Localization of the alpha-chain cross-link acceptor sites of human fibrin

The potential cross-link acceptor sites of fibrin were specifically labeled with the fluorescent, substitute cross-link donor monodansyl cadaverine (MDC). Several fluorescent alpha-chain peptides generated from enzymatic and cyanogen bromide (CNBr) cleavage of the labeled fibrin were identified by sodium dodecyl sulfate disc gel electrophoresis; they were isolated and then characterized by amino acid analysis, NH2-terminal sequence analysis, and chromatographic and electrophoretic analyses of their digestion products. Ancrod cleavage of MDC-labeled fibrin produced a series of six alpha-chain peptides of molecular weights 34,000 to 12,000, each of which contained an MDC-labeled acceptor site, and an NH2-terminal alpha-chain derivative of molecular weight 37,500. The latter remains disulfide bound in the residual fibrin and has two MDC-labeled sit-s which are separable by CNBr cleavage. Mild plasmin digestion of MDC-labeled fibrin generated fluorescent alpha-chain peptides of molecular weights 45,000, 42,000, 35,000, 23,000, 21,000, and 2,500 in the supernatant and a nonfluorescent NH2-terminal alpha-chain derivative of molecular weight 25,000 which remained in the insoluble residual fibrin. The alignment of these plasmic supernatant peptides was determined from NH2-terminal sequence analyses which indicated that an MDC acceptor site was located at approximately residue 255 of the Aalpha-chain. Cleavage of the MDC-labeled alpha-chain by CNBr, however, localized most of its fluorescence (approximately 80%) to a fragment of molecular weight 29,000 which had the same NH2-terminal sequence as the labeled plasmic peptide of molecular weight 21,000. Both peptides were cleaved by ancrod into two acceptor site-containing peptides of approximately equal fluorescence. The preliminary NH2-terminal sequence analyses of these peptides, when combined with the above findings, indicated that these two other cross-link acceptor sites are in a peptide segment which comprises the middle 17% of the Aalpha-chain.     

Primary structure of human fibrinogen and fibrin. Isolation and partial characterization of chains of fragment D.

Fragment D has been isolated as an apparently single molecular weight species (molecular weight about 100,000) from plasmin digests of humman fibrinogen, using a combination of affinity chromatography on insolubilized "fibrin monomer" and gel filtration. This fragment consists of three chains with molecular weights of 15,000 (Dbeta), 42,500 (Dgamma1) or 39,500 (Dgamma2), and 14,000 (Dalpha) held together by disulfide bonds. The S-carboxymethyl derivatives of the chains have been separated by gel filtration and ion exchange chromatography, and their identity has been confirmed by peptide mapping and immunological analysis. The chain with a molecular weight of 45,000 is a fragment of the Bbeta chain of fibrinogen. The chain derived from the gamma chain of fibrinogen occurred in two molecular forms having molecular weight 42,500 and 39,500. The chain derivative with molecular weight 14,000 is most likely derived from the Aalpha chain of fibrinogen. The chains were characterized by NH2-terminal sequence analysis, amino acid composition, and carbohydrate staining. The two molecular analysis, amino acid composition, and carbohydrate staining. The two molecular forms of the gamma chain appeared to be identical except for an NH2-terminal peptide extension of 23 amino acid residues in the longer chain. The latter has sequences in common with the COOH-terminal part of the gamma chain of the NH2-terminal disulfide knot (BROMBACK, B., BRONDAHL, N. J., HESSEL, B., IWANAGA, S., and WALLEN, P. (1973) J. Biol. Chem. 248, 5806-5820); its NH2-terminal residue being Ala-63 of the gamma chain of fibrinogen.     

