, but the results have been controversial. Objective: The objective was to evaluate the effects of chronic cocoa consumption on cellular and serum biomarkers related to atherosclerosis in high-risk patients. Design: Forty-two high-risk volunteers (19 men and 23 women; mean 6 SD age: 69.7 6 11.5 y) were included in a randomized crossover feeding trial. All subjects received 40 g cocoa powder with 500 mL skim milk/d (C+M) or only 500 mL skim milk/d (M) for 4 wk. Before and after each intervention period, cellular and serum inflammatory biomarkers related to atherosclerosis were evaluated. Results: Adherence to the dietary protocol was excellent. No significant changes in the expression of adhesion molecules on T lymphocyte surfaces were found between the C+M and M groups. However, in monocytes, the expression of VLA-4, CD40, and CD36 was significantly lower (P = 0.005, 0.028, and 0.001, respectively) after C+M intake than after M intake. In addition, serum concentrations of the soluble endothelium-derived adhesion molecules P-selectin and intercellular adhesion molecule-1 were significantly lower (both P = 0.007) after C+M intake than after M intake. Conclusions: These results suggest that the intake of cocoa polyphenols may modulate inflammatory mediators in patients at high risk of cardiovascular disease. These antiinflammatory effects may contribute to the overall benefits of cocoa consumption against atherosclerosis. This trial was registered in the Current Controlled Trials at London, International Standard Randomized Controlled Trial Number, at controlled-trials.com as ISRCTN75176807.

Rosuvastatin and vascular dysfunction markers in pulmonary arterial hypertension: a placebo-controlled study

We investigated whether chronic rosuvastatin administration could improve the abnormalities of the circulating levels of vascular dysfunction markers in pulmonary arterial hypertension (PAH). Sixty patients, aged 13 to 60 years, with idiopathic (N = 14) or congenital heart disease-associated PAH (N = 46) were equally but randomly assigned to rosuvastatin treatment (10 mg a day, orally) or placebo for 6 months in a blind fashion. Plasma levels of P-selectin, tissue-plasminogen activator and its inhibitor as well as von Willebrand factor antigen were measured by enzyme-linked immunoassay before and after 1, 3, and 6 months of treatment. Baseline levels of biomarkers were elevated (68, 16, 45 and 46% increase relative to controls, for P-selectin, von Willebrand factor antigen, tissue-plasminogen activator and its inhibitor, respectively; P < 0.001). P-selectin values at baseline, 1, 3, and 6 months were 39.9 ± 18.5, 37.6 ± 14.6, 34.8 ± 14.6, and 35.4 ± 13.9 ng/mL, respectively, for the rosuvastatin group and 45.7 ± 26.8, 48.0 ± 26.9, 48.1 ± 25.7, and 45.7 ± 25.6 ng/mL for the placebo group. The P-selectin level was lower in the rosuvastatin group compared with placebo throughout treatment (P = 0.037, general linear model). A trend was observed towards a decrease in tissue-plasminogen activator in the statin group (16% reduction, P = 0.094), with no significant changes in the other markers. Since P-selectin is crucial in inflammation and thrombosis, its reduction by rosuvastatin is potentially relevant in the pathophysiological scenario of PAH.

Effect of oral sirolimus therapy on inflammatory biomarkers following coronary stenting

