Analysis of Aα251 Fibrinogen:  The αC Domain Has a Role in Polymerization, Albeit More Subtle Than Anticipated from the Analogous Proteolytic Fragment X

Abstract

Numerous experiments have demonstrated that the C-terminal domain of the fibrinogen Aalpha-chain, the alphaC domain, has a role in polymerization. To further examine the role of this domain, we synthesized a recombinant fibrinogen, Aalpha251 fibrinogen, that lacks the alphaC domain. We examined thrombin-catalyzed fibrinopeptide release and found that the rate of FpB release from Aalpha251 fibrinogen was 2.5-fold slower than FpB release from normal fibrinogen, while the rate of FpA release was the same for both proteins. We examined thrombin-catalyzed polymerization and found that the rates of protofibril formation and lateral aggregation were similar for both proteins, although discernible differences in lateral aggregation were clear. The rate of protofibril formation for Aalpha251 fibrinogen was never less than 85% of normal fibrinogen, while the rate of lateral aggregation for Aalpha251 fibrinogen varied from 64 to 74% of normal. We examined polymerization of fibrin monomers and found that polymerization of Aalpha251 fibrin was similar to normal fibrin at 0.4 M NaCl, but clearly different from normal at 0.05 M NaCl. These results indicate that the alphaC domain has a role in lateral aggregation, but this role is more subtle than anticipated from previous experiments, particularly those with fibrinogen fragment X. We interpret this unanticipated finding as indicative of an important contribution from the N-terminus of the beta-chain, such that protein heterogeneity that includes small amounts of fibrin lacking that N-terminus of the beta-chain leads to markedly altered lateral aggregation.

The Assembly of Nonadhesive Fibrinogen Matrices Depends on the αC Regions of the Fibrinogen Molecule

“…Fibrinogen was labeled with 125 I using IODO-GEN (Thermo Scientific Pierce), dialyzed against PBS, and stored at Ϫ20°C. Recombinant normal (rFg; 340 kDa) and fibrinogen with the truncated ␣C regions (FgA␣251; 260 kDa) in which the A␣ chain is terminated at Thr-251, were produced as previously described (23). The protein concentration was determined by spectrophotometry at 280 nm using the extinction coefficient 1.51 at 1 mg/ml for hFg and 1.6 for rFg and FgA␣251.…”

Fibrinogen splice variation and cross-linking: Effects on fibrin structure/function and role of fibrinogen γ′ as thrombomobulin II

“…The first, fibrinogen -251, described in 1998, contained a deletion of -chain amino acids 251-610 where most of cross-linking site are located [28]. Collet et al recently showed that in the presence of FXIII, clots made of fibrin -251 were denser, with the pore size decreasing by 1.8-fold, compared to wild-type fibrin [29].…”

A Comprehensive Mechanism of Fibrin Network Formation Involving Early Branching and Delayed Single- to Double-Strand Transition from Coupled Time-Resolved X-ray/Light-Scattering Detection

“…We point out that although the snake venom enzyme Ancrod was used to avoid complicating the interpretation by even limited FpB release, the present data are quite similar to those we previously collected using thrombin and only a WA-MALS with a more limited angular range. 21,22 Further work with recombinant FG species with missing 48 or human/chicken hybrid 49 αC regions, and with either uncleavable FpB's or nonbinding B knobs/ unavailable b holes and thrombin as the enzyme, is planned. It should help to clarify the role played by the largely unstructured Aα chain appendages, which are also functionally important for FG adhesion, 50,51 …”