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.     

Comparative evaluation of fibrinogen and fibrin hydrolysis with plasmin

A high-sensitive method is developed for determining the degree of plasmin-catalyzed fibrinogen hydrolysis by the released amino groups stained with trinitrobenzene sulphoacid. The method permits determining 0.02-0.08 casein units of plasmin. The method made it possible to establish that after streptokinase activation plasmin hydrolyzes equally fibrinogen and fibrin in solution and as gel. When a tissue activator is used, fibrin intensifies significantly the plasminogen activation. Inhibition of plasmin by an inhibitor produced from soya is considerably slowed down in fibrin gel.     

Inhibition of plasmin by fibrinogen.

The kinetics of inhibition of the amidolytic activity of plasmin on D-Val-L-Leu-L-Lys p-nitroanilide hydrochloride (S-2251) by fibrinogen and fibrin were determined. Reciprocal (1/v versus 1/[S]) plots of plasmin inhibition by 0.50 microM-fibrinogen showed a non-linear downward curve. The Hill coefficient (h) was 0.68, suggesting negative co-operativity. By contrast, fibrin produced a simple competitive inhibition of plasmin (Ki = 12 micrograms/ml). Addition of 0.1 mM-6-aminohexanoic acid shifted the non-linear curve obtained in the presence of fibrinogen to a straight line as for controls, indicating that 6-aminohexanoic acid abolishes the fibrinogen-induced inhibition. Transient exposure of the enzyme to pH 1.0 abrogates the ability of fibrinogen to inhibit plasmin activity. Acidification had no effect on the Vmax but increased the Km of plasmin. The present evidence for modulation of plasmin reveals a novel mechanism for control of fibrinolysis by fibrinogen, a component of the coagulation system and the precursor of the physiological substrate of plasmin.     

Structure-activity relationships of vasoactive peptides derived from fibrin or fibrinogen degraded by plasmin

The amino acid sequences of three vasoactive peptides which we previously isolated from fibrin(ogen) degraded by plasmin (EC 3.4.21.7) have been determined. Each peptide originates from a different chain of the fibrinogen molecule. Comparison with other studies shows that the two peptides having the permeability-increasing effect arise from regions of the fibrinogen' molecule that are easily accessible to plasmin attack. The third peptide, which has a slight vasoconstrictor activity, is part of fragment E released from the fibrinogen molecule after plasmin degradation. The two peptides having permeability-increasing effect lose their carboxyl-terminal lysine after interaction with carboxypeptidase B (EC 3.4.12.3) with a concomitant loss of activity. One of these peptides (6A) is resistant to tryptic digestion, while the second peptide (6D) is easily split into two inactive fragments. Complete deamination or modification of the free amino groups (carbamylation, methylation) almost completely abolishes their activity, whereas selective modification of only the free ϵ-amino groups of lysine does not. Modification of the Trp residue in one of these peptides (6D) increased its activity. These findings show that a carboxylterminal lysine with a free ϵ-amino group as well as an unblocked N-terminal residue are essential for their activity. Proline, which is situated near or at the middle of both peptides, is also apparently essential indicating that a specific conformation might be required for physiological activity.     

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.     

Fibronectin decreases the stimulatory effect of fibrin and fibrinogen fragment FCB-2 on plasmin formation by tissue plasminogen activator.

Fibronectin is a dimeric glycoprotein (Mr 440,000) involved in many adhesive processes. During blood coagulation it is bound and cross-linked to fibrin. Fibrin binding is achieved by structures (type I repeats) which are homologous to the "finger" domain of tissue plasminogen activator. Tissue plasminogen activator also binds to fibrin via the finger domain and additionally via the "kringle 2" domain. Fibrin binding of tissue plasminogen activator results in stimulation of its activity and plays a crucial role in fibrinolysis. Since fibronectin might interfere with this binding, we studied the effect of fibronectin on plasmin formation by tissue plasminogen activator. In the absence of fibrin, fibronectin had no effect on plasminogen activation. In the presence of stimulating fibrinogen fragment FCB-2, fibronectin increased the duration of the initial lag phase (= time period until maximally stimulated plasmin formation occurs) and decreased the rate of maximal plasmin formation which occurs after that lag phase mainly by increasing the Michaelis constant (Km). These effects of fibronectin were dose-dependent and were similar with single- and two-chain tissue plasminogen activator. They were also observed with plasmin-pretreated FCB-2. An apparent Ki of 43 micrograms/ml was calculated for the inhibitory effect of fibronectin when plasminogen activation by recombinant single-chain tissue plasminogen activator was studied in the presence of 91 micrograms/ml FCB-2. When a recombinant tissue plasminogen activator mutant lacking the finger domain was used in a system containing FCB-2, no effect of fibronectin was seen, indicating that the inhibitory effect of fibronectin might in fact be due to competition of fibronectin and tissue plasminogen activator for binding to fibrin(ogen) via the finger domain.     

