The Effect of Soluble P-Selectin in Coagulation

Abstract

P-selectin, a member of the selectin family, is a vascular cell adhesion molecule. It is expressed and stored in alpha granules of platelets and in Weibel-Palade bodies of endothelial cells, and upon activation, P-selectin is translocated/transferred to the membrane surface. A key function of the P-selectin is to mediate leukocyte, lymphocyte and platelet interactions in inflammation and possibly in the thrombus formation. A soluble variant, S-Psel, comprising the extracellular domain of P-selectin, has been identified in healthy individuals, but is markedly elevated in patient with vascular disorders. Recent work on S-Psel suggested that S-Psel may play a role in hemostasis/coagulation through the generation of procoagulant TF-bearing microparticles (MP), and therefore, has potential in treating patients with the bleeding disorders, like hemophilia. The aim of this study is to verify studies reporting that S-Psel exhibits in vitro and in vivo pro-coagulant activity. S-Psel and S-Psel-Fc (IgG) fusion were purchased commercially or prepared at Novo Nordisk Research US (NNRUS) and their biological activity was verified by P-selectin/Pselectin glycoprotein ligand-1(PSGL-1) interaction in vitro. The clotting times of human whole blood and plasma treated with S-Psel or S-Psel-Fc, or with an irrelevant human IgG control protein, were measured by thromboelastography and aggregometry respectively. After up to 8 hours of incubation with S-Psel and S-Psel-Fc at a concentration of 15ug/ml, we found no significant difference between samples treated with S-Psel, S-Psel-Fc and the IgG controls. The ability of S-Psel to generate TF-bearing microparticles in human whole blood was examined in a FXa substrate cleavage assay; however, no significant difference in cleavage was observed. Finally, we evaluated S-Psel in vivo. Hemophilia A mice were injected with recombinant mouse S-Psel-IgG or S-Psel-Fc (IgG) at the concentration of 1.2 mg/Kg body weight and human IgG was used as control. As suggested from published results, the effect of S-Psel was determined 6 h after the treatment. Contrary to previous reports, the results revealed no significant difference in bleed time and blood loss between the experimental and control group. In conclusion, we were unable to demonstrate the procoagulant activity of S-Psel in our laboratory either in vitro or in vivo.