Extracellular Protein Disulfide Isomerase Cleaves Allosteric Disulfides in Histidine-Rich Glycoprotein to Regulate Thrombus Formation

Introduction

The fine-tuning of thrombus formation is influenced by multiple factors among which extracellular protein disulfide isomerase (PDI) released by activated platelets and endothelial cells plays critical roles. However, the precise mechanisms whereby PDI modulates the kinetics of thrombosis remain elusive. Using mechanism-based kinetic trapping strategy, we identified plasma histidine-rich glycoprotein (HRG) as a substrate of extracellular PDI during thrombus formation. HRG exerts both anticoagulant and procoagulant functions. On one hand, HRG inhibits the contact pathway by binding to activated factor XII (fXIIa); on the other hand, HRG attenuates the anticoagulant activity of antithrombin (AT) by competing with AT binding to endothelial heparan sulfate. Both functions are dependent on zinc ions. In this study, we characterized the effects of PDI-mediated disulfide bond cleavage on HRG functions in the context of thrombosis.

Methods

Recombinant PDI variant with the C-terminal catalytic Cys of the CGHC motif replaced with Ala (PDI-CA) was used to trap its redox substrates in platelet rich plasma (PRP). Dual fluorescent immunoblotting was utilized to detect the stabilized intermediate complex between PDI-CA and HRG. Differential cysteine alkylation and mass spectrometry was performed using purified plasma HRG to identify the disulfide bonds targeted by PDI. ELISA was performed to determine the effects of PDI treatment on HRG binding to heparin, an analog of endothelial heparan sulfate, and fXIIa. Cell-based ELISA, immunofluorescent imaging, and immunohistochemistry were employed to examine in vitro and in vivo binding of HRG and AT on endothelial cells. HRG-mediated inhibition of fXIIa activity was determined using the chromogenic substrate S-2302. The kinetics of HRG accumulation during thrombus formation were examined using high-speed intravital microscopy in the cremasteric arterioles. The effects of HRG on thrombus formation were examined in the laser injury thrombosis model in the presence (wild-type mice) or absence of fXII (f12^-/- mice).

Results

The trapping mutant PDI-CA, but not variants of endoplasmic protein 57 (ERp57), a close member in the PDI family with similar domain structure, formed disulfide-linked complexes with HRG in PRP. Mass spectrometry showed that PDI cleaves three disulfide bonds, C306-C309, C390-C434 and C409-C410, in the histidine-rich region of HRG that is important for its binding to heparan sulfate and fXIIa. Compared to inert-PDI (PDI-AA), where both catalytic Cys were substituted with Ala, wild-type PDI (PDI-CC) increased HRG binding to heparin in a Zn²⁺-dependent manner. Plasma treated with PDI-CC had increased HRG binding but decreased AT binding to cultured endothelial cells compared to PDI-AA treated control. Further, PDI-CC increased HRG binding to fXIIa and enhanced its inhibitory effect on fXIIa activity.

Following laser injury of cremaster arterioles, plasma HRG accumulates rapidly at the injury site preceding the main platelet signal. When mice were treated with Eptifibatide, an integrin α_IIbβ₃ antagonist that eliminates platelet deposition and Zn²⁺release, plasma HRG accumulation at the site of vessel injury was reduced, indicating a critical role of Zn²⁺ for HRG binding in vivo. Intravenous treatment with a PDI inhibitor, isoquercetin, also inhibited HRG accumulation in the growing thrombus. In addition, following FeCl₃-induced carotid injury, PDI inhibition by isoquercetin was found to reduce HRG binding but sustain AT binding on the injured artery as determined by immunohistochemistry. Finally, knockdown of plasma HRG with vivo-siRNA in f12^-/- mice attenuated thrombus formation compared to scramble siRNA treatment thus suggesting a procoagulant role of HRG independent of fXIIa.

Conclusion

PDI cleavage of allosteric disulfide bonds in HRG represents a novel regulatory mechanism that fine-tunes the kinetics of thrombus formation. Our results indicate that at the early stage of thrombosis, PDI promotes HRG binding to endothelial cells to suppress the anticoagulant activity of AT and allow the rapid initiation of thrombosis; at the later stage, PDI reduction of HRG enhances its binding to fXIIa leading to inhibition of fXIIa activity to prevent excessive clot formation.