Identification of extant vertebrate Myxine glutinosa VWF: evolutionary conservation of primary hemostasis

Hemostasis in vertebrates involves both a cellular and protein component. Previous studies in jawless vertebrates (cyclostomes) suggest that the protein response, which involves thrombin-catalyzed conversion of a soluble plasma protein, fibrinogen, into a polymeric fibrin clot, is conserved in all vertebrates. However, similar data are lacking for the cellular response, which in gnathostomes, is regulated by von Willebrand factor (VWF), a glycoprotein that mediates the adhesion of platelets to the subendothelial matrix of injured blood vessels. To gain evolutionary insights into the cellular phase of coagulation, we asked whether a functional vwf gene is present in the Atlantic hagfish, Myxine glutinosa . We found a single vwf transcript that encodes a simpler protein compared to higher vertebrates, the most striking difference being the absence of an A3 domain, which otherwise binds collagen under high flow conditions. Immunohisto-chemical analyses of hagfish tissues and blood revealed Vwf expression in endothelial cells and thrombocytes. Electron microscopic studies of hagfish tissues demonstrated the presence of Weibel-Palade bodies (WPB) in the endothelium. Hagfish Vwf formed high molecular weight multimers in hagfish plasma and in stably transfected CHO cells. In functional assays, botrocetin promoted VWF-dependent thrombocyte aggregation. A search for vwf sequences in the genome of sea squirts, the closest invertebrate relatives of hagfish, failed to reveal evidence of an intact vwf gene. Together, our findings suggest that VWF evolved in the ancestral vertebrate following the divergence of the urochordates some 500 million years ago and that it acquired increasing complexity though sequential insertion of functional modules.