Response variability to aspirin as assessed by the platelet function analyzer (PFA)-100. A systematic review.

It was the aim of the present study to perform a systematic review of the published studies that estimated the prevalence of non-responders to aspirin, as assessed by the closure time of PFA-100, a point-of-care device, and to analyse: 1) some major clinical and methodological factors that can influence it and 2) its possible association with vascular outcomes. The prevalence of non-responders to aspirin in 64 populations from 53 studies, comprising 6,450 subjects, had a median value of 0.27. A higher number of aspirin non-responders was found among older patients, those with acute vascular events, or those treated for more than one month. Aspirin non-response was more frequently associated with the use of "home-established" cut-offs or when closure time was only assessed after aspirin (rather than both before and after). Among risk factors, type 2 diabetes appeared to be associated with a higher prevalence of aspirin non-responders. The latter was also higher in less recent publications and in studies that used 3.2% rather than 3.8% Na-citrate as an anticoagulant. In eight studies comprising 847 subjects, aspirin non-responders were more likely to have vascular events than responders (relative risk: 1.63; 95% CI 1.16-2.28). In conclusion, although there appears to be heterogeneity among the studies analysed, this review indicates that about one quarter of people receiving aspirin would be identified--as an average--as aspirin non-responders by PFA-100. As this is a simple, widely available point-of-care test, efforts to better standardize it and to control for its major methodological variables might be useful to improve monitoring of platelet performance under aspirin treatment and to firmly establish the observed association with clinical vascular events.

The pituitary adenylate cyclase-activating polypeptide is a physiological inhibitor of platelet activation.

The pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide of the vasoactive intestinal peptide/secretin/glucagon superfamily. Studies in two related patients with a partial trisomy 18p revealed three copies of the PACAP gene and elevated PACAP concentrations in plasma. The patients suffer from severe mental retardation and have a bleeding tendency with mild thrombocytopenia, and their fibroblasts show increased PACAP mRNA levels. The PACAP receptor (vasoactive intestinal peptide/pituitary adenylate cyclase-activating peptide receptor 1 [VPAC1]) in platelets and fibroblasts is coupled to adenylyl cyclase activation. Accordingly, we found increased basal cAMP levels in patients' platelets and fibroblasts, providing a basis for the reduced platelet aggregation in these patients. Megakaryocyte-specific transgenic overexpression of PACAP in mice correspondingly increased PACAP release from platelets, reduced platelet activation, and prolonged the tail bleeding time. In contrast, the PACAP antagonist PACAP(6-38) or a monoclonal PACAP antibody enhanced the collagen-induced aggregation of normal human platelets, and in PACAP knockout mice, an increased platelet sensitivity toward collagen was found. Thus, we found that PACAP modulates platelet function and demonstrated what we believe to be the first hemostatic defect associated with PACAP overexpression; our study suggests the therapeutic potential to manage arterial thrombosis or bleeding by administration of PACAP mimetics or inhibitors, respectively.

-391 C to G substitution in the regulator of G-protein signalling-2 promoter increases susceptibility to the metabolic syndrome in white European men: consistency between molecular and epidemiological studies.

BACKGROUND: The regulator of G-protein signalling-2 (RGS2) is a key factor in adipogenesis. We hypothesized that the metabolic syndrome, of which obesity is an important component, might be related to genetic variation in RGS2. METHODS AND RESULTS: We screened the human RGS2 gene. We tested the functionality of a common genetic variant in vitro, ex vivo, and in epidemiological study involving six European populations. The C to G substitution at position -391 in the RGS2 promoter was associated with enhanced RGS2 expression in vitro in transfected 3T3-L1 adipocytes and Chinese hamster cells and ex vivo in adipocytes from male, but not female, volunteers. In 2732 relatives from 512 families and 348 unrelated individuals, randomly recruited from six European populations, the prevalence of GG homozygosity was 54.1%. The metabolic syndrome score, a composite of six continuous traits making up this clinical entity, was 0.27 standardized units higher (P < 0.001) in 795 GG homozygous men compared with 683 men carrying the C allele. Transmission of the -391 G allele to male offspring was associated with a 0.20 unit increase in the score (P=0.039). These epidemiological relations were not significant in 1602 women. CONCLUSIONS: The C to G substitution at position -391 in the RGS2 promoter increases RGS2 expression in adipocytes and is associated with the metabolic syndrome in white European men. Further experimental and clinical research should establish whether this common polymorphism might be a target for preventive or therapeutic intervention.

