Platelet CD36 surface expression levels affect functional responses to oxidized LDL and are associated with inheritance of specific genetic polymorphisms

Abstract

CD36 modulates platelet function via binding to oxidized LDL (oxLDL), cell-derived microparticles, and thrombospondin-1. We hypothesized that the level of platelet CD36 expression may be associated with inheritance of specific genetic polymorphisms and that this would determine platelet reactivity to oxLDL. Analysis of more than 500 subjects revealed that CD36 expression levels were consistent in individual donors over time but varied widely among donors (200-14,000 molecules per platelet). Platelet aggregometry and flow cytometry in a subset of subjects with various CD36 expression levels revealed a high level of correlation (r² = 0.87) between platelet activation responses to oxLDL and level of CD36 expression. A genome-wide association study of 374 white subjects from the Cleveland Clinic ASCLOGEN study showed strong associations of single nucleotide polymorphisms in CD36 with platelet surface CD36 expression. Most of these findings were replicated in a smaller subset of 25 black subjects. An innovative gene-based genome-wide scan provided further evidence that single nucleotide polymorphisms in CD36 were strongly associated with CD36 expression. These studies show that CD36 expression on platelets varies widely, correlates with functional responses to oxLDL, and is associated with inheritance of specific CD36 genetic polymorphisms, and suggest that inheritance of specific CD36 polymorphisms could affect thrombotic risk.

Figure 1

Variation of platelet CD36 surface expression in a normal population. PRP from human subjects was incubated with either PE-conjugated anti-CD36 IgG or its isotype control and then analyzed by flow cytometry. (A) Left panel: representative histogram from a subject expressing high CD36 levels. Middle panel: Representative histogram from a subject expressing low CD36 levels. Right panel: histogram from a CD36 null subject. (B) CD36 expression levels on platelets from 32 normal volunteer donors were quantified as described in the “Quantitative assay of platelet CD36 expression.” Each triangle represents a single donor at one point in time. (C) Four donors were analyzed at least 5 times over time periods of 3 to 6 months. Bar graph represents the mean and coefficient of variance of platelet CD36 expression.

Figure 2

Variation of platelet CD36 surface expression in a larger population. Platelet CD36 expression was quantified by flow cytometry as in Figure 1 in a population of 500 patients presenting to the cardiac catheterization laboratory of Cleveland Clinic Foundation. (A) Each diamond represents a single patient. (B) CD36 expression levels in male and female patients. No significant difference was seen (P = .31).

Figure 3

Platelet CD36 expression levels correlate with activation response to oxLDL. (A) PRP from 4 different healthy subjects expressing high, medium, low, or no platelet CD36 were incubated with oxLDL 50 μg/mL for 30 minutes and then stimulated with low-dose ADP (2μM). Aggregation was assessed turbidometrically with a dual-chamber aggregometer. Tracings show representative aggregometry curves from triplicate assays performed on platelets from 2 CD36 null donors and from 5 or more donors from each of the 3 other groups. oxLDL at this concentration by itself did not effect platelet aggregation. (B) Washed platelets from 7 normal healthy subjects with levels of CD36 expression ranging from 0 to more than 12 000 molecules/platelet were incubated with oxLDL 50 μg/mL for 30 minutes, and platelet α-granule release was quantified by flow cytometry using anti–P-selectin IgG. The graph plots mean fluorescence intensity of anti–P-selectin binding against surface expression of CD36 and shows a correlation coefficient (r²) of 0.87.

Figure 4

Manhattan plot of SNP-CD36 MFI associations from the white cohort. Chromosome and position information from NCBI Build 37/hg19. Black horizontal line indicates the 0.10 Bonferroni threshold.

Figure 5

Manhattan plot of gene-CD36 MFI associations from the LSKM genome-wide scan from the white cohort. Chromosome and gene position information from hg19. Dark gray horizontal line indicates the 0.05 Bonferroni threshold.

Figure 6

CD36 SNP P values for association with CD36 MFI. P values are listed in supplemental Table 1. CAF indicates coded allele frequency; and R2, estimated r² of SNP with top SNP rs3211870, estimated separately for each subset (white and black). Dark gray horizontal line represents the Bonferroni 0.05 threshold for the CD36 cis analysis. SNPs with dark labels have been associated with lipid, metabolic, or CVD phenotypes in the literature (supplemental Table 2).