Analysis of soluble fibrin complexes by agarose gel chromatography and protamine sulfate gelation

The molecular makeup of soluble fibrin complexes was studied by gel exclusion chromatography using radio-labelling to characterize individual components in protein mixtures. Products of limited plasmin degradation of fibrinogen and mixtures of fibrinogen and “early” fibrinogen digests formed high molecular weight soluble fibrin complexes upon incubation with thrombin. Purified, nonclottable fragment Y did not incorporate into soluble fibrin complexes, nor could we demonstrate incorporation of fragments D and E as previously described from our laboratory. Thus, under the conditions of these experiments, soluble fibrin complexes have two identifiable components, fibrin monomer and clottable fragment X monomer, although incorporation of native fibrinogen or fragment X unreacted by thrombin into soluble fibrin complexes cannot be excluded. Individual fractions of thrombin-treated early fibrinogen digests isolated by agarose gel chromatography were treated with protamine sulfate at 37 °C resulting in precipitation-gelation of greater than 90 per cent of high molecular weight soluble fibrin complexes; whereas, less than 10 per cent of lower molecular weight fibrinogen degradation products precipitated by protamine sulfate. These findings do not support the widely held concept that soluble fibrin complexes incorporate nonclottable degradation products of fibrinogen proteolysis, nor do they support the notion that the so-called paracoagulation reaction induced by protamine sulfate results from the splitting of complexes between fibrin monomer and nonclottable fibrinogen degradation products.     

Analysis of soluble fibrin complexes by agarose gel chromatography and protamine sulfate gelation.

The molecular makeup of soluble fibrin complexes was studied by gel exclusion chromatography using radio-labelling to characterize individual components in protein mixtures. Products of limited plasmin degradation of fibrinogen and mixtures of fibrinogen and "early" fibrinogen digests formed high molecular weight soluble fibrin complexes upon incubation with thrombin. Purified, nonclottable fragment Y did not incorporate into soluble fibrin complexes, nor could we demonstrate incorporation of fragments D and E as previously described from our laboratory. Thus, under the conditions of these experiments, soluble fibrin complexes have two identifiable components, fibrin monomer and clottable fragment X monomer, although incorporation of native fibrinogen or fragment X unreacted by thrombin into soluble fibrin complexes cannot be excluded. Individual fractions of thrombin-treated early fibrinogen digests isolated by agarose gel chromatography were treated with protamine sulfate at 37 degrees C resulting in precipitation-gelation of greater than 90 per cent of high molecular weight soluble fibrin complexes; whereas, less than 10 per cent of lower molecular weight fibrinogen degradation products precipitated by protamine sulfate. These findings do not support the widely held concept that soluble fibrin complexes incorporate nonclottable degradation products of fibrinogen proteolysis, nor do they support the notion that the so-called paracoagulation reaction induced by protamine sulfate results from the splitting of complexes between fibrin monomer and nonclottable fibrinogen degradation products.     

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.     

Incorporation of fragment X into fibrin clots renders them more susceptible to lysis by plasmin

Bleeding, the most serious complication of thrombolytic therapy with tissue-type plasminogen activator (t-PA), is thought to result from lysis of fibrin in hemostatic plugs and from the systemic lytic state caused by unopposed plasmin. One mechanism by which systemic plasmin can impair hemostasis is by partially degrading fibrinogen to fragment X, a product that retains clottability but forms clots with reduced tensile strength that stimulate plasminogen activation by t-PA more than fibrin clots. The purpose of this study was to elucidate potential mechanisms by which fragment X accelerates t-PA-mediated fibrinolysis. In the presence of t-PA, clots containing fragment X were degraded faster than fibrin clots and exhibited higher rates of plasminogen activation. Although treatment with carboxypeptidase B, an enzyme that reduces plasminogen binding to fibrin, prolonged the lysis times of fragment X and fibrin clots, clots containing fragment X still were degraded more rapidly. Furthermore, plasmin or trypsin also degraded clots containing fragment X more rapidly than fibrin clots, suggesting that this effect is largely independent of plasminogen activation. Fragment X-derived degradation products were not preferentially released by plasmin from clots composed of equal concentrations of fibrinogen and fragment X, indicating that fragment X does not constitute a preferential site for proteolysis. These data suggest that structural changes resulting from incorporation of fragment X into clots promote their lysis. Thus, attenuation of thrombolytic therapy-induced fragment X formation may reduce the risk of bleeding.     