Sites of D-domain interaction in fibrin-derived D dimer

We have examined the plasmic digestion products of fibrin formed in the presence of dansylcadaverine, the fluorescent D dimer, to determine whether they are held together not only by the cross-link region on the gamma chain but also by other interactions on the D domain. Antibodies to the D dimer reacted 8X more strongly with sites on the D dimer (purified or in the presence of E) than with sites on fibrinogen or plasmin-digested fibrinogen. The reactivity of this surface site was lost when the gamma chain was cleaved by plasmin after the molecule had been destabilized by the removal of calcium ions, thus breaking the covalent linkage of the homodimer. The noncovalent D dimer retained its dimeric structure by the criteria of molar volume, measured by fluorescence polarization, and molecular sieving. The noncovalently attached, cross-link-containing peptide bound tightly to the parent molecules at higher temperatures but rotated more freely below 15 degrees C, and could be lost from the parent molecules without destroying the dimeric structure. We therefore propose that the forces maintaining the dimeric structure of the noncovalently joined molecule are not solely located at the gamma-chain cross-link region. These other sites on the D domain are therefore candidates for the initial fibrinogen polymerization site and may also have a role in fibrinogen half-molecule assembly.     

Crystallization of fragment D from human fibrinogen.

Fragment D from human fibrinogen has been crystallized. The fragment, which is composed of three disulfide-linked chains (alpha' beta' gamma' = 88,000), was generated with either plasmin or mild trypsin digestion. The crystals diffracted out to 3.5 A; the space group is P2(1), unit cell dimensions a = 108 A, b = 48 A, c = 167 A, beta = 106 degrees. Fragment D was also co-crystallized with the ligand GPRP-amide, in which case the space group is consistent with P212121, unit cell dimensions a = 476 A, b = 82 A, c = 432 A.     

Binding phenomena of isolated unique plasmic degradation products of human cross-linked fibrin.

Proteolysis of human cross-linked fibrin by plasmin results in the formation of a DD . E complex, and Fragments DD and E as the major degradation products. Three species of Fragment E, which differ both in molecular weights (E1, Mr = 60,000; E2, Mr = 55,000; E3, Mr = 50,000) and in charge, have been isolated from a digest of cross-linked fibrin. Each Fragment E species reacts with monospecific anti-E antiserum. Fragments E1 and E2 bind with Fragment DD to form a DD . E complex but Fragment E3 is inactive. This binding is specific since these Fragments E do not bind to fibrinogen or to degradation products of fibrinogen or of noncross-linked fibrin. Fragments E1 and E2 incubated with plasmin are degraded to Fragment E3, suggesting that the three species represent sequential degradation products. Plasmin-treated Fragments E1 and E2 no longer bind with Fragment DD; therefore, it appears that the peptides cleaved from Fragment E2 by plasmin contain or modify the sites responsible for complex formation. On the other hand, Fragment DD binds not only to Fragments E1 and E2, but also to fibrinogen, Fragments X (Stage 1), X (Stage 2), Y, and NH2-terminal disulfide knot, but only after thrombin treatment, suggesting that Fragment DD binds to complementary sites on the NH2-terminal region of fibrinogen which are exposed after thrombin treatment.     

Recombinant human fibrinogen and sulfation of the gamma' chain

Human fibrinogen and the homodimeric gamma'-chain-containing variant have been expressed in BHK cells using cDNAs coding for the alpha, beta, and gamma (or gamma') chains. The fibrinogens were secreted at levels greater than 4 micrograms (mg of total cell protein)-1 day-1 and were biologically active in clotting assays. Recombinant fibrinogen containing the gamma' chain incorporated 35SO4 into its chains during biosynthesis, while no incorporation occurred in the protein containing the gamma chain. The identity of the sulfated gamma' chain was verified by its ability to form dimers during clotting. In addition, carboxypeptidase Y digestion of the recombinant fibrinogen containing the gamma' chain released 96% of the 35S label from the sulfated chain, and the radioactive material was identified as tyrosine O-sulfate. These results clarify previous findings of the sulfation of tyrosine in human fibrinogen.     

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.     

Modification of high molecular weight plasmic degradation products of human crosslinked fibrin.

