Abstract 039: A Small Fragment Derived from von Willebrand Factor Improves Survival in a Mouse Model of Endotoxemia-Induced Disseminated Intravascular Coagulation

Abstract

Introduction: Disseminated intravascular coagulation (DIC) is a life-threatening condition that results in wide-spread clot formation and organ failure. Sepsis-induced DIC is associated with a high mortality rate, prompting the need for an effective therapeutic intervention. Our lab has identified the A2 domain of the von Willebrand factor (VWF) protein as a potent treatment for sepsis-induced DIC. Historically, VWF has only been described as a contributor of clot formation; however, our lab has demonstrated that purified recombinant full length A2 domain of the VWF protein can dampen fibrin-rich microthrombi formation in mice with endotoxin. The mode of action appears to be via the interaction with the cell-surface expressed vimentin on endothelium, inhibiting VWF strings formation and thus, platelet adhesion. Goal: Since the VWF protein is naturally cleaved in circulation by the enzyme ADAMTS-13 via the A2 domain, one can argue whether the beneficial effect observed with full len gth A2 domain is terminated by the cleavage of ADAMTS-13. We produced recombinant proteins encompassing the sequences of the two proteolytic products of the A2 domain; the A2Nt and A2Ct proteins. I will identify which fragment retains the mode of action of the full length A2 domain in a mouse model of endotoxin-induced DIC. Methods: The recombinant A2 and vimentin proteins were produced using the bacterial expression system. Protein: protein interactions were determined by optical interferometry. The endotoxin-induced DIC model was achieved by injecting C57B/6 mice with lipopolysaccharide (LPS). Treatment with A2 or its fragments was started approximately 2 hours after LPS insult. Results: The A2N t protein has a higher affinity for vimentin than the A2Ct. Treatment of A2Nt in a LPS-induced DIC mouse model was effective in maintaining survival in males, a comparable finding that mimics treatment of mice with full-length recombinant A2. Interestingly, A2Nt showed less survival than full length A2 in females. These findings highlight the potential differences in therapy responses between genders, an occurrence that some studies have described to also occur in humans.