Analysis of Engineered Fibrinogen Variants Suggests That an Additional Site Mediates Platelet Aggregation and That “B−b” Interactions Have a Role in Protofibril Formation

Abstract

The C-terminal domain of the fibrinogen gamma-chain includes multiple functional sites that have been defined in high-resolution structures and biochemical assays. Calcium binds to this domain through the side chains of gammaD318 and gammaD320 and the backbone carbonyls of gammaF322 and gammaG324. We have examined variant fibrinogens with alanine at position gamma318 and/or gamma320 and found that calcium binding, fibrin polymerization, and fibrinogen-mediated platelet aggregation, but not FXIIIa-catalyzed cross-linking, were abnormal. When measured by turbidity, thrombin-catalyzed polymerization was severely reduced, and batroxobin-catalyzed polymerization was completely obliterated. Moreover, thrombin-catalyzed polymerization was abolished by the peptide GHRP, which binds to the polymerization site in the beta-chain but does not inhibit polymerization of normal fibrinogen. ADP-induced platelet aggregation was also severely impaired. In contrast, as measured by SDS-PAGE, FXIIIa introduced cross-links between gamma-chains for all three variants, as expected if the gamma-chain C-terminal sites were normal. In addition, binding of the monoclonal antibody 4A5, which recognizes the C-terminal residues, was not different from normal. These data suggest two specific conclusions: (1) a site in the gamma-module other than the C-terminus is critical for platelet aggregation and (2) "B-b" interactions have a role in protofibril formation.

Citrullinated fibrinogen shows defects in FPA and FPB release and fibrin polymerization catalyzed by thrombin

“…3). These results demonstrate that neither the binding of the GPRP peptide to the "a-hole" nor the binding of Ca ions to the low affinity Ca-binding sites in the C-terminal module of the γ-chain are affected by the citrullination of fibrinogen [7].…”

“…Fibrinogen (0.2 mg/mL) in HEPES buffer containing 5 mM EDTA, 5 mM CaCl 2 , or 2 mM Gly-Pro-Arg-Pro-peptide supplemented with 1 mM EDTA was incubated with 0.2 casein-unit/mL plasmin for 4 h at 37 ºC [7]. The reactions were stopped by the addition of an equal volume of sodium dodecyl sulfate (SDS)-sample buffer and heating at 100 ºC for 5…”

“…To analyze the other functions of the C-terminal module of the fibrinogen γ-chain, the "a-hole", and the low affinity Ca-binding sites, protection against plasmin digestion of C-Fig was performed [7]. In this assay, in the presence of 5 mM CaCl 2 or 2 mM…”

αIIbβ3 binding to a fibrinogen fragment lacking the γ-chain dodecapeptide is activation dependent and EDTA inducible

“…Specifically, potential ancillary sites have been identified in the fibrinogen g-module (g-148-411), including g-316-322 and g-370-381. [44][45][46][47] Such sites are difficult to study using intact fibrinogen because of the confounding effect of the interaction of aIIbb3 with g-404-411. As a result, to identify additional sites of interaction between fibrinogen and aIIbb3, we studied the adhesion of platelets and HEK293 cells expressing either normal aIIbb3 or mutant aIIbb3 to fibrinogen and plasmin fragments of fibrinogen that either contain or lack intact g-404-411 (D100 and 'D98,' respectively), in the presence or absence of EDTA.…”

Platelet Factor 4 (CXCL4) Seals Blood Clots by Altering the Structure of Fibrin

“…Although the role of the A-knob/hole a interaction is well established, that of the B-knob/hole b interaction is unclear since it is not critical for network formation, although it presumably enhances lateral aggregation and assembly of protofibrils (17-19). Snake venom toxins such as reptilase (Ancrod) that remove only fibrinopeptide A from fibrinogen (leaving fibrinopeptide B intact), trigger fibrin network assembly resembling that induced by thrombin (18,20). Accordingly, Gly-ProArg-Pro-amide (GPRPam), which mimics the A-knob, impedes polymerization, whereas Gly-His-Arg-Pro-amide (GHRPam), which mimics the B-knob, does not (21,22).…”