Functional effects of asparagine-linked oligosaccharide on natural and variant human tissue-type plasminogen activator

The role of Asn-linked oligosaccharide in the functional properties of both human tissue-type plasminogen activator (t-PA) and a genetic variant of t-PA was studied. Nonglycosylated and glycosylated wild-type t-PA were produced in mammalian cells which express recombinant t-PA. These proteins were compared in fibrin binding and 125I-labeled fibrin clot lysis assays, using purified components. The nonglycosylated form showed higher fibrin binding, as well as higher fibrinolytic potency than the glycosylated form. Subsequently, prevention of glycosylation of a t-PA variant which lacked the finger and epidermal growth factor domains (delta FE), was carried out in an attempt to enhance its fibrinolytic activity. Glycosylation was prevented by changing Asn to Gln; at Asn-117 to produce delta FE1X t-PA, and at Asn-117, -184, and -448 to produce delta FE3X t-PA. All variants were similar to wild-type t-PA in their catalytic dependence on fibrinogen fragments, fibrinolytic activity in fibrin autography analysis, and plasminogen activator activity. In a clot lysis assay, using citrated human plasma, the fibrinolytic potency of the variants were comparable to that of wild-type t-PA at activator concentrations of 17-51 nM (approximately 1-3 micrograms/ml). At 0.5-5.1 nM (approximately 0.03-0.3 micrograms/ml), however, the variant proteins had lower fibrinolytic potency than wild-type t-PA. Fifty percent lysis in 1.5 h for wild-type, delta FE, delta FE1X, and delta FE3X t-PA, required 2.5, 10, 7.5, and 5.5 nM t-PA, respectively. The fibrinogenolytic activity in human plasma was measured for wild-type, delta FE, delta FE1X, and delta FE3X t-PA, and showed significant fibrinogen depletion after 3 h of incubation at 51 nM, decreasing to 11, 11, 50, and 72% of basal levels, respectively. These data indicate that partial or total nonglycosylated t-PA variants have a higher fibrinolytic versus fibrinogenolytic ratio than their fully glycosylated counterparts.     

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.     

Kinetics of the activation of plasminogen by natural and recombinant tissue-type plasminogen activator

The kinetics of the activation of plasminogen by tissue-type plasminogen activator were studied in the presence and the absence of CNBr-digested fibrinogen as a soluble cofactor. Michaelis-Menten kinetics applied and the kinetic parameters obtained were very similar to those previously reported for the activation in the presence of solid phase fibrin (Hoylaerts, M., Rijken, D. C., Lijnen, H. R., and Collen, D. (1982) J. Biol. Chem. 257, 2912-2919). The affinity of the enzyme for plasminogen dramatically increases in the presence of the soluble cofactor while the catalytic rate constant does not change significantly (KM drops from 83 to 0.18 microM and kcat increases from 0.07 to 0.28 s-1 for tissue-type plasminogen activator of melanoma origin). Fragments containing the lysine-binding sites of plasminogen compete with plasminogen for interaction with CNBr-digested fibrinogen. The dissociation constant of this interaction was found to be 4.5 microM for the high affinity lysine-binding site. No difference was found in the kinetic parameters for the activation of plasminogen by either tissue-type plasminogen activator of melanoma origin or by glycosylated forms of tissue-type plasminogen activator obtained by recombinant DNA technology. The present findings obtained in a homogenous liquid milieu support the previously proposed mechanism of the activation of plasminogen by tissue-type plasminogen activator in the presence of fibrin. This mechanism involves binding of both tissue-type plasminogen activator and plasminogen to fibrin.     

