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Fibrin, but not fibrinogen, enhances the rate of activation of plasminogen by tissue type plasminogen activator (t-PA). Studies with enzymatic and chemical fragments of fibrinogen showed that several sites in fibrinogen are involved in this rate enhancement; these are, A alpha 148-160 (located in CNBr fragment FCB-2), and FCB-5 (a CNBr fragment comprising gamma 312-324), and recently discovered sites in the fibrinogen alpha C domains. All these sites are buried in fibrinogen, but exposed in fibrin and some fibrinogen fragments. For the first two of these, located in the D-domains, this was shown by the fact that monoclonal antibodies against A alpha 148-160 and gamma 312-324 bind to fibrin and rate enhancing fibrin(ogen) fragments, but not to fibrinogen. Direct binding studies indicate that at physiological concentrations plasminogen binds to FCB-2, and t-PA to FCB-5. More detailed studies have demonstrated the importance of residues A alpha-157 and A alpha-152, and that the minimum stretch with rate enhancing properties is A alpha 154-159. The sites in the alpha C domains await further identification. With the recently reported three-dimensional structure of fragments D and D-dimer it is now possible to explain these findings at the molecular level. Molecular calculations and experimental data show that the site A alpha 148-160 in fibrinogen is covered among others by a part of the A alpha chain (A alpha 166-195) that forms an alpha-helix, and by a globular domain formed by the beta-chain. On fibrin formation, the last two may move away, and give access to A alpha 148-160. It is conceivable that in the alpha C domain sites are involved in the early phases of fibrinolysis. The site A alpha 148-160 and that in FCB-5 may be more important at later stages. It is also clear that fibrin polymerization is important. This polymerization has probably several effects: exposure of the rate enhancing sites; mutual positioning of the t-PA and plasminogen binding sites; a concentrating effect of t-PA and plasminogen on the fibrin surface; effects on the kinetic properties of t-PA and plasminogen. These effects together explain the rate enhancement.

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.

Polymerized fibrin strongly enhances tissue plasminogen activator (tPA)-mediated plasminogen activation, concomitant with exposure of 'fibrin-specific' epitopes at 'Aalpha148-160' and 'gamma312-324'. To investigate which aspects of polymerization are involved in these activities, we explored the fibrin polymerization process by evaluating the ability of factor XIIIa-crosslinked fibrinogen polymers to expose 'fibrin-specific' epitopes and enhance plasminogen activation. Crosslinked normal fibrinogen, fibrinogen with deficient [des Bbeta1-42] or defective [Birmingham (AalphaR16H)] fibrin 'D:E' assembly sites ('E(A)'), or with defective end-to-end self-association sites ('D:D') [Cedar Rapids (gammaR275C)], exposed both 'fibrin-specific' epitopes and enhanced tPA-dependent plasminogen activation, whereas non-crosslinked fibrinogens showed minimal or no such activities. Epitope expression in crosslinked fibrinogen was retained in the presence of the fibrin E(A) site peptide homolog, gly-pro-arg-pro (GPRP), which inhibits fibrin D:E association, except for the Aalpha148-160 epitope in des Bbeta1-42 fibrinogen, which was not expressed. Fibrin prepared from crosslinked normal or abnormal fibrinogen, except for the des Bbeta1-42 fibrin epitopes, which were reduced or absent, expressed 'fibrin-specific' epitopes even in the presence of GPRP, which otherwise impairs such expression in non-crosslinked fibrin. Epitope exposure in fibrin prepared from non-crosslinked fibrinogen was nearly normal in Cedar Rapids fibrin (heterozygous D:D defect), but reduced in Birmingham fibrin (heterozygous E(A) defect), nil in des Bbeta1-42 fibrin (E(A) deficient), and absent in all cases in the presence of GPRP. In contrast, plasminogen activation stimulatory activity that had been exposed in crosslinked normal fibrinogen or in crosslinked des Bbeta1-42 or Cedar Rapids fibrin, was preserved to a large extent in the presence of GPRP, suggesting that once enhanced stimulatory activity and epitopes are exposed, they are not completely reversible. The findings indicate that end-to-end intermolecular associations (D:D) are not critical for 'fibrin-specific' epitope exposure, but that polymerization brought about in fibrinogen through factor XIIIa crosslinking, or in fibrin through 'D:E' interactions, is necessary for 'fibrin-specific' (more correctly, 'polymerization-specific') epitope exposure and enhancement of plasminogen activation.