The predominant high molecular weight products of plasmic digestion of human crosslinked fibrin Fragments DD, E and (DD)E complex were purified by column gel filtration in a non-dissociating buffer or by ion-exchange chromatography on DEAE-cellulose. The structure of the degradation products was studied by proteolytic degradation, polyacrylamide gel electrophoresis immunodiffusion and sucrose density gradient centrifugation. Unaltered derivatives were very resistant to proteolytic degradation by plasmin. In the the presence of 10 mM EDTA the (DD)E complex did not dissociate, but similar to Fragment DD, became susceptible to plasmic degradation forming Fragment D derivatives. The (DD)E complex dissociated in 3 M urea at pH 5.5, had an altered conformation as evidenced by its aggregability and by its increased susceptibility to degradation by plasmin resulting in the formation of Fragment d. The gammagamma chain remnants of Fragment DD were attacked first, followed by cleavage of the beta chain remnants. It is concluded that plasmin resistance is a function of the intact structure and it is not directly dependent on the presence of the crosslink bonds or calcium ions.     

Position of gamma-chain carboxy-terminal regions in fibrinogen/fibrin cross-linking mixtures

There are conflicting ideas regarding the location of the carboxyl-terminal regions of cross-linked gamma-chain dimers in double-stranded fibrin fibrils. Some investigators believe that the chains are always oriented longitudinally along each fibril strand and traverse the contacting ends of abutting fibrin D domains ("DD-long" cross-linking). Other investigations have indicated instead that the chains are situated transversely between adjacent D domains in opposing fibril strands (transverse cross-linking). To distinguish between these two possibilities, the gamma dimer composition of factor XIIIa-cross-linked fibrin/fibrinogen complexes that had been formed through noncovalent D/E interactions between fibrinogen D domains and fibrin E domains was examined. Two factor XIIIa-mediated cross-linking conditions were employed. In the first, fibrin/fibrinogen complexes were formed between (125)I-labeled fibrinogen 2 ("peak 2" fibrinogen), each heterodimeric molecule containing one gamma(A) and one larger gamma' chain, and nonlabeled fibrin 1 molecules ("peak 1" fibrin), each containing two gamma(A) chains. If DD-long cross-linking occurred, (125)I-labeled gamma(A)-gamma(A), gamma(A)-gamma', and gamma'-gamma'dimers in a 1:2:1 ratio would result. Transverse cross-linking would yield a 1:1 mixture of (125)I-labeled gamma(A)-gamma(A) and gamma(A)-gamma' dimers, without any gamma'-gamma' dimers. Autoradiographic analyses of reduced SDS-PAGE gels from protocol 1 revealed (125)I-labeled gamma(A)-gamma(A) and gamma(A)-gamma' dimers at a ratio of approximately 1:1. No labeled gamma'-gamma' dimers were detected. Protocol 2 used a converse mixture, (125)I-fibrin 2 and nonlabeled fibrinogen 1. DD-long cross-linking of this mixture would yield only nonradioactive gamma(A)-gamma(A) dimers, whereas transverse cross-linking would yield a 1:1 mixture of (125)I-labeled gamma(A)-gamma(A) and gamma(A)-gamma' dimers. Autoradiographic analyses of this mixture yielded (125)I-labeled gamma(A)-gamma(A) and gamma(A)-gamma' dimers in a 1:1 ratio. These findings provide no evidence that longitudinal (DD-long) gamma chain positioning occurs in cross-linked fibrin and indicate instead that most, if not all, gamma-chain positioning in an assembled fibrin polymer is transverse.     

The antifibrinolytic action of the D-dimer fragment

The effect of D-dimer on the process of plasmin hydrolysis of unstabilized and crosslinked fibrin has been studied. Less degraded early, intermediate, and late products of fibrin cleavage have been revealed by electrophoresis of reduced and nonreduced samples. The molecular mechanism of antifibrinolytic effect of the D-dimer is supposed to be determined by shielding of peptide regions of monomer fibrin, localized both in N-terminal area of beta chain and in alpha, beta, and gamma chains between D and E domains. A notion has been proposed of autoinhibition of fibrinolytic reaction as a phenomenon related to the physical-chemical regulation of fibrinogen transformation into fibrin.     

