Histidine-rich glycoprotein binds factor XIIa with high affinity and inhibits contact-initiated coagulation

Abstract

Histidine-rich glycoprotein (HRG) circulates in plasma at a concentration of 2M and binds plasminogen, fibrinogen, and thrombospondin. Despite these interactions, the physiologic role of HRG is unknown. Previous studies have shown that mice and humans deficient in HRG have shortened plasma clotting times. To better understand this phenomenon, we examined the effect of HRG on clotting tests. HRG prolongs the activated partial thromboplastin time in a concentrationdependent fashion but has no effect on tissue factor-induced clotting, localizing its effect to the contact pathway. Plasma immunodepleted of HRG exhibits a shortened activated partial thromboplastin time that is restored to baseline with HRG replenishment. To explore how HRG affects the contact pathway, we examined its binding to factors XII, XIIa, XI, and XIa. HRG binds factor XIIa with high affinity, an interaction that is enhanced in the presence of Zn 2؉ , but does not bind factors XII, XI, or XIa. In addition, HRG inhibits autoactivation of factor XII and factor XIIa-mediated activation of factor XI. These results suggest that, by binding to factor XIIa, HRG modulates the intrinsic pathway of coagulation, particularly in the vicinity of a thrombus where platelet release of HRG and Zn 2؉ will promote this interaction.(Blood. 2011;117(15): 4134-4141) IntroductionDespite the capacity of the intrinsic pathway to enhance thrombin generation, patients deficient in factor XII (FXII) do not bleed. 1 Even patients with FXI deficiency rarely have hemorrhagic complications, except with surgery or major trauma. 2 Because of these observations, it is well accepted that the contact system plays little part in hemostasis; and, by extension, it was also thought to be unimportant for thrombosis. However, a number of recent studies challenge this thinking. First, mice with deficiencies of highmolecular-weight kininogen (HK), bradykinin B2 receptor, FXII, or FXI are protected against injury-induced thrombosis, and an antibody against FXI inhibits thrombus formation in a baboon arteriovenous shunt model. [3][4][5][6][7] These observations raise the possibility that the contact pathway contributes to thrombogenesis. 8 Second, potential physiologic activators of FXII have been identified, which could initiate the contact system at sites of vascular injury. In addition to glycosaminoglycans and collagen, novel activators include polyphosphates and RNA. 9,10 Polyphosphates, which are released from the dense granules of platelets activated at sites of injury, trigger coagulation in a FXII-dependent fashion. Likewise, RNA released from the damaged vessel wall also can activate FXII, and RNAase administration to animals attenuates thrombosis at sites of injury, observations that have sparked a renewed interest in the contact pathway. 10 Consequently, it is important to better understand this pathway and how it is regulated.Histidine-rich glycoprotein (HRG) is an abundant plasma protein whose role is largely unknown. 11 HRG circulates at a concentration of approximately 2M. I...

Histidine-rich Glycoprotein Binds Fibrin(ogen) with High Affinity and Competes with Thrombin for Binding to the γ′-Chain

“…Zn 2ϩ is important for the binding of HRG to bacteria, cells, and hemostatic factors, such as glycosaminoglycans, plasminogen, and factor XIIa (12,19,24,34). It is likely that there are still other HRG interactions that have been overlooked because of the widespread use of citrate as an anticoagulant.…”

“…HRG was purified by metal-chelate chromatography, and HRG-deficient plasma was prepared as described previously (24). Non-immune sheep IgG and a human HRGdirected IgG from sheep were prepared by Affinity Biologicals, and the HRG-specific IgG fraction was isolated by affinity chromatography using immobilized HRG (24). Unless otherwise specified, other reagents were from Sigma.…”

“…Fibrinogen was rendered factor XIII-free, and ␥ A /␥ A -and ␥ A /␥Ј-fibrinogen were separated by fractionation on DEAESepharose (GE Healthcare) and characterized as previously described (10,23). HRG was purified by metal-chelate chromatography, and HRG-deficient plasma was prepared as described previously (24). Non-immune sheep IgG and a human HRGdirected IgG from sheep were prepared by Affinity Biologicals, and the HRG-specific IgG fraction was isolated by affinity chromatography using immobilized HRG (24).…”

“…The interaction of HRG with immobilized ␥ A /␥ A -or ␥ A /␥Ј-fibrin(ogen), biotinylated-␥Ј-peptide, or fragments X, D, or E was assessed by SPR using a BIAcore 1000 (GE Healthcare) as previously described (24,30) but with some modifications. Briefly, proteins were covalently linked to separate flow cells of a carboxymethylated dextran (CM4) biosensor chip at a flow rate of 5 l/min using an amine coupling kit (GE Healthcare).…”

“…SPR Data Analysis-K d values were determined by kinetic analysis of on-and off-rates of HRG binding to immobilized ligands using Scrubber2 version 2.0a (Bio-Logic Software Co., Campbell, Australia) as described previously (24,30). For further assessment of binding, the amount of HRG bound at the equilibrium position (Req) was determined using the Langmuir 1:1 binding model (BIAEvaluation software Version 3.2) and was plotted against the titrant concentration.…”