The present paper shows that conformationally changed fibrinogen can expose the sites A alpha-(148-160) and gamma-(312-324) involved in stimulation of the tissue-type plasminogen activator (t-PA)-catalysed plasminogen activation. The exposure of the stimulating sites was determined by ELISA using mABs directed to these sites, and was shown to coincide with stimulation of t-PA-catalysed plasminogen activation as assessed in an assay using a chromogenic substrate for plasmin. Gel permeation chromatography of fibrinogen conformationally changed by heat (46.5 degrees C for 25 min) demonstrated the presence of both aggregated and monomeric fibrinogen. The aggregated fibrinogen, but not the monomeric fibrinogen, has exposed the epitopes A alpha-(148-160) and gamma-(312-324) involved in t-PA-stimulation. Fibrinogen subjected to heat in the presence of 3 mM of the tetrapeptide GPRP neither aggregates nor exposes the rate-enhancing sites. Thus, aggregation and exposure of t-PA-stimulating sites in fibrinogen seem to be related phenomena, and it is tempting to believe that the exposure of stimulating sites is a consequence of the conformational changes that occur during aggregation, or self-association. Fibrin monomers kept in a monomeric state by a final GPRP concentration of 3 mM do not expose the epitopes A alpha-(148-160) and gamma-(312-324) involved in t-PA-stimulation, whereas dilution of GPRP to a concentration that is not longer anti-polymerizing, results in exposure of these sites. Consequently, the exposure of t-PA-stimulating sites in fibrin as well is due to the conformational changes that occur during self-association.

Polymeric fibrin is a strong enhancer of the activation of plasminogen by t-PA. At least two types of sites are involved in this enhancement i.e. a site within A alpha-(148-160), and a site within gamma-(311-379). These sites are not accessible in fibrinogen, but are exposed upon conversion of fibrinogen to fibrin. This explains why fibrinogen has no rate-enhancing properties, and helps to explain the effects of fibrin. Fibrin with its ordered structure appears to exert its rate-enhancing effect by presenting the above sites for interaction with t-PA and plasminogen; thus concentrating and correctly orienting these two reactants on its surface and inducing conformational changes which lead to higher catalytic efficiencies.

A well characterized model of an intact and a degraded surface of fibrin that represents the states of fibrin during the initiation and the progression of fibrinolysis was used to quantitatively characterize the molecular interplay between tissue-type plasminogen activator (t-PA), plasminogen and fibrin. The molecular assembly of t-PA and plasminogen on these surfaces was investigated using combinations of proteins that preclude complications due to side reactions caused by generated plasmin: native plasminogen with di-isopropylphosphofluoridate-inactivated t-PA, and a recombinant human plasminogen with the active-site Ser741 mutagenized to Ala which renders the catalytic site inactive. Under these conditions, neither the affinity nor the maximal number of binding sites for plasminogen were modified by the presence of t-PA, indicating that binding sites for plasminogen pre-exist in intact fibrin and are not dependent on the presence of t-PA. In contrast, when plasminogen activation is allowed, increasing binding of plasminogen to the progressively degraded fibrin surface is directly correlated (r = 0.98) to the appearance of the fibrin E-fragment as shown using a monoclonal antibody (FDP-14) that has its epitope in the E domain of fibrin. t-PA was shown to bind with a high affinity to both the intact (Kd = 3.3 +/- 0.6 nM) and the degraded surface of fibrin (Kd = 1.2 +/- 0.4 nM). Binding of t-PA to carboxy-terminal lysine residues of degraded fibrin was shown to be efficiently competed by physiological concentrations of plasminogen (2 microM), indicating that the affinity of t-PA for these residues was lower than that of plasminogen (Kd = 0.66 +/- 0.22 microM) and unrelated to the high affinity of t-PA for specific binding sites on intact fibrin. These data confirm and establish that the generation of carboxy-terminal lysine residues on fibrin during ongoing fibrinolysis, and the binding of plasminogen to these sites, is an important pathway in the acceleration of clot dissolution.