We studied the effect of oral sirolimus, administered to prevent and treat in-stent restenosis (ISR), on the variation of serum levels of inflammatory markers following coronary stenting with bare metal stents. The mean age of the patients was 56 ± 13 years, 65% were males and all had clinically manifested ischemia. Serum levels of high sensitivity C-reactive protein (hs-CRP) concentration were determined by chemiluminescence and serum levels of all other biomarkers by ELISA. One group of patients at high risk for ISR received a loading oral dose of 15 mg sirolimus and 5 mg daily thereafter for 28 days after stenting (SIR-G). A control group (CONT-G) was submitted to stenting without sirolimus therapy. The increase in hs-CRP concentration was highest at 24 h after stenting in both groups. A significant difference between SIR-G and CONT-G was observed at 4 weeks (-1.50 ± 5.0 vs -0.19 ± 0.4, P = 0.008) and lost significance 1 month after sirolimus discontinuation (-1.73 ± 4.3 vs -0.01 ± 0.7, P = 0.0975). A continuous fall in MMP-9 concentration was observed in SIR-G, with the greatest reduction at 4 weeks (-352.9 ± 455 vs +395.2 ± 377, P = 0.0004), while a positive variation was noted 4 weeks after sirolimus discontinuation (227 ± 708 vs 406.2 ± 472.1, P = 0.0958). SIR-G exhibited a higher increase in P-selectin after sirolimus discontinuation at week 8 (46.1 ± 67.9 vs 5.8 ± 23.7, P = 0.0025). These findings suggest that the anti-restenotic actions of systemic sirolimus include anti-proliferative effects and modulation of the inflammatory response with inhibition of adhesion molecule expression.

Plasma von Willebrand factor as a predictor of survival in pulmonary arterial hypertension associated with congenital heart disease

Biomarkers have been identified for pulmonary arterial hypertension, but are less well defined for specific etiologies such as congenital heart disease-associated pulmonary arterial hypertension (CHDPAH). We measured plasma levels of eight microvascular dysfunction markers in CHDPAH, and tested for associations with survival. A cohort of 46 inoperable CHDPAH patients (age 15.0 to 60.2 years, median 33.5 years, female:male 29:17) was prospectively followed for 0.7 to 4.0 years (median 3.6 years). Plasma levels of von Willebrand factor antigen (VWF:Ag), tissue plasminogen activator (t-PA) and its inhibitor (PAI-1), P-selectin, reactive C-protein, tumor necrosis factor alpha, and interleukin-6 and -10 were measured at baseline, and at 30, 90, and 180 days in all subjects. Levels of six of the eight proteins were significantly increased in patients versus controls (13 to 106% increase, P < 0.003). Interleukin-10 level was 2.06 times normal (P = 0.0003; Th2 cytokine response). Increased levels of four proteins (t-PA, PAI-1, P-selectin, and interleukin-6) correlated with disease severity indices (P < 0.05). Seven patients died during follow-up. An average VWF:Ag (mean of four determinations) above the level corresponding to the 95th percentile of controls (139 U/dL) was independently associated with a high risk of death (hazard ratio = 6.56, 95%CI = 1.46 to 29.4, P = 0.014). Thus, in CHDPAH, microvascular dysfunction appears to involve Th2 inflammatory response. Of the biomarkers studied, plasma vWF:Ag was independently associated with survival.

Exogenous normal lymph reduces liver injury induced by lipopolysaccharides in rats

The liver is one of the target organs damaged by septic shock, wherein the spread of endotoxins begins. This study aimed to investigate the effects of exogenous normal lymph (ENL) on lipopolysaccharide (LPS)-induced liver injury in rats. Male Wistar rats were randomly divided into sham, LPS, and LPS+ENL groups. LPS (15 mg/kg) was administered intravenously via the left jugular vein to the LPS and LPS+ENL groups. At 15 min after the LPS injection, saline or ENL without cell components (5 mL/kg) was administered to the LPS and LPS+ENL groups, respectively, at a rate of 0.5 mL/min. Hepatocellular injury indices and hepatic histomorphology, as well as levels of P-selectin, intercellular adhesion molecule 1 (ICAM-1), myeloperoxidase (MPO), and Na+-K+-ATPase, were assessed in hepatic tissues. Liver tissue damage occurred after LPS injection. All levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in plasma as well as the wet/dry weight ratio of hepatic tissue in plasma increased. Similarly, P-selectin, ICAM-1, and MPO levels in hepatic tissues were elevated, whereas Na+-K+-ATPase activity in hepatocytes decreased. ENL treatment lessened hepatic tissue damage and decreased levels of AST, ALT, ICAM-1, and MPO. Meanwhile, the treatment increased the activity of Na+-K+-ATPase. These results indicated that ENL could alleviate LPS-induced liver injury, thereby suggesting an alternative therapeutic strategy for the treatment of liver injury accompanied by severe infection or sepsis.

