Interactions Mediated by the N-Terminus of Fibrinogen’s B Chain ² Oleg V. Gorkun,* ,‡ Rustem I. Litvinov, § Yuri I. Veklich, § and John W. Weisel § Department of Pathology and Laboratory Medicine, UniVersity of North Carolina School of Medicine, Chapel Hill, North Carolina 27599, and Department of Cell and DeVelopmental Biology, UniVersity of PennsylVania School of Medicine, Philadelphia, PennsylVania 19104-6058 ReceiVed July 14, 2006; ReVised Manuscript ReceiVed September 29, 2006 ABSTRACT: Specific molecular interactions mediated by the N-terminus of fibrinogen’s B chain were revealed using laser tweezers-based force spectroscopy. We examined interactions between fibrinogen fragments representing the center of the molecule, NDSK, desA-NDSK, and desAB-NDSK, and two recombinant fibrinogens, γD364H and γD364A, which have nonfunctional γ-chain polymerization sites to prevent the dominant knob-hole binding. Interactions between desA-NDSK, where the N-terminus of the B chain is present, and the fibrinogen variants showed a complex spectrum of rupture forces which disappeared with desAB-NDSK, lacking both FpA and FpB. The interactions between desA-NDSK and γD364H or γD364A were inhibited by addition of soluble FpB, but not FpA or the polymerization inhibitor peptides GPRP and GHRP. When γD364H fibrinogen was replaced with its X-fragment lacking RC- domains or with fragment D, the strongest component of the rupture force spectrum disappeared, suggesting interactions between the uncleaved FpB and the RC-domain. Electron microscopy confirmed the binding of desA-NDSK to either D or E regions of fibrinogen as well as to RC-domains. The data demonstrate the existence of weak transient interactions within and between fibrin molecules mediated by the N-terminus of the fibrinogen B chain. Fibrinogen is a key player in hemostasis and wound healing (1-3). The fibrinogen molecule consists of three pairs of nonidentical polypeptide chains, AR,B, and γ, linked together by 29 disulfide bonds (4). The N-termini of all six chains come together in the central E region of the molecule, while the C-termini of each pair of AR,B, and γ chains extend outward to form independently folded C and γC modules at the distal ends of the molecule, called the D regions (Figure 1a). A coiled-coil consisting of all three chains links the globular domains in the middle and ends of the molecule (5). Thrombin converts fibrinogen into fibrin monomer by cleaving short N-terminal sequences of the AR and B chains called fibrinopeptide A (FpA) 1 and fibrinopeptide B (FpB). The removal of fibrinopeptides exposes the polymerization sites called the ‘A’ knobs, represented by the GPRV sequence of the R chain, and the ‘B’ knobs, represented by the GHRP sequence of the  chain (6). During clot formation, the newly ² This work was supported by National Institutes of Health Grants HL30954 (J.W.W.) and HL 31048 (Susan T. Lord). * To whom correspondence should be addressed: Department of Pathology and Laboratory Medicine, University of North Carolina School of Medicine, CB#7525 BBB 821, Chapel Hill, NC 27599-7525. Telephone: (919)966-2617.Fax: (919)966-6718.E-mail: ovg@med.unc.edu. ‡ University of North Carolina School of Medicine. § University of Pennsylvania School of Medicine. 1 Abbreviations: NDSK, N-terminal disulfide knot fragment of fibrinogen; desA-NDSK, N-terminal disulfide knot fragment of fibrin with FpA cleaved; desAB-NDSK, N-terminal disulfide knot fragment of fibrin with FpA and FpB cleaved; FpA, fibrinopeptide A; FpB, fibrinopeptide B; γD364H or γD364A, recombinant fibrinogens with Asp substituted with His or Ala, respectively, at position 364 in the γ chain; GPRP, Gly-Pro-Arg-Pro-amide peptide; GHRP, Gly-His-Arg- Pro-amide peptide; CHO, Chinese hamster ovary; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; HEPES, N-(2- hydroxyethyl)piperazine-N-2-ethanesulfonic acid; HBS, 20 mM HEPES (pH 7.4), 150 mM NaCl buffer; Tris, 2-amino-2-hydroxymethyl-1,3- propanediol; D-γD364H, fragment D of recombinant fibrinogen γD364H; X-γD364H, fragment X of recombinant fibrinogen γD364H; BSA, bovine serum albumin. FIGURE 1: Fibrinogen molecule (a) depicted in D-E-D linear arrangement. The two distal D regions consist of γC and C modules and are connected to the central region E by coiled-coil connectors. The positions of the RC-domains, FpA, and FpB in the center of the molecule are denoted with arrows. Compared to fibrinogen, the X fragment (b) lacks both RC-domains (marked with two asterisks) and parts of the N-terminus of the B chain, including FpB (marked with one asterisk), but preserves the D-E-D arrangement and the N-terminus of the AR chain, including FpA. The D fragment (c) consists of the C and γC modules and the coiled-coil connector. The N-terminal disulfide knot (NDSK) represents the E region and consists of the intact N-termini of all six fibrinogen chains (d). Unlike in fibrinogen, X, and D fragments, the coiled-coil connector in NDSK is composed of the B and γ chains only, because the R chain is not long enough to be a part of it. 14843 Biochemistry 2006, 45, 14843-14852 10.1021/bi061430q CCC: $33.50 © 2006 American Chemical Society Published on Web 11/16/2006