Expression of primary polymerization sites in the D domain of human fibrinogen depends on intact conformation

Fragments D1 and DD, plasmic degradation products of human fibrinogen and cross-linked fibrin, respectively, originate from the COOH-terminal domain of the parent molecule. Since a specific binding site for fibrin resides in the COOH-terminal region of the gamma chain, the primary structure of the two fragments was compared and their affinity for fibrin monomer measured. Fragments D1 and DD contained the same segments of the three fibrinogen chains, corresponding to the sequences alpha 105-206, beta 134-461, and gamma 63-411. Fragment DD had a double set of the same chain remnants. Fragments D1 and DD inhibited polymerization of fibrin monomer in a dose-dependent manner; 50% inhibition occurred at a molar ratio of fragment to monomer of 1:1 and 0.5:1, respectively. To prevent fibrin monomer polymerization and render it suitable for binding studies in the liquid phase, fibrinogen was decorated with Fab fragments isolated from rabbit antibodies to human fragment D1. Fibrinogen molecules decorated with 6 molecules of this Fab fragment did not clot after incubation with thrombin, and the decorated fibrin monomer could be used to measure binding of fragments D1 and DD in a homogeneous liquid phase. The data analyzed according to the Scatchard equation and a double-reciprocal plot gave a dissociation constant of 12 nM for fragment D1 and 38 nM for fragment DD. There were two binding sites/fibrin monomer molecule for each fragment. After denaturation in 5 M guanidine HCl, the inhibitory function on fibrin polymerization was irreversibly destroyed. Denatured fragments also lost binding affinity for immobilized fibrin monomer. The preservation of the native tertiary structure in both fragments was essential for the expression of polymerization sites in the structural D domain.     

Immunochemical determination of conformational equilibria for fragments of the B beta chain of fibrinogen

The conformations of the B beta chain of the intact fibrinogen molecule and of various fragments of the B beta chain of fibrinogen that contain the region that is hydrolyzed by thrombin have been compared by an immunochemical method [Sachs, D. H., Schechter, A. N., Eastlake, A., & Anfinsen, C. B. (1972) Proc. Natl. Acad. Sci. U.S.A. 69, 3790]. Anti-fibrinogen antibodies were induced in rabbits by immunization with native bovine fibrinogen. An antibody population specific for the native antigenic determinant within the B beta fragment 20-28 was isolated by immunoadsorption. This preparation was to determine the value of Kconf, the equilibrium constant for the interconversion of the nonnative and native conformations of this determinant. Values of Kconf were measured for this determinant within native fibrinogen, the disulfide knot (DSK), CNBrB beta, B beta fragment 16-28, B beta fragment 20-28, and fibrinopeptide B (FpB). 125I-Labeled fibrinogen (125I-F) was used in the determination of Kconf by measuring the competition between 125I-F and the fibrinogen derivatives under study for binding to the purified antibody. For the antigenic region in F, the DSK, and CNBrB beta, the values of Kconf at 4 degrees C were infinity, (5.9 +/- 3.5) X 10(-3), and (1.2 +/- 0.7) X 10(-3), respectively. The values of Kconf for B beta fragment 16-28, B beta fragment 20-28, and FpB at 4 degrees C were less than (6.0 +/- 3.9) X 10(-7).(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunochemical mapping of the conformation of human fibrinogen. The gamma 95-264 segment in inaccessible to antibody in native fibrinogen but progressively exposed by plasmic cleavage

The accessibility of the gamma 95-264 sequence to specific antibody probes in the native fibrinogen molecule and its plasmic cleavage fragments have been investigated. The gamma 95-264 segment was generated by cyanogen bromide cleavage of the gamma chain and isolated by gel filtration and ion exchange chromatography. Rabbit antisera to this peptide and to gamma chain recognized at least five antigenic loci uniformly distributed throughout this segment. In primary binding assays, antibodies to gamma 95-264 bound gamma 95-264, free gamma chain, and fibrinogen fragment D, but not native fibrinogen. Also, gamma 95-264 was bound by antibodies to gamma chain and fibrinogen fragment D, but not by antibodies generated to native fibrinogen. Thus, the gamma 95-264 sequence was not accessible to antibody in the native structure. In competitive equilibrium radioimmunoassays, neither native fibrinogen nor highly soluble fibrinogen fraction I-9 inhibited the binding of gamma 95-264 by its antiserum or anti-gamma chain. With plasmic cleavage, however, the gamma 95-264 sequence became accessible to antibody and the series of fragments D greater than Y greater than D:E = X describes the relative reactivity of the gamma chain sequence in fibrinogen degradation products. Differential expression of gamma 95-264 antigenic loci was also observed with D fragments differing in molecular weight. Plasmic cleavage of cross-linked and noncross-linked fibrin generated D fragments which did not express gamma 95-264 as well as fibrinogen D derivatives, indicating that the D domains of fibrinogen and fibrin are immunochemically distinguishable. These findings indicate that the central segment of the gamma chain is inaccessible to antibody in native fibrinogen, but that proper surface orientation is achieved upon plasmic degradation.     

