Conversion of fibrinogen into fibrin results in the exposure of cryptic interaction sites and modulation of various activities. To elucidate the mechanism of this exposure, we tested the accessibility of the Aalpha148-160 and gamma312-324 fibrin-specific epitopes that are involved in binding of plasminogen and its activator tPA, in several fragments derived from fibrinogen (fragment D and its subfragments) and fibrin (cross-linked D-D fragment and its noncovalent complex with the E(1) fragment, D-D. E(1)). Neither D nor D-D bound tPA, plasminogen, or anti-Aalpha148-160 and anti-gamma312-324 monoclonal antibodies, indicating that their fibrin-specific epitopes were inaccessible. The Aalpha148-160 epitope became exposed only upon proteolytic removal of the beta- and gamma-modules from D. At the same time, both epitopes were accessible in the D-D.E(1) complex, indicating that the DD.E interaction resulted in their exposure. This exposure was reversible since the dissociation of the D-D.E(1) complex made the sites unavailable, while reconstitution of the complex made them exposed. The results indicate that upon fibrin assembly, driven primarily by the interaction between complementary sites of the D and E regions, the D regions undergo conformational changes that cause the exposure of their plasminogen- and tPA-binding sites. These changes may be involved in the regulation of fibrin assembly and fibrinolysis.

While testing the effect of the (␤15-66) 2 fragment, which mimics a pair of fibrin ␤N-domains, on the morphology of endothelial cells, we found that this fragment induces redistribution of vascular endothelial-cadherin in a process that is inhibited by the receptor-associated protein (RAP). Based on this finding, we hypothesized that fibrin may interact with members of RAP-dependent low-density lipoprotein (LDL) receptor family. To test this hypothesis, we examined the interaction of (␤15-66) 2 , fibrin, and several fibrin-derived fragments with 2 members of this family by ELISA and surface plasmon resonance. The experiments showed that very LDL (VLDL) receptor (VLDLR) interacts with high affinity with fibrin through its ␤N-domains, and this interaction is inhibited by RAP and (␤15-66) 2 . Furthermore, RAP inhibited transendothelial migration of neutrophils induced by fibrin-derived NDSK-II fragment containing ␤N-domains, suggesting the involvement of VLDLR in fibrin-dependent leukocyte transmigration. Our experiments with VLDLR-deficient mice confirmed this suggestion by showing that, in contrast to wild-type mice, fibrin-dependent leukocyte transmigration does not occur in such mice. Altogether, the present study identified VLDLR as a novel endothelial cell receptor for fibrin that promotes fibrin-dependent leukocyte transmigration and thereby inflammation. Establishing the molecular mechanism underlying this interaction may result in the development of novel inhibitors of fibrin-dependent inflammation. (Blood. 2012; 119(2):637-644) IntroductionThrombin-mediated conversion of fibrinogen into fibrin plays a prominent role in preventing the loss of blood on vascular injury. During this conversion, thrombin removes from fibrinogen 2 pairs of N-terminal fibrinopeptides, fibrinopeptide A (FpA) and fibrinopeptide B (FpB), enabling spontaneous polymerization of fibrin monomers into an insoluble fibrin clot. The clot seals the injured vasculature and serves as a provisional matrix that participates in subsequent wound healing and other important physiologic and pathologic processes through the interaction with various plasma proteins and cell types. Specifically, the interaction of fibrin with endothelial cells contributes to anchoring of the clot to places of vascular injury and then promotes transendothelial migration of leukocytes and thereby inflammation and capillary tube formation, that is, angiogenesis. [1][2][3][4] Several receptors on the endothelial cell surface mediate the interaction of fibrin(ogen) with endothelial cells. They include integrins ␣V␤3, ␣V␤5, and ␣5␤1, 5-7 and nonintegrin receptors ICAM-1, vascular endothelial (VE)-cadherin, and proteoglycans. [8][9][10] The interaction of endothelial cell integrins with fibrin-(ogen) occurs mainly through the fibrin(ogen) RGD sequences and promotes endothelial cell adhesion and spreading. 5,7 This interaction may also promote cell migration and angiogenesis. 3,7 The interaction with proteoglycans mediates binding of fibrin(ogen) to endothelial cells, 10 whe...

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 ...