Pharmacological stabilization of mast cells abrogates late thrombotic events induced by diesel exhaust particles in hamsters.

BACKGROUND: Particulate air pollution is associated with cardiovascular diseases and myocardial infarction (MI). METHODS AND RESULTS: We investigated the relationship between airway inflammation and thrombosis 24 hours after intratracheal (IT) instillation of diesel exhaust particles (DEP; 50 microg/hamster). Mild thrombosis was induced in the femoral vein by endothelial injury, and the consequences of airway inflammation on thrombogenicity were studied via online video microscopy. Lung inflammation and histamine analysis in bronchoalveolar lavage (BAL) and plasma were performed after pretreatment with dexamethasone (DEX) or sodium cromoglycate (SC). DEP induced airway inflammation and histamine release in BAL and in plasma, and increased thrombosis, without elevating plasma von Willebrand factor (vWF) levels. The IT instillation of 400-nm positively charged polystyrene particles (500 microg/hamster), serving as particles that do not penetrate into the circulation, equally produced airway inflammation, histamine release, and enhanced thrombosis. Histamine in plasma resulted from basophil activation. Intraperitoneal (IP) pretreatment with DEX (5 mg/kg) abolished the DEP-induced histamine increase in BAL and plasma and abrogated airway inflammation and thrombogenicity. The IT pretreatment with DEX (0.5 mg/kg) showed a partial but parallel inhibition of all of these parameters. Pretreatment with SC (40 mg/kg, IP) strongly inhibited airway inflammation, thrombogenicity, and histamine release. CONCLUSIONS: Our results are compatible with the triggering of mast cell degranulation and histamine release by DEP. Histamine plays an initial central role in airway inflammation, further release of histamine by circulating basophils, and peripheral thrombotic events. Antiinflammatory pretreatment can abrogate the peripheral thrombogenicity by preventing histamine release from mast cells.

Quality assessment of oral anticoagulation in Belgium, as practiced by a group of general practitioners.

OBJECTIVE: In Belgium oral anticoagulation therapy is mainly supervised by general practitioners (GPs). This study aims to evaluate the quality of management of oral anticoagulation by Belgian GPs and to verify the relation between time in range and a set of potentially influencing co-variables. METHODS: In a retrospective cross-sectional study, involving 66 GP-practices, the INR-values obtained over a 6-month period were analysed. All INR-values were determined by a single clinical laboratory and additional medical information was provided by the GPs. Linear mixed models have been used to model the patient-specific percentage INR in target as a function of different co-variables. RESULTS: 737 patients were included in the study. Patients who underwent a surgical intervention with an interruption of the anticoagulation during the study were excluded. Patients were only included after the initial starting-up period. 5890 INR-values were obtained. A total of 92,566 days of therapy was evaluated. 50% of the day values were within 0.5 INR-units from target (and 66% within 0.75 INR-units from target). In a multiple regression model, a significant relation between the percentage of time in range and the target INR (2.5 or 3.5) and the gender of the patient was shown. The incidence rate for major bleeding was 5.5/100 patient years (and 3.5/100 patient years for thrombo-embolic events). CONCLUSION: The quality of management of oral anticoagulation by the GPs in Belgium is suboptimal. It is unknown whether interventions such as guidelines, feedback, point-of-care monitoring and computer-assisted anticoagulation monitoring could improve the results.