High levels of LDL-C combined with low levels of HDL-C further increase platelet activation in hypercholesterolemic patients

High levels of low-density lipoprotein cholesterol (LDL-C) enhance platelet activation, whereas high levels of high-density lipoprotein cholesterol (HDL-C) exert a cardioprotective effect. However, the effects on platelet activation of high levels of LDL-C combined with low levels of HDL-C (HLC) have not yet been reported. We aimed to evaluate the platelet activation marker of HLC patients and investigate the antiplatelet effect of atorvastatin on this population. Forty-eight patients with high levels of LDL-C were enrolled. Among these, 23 had HLC and the other 25 had high levels of LDL-C combined with normal levels of HDL-C (HNC). A total of 35 normocholesterolemic (NOMC) volunteers were included as controls. Whole blood flow cytometry and platelet aggregation measurements were performed on all participants to detect the following platelet activation markers: CD62p (P-selectin), PAC-1 (GPIIb/IIIa), and maximal platelet aggregation (MPAG). A daily dose of 20 mg atorvastatin was administered to patients with high levels of LDL-C, and the above assessments were obtained at baseline and after 1 and 2 months of treatment. The expression of platelets CD62p and PAC-1 was increased in HNC patients compared to NOMC volunteers (P<0.01 and P<0.05). Furthermore, the surface expression of platelets CD62p and PAC-1 was greater among HLC patients than among HNC patients (P<0.01 and P<0.05). Although the expression of CD62p and PAC-1 decreased significantly after atorvastatin treatment, it remained higher in the HLC group than in the HNC group (P<0.05 and P=0.116). The reduction of HDL-C further increased platelet activation in patients with high levels of LDL-C. Platelet activation remained higher among HLC patients regardless of atorvastatin treatment.

Rôle des Cellules Endothéliales Progénitrices dans la Régulation de la Fonction Plaquettaire

