Role of CD36 in Platelet Function

CD36 is a Class B scavenger receptor expressed on platelets, erythrocyte precursors, monocytes, microvascular endothelial cells, epithelial cells, adipocytes, and cardiac and skeletal myocytes. It recognizes multiple ligands including thrombospondins, oxidized phospholipids and apoptotic cells, and has been shown to play a role in phagocytosis, angiogenesis and atherosclerosis. The function of CD36 on platelets is incompletely characterized, but our group has recently identified CD36 on platelets as a signaling receptor which can modulate platelet function by binding to ligands such as oxidized LDL. Endothelial cell (EC) derived microparticles (MP) have been identified in the circulation of patients with diseases such as diabetes, anti-phospholipid syndrome and acute coronary syndrome in which patients are prone to arterial thrombosis, and thus platelet activation and aggregation play a pivotal role. Because EC MP express phosphatidyl serine (PS) on their surfaces, a potential CD36 ligand, we hypothesize that MP may bind to platelets via a PS-CD36 interaction and function to transmit an activating signal, thereby promoting a prothrombotic state. vii To test this hypothesis, we first isolated EC-derived MP by stimulating human umbilical vein EC with TNFα and cyclohexamide according to a previously published protocol. MP were characterized and quantified by flow cytometry and shown to express CD105 and PS. Binding of MP to platelets was detected and quantified by flow cytometry and immunofluorescence microscopy. Platelet activation was assessed by aggregometry and flow cytometry. Washed human platelets (CD105 negative) were incubated with EC-derived MP at a ratio of 1:9 and analyzed by flow cytometry with a fluorescence tagged anti-CD105 dye) positive MP formed rosettes around (Calcein-Green Tagged) platelets. With both the flow cytometry and microscopy assays, platelet-MP association was inhibited by addition of anti-CD36 antibody or by using platelets from CD36 null donors. This inhibition by CD36 antibody was statistically significant (p=0.02). Furthermore, pretreatment of platelets with other CD36 ligands such as oxLDL inhibited MP-platelet association by more than 50%. Next we determined the functional effect of the MP-platelet association. We observed a significant increase in the rate and extent of platelet aggregation to low concentrations (2μM) of ADP and an increase in platelet secretion (measured as surface Pselectin expression) when platelets were incubated with EC-derived MP prior to addition of agonist. This effect was markedly diminished in platelets from CD36 null donors and also inhibited by pre-incubation with anti CD36 antibody. To test the MP-platelet interaction in vivo, carotid arteries were injured by FeCl3 in wild type and CD36 knock out mice. The thrombosed arteries were sectioned and immunostained with an endothelial cell specific antibody to CD105 (red) and a viii platelet specific antibody to CD61 (green). We reasoned that CD105 staining of thrombi would reflect incorporation of EC-derived MP into the thrombi. We observed significantly more CD105 staining within the thrombi from wild type mice compared to CD36 null mice, We thus propose a model where CD36 ligands presented to platelets renders them “hyperactive” predisposing patients to pathological thrombosis. It is also possible that CD36 ligands such as EMPs, generated during an acute thrombotic event, could increase the thrombotic response in a CD36 dependent manner by signaling platelets in a positive feedback loop. CD36 expression levels have been reported to vary significantly among normal human subjects. We thus hypothesized that levels of expression in an individual donor would correlate with platelet reactivity in response to CD36 ligands. We developed a quantitative flow cytometric technique to measure CD36 surface expression on platelets and studied 32 normal healthy volunteers. We found that expression levels were highly variable ranging from as low as less than 2000 molecules per platelet to more than 14000 molecules per platelet. We then replicated this study in a larger population (567) of subjects who came for screening at the Cleveland Clinic Cardiac Catheterization lab. To assess whether this variability of CD36 expression had a functional effect, donors were selected from high, medium and low expressing groups, and their platelet reactivity to oxLDL was analyzed by flow cytometric techniques. Results showed a very good correlation of platelet activation by oxidized LDL with levels of CD36 expression. ix Having shown that CD36 expression levels varied among individuals but was consistent over time in any single donor we hypothesized that a component of the variance was heritable. We identified 10 tagged SNPs in the CD36 gene from the International HapMap consortium website. DNA from the 550 subjects described above were then genotyped for the tagged SNPs and 3 SNPs were found to be significantly associated with expression level (rs3211864, p value 0.023, OR=0.55; rs3211932, p value=0.02, OR=0.617 and rs1537593, p value=0.03, OR=1.067). For all of these SNPs the minor allele was associated with lower levels of CD36 expression. These data suggest that the variability of CD36 expression on platelets is at least in part genetically determined and together this phenotype-genotype can affect platelet function.