Fibrin‐targeted plasminogen activation by plasminogen activator,PadA, from Streptococcus dysgalactiae

Bacterial plasminogen activators differ from each other in their mechanism of plasminogen activation besides their host specificity. Three‐domain streptokinase (SK) and two‐domain PauA generate nonproteolytic active site center in their cognate partner plasminogen but their binary activator complexes are resistant to α2‐antiplasmin (a2AP) inhibition causing nonspecific plasminogen activation in plasma. In contrast, single‐domain plasminogen activator, staphylokinase (SAK), requires proteolytic cleavage of human plasminogen into plasmin for the active site generation, and this activator complex is inhibited by a2AP. The single‐domain plasminogen activator, PadA, from Streptococcus dysgalatiae, having close sequence and possible structure homology with SAK, was recently reported to activate bovine Pg in a nonproteolytic manner similar to SK. We report hereby that the binary activator complex of PadA with bovine plasminogen is inhibited by a2AP and PadA is recycled from this complex to catalyze the activation of plasminogen in the clot environment, where it is completely protected from a2AP inhibition. Catalytic efficiency of the activator complex formed by PadA and bovine plasminogen is amplified several folds in the presence of cyanogen bromide digested fibrinogen but not by intact fibrinogen indicating that PadA may be highly efficient at the fibrin surface. The present study, thus, demonstrates that PadA is a unique single‐domain plasminogen activator that activates bovine plasminogen in a fibrin‐targeted manner like SAK. The sequence optimization by PadA for acquiring the characteristics of both SK and SAK may be exploited for the development of efficient and fibrin‐specific plasminogen activators for thrombolytic therapy.     

Kinetic studies on novel plasminogen activators. Demonstration of fibrin enhancement for hybrid enzymes comprising the A-chain of plasmin (Lys-78) and B-chain of tissue-type plasminogen activator (Ile-276) or urokinase (Ile-159).

The activation of plasminogen by two novel hybrid enzymes, constructed from the A-chain of plasmin and the B-chains of tissue-type plasminogen activator (t-PA) or urokinase, was compared with the activation by the parent enzymes. Basal kinetic constants for 'Lys-plasminogen' (human plasminogen with N-terminal lysine) and 'Glu-plasminogen' (human plasminogen with N-terminal glutamic acid) activation were similar to those of the parent activators. The Km for plasminogen turnover for both hybrid enzymes was considerably decreased in the presence of both soluble fibrin and a mimic, a CNBr digest of fibrinogen. These enhancements and the related apparent negative co-operativity are similar to the behaviour of t-PA itself. The results are discussed with regard to the molecular features involved in the mechanism of fibrin stimulation.     

Vampire bat salivary plasminogen activator exhibits a strict and fastidious requirement for polymeric fibrin as its cofactor, unlike human tissue-type plasminogen activator. A kinetic analysis.

The vampire bat salivary plasminogen activator (BatPA) is virtually inactive toward Glu-plasminogen in the absence of a fibrin-like cofactor, unlike human tissue-type plasminogen activator (tPA) (the kcat/Km values were 4 and 470 M-1 s-1, respectively). In the presence of fibrin II, tPA and BatPA activated Glu-plasminogen with comparable catalytic efficiencies (158,000 and 174,000 M-1 s-1, respectively). BatPA's cofactor requirement was partially satisfied by polymeric fibrin I (54,000 M-1 s-1), but monomeric fibrin I was virtually ineffective (970 M-1 s-1). By comparison, a variety of monomeric and polymeric fibrin-like species markedly enhanced tPA-mediated activation of Glu-plasminogen. Fragment X polymer was 2-fold better but 9-fold worse as cofactor for tPA and BatPA, respectively, relative to fibrin II. Fibrinogen, devoid of plasminogen, was a 10-fold better cofactor for tPA than fibrinogen rigorously depleted of plasminogen, Factor XIII, and fibronectin; the enhanced stimulatory effect of the less-purified fibrinogen was apparently due to the presence of Factor XIII. By contrast, the two fibrinogen preparations were equally poor cofactors of BatPA-mediated activation of Glu-plasminogen. BatPA possessed only 23 and 4% of the catalytic efficiencies of tPA and two-chain tPA, respectively, in hydrolyzing the chromogenic substrate Spectrozyme tPA. However in the presence of fibrin II, BatPA and tPA exhibited similar kcat/Km values for the hydrolysis of Spectrozyme tPA. Our data revealed that BatPA, unlike tPA, displayed a strict and fastidious requirement for polymeric fibrin I or II. Consequently, BatPA may preferentially promote plasmin generation during a narrow temporal window of fibrin formation and dissolution.     