Cross-linked hybrid oligomers of fibrinogen and fibrin are found in plasma from fibrinaemic patients and normal individuals as well as in preparations of purified human fibrinogen. The present study was undertaken to see if such hybrid oligomers have the same stimulatory effect on tissue plasminogen activator (t-PA) conversion of plasminogen as do polymeric and monomeric fibrin. Hybrid oligomeric fibrin(ogen) material was provided by subjecting purified human fibrinogen to gel filtration in urea-containing buffer at pH 5.6. Well separated fractions of hybrid oligomeric material and monomeric fibrinogen were thus obtained. Some of this material was converted to soluble polymeric or monomeric fibrin using insolubilized thrombin. Hybrid polymeric fibrin, polymeric fibrin or monomeric fibrin were then added to citrated, normal plasma to 2.5 or 5 per cent of the plasma fibrinogen concentration. The added material was kept in solution by plasma fibrinogen. The "COA-SET Fibrin Monomer Test" (Kabi,Stocholm,Sweden), based on the ability of fibrin monomers to enhance t-PA mediated plasminogen-plasmin conversion, was used to compare the potential stimulatory effect of the preparations above. The results led to the following conclusions: 1) Cross-linked, soluble fibrin(ogen) hybrid polymers in a concentration of 5 per cent of plasma fibrinogen concentration (w/w) do not stimulate t-PA. 2) Thrombin conversion of the fibrin-fibrinogen hybrid material resulted in an increase in the rate of t-PA mediated plasminogen conversion, corresponding to the one observed with equivalent (w/w) amounts of fibrin monomers. Compared on a mole to mole basis, fibrin oligomers are more powerful than fibrin monomers as stimulators of t-PA activity.

In previous publications [e.g. Voskuilen, Vermond, Veeneman, Van Boom, Klasen, Zegers & Nieuwenhuizen (1987) J. Biol. Chem. 262, 5944-5946] we have shown that fibrin(ogen) chain fragment A alpha-(148-160) contains a site that contributes to the acceleration of Glu-plasminogen activation by tissue-type plasminogen activator (t-PA). In contrast with fibrin, this peptide, however, does not enhance the rate of mini-plasminogen activation. Therefore, possibly more stimulatory sites than A alpha-(148-160) are present in fibrin. In the present investigation we have localized a possible second type of stimulatory site in the fibrin(ogen) molecule. A whole CNBr digest of fibrinogen was applied to a Bio-Gel P-2 column run in water, pH 4. Two peaks with stimulatory activity were observed, one at the void volume and one between the void volume and the total volume. The former contained the previously described stimulating fragment FCB-2 [which comprises A alpha-(148-160)]; the latter had not been observed before and was characterized further. The stimulating material in the low-M(r) fraction of the Bio-Gel P-2 column was precipitated at pH 8.3 in a virtually pure form. It has a high tryptophan content, and an M(r) of 6500 as assessed by SDS/PAGE. On reduction, a main band of M(r) 2500 is seen, plus a weakly staining band of M(r) 4000. These properties plus the amino acid sequence data identify the fragment as FCB-5. FCB-5 consists of two chains, i.e. gamma-(311-336) and gamma-(337-379), linked by a single disulphide bond between Cys-gamma-326 and Cys-gamma-339. Both these chains and the disulphide bond appear to be essential for rate enhancement. FCB-5 enhances the activation rates of Glu-, mini- and micro-plasminogen, with all five kringles, only kringle V and without kringles respectively. FCB-5 binds t-PA, but none of the plasminogen forms binds to FCB-5. This indicates that the rate enhancements induced by FCB-5 are due to an effect on t-PA.

The sequence fibrinogen-A alpha-(148-160) can mimic part of the fibrin-induced rate enhancement of the activation of plasminogen by tissue-type plasminogen activator. Previously we have reported that the lysine residue at position A alpha-157 is crucial. During our further investigations on A alpha-157 we found that lysine at position A alpha-157 may be replaced by glutamic acid. This unexpected finding prompted us to re-investigate the requirements of this position. We prepared analogues of A alpha-(148-160) in which the lysine residue at position A alpha-157 was replaced by lysine derivatives (acetyl-lysine, benzyloxycarbonyl-lysine and methanesulphonylethyloxycarbonyl-lysine), acidic residues (aspartic acid and glutamic acid), basic residues (arginine and ornithine), polar residues (glutamine and methanesulphonylethyloxycarbonylornithine), apolar residues (alanine, valine, norleucine and glutamic acid 4-nitrobenzyl ester) and glycine. These analogues were tested for their stimulatory activity. When aspartic acid, glutamic acid 4-nitrobenzyl ester or norleucine is present at position A alpha-157 in A alpha-(148-160) virtually all stimulatory capacity is lost. With valine at position A alpha-157 the stimulatory activity is marginal. None of the other replacements at position A alpha-157 caused loss of rate-enhancing properties. From these results we conclude that for the rate-enhancing effect of A alpha-(148-160) the side chain of the amino acid residue at position A alpha-157 must fulfill certain requirements: there must be one (as in alanine) or no (as in glycine) carbon atom in the side chain, or at least two carbon atoms and a polar group (charged or uncharged) to which a rather bulky group (such as the benzyloxycarbonyl group) or a polar group (such as the methanesulphonylethyloxycarbonyl group) may be attached. The highest activity [even higher than native A alpha-(148-160)] was obtained with ornithine, methanesulphonylethyloxycarbonylornithine or methanesulphonylethyloxycarbonyl-lysine at position A alpha-157.

