B:b interactions are essential for polymerization of variant fibrinogens with impaired holes ‘a’

Abstract

Summary. Background: Fibrin polymerization is mediated by interactions between knobs ÔAÕ and ÔBÕ exposed by thrombin cleavage, and holes ÔaÕ and ÔbÕ always present in fibrinogen. The role of A:a interactions is well established, but the roles of knob:hole interactions A:b, B:b or B:a remain ambiguous. Objectives: To determine whether A:b or B:b interactions have a role in thrombin-catalyzed polymerization, we examined a series of fibrinogen variants with substitutions altering holes ÔaÕ: c364Ala, c364His or c364Val. Methods: We examined thrombin-and reptilase-catalyzed fibrinopeptide release by high-performance liquid chromatography, fibrin clot formation by turbidity, fibrin clot structure by scanning electron microscopy (SEM) and factor (F) XIIIa-catalyzed crosslinking by sodium dodecylsulfate polyacrylamide gel electrophoresis. Results: Thrombin-catalyzed fibrinopeptide A release was normal, but fibrinopeptide B release was delayed for all variants. The variant fibrinogens all showed markedly impaired thrombin-catalyzed polymerization; polymerization of c364Val and c364His were more delayed than c364Ala. There was absolutely no polymerization of any variant with reptilase, which exposed only knobs ÔAÕ. SEM showed that the variant clots formed after 24 h had uniform, ordered fibers that were thicker than normal. Polymerization of the variant fibrinogens was inhibited dose-dependently by the addition of either Gly-Pro-Arg-Pro (GPRP) or Gly-His-Arg-Pro (GHRP), peptides that specifically block holes ÔaÕ and ÔbÕ, respectively. FXIIIa-catalyzed crosslinking between c-chains was markedly delayed for all the variants. Conclusion: These results demonstrate that B:b interactions are critical for polymerization of variant fibrinogens with impaired holes ÔaÕ. Based on these data, we propose a model wherein B:b interactions participate in protofibril formation.

Mice expressing a mutant form of fibrinogen that cannot support fibrin formation exhibit compromised antimicrobial host defense

“…However, consistent with findings with other fibrinogen variants, [24][25][26][27][28] HPLC analyses suggested that FpB release from fibrinogen AEK was delayed relative to thrombin-mediated release of FpB from WT fibrinogen (Figure 4B-C). The calculated specificity constants (k cat /K M ) confirmed a significant diminution in the efficiency of FpB release from fibrinogen AEK relative to WT fibrinogen ( Figure 4D).…”

“…Both turbidity measurements and scanning electron microscopy studies revealed robust polymerization of fibrinogen AEK following incubation with EK enzyme (supporting FpA, but not FpB, release), but a fundamental failure of polymerization following incubation with thrombin (supporting FpB, but not FpA, release). Our findings are compatible with prior biochemical analyses using snake venom proteases favoring FpA or FpB release, 27,[38][39][40][41] as well as previous reports indicating that polymerization is compromised in the fibrinogen variant g Asp364His , which alters the "a" binding pocket, whereas polymerization is similar to normal in the fibrinogen variant Bb Arg432Ala , which disrupts the "b" binding pocket. [41][42][43][44] B:b interactions are reported to be exceptionally weak as characterized by high affinity constants and a low strength force to rupture the bonds.…”

Mechanisms of fibrin polymerization and clinical implications

“…11 Furthermore, recombinant mutants of the g364 residue that have no functional holes 'a' and cannot build A:a bonds form clots slowly with thrombin but not with reptilase. 12 On the other hand, fibrinogen variant BbD432A with impaired hole 'b' displays normal polymerization. 13 Therefore, it appears that B:b bonds can occur at least when A:a interactions are compromised, but their normal functional role is still mostly unknown.…”

Effect of Plasmin Treatment on the Fibrin Gel Formation

“…In the recombinant variant fibrinogen, in which the binding of A-knob to a-hole is inhibited, the B:b interactions result in the delayed fibrin polymerization, and reptilase could not result in a fibrin gel [20]. When b-holes are blocked further by B-knob analog peptide GHRPam, thrombin-catalyzed polymerization was destructively impaired.…”