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.     

Inhibitory effect of angiostatins on activity of the plasminogen/plasminogen activator system

Angiostatins, kringle-containing fragments of plasminogen, are potent inhibitors of angiogenesis. Effects of three angiostatin forms, K1–3, K1–4, and K1-4.5 (0–2 μM), on the rate of native Glu-plasminogen activation by its physiological activators in the absence or presence of soluble fibrin were investigated in vitro. Angiostatins did not affect the intrinsic amidolytic activities of plasmin and plasminogen activators of tissue type (tPA) and urokinase type (single-chain scuPA and two-chain tcuPA), but inhibited conversion of plasminogen to plasmin in a dose-dependent manner. All three angiostatins suppressed Glu-plasminogen activation by tcuPA independently of the presence of fibrin, and the inhibitory effect increased in the order: K1-3 < K1-4 < K1-4.5. The inhibitory effects of angiostatins on the scuPA activator activity were lower and further decreased in the presence of fibrin. Angiostatin K1-3 (up to 2 μM) had no effect, while 2 μM angiostatins K1-4 and K1-4.5 inhibited the fibrin-stimulated Glu-plasminogen activation by tPA by 50 and 100%, respectively. The difference in effects of the three angiostatins on the Glu-plasminogen activation by scuPA, tcuPA, and tPA in the absence or presence of fibrin is due to the differences in angiostatin structures, mechanisms of action, and fibrin-specificity of plasminogen activators, as well as due to the influence of fibrin on the Glu-plasminogen conformation. Angiostatins in vivo, which mimic plasminogen-binding activity, can inhibit plasminogen activation stimulated by various proteins (including fibrin) of extracellular matrix, thereby blocking cell migration and angiogenesis. The data of this work indicate that the inhibition of Glu-plasminogen activation under the action of physiological plasminogen activators by angiostatins can be implicated in the complex mechanism of their antiangiogenic and antitumor action.     

Characterization of a recombinant chimeric plasminogen activator with enhanced fibrin binding

A recombinant chimeric plasminogen activator (GHRP-scu-PA-32K), consisting of the tetrapeptide Gly-His-Arg-Pro fused to the N-terminus of the low-molecular single-chain urokinase-type plasminogen activator (Leu144-Leu411), was produced by expression in CHO cells. The stable expression cell line was selected for large-scale expression. The product was purified by antibody-Sepharose affinity chromatography with a recovery of 67%. The apparent molecular weight of purified GHRP-scu-PA-32K was 33 kDa according to SDS-PAGE. Its specific activity was 150000 IU/mg protein according to fibrin plate determination. The conversion of single-chain to two-chain molecules mediated by plasmin was comparable for GHRP-scu-PA-32K (K(m)=4.9 microM, k(2)=0.35 s(-1)) and scu-PA-32K. The activation of plasminogen by GHRP-scu-PA-32K (K(m)=1.02 microM, k(2)=0.0028 s(-1)) was also similar to that of scu-PA-32K. The fibrin binding of GHRP-scu-PA-32K was 2.5 times higher than that of scu-PA-32K at a fibrin concentration of 3.2 mg/ml. In contrast to scu-PA-32K in vitro 125I-fibrin-labeled plasma clot lysis, GHRP-scu-PA had a higher thrombolytic potency, whereas it depleted less fibrinogen in plasma. These results show that GHRP-scu-PA-32K as expected is a potential thrombolytic agent.     