Structure of the gamma heavy chain of the outer arm dynein from Chlamydomonas flagella

We describe here the vanadate-dependent photocleavage of the gamma heavy chain from the Chlamydomonas outer arm dynein and the pathways by which this molecule is degraded by endoproteases. UV irradiation in the presence of ATP, Mg2+, and vanadate cleaves the gamma chain at a single site (termed V1) to yield fragments of Mr 235,000 and 180,000. Irradiation in the presence of vanadate and Mn2+ results in cleavage of the gamma chain at two other sites (termed V2a and V2b) to yield fragment pairs of Mr 215,000/200,000 and 250,000/165,000. The mass of the intact chain is therefore estimated to be 415,000 D. We have located the major tryptic and staphylococcal protease cleavage sites in the gamma chain, determined the origins of the resulting fragments, and identified the regions which contain the epitopes recognized by two different monoclonal antibodies. Both antibodies react with the smaller V1 fragment; the epitope recognized by antibody 25-8 is within 9,000-52,000 D of the original gamma-chain terminus contained in that fragment, whereas that recognized by antibody 12 gamma B is within 16,000 D of the V1 site. The data permit the construction of a linear map showing the structural organization of the polypeptide. The substructure of the gamma chain is similar to that of the alpha and beta chains of the outer arm dynein with regard to polarity as defined by the sites of vanadate-dependent photocleavage, and to that of the beta chain with regard to a highly sensitive protease site located approximately 10,000 D from the original terminus contained in the smaller V1 fragment.     

Anti-idiotypic antibodies against an antibody to the platelet glycoprotein (GP) IIb-IIIa complex mimic GP IIb-IIIa by recognizing fibrinogen.

Binding of the adhesive ligand fibrinogen and the monoclonal antibody PAC1 to platelet glycoprotein (GP) IIb-IIIa is dependent on cell activation and inhibited by Arg-Gly-Asp (RGD)-containing peptides. Previously, we identified a sequence in a hypervariable region of PAC1 (mu-CDR3) that mimics the activity of the antibody. Here we examine whether monoclonal antibodies to this idiotypic determinant in PAC1 can mimic GP IIb-IIIa by binding to fibrinogen. Mice were immunized with a peptide derived from the mu-CDR3 of PAC1. Four antibodies were obtained that recognized fibrinogen as well as a recombinant form of the variable region of PAC1. However, they did not bind to other RGD-containing proteins, including von Willebrand factor, fibronectin, and vitronectin. Several studies suggested that these anti-PAC1 peptide antibodies were specific for GP IIb-IIIa recognition sites in fibrinogen. Three such sites have been proposed: two RGD-containing regions in the A alpha chain, and the COOH terminus of the gamma chain (gamma 400-411). Two of the antibodies inhibited fibrinogen binding to activated platelets, and all four antibodies bound to the fibrinogen A alpha chain on immunoblots. Antibody binding to immobilized fibrinogen was partially inhibited by monoclonal antibodies specific for the two A alpha chain RGD regions. However, the anti-PAC1 peptide antibodies also bound to plasmin-derived fibrinogen fragments X and D100, which contain gamma 400-411 but lack one or both A alpha RGD regions. This binding was inhibited by an antibody specific for gamma 400-411. When fragment D100 was converted to D80, which lacks gamma 400-411, antibody binding was reduced significantly (p less than 0.01). Electron microscopy of fibrinogen-antibody complexes confirmed that each antibody could bind to sites on the A alpha and gamma chains. These studies demonstrate that certain anti-PAC1 peptide antibodies mimic GP IIb-IIIa by binding to platelet recognition sites in fibrinogen. Furthermore, they suggest that the gamma 400-411 region of fibrinogen may exist in a conformation similar to that of an A alpha RGD region of the molecule.     