Les Cellules Endothéliales Progénitrices ("Endothelial Progenitor Cells", EPCs) sont des précurseurs endothéliaux qui jouent un rôle émergeant en biologie vasculaire. Les EPCs ont été localisées dans le cordon ombilical, la moelle osseuse, le sang périphérique et dans certains tissus régénérateurs. Les interactions des EPCs avec les cellules sanguines et vasculaires peuvent largement influencer leurs propriétés biologiques et dicter leur fonctionnement pendant la réparation endothéliale. Plus spécifiquement, les interactions des EPCs avec les plaquettes circulantes induisent leur migration, leur recrutement et leur différentiation en cellules endothéliales aux sites de lésions vasculaires. Cependant, l’impact d’une telle interaction sur la fonction plaquettaire n’a pas été recherché. Le but de mon projet était de :1) générer des EPCs à partir des cellules mononucléaires du sang humain périphérique ("Peripheral Blood Mononuclear Cells", PBMCs); 2) étudier les interactions adhésives entre les EPCs et les plaquettes; 3) déterminer leur impact sur la fonction plaquettaire et la formation du thrombus et 4) décrire le mécanisme d’action des EPCs sur les plaquettes et le thrombus. Mises en culture sur une surface de fibronectine dans un milieu conditionné, les PBMCs fraîchement isolées possédaient une morphologie ronde et une petite taille. Après cinq jours, les PBMCs adhérentes donnaient naissance à des colonies, puis formaient une monocouche de cellules aplaties caractéristiques des EPCs après dix jours de culture. Les EPCs différenciées étaient positives pour l’Ulex-lectine et l’Acétyle des lipoprotéines de faible densité ("Acetylated Low Density Lipoprotein", Ac-LDL), exprimaient les marqueurs progéniteurs (CD34, P-sélectine, VEGFR2, vWF et VE-Cadhérine) tandis que les marqueurs leucocytaires (CD14, PSGL-1 et L-sélectine) étaient absents. Ces EPCs interagissaient avec les plaquettes activées par un mécanisme dépendant de la P-sélectine plaquettaire, inhibaient l’activation et l’agrégation plaquettaire et réduisaient significativement l’adhésion plaquettaire, principalement par l’action de prostacycline (PGI2). En fait, ceci était associé avec une augmentation de l’expression de la cyclooxygénase-2 (COX-2) et du monoxyde d’azote (NO) synthéthase inductible (iNOS). Toutefois, les effets inhibiteurs des EPCs sur la fonction plaquettaire ont été renversés par une inhibition de la COX et non pas du NO. Bien que les EPCs fussent en mesure de lier les plaquettes via la P-sélectine, leurs effets prédominants étaient médiés essentiellement par une sécrétion paracrine, impliquant la PGI2. Néanmoins, un rapprochement étroit ou un bref contact entre les EPCs et les plaquettes était requis pour que cette fonction soit complètement réalisée. D’ailleurs, cet aspect a été investigué chez des souris déficientes en P-sélectine (P-sel-/-) et chez leurs congénères de phénotype sauvage (Wild Type, WT). Chez les souris WT, les EPCs inhibaient l’agrégation plaquettaire dans le sang complet de manière concentration-dépendante alors que dans les souris P-sel-/-, l’action des EPCs n’avait pas d’effet significatif. De plus, en utilisant un modèle murin de thrombose artérielle, nous avons démontré que l’infusion systémique des EPCs altéraient la formation du thrombus et réduisaient significativement sa masse chez les souris WT, mais non pas chez les souris P-sel-/-. En outre, le nombre des EPCs incorporées au niveau du thrombus et de la paroi vasculaire était visiblement réduit chez les P-sel-/- par rapport aux souris WT. Dans cette étude, nous sommes parvenus à différentier adéquatement des EPCs à partir des PBMCs, nous avons étudié les interactions adhésives entre les EPCs et les plaquettes, et nous avons décrit leur impact sur la fonction plaquettaire et la formation du thrombus. De plus, nous avons identifié la PGI2 comme étant le principal facteur soluble sécrété par les EPCs en culture et responsable de leurs effets inhibiteurs sur l’activation, l’adhésion et l’agrégation plaquettaire in vitro. De surcroît, nous avons élucidé le mécanisme d’action des EPCs sur l’agrégation plaquettaire et la formation du thrombus, in vivo, et nous avons souligné le rôle de la P-sélectine plaquettaire dans ce processus. Ces résultats ajoutent de nouvelles connaissances sur la biologie des EPCs et définissent leur rôle potentiel dans la régulation de la fonction plaquettaire et la thrombogenèse.

Régulation de la fonction plaquettaire par un aptamère dirigé contre le domaine A1 du facteur Von-Willebrand

Drs Dandachli and Arzamendi contributed equally to this work.