Fibrin and plasminogen structures essential to stimulation of plasmin formation by tissue-type plasminogen activator

Plasminogen activation catalysed by tissue-type plasminogen activator (t-PA) has been examined in the course of concomitant fibrin formation and degradation. Plasmin generation has been measured by the spectrophotometric method of Petersen et al. (Biochem. J. 225 (1985) 149-158), modified so as to allow for light scattering caused by polymerized fibrin. Glu1-, Lys77- and Val442-plasminogen are activated in the presence of fibrinogen, des A- and des AB-fibrin and the rate of plasmin formation is found to be greatly enhanced by both des A- and des AB-fibrin polymer. Plasmin formation from Glu1- and Lys77-plasminogen yields a sigmoidal curve, whereas a linear increase is obtained with Val442-plasminogen. The rate of plasmin formation from Glu1- and Lys77-plasminogen declines in parallel with decreasing turbidity of the fibrin polymer effector. In order to study the effect of polymerization, this has been inhibited by the synthetic polymerization site analogue Gly-Pro-Arg-Pro, by fibrinogen fragment D1 or by prior methylene blue-dependent photooxidation of the fibrinogen used. Inhibition of polymerization by Gly-Pro-Arg-Pro reduces plasmin generation to the low rate observed in the presence of fibrinogen. Antipolymerization with fragment D1 or photooxidation has the same effect on Glu1-plasminogen activation, but only partially reduces and delays the stimulatory effect on Lys77- and Val442-plasminogen activation. The results suggest that protofibril formation (and probably also gelation) of fibrin following fibrinopeptide release is essential to its stimulatory effect. The gradual increase and subsequent decline in the rate of plasmin formation from Glu1- or Lys77-plasminogen during fibrinolysis may be explained by sequential exposure, modification and destruction of different t-PA and plasminogen binding sites in fibrin polymer.     

Characterization of the inhibition of fibrin assembly by fibrinogen fragment D.

Fragment D (Mr 100 000) prepared from a terminal plasmin digest of fibrinogen was isolated and used to study its effect on fibrin formation. Increasing amounts of fragment D added to a solution of fibrinogen and thrombin decrease the rigidity of the resultant gel (10% of control at 2 mol of fragment D/mol of fibrinogen). Half-maximal inhibition is achieved at 1 mol of fragment D/mol of fibrinogen for non-cross-linked clots and at 1/2 mol of fragment D/mol of fibrinogen for cross-linked clots. "Clottability' decreases concomitantly with the rigidity. Only small amounts of fragment D (less than 10% for non-cross-linked gels) are incorporated into the gel. Light-scattering shows an increase in the final fibre thickness at fragment D concentrations up to 2 mol of fragment D/mol of fibrinogen, from 60 molecules/cross-section for the control to 120 molecules/cross-section. Higher fragment D concentrations lead to a decrease in the final fibre thickness. The limit fibre thickness is 8 nm, with a length of 80 nm, which is equivalent to a fibrin trimer. On the basis of results of synthetic-substrate and fibrinopeptide-release assays, it is clear that thrombin inactivation is not responsible for this effect. These data suggest that fragment D may inhibit fibrin formation by blocking the bimolecular polymerization of activated fibrin monomer molecules to form protofibrils, although additional effects on subsequent assembly steps may also be involved.     

Interaction of heavy and light chains of plasmin with fibrinogen E and D fragments

It was demonstrated that plasminogen and the plasmin heavy chain form a complex with an immobilized fibrinogen fragment E. The E-fragment interacts, in its turn, with the immobilized heavy chain; this interaction is provided for by the lysin binding sites of the plasminogen molecule. The plasmin light chain having no lysin binding sites is specifically absorbed on the immobilized fragment D, whereas the D-fragment--on the immobilized light chain. The elution is caused by arginine or benzamidine; 6-aminohexanoic acid does not affect this interaction. It is assumed that the interaction of plasminogen and plasmin with fibrin is provided for not only by the lysine binding but also by the benzamidine binding sites of the plasminogen molecule.     

A re-examination of the cleavage of fibrinogen and fibrin by plasmin.