Folding of thermolysin fragments. Correlation between conformational stability and antigenicity of carboxyl-terminal fragments

Circular dichroism (CD) and immunochemical measurements have been used to examine conformational properties of COOH-terminal fragments 121-316, 206-316 and 225(226)-316 of thermolysin, and to compare these properties to those of native thermolysin and thermolysin S, the stable partially active two-fragment complex composed of fragments 5-224(225) and 225(226)-316. In aqueous solution at neutral pH, all the COOH-terminal fragments attain a native-like conformation, as judged both by the content of secondary structure deduced from far-ultraviolet CD spectra and by the recognition of rabbit polyclonal antibodies specific for the COOH-terminal region in native thermolysin. The three fragments showed reversible cooperative unfolding transitions mediated by both heat and guanidine hydrochloride (Gdn X HCl). The phase transition curves were analyzed for Tm (temperature of half-denaturation) and Gibbs free energies (delta GD) of unfolding from native to denatured state. The observed order of thermal stability is 225(226)-316 less than or equal to 206-316 less than 121-316 less than thermolysin S less than thermolysin. The ranking of delta GD values for the three fragments correlates with the size of each fragment. Competitive binding studies by radioimmunoassay using 14C-labeled thermolysin and affinity purified antibodies specific for native antigenic determinants in segment 206-316 of native thermolysin indicate that the COOH-terminal fragments adopt native-like conformations which are in equilibrium with non-native conformations. These equilibria are shifted towards the native state as the fragment size increases from 225(226)-316, to 206-316, to 121-316. Fragment 225(226)-316, when combined with fragment 5-224(225) in the thermolysin S complex, adopts a more stable native-like conformation and becomes much more antigenic. It has been shown that the degree of antigenicity of COOH-terminal fragments towards thermolysin antibodies correlates directly with their conformational stability. The results of this study are discussed in relation to the recently proposed correlation between antigenicity and segmental mobility of globular proteins.     

Characterization of an apparently lower molecular weight gamma-chain variant in fibrinogen Kyoto I. The replacement of gamma-asparagine 308 by lysine which causes accelerated cleavage of fragment D1 by plasmin and the generation of a new plasmin cleavage site

Congenitally abnormal fibrinogen Kyoto I with impaired fibrin monomer polymerization contains a normal gamma-chain and a gamma-chain variant (gamma Kyoto I) that has an apparently lower Mr on sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the Laemmli system (Laemmli, U. K. (1970) Nature 227, 680-685) but migrates with apparently normal Mr in the Weber and Osborn system (Weber, K., and Osborn, M. (1969) J. Biol. Chem. 244, 4406-4412). Reverse-phase high performance liquid chromatographic analyses of the cyanogen bromide or lysyl endopeptidase cleavage fragments of the purified gamma-chains of fibrinogen Kyoto I showed the presence of peptides not seen from normal fibrinogen. Amino acid sequence analysis of these peptides indicated that gamma Asn308 of the gamma-chain variant is replaced by lysine. Purified fragment D1 of fibrinogen Kyoto I also contains two types of D1 gamma-remnants: normal and apparently lower Mr types. Abnormal fragment D1 is cleaved faster to fragments D2 and D3 by plasmin in the presence of [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA) than normal fragment D1, as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by immunoblotting using anti-gamma-chain monoclonal antibody. Analysis of peptides released from fragment D1 by plasmin in the presence of EGTA demonstrated the cleavage of the gamma Lys308-Gly309 bond. Fragment D1 of fibrinogen Kyoto I has normal calcium binding properties. The data suggest that a region or conformation containing gamma Asn308 affects the polymerization of fibrin monomers and that the gamma Asn308----Lys replacement causes a conformational change in the gamma-chain which results in the accelerated cleavage of gamma Lys356-Ala357 and gamma Lys302-Phe303 bonds by plasmin and also results in the generation of a new plasmin cleavage site between Lys308 and Gly309 in the presence of EGTA. During these studies, we found that part of the gamma Lys212-Glu213 bond in fragment D1 is cleaved by plasmin in the presence of EGTA.     

