The Thrombospondin-1 N700S Polymorphism Does Not Alter von Willebrand Factor Multimer Size in Patients Suffering an Acute Myocardial Infarction.

Abstract

An uncommon missense variant of thrombospondin-1 representing an asparagine (Asn-700) to serine substitution (Ser-700) has been identified as a potential genetic risk factor for myocardial infarction (MI). Thrombospondin-1 (TSP-1) is a glycoprotein stored in human platelet alpha granules and has been shown to regulate von Willebrand factor (VWF) multimer size. The TSP-1 domain responsible for VWF-reducing activity was localized to the calcium-binding C-terminal sequence at position Cys 974. Considering the proximity of the N700S TSP-1 polymorphism and the demonstration that the Ser-700 polymorphism alters the calcium binding affinity of this domain, we investigated whether altered VWF-reducing activity of Ser-700 polymorphism underlies the observed prothrombotic phenotype. Plasma samples were analyzed from a cohort of patients (N=31) who survived an acute myocardial infarction at age 45 or younger compared with age-matched controls (N=19). The relative percentage of high molecular weight VWF multimers did not differ significantly between individuals homozygous for either Ser-700 or Asn-700 (p=.20). Similarly, the relative percentage of high molecular weight multimers was 31.2% for the Ser-700 homozygous MI patients (N=21) versus 31.1% (p=.95) in the Asn-700 healthy controls (N=10). These findings were confirmed using the collagen binding assay to assess VWF activity relative to antigen concentrations. The mean ratio of CBA/Ag was .98 in the Ser-700 patients versus 1.0 in the Asn-700 control group (p=.22). Mean plasma thrombospondin-1 concentrations did not differ between the two groups. The VWF reducing capacity of TSP-1 was also assessed in vitro using recombinant TSP-1 N700S constructs containing the last EGF-like repeat, all type-3 repeats (calcium-binding domains), and C-terminal sequence. Incubation of Ser-700 peptide, Asn-700 peptide, or platelet purified full-length TSP-1 with ultra-large VWF (using either TTP plasma or recombinant ultra-large VWF) did not result in any significant reduction of VWF multimer size under static conditions. In conclusion, the association between coronary artery thrombosis and the TSP-1 Ser-700 polymorphism is not mediated through altered VWF reducing activity.