A synthesis approach of mouse studies to identify genes and proteins in arterial thrombosis and bleeding.

Antithrombotic therapies reduce cardiovascular diseases by preventing arterial thrombosis and thromboembolism, but at expense of increased bleeding risks. Arterial thrombosis studies using genetically modified mice have been invaluable for identification of new molecular targets. Because of low sample sizes and heterogeneity in approaches or methodologies, a formal meta-analysis to compare studies of mice with single-gene defects encountered major limitations. To overcome these, we developed a novel synthesis approach to quantitatively scale 1514 published studies of arterial thrombus formation (in vivo and in vitro), thromboembolism, and tail-bleeding of genetically modified mice. Using a newly defined consistency parameter (CP), indicating the strength of published data, comparisons were made of 431 mouse genes, of which 17 consistently contributed to thrombus formation without affecting hemostasis. Ranking analysis indicated high correlations between collagen-dependent thrombosis models in vivo (FeCl3 injury or ligation/compression) and in vitro. Integration of scores and CP values resulted in a network of protein interactions in thrombosis and hemostasis (PITH), which was combined with databases of genetically linked human bleeding and thrombotic disorders. The network contained 2946 nodes linked to modifying genes of thrombus formation, mostly with expression in megakaryocytes. Reactome pathway analysis and network characteristics revealed multiple novel genes with potential contribution to thrombosis/hemostasis. Studies with additional knockout mice revealed that 4 of 8 (Apoe, Fpr2, Ifnar1, Vps13a) new genes were modifying in thrombus formation. The PITH network further: (i) revealed a high similarity of murine and human hemostatic and thrombotic processes and (ii) identified multiple new candidate proteins regulating these processes.

Altered platelet contents in survivors of early ischemic ventricular fibrillation: preliminary findings.

Early ischemic ventricular fibrillation (VF) in the setting of an acute myocardial infarction (AMI) due to thrombotic coronary occlusion remains a major health problem. Several animal studies have shown that platelet-dense granule contents released during thrombus formation can induce arrhythmias. We hypothesize that the platelet release reaction is involved in the predisposition to early ischemic VF. A case-control study was performed in patients who survived VF during a first AMI ("cases," n = 26) and in patients with one previous AMI without arrhythmias ("controls," n = 24). All patients were on aspirin 100 mg OD. Baseline platelet activation was assessed with flow cytometry. Response to activation was assessed with aggregometry, flow cytometry and PFA-100 analysis. Differences in platelet contents and content release were assessed by labeling platelet-dense granules with mepacrine and by measuring serotonin and ADP/ATP content. Patient and infarct characteristics and baseline platelet function tests were similar between groups. The mean time from event was 4.9 (±3.2) years among cases and 4.7 (±2.7) years among controls. Dense granule release was similar in cases versus controls. Platelet serotonin content in cases was higher than in controls (611 ± 118 ng/10E(9) platelets vs. 536 ± 141 ng/10(9), p = 0.048). Even years after the event, elevations in the platelet dense granule contents between VF survivors and controls may be detected. These preliminary findings shed new light on the pathophysiological mechanisms underlying ischemic VF, as platelet-dense granules may contain mediators of early ischemic VF risk.

Platelet interaction with von Willebrand factor is enhanced by shear-induced clustering of glycoprotein Ibα.

Initial platelet arrest at the exposed arterial vessel wall is mediated through glycoprotein Ibα binding to the A1 domain of von Willebrand factor. This interaction occurs at sites of elevated shear force, and strengthens upon increasing hydrodynamic drag. The increased interaction requires shear-dependent exposure of the von Willebrand factor A1 domain, but the contribution of glycoprotein Ibα remains ill defined. We have previously found that glycoprotein Ibα forms clusters upon platelet cooling and hypothesized that such a property enhances the interaction with von Willebrand factor under physiological conditions. We analyzed the distribution of glycoprotein Ibα with Förster resonance energy transfer using time-gated fluorescence lifetime imaging microscopy. Perfusion at a shear rate of 1,600 s(-1) induced glycoprotein Ibα clusters on platelets adhered to von Willebrand factor, while clustering did not require von Willebrand factor contact at 10,000 s(-1). Shear-induced clustering was reversible, not accompanied by granule release or αIIbß3 activation and improved glycoprotein Ibα-dependent platelet interaction with von Willebrand factor. Clustering required glycoprotein Ibα translocation to lipid rafts and critically depended on arachidonic acid-mediated binding of 14-3-3ζ to its cytoplasmic tail. This newly identified mechanism emphasizes the ability of platelets to respond to mechanical force and provides new insights into how changes in hemodynamics influence arterial thrombus formation.