Thrombin binding to the A alpha-, B beta-, and gamma-chains of fibrinogen and to their remnants contained in fragment E

In order to study thrombin interaction with fibrinogen, thrombin binding to fragments D and E (prepared by plasmin digestion of fibrinogen) and to intact S-carboxymethylated chains of fibrinogen (A alpha, B beta, and gamma) was analyzed by autoradiography, immunoblotting, and affinity chromatography. Complex formation was observed between late fragment E and thrombin but not with fragment D. The three reduced chain remnants of fragment E all formed complexes with thrombin. Also, thrombin bound to the intact, separated A alpha, B beta, and gamma chains of fibrinogen as well as to the alpha and beta chains of fibrin. In these experiments the extended substrate-binding site, but not the catalytic-binding site, was being examined because fragment E had as its amino-terminal amino acids Val20 in the alpha chain, Lys54 in the beta chain, and Tyr1 in the gamma chain. Also, thrombin inhibited in its active center by D-phenyl-alanyl-L-prolyl-L-arginine-chloromethyl ketone bound to fragment E and to the separated chains in the same manner as unmodified thrombin. A lysine residue to thrombin was essential for its binding to fibrinogen. Thrombin attached to CNBr-activated Sepharose through its amino groups did not bind to fragment E, but when thrombin was attached through its carboxyl groups, it bound fragment E.     

Conversion of fibrinogen to fibrin: mechanism of exposure of tPA- and plasminogen-binding sites

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.     

Characterization of peptides cleaved by plasmin from the C-terminal polymerization domain of human fibrinogen

The C-terminal region of the fibrinogen gamma chain is known to participate in several functional interactions including fibrin polymerization. This part of the molecule is retained on the gamma chain of fragment D (FgD) when fibrinogen is digested by plasmin in the presence of calcium to produce the fragment D-fragment E (FgD X FgE) complex but is lost if FgD is prepared in the absence of calcium. In an attempt to characterize the C-terminal polymerization domain we have used three techniques to examine this further degradation of FgD following the addition of EDTA and plasmin. Analysis of the digestion by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a progressive cleavage of the gamma chain to two small remnants. The polymerization-inhibitory activity of the whole digest was studied using acid-solubilized fibrin. A progressive loss of inhibitory activity was associated with gamma chain shortening, reaching greater than a 120-fold reduction at the end of digestion. The cleavage of peptides was followed by reverse-phase high performance liquid chromatography and the release of a characteristic peptide triplet was associated with gamma chain cleavage. Manual sequencing, amino acid analysis, and fast atom bombardment mass spectrometry established the three peptides as gamma 303-356, 357-373, and 374-405. These peptides have sequences in common with those peptides recently reported by other investigators to be potent polymerization inhibitors. However, when a mixture of the three peptides was added in a 200-fold molar excess to polymerizing fibrin, no inhibitory activity could be demonstrated. It is concluded that the C-terminal polymerization domain of fibrinogen may be an extended region which includes the sequence gamma 303-405, when this is contiguous with the remainder of the gamma chain.     

A receptor-induced binding site in fibrinogen elicited by its interaction with platelet membrane glycoprotein IIb-IIIa.

The interaction of fibrinogen with membrane glycoprotein GPIIb-IIIa regulates platelet aggregation. This ligand:integrin receptor interaction elicits conformational changes in GPIIb-IIIa as evidenced by the induction of ligand-induced binding sites which are recognized by antibodies that react selectively with the occupied receptor. The dynamic nature of these conformational changes is now demonstrated by the identification and characterization of a receptor-induced binding site (RIBS) elicited in fibrinogen bound to GPIIb-IIIa. A monoclonal antibody to fibrinogen, anti-Fg-RIBS-I, failed to bind to nonstimulated platelets in the presence or absence of fibrinogen. However, when platelets were stimulated with an agonist, the antibody reacted with platelet-bound fibrinogen even in the presence of a marked excess of unbound fibrinogen. A key element of the RIBS epitope has been precisely localized to residues 373-385 of the gamma chain of fibrinogen. Conformational elements also are important in defining the epitope. Fab fragments of the antibody inhibited platelet aggregation. As these fragments also inhibited fibrin polymerization, a commonality between these two diverse functions of fibrinogen in thrombus formation is indicated. In general, antibodies to RIBS and ligand-induced binding site provide unique probes for characterizing ligand:receptor interactions.     