Characterization of the terminal degradation products of canine fibrinogen by plasmin.

Fibrinogen, isolated from canine plasma by the successive procedures of (1) freezing and thawing, (2) fractional precipitation with 25% saturated (HN4)2SO4 and (3) Sepharose 6B gel-filtration, had a molecular weight of 282 000 by the rapid sedimentation equilibrium method. However, a molecular weight for canine fibrinogen of 332 000, which is closer to that reported for human and bovine fibrinogens (340 000 plus or minus 20 000), was obtained from the sum of the molecular weights of the Aalpha, Bbeta and gamma chains, determined from dodecylsulfate gel electrophoretic patterns of reduced fibrinogen. Canine fibrinogen, subjected to proteolysis by urokinase-activated plasminogen for 24 h, contained degradation fragments D and E which were isolated by starch block electrophoresis and Sephadex G-200 gel-filtration. The purified D and E fragments with sedimentation coefficients of 5.0 S and 2.5 S had weight average molecular weights of 89 000 and 42 000, respectively by the rapid sedimentation equilibrium method. The ratio of D to E was 2:1 per parent fibrinogen molecule. Antigenic analysis according to anti-fibrinogen antiserum showed that both D and E fragments were antigenically deficient to native fibrinogen and revealed a reaction of non-identity with each other. Upon immunoelectrophoresis at pH 8.2, D and E had different electrophoretic mobilities. Preliminary studies indicate that based on thrombin time alone, D has anticoagulant activity while E appears to be a coagulation potentiator. Canine fibrinogen apparently consist of two core fragments with dissimilar chemical characteristics in common with the fundamental structures of human and bovine fibrinogens.     

A method for determining plasmin by the rate of fibrin gel lysis

A simple and sensitive method has been developed to assess the fibrinolytic activity of plasmin from the change in the column height of fibrin gel. Two conditions were used: 1) 37 degrees C and 16 h incubation at plasmin concentrations of 0.5-50 micrograms/ml and 2) 25 degrees C and 1-2.5 h incubation at plasmin concentrations of 50-1000 micrograms/ml. The method permits to observe the kinetics of fibrinolysis at plasmin concentrations higher that 10 micrograms/ml. The results have shown that the method is applicable for quantitation of plasminogen in human plasma. The method is precise and well reproducible.     

Terminal plasmin degradation products D and E of duck fibrinogen and their effect on polymerization of fibrin

Duck fibrinogen (Mr 320 000) treated with streptokinase-activated human plasminogen in the presence of calcium ions was hydrolysed to terminal core fragments D and E. They were isolated from the digest by: (1) ion-exchange chromatography on DEAE-cellulose, (2) gel filtration on Sephadex G-100, and (3) affinity chromatography with the use of fibrin monomers coupled to CNBr-activated Sepharose. When the native D fragment, D1 was additionally digested by plasmin in the presence of EDTA, more degraded forms D2 and D3 appeared. Molecular weight of D1, D2, D3 and E estimated on SDS-polyacrylamide gel electrophoresis is 100 000, 89 000, 80 000 and 50 000, respectively. It was found that after reduction with 2-mercaptoethanol the fragments D1 and D3 consisted each of three polypeptide chains: alpha, beta, gamma: the gamma-chain of D3 remnant was more degraded (Mr 24 000) as compared with the gamma-chain of D1 remnant (Mr 42 000). Polymerization of both duck and pig fibrin monomers was inhibited by fragments D1 but not by D3.     

The role of DL and DH degradation products in the reactions of the plasmin hydrolysis of fibrinogen and fibrin]

The mechanism of effect of plasmin hydrolysis degradation products, fragments DL and DH, on fibrinolysis and fibrinogenolysis processes was investigated on the basis of their various structural and functional characteristics. Using electrophoresis of unreduced samples and degradation products concentrations changing kinetics, DH was shown to be the only fragment which possessed an antifibrinolytic effect. Rauleigh's light scattering analysis indicated the ability of fragments DL and DH to co-form with plasminogen reversible equimolar complexes.     

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.     

Platelet fibrinogen. Identity and initial observations on the mode of its degradation by plasmin.

Fibrinogen has been purified from human platelets. Platelet fibrinogen exhibits a characteristic pattern in agar gel immunoelectrophoresis different from that of plasma fibrinogen. Stepwise plasmin degradation has been used in further elucidation of the molecular properties of the platelet protein. Examination of comparative digests by immunologic and gel electrophoretic methods has revealed that (1) the platelet protein is more resistant to plasmin degradation, (2) the plasmin-produced fragments of platelet fibrinogen differ consistently from those of its plasma counterpart, and (3) platelet fibrinogen is different from fragment X of plasma fibrinogen. It is suggested that platelet fibrinogen may contribute to the stability of the thrombus.