Arachidonic acid depletion extends survival of cold-stored platelets by interfering with the [glycoprotein Ibα--14-3-3ζ] association.

BACKGROUND: Cold storage of platelets reduces bacterial growth and preserves their hemostatic properties better than current procedures do. However, storage at 0°C induces [14-3-3ζ-glycoprotein Ibα] association, 14-3-3ζ release from phospho-Bad, Bad activation and apoptosis. DESIGN AND METHODS: We investigated whether arachidonic acid, which also binds 14-3-3ζ, contributes to coldinduced apoptosis. RESULTS: Cold storage activated P38-mitogen-activated protein kinase and released arachidonic acid, which accumulated due to cold inactivation of cyclooxygenase-1/thromboxane synthase. Accumulated arachidonic acid released 14-3-3ζ from phospho-Bad and decreased the mitochondrial membrane potential, which are steps in the induction of apoptosis. Addition of arachidonic acid did the same and its depletion made platelets resistant to cold-induced apoptosis. Incubation with biotin-arachidonic acid revealed formation of an [arachidonic acid-14-3-3ζ-glycoprotein Ibα] complex. Indomethacin promoted complex formation by accumulating arachidonic acid and released 14-3-3ζ from cyclo-oxygenase-1. Arachidonic acid depletion prevented the cold-induced reduction of platelet survival in mice. CONCLUSIONS: We conclude that cold storage induced apoptosis through an [arachidonic acid-14-3-3ζ-glycoprotein Ibα] complex, which released 14-3-3ζ from Bad in an arachidonic acid-dependent manner. Although arachidonic acid depletion reduced agonist-induced thromboxane A(2) formation and aggregation, arachidonic acid repletion restored these functions, opening ways to reduce apoptosis during storage without compromising hemostatic functions post-transfusion.

Induction of insulin resistance by the adipokines resistin, leptin, plasminogen activator inhibitor-1 and retinol binding protein 4 in human megakaryocytes.

BACKGROUND: In normal platelets, insulin inhibits agonist-induced Ca(2+) mobilization by raising cyclic AMP. Platelet from patients with type 2 diabetes are resistant to insulin and show increased Ca(2+) mobilization, aggregation and procoagulant activity. We searched for the cause of this insulin resistance. DESIGN AND METHODS: Platelets, the megakaryocytic cell line CHRF-288-11 and primary megakaryocytes were incubated with adipokines and with plasma from individuals with a disturbed adipokine profile. Thrombin-induced Ca(2+) mobilization and signaling through the insulin receptor and insulin receptor substrate 1 were measured. Abnormalities induced by adipokines were compared with abnormalities found in platelets from patients with type 2 diabetes. RESULTS: Resistin, leptin, plasminogen activator inhibitor-1 and retinol binding protein 4 left platelets unchanged but induced insulin resistance in CHRF-288-11 cells. Interleukin-6, tumor necrosis factor-α and visfatin had no effect. These results were confirmed in primary megakaryocytes. Contact with adipokines for 2 hours disturbed insulin receptor substrate 1 Ser(307)-phosphorylation, while contact for 72 hours caused insulin receptor substrate 1 degradation. Plasma with a disturbed adipokine profile also made CHRF-288-11 cells insulin-resistant. Platelets from patients with type 2 diabetes showed decreased insulin receptor substrate 1 expression. CONCLUSIONS: Adipokines resistin, leptin, plasminogen activator-1 and retinol binding protein 4 disturb insulin receptor substrate 1 activity and expression in megakaryocytes. This might be a cause of the insulin resistance observed in platelets from patients with type 2 diabetes.

Deletion of the high-density lipoprotein receptor scavenger receptor BI in mice modulates thrombosis susceptibility and indirectly affects platelet function by elevation of plasma free cholesterol.