The intracellular tyrosine residues of the ATP-gated P2X(1) ion channel are essential for its function.

The four highly conserved intracellular tyrosine residues of the P2X(1) ion channel were mutated into phenylalanine. Simultaneous electrophysiological and calcium measurements in transfected human embryonic kidney (HEK 293) cells indicated that Y362F and Y370F mutants were non-functional, despite their proper plasma membrane expression. The Y16F and Y363F mutants retained 2.2% and 26% of the wild-type P2X(1) activity, respectively. However, no tyrosine phosphorylation was detected on Western blots of P2X(1) immunoprecipitates derived either from HEK 293 cell lysates or from human platelets, expressing P2X(1) endogenously. Thus, Y16, Y362, Y363 and Y370 are required for the appropriate three-dimensional structure and function of the intracellular P2X(1) domains.

Inhibition of von Willebrand factor-GPIb/IX/V interactions as a strategy to prevent arterial thrombosis.

Although drugs exist for the primary and secondary prevention of thrombosis, more potent antiplatelet drugs with sufficiently wide therapeutic windows to avoid bleeding complications are needed. Both academic and pharmaceutical laboratories are working to develop such drugs. This chapter reviews the potential of inhibiting interactions between von Willebrand factor (vWF) and the second most abundant receptor on the platelet, the glycoprotein (GP) Ib/IX/V complex, interactions that are essential for the activation of circulating platelets, contacting a vessel wall injury. Although still at the level of preclinical testing, this area is expected to progress quickly during the next few years, also in view of the three-dimensional structural information that has recently become available and that allows a molecular understanding of vWF binding to the GPIbalpha chain of the GPIb complex.

PACAP and its receptor VPAC1 regulate megakaryocyte maturation: therapeutic implications.

Megakaryocytes and platelets express the Gs-coupled VPAC1 receptor, for which the pituitary adenylyl cyclase-activating peptide (PACAP) and the vasointestinal peptide (VIP) are agonists. We here demonstrate a regulatory role for VPAC1 signaling during megakaryopoiesis. A total of 2 patients with trisomy 18p with PACAP overexpression and transgenic mice overexpressing PACAP in megakaryocytes have thrombopathy, a mild thrombocytopenia, and a reduced number of mature megakaryocytes in their bone marrow. In vitro differentiation of hematopoietic stem cells from the patient and transgenic mice shows a reduced number of megakaryocyte colonies compared with controls. The addition of PACAP, VIP, or the adenylyl cyclase activator forskolin to CD34(+) cells inhibits megakaryocyte differentiation. In contrast, neutralizing monoclonal anti-PACAP (PP1A4) or anti-VPAC1 (23A11) antibodies inhibit cAMP formation and stimulate megakaryopoiesis in a thrombopoietin-independent manner. Moreover, wild-type mice obtain an increased platelet count after subcutaneous injection of PP1A4 or 23A11. These antibodies also elevate platelet numbers in animal models of myelosuppressive therapy and in GATA1-deficient mice with congenital thrombocytopenia. Furthermore, 23A11 stimulates the in vitro megakaryocyte differentiation of both normal and GATA1-deficient human CD34(+) cells. Together, our data strongly suggest that VPAC1 signaling tempers normal megakaryopoiesis, and that inhibition of this pathway stimulates megakaryocyte differentiation, enhancing platelet recovery after myelosuppressive therapy and in GATA1 deficiency.

Thrombospondin-1 controls vascular platelet recruitment and thrombus adherence in mice by protecting (sub)endothelial VWF from cleavage by ADAMTS13.