On the mechanism of fibrin-specific plasminogen activation by staphylokinase

The mechanism of plasminogen activation by recombinant staphylokinase was studied both in the absence and in the presence of fibrin, in purified systems, and in human plasma. Staphylokinase, like streptokinase, forms a stoichiometric complex with plasminogen that activates plasminogen following Michaelis-Menten kinetics with Km = 7.0 microM and k2 = 1.5 s-1. In purified systems, alpha 2-antiplasmin inhibits the plasminogen-staphylokinase complex with k1(app) = 2.7 +/- 0.30 x 10(6) M-1 s-1 (mean +/- S.D., n = 12), but not the plasminogen-streptokinase complex. Addition of 6-aminohexanoic acid induces a concentration-dependent reduction of k1(app) to 2.0 +/- 0.17 x 10(4) M-1 s-1 (mean +/- S.D., n = 5) at concentrations greater than or equal to 30 mM, with a 50% reduction at a 6-aminohexanoic acid concentration of 60 microM. Staphylokinase does not bind to fibrin, and fibrin stimulates the initial rate of plasminogen activation by staphylokinase only 4-fold. Staphylokinase induces a dose-dependent lysis of a 0.12-ml 125I-fibrin-labeled human plasma clot submersed in 0.5 ml of citrated human plasma; 50% lysis in 2 h is obtained with 17 nM staphylokinase and is associated with only 5% plasma fibrinogen degradation. Corresponding values for streptokinase are 68 nM and more than 90% fibrinogen degradation. In the absence of a fibrin clot, 50% fibrinogen degradation in human plasma in 2 h requires 790 nM staphylokinase, but only 4.4 nM streptokinase. These results suggest the following mechanism for relatively fibrin-specific clot lysis with staphylokinase in a plasma milieu. In plasma in the absence of fibrin, the plasminogen-staphylokinase complex is rapidly neutralized by alpha 2-antiplasmin, thus preventing systemic plasminogen activation. In the presence of fibrin, the lysine-binding sites of the plasminogen-staphylokinase complex are occupied and inhibition by alpha 2-antiplasmin is retarded, thus allowing preferential plasminogen activation at the fibrin surface.     

Tissue plasminogen activator and urokinase mediate the binding of Glu-plasminogen to plasma fibrin I. Evidence for new binding sites in plasmin-degraded fibrin I

The effect of tissue plasminogen activator (TPA) or urokinase on the specific binding of human Glu-plasminogen to fibrin I formed in plasma by clotting with Reptilase was studied using 125I-plasminogen and 131I-fibrinogen. In the absence of TPA, small amounts of plasminogen were bound to fibrin I. TPA induced binding of plasminogen to plasma fibrin I that was dependent upon the concentrations of TPA and plasminogen as well as upon the time of incubation. Plasminogen binding occurred in association with fibrin clot lysis and the formation in the clot supernatant of alpha 2-plasmin inhibitor-plasmin complexes. Urokinase also induced binding of plasminogen to plasma fibrin I that was concentration- and time-dependent. The molecular form of plasminogen bound to the fibrin I plasma clot was identified as Glu-plasminogen by dodecyl sulfate-polyacrylamide gel electrophoresis and by fast performance liquid chromatography. Further studies demonstrated that fibrin I formed from fibrinogen that had been progressively degraded by plasmin-bound Glu-plasminogen. The mole ratio of plasminogen bound increased with the time of plasmin digestion. Glu-plasminogen did not bind to fibrin I formed from fibrinogen progressively digested by human leukocyte elastase, thereby demonstrating the specificity of plasmin. These studies demonstrate that plasminogen activators regulate the binding of Glu-plasminogen to fibrin I by catalyzing plasmin-mediated modifications in the fibrin substrate.     