OBJECTIVE: Scavenger receptor BI (SR-BI) is a cell surface receptor that promotes the selective uptake of cholesteryl esters from high-density lipoprotein (HDL) by the liver. In mice, SR-BI deficiency results in increased plasma HDL cholesterol levels and enhanced susceptibility to atherosclerosis. The aim of this study was to investigate the role of SR-BI deficiency on platelet function. METHODS AND RESULTS: SR-BI-deficient mice were thrombocytopenic, and their platelets were abnormally large, probably because of an increased cholesterol content. The FeCl(3) acute injury model to study arterial thrombosis susceptibility showed that SR-BI wild-type mice developed total arterial occlusion after 24±2 minutes. In SR-BI-deficient mice, however, the time to occlusion was reduced to 13±1 minutes (P=0.02). Correspondingly, in SR-BI-deficient mice, platelets circulated in an activated state and showed increased adherence to immobilized fibrinogen. In contrast, platelet-specific disruption of SR-BI by bone marrow transplantation in wild-type mice did not alter plasma cholesterol levels or affect platelet count, size, cholesterol content, or reactivity, suggesting that changes in plasma cholesterol levels were responsible for the altered responsiveness of platelets in SR-BI-deficient mice. CONCLUSIONS: The function of SR-BI in HDL cholesterol homeostasis and prevention of atherosclerosis is indirectly also essential for maintaining normal platelet function and prevention of thrombosis.

The platelet P2Y12 receptor contributes to granule secretion through Ephrin A4 receptor.

The main responses of P2Y(1) ligation are platelet shape change and transient aggregation while P2Y(12) ligation amplifies P2Y(1)-induced aggregation and accelerates aggregation, secretion and thromboxane A(2) production induced by other agonist-receptor complexes. We searched for new targets of P2Y signalling using micro-arrays with 144 peptides representing known phosphosites of protein tyrosine kinases. ADP induced phosphorylation of peptides representing surface receptors, second messenger enzymes and cytoskeletal proteins. Strong phosphorylation was found in peptides representing Ephrin-receptor family members. Blockade of P2Y(1/12) inhibited phosphorylation of EphA4- and EphB1-peptides on micro-arrays. The EphA2/4 inhibitor 2,5-dimethylpyrrolyl benzoic acid derivative interfered with P2Y(1/12)-induced EphA4 phosphorylation, left P2Y(1)-induced aggregation unchanged but inhibited with P2Y(12)-induced secretion, second phase aggregation and thrombus formation on collagen at 1600 s(-1). These results show that platelet EphA4 is an important intermediate in P2Y(12)-induced granule secretion.

Activated human platelet products induce proarrhythmic effects in ventricular myocytes.

Sudden cardiac death remains one of the most prevalent modes of death and is mainly caused by ventricular fibrillation (VF) in the setting of acute ischemia resulting from coronary thrombi. Animal experiments have shown that platelet activation may increase susceptibility of ischemic myocardium to VF, but the mechanism is unknown. In the present study, we evaluated the effects of activated blood platelet products (ABPPs) on electrophysiological properties and intracellular Ca(2+) (Ca(2+)(i)) homeostasis. Platelets were collected from healthy volunteers. After activation, their secreted ABPPs were added to superfusion solutions. Rabbit ventricular myocytes were freshly isolated, and membrane potentials and Ca(2+)(i) were recorded using patch-clamp methodology and indo-1 fluorescence measurements, respectively. ABPPs prolonged action potential duration and induced early and delayed afterdepolarizations. ABPPs increased L-type Ca(2+) current (I(Ca,L)) density, but left densities of sodium current, inward rectifier K(+) current, transient outward K(+) current, and rapid component of the delayed rectifier K(+) current unchanged. ABPPs did not affect kinetics or (in)activation properties of membrane currents. ABPPs increased systolic Ca(2+)(i), Ca(2+)(i) transient amplitude, and sarcoplasmic reticulum Ca(2+) content. ABPPs did not affect the Na(+)-Ca(2+) exchange current (I(NCX)) in Ca(2+)-buffered conditions. Products secreted from activated human platelets induce changes in I(Ca,L) and Ca(2+)(i), which result in action potential prolongation and the occurrence of early and delayed afterdepolarizations in rabbit myocytes. These changes may trigger and support reentrant arrhythmias in ischemia models of coronary thrombosis.

Co-expression of the collagen receptors leukocyte-associated immunoglobulin-like receptor-1 and glycoprotein VI on a subset of megakaryoblasts.