The function of thrombospondin-1 (TSP-1) in hemostasis was investigated in wild-type (WT) and Tsp1-/- mice, via dynamic platelet interaction studies with A23187-stimulated mesenteric endothelium and with photochemically injured cecum subendothelium. Injected calcein-labeled WT platelets tethered or firmly adhered to almost all A23187-stimulated blood vessels of WT mice, but Tsp1-/- platelets tethered to 45% and adhered to 25.8% of stimulated Tsp1-/- vessels only. Stimulation generated temporary endothelium-associated ultralarge von Willebrand factor (VWF) multimers, triggering platelet string formation in 48% of WT versus 20% of Tsp1-/- vessels. Injection of human TSP-1 or thrombotic thrombocytopenic purpura (TTP) patient-derived neutralizing anti-ADAMTS13 antibodies corrected the defective platelet recruitment in Tsp1-/- mice, while having a moderate effect in WT mice. Photochemical injury of intestinal blood vessels induced thrombotic occlusions with longer occlusion times in Tsp1-/- venules (1027 +/- 377 seconds) and arterioles (858 +/- 289 seconds) than in WT vessels (559 +/- 241 seconds, P < .001; 443 +/- 413 seconds, P < .003) due to defective thrombus adherence, resulting in embolization of complete thrombi, a defect restored by both human TSP-1 and anti-ADAMTS13 antibodies. We conclude that in a shear field, soluble or local platelet-released TSP-1 can protect unfolded endothelium-bound and subendothelial VWF from degradation by plasma ADAMTS13, thus securing platelet tethering and thrombus adherence to inflamed and injured endothelium, respectively.

Thalamic stimulation as a treatment for primary erythromelalgia: technical case report.

OBJECTIVE AND IMPORTANCE: We report the use of bilateral thalamic stimulation in a case of primary erythromelalgia with immediate and important pain relief for 3 years. CLINICAL PRESENTATION: A 12-year-old boy experiencing primary erythromelalgia had a 4-year history of recurrent attacks of severe burning pain in both feet, accompanied by local reddening, swelling, and heating of the skin. The attacks were triggered by warmth and exercise. The pain was relieved only by elevation and cooling of the lower limbs, which he achieved by immersing his legs in a bucket of ice water, resulting in severe ulceration of the skin. INTERVENTION: Because of the gradual aggravation of the signs and symptoms and resistance of the patient's condition to several medical therapies, the patient received spinal cord stimulation. The implants were removed twice because of recurrent infection. Finally, the patient was treated with bilateral electrical stimulation of the ventral posterolateral thalamic nucleus, which resulted in important pain control until 3 years later. The patient was able to avoid water immersions, and all ulcerations disappeared. CONCLUSION: We conclude that thalamic stimulation was successful in this case of primary erythromelalgia.

The Belgian Improvement Study on Oral Anticoagulation Therapy: a randomized clinical trial.

AIMS: In Belgium, general practitioners (GPs) mainly manage oral anticoagulation therapy. To improve the quality of oral anticoagulation management by GPs and to compare different models and interventions, a randomized clinical trial was performed. METHODS AND RESULTS: Stratified randomization divided 66 GP-practices into four groups. A 6-month retrospective analysis assessed the baseline quality. In the prospective study, each group received education on oral anticoagulation, anticoagulation files, and patient information booklets (groups A, B, C, and D). Group B additionally received feedback every 2 months on their anticoagulation performance; group C determined the international normalized ratio (INR) with a CoaguChek device in the doctor's office or at the patient's home; and group D received Dawn AC computer assisted advice for adapting oral anticoagulation. For the different groups, the time spent in target INR range (Rosendaal's method) and adverse events related to anticoagulation were determined and compared with the same quality indicators at baseline. There was a significant increase in per cent of time within 0.5 INR from target, from 49.5% at baseline to 60% after implementing the different interventions. However, neither the per cent in target range nor the event rates differed among the four groups. CONCLUSION: The interventions significantly improved the quality of management of oral anticoagulation by Belgian GPs, mainly as a result of an education and support programme.

The TUBB1 Q43P functional polymorphism reduces the risk of cardiovascular disease in men by modulating platelet function and structure.