Rôle de l’axe CD40L/CD40 dans les cellules endothéliales progénitrices

Les cellules endothéliales progénitrices («Endothelial Progenitor Cells», EPCs) sont des précurseurs endothéliaux qui possèdent un potentiel considérable dans la réparation et la régénération vasculaire. Dans le contexte des maladies cardiovasculaires, la compréhension du rôle des EPCs dans la régulation de la thrombogenèse et la réparation endothéliale est pertinente et nécessaire pour comprendre leur potentiel thérapeutique. Nous avons rapporté que les EPCs interagissent avec les plaquettes via la P-sélectine et inhibent l’adhésion, l’activation et l’agrégation des plaquettes ainsi que la formation de thrombus. Plus récemment, nous avons démontré que les EPCs expriment le récepteur inflammatoire CD40 et il est bien connu que les plaquettes constituent la source principale de la forme soluble de son agoniste le CD40L («soluble CD40 Ligand», sCD40L). Ainsi, nous avons émis l’hypothèse principale que l’axe CD40L/CD40 dans les EPCs influence leurs fonctions anti-thrombotique et pro-angiogénique. Pour vérifier cette hypothèse, nous avons réussi à générer des «early» et «late» EPCs à partir de cellules mononucléaires du sang périphérique («Peripheral Blood Mononuclear Cells», PBMCs) en culture. Nous avons mis en évidence l’existence de l’axe CD40L/CD40 dans ces EPCs en démontrant l’expression des protéines adaptatrices, nommées les facteurs associés au récepteur du facteur de nécrose tumorale («TNF Receptor Associated Factors», TRAFs). Dans une première étude, nous avons investigué l’effet du sCD40L sur la fonction des «early» EPCs dans l’agrégation plaquettaire. En effet, nous avons démontré que le sCD40L renverse leur effet inhibiteur sur l’agrégation plaquettaire, et ce sans avoir un effet significatif sur la sécrétion de prostacycline (PGI2) et d’oxyde nitrique («Nitric Oxide», NO) par ces cellules. De plus, aucun effet du sCD40L n’a été noté sur l’apoptose et la viabilité de ces cellules. Par contre, nous avons noté une augmentation importante du stress oxydatif dans les «early» EPCs suite à leur stimulation avec le sCD40L. L’inhibition du stress oxydatif renverse l’effet du sCD40L sur les «early» EPCs dans l’agrégation plaquettaire. Ces résultats pourraient expliquer, en partie, la fonction réduite des EPCs chez les individus présentant des niveaux élevés de sCD40L en circulation. Dans une deuxième étude, nous avons étudié l’effet de sCD40L dans la fonction des «early» EPCs en relation avec l’angiogenèse. Nous avons identifié, dans un premier temps,les métalloprotéinases de la matrice («Matrix Metalloproteinases», MMPs) qui sont sécrétées par ces cellules. Nous avons trouvé que les «early» EPCs relâchent principalement la MMP-9 et que cette relâche est augmentée par le sCD40L. Le sCD40L induit aussi la phosphorylation de la p38 MAPK qui contribue à augmenter la sécrétion de MMP-9. Des études fonctionnelles ont démontré que le prétraitement des «early» EPCs au sCD40L potentialise la réparation endothéliale des HUVECs. En conclusion, l’ensemble de nos travaux, dans le cadre de ce projet de doctorat, nous a permis d’élucider les mécanismes responsables de l’action du sCD40L sur les effets inhibiteur et angiogénique des «early» EPCs dans l’agrégation plaquettaire et l’angiogenèse, respectivement. Ces résultats ajoutent de nouvelles connaissances sur le rôle des EPCs et pourront constituer la base pour des études futures permettant de corréler les niveaux élevés du sCD40L circulant et l’incidence des maladies cardiovasculaires, particulièrement l’athérothrombose.

A functional genomics approach reveals novel quantitative trait loci associated with platelet signalling pathways

Platelet response to activation varies widely between individuals but shows interindividual consistency and strong heritability. The genetic basis of this variation has not been properly explored. We therefore systematically measured the effect on function of sequence variation in 97 candidate genes in the collagen and adenosine-diphosphate (ADP) signaling pathways. Resequencing of the genes in 48 European DNA samples nearly doubled the number of known single nucleotide polymorphisms (SNPs) and informed the selection of 1327 SNPs for genotyping in 500 healthy Northern European subjects with known platelet responses to collagen-related peptide (CRP-XL) and ADP. This identified 17 novel associations with platelet function (P < .005) accounting for approximately 46% of the variation in response. Further investigations with platelets of known genotype explored the mechanisms behind some of the associations. SNPs in PEAR1 associated with increased platelet response to CRP-XL and increased PEAR1 protein expression after platelet degranulation. The minor allele of a 3' untranslated region (UTR) SNP (rs2769668) in VAV3 was associated with higher protein expression (P = .03) and increased P-selectin exposure after ADP activation (P = .004). Furthermore the minor allele of the intronic SNP rs17786144 in ITPR1 modified Ca2+ levels after activation with ADP (P < .004). These data provide novel insights into key hubs within platelet signaling networks.