BACKGROUND: The collagen receptor glycoprotein VI generates activating signals through an immunoreceptor tyrosine-based activating motif on the co-associated Fc receptor gamma chain. Leukocyte-associated immunoglobulin-like receptor-1 also ligates collagen but generates inhibitory signals through immunoreceptor tyrosine-based inhibitory motifs. Thus far, the cellular expression of glycoprotein VI and leukocyte-associated immunoglobulin-like receptor-1 appears mutually exclusive. DESIGN AND METHODS: Using flow cytometry, we studied expression of collagen receptors on differentiating human megakaryocytes. CD34(+) cells were isolated from umbilical cord blood and matured to megakaryocytes in vitro. Freshly isolated bone marrow cells were used to study primary megakaryocytes. Upon cell sorting, cytospins were made to examine cytological characteristics of differentiation. RESULTS: Megakaryocyte maturation is accompanied by up-regulation of glycoprotein VI and down-regulation of leukocyte-associated immunoglobulin-like receptor-1. Interestingly, both in cultures from hematopoietic stem cells and primary cells obtained directly from bone marrow, we identified a subset of morphologically distinct megakaryocytes which co-express glycoprotein VI and leukocyte-associated immunoglobulin-like receptor-1. CONCLUSIONS: This is the first report of a primary cell that co-expresses these collagen receptors with opposite signaling properties. Since megakaryocytes mature in the collagen-rich environment of the bone marrow, these findings may point to a role for leukocyte-associated immunoglobulin-like receptor-1 in the control of megakaryocyte maturation/migration.

Downregulation of platelet responsiveness upon contact with LDL by the protein-tyrosine phosphatases SHP-1 and SHP-2.

OBJECTIVE: The sensitivity of platelets to aggregating agents increases when low-density lipoprotein (LDL) binds to apolipoprotein E receptor 2' (apoER2'), triggering activation of p38MAPK and formation of thromboxane A2. LDL signaling is terminated by PECAM-1 through recruitment and activation of the Ser/Thr protein phosphatase PP2A, but platelets remain unresponsive to LDL when PECAM-1 activation disappears. We report a second mechanism that halts LDL signaling and in addition lowers platelet responsiveness to aggregating agents. METHODS AND RESULTS: After a first stimulation with LDL, platelets remain unresponsive to LDL for 60 minutes, despite normal apoER2' activation by a second dose of LDL. A possible cause is persistent activation of the tyrosine phosphatases SHP-1 and SHP-2, which may not only block a second activation of p38MAPK, PECAM-1, and PP2A by LDL but also seem to reduce aggregation by TRAP, collagen, and ADP. CONCLUSION: These findings reveal that p38MAPK phosphorylation and platelet activation by LDL are suppressed by two mechanisms: (1) short activation of PECAM-1/PP2A, and (2) prolonged activation of SHP-1 and SHP-2. Activation of SHP-1 and SHP-2 is accompanied by reduced responsiveness to aggregating agents, which--if present in vivo--would make LDL an aggregation inhibitor during prolonged contact with platelets.

From low-density lipoprotein to platelet activation.

There is a strong correlation between the level of plasma low-density lipoprotein (LDL) and death by cardiovascular disease (CVD). As a main carrier of cholesterol, a high low-density lipoprotein concentration stimulates atherogenesis by its capacity to become oxidized and to become endocytosed by macrophages in the vessel wall forming cholesterol-rich plaques that are sites for arterial occlusion. New evidence points at a second role of low-density lipoprotein in increasing cardiovascular disease-risk. Contact with low-density lipoprotein induces platelet hypersensitivity to agonists that initiate platelet functions thereby enhancing adhesion, aggregation and secretion of granule contents. The signalling pathways that mediate the priming of platelets by native and oxidized low-density lipoprotein have now been characterized.

Role of membrane cholesterol in platelet calcium signalling in response to VWF and collagen under stasis and flow.

Several studies have highlighted a specific role for membrane cholesterol domains in platelet signalling. Upon adhesion to von Willebrand factor (VWF) or collagen, cholesterol-rich domains (CRDs) accumulate in filopodial extensions and selectively harbour counterpart receptors (GPIb and GPVI) and associated signalling molecules. In the present study we have addressed the role of membrane cholesterol in Ca(2+) signalling and secretion during the interaction of platelets with VWF and collagen. VWF/ristocetin-induced platelet aggregation was delayed after treatment with methyl beta-cyclodextrin (mbCD), but the maximal aggregation response was not affected. Platelet spreading but not adhesion to immobilised VWF under flow was attenuated by cholesterol removal, and accompanied by moderate lowering in the spiking Ca(2+) response. On the other hand, platelet interaction with collagen was quite sensitive to cholesterol depletion. Platelet aggregation decreased after treatment with mbCD, and Ca(2+) responses were decreased, both under static and flow conditions. Cholesterol depletion affected the secondary feedback activation via release of thromboxane A(2) and ADP. The collagen-induced secretion of alpha granules and surface translocation of P-selectin and CD63 was also critically affected by cholesterol depletion. Confocal microscopy showed localization of p-Tyr at sites of contact with substrate and other platelets, where also CRDs accumulate. Our data thus reveal a more critical role for membrane cholesterol in collagen-induced than in VWF-induced Ca(2+) signalling, and furthermore support the concept that secondary activation responses are dependent on intact CRDs.

