Characterization of platelet glycoprotein Ib-IX-V: von Willebrand factor interaction under shear conditions

Arterial thrombosis is one of the important pathophysiological mechanisms that lead to cardiovascular diseases. In this thesis, we have made an attempt to better characterize the kinetic and molecular mechanisms that underlie the critical first step in arterial thrombosis, namely, the interaction between platelet glycoprotein (GP) Ib and von Willebrand factor (VWF).; In the first part of the work, we evaluated the kinetics of interaction between platelet GP Ib-IX-V complex and VWF under arterial flow conditions. The GP Ibalpha subunit of GP Ib complex binds to VWF through the Al domain of VWF. Impaired GP Ib-VWF interaction due to GP Ibalpha mutations can result in bleeding abnormalities including platelet-type von Willebrand disease (ptVWD). We measured the cellular on- and off-rate constants of CHO cells expressing wild-type or gain- or loss-of-function mutant GP Ibalpha interacting with VWF-Al-coated surfaces at different shear stresses. We found that the gain-of-function mutant, K237V, rolled very slowly and continuously on VWF-Al surface while the loss-of-function mutant, Q232V, showed fast, saltatory movement compared to the wild-type (WT). The off-rate constants, calculated based on the analysis of lifetimes of transient tethers formed on surfaces coated with limiting densities of VWF-Al, revealed that the Q232V and K237V dissociated 1.25-fold faster and 2.2-fold slower than the WT. The cellular on-rate constant of WT, measured in terms of tethering frequency was 3-fold more and 3-fold less than Q232V and K237V, respectively. Thus, the gain- and loss-of-function mutations in GP Ibalpha affect both the association and dissociation kinetics of the GP Ibalpha-VWF-Al bond.; In the second part of the work, we compared the interaction of unusually large multimers of VWF (ULVWF) and that of the normal plasma multimers of VWF (P-VWF) platelets. ULVWF multimers are implicated in the pathology of a thrombotic disorder, thrombotic thrombocytopenic purpura (TTP) due to their increased affinity for platelets. We found that the ULVWF multimers are more effective than the normal P-VWF multimers in mediating (a) platelet aggregation in solution at high shear stress; (b) ristocetin-modulated platelet agglutination and (c) platelet adhesion to immobilized VWF under arterial shear conditions.