The rate of activation of plasminogen by tissue-type plasminogen activator (t-PA) is greatly increased by fibrin, but much less by fibrinogen. Fibrin(ogen) fragments such as the fibrin(ogen) cyanogen bromide fragment FCB-2 and FCB-5, and a synthetic peptide with the sequence of fibrinogen A alpha-(148-160), a constituent of FCB-2, also have rate-enhancing properties. In order to find a possibly smaller, still stimulating site within A alpha-(148-160) we synthesized successive linear amino-terminally acylated hexapeptides [i.e. A alpha-(148-153), A alpha-(149-154)'d, .... A alpha-(155-160)] from the sequence A alpha-(148-160). The only hexapeptide within the sequence A alpha-(148-160) capable of enhancing the rate of plasminogen-to-plasmin conversion by t-PA appears to be the amino-terminally acylated peptide comprising the sequence A alpha-(154-159). This peptide enhances the plasminogen activation rate six-fold; half-maximal activation rate is reached at a peptide concentration of 56 microM.

Fibrin, not fibrinogen, enhances the rate of tPA catalysed plasminogen activation. In earlier studies we have shown that a site involved in this rate enhancement is located in a tridecapeptide, i.e. fibrinogen A alpha-(148-160). This sequence comprises a special charge distribution in which a stretch with alternating neutral and acidic amino acids is embraced by basic amino acids. In this study we found that the disruption of charge distribution as caused by replacing valine 152 by other (charged and/or polar) amino acids leads to loss of rate-enhancing capacity. Also lysine at position A alpha-157 was replaced by lysine derivatives and other amino acids. We found that the side chain of the amino acid at position A alpha-157 must contain no (as in glycine) or one carbon atom without substitution (alanine). When the side chain contains two or more carbon atoms, there should also be a polar group in the side chain. We also synthesized a series of hexapeptides covering the sequence of A alpha-(148-160), and found that only A alpha-(154-159) is stimulatory, notwithstanding the fact that the peptides A alpha-(152-157), A alpha-(153-158) and A alpha-(155-160) also contain lysine A alpha-157. We conclude that the shortest peptide with stimulation activity is A alpha-(154-159); that the charge distribution in A alpha-(148-160) is important; that it is not lysine A alpha-157 per se that is crucial, but rather the properties and orientation of the side chain of A alpha-157.

Tissue activator-mediated plasminogen activation is potentiated both by fibrin and by some soluble fibrin(ogen) fragments. The potentiating effect of the different fragments decreases in the order fibrin monomer greater than D-dimer greater than Y greater than D EGTA greater than Dcate greater than X. Fibrinogen and the fragments Ecate, E EGTA and E fibrin have almost no effect. The existence of a fibrin polymer is apparently not a prerequisite for this potentiating effect. The plasminogen activation by various urokinase preparations is not potentiated by fibrin and fibrin(ogen) fragments.

The activation rate of plasminogen by tissue-type plasminogen activator can be increased by fibrin(ogen) fragments. There is a remarkable difference in the effect of these fragments on the stimulation of 1-glu-plasminogen activation and 442-val-plasminogen (mini-plasminogen) activation. Fibrin monomer as well as plasmic fragments Y, D EGTA and D-dimer have a stimulating effect on both 1-glu-plasminogen and 442-val-plasminogen activation, whereas cyanogen bromide fragment FCB2 stimulates only the activation of 1-glu-plasminogen. Results indicate that two types of sites may be operational in fibrin and fibrin(ogen) fragments Y, D EGTA and D-dimer. One type of site (FCB2-related) interacts probably with plasminogen and may be dependent on the kringle 1-4 region; the other type of site probably interacts either with plasminogen in a non-kringle 1-4 region-dependent manner or with tissue-type plasminogen activator.