Platelet activation by oxidized low density lipoprotein is mediated by CD36 and scavenger receptor-A.

OBJECTIVE: The interaction of platelets with low density lipoprotein (LDL) contributes to the development of cardiovascular disease. Platelets are activated by native LDL (nLDL) through apoE Receptor 2' (apoER2')-mediated signaling to p38(MAPK) and by oxidized LDL (oxLDL) through lysophosphatidic acid (LPA) signaling to Rho A and Ca2+. Here we report a new mechanism for platelet activation by oxLDL. METHODS AND RESULTS: Oxidation of nLDL increases p38(MAPK) activation through a mechanism that is (1) independent of LPA, and (2) unlike nLDL-signaling not desensitized by prolonged platelet-LDL contact or inhibited by receptor-associated protein or chondroitinase ABC. Antibodies against scavenger receptors CD36 and SR-A alone fail to block p38(MAPK) activation by oxLDL but combined blockade inhibits p38(MAPK) by >40% and platelet adhesion to fibrinogen under flow by >60%. Mouse platelets deficient in either CD36 or SR-A show normal p38(MAPK) activation by oxLDL but combined deficiency of CD36 and SR-A disrupts oxLDL-induced activation of p38(MAPK) by >70%. CONCLUSION: These findings reveal a novel platelet-activating pathway stimulated by oxLDL that is initiated by the combined action of CD36 and SR-A.

Activation of human platelets by misfolded proteins.

OBJECTIVE: Protein misfolding diseases result from the deposition of insoluble protein aggregates that often contain fibrils called amyloid. Amyloids are found in Alzheimer disease, atherosclerosis, diabetes mellitus, and systemic amyloidosis, which are diseases where platelet activation might be implicated. METHODS AND RESULTS: We induced amyloid properties in 6 unrelated proteins and found that all induced platelet aggregation in contrast to fresh controls. Amyloid-induced platelet aggregation was independent of thromboxane A2 formation and ADP secretion but enhanced by feedback stimulation through these pathways. Treatments that raised cAMP (iloprost), sequestered Ca2+ (BAPTA-AM) or prevented amyloid-platelet interaction (sRAGE, tissue-type plasminogen activator [tPA]) induced almost complete inhibition. Modulation of the function of CD36 (CD36-/- mice), p38(MAPK) (SB203580), COX-1 (indomethacin), and glycoprotein Ib alpha (Nk-protease, 6D1 antibody) induced approximately 50% inhibition. Interference with fibrinogen binding (RGDS) revealed a major contribution of alphaIIb beta3-independent aggregation (agglutination). CONCLUSIONS: Protein misfolding resulting in the appearance of amyloid induces platelet aggregation. Amyloid activates platelets through 2 pathways: one is through CD36, p38(MAPK), thromboxane A2-mediated induction of aggregation; the other is through glycoprotein Ib alpha-mediated aggregation and agglutination. The platelet stimulating properties of amyloid might explain the enhanced platelet activation observed in many diseases accompanied by the appearance of misfolded proteins with amyloid.

Platelets take up the monoclonal antibody bevacizumab.