Three Fragment D species (D1, D2, D3) were isolated with time from a plasmin digest of fibrinogen and had molecular weights of 92,999, 86,000 and 82,000 by summation of subunit molecular weights from sodium dodecyl sulfate polyacrylamide gel electrophoresis. Their molecular weights by sedimentation equilibrium ultracentrifugation were 94,000 t87,000, 88,000 to 82, 000, and 76,000 to 70,000 depending on the values calculated for the partial specific volumes. Each of the Fragment D species contained three disulfide-linked subunits derived from the Aalpha, Bbeta, and gamma chains of fibrinogen and differed only in the extent of COOH-terminal degradation of their gamma chain derivatives. Plasmin cleaved Fragment D1 to release the cross-link sites from its gamma' subunit of 38,000 molecular weight; however, the beta' subunit of 42,000 molecular weight and the alpha' subunit of 12,000 molecular weight were resistant to further digestion by plasmin. Fragment D isolated from highly cross-linked fibrin had a dimeric structure due to cross-link formation between the gamma' subunits of two fibrinogen Fragment D species. The molecular weight of fibrin Fragment D was 184,000 by summation of subunit molecular weights and 190,000 to 175,000 by sedimentation equilibrium. Cross-linking the gamma chain, as well as incorporating the site-specific fluorescent label monodansyl cadaverine into the gamma chain cross-link acceptor site, prevented its COOH-terminal degradation by plasmin. Therefore, only one species of fibrin Fragment D, as well as only one species of monodansyl cadaverine-labeled fibrin Fragment D monomer, was generated during plasmin digestion. These results show unequivocally that each fibrinogen Fragment D contains only three subunit chains and therefore the digestion of fibrinogen by plasmin must result in the production of two Fragment D molecules from each fibrinogen molecule. The recently proposed model of fibrinogen cleavage that postulates the generation of a single Fragment D with three pairs of subunit chains from each fibrinogen molecule is incorrect. Incorporation of monodansyl cadaverine into the cross-link acceptor sites of the alpha chain did not alter its cleavage by plasmin detectably. A series of monodansyl cadaverine-labeled peptides, which ranged in molecular weight from 40,000 to 23,000, were cleaved from the alpha chain of monodansyl cadaverine-labeled fibrin monomer during the early stages of plasmin digestion. These peptides were degraded progressively to a brightly fluorescent plasmin-resistant peptide of 21,000 molecular weight and a weakly fluorescent peptide of 2,500 molecular weight. Thus both alpha chain cross-link acceptor sites are contained within a peptide segment of 23,000 molecular weight.     

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.     

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.     

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.     

Factors influencing the structure of terminal plasmin degradation products of human fibrinogen and fibrin

Experiments have been carried out with fibrinogen and with purified degradation products of fibrinogen and fibrin which demonstrate that the structure of D fragments obtained after prolonged plasmin digestion is influenced by several factors in the media. The previously described protective effect of calcium ions on the gamma-chain carboxy-terminals of fibrinogen against attack has been confirmed by working at high plasmin concentrations and/or in the presence of 2 M urea. Several compounds such as EDTA, EGTA, citrate and iminodiacetic acid appear to have a separate effect. In the absence of calcium ions these compounds appear to make the gamma-chain carboxy-terminal ends of the D and D-dimer fragments more susceptible to plasmin digestion. Finally, as demonstrated by experiments with purified D-E complexes from fibrinogen and with whole fibrinogen digests, the E moiety of the D-E complexes appears to be capable of protecting the D moiety against low plasmin concentrations also in the absence of calcium ions.     

Characterization of stromelysin 1 (MMP-3), matrilysin (MMP-7), and membrane type 1 matrix metalloproteinase (MT1-MMP) derived fibrin(ogen) fragments D-dimer and D-like monomer: NH2-terminal sequences of late-stage digest fragments.