Activation of human plasminogen by human tissue-type plasminogen activator (t-PA) is accelerated in the presence of cyanogen bromide digests of human fibrin(ogen). In the present study a possible species specificity of this phenomenon was investigated. All combinations of the plasminogens, fibrin monomers and cyanogen bromide digests of the fibrinogens of man, pig, rat, cat and monkey (Macaca mulatta), and three t-PA species (man, rat and pig) were studied. No species differences were noted with the fibrin monomers i.e. the activation rate of all five plasminogens increased more than 20-fold in the presence of all five fibrin monomer species, irrespective if man, rat or pig t-PA was used. However, we found that species specificities come to expression when cyanogen bromide digests of the corresponding fibrinogens were used as accelerators. Our results indicate that the plasminogen species and not the source of t-PA or fibrinogen dictates if accelerated activation occurs in the presence of a fibrinogen CNBr digest. The plasminogens can be roughly divided in two groups: --One group, comprising human, monkey and cat plasminogen, which are activated at a higher rate by all three t-PA species in the presence of fibrinogen digest independent on the fibrinogen species from which the digest was prepared. --Another group, comprising pig and rat plasminogen, which is not or only marginally more quickly activated by the 3 t-PA species, irrespective of the fibrinogen species from which the CNBr digest was prepared.

At least four molecular forms of plasminogen are known. Two of those forms have glutamic acid at their amino-terminal end, and are designated as glu-plasminogen. The other two have lysine, methionine and/or valine as amino-terminal amino acid and are collectively designated as lys-plasminogen. Two subforms (I and II) each of glu- and lys-plasminogen exist. The I-forms are glycosylated at asn-288 and thr-345, whereas the II-forms are only glycosylated at thr-345. In a previous publication (Thromb Haemostas 1984; 52: 347-349) we have described the separation of the I- and II-forms of plasminogen in lysine-Sepharose in phosphate buffers. Now we have combined those findings with the differential affinity of glu- and lys-plasminogen for aminohexyl-Sepharose through their aminohexyl-sites, recently described by Christensen (Biochem J 1984; 223: 431-421). Acid/urea electrophoresis, end-group determination and carbohydrate analysis show that the combination of affinity chromatography on lysine-Sepharose in phosphate buffers, and on aminohexyl-Sepharose provides an efficient procedure to separate the four molecular forms of plasminogen.

In the present work we have determined Km,app and kcat,app values for tissue-type plasminogen-activator-catalyzed activation of Glu-plasminogen, Lys-plasminogen and mini-plasminogen in the absence and in the presence of fibrinogen-derived fragments. These were CNBr fragment 2, the A alpha chain remnant of CNBr fragment 2 (A alpha 148-207) and plasmin-generated fragment D-EGTA. The time course of plasmin formation from the various types of plasminogen (plg) was measured spectrophotometrically in a coupled assay system where D-valyl-L-leucyl-L-lysine p-nitroanilide served as a plasmin substrate. The kinetic constants are summarized as follows. (Values in parentheses are concentrations at which the minimum Km,app and maximum kcat,app value is reached.) (Table: see text). In conclusion our results show that CNBr fragment 2, A alpha 148-207 and to some extent D-EGTA mimic the accelerating effect of fibrin. The first two of these fragments did not accelerate activation of mini-plasminogen, lacking the kringle structures I-IV. This suggests that the stimulating effects of these two fragments were dependent on the presence of kringles I-IV of the plasminogen molecule.

Native human plasminogen, the proenzyme of plasmin (E.C. 3.4.21.7) occurs in blood in two well defined forms, affinity forms I and II. In this paper, the feasibility of separating these forms of human native plasminogen by affinity chromatography, is shown to be dependent on two factors: 1) the ionic composition of the buffer containing the displacing agent: buffers of varying contents of sodium, Tris, phosphate and chloride ions were compared, and 2) the type of adsorbent. Two adsorbents were compared: Sepharose-lysine and Sepharose-bisoxirane-lysine. Only in the phosphate containing buffers, irrespective of the type of adsorbent, the affinity forms can be separated. The influence of the adsorbent can be accounted for by a large difference in dissociation constants of the complex between plasminogen and the immobilized lysine.