Histidine-rich glycoprotein (HRG) is an abundant plasma protein that binds factor (F) XIIa and inhibits FXII autoactivation and FXIIa-mediated activation of FXI. Polyphosphate (polyP), a potent procoagulant released from activated platelets, may serve as a physiological activator of the contact system. Previously, we showed that HRG binds DNA and neutralizes its procoagulant activity. Consequently, we set out to determine whether the capacity of HRG to bind polyanions enables it to regulate polyP-induced thrombosis. In a plate-based assay, immobilized polyP bound HRG, FXII, and FXIIa in a zinc-dependent manner. Basal and polyP-induced thrombin generation were greater in plasma from HRG-deficient mice than in plasma from wild-type mice. Intraperitoneal injection of polyP shortened the activated partial thromboplastin time, enhanced thrombin generation, increased thrombin-antithrombin (TAT) levels, reduced lung perfusion, and promoted pulmonary fibrin deposition to a greater extent in HRG-deficient mice than in wild-type mice, effects that were abrogated with FXII knockdown. Therefore, HRG attenuates the procoagulant and prothrombotic effects of polyP in a FXII dependent manner by modulating the contact system.

Background: Previously, we showed that histidine-rich glycoprotein (HRG) binds factor (F) XIIa with high affinity, inhibits FXII autoactivation and FXIIa-mediated activation of FXI, and attenuates ferric chloride-induced arterial thrombosis in mice.Therefore, HRG downregulates the contact pathway in vitro and in vivo.Objective: To identify the domains on HRG responsible for contact pathway inhibition. Methods: Recombinant HRG domain constructs (N-terminal [N1, N2, and N1N2],proline-rich regions, histidine-rich region [HRR], and C-terminal) were expressed and purified. The affinities of plasma-derived HRG, HRG domain constructs, and synthetic HRR peptides for FXII, FXIIa, β-FXIIa, and polyphosphate (polyP) were determined using surface plasmon resonance, and their effects on polyP-induced FXII autoactivation, FXIIa-mediated activation of FXI and prekallikrein, the activated partial thromboplastin time (APTT), and thrombin generation were examined.Results: HRG and HRG domain constructs bind FXIIa, but not FXII or β-FXII. HRR, N1, and N1N2 bind FXIIa with affinities comparable with that of HRG, whereas the remaining domains bind with lower affinity. Synthetic HRR peptides bind FXIIa and polyP with high affinity. HRG and HRR significantly inhibit FXII autoactivation and prolong the APTT. Like HRG, synthetic HRR peptides inhibit FXII autoactivation, attenuate FXIIa-mediated activation of prekallikrein and FXI, prolong the APTT, and attenuate thrombin generation. Conclusion:The interaction of HRG with FXIIa and polyP is predominantly mediated by the HRR domain. Like intact HRG, HRR downregulates the contact pathway and contributes to HRG-mediated down regulation of coagulation.

Factor (F) XII knockdown attenuates catheter thrombosis in rabbits. Because histidine-rich glycoprotein (HRG) modulates FXIIa activity, we hypothesized that HRG depletion would promote catheter thrombosis. To test this, rabbits were given antisense oligonucleotides (ASOs) against HRG, FXII, or a control ASO or saline. The activated partial thromboplastin time (aPTT), prothrombin time (PT), and catheter-induced thrombin generation were determined in blood collected before and after treatment. Compared with the controls, the HRG- and FXII-directed ASOs reduced hepatic mRNA and plasma levels of HRG and FXII, respectively, by over 90%. Whereas HRG knockdown shortened the aPTT by 2.5-fold, FXII knockdown prolonged it by 4-fold; neither ASOs affected the PT. Catheter segments shortened the lag time and increased peak thrombin in plasma from control rabbits; effects significantly enhanced and attenuated in plasma from rabbits given the HRG- and FXII-directed ASOs, respectively. Catheters were then inserted into the right external jugular vein of the rabbits and the time for catheter occlusion was determined. The catheter occlusion times with the control ASO or saline were 62 ± 8 and 60 ± 11 min, respectively. The occlusion time was significantly reduced to 34 ± 9 min with HRG knockdown and significantly prolonged to 128 ± 19 min with FXII knockdown. HRG levels are decreased with sepsis or cancer and such patients are prone to catheter thrombosis. Because HRG modulates catheter thrombosis, our findings suggest that HRG supplementation may prevent this problem.

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