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

Abstract

Histidine-rich glycoprotein (HRG) is an abundant protein that binds fibrinogen and other plasma proteins in a Zn2؉ -dependent fashion but whose function is unclear. HRG has antimicrobial activity, and its incorporation into fibrin clots facilitates bacterial entrapment and killing and promotes inflammation. Although these findings suggest that HRG contributes to innate immunity and inflammation, little is known about the HRGfibrin(ogen) interaction. By immunoassay, HRG-fibrinogen complexes were detected in Zn 2؉ -supplemented human plasma, a finding consistent with a high affinity interaction. Fibrinogen is the soluble precursor of fibrin, a critical component of blood clots that endows them with strength and elasticity. Fibrinogen is a glycoprotein composed of three pairs of polypeptide chains, termed A␣, B␤, and ␥, that are connected by disulfide bonds (1). Approximately 10 -15% of circulating fibrinogen has a variant ␥-chain termed the ␥Ј-chain, which results from differential processing of the ␥ A -chain mRNA transcript (2-4). The ␥Ј-chain is distinguished from the ␥ A -chain by the presence of an acidic 20-residue extension at its COOH terminus (2, 5).Thrombin catalyzes the conversion of fibrinogen to insoluble fibrin, and during this process some thrombin remains bound to the fibrin network (6). Exosites 1 and 2 are two regulatory domains that flank the active site of thrombin and mediate its binding to fibrin (7,8). Exosite 1 of thrombin interacts with the central E-domain of fibrin, whereas exosite 2 binds only to the COOH terminus of the ␥Ј-chain. Consequently, thrombin binds ␥ A /␥ A -fibrin in a univalent fashion with a K d value of 2-4 M. In contrast, both exosites are engaged when thrombin binds to ␥ A /␥Ј-fibrin, resulting in a higher affinity interaction (K d value of 0.08 -0.18 M) (5, 9). Fibrin-bound thrombin remains active, and the protease is protected from inhibition by fluid-phase inhibitors, such as antithrombin and heparin cofactor II (6). Because of its bivalent interaction with ␥ A /␥Ј-fibrin, thrombin bound to ␥ A /␥Ј-fibrin is more protected from inhibition by fluid-phase inhibitors than thrombin bound to ␥ A /␥ Afibrin (10).Like thrombin, histidine-rich glycoprotein (HRG) 3 binds to fibrinogen and is incorporated into fibrin clots (11). Although the plasma concentration of HRG ranges from 1.6 to 2 M, the concentration in platelet-rich thrombi may be higher because HRG is stored in the alpha granules of platelets and is released when platelets are activated (12, 13). A 75-kDa glycoprotein, HRG, is composed of two NH 2 -terminal cystatin-like domains, a central histidine-rich region (HRR) flanked by two prolinerich regions, and a COOH-terminal domain (14). In addition to fibrinogen, HRG also binds plasminogen, heparan sulfate, and divalent cations, such as Zn 2ϩ (12,15,16). Therefore, HRG is hypothesized to be an important accessory or adapter protein that brings different ligands together under specific conditions (14).HRG-deficient mice exhibit a shorter prothrombin time and accelerat...

A High Affinity Interaction of Plasminogen with Fibrin Is Not Essential for Efficient Activation by Tissue-type Plasminogen Activator

“…In previous reports, the affinity of Glu-Pg for Fn ranged from 13 to 38 M and there was low or no measurable affinity of Glu-Pg for Fg (14,22). Therefore, the higher affinity of Glu-Pg for Fn relative to Fg is consistent with previous work, which validates the use of SPR for determination of the affinity for Fg and Fn, as observed previously with thrombin (18,19). Affinities of Various Pg Variants for tPA-The Pg/tPA interaction was studied using SPR to explore the influence of this binary interaction on Pg activation.…”

“…For studies with Fn, immobilized Fg was converted to Fn by injecting 0.5 M thrombin in HBSC-Tween 20 at a rate of 10 l/min for 30 min as described previously (18,19). This procedure was repeated at least twice and was continued until there was no further reduction in RU, indicating complete conversion of Fg to Fn.…”

Batroxobin Binds Fibrin with Higher Affinity and Promotes Clot Expansion to a Greater Extent than Thrombin

“…Modification of Proteins and Peptides-Batroxobin and thrombin were radiolabeled by reaction with 125 I-YPR, prepared using Na 125 I (McMaster University Nuclear Reactor, Hamilton, Ontario, Canada) and IODO-BEADs (Pierce) as described (18,30).…”

“…Des-B␤(1-42) fibrinogen was prepared by incubating ␥ A /␥ Afibrinogen with protease III fraction from Crotalus atrox venom and purified using ethanol precipitation as described (16). Fragments D and E were generated by plasmin digestion and purified as reported previously (30). The NH 2 -terminal disulfide knot (NDSK) was obtained with cyanogen bromide treatment and purified as described (32).…”

“…The ␥Ј-peptide-directed IgG was subjected to affinity chromatography using immobilized ␥Ј-peptide as described previously (30). The ␣(17-51)-peptide (GPRVVERHQSAAKDSDWPF-ASDEDWNYKAPSGCRM) with Cys-28, Cys-36, and Cys-45 changed to Ala and an NH 2 -terminal Cys residue was synthesized by LifeTein (Plainfield, NJ).…”