A Novel Role of Protein Disulfide Isomerase in Calpain Regulated Hypoxia Induced Prothrombotic Phenotype
Authors: Tyagi Tarun , Prabhakar Amit , Sengupta Shantanu , Ashraf Mohammad Z 
Published: October 2019

Abstract
Introduction The increased risk of thromboembolic events under low oxygen environments such as high altitude regions is a well known phenomenon but with little knowledge about molecular events underlying its pathogenesis. We have recently reported the proteomic changes in hyperreactive platelets under hypoxia and demonstrated that increased activity of protease calpain is crucial for the induction of prothrombotic phenotype under hypoxic environment (Tyagi et al, 2014). However, considering the complex nature of hemostatic reactions, multiple aspects of the regulation of hemostatic balance under hypoxia need to be understood. Hypoxic environment creates oxidative stress in biological system which needs to be contained by counteractive factors. One of such factor, the Protein disulfide isomerase (PDI), which catalyzes the formation and rearrangements of disulfide bonds on proteins, has been found to be upregulated in multiple cell types and is believed to be protective under oxidative stress. The extracellular PDI has been recently reported to play a key role in initial thrombotic events in vivo and in vitro, but whether it affects the hemostasis under hypoxia remains unknown. We analyzed the role played by PDI in calpain regulated hypoxia induced prothrombotic phenotype by using specific PDI inhibitor Quercetin-3-rutinoside (Q-3R) in an animal model.
Method:The Sprague-Dawley male rats were exposed to simulated hypobaric hypoxia in a specially designed animal decompression chamber maintained at pressure of 366 torr (equivalent to altitude of 6096m) for 3h duration. The exposed group of animals was infused with either Q-3R or vehicle, via tail vein injection prior to hypoxia exposure. The effect of PDI inhibitor on coagulation was analyzed by measuring clotting time and Prothrombin time. The PDI activity was measured in plasma by insulin reduction assay. The plasma calpain activity was measured by fluorescence based assay. The status of oxidative damage was assessed by measuring MDA levels which are hallmark of oxidative stress. We also analyzed the factor V activity in plasma of animals by standard clot based assay using factor V deficient plasma.
Results: As previously reported by us, the animals exposed to hypoxic conditions demonstrated prothrombotic tendency as evident in significant shortening of clotting times (PT 84% of control, p < .03) as compared to control animals. The exposed animals preinfused with PDI inhibitors demonstrated further reductions in clotting times. The preinfusion of Q-3R (0.25 to 1.5 mg/kg body wt) significantly shortened the Prothrombin times (71%, p < .01) as compared to respective vehicle control. The hypercoagulative tendency triggered by Q-3R was also evident in drastically reduced activated clotting times in whole blood. Hypoxia induced the activation of PDI which was reflected by significantly elevated activity as compared to control animals. The dose dependent PDI inhibition in Q-3R preinfused animals was confirmed by the fallen activity of PDI in plasma from exposed animals. The calpain activity remained elevated in hypoxic animals (compared to controls) as observed previously. The oxidative damage as measured by MDA levels, was found to be much higher in exposed animals preinfused with Q-3R as compared to the vehicle group. Interestingly, the active factor V was observed to be significantly higher in case of PDI inhibition with largest activity increase in animals infused with highest dose of Q-3R.
Conclusion:These results demonstrate the critical role of PDI in regulation of hypoxia induced prothrombotic state. The prevention of increase in activity of PDI in hypoxic animals by using specific PDI inhibitor accelerated the prothrombotic effect of hypoxia. These results appear to be in contrast with recently reported in vivo antithrombotic effect of PDI inhibition. However, as PDI upregulation is considered largely as a protective mechanism, this prothrombotic effect of PDI inhibition under hypoxia seems to be in part due to uncontrolled oxidative stress as shown by higher MDA levels. Also, as factor V activity was shot up by PDI inhibitors, this shows the activation of factor V to be under control of PDI, which can also be explained by role of disulfide linkages in activation of released factor V. Together, these results suggests a novel role of PDI, along with calpain, in regulating hypoxia induced prothrombotic phenotype.

Disclosures
No relevant conflicts of interest to declare.