Identification of the mechanism responsible for the increased fibrin specificity of TNK-tissue plasminogen activator relative to tissue plasminogen activator

TNK-tissue plasminogen activator (TNK-t-PA), a bioengineered variant of tissue-type plasminogen activator (t-PA), has a longer half-life than t-PA because the glycosylation site at amino acid 117 (N117Q, abbreviated N) has been shifted to amino acid 103 (T103N, abbreviated T) and is resistant to inactivation by plasminogen activator inhibitor 1 because of a tetra-alanine substitution in the protease domain (K296A/H297A/R298A/R299A, abbreviated K). TNK-t-PA is more fibrin-specific than t-PA for reasons that are poorly understood. Previously, we demonstrated that the fibrin specificity of t-PA is compromised because t-PA binds to (DD)E, the major degradation product of cross-linked fibrin, with an affinity similar to that for fibrin. To investigate the enhanced fibrin specificity of TNK-t-PA, we compared the kinetics of plasminogen activation for t-PA, TNK-, T-, K-, TK-, and NK-t-PA in the presence of fibrin, (DD)E or fibrinogen. Although the activators have similar catalytic efficiencies in the presence of fibrin, the catalytic efficiency of TNK-t-PA is 15-fold lower than that for t-PA in the presence of (DD)E or fibrinogen. The T and K mutations combine to produce this reduction via distinct mechanisms because T-containing variants have a higher K(M), whereas K-containing variants have a lower k(cat) than t-PA. These results are supported by data indicating that T-containing variants bind (DD)E and fibrinogen with lower affinities than t-PA, whereas the K and N mutations have no effect on binding. Reduced efficiency of plasminogen activation in the presence of (DD)E and fibrinogen but equivalent efficiency in the presence of fibrin explain why TNK-t-PA is more fibrin-specific than t-PA.     

Kinetic analysis of the interaction between plasminogen activator inhibitor-1 and tissue-type plasminogen activator.

The kinetics of inhibition of tissue-type plasminogen activator (t-PA) by the fast-acting plasminogen activator inhibitor-1 (PAI-1) was investigated in homogeneous (plasma) and heterogeneous (solid-phase fibrin) systems by using radioisotopic and spectrophotometric analysis. It is demonstrated that fibrin-bound t-PA is protected from inhibition by PAI-1, whereas t-PA in soluble phase is rapidly inhibited (K1 = 10(7) M-1.s-1) even in the presence of 2 microM-plasminogen. The inhibitor interferes with the binding of t-PA to fibrin in a competitive manner. As a consequence the Kd of t-PA for fibrin (1.2 +/- 0.4 nM) increases and the maximal velocity of plasminogen activation by fibrin-bound t-PA is not modified. From the plot of the apparent Kd versus the concentration of PAI-1 a Ki value of 1.3 +/- 0.3 nM was calculated. The quasi-similar values for the dissociation constants between fibrin and t-PA (Kd) and between PAI-1 and t-PA (Ki), as well as the competitive type of inhibition observed, indicate that the fibrinolytic activity of human plasma may be the result of an equilibrium distribution of t-PA between both the amount of fibrin generated and the concentration of circulating inhibitor.     

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.     

A chromogenic assay for the detection of plasmin generated by plasminogen activator immobilized on nitrocellulose using a para-nitroanilide synthetic peptide substrate

A direct solid phase chromogenic assay has been developed for the detection of plasmin (EC 3.4.21.7), generated by the interaction of a nitrocellulose-bound plasminogen activator, using the plasmin specific tripeptide substrate, H-D-valyl-leucyl-lysine - p-nitroaniline. para-Nitroaniline released by the cleavage of the lysine - p-nitroaniline bound by plasmin was derivatized to its diazonium salt and subsequently coupled to N-1-napthylethylenediamine in situ to form a diazoamino of an intense red color at the site of the plasminogen activator. This method was used to assay for the streptococcal plasminogen activator, streptokinase, not only in crude bacterial supernatants, but also to detect streptokinase secreted by individual bacterial colonies. In addition, this solid phase assay was used to identify monoclonal antibodies specific for streptokinase which could inhibit the activation of human plasminogen by streptokinase. This method also permitted simultaneous immunological and biochemical identification of the plasminogen activator, thus permitting unequivocal comparative observations. This assay is quantitative and sensitive to nanogram amounts of activator comparable to those obtained with soluble assays. This method may also be applicable for the detection of other plasminogen activators, such as tissue plasminogen activator, urokinase, and staphylokinase, and also for the detection of immobilized proteases which can cleave other substrates derivatized with p-nitroaniline. The reagents used in this assay are inexpensive and easy to prepare.     