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.     

Expression of the fibrinogen genes in rat megakaryocytes

A variety of evidence suggests that megakaryocytes synthesize fibrinogen and comparative immunochemical and structural studies indicate that fibrinogen produced in or associated with megakaryocytes may be different than fibrinogen produced in the liver. Two studies have reported that the gamma' chain, which is produced from the gamma chain gene by alternative splicing, is absent from fibrinogen produced in the megakaryocyte. Since there is only a single gene for each of the three fibrinogen chains the reported structural differences suggest different mechanisms for production of hepatic and megakaryocytic fibrinogen. We have begun an investigation of the varying mechanisms for expression of the fibrinogen genes by examining the structure of fibrinogen mRNA's in the two tissues. Fibrinogen mRNA's of identical length are found in both liver and megakaryocytes. Furthermore, despite the reported absence of the gamma' chain in platelet-associated fibrinogen, we have used a probe specific for the alternative spliced region of the gamma' mRNA to clearly demonstrate this chain in megakaryocyte mRNA. These studies indicate that the gamma' mRNA is either not translated in platelets or that the gamma' chain is unable to associated with the alpha and beta chains to form a mature molecule.     

Plasmic degradation of human fibrinogen. IV. Identification of subunit chain remnants in fragment Y.

A method is presented for detection of cross-linking acceptor sites on fibrinogen chains, using monodansyl-cadaverine labeling in the presence of activated fibrin stabilizing factor, and polyacrylamide electrophoresis in the presence of sodium dodecyl sulfate. Fluorescent gamma-chain monomers and dimers were produced at a considerably faster rate than the labeled alpha-chain derivative. Purified fragments X, Y and D were prepared all from the same plasmic digest of fibrinogen. Following incubation with fibrin stabilizing factor, thrombin and monodansyl-cadaverine, they were reduced with beta-mercaptoethanol and examined by sodium dodecyl sulfate/acrylamide electrophoresis. Three gamma-chains (mol. wts 49 000, 42 000 and 39 000) had reacted with dansyl-cadaverine while no alpha-chain remnant took up the label. Additional protein and carbohydrate staining further facilitated identification of the individual subunit chains. At least three critical peptide bonds, located on alpha, beta- and gamma-chain remnants, must be broken during conversion of fragment Y into D and E. Sequential cleavage results in heterogeneous appearance of reduced subunit chains. As a consequence, there exist several molecular entities of fragment Y, all of which may have the same molecular weight though they represent various products of progressive plasmic digestion. Our results are compatible with the model of asymmetric degradation of fibrinogen, according to which fragment X produces 1 mol of fragment E e and 2 mol of the monomeric fragment D.     

Localization of a fibrin polymerization site

The formation of a fibrin clot is initiated after the proteolytic cleavage of fibrinogen by thrombin. The enzyme removes fibrinopeptides A and B and generates fibrin monomer which spontaneously polymerizes. Polymerization appears to occur though the interaction of complementary binding sites on the NH2-terminal and COOH-terminal (Fragment D) regions of the molecule. A peptide has been isolated from the gamma chain remnant of fibrinogen Fragment D1 which has the ability to bind to the NH2-terminal region of fibrinogen as well as to inhibit fibrin monomer polymerization. The peptide reduces the maximum rate and extent of the polymerization of thrombin or batroxobin fibrin monomer and increases the lag time. The D1 peptide does not interact with disulfide knot, fibrinogen, or Fragment D1, but it binds to thrombin-treated disulfide knot with a Kd of 1.45 X 10(-6) M at approximately two binding sites per molecule of disulfide knot. Fibrin monomer formed either by thrombin or batroxobin binds approximately two molecules of D1 peptide per molecule of fibrin monomer, indicating that the complementary site is revealed by the loss of fibrinopeptide A. The NH2-terminal sequence (Thr-Arg-Trp) and COOH-terminal sequence (Ala-Gly-Asp-Val) of the D1 peptide were determined. Therefore the gamma 373-410 region of fibrinogen contains a polymerization site which is complementary to the thrombin-activated site on the NH2-terminal region of fibrinogen.     

