Impaired Protofibril Formation in Fibrinogen γN308K Is Due to Altered D:D and “A:a” Interactions ,

“A:a” knob–hole interactions and D:D interfacial interactions are important for fibrin polymerization. Previous studies with recombinant γN308K fibrinogen, a substitution at the D:D interface, showed impaired polymerization. We examined the molecular basis for this loss of function by solving the crystal structure of γN308K fragment D. In contrast to previous fragment D crystals, the γN308K crystals belonged to a tetragonal space group with an unusually long unit cell (a = b =95Å, c = 448.3 Å). Alignment of the normal and γN308K structures showed the global structure of the variant was not changed and the knob “A” peptide GPRP was bound as usual to hole “a”. The substitution introduced an elongated positively charged patch in the D:D region. The structure showed novel, symmetric D:D crystal contacts between γN308K molecules, indicating the normal asymmetric D:D interface in fibrin would be unstable in this variant. We examined GPRP binding to γN308K in solution by plasmin protection assay. The results showed weaker peptide binding, suggesting that “A:a” interactions were altered. We examined fibrin network structures by scanning electron microscopy and found the variant fibers were thicker and more heterogeneous than normal fibers. Considered together, our structural and biochemical studies indicate both “A:a” and D:D interactions are weaker. We conclude that stable protofibrils cannot assemble from γN308K monomers, leading to impaired polymerization

Protein disulfide isomerase secretion following vascular injury initiates a regulatory pathway for thrombus formation

Protein disulfide isomerase (PDI), secreted by platelets and endothelial cells on vascular injury, is required for thrombus formation. Using PDI variants that form mixed disulfide complexes with their substrates, we identify by kinetic trapping multiple substrate proteins, including vitronectin. Plasma vitronectin does not bind to αvβ3 or αIIbβ3 integrins on endothelial cells and platelets. The released PDI reduces disulfide bonds on plasma vitronectin, enabling vitronectin to bind to αVβ3 and αIIbβ3. In vivo studies of thrombus generation in mice demonstrate that vitronectin rapidly accumulates on the endothelium and the platelet thrombus following injury. This process requires PDI activity and promotes platelet accumulation and fibrin generation. We hypothesize that under physiologic conditions in the absence of secreted PDI, thrombus formation is suppressed and maintains a quiescent, patent vasculature. The release of PDI during vascular injury may serve as a regulatory switch that allows activation of proteins, among them vitronectin, critical for thrombus formation

Protein disulfide isomerase inhibitors constitute a new class of antithrombotic agents

Thrombosis, or blood clot formation, and its sequelae remain a leading cause of morbidity and mortality, and recurrent thrombosis is common despite current optimal therapy. Protein disulfide isomerase (PDI) is an oxidoreductase that has recently been shown to participate in thrombus formation. While currently available antithrombotic agents inhibit either platelet aggregation or fibrin generation, inhibition of secreted PDI blocks the earliest stages of thrombus formation, suppressing both pathways. Here, we explored extracellular PDI as an alternative target of antithrombotic therapy. A high-throughput screen identified quercetin-3-rutinoside as an inhibitor of PDI reductase activity in vitro. Inhibition of PDI was selective, as quercetin-3-rutinoside failed to inhibit the reductase activity of several other thiol isomerases found in the vasculature. Cellular assays showed that quercetin-3-rutinoside inhibited aggregation of human and mouse platelets and endothelial cell–mediated fibrin generation in human endothelial cells. Using intravital microscopy in mice, we demonstrated that quercetin-3-rutinoside blocks thrombus formation in vivo by inhibiting PDI. Infusion of recombinant PDI reversed the antithrombotic effect of quercetin-3-rutinoside. Thus, PDI is a viable target for small molecule inhibition of thrombus formation, and its inhibition may prove to be a useful adjunct in refractory thrombotic diseases that are not controlled with conventional antithrombotic agents