Matrix metalloproteinases (MMPs) participate in physiological remodeling of the extracellular matrix. Recently we determined that both fibrinogen (Fg) and cross-linked fibrin (XL-Fb) are substrates for selected MMPs. Specifically, XL-Fb clots were solubilized by MMP-3 (stromelysin 1) by cleavage at gamma Gly 404-Ala 405, resulting in a D-like monomer fragment. Similarly, MMP-7 (matrilysin) and MT1-MMP (membrane type 1 matrix metalloproteinase) solubilized XL-Fb clots. However, the molecular mass of fragment D-dimer, obtained after MMP-7 and MT1-MMP degradation of XL-Fb, is similar to that of fragment D-dimer from plasmin degradation ( approximately 186 kDa). In contrast, fragment D-like monomer, from MMP-3 degradation of both fibrinogen (Fg) and XL-Fb, is similar to fragment D from plasmin degradation of Fg ( approximately 94 kDa). Reduced chains from MMP-3, MMP-7, and MT1-MMP digests of Fg and XL-Fb were subjected to direct sequence analyses and D/D-dimer alpha-chain showed cleavage at both alpha Asp 97-Phe 98 and alpha Asn 102-Asn 103. Degradation of the beta-chain resulted in microheterogeneity of cleavage sites at beta Asp 123-Leu 124, beta Asn 137-Val 138, and beta Glu 141-Tyr 142, whereas all three enzymes cleaved the gamma-chain at gamma Thr 83-Leu 84. In both Fg and XL-Fb, several cleavage sites obtained by proteolysis with MMP-3, MMP-7, and MT1-MMP were found to be in very close proximity to those obtained by plasmin on these same substrates. That does not occur with other MMPs such as MMP-1, -2, and -9 and MT2-MMP. The degradation of XL-Fb by MMPs suggests both plasmin-dependent and independent mechanisms of fibrinolysis that might be relevant in inflammation, angiogenesis, arthritis, and atherosclerosis.     

Significance of tryptophan residues in the D-domain of the fibrin molecule in fibrin polymer formation

The modification of fibrin monomer with H2O2 caused reduction of the association activity of fibrin monomer. The association activity was not reduced even by modification of approx. 16 out of the total 64 tryptophan residues in the fibrin molecule; it was then abolished by further modification of the following several residues. Fragment D obtained by proteolysis of fibrinogen with plasmin, inhibited the association activity of fibrin monomer and the modification of approx. six out of the total 21 tryptophan residues in the fragment led to the complete loss of the inhibitory effect. It was concluded from these studies that about six tryptophan residues in the D-domain of fibrin are important for the association of fibrin monomer.     

Different molecular forms of plasminogen and plasmin produced by urokinase in human plasma and their relation to protease inhibitors and lysis of fibrinogen and fibrin

Urokinase-activated human plasma was studied by gel electrophoresis, gel filtration, crossed immunoelectrophoresis and electroimmunoassay with specific antibodies and by assay of esterase and protease activity of isolated fractions. Urokinase induced the formation of different components with plasminogen+plasmin antigenicity. At low concentrations of urokinase, a component with a K(D) value of 0.18 by gel filtration and post beta(1) mobility by gel electrophoresis was detected. The isolated component had no enzyme or plasminogen activity. In this plasma sample fibrinogen was not degraded for 10h, but when fibrin was formed, by addition of thrombin, fibrin was quickly lysed, and simultaneously a component with a K(D) value of 0 and alpha(2) mobility appeared, which was probably plasmin in a complex with alpha(2) macroglobulin. This complex showed both esterase and protease activity. After gel filtration with lysine buffer of the clotted and lysed plasma another two components were observed with about the same K(D) value by gel filtration as plasminogen (0.35), but beta(1) and gamma mobilities by gel electrophoresis. They appeared to be modified plasminogen molecules, and possibly plasmin with gamma mobility. Similar processes occurred without fibrin at higher urokinase concentrations. Here a relatively slow degradation of fibrinogen was correlated to the appearance of the plasmin-alpha(2) macroglobulin complex. The fibrin surface appeared to catalyse the ultimate production of active plasmin with a subsequent preferential degradation of fibrin and the formation of a plasmin-alpha(2) macroglobulin complex. The gel filtration and electrophoresis of the plasma protease inhibitors, alpha(1) antitrypsin, inter-alpha-inhibitor, antithrombin III, and C(1)-esterase inhibitor indicated that any complex between plasmin and these inhibitors was completely dissociated. The beta(1) and post beta(1) components appear to lack correlates among components occurring in purified preparations of plasminogen and plasmin.     

Characterization of an abnormal fibrinogen Osaka V with the replacement of gamma-arginine 375 by glycine. The lack of high affinity calcium binding to D-domains and the lack of protective effect of calcium on fibrinolysis.

