The thrombospondin-1 N700S polymorphism is associated with early myocardial infarction without altering von Willebrand factor multimer size

Abstract

The N700S polymorphism of thrombospondin-1 (TSP-1) has been identified as a potential genetic risk factor for myocardial infarction (MI). In a large case-control study of 1425 individuals who survived a myocardial infarction prior to age 45, the N700S polymorphism was a significant risk factor for myocardial infarction in both homozygous (odds ratio [OR] 1.9, 95% confidence interval [CI] 1.1-3.3, P = .01) and heterozygous carriers of the S700 allele (OR 1.4, 95% CI 1.1-3.3, P = .01). TSP-1 has been shown to reduce von Willebrand factor (VWF) multimer size, and the domain responsible for VWF-reducing activity has been localized to the calcium-binding C-terminal sequence. As the N700S polymorphism was previously shown to alter the function of this domain, we investigated whether the altered VWF-reducing activity of TSP-1 underlies the observed prothrombotic phenotype. The TSP1 N700S polymorphism did not influence VWF multimer size in patients homozygous for either allele nor was there a significant reduction of VWF multimer size following incubation with recombinant N700S fragments or platelet-derived TSP-1.

Figure 1.

VWF multimer analysis following incubation with TSP-1 proteins. (A) 100 nM of TSP-1 in HEPES buffer (50 mM HEPES, 0.125 M NaCl, and 0.1 mM or 2 mM CaCl2) was incubated with TTP plasma for 1 hour at 37°C and then directly visualized by gel electrophoresis. The percentage of high molecular weight multimers was not affected by the addition of TSP-1 (P = .50) as measured by densitometry. (B-C) Recombinant VWF (0.4 U/mL) was incubated with full-length TSP-1 or N700S recombinant protein (10 nM or 100 nM) for 1 hour at 37°C. Controls were incubated with HEPES buffer alone. No significant reduction was observed in VWF multimer size by gel electrophoresis or as measured by collagen-binding activity relative to VWF antigenic concentrations (ANOVA 1-way analysis, P = .08). Error bars represent standard deviations. (D) rVWF (0.4 U/mL) was incubated overnight with recombinant ADAMTS13 (10 μg/mL) or platelet TSP-1 (100 nM) at 37°C in Tris (tris[hydroxymethyl]aminomethane) buffer (10 mM Tris, 1.5 mM urea, 3 mM BaCl2, 0.1 mM CaCl2).