PDI Inhibition Blocks Thrombin Generation in Humans By Interfering with Platelet Factor V Activation

Protein disulfide isomerase (PDI) is a ubiquitously expressed oxidoreductase that serves an essential role in protein folding in the endoplasmic reticulum by reshuffling disulfide bonds within nascent proteins. PDI can be released from vascular cells, including platelets, and inhibition or platelet-specific deletion of PDI blocks thrombus formation in vivo. However, the specific function of PDI in thrombus formation is poorly understood. Unlike the role of proteases in blood coagulation, which have been studied in depth, little is known about PDI substrates in the vasculature. Several platelet and endothelial integrins have been identified as putative substrates for PDI, but whether coagulation factors are directly targeted by extracellular PDI has not been established. We now identify platelet factor V as a principal coagulation substrate of extracellular PDI. We developed an unbiased strategy to identify novel substrates of PDI in washed platelets using PDI variants capable of trapping substrates: FLAG-tagged PDI mutants modified by a substitution of arginine or proline for histidine (CGHC → CGRC; CGHC → CGPC) in the catalytic motif of both the a and a' domains. Whereas the AGHA-PDI variant which has no catalytic activity serves as a control. The CGRC-PDI variant co-precipitated with platelet factor V in a redox-sensitive manner while there was no platelet factor V detected with the AGHA-PDI variant, thus confirming that binding of PDI to platelet-derived factor V occurs through disulfide bond exchange. Platelet factor V associates with multimerin-1 through a disulfide bond. Trapping PDI mutants also bind to multimerin-1 in a reaction requiring disulfide bond exchange. To evaluate the effect of PDI inhibition on the activation of platelet factor V, washed platelets from healthy donors were stimulated with 0.1 U/mL of thrombin in the presence of varying concentrations of isoquercetin (0 to 50 µM), which has previously been shown to inhibit PDI function. We observed a dose-dependent reduction of factor Va following platelet activation despite the fact that isoquercetin did not inhibit platelet release of PF4 or block Xa or thrombin enzymatic activity directly. We next performed a clinical study designed to determine whether oral isoquercetin inhibits thrombin generation in human subjects via its ability to inhibit platelet Va generation. Plasma samples collected from healthy participants before and 4 hours after ingestion of 1000 mg of isoquercetin (N=17). In plasma samples, post-isoquercetin platelet-dependent thrombin generation decreased by 51% compared with pre-ingestion controls (P=0.0004). Furthermore, we observed an overall 26% reduction in FVa in non-FV depleted plasma (P<0.001), which corresponded with a 53% decrease in FVa generated from platelets (P<0.001). These data confirm a significant effect of PDI inhibition on the generation of FVa following platelet activation. Considering that isoquercetin reduces platelet FVa generation and similarly inhibits platelet-dependent thrombin generation in a PDI-dependent manner, we investigated whether the addition of FVa in vitro restored platelet-dependent thrombin generation. The pre-incubation of 7 µg/mL FVa prior to stimulation with low dose thrombin restored platelet-dependent thrombin generation to within 80% baseline of pre-treatment levels. We conclude that platelet factor V is an essential substrate in mediating PDI-dependent thrombin generation on platelets and propose that PDI cleaves a disulfide bond that links platelet factor V to multimerin-1, thereby releasing platelet factor V for activation and subsequent thrombin generation.