Molecular Studies of Hemolytic Thrombosis

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A major complication of mechanical circulatory support devices (MCSD) for multi-organ support is the dichotomous pathology of thrombosis and bleeding. Intravascular hemolysis associated with MCSD produces free hemoglobin (fHb). Adsorption of fibrinogen and von Willebrand factor (VWF) onto non-biological surfaces captures platelets that may aggregate. We reported that high levels of fHb increased VWF- mediated platelet adhesion and thrombus formation on fibrin(ogen)-coated surfaces at high shear stress. Importantly, fHb increased the flow-dependent adhesion of formalin fixed platelets to a VWF coated surface, suggesting that fHb directly enhances the VWF- glycoprotein (GP)Ib interaction. VWF deficiency or antibodies against GPIb block the enhancement of platelet adhesion by fHb, validating the role of VWF. Lastly, our identification of ultra large VWF (ULVWF) in whole blood exposed to fHb and high shear rates suggests that platelet derived ULVWF may contribute to the pathology of thrombosis in the presence of high levels of fHb. Together, these findings provide the first evidence that high levels of fHb are critical for VWF-mediated thrombosis on surfaces coated with fibrin(ogen), and that ULVWF multimers from shear-activated platelets may enhance thrombosis more robustly than plasma VWF in the presence of fHb. Our overarching hypothesis is that fHb targets VWF, including the platelet-derived VWF, to promote platelet adhesion via GPIb, and potentiate fibrin formation by facilitating the binding of VWF to fibrinogen. We propose molecular, biochemical, and structural studies to investigate the mechanisms by which fHb dysregulates the interactions between VWF, fibrin(ogen), and platelets. Aim 1 will investigate the contributions of platelet VWF vs. plasma VWF to thrombosis. We will test the hypotheses that fHb interaction with platelet VWF enhances platelet adhesion to fibrin(ogen) and potentiates fibrin formation more robustly than plasma VWF. Aim 2 will test the hypothesis that fHb-bound VWF has a conformation that favors platelet adhesion. Aim 3 will determine the mechanism by which fHb dysregulate VWF-fibrin(ogen) interaction. We will test the hypotheses that fHb promotes VWF-fibrin(ogen) association and modulates the VWF-mediated fibrin formation. These studies will describe new mechanisms related to thrombosis in patients on MCSD and identify new, potential targets for therapeutic interventions in fHb-induced thrombosis.