Prolonged thrombin time was completely corrected by the addition of millimolar concentrations of calcium in a new abnormal fibrinogen, Osaka V. Analysis of lysyl endopeptidase digests of A alpha-, B beta-, or gamma-chains by high performance liquid chromatography, and the following amino acid sequence analysis of relevant peptides revealed that about 50% of the gamma-chain has a replacement of gamma-arginine 375 by glycine. When fibrinogen was digested with plasmin in the presence of millimolar concentration of calcium, the amount of fragment D1 was about 50% of the normal control, and the rest was further cleaved to fragment D2, D3, or D62 with an apparent Mr of 62,000. Plasmic digestion of cross-linked fibrin in the presence of calcium resulted in the appearance of an abnormal fragment with an apparent Mr of 123,000 as well as fragments D2, D3, and D62, concomitant with the decrease of D dimer. The gamma-remnant of the abnormal fragment proved to be a cross-linked complex of the normal D1 gamma-remnant and residues 374-406/411 of the abnormal gamma-chain. The number of high affinity Ca(2+)-binding sites for the normal fibrinogen and fibrinogen Osaka V obtained by equilibrium dialysis was 2.88 (about 3) and 1.85, respectively, and that for the abnormal molecules was calculated as 0.9 (about 1) from their relative amounts in the samples, suggesting the lack of two Ca(2+)-binding sites in the D-domains. These data suggest that the normal structure of the COOH-terminal portion of the gamma-chain including residue 375 is required for the full expression of high affinity calcium binding to D-domains, the ability to be protected by calcium against plasmic digestion, and fibrin polymerization. During these studies, we found that the NH2-terminal amino acid of the gamma-remnant in fragments D or D dimer which were obtained after prolonged digestion with plasmin is gamma-Met89.     

Proteolytic activity of the Glu-plasminogen complex with fibrinogen fragment E

Glu-plasminogen interaction with fibrinogen fragment E results in the alteration of its adsorptive capacity. During this interaction in the absence of plasmin and tissue activator of plasminogen, Glu-plasminogen is transformed into a partly degraded form. Glu-plasminogen complexes with soluble and immobilized fibrinogen fragment E. contain a serine proteinase-specific activity which is inhibited by diisopropylfluorophosphate. The complexes under study are active towards fibrin and the plasmin-specific tripeptide substrate, D-Val-L-Leu-L-Lys-p-nitroanilide. It is concluded that fibrinogen fragment E induces structural changes in the enzyme molecule which eventually result in the formation of an active center.     

The course and prerequisites of Lys-plasminogen formation during fibrinolysis

Plasmin-catalyzed modification of the native plasma zymogen Glu1-plasminogen to its more reactive Lys78 form has been shown to be enhanced in the presence of fibrin. The aim of the present work has been to characterize the influence of fibrinopeptide release, fibrin polymerization, and plasmin cleavage of fibrin on the rate of Lys78-plasminogen formation. 125I-Labeled Glu1- to Lys78-plasminogen conversion was catalyzed by performed Lys78-plasmin, or by plasmin generated during plasminogen activation with tissue plasminogen activator or urokinase. The two forms of plasminogen were quantitated following separation by polyacrylamide gel electrophoresis in acetic acid/urea. Plasmin generated by plasminogen activator was monitored by a fixed-time amidolytic assay. The rate of Lys78-plasminogen formation was correlated, in separate experiments, to the simultaneous, plasmin-catalyzed cleavage of 125I-labeled fibrinogen or fibrin to fragments X, Y, and D. The radiolabeled components were quantitated after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The results show that the formation of both bathroxobin-catalyzed des-A-fibrin and thrombin-catalyzed des-AB-fibrin leads to marked stimulation of Lys78-plasminogen formation, whereas inhibition of fibrin polymerization, with Gly-Pro-Arg-Pro, abolishes the stimulatory effect. The rate of Lys78-plasminogen formation varies markedly in the course of fibrinolysis. The apparent second-order rate constant of the reaction undergoes a transient increase upon transformation of fibrin to des-A(B) fragment X polymer and decreases about 10-fold to the level observed during fibrinogenolysis upon further degradation to soluble fragments Y and D.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.