Platelet Reactivity Is Associated with VAMP8 Expression and a VAMP8 3′UTR Polymorphism

Abstract

Variations in platelet reactivity can promote either a hemorrhagic or thrombotic phenotype, and platelet granule secretion is essential for platelet activation and normal hemostasis. We have earlier rigorously phenotyped platelet reactivity in 337 healthy subjects and found excellent reproducibility for secretion-dependent platelet functions. Vesicle associated membrane protein (VAMP)-8, a v-SNARE, is the primary regulator of platelet secretion. Our goals were to determine whether the expression of 9 key molecules involved in the platelet secretory pathway – SNAP23, SNAP25, VAMP3, VAMP8, STX2, STX4, STX7, STXBP3, and RAB27B are associated with the well-known variability in platelet reactivity and investigate potential mechanisms. Leukocyte-depleted platelets were prepared from 10 subjects with hyperreactive platelets and 6 subjects with hyporeactive platelets. No significant variations were observed for SNAP25, STX2, STX4 and RAB27B mRNA levels. Although small variations were observed for other genes examined VAMP8 mRNA levels showed the highest variation. VAMP8 mRNA levels were 4.8-fold higher in hyperreactive platelets than hyporeactive platelets (P=0.0023). Immunoblotting showed that VAMP8 proteins were also significantly elevated by 2.5-fold in the hyperresponsive subjects (P = 0.04). An A-to-G transition in the 3′UTR region of the VAMP8 gene (rs1010) has been consistently shown to be associated with a high risk of myocardial infarction (MI) in European Americans [EA]. We genotyped all 337 subjects (182 EA and 155 African Americans [AA]) in our cohort for this rs1010 SNP. The ‘G’ allele frequency was 0.44 and 0.62 in EA and AA, respectively. The ‘G’ allele was associated with enhanced aggregation to epinephrine (P=0.001) and ADP (P=0.001) in an age dependent manner. The 3′ UTR of VAMP8 has at least 10 predicted microRNAs (miRNAs), 8 of which we have identified in platelets and 5 of which encompass the rs1010 SNP. In summary, variable VAMP8 expression levels appear to be a major regulator of platelet reactivity, and a common VAMP8 SNP known to be associated with MI is associated with the platelet aggregation response to epinephrine and ADP. Studies are currently on-going to assess whether this SNP modifies miRNA binding and whether this miRNA regulates VAMP8 expression.