PURPOSE: One of the key factors that promotes angiogenesis is vascular endothelial growth factor (VEGF). Platelets are the main source of VEGF in blood and contribute to angiogenesis by release of growth factors, including VEGF, from their alpha-granules on activation. The monoclonal antibody bevacizumab blocks VEGF in the blood of patients within hours after administration. Platelets are known to endocytose plasma proteins including immunoglobulins. We tested the hypothesis that platelets take up bevacizumab. EXPERIMENTAL DESIGN: Fluorescence-activated cell sorting analysis, immunofluorescence imaging, and Western blotting were used to study uptake and release of bevacizumab by platelets in vitro and in vivo. The angiogenic activity of platelets preincubated with bevacizumab was studied in endothelial proliferation assays. Finally, we determined whether treatment with bevacizumab neutralizes VEGF in platelets from cancer patients. RESULTS: We found that platelets are able to take up bevacizumab. Activation of platelets preincubated with bevacizumab resulted in release of the antibody and release of VEGF neutralized by bevacizumab. Immunofluorescence microscopy revealed that FITC-labeled bevacizumab and P-selectin colocalize, indicating alpha-granule localization. In addition, bevacizumab uptake inhibited platelet-induced human endothelial cell proliferation. In in vivo rabbit experiments, FITC-labeled bevacizumab was present in platelets after 2 h and up to 2 weeks following i.v. administration. Finally, we found that platelets take up bevacizumab in patients receiving bevacizumab treatment. Within 8 h after bevacizumab administration, platelet VEGF was almost completely neutralized due to this uptake. CONCLUSION: These studies show that bevacizumab is taken up by platelets and may explain its clinical effect on wound healing and tumor growth.

Hemostatic and signaling functions of transfused platelets.

Metabolic studies have revealed a gradual impairment in platelet integrity during storage, a process termed the platelet storage lesion. Recent evidence shows that stored platelets also lose signaling responses to physiological agonists with impaired integrin activation, secretion, and aggregation of the cells. On the other hand, storage leads to a gain in platelet activation properties, such as release of microparticles and appearance of surface epitopes for their clearance by macrophages. New techniques for measuring flow-induced thrombus formation and platelet-dependent coagulation provide evidence that the hemostatic activity of platelets decreases during storage. Besides pharmacological inhibition, novel storage strategies, like metabolic suppression, should be considered to better preserve platelet functionality while limiting the expression of clearance markers. Understanding the changes that occur in association with the platelet storage lesion and the use of updated storage methods will help to generate platelets for transfusion with optimal hemostatic function and a long circulation time after transfusion.

Platelet inhibition by insulin is absent in type 2 diabetes mellitus.

OBJECTIVE: ADP-induced P2y12 signaling is crucial for formation and stabilization of an arterial thrombus. We demonstrated recently in platelets from healthy subjects that insulin interferes with Ca2+ increases induced by ADP-P2y1 contact through blockade of the G-protein Gi, and thereby with P2y12-mediated suppression of cAMP. METHODS AND RESULTS: Here we show in patients with type 2 diabetes mellitus (DM2) that platelets have lost responsiveness to insulin leading to increased adhesion, aggregation, and procoagulant activity on contact with collagen. Using Ser473 phosphorylation of protein kinase B as output for insulin signaling, a 2-fold increase is found in insulin-stimulated normal platelets, but in DM platelets there is no significant response. In addition, DM2 platelets show increased P2y12-mediated suppression of cAMP and decreased P2y12 inhibition by the receptor antagonist AR-C69931MX. CONCLUSIONS: The loss of responsiveness to insulin together with increased signaling through P2y12 might explain the hyperactivity of platelets in patients with DM2.

Effect of oxidation on the platelet-activating properties of low-density lipoprotein.

OBJECTIVE: Because of the large variation in oxidizing procedures and susceptibility to oxidation of low-density lipoprotein (LDL) and the lack in quantification of LDL oxidation, the role of oxidation in LDL-platelet contact has remained elusive. This study aims to compare platelet activation by native LDL (nLDL) and oxidized LDL (oxLDL). METHODS AND RESULTS: After isolation, nLDL was dialyzed against FeSO4 to obtain LDL oxidized to well-defined extents varying between 0% and >60%. The oxLDL preparations were characterized with respect to their platelet-activating properties. An increase in LDL oxidation enhances platelet activation via 2 independent pathways, 1 signaling via p38(MAPK) phosphorylation and 1 via Ca2+ mobilization. Between 0% and 15% oxidation, the p38(MAPK) route enhances fibrinogen binding induced by thrombin receptor (PAR-1)-activating peptide (TRAP), and signaling via Ca2+ is absent. At >30% oxidation, p38(MAPK) signaling increases further and is accompanied by Ca2+ mobilization and platelet aggregation in the absence of a second agonist. Despite the increase in p38(MAPK) signaling, synergism with TRAP disappears and oxLDL becomes an inhibitor of fibrinogen binding. Inhibition is accompanied by binding of oxLDL to the scavenger receptor CD36, which is associated with the fibrinogen receptor, alpha(IIb)beta3. CONCLUSIONS: At >30% oxidation, LDL interferes with ligand binding to integrin alpha(IIb)beta3, thereby attenuating platelet functions.