The discoid form of platelets is maintained by a marginal band of tightly coiled microtubules. beta1-tubulin is the major isoform within platelet and megakaryocyte microtubules. In 24.2% of 33 unrelated inherited macrothrombocytopenia patients and in 10.6% of 272 subjects of a healthy population a P for Q substitution in beta1-tubulin was found in the highly conserved residue 43. Heterozygous carriers of the Q43P variant showed a reduced platelet protein beta1-tubulin expression. Transfection of green fluorescent protein (GFP)-tagged Q43P beta1-tubulin in megakaryocytic MEG01 cells resulted in a disturbed tubulin organization. Electron microscopy revealed enlarged spherocytic platelets with a disturbed marginal band and organelle-free zones. In addition, platelets with the Q43P beta1-tubulin variant had reduced adenosine triphosphate (ATP) secretion, thrombin receptor activating peptide (TRAP)-induced aggregation and collagen adhesion. The prevalence of the Q43P beta1-tubulin variant was also 2 times higher (odds ratio, [OR] = 2.1;95% confidence interval [CI], 1.22-3.59) among control subjects than among patients with cardiovascular disease (10.4% versus 5.2%, P < .001). By analyzing this protective factor in men and women separately, this association was only found in men. This study thus presents the functional consequences of the platelet Q43P beta1-tubulin substitution that is frequent in the healthy population and may protect men against arterial thrombosis.

ATP augments von Willebrand factor-dependent shear-induced platelet aggregation through Ca2+-calmodulin and myosin light chain kinase activation.

Shear stress triggers von Willebrand factor (VWF) binding to platelet glycoprotein Ibalpha and subsequent integrin alpha(IIb)beta(3)-dependent platelet aggregation. Concomitantly, nucleotides are released from plateletdense granules, and ADP is known to contribute to shear-induced platelet aggregation (SIPA). We found that the impaired SIPA of platelets from a Hermansky-Pudlak patient lacking dense granules was restored by exogenous l-beta,gamma-methylene ATP, a stable P2X(1) agonist, as well as by ADP, confirming that in addition to ADP (via P2Y(1) and P2Y(12)), ATP (via P2X(1)) also contributes to SIPA. Likewise, SIPA of apyrase-treated platelets was restored upon P2X(1) activation with l-beta,gamma-methylene ATP, which promoted granule centralization within platelets and stimulated P-selectin expression, which is a marker of alpha-granule release. In addition, during SIPA, platelet degranulation required both extracellular Ca(2+) and VWF-glycoprotein Ibalpha interactions without involving alpha(IIb)beta(3). Neither platelet release nor SIPA was affected by protein kinase C inactivation, even though protein kinase C blockade inhibits platelet responses to collagen and thrombin in stirring conditions. In contrast, inhibiting myosin light chain (MLC) kinase with ML-7 reduced platelet release and SIPA by 30%. Accordingly, the potentiating effect of P2X(1) stimulation on the aggregation of apyrase-treated platelets coincided with intensified phosphorylation of MLC and was abrogated by ML-7. SIPA-induced MLC phosphorylation occurred exclusively through released nucleotides and selective antagonism of P2X(1) with MRS2159-reduced SIPA, ATP release, and potently inhibited MLC phosphorylation. We conclude that the P2X(1) ion channel induces MLC-mediated cytoskeletal rearrangements, thus contributing to SIPA and degranulation during VWF-triggered platelet activation.

Shielding the front-strand beta 3 of the von Willebrand factor A1 domain inhibits its binding to platelet glycoprotein Ibalpha.