gamma and gamma' chains of human fibrinogen are produced by alternative mRNA processing

cDNAs and the genomic DNA coding for the gamma and gamma' chains of human fibrinogen have been isolated and characterized by sequence analysis. The cDNAs coding for the gamma and gamma' chains share a common nucleotide sequence coding for the first 407 amino acid residues in each polypeptide chain. The predominant gamma chain contains an additional four amino acids on its carboxyl-terminal end (residues 408-411). These four amino acids, together with the 3' noncoding sequences, are encoded by the tenth exon. Removal of the ninth intervening sequence following the processing and polyadenylation reactions yields a mature mRNA coding for the predominant gamma chain. The less prevalent gamma' chain contains 20 amino acids at its carboxyl-terminal end (residues 408-417). These 20 amino acids are encoded by the immediate 5' end of the ninth intervening sequence. This results from an occasional processing and polyadenylation reaction that occurs within the region normally constituting the ninth intervening sequence. Accordingly, the gene for the gamma chain of human fibrinogen gives rise to two mRNAs that differ in sequence on their 3' ends. These mRNAs code for polypeptide chains with different carboxyl-terminal sequences. Both of these polypeptides are incorporated into the fibrinogen molecule present in plasma.     

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.     

The role of gamma A/gamma ' fibrinogen in plasma factor XIII activation

Factor XIII zymogen activation is a complex series of events that involve fibrinogen acting in several different roles. This report focuses on the role of fibrinogen as a cofactor in factor XIII activation by thrombin. We demonstrate that fibrinogen has two distinct activities that lead to an increased rate of factor XIII activation. First, the thrombin proteolytic activity is increased by fibrin. The cleavage rates of both a small chromogenic substrate and the factor XIII activation peptide are increased in the presence of either the major fibrin isoform, gammaA/gammaA fibrin, or a minor variant form, gammaA/gamma' fibrin. This enhancement of thrombin activity by fibrin is independent of fibrin polymerization and requires only cleavage of the fibrinopeptides. Subsequently, gammaA/gamma' fibrinogen accelerates plasma factor XIII activation by a non-proteolytic mechanism. This increased rate of activation results in a slightly more rapid cross-linking of fibrin gammaA and gamma' chains and a significantly more rapid cross-linking of fibrin alpha chain multimers. Together, these results show that although both forms of fibrin increase the rate of activation peptide cleavage by thrombin, gammaA/gamma' fibrinogen also increases the rate of factor XIII activation in a non-proteolytic manner. A revised model of factor XIII activation is presented below.     

A subfraction of fragment D isolated from a plasmin hydrolysate of human fibrinogen.

Fragment D from a 4-hour plasminolysate of human fibrinogen was chromatographed on DEAE-cellulose and a nearly homogeneous subfraction obtained. It migrated as a single band in dodecylsulfate gel electrophoresis. Reduction yielded three peptide chains with approximate molecular weights of 45000, 295000 and 13000 as estimated from the electrophoretic migration rate in dodecylsulfate acrylamide gels. From these data the molecular weight of the Fragment D subfraction was calculated to be ca. 87500. The S-carboxymethylated peptide chains were separated by chromatography on DEAE-cellulose. They were correlated electrophoretically and their amino acid composition was determined. The peptide chains of molecular weight 45000 and 29500 showed a chromatographic microheterogeneity. The subfractions of these two chains, however, were not distinguished by their electrophoretic mobility in dodecylsulfate acry lamide gels and showed only insignificant differences in their amino acid composition.     