A role for the thiol isomerase. protein ERP5 in platelet function

Formation and rearrangement of disulfide bonds during the correct folding of nascent proteins is modulated by a family of enzymes known as thiol isomerases, which include protein disulfide isomerase (PDI), endoplasmic reticulum protein 5 (ERP5), and ERP57. Recent evidence supports an alternative role for this family of proteins on the surface of cells, where they are involved in receptor 'remodeling and recognition. In platelets, blocking PDI with inhibitory antibodies inhibits a number of platelet activation pathways, including aggregation, secretion, and fibrinogen binding. Analysis of human platelet membrane fractions identified the presence of the thiol isomerase protein ERP5. Further study showed that ERP5 is resident mainly on platelet intracellular membranes, although it is rapidly recruited to the cell, surface in response to a range of platelet agonists. Blocking cell-surface ERP5 using inhibitory antibodies leads to a decrease in platelet aggregation in response to agonists, and a decrease in fibrinogen binding and P-selectin exposure. It is Possible that this is based on the disruption of integrin function, as we observed that ERP5 becomes physically associated with the integrin beta(3) subunit during platelet stimulation. These results provide new insights into the involvement of thiol isomerases and regulation of platelet activation. (C) 2005 by The American Society of Hematology.

Non-genomic effects of PPARgamma ligands: inhibition of GPVI-stimulated platelet activation

BACKGROUND: Peroxisome proliferator-activated receptor-(gamma) (PPAR(gamma)) is expressed in human platelets although in the absence of genomic regulation in these cells, its functions are unclear. OBJECTIVE: In the present study, we aimed to demonstrate the ability of PPAR(gamma) ligands to modulate collagen-stimulated platelet function and suppress activation of the glycoprotein VI (GPVI) signaling pathway. METHODS: Washed platelets were stimulated with PPAR(gamma) ligands in the presence and absence of PPAR(gamma) antagonist GW9662 and collagen-induced aggregation was measured using optical aggregometry. Calcium levels were measured by spectrofluorimetry in Fura-2AM-loaded platelets and tyrosine phosphorylation levels of receptor-proximal components of the GPVI signaling pathway were measured using immunoblot analysis. The role of PPAR(gamma) agonists in thrombus formation was assessed using an in vitro model of thrombus formation under arterial flow conditions. RESULTS: PPAR(gamma) ligands inhibited collagen-stimulated platelet aggregation that was accompanied by a reduction in intracellular calcium mobilization and P-selectin exposure. PPAR(gamma) ligands inhibited thrombus formation under arterial flow conditions. The incorporation of GW9662 reversed the inhibitory actions of PPAR(gamma) agonists, implicating PPAR(gamma) in the effects observed. Furthermore, PPAR(gamma) ligands were found to inhibit tyrosine phosphorylation levels of multiple components of the GPVI signaling pathway. PPAR(gamma) was found to associate with Syk and LAT after platelet activation. This association was prevented by PPAR(gamma) agonists, indicating a potential mechanism for PPAR(gamma) function in collagen-stimulated platelet activation. CONCLUSIONS: PPAR(gamma) agonists inhibit the activation of collagen-stimulation of platelet function through modulation of early GPVI signalling.

Platelet protein disulfide isomerase is required for thrombus formation but not essential for hemostasis in mice

Protein disulfide isomerase (PDI) derived from intravascular cells is required for thrombus formation. However, it remains unclear whether platelet PDI contributes to the process. Using platelet-specific PDI-deficient mice, we demonstrate that PDI-null platelets have defects in aggregation and ATP secretion induced by thrombin, collagen, and ADP. Such defects were rescued by exogenously-added wild-type but not mutant PDI, indicating that the isomerase activity of platelet surface PDI is critical for the regulatory effect. PDI-deficient platelets expressed increased levels of intracellular ERp57 and ERp72. Platelet PDI regulated αIIbβ3 integrin activation but not P-selectin exposure, Ca2+ mobilization, β3-talin interaction, and platelet spreading on immobilized fibrinogen. Inhibition of ERp57 further diminished αIIbβ3 integrin activation, aggregation and ATP secretion of activated PDI-deficient platelets, suggesting distinct roles of PDI and ERp57 in platelet functions. We found that platelet PDI is important for thrombus formation on collagen-coated surfaces under arteriolar shear. Intravital microscopy demonstrates that platelet PDI is important for platelet accumulation but not initial adhesion and fibrin generation following laser-induced arteriolar injury. Tail bleeding time and blood loss in platelet-specific PDI-deficient mice were not significantly increased. Our results provide important evidence that platelet PDI is essential for thrombus formation but not for hemostasis in mice.