Platelet adhesion to damaged vessel wall and shear-induced platelet aggregation necessitate binding of the von Willebrand factor (VWF) A1 domain to platelet GPIbalpha. Blocking this interaction represents a promising approach to the treatment of arterial thrombosis. Comparison of amino acid sequences of the VWF A1 domain in several species, expressing VWF recognized by the blocking monoclonal antibody AJvW-2, suggested 9 residues (His563, Ile566, Asp570, Ala581, Val584, Ala587, Arg616, Ala618, and Met622) to contribute to the epitope for AJvW-2 or to be part of the GPIbalpha-binding site. Glutathione-S-transferase (GST)-human VWF A1 fusion proteins, in which these amino acids were mutated to their murine counterparts, were tested for their capacity to bind AJvW-2 or heparin, to interfere with botrocetin- or ristocetin-mediated VWF binding to GPIb, or to induce flow-dependent platelet tethering in a perfusion chamber. Thus, mutations His563Arg, Ile566Leu, Asp570Ala, and Ala587Thr, clustered on the outer surface of the A1 domain, dramatically impaired binding of AJvW-2 to A1. The His563Arg, Ile566Leu, and Asp570Ala mutations also impaired the binding of heparin, which competes with AJvW-2 for binding to A1. Perfusion studies revealed that His563, Ile566, Asp570, Arg616, and Ala618 take part in GPIbalpha binding, their mutation-impairing platelet recruitment. In agreement with the surface distribution of VWF type 2M mutations, this study demonstrates overlapping of the epitope for AJvW-2 and the GPIbalpha-binding site, located around the front pocket of the A1 domain and defined by strands beta3, beta4, and helix alpha3, and it provides a mechanistic basis for VWF neutralization by this antibody.

Pulmonary inflammation and thrombogenicity caused by diesel particles in hamsters: role of histamine.

Short-term increases in particulate air pollution are associated with increased incidence of cardiovascular events. Previously, we showed that intratracheally instilled diesel exhaust particles (DEPs) are prothrombotic. Here, we investigated the time course and the mechanisms. At 1, 6, and 24 hours after instillation of 50 microg DEPs per hamster, the mean size of in vivo-induced and quantified venous thrombosis was increased by 480%, 770%, and 460%, respectively. Platelets activation in blood was confirmed by a shortened closure time in the platelet function analyzer (PFA-100). In bronchoalveolar lavage, neutrophils and histamine levels were increased at all time points. In plasma, histamine was increased at 6 and 24 hours but not at 1 and 3 hours. Pretreatment with a histamine H1-receptor antagonist (diphenhydramine, 30 mg/kg intraperitoneally) abolished the DEP-induced neutrophil influx in bronchoalveolar lavage at all time points. However, diphenhydramine pretreatment did not affect DEP-induced thrombosis or platelet activation at 1 hour, whereas both were markedly reduced at 6 and 24 hours. In conclusion, pulmonary inflammation and peripheral thrombosis are correlated at 6 and 24 hours, but at 1 hour, the prothrombotic effects do not appear to result from pulmonary inflammation but possibly from the blood penetration of DEP-associated components or by DEP particles themselves.

P2X(1)-mediated activation of extracellular signal-regulated kinase 2 contributes to platelet secretion and aggregation induced by collagen.

Adenosine triphosphate (ATP) and its stable analog, alpha,beta-methylene ATP, activate the platelet P2X(1) ion channel, causing a rapid Ca(++) influx. Here, we show that, in washed apyrase-treated platelets, alpha,beta-methylene ATP elicits reversible extracellular signal-regulated kinase 2 (ERK2) phosphorylation through a Ca(++)- and protein kinase C-dependent pathway. In contrast, high-performance liquid chromatography-purified adenosine diphosphate (ADP) did not trigger ERK2 phosphorylation. alpha,beta-Methylene ATP also activated the ERK2 pathway in P2X(1)-transfected HEK293 cells but not in cells expressing mutated P2X(1)delL nonfunctional channels. Because ATP released from the dense granules during platelet activation contributes to platelet aggregation elicited by low doses of collagen, and because collagen causes ERK2 phosphorylation, we have investigated the role of P2X(1)-mediated ERK2 activation in these platelet responses. We found that the antagonism of P2X(1) with ADP or desensitization of this ion channel with alpha,beta-methylene ATP both resulted in impaired ERK2 phosphorylation, ATP secretion, and platelet aggregation induced by low concentrations of collagen (< or = 1 microg/mL) without affecting the minor early dense granule release. Selective MEK1/2 inhibition by U-0126 and Ca(++) chelation with EGTA (ethyleneglycoltetraacetic acid) behaved similarly, whereas the PKC inhibitor GF109203-X totally prevented collagen-induced secretion and ERK2 activation. In contrast, when elicited by high collagen concentrations (2 microg/mL), platelet aggregation and secretion no longer depended on P2X(1) or ERK2 activation, as shown by the lack of their inhibition by alpha,beta-methylene ATP or U-0126. We thus conclude that mild platelet stimulation with collagen rapidly releases ATP, which activates the P2X(1)-PKC-ERK2 pathway. This process enhances further degranulation of the collagen-primed granules allowing platelet aggregation to be completed.