Evidence that catalytically-inactivated thrombin forms non-covalently linked dimers that bridge between fibrin/fibrinogen fibers and enhance fibrin polymerization

Phe-pro-arg-chloromethyl ketone-inhibited alpha-thrombin [FPR alpha-thr] retains its fibrinogen recognition site (exosite 1), augments fibrin/fibrinogen [fibrin(ogen)] polymerization, and increases the incorporation of fibrin into clots. There are two 'low-affinity' thrombin-binding sites in each central E domain of fibrin, plus a non-substrate 'high affinity' gamma' chain thrombin-binding site on heterodimeric 'fibrin(ogen) 2' molecules (gamma(A), gamma'). 'Fibrin(ogen) 1' (gamma(A), gamma(A)) containing only low-affinity thrombin-binding sites, showed concentration-dependent FPR alpha-thr enhancement of polymerization, thus indicating that low-affinity sites are sufficient for enhancing polymerization. FPR gamma-thr, whose exosite 1 is non-functional, did not enhance polymerization of either fibrin(ogen)s 1 or 2 and DNA aptamer HD-1, which binds specifically to exosite 1, blocked FPR alpha-thr enhanced polymerization of both types of fibrin(ogen) (1>2). These results showed that exosite 1 is the critical element in thrombin that mediates enhanced fibrin polymerization. Des B beta 1-42 fibrin(ogen) 1, containing defective 'low-affinity' binding sites, was subdued in its FPR alpha-thr-mediated reactivity, whereas des B beta 1-42 fibrin(ogen) 2 (gamma(A), gamma') was more reactive. Thus, the gamma' chain thrombin-binding site contributes to enhanced FPR alpha-thr mediated polymerization and acts through a site on thrombin that is different from exosite 1, possibly exosite 2. Overall, the results suggest that during fibrin clot formation, catalytically-inactivated FPR alpha-thr molecules form non-covalently linked thrombin dimers, which serve to enhance fibrin polymerization by bridging between fibrin(ogen) molecules, mainly through their low affinity sites.     

Incorporation of vitronectin into fibrin clots. Evidence for a binding interaction between vitronectin and gamma A/gamma' fibrinogen.

Vitronectin is an abundant plasma protein that regulates coagulation, fibrinolysis, complement activation, and cell adhesion. Recently, we demonstrated that plasma vitronectin inhibits fibrinolysis by mediating the interaction of type 1 plasminogen activator inhibitor with fibrin (Podor, T. J., Peterson, C. B., Lawrence, D. A., Stefansson, S., Shaughnessy, S. G., Foulon, D. M., Butcher, M., and Weitz, J. I. (2000) J. Biol. Chem. 275, 19788-19794). The current studies were undertaken to further examine the interactions between vitronectin and fibrin(ogen). Comparison of vitronectin levels in plasma with those in serum indicates that approximately 20% of plasma vitronectin is incorporated into the clot. When the time course of biotinylated-vitronectin incorporation into clots formed from (125)I-fibrinogen is monitored, vitronectin incorporation into the clot parallels that of fibrinogen in the absence or presence of activated factor XIII. Vitronectin binds specifically to fibrin matrices with an estimated K(d) of approximately 0.6 microm. Additional vitronectin subunits are assembled on fibrin-bound vitronectin multimers through self-association. Confocal microscopy of fibrin clots reveals the globular vitronectin aggregates anchored at intervals along the fibrin fibrils. This periodicity raised the possibility that vitronectin interacts with the gamma A/gamma' variant of fibrin(ogen) that represents about 10% of total fibrinogen. In support of this concept, the vitronectin which contaminates fibrinogen preparations co-purifies with the gamma A/gamma' fibrinogen fraction, and clots formed from gamma A/gamma' fibrinogen preferentially bind vitronectin. These studies reveal that vitronectin associates with fibrin during coagulation, and may thereby modulate hemostasis and inflammation.