Mapping the platelet profile for functional genomic studies and demonstration of the effect size of the GP6 locus

BACKGROUND: Evidence suggests the wide variation in platelet response within the population is genetically controlled. Unraveling the complex relationship between sequence variation and platelet phenotype requires accurate and reproducible measurement of platelet response. OBJECTIVE: To develop a methodology suitable for measuring signaling pathway-specific platelet phenotype, to use this to measure platelet response in a large cohort, and to demonstrate the effect size of sequence variation in a relevant model gene. METHODS: Three established platelet assays were evaluated: mobilization of [Ca(2+)](i), aggregometry and flow cytometry, each in response to adenosine 5'-diphosphate (ADP) or the glycoprotein (GP) VI-specific crosslinked collagen-related peptide (CRP). Flow cytometric measurement of fibrinogen binding and P-selectin expression in response to a single, intermediate dose of each agonist gave the best combination of reproducibility and inter-individual variability and was used to measure the platelet response in 506 healthy volunteers. Pathway specificity was ensured by blocking the main subsidiary signaling pathways. RESULTS: Individuals were identified who were hypo- or hyper-responders for both pathways, or who had differential responses to the two agonists, or between outcomes. 89 individuals, retested three months later using the same methodology, showed high concordance between the two visits in all four assays (r(2) = 0.872, 0.868, 0.766 and 0.549); all subjects retaining their phenotype at recall. The effect of sequence variation at the GP6 locus accounted for approximately 35% of the variation in the CRP-XL response. CONCLUSION: Genotyping-phenotype association studies in a well-characterized, large cohort provides a powerful strategy to measure the effect of sequence variation in genes regulating the platelet response.

Platelet endothelial cell adhesion molecule-1 inhibits platelet response to thrombin and von Willebrand factor by regulating the internalization of glycoprotein Ib via AKT/glycogen synthase kinase-3/dynamin and integrin αIIbβ3

OBJECTIVE: Platelet endothelial cell adhesion molecule-1 (PECAM-1) regulates platelet response to multiple agonists. How this immunoreceptor tyrosine-based inhibitory motif-containing receptor inhibits G protein-coupled receptor-mediated thrombin-induced activation of platelets is unknown. APPROACH AND RESULTS: Here, we show that the activation of PECAM-1 inhibits fibrinogen binding to integrin αIIbβ3 and P-selectin surface expression in response to thrombin (0.1-3 U/mL) but not thrombin receptor-activating peptides SFLLRN (3×10(-7)-1×10(-5) mol/L) and GYPGQV (3×10(-6)-1×10(-4) mol/L). We hypothesized a role for PECAM-1 in reducing the tethering of thrombin to glycoprotein Ibα (GPIbα) on the platelet surface. We show that PECAM-1 signaling regulates the binding of fluorescein isothiocyanate-labeled thrombin to the platelet surface and reduces the levels of cell surface GPIbα by promoting its internalization, while concomitantly reducing the binding of platelets to von Willebrand factor under flow in vitro. PECAM-1-mediated internalization of GPIbα was reduced in the presence of both EGTA and cytochalasin D or latrunculin, but not either individually, and was reduced in mice in which tyrosines 747 and 759 of the cytoplasmic tail of β3 integrin were mutated to phenylalanine. Furthermore, PECAM-1 cross-linking led to a significant reduction in the phosphorylation of glycogen synthase kinase-3β Ser(9), but interestingly an increase in glycogen synthase kinase-3α pSer(21). PECAM-1-mediated internalization of GPIbα was reduced by inhibitors of dynamin (Dynasore) and glycogen synthase kinase-3 (CHIR99021), an effect that was enhanced in the presence of EGTA. CONCLUSIONS: PECAM-1 mediates internalization of GPIbα in platelets through dual AKT/protein kinase B/glycogen synthase kinase-3/dynamin-dependent and αIIbβ3-dependent mechanisms. These findings expand our understanding of how PECAM-1 regulates nonimmunoreceptor signaling pathways and helps to explains how PECAM-1 regulates thrombosis.