The mechanism of a bacterial plasminogen activator intermediate between streptokinase and staphylokinase

The therapeutic properties of plasminogen activators are dictated by their mechanism of action. Unlike staphylokinase, a single domain protein, streptokinase, a 3-domain (alpha, beta, and gamma) molecule, nonproteolytically activates human (h)-plasminogen and protects plasmin from inactivation by alpha(2)-antiplasmin. Because a streptokinase-like mechanism was hypothesized to require the streptokinase gamma-domain, we examined the mechanism of action of a novel two-domain (alpha,beta) Streptococcus uberis plasminogen activator (SUPA). Under conditions that quench trace plasmin, SUPA nonproteolytically generated an active site in bovine (b)-plasminogen. SUPA also competitively inhibited the inactivation of plasmin by alpha(2)-antiplasmin. Still, the lag phase in active site generation and plasminogen activation by SUPA was at least 5-fold longer than that of streptokinase. Recombinant streptokinase gamma-domain bound to the b-plasminogen.SUPA complex and significantly reduced these lag phases. The SUPA-b.plasmin complex activated b-plasminogen with kinetic parameters comparable to those of streptokinase for h-plasminogen. The SUPA-b.plasmin complex also activated h-plasminogen but with a lower k(cat) (25-fold) and k(cat)/K(m) (7.9-fold) than SK. We conclude that a gamma-domain is not required for a streptokinase-like activation of b-plasminogen. However, the streptokinase gamma-domain enhances the rates of active site formation in b-plasminogen and this enhancing effect may be required for efficient activation of plasminogen from other species.     

Mechanisms of plasminogen activation by mammalian plasminogen activators

Plasminogen activators convert the proenzyme plasminogen to the active serine protease plasmin by hydrolysis of the Arg560-Val561 peptide bond. Physiological plasminogen activation is however regulated by several additional molecular interactions resulting in fibrin-specific clot lysis. Tissue-type plasminogen activator (t-PA) binds to fibrin and thereby acquires a high affinity for plasminogen, resulting in efficient plasmin generation at the fibrin surface. Single-chain urokinase-type plasminogen activator (scu-PA) activates plasminogen directly but with a catalytic efficiency which is about 20 times lower than that of urokinase. In plasma, however, it is inactive in the absence of fibrin. Chimeric plasminogen activators consisting of the NH2-terminal region of t-PA (containing the fibrin-binding domains) and the COOH-terminal region of scu-PA (containing the active site), combine the mechanisms of fibrin specificity of both plasminogen activators. Combination of t-PA and scu-PA infusion in animal models of thrombosis and in patients with coronary artery thrombosis results in a synergic effect on thrombolysis, allowing a reduction of the therapeutic dose and elimination of side effects on the hemostatic system.     

