In Vivo Interrogation of a Human Von Willebrand Type A Domain.

Abstract

Abstract 2176Proteins containing Von Willebrand A domains play critical roles in hemostasis and arterial thrombosis by promoting cell-cell and cell-ligand interactions. Central to these processes is the formation of an adhesive bond between the A1 domain containing protein Von Willebrand Factor (VWF) and its receptor on platelets known as GPIbα. Although ex-vivo approaches have broadened our understanding of the biophysical properties that govern the interaction between this human receptor–ligand pair, validation of hypotheses based on these intriguing studies will require an appropriate biological model that permits the study of such complex adhesive interactions under appropriate hemodynamic conditions; only then will it be possible to truly ascertain the biological relevance of in vitro observations as they pertain to hemostasis and thrombosis in humans. Moreover, a biological model that permits the study of the function of the human VWF-A1 domain in vivo will be of tremendous value in the development of drugs for use in disorders associated with thrombotic microangiopathy.Given the importance of the interaction between GPIbα and the A1 domain of VWF in hemostasis and thrombus formation in humans, we genetically modified the murine VWF genomic sequence so that it contains the majority of the human A1 domain (VWF HA1) in lieu of its murine counterpart. Mutant mice were viable, born in expected mendelian ratio, and had platelet counts comparable to WT littermates. Importantly, VWF gene transcription, antigen levels, and multimer pattern were also equivalent to WT controls. In contrast, hemostasis was significantly disrupted in homozygous VWF HA1 mice with majority of animals continuing to bleed for >10 minutes after removal of the distal tip of the tail. Evidence that the defect in hemostasis was due to the inability of murine platelets to accumulate at sites of vascular damage is demonstrated by a 4-fold reduction in thrombus size in response to laser-induced arteriolar injury in the microcirculation of the cremaster muscle. To further evaluate the ability of this modified VWF to support interactions with murine platelets, we surfaced immobilized plasma VWF from these animals onto a glass cover slip, which was then incorporated into a parallel plate flow system. WT mouse blood infused over the immobilized substrate was unable to support any significant interactions with plasma VWF HA1 (wall shear rate of 1,600s−1). By contrast, human platelets rapidly accumulated on surface-immobilized VWF HA1 at levels observed for its human plasma counterpart; human platelets administered to VWF HA1 mice were also capable of restoring hemostasis in these animals and formed occlusive thrombi in response to laser-induced arterial injury. Remarkably, the human A1 domain contained within murine VWF was able to support ristocetin-induced human platelet agglutination whereas wild-type murine plasma VWF did not. We are now taking the next important steps in defining the in vivo consequences of altered bond formation between the human VWF-A1 domain and GPIbα as well as evaluating the anti-thrombotic effects of compounds designed to disrupt this interaction in patients with disease-associated thrombotic microangiopathy. Disclosures:No relevant conflicts of interest to declare.