Protective effect of divalent cations in the plasmin degradation of fibrinogen

Calcium limits the plasmic proteolysis of fibrinogen fragment D by binding to a specific site on the carboxy-terminal segment of the D gamma chain. Employing sodium dodecyl sulfate-polyacrylamide gel electrophoresis to visualize plasmic fragments, Sr2+, Ba2+, and Mn2+ were found to have an equivalent capacity to limit the degradation of fibrinogen fragment D (Mr 94,000). Mg2+, Fe2+, Co2+, and Zn2+ did not comparably limit the digestion of fragment D. Equilibrium dialysis demonstrated that Ba2+ competitively inhibited Ca2+ binding to fibrinogen, suggesting that the ions occupied the Ca2+ binding site of fibrinogen and thereby limited the plasmic digestion of fragment D. The results suggest that Ca2+, Sr2+, Ba2+, and Mn2+ limit plasmin digestion of fragment D by interacting with a Ca2+ binding site in the D domain of the fibrinogen molecule.     

Conformational and structural modulation of the NH2-terminal regions of fibrinogen and fibrin associated with plasmin cleavage.

Conformational and structural modulations of the NH2-terminal region of fibrinogen and fibrin associated with plasmin cleavage have been examined utilizing specific antibody probes. The E region derived from the NH2-terminal aspects of fibrinogen undergoes complex structural and conformational changes throughout the cleavage process as indicated by differences in the quantitative and qualitative expression of antigenic determinants by the E region of each isolated cleavage fragment. When the range of antigenic determinants recognized by the antibody probe is limited to a specific molecular marker on the gamma chain within the E region, fg-E-neo, evidence for a systematic and progressive modulation of this site during plasmin cleavage is observed. Fg-E-neo undergoes progressive exposure as the cleavage of fibrinogen proceeds from X to Y to D:E complex. Separation of the D:E complex into its constituent, D and E fragments, is associated with further exposure of fg-E-neo determinants. The sequential cleavage of fibrin by plasmin also leads to progressive exposure of the fg-E-neo site; however, comparison of corresponding fragments derived from fibrinogen and fibrin reveals significant differences in the character of fg-E-neo expression. Immunochemical differences between fibrin and fibrinogen E fragments are not abolished by further exposure of the fragments to plasmin, are apparently not due to the presence or absence of fibrinopeptides, and are maintained following denaturation and renaturation of the fragments. These results suggest that the differential expression of fg-E-neo by the E fragments may be primarily dependent upon differences in amino acid compositions of the fragments.     

Identification of a site in fibrin(ogen) which is involved in the acceleration of plasminogen activation by tissue-type plasminogen activator

The rate of activation of plasminogen by tissue-type plasminogen activator is greatly increased by fibrin, but not by fibrinogen. A possible explanation for this phenomenon could be that conformational changes take place during the transformation of fibrinogen to fibrin which lead to exposure of sites involved in the accelerated plasmin formation. This is also supported by our recent observation that some enzymatically prepared fragments of fibrinogen and fibrin (D EGTA, D-dimer, Y) and also CNBr fragment 2 from fibrinogen have this property. CNBr fragment 2 consists of amino acid residues A alpha (148-207), B beta (191-224) + (225-242) + (243-305) and gamma 95-265, kept together by disulphide bonds. In order to study the localization of a stimulating site within this structure we purified the chain remnants of CNBr fragment 2 after reduction and carboxymethylation, and found that only A alpha 148-207 was stimulating. This was further confirmed by digesting pure A alpha-chains with CNBr and purifying the resulting A alpha-chain fragments. CNBr digests of B beta- and gamma-chains were not stimulatory. The A alpha-chain remnant (residues 111-197) in D EGTA and D-dimer also comprise the major part (residues A alpha 148-197) of the CNBr A alpha-chain fragment. We conclude that a site capable of accelerating the plasminogen activation by tissue-type plasminogen activator preexists in fibrinogen, that this site becomes exposed upon fibrin formation or disruption of fibrinogen by plasmin or CNBr and that this site is within the stretch A alpha 148-197, which is retained in the A alpha-chain remnants of fibrinogen degradation products.     

