Direct Evidence for Specific Interactions of the Fibrinogen αC-Domains with the Central E Region and with Each Other

Abstract

The carboxyl-terminal regions of the fibrinogen Aα chains (αC regions) form compact αC-domains tethered to the bulk of the molecule with flexible αC-connectors. It was hypothesized that in fibrinogen two αC-domains interact intramolecularly with each other and with the central E region preferentially through its N-termini of Bβ chains, and that removal of fibrinopeptides A and B upon fibrin assembly results in dissociation of the αC regions and their switch to intermolecular interactions. To test this hypothesis, we studied the interactions of the recombinant αC region (Aα221-610 fragment) and its sub-fragments, αC-connector (Aα221-391) and αC-domain (Aα392-610), between each other and with the recombinant (Bβ1-66) 2 and (β15-66) 2 fragments and NDSK corresponding to the fibrin(ogen) central E region, using laser tweezers-based force spectroscopy. TheαC-domain, but not the αC-connector, bound to NDSK, which contains fibrinopeptides A and B, and less frequently to desA-NDSK and (Bβ1-66) 2 containing only fibrinopeptides B; it was poorly reactive with desAB-NDSK and (β15-66) 2 both lacking fibrinopeptides B. The interactions of the αC-domains with each other and with the αC-connector were also observed, although they were weaker and heterogeneous in strength. These results provide the first direct evidence for the interaction between the αC-domains and the central E region through fibrinopeptides B, in agreement with the above hypothesis, and indicate that fibrinopeptides A are also involved. They also confirm the hypothesized homomeric interactions between the αC-domains and display their interaction with the αC-connectors, which may contribute to covalent cross-linking of α polymers in fibrin. KeywordsBlood coagulation; protein interactions; fibrinogen; fibrin Fibrinogen is a blood plasma protein involved in a number of (patho)physiological processes such as hemostasis, fibrinolysis, inflammation, angiogenesis, wound healing, and neoplasia *To whom correspondence should be addressed: Dr. Rustem I. Litvinov, Department of Cell and Developmental Biology, University of Pennsylvania, School of Medicine, 421 Curie Blvd., 1040 BRB II/III, Philadelphia, PA 19104-6058, USA. Tel.: 215-898-9141; Fax: 215-898- NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript (1,2). This polyfunctionality is due to the complex structure of fibrinogen molecules that have multiple binding sites, either constitutively open or exposed after precise enzymatic cleavage and/or conformational rearrangement. The ability to polymerize upon the action of thrombin is the unique property of fibrinogen that mainly determines its physiological significance.Structurally, fibrinogen is a 45 nm-long elongated dimer composed of three pairs of nonidentical polypeptide chains, designated Aα, Bβ, and γ (Fig. 1). The N-termini of the six chains, cross-linked by a cluster of disulfide bonds, form a central part, hence named "N-terminal disulfide knot" (3). The C-termini of Bβ and γ chains form globular modules on each end of the ...

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

“…The binding sites within the ␣C regions involved in the interactions with fibrinopeptides and self-association have not been established. Nevertheless, the ability of the fragments representing the entire ␣C region or the ␣C domain to form oligomers, as well as the reports of ␣C-␣C interactions at the single-molecule level provide direct evidence that they contain the self-association sites (19,33). The release of fibrinopeptides B by thrombin during the conversion of fibrinogen to fibrin is accompanied by the dissociation of the ␣C regions from the E region (Fig.…”

“…8A). The interaction of the ␣C regions with the E region is mediated mainly via fibrinopeptides B, although the contribution of fibrinopeptides A has also been shown (19). The binding sites within the ␣C regions involved in the interactions with fibrinopeptides and self-association have not been established.…”

Effect of Plasmin Treatment on the Fibrin Gel Formation

“…Additionally, since the region contains the β-wall to which thrombin binds [17], FpA release is likely to be impaired. Besides, the deletion of Bβ9-72 could affect the function of αC domain, because FpA is involved in the interaction with αC domain together with BβN region [11]. Thus, the deletion of Bβ9-72 results in not only the impaired function of BβN region, but also affects the release of αC-domain.…”

“…Bowley and Lord have reported that b-hole and binding between B-knob and b-hole per se have no influence on fibrin polymerization [10]. Because the interaction between the αC-domain and central E region through FpB and the interaction between αC-domains have been ascertained [11], the importance of the αC-domain is clear, but another substantial factor for polymerization should be involved especially in the lateral aggregation process.…”

Characterizing reactive glutamines in fibrinogen and elucidating factor XIII substrate specificity.

“…Prior to fibrin polymerization, the αC region is tethered to the central E region. 30,31 Fibrinogen is converted to active fibrin upon thrombin cleavage of fibrinopeptides A and B, on the Aα and Bβ chains respectively. Cleavage of fibrinopeptides A and B initiates fibrin polymerization by exposing knobs "a" and "b" in the central E region which interact with the complementary holes "A" and "B" in the D region of another fibrin molecule.…”