Localization of an Effective Fibrin β-Chain Polymerization Site:  Implications for the Polymerization Mechanism

Abstract

To examine whether fibrin N-terminal Aalpha 17-23 and Bbeta 15-25 may contain high-affinity polymerization sites, GPRVVER and GHRPLDKKREE analogs were prepared, and their abilities to inhibit fibrin monomers from repolymerizing were compared in turbidity and clottability assays. Within Aalpha 17-23, GPR is the most active site (IC30 of 0.95-1.36 mM). Its extension into GPRVVER (IC30 of 1.75-2.3 mM) reduced activity. Within Bbeta 15-25, acyl-DKKREE (IC30 of 0.30-0.53 mM) can account for GHRPLDKKREE activity (IC30 of 0. 33-0.44 mM). Comparison of the assays showed that calcium, whose presence induces thick fibrin fibers, elicited a higher turbidity than clottability inhibition. Similarly, the lateral-association-promoting GHRP (IC30 of 1.25-1.43 mM) gave a high turbidity vs clottability inhibition ratio (137%). In contrast, low ratios were found for the linear-association-initiating GPR (73%) and for acyl-DKKREE (34%). Structure-activity correlation showed that fibrinogen-like acyl-GPRP and acyl-GHRP could inhibit D. E association at the millimolar range, but in a manner different from fibrin-related GPR peptides did, which required the NH2 as well as Arg presence. To explain Bbeta 20-25 masking, it is proposed that DKKREE in fibrinogen may engage in ionic and hydrogen bonds with KDSDW, the Aalpha 29-33 sequence implicated in thrombin binding. To explain acyl-GPRP and acyl-GHRP inhibition of D.E association, it is proposed that fibrinogen packing may be mediated by E domain association with alphaC (Aalpha 220-609) fragments of adjacent molecules, and by alphaC-alphaC association. A modified polymerization mechanism is deduced by taking into account fibrinogen N-terminal conformation as well as E domain binding to thrombin vs alphaC fragments. This model proposes the following. (1) Upon thrombin binding to fibrinogen KDSDW, DKKREE may become exposed. (2) Fibrinopeptide A cleavage further unmasks the NH2 and Arg group of GPR, leading to DKKREE and GPR initiation of polymerization. (3) The micromolar-effective thrombin-fibrin(ogen) binding may initiate a partial alphaC repulsion. Subsequent DKKREE and GPR binding to D domains of other fibrin(ogen) will lead to the formation of the trimer and bring additional molecules to fibrin N-terminal region, and the combined steric congestion may lead to a complete alphaC repulsion from the overcrowded E domain. (4) Repulsion of the large Aalpha 220-609 fragments may unmask multiple polymerization sites beyond the fibrin N-terminal region.

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

“…It is reasonable to conclude that any or all of the chain heterogeneities contribute to these differences. We favor an important contribution from the N-terminal residues of the β-chain (48)(49)(50). It is known that β15-18 participates in fibrin polymerization, so loss of these residues in X 1 and X 2 could have a dramatic effect.…”

Fibrinogen C-terminal peptidic sequences (Haptides) modulate fibrin polymerization

“…Prior work revealed the reduced clot turbidity, modified clot ultra-structure and altered mechanical properties of fibrinogen Osaka VI, which contains a short 12 amino acid extension on the β-chain, and thereby indicated that the β-chain terminus was involved in fibrin polymerization (23). Other observations showed that a 37-amino acid peptide equivalent to the sequence γ374-411 of the γ-chain bound inhibited the polymerization of fibrin oligomers and decreased the maximal turbidity of the fibrin clots (10)(11)(12). Calorimetry, fluorescence, HPLC and size exclusion techniques showed that a 32-amino acid peptide equivalent to the γ374-405 could non-covalently attach to fibrinogen fragment D3.…”

“…Calorimetry, fluorescence, HPLC and size exclusion techniques showed that a 32-amino acid peptide equivalent to the γ374-405 could non-covalently attach to fibrinogen fragment D3. In free form, wild and truncated versions of the γ374-405 peptide tended to self-associate and spontaneously formed oligomers (10)(11)(12)(13). Combined, these reports suggested that a large portion of the C-termini of both β and γ chains of normal fibrin(ogen) were involved in the assembling the monomers into a clot.…”

“…α14-35 and β15-52) can become prime polymerization foci ("knobs") by binding to complimentary sites ("holes") on the D-domains of adjacent fibrin molecule. The D-D and D-E contacts between different fibrin(ogen) molecules help align the molecules to form linear protofibrils (10)(11)(12)(13)(14)(15)(16). Lateral contacts lead to branching that ultimately result in a phase-change and the formation of the 3-dimensional gel network that constitutes the fibrin clot.…”

“…It was also shown that a 32 amino acid peptide, equivalent to the fibrinogen γ-chain (γ374-405), could non-covalently associate with the D3 fragment which contains the γ C-terminal module of fibrinogen (13). In another example, peptides GPRVVER (equivalent to α17-23) and GHRPLDKKREE (equivalent to β15-25) could effectively inhibit thrombin-induced fibrin polymerization, as monitored by decreased clot turbidity (11).…”