Anti-(Arg-Gly-Asp-Ser) antibody and its interaction with fibronectin, fibrinogen and platelets

An antibody population recognizing the sequence Arg-Gly-Asp-Ser (RGDS) in fibronectin, anti-(RGDS)N, was isolated by immunoadsorption. Between 2.5% and 4.9% of antibodies were obtained from two different anti-fibronectin sera indicating that this region represents an antigenic epitope in native fibronectin. Complete inhibition of binding of 125I-fibronectin to anti-(RGDS)N was produced only by nonreduced and reduced fibronectin. Fibrinogen and synthetic RGDS tetrapeptide, each at concentration of 10 microM, showed only a slight inhibition of 22% and 17%, respectively. Measurements of the conformational constant, the equilibrium constant for the interconversion of the non-native and native conformations of this epitope, showed that less than 0.0001% of the RGDS molecules adopt the native conformation in aqueous solutions. It indicates that long-range interactions in fibronectin and fibrinogen result in different conformations of the RGDS sequence in both proteins. Anti-(RGDS)N antibodies purified from anti-fibronectin serum had a strong inhibitory effect on thrombin-stimulated platelet aggregation. They also inhibited binding of fibronectin and fibrinogen to thrombin-stimulated platelets, supporting the primary role of the RGDS sequence in the direct interaction of these proteins with platelet membrane receptors.     

Localization of a fibrinogen calcium binding site between gamma-subunit positions 311 and 336 by terbium fluorescence

Calcium is required for effective fibrin polymerization. The high affinity Ca2+ binding capacity of fibrinogen was directly localized to the gamma-chain by autoradiography of nitrocellulose membrane blots of fibrinogen subunits incubated with 45Ca2+. Terbium (Tb3+) competitively inhibited 45Ca2+ binding to fibrinogen during equilibrium dialysis, accelerated fibrin polymerization, and limited fibrinogen fragment D digestion by plasmin. The intrinsic fluorescence of Ca2+-depleted fibrinogen was maximally enhanced by Ca2+ and Tb3+, but not by Mg2+, at about 3 mol of cation/mol of fibrinogen. Protein-bound Tb3+ fluorescence at 545 nm was maximally enhanced by resonance energy transfer from tryptophan (excitation at 290 nm) at about 2 mol of Tb3+mol of fibrinogen and about 1 mol of Tb3+/mol of plasmic fragment D94 (Mr 94,000). Fibrinogen fragments D78 (Mr 78,000) and E did not show effective enhancement of Tb3+ fluorescence, suggesting that the Ca2+ site is located within gamma 303 to gamma 411, the peptide which is absent in fragment D78 but present in D94. When CNBr fragments of the carboxyamidated gamma-subunit were assayed for enhancement of Tb3+ fluorescence, peptide CBi (gamma 311-336) bound 1 mol of Tb3+/mol of CBi. Thus, the Ca2+ site is located within this peptide. The sequence between gamma 315 and gamma 329 is homologous to the calmodulin and parvalbumin Ca2+ binding sites.     

Localization of the binding site on fibrin for the secondary binding site of thrombin

Affinity chromatography of active site inhibited thrombin on immobilized fragments derived from the central (desAB-NDSK) and terminal (D1) globular domains of fibrinogen revealed that the site responsible for the binding of thrombin at its secondary fibrin binding site is located in the central domain. Chromatography of various domains of the central nodule (desAB-NDSK, fibrinogen E, and fibrin E) having nonidentical amino acid sequences showed that all of these fragments are capable of binding to PMSF-thrombin-Sepharose, suggesting that the thrombin binding site resides within the peptide regions common to all of these fragments: alpha(Gly17-Met51), beta(Val55-Met118), and gamma(Tyr1-Lys53). Competitive affinity chromatography of the same binding domains revealed that there is no detectable difference in their binding constants to PMSF-thrombin-Sepharose, indicating that the alpha(Lys52-Lys78), beta(Gly15-Lys54)/(Tyr119-Lys122), and gamma(Thr54-Met78) peptide segments do not contribute significantly to the binding of thrombin. Chromatography of the isolated chains of fibrinogen E showed that the alpha(Gly17-Lys78) peptide region itself contains a strong binding site for PMSF-thrombin-Sepharose. The location of the binding site suggests that the secondary site interaction may play an important role in determining the cleavage specificity of thrombin on fibrinogen and can affect the rate of release of the fibrinopeptides. Affinity chromatography of fragments prepared from polymerized fibrin showed that cross-linked DD (D x D) itself does not bind to thrombin, whereas the D x DE complex remained attached to the column, suggesting that the binding site on fragment E for thrombin is distinct from its binding site for D x D.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction between complementary polymerization sites in the structural D and E domains of human fibrin.

