Molecular mechanisms of platelet thrombosis under arterial shear conditions
Fredrickson, Becky Jo
McIntire, Larry V.
Doctor of Philosophy
Pathologic arterial thrombosis is the leading cause of death in many countries. Current therapies provide only moderate protection from heart attack, stroke, and other clinical manifestations of thrombosis. To develop more potent therapeutics, molecular mechanisms that mediate platelet thrombosis are being researched. Thrombosis depends on complex interactions between the vascular wall, circulating components, and blood flow conditions. Under arterial shear conditions, platelet thrombosis requires von Willebrand factor (vWf) binding to platelet glycoprotein (GP) Ib-IX-V and GP IIb-IIIa complexes. In this work, dynamic experimental models of the vascular system are used in three studies investigating the role of GP Ib-IX-V and GP IIb-IIIa in arterial thrombosis. First, experimental results indicate two compounds that inhibit GP Ib-vWf binding have strong potential as anti-thrombotic agents. Findings with these compounds also suggest that soluble and immobilized vWf have different structural conformations. Second, a novel experimental model was developed to examine vWf-GP Ib-IX-V interactions that mediate platelet adhesion to exposed subendothelium. The system consists of vWf-coated glass slides, mammalian cells expressing full or partial GP Ib-IX-V complexes, and a parallel plate flow chamber with phase contrast video microscopy and digital image processing. Results with this system suggest the novel finding that optimal binding between immobilized vWf and GP Ib$\alpha$ requires the presence of GP V within the GP Ib-IX-V complex. The results also demonstrate that the role of immobilized vWf with respect to GP Ib-IX-V binding does not change after exposure to high shear. Third, platelet and leukocyte function were evaluated using whole blood samples from patients undergoing percutaneous transluminal coronary angioplasty (PTCA) and receiving one of three standard therapies: abciximab, a GP IIb-IIIa inhibitor, ticlopidine, an orally active anti-platelet agent, or both treatments. Combined abciximab/ticlopidine therapy produces the most prolonged inhibition of in vitro mural thrombosis and the most consistent reduction in shear-induced platelet aggregation. At 2 hours post-PTCA, abciximab therapy, with or without ticlopidine, promotes leukocyte rolling on the collagen/vWf-platelet surface. However, abciximab-enhanced leukocyte rolling is almost completely inhibited by monoclonal antibodies to P-selectin or P-selectin glycoprotein ligand-1. Basic mechanism studies like these increase understanding of thrombosis pathophysiology and accelerate anti-thrombotic agent development.
Physiology; Biomedical engineering; Chemical engineering; Pathology