Genetic Variants In Platelet Supervillin but Not Archvillin Are Associated with PFA-100 Closure Times In African Americans

Abstract 4311

Myocardial infarction typically occurs after atherosclerotic plaque rupture that exposes collagen under high shear stress, favoring platelet adhesion, activation with ADP release, aggregation and platelet thrombus formation. There is a well-established genetic contribution to myocardial infarction, and we have previously shown a shear-dependent assay of platelet function is heritable (JTH 2007;5:1617). Collagen-induced platelet aggregation is highly heritable in African Americans (AAs) but not in European Americans, which may be relevant since AAs have the highest overall coronary heart disease mortality rate of all ethnic groups in the US. The mechanisms for these ethnic differences in platelet function are not clear. The goal of the current study was to identify genetic variants in AAs affecting the Platelet Function Analyzer (PFA)-100 (ADP cartridge) that is both collagen- and shear-dependent. We recruited healthy AA subjects and measured closure times in the PFA-100. Subjects were excluded if there was evidence of exposure to anti-platelet therapy (absent response to arachidonic acid). Closure times showed a normal distribution (mean CT = 96 sec +/− 20 sec [SD]). Genome-wide genotyping with the Infinium II Assay using the Illumina BeadStation 500 system was performed. After controlling for population admixture and excluding markers with a less than 95% call rate, 131 subjects passed criteria for ethnicity and successful genotyping of 956,604 tagSNPs. Statistical analysis identified a locus on chromosome 10 that appeared to be associated with PFA-100 closure times. Of the 7 most significantly associated SNPs, 5 were in SVIL, the gene encoding supervillin. The closest flanking genes to the SNP with the best P value were >77 kb upstream (2 pseudogenes) and >214 kb downstream (LTZ1; lysozyme-like 1). After adjusting for VWF and fibrinogen levels, rs10826650 in SVIL was associated with PFA-100 closure times (P = 5.92E–07). We pursued SVIL (chr 10p11.23) because it is known to form a high-affinity link between the actin cytoskeleton and the plasma membrane during cell spreading and disassembly of focal adhesions. Supervillin slows the rate of cell spreading and increases myosin contractility by linking the myosin II heavy chain with myosin light chain kinase. Supervillin also decreases the stability of focal adhesions and co-localizes with signaling molecules in cholesterol-rich lipid rafts. Prior work has identified 2 transcript variants encoding different isoforms of supervillin. Isoform 1 (supervillin) is reported to be most abundantly expressed in muscle, bone marrow, thyroid gland and salivary gland; isoform 2 (archvillin) is reported as muscle specific. Because neither isoform has been characterized in platelets, we performed gene expression analysis with 23 leukocyte depleted platelet (LDP) RNA samples (<1 leukocyte per 5 million platelets) using the Sentrix BeadChip and BeadStation system from Illumina, Inc. A probe recognizing both isoforms was highly expressed in these samples, whereas a probe specific for the archvillin isoform was barely detectable. The level of SVIL mRNA expression was higher than PECAM1 but less than GPIbα. These microarray data were confirmed by RT-PCR using LDP RNA and PCR primers specific for supervillin. Notably, SVIL was not detected in megakaryocyte lines, HEL or Meg-01. Anti-supervillin antisera recognized the appropriate sized ∼205 kD polypeptide by immunoblotting. PFA-100 closure times were found to directly correlate with supervillin (r = 0.481, P = 0.02) but not with archvillin (r = 0.11, P=n.s.) mRNA levels. In summary, we have demonstrated for the first time that platelets contain supervillin but not the alternately spliced isoform archvillin, and that expression is associated with PFA-100 closure times in AAs. Because higher supervillin expression was associated with longer closure times, this finding is consistent with an inhibitory role for supervillin in regulating the rapid actin cytoskeletal rearrangement required to form platelet thrombi on collagen under shear stress.