Molecular cloning and characterization of the GATA1 cofactor human FOG1 and assessment of its binding to GATA1 proteins carrying D218 substitutions.

Erythroid and megakaryocytic lineage differentiation and maturation are regulated via cooperation between transcription factor GATA1 and its essential cofactor friend-of-GATA1 (FOG1). The interaction between these two murine proteins is well studied in vitro and depends on the binding of Fog1 to the N-terminal zinc finger (N-finger) of Gata1. We identified the human FOG1 gene on chromosome 16q24 and found expression mainly in hematopoietic cells and also in several other tissues. Sequencing of FOG1 cDNA revealed a 1006 amino-acid protein that contained nine zinc fingers, highly homologous to murine Fog1 fingers. The amino acid sequence and the GATA1-binding capacity of the human and murine finger 5 are however different. Ex vivo binding studies demonstrated that FOG1 interacts with both GATA1 and GATA2. We and others have described patients with mutations in the GATA1 N-finger (V205 M, D218G, D218Y, or G208S), who suffer from macrothrombocytopenia and erythrocyte abnormalities. We now show ex vivo that the interaction between GATA1 and FOG1 is indeed disturbed in platelets and erythrocytes of those patients carrying D218 GATA1 mutations. The identification of the human FOG1 gene will enable the genetic screening of patients with non X-linked thrombocytopenia and dyserythropoiesis.

Endothelial von Willebrand factor recruits platelets to atherosclerosis-prone sites in response to hypercholesterolemia.

Platelets are thought to play a causal role during atherogenesis. Platelet-endothelial interactions in vivo and their molecular mechanisms under shear are, however, incompletely characterized. Here, an in vivo platelet homing assay was used in hypercholesterolemic rabbits to track platelet adhesion to plaque predilection sites. The role of platelet versus aortic endothelial cell (EC) activation was studied in an ex vivo flow chamber. Pathways of human platelet immobilization were detailed during in vitro perfusion studies. In rabbits, a 0.125% cholesterol diet induced no lesions within 3 months, but fatty streaks were found after 12 months. ECs at segmental arteries of 3- month rabbits expressed more von Willebrand factor (VWF) and recruited 5-fold more platelets than controls (P <.05, n = 5 and 4, respectively). The 3-month ostia had an increased likelihood to recruit platelets compared to control ostia (56% versus 18%, P <.0001, n = 89 and 63, respectively). Ex vivo, the adhesion of 3-month platelets to 3-month aortas was 8.4-fold increased compared to control studies (P <.01, n = 7 and 5, respectively). In vitro, endothelial VWF-platelet glycoprotein (GP) Ib and platelet P-selectin- endothelial P-selectin glycoprotein ligand 1 interactions accounted in combination for 83% of translocation and 90% of adhesion (P <.01, n = 4) of activated human platelets to activated human ECs. Platelet tethering was mainly mediated by platelet GPIb alpha, whereas platelet GPIIb/IIIa contributed 20% to arrest (P <.05). In conclusion, hypercholesterolemia primes platelets for recruitment via VWF, GPIb alpha, and P-selectin to lesion-prone sites, before lesions are detectable.