Plasmic degradation products of human fibrin, fragments DD, D, and E, bind to fibrin. It has been inferred from this observation that the binding occurs by attraction of complementary sites located in the NH2- and COOH-terminal domains of the fibrin molecule. The interaction between fragments D1 and E1 has been investigated in this work since it represents the first step in the process of fibrin clot formation. Fragment D1, that was initially as active as fragment DD, lost most of its anticoagulant activity after purification by cation-exchange chromatography. The lability of fragment D1 function explained the previous unsuccessful attempts to form a complex between fragments D1 and E1. The loss of fragment D1 anticoagulant activity was not associated with the cleavage of the gamma 63-85 chain segment, since fragments D1A and D1 identically inhibited the fibrin monomer polymerization rate. In order to demonstrate the formation of a complex between fragments D1 and E1, three lines of experiments were advanced. First, the anticoagulant activity of fragment D1 was neutralized by fragment E1 in a dose-dependent manner, demonstrating that the association between these fragments involved polymerization sites. Second, two products, D1.E1 and D1.E1.D1, were stabilized in a reaction with bifunctional cross-linking reagents, proving the formation of D.E complexes in aqueous solution. Third, immobilized fragment D1 bound fragments E1 and E2, but not fragment E3, showing that fragments E1 and E2 attached via a polymerization site to the complementary one in fragment D1, since this association was disrupted by fibrin polymerization inhibitory peptide GPRP. These results provided direct evidence for specific binding between the structural D and E domains of fibrin mediated through complementary polymerization sites. Thus, the initial formation of fibrin clot fibers appears to be driven by specific association of these sites.     

Bacteroides gingivalis and Bacteroides intermedius recognize different sites on human fibrinogen.

Bacteroides (Porphyromonas) gingivalis and Bacteroides (Porphyromonas) intermedius have been implicated in the etiology of human periodontal diseases. These organisms are able to bind and degrade human fibrinogen, and these interactions may play a role in the pathogenesis of periodontal disease. In attempts to map the bacterial binding sites along the fibrinogen molecule, we have found that strains of B. gingivalis and B. intermedius, respectively, recognize spatially distant and distinct sites on the fibrinogen molecule. Isolated reduced and alkylated alpha-, beta-, and gamma-fibrinogen chains inhibited binding of 125I-fibrinogen to both Bacteroides species in a concentration-dependent manner. Plasmin fragments D and to some extent fragment E, however, produced a concentration-dependent inhibition of 125I-fibrinogen binding to B. intermedius strains but did not affect binding of 125I-fibrinogen to B. gingivalis strains. Radiolabeled fibrinogen chains and fragments were compared with 125I-fibrinogen with respect to specificity and reversibility of binding to bacteria. According to these criteria, gamma chain most closely resembled the native fibrinogen molecule in behavior toward B. gingivalis strains and fragments D most closely resembled fibrinogen in behavior toward B. intermedius strains. The ability of anti-human fibrinogen immunoglobulin G (IgG) to inhibit binding of 125I-fibrinogen to B. intermedius strains was greatly reduced by absorbing the IgG with fragments D. Absorbing the IgG with fragments D had no effect on the ability of the antibody to inhibit binding of 125I-fibrinogen to B. gingivalis strains. A purified staphylococcal fibrinogen-binding protein blocked binding of 125I-fibrinogen to B. intermedius strains but not to B. gingivalis strains.     

Substitution of gamma Arg-275 by Cys in an abnormal fibrinogen, "fibrinogen Osaka II". Evidence for a unique solitary cystine structure at the mutation site

In an abnormal fibrinogen with impaired fibrin monomer polymerization designed as fibrinogen Osaka II, we have identified substitution of Arg by Cys at position 275 of the gamma chain. This Cys is linked to a free cysteine molecule by a disulfide link as evidenced by fast atom bombardment mass spectrometry. This finding was supported by identification of a single cysteine released from isolated abnormal fragment D1 upon reduction. This unique cystine structure at the mutation site has not been reported heretofore in any abnormal protein including fibrinogen. The substitution may well perturb the structure required for fibrin monomer polymerization, specifically that assigned to the carboxyl-terminal D domain of fibrinogen. Indeed, isolated fragment D1 with the Cys substitution failed to inhibit thrombin-mediated clotting of normal fibrinogen and normal fibrin monomer polymerization, while normal fragment D1 inhibited them markedly. Our data seem to provide supporting evidence that the putative polymerization site(s) assigned to the D domain of fibrinogen may be structure-dependent, including the carboxyl-terminal segment of the gamma chain as well as a contiguous region that contains the gamma 275 residue.