Interaction of plasminogen and fibrin in plasminogen activation

Glu1-, Lys77-, miniplasminogens, kringle 1-3, kringle 1-5A, and kringle 1-5R were able to bind with fibrin, while microplasminogen and kringle 4 did not bind significantly. Kringle 1-5A, but not kringle 1-3, effectively inhibited the binding of Glu1-, Lys77-, and miniplasminogens with fibrin. Miniplasminogen also inhibited the binding of Glu1-plasminogen with fibrin. The binding of kringle 1-3 with fibrin was blocked by mini- or Glu1-plasminogen. It is therefore evident that there are two fibrin-binding domains in plasminogen and that the one in kringle 5 is of higher affinity than that in kringle 1-3. CNBr cleavage products of fibrinogen effectively enhanced the activation of Glu1-, Lys77-, or miniplasminogens, but not microplasminogen, by tissue-type plasminogen activator. Kringle 1-5, but not kringle 1-3, dose-dependently inhibited the enhancement by fibrinogen degradation products of Glu1-plasminogen activation by the activator. Lysine and epsilon-aminocaproic acid could inhibit the binding of plasminogens and plasminogen derivatives with fibrin and block the enhancement effect of fibrinogen degradation products on plasminogen activation. The data clearly illustrate that the binding of plasminogen with fibrin, mainly determined by kringle 5, is essential for effective activation by tissue-type plasminogen activator. However, the presence of kringle 1-4 in the plasminogen molecule is required for the full enhancing effect since the kcat/Km of miniplasminogen activation in the presence of fibrinogen degradation products was 8.2 microM-1 min-1 which is significantly less than 52.0 microM-1 min-1 of Glu1-plasminogen.     

A region of tissue plasminogen activator that affects plasminogen activation differentially with various fibrin(ogen)-related stimulators.

The dissolution of blood clots by plasmin is normally initiated in vivo by the activation of plasminogen to plasmin through the activity of tissue plasminogen activator (t-PA). The rate of plasminogen activation can be stimulated several orders of magnitude by the presence of fibrin-related proteins. Here we describe the kinetic analysis of both recombinant human t-PA (wild-type) and a t-PA variant produced by site-directed mutagenesis in which the original sequence from amino acids 296 to 299, KHRR, has been altered to AAAA. This tetra-alanine variant form of t-PA, K296A/H297A/R298A/R299A t-PA, we refer to as "KHRR" t-PA here. The plasminogen activating kinetics of wild-type t-PA (Activase alteplase) showed a catalytic efficiency which changed over 100-fold dependent on the stimulator in the assay. The lowest rate was in the absence of a stimulator. The following stimulators showed increasing ability to accelerate the catalytic efficiency of the reaction: fibrinogen, fragments of fibrinogen obtained by digestion with plasmin, fibrin, and slightly degraded fibrin. This increase in efficiency was driven primarily by decreases in the Michaelis constant (KM) of the reaction, whereas the catalytic rate constant (kcat) of the reaction did not change significantly. The "KHRR" variant of t-PA displayed novel kinetics with all stimulators tested. In the absence of a stimulator or with the poorer stimulators (fibrinogen and fibrinogen fragments), the KM values of the reaction with Activase alteplase and "KHRR" t-PA were similar. The kcat however, was lower with "KHRR" t-PA than with wild-type t-PA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Slow and fast rat skeletal muscles differ in their plasminogen activator activities

Slow and fast contracting muscles differ in their innervation and electrophysiological properties as well as in their regenerating potentialities. The purpose of the present work was to investigate the expression of plasminogen activators and its possible relation to each type of muscle. Slow (Soleus) and fast (Extensor Digitorum Longus) muscles were obtained from white Wistar rats. Before sectioning the muscles, the euthanized rats were perfused with cold phosphate buffer saline to avoid interference by circulating proteases and inhibitors. Muscle extracts were pounded in an ice-cold Potter tube. Plasminogen activators (PAs) were assayed by fibrin zymography and by both liquid and solid-phase fibrin spectrophotometric assays for the detection of PAs activity. Both urokinase (uPA) and tissue-type plasminogen activator (tPA) activities corresponding to proteins of 38 kDa and 65 kDa molecular masses, were detected in the extracts. Slow muscles contained higher amounts of both activators than fast muscles, but the relative amount of uPA was higher in both types of muscles. In addition, the characteristics of each type of extracts differed somewhat: the fast muscle activity curve was typical of an accelerating process, while the slow muscle curve showed an activity probably related to already formed plasmin or to some other trypsin-like enzyme. These results suggest that the amount of plasminogen activators could be a new criterion of discrimination between slow and fast skeletal muscles.