Protein disulfide isomerase cleaves allosteric disulfides in histidine-rich glycoprotein to regulate thrombosis.

The essence of difference between hemostasis and thrombosis is that the clotting reaction is a highly fine-tuned process. Vascular protein disulfide isomerase (PDI) represents a critical mechanism regulating the functions of hemostatic proteins. Herein we show that histidine-rich glycoprotein (HRG) is a substrate of PDI. Reduction of HRG by PDI enhances the procoagulant and anticoagulant activities of HRG by neutralization of endothelial heparan sulfate (HS) and inhibition of factor XII (FXIIa) activity, respectively. Murine HRG deficiency (Hrg-/-) leads to delayed onset but enhanced formation of thrombus compared to WT. However, in the combined FXII deficiency (F12-/-) and HRG deficiency (by siRNA or Hrg-/-), there is further thrombosis reduction compared to F12-/- alone, confirming HRG's procoagulant activity independent of FXIIa. Mutation of target disulfides of PDI leads to a gain-of-function mutant of HRG that promotes its activities during coagulation. Thus, PDI-HRG pathway fine-tunes thrombosis by promoting its rapid initiation via neutralization of HS and preventing excessive propagation via inhibition of FXIIa.

Hemostatic efficacy of marstacimab alone or in combination with bypassing agents in hemophilia plasmas and a mouse bleeding model.

Background: Patients with hemophilia have deficiencies in intrinsic coagulation factors and can develop inhibitors that limit the effectiveness of replacement coagulation factors. Marstacimab, a human monoclonal antibody, binds and inhibits the human tissue factor pathway inhibitor. Marstacimab is currently under development as a potential prophylactic treatment to prevent bleeding episodes in patients with hemophilia A and B. Objective: To assess the effects of marstacimab alone or in combination with the bypassing agent recombinant factor FVIIa (rFVIIa) or activated prothrombin complex concentrate (aPCC) on thrombin generation and bleeding. Methods: Marstacimab and/or rFVIIa or aPCC were added to hemophilic A or B plasma or nonhemophilic plasma in vitro. Hemostatic activity was measured using the thrombin generation assay. In vivo effects were assessed using a mouse acute bleeding model. Male hemophilia A mice were dosed with marstacimab plus aPCC before tail clip; blood loss was quantified by measuring hemoglobin. Results: Marstacimab plus rFVIIa or aPCC slightly increased peak thrombin levels compared with either agent alone. This increase was within the reported range for nonhemophilic plasma and did not exceed levels observed in nonhemophilic plasma treated with marstacimab alone. Hemophilia A mice that received 200 U/kg aPCC had significantly reduced bleeding (62%) compared with vehicle-treated mice (p < 0.05), and marstacimab plus aPCC reduced bleeding by 83.3% compared with vehicle (p= 0.0009). Conclusions: Marstacimab alone or with bypassing agents increased hemostasis in hemophilia plasma without generating excessive thrombin. The hemostatic activity of marstacimab plus aPCC was confirmed in hemophilia A mice.

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 αvß3 or αIIbß3 integrins on endothelial cells and platelets. The released PDI reduces disulfide bonds on plasma vitronectin, enabling vitronectin to bind to αVß3 and αIIbß3. 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.

Quercetin-3-rutinoside Inhibits Protein Disulfide Isomerase by Binding to Its b'x Domain.

Quercetin-3-rutinoside inhibits thrombus formation in a mouse model by inhibiting extracellular protein disulfide isomerase (PDI), an enzyme required for platelet thrombus formation and fibrin generation. Prior studies have identified PDI as a potential target for novel antithrombotic agents. Using a fluorescence enhancement-based assay and isothermal calorimetry, we show that quercetin-3-rutinoside directly binds to the b' domain of PDI with a 1:1 stoichiometry. The binding of quercetin-3-rutinoside to PDI induces a more compact conformation and restricts the conformational flexibility of PDI, as revealed by small angle x-ray scattering. The binding sites of quercetin-3-rutinoside to PDI were determined by studying its interaction with isolated fragments of PDI. Quercetin-3-rutinoside binds to the b'x domain of PDI. The infusion of the b'x fragment of PDI rescued thrombus formation that was inhibited by quercetin-3-rutinoside in a mouse thrombosis model. This b'x fragment does not possess reductase activity and, in the absence of quercetin-3-rutinoside, does not affect thrombus formation in vivo. The isolated b' domain of PDI has potential as an antidote to reverse the antithrombotic effect of quercetin-3-rutinoside by binding and neutralizing quercetin-3-rutinoside.

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.

Impaired protofibril formation in fibrinogen gamma N308K 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 gammaN308K 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 gammaN308K fragment D. In contrast to previous fragment D crystals, the gammaN308K crystals belonged to a tetragonal space group with an unusually long unit cell (a = b = 95 A, c = 448.3 A). Alignment of the normal and gammaN308K 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 gammaN308K molecules, indicating the normal asymmetric D:D interface in fibrin would be unstable in this variant. We examined GPRP binding to gammaN308K 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 gammaN308K monomers, leading to impaired polymerization.

Fibrinogen variant BbetaD432A has normal polymerization but does not bind knob "B".

Fibrinogen residue Bbeta432Asp is part of hole "b" that interacts with knob "B," whose sequence starts with Gly-His-Arg-Pro-amide (GHRP). Because previous studies showed BbetaD432A has normal polymerization, we hypothesized that Bbeta432Asp is not critical for knob "B" binding and that new knob-hole interactions would compensate for the loss of this Asp residue. To test this hypothesis, we solved the crystal structure of fragment D from BbetaD432A. Surprisingly, the structure (rfD-BbetaD432A+GH) showed the peptide GHRP was not bound to hole "b." We then re-evaluated the polymerization of this variant by examining clot turbidity, clot structure, and the rate of FXIIIa cross-linking. The turbidity and the rate of gamma-gamma dimer formation for BbetaD432A were indistinguishable compared with normal fibrinogen. Scanning electron microscopy showed no significant differences between the clots of BbetaD432A and normal, but the thrombin-derived clots had thicker fibers than clots obtained from batroxobin, suggesting that cleavage of FpB is more important than "B:b" interactions. We conclude that hole "b" and "B:b" knob-hole binding per se have no influence on fibrin polymerization.

Polymerization-defective fibrinogen variant gammaD364A binds knob "A" peptide mimic.

Fibrin polymerization is supported in part by interactions called "A:a". Crystallographic studies revealed gamma364Asp is part of hole "a" that interacts with knob "A" peptide mimic, GPRP. Biochemical studies have shown gamma364Asp is critical to polymerization, as polymerization of variants gammaD364A, gammaD364H, and gammaD364V is exceptionally impaired. To understand the molecular basis for the aberrant function, we solved the crystal structure of fragment D from gammaD364A. Surprisingly, the structure (rfD-gammaD364A+GP) showed near normal "A:a" interactions with GPRP bound to hole "a" and no change in the overall structure of gammaD364A. Of note, inspection of the structure showed negative electrostatic potential inside hole "a" was diminished by this substitution. We examined GPRP binding to the gamma364Asp variants in solution by plasmin protection assay. We found no protection of either gammaD364H or gammaD364V but partial protection of gammaD364A, indicating the peptide does not bind to either gammaD364H or gammaD364V and binds more weakly than normal to gammaD364A. We also examined protection by calcium and found all variants were indistinguishable from normal, suggesting the global structures of the variants are not markedly different from normal. Our data imply that gamma364Asp per se is not required for knob "A" binding to hole "a"; rather, this residue's negative charge has a critical role in the electrostatic interactions that facilitate the important first step in fibrin polymerization.