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.     

Replacement of finger and growth factor domains of tissue plasminogen activator with plasminogen kringle 1. Biochemical and pharmacological characterization of a novel chimera containing a high affinity fibrin-binding domain linked to a heterologous protein.

A novel triple-kringle plasminogen activator protein, PK1 delta FE1X, has been produced which is a genetic chimera between the fibrin binding kringle 1 domain of plasminogen and the two kringles and serine protease domains of naturally occurring wild-type tissue plasminogen activator (wt t-PA). This chimera also contains a modification to prevent high mannose type N-linked glycosylation on kringle 1 of t-PA. PK1 delta FE1X is biochemically and fibrinolytically similar to wt t-PA in vitro but retains the decreased plasma clearance rate characteristic of other t-PA variants which lack fibronectin finger-like and epidermal growth factor domains. The serine protease domain of PK1 delta FE1X exhibits the amidolytic activity characteristic of wt t-PA. In an indirect coupled plasminogen activator assay, the specific activity of PK1 delta FE1X is approximately 1.4 times greater than that of wt t-PA. In a fibrin film-binding assay, greater binding to untreated fibrin is observed with wt t-PA than with PK1 delta FE1X. However, following limited plasmin digestion of the fibrin film, PK1 delta FE1X binding increases to the level observed with wt t-PA. The incremental binding to plasmin-digested fibrin observed with PK1 delta FE1X is eliminated if plasmin digestion of the fibrin film is followed by carboxypeptidase B treatment. This result suggests that plasminogen kringle 1 binds plasmin-digested fibrin even after recombination with a heterologous protein. The fibrinolytic activity of PK1 delta FE1X in human plasma clot lysis assays was similar to that of wt t-PA at activator concentrations of approximately 1 microgram/ml. At substantially lower concentrations, approximately 0.1 microgram/ml, PK1 delta FE1X was only slightly less active than wt t-PA. Pharmacokinetic analysis showed that wt t-PA activity is cleared approximately 15 times as rapidly as PK1 delta FE1X following intravenous bolus injection. In a rabbit jugular vein clot lysis model, intravenous bolus injection of 0.06 mg/kg of PK1 delta FE1X showed greater thrombolytic potency than a similar administration of 0.5 mg/kg of wt t-PA. Thus it appears that in vitro exon shuffling techniques can be used to generate novel fibrinolytic agents which biochemically and pharmacologically represent the combination of individual domains of naturally occurring proteins.     

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.     

Characterization of a chimeric plasminogen activator consisting of amino acids 1 to 274 of tissue-type plasminogen activator and amino acids 138 to 411 of single-chain urokinase-type plasminogen activator

A chimeric plasminogen activator (t-PA/scu-PA-s), consisting of amino acids 1-263 of tissue-type plasminogen activator (t-PA) and 144-411 of single-chain urokinase-type plasminogen activator (scu-PA), was previously shown to maintain the enzymatic properties of scu-PA but to have only partially acquired the fibrin affinity of t-PA, possibly as a result of steric interaction between the functional domains of t-PA and scu-PA (Nelles, L., Lijnen, H. R., Collen, D., and Holmes, W.E. (1987) J. Biol. Chem. 262, 10855-10862). Therefore, we now have constructed an extended chimeric t-PA/scu-PA protein, consisting of amino acids 1-274 of t-PA and 138-411 of scu-PA, which thus has an additional sequence of 17 residues in the region joining the two proteins. The highly purified extended chimeric protein (t-PA/scu-PA-e) was found to have similar specific activity on fibrin film (65,000 IU/mg), kinetic constants for the activation of plasminogen (Km = 1 microM, k2 = 0.0026 s-1), fibrin affinity (50% binding at a fibrin concentration of 3.3 g/liter), and fibrin specificity of clot lysis in a plasma environment (50% lysis in 2 h with 8 nM of the chimer) as the previously characterized chimeric protein (t-PA/scu-PA-s). Thus, unexpectedly, the fibrin affinity of t-PA is also only partially expressed in this extended chimeric protein. Therefore, the NH2-terminal chains (A-chains) of the plasmin-generated two-chain derivatives t-PA/tcu-PA-e, t-PA/tcu-PA-s, and of t-PA were isolated. These A-chain structures of the chimers were found to have lost most of their fibrin affinity, whereas the fibrin affinity of the A-chain of native t-PA was maintained. Differential reactivity of the A-chain structures of both chimeric molecules with monoclonal antibodies directed against the A-chain of t-PA suggested that they were conformationally altered. Sequential fibrin binding experiments with t-PA/scu-PA-e and t-PA/scu-PA-s yielded 45 +/- 8 (n = 11) and 43 +/- 5% (n = 8), respectively, binding in the first cycle and 44 +/- 7 (n = 11) and 27 +/- 10% (n = 8), respectively, binding in the second cycle. This suggests that the low affinity of the chimeric molecules for fibrin is not due to the occurrence of subpopulations of molecules with different fibrin affinity but, instead, to a uniformly decreased fibrin affinity in all molecules.     

A randomised dose ranging study of recombinant tissue plasminogen activator in acute myocardial infarction

To assess the thrombolytic efficacy and the effect on the systemic fibrinolytic system of recombinant tissue plasminogen activator doses of 20 mg, 50 mg, and 100 mg were compared in a randomised study. Tissue plasminogen activator was infused intravenously over 90 minutes in 50 consecutive patients with acute myocardial infarction of four hours'' duration or less; on average the infusion was started 135 minutes (range 20 to 240) after the onset of pain. The affected artery was patent at the end of the 90 minute infusion in 14/17 (82%) of those who received 100 mg, 12/17 (71%) of those who received 50 mg, and 8/16 (50%) of those who received 20 mg. Regardless of dose, reperfusion rates were significantly better for patients treated within two hours of the onset of symptoms (81%) than for those treated in the third and fourth hours (54%). At the end of the infusion serum fibrinogen concentrations fell to 86% of the preinfusion value after 20 mg, 75% after 50 mg, and 63% after 100 mg, and similar dose dependent changes occurred in plasminogen, α₂ anti-plasmin, and fibrinogen and fibrin degradation products. The mean infarct related regional third ejection fraction was 46% for patients with grade 2 or 3 reperfusion and 35% for those with grade 0 or 1. Ventricular fibrillation occurred in six (12%) patients during the infusion of tissue plasminogen activator, but no late ventricular fibrillation occurred. Bleeding was minimal, reocclusion occurred in three patients, and four patients died from cardiac causes.Recombinant tissue plasminogen activator is an effective thrombolytic agent which produces better reperfusion rates after a 50 or 100 mg dose than after a 20 mg dose. The effect on the systemic fibrinolytic system is dose dependent. Successful reperfusion results in improvement of left ventricular function.     

A rapid and strong increase of plasminogen activator induced by experimental anaphylaxis in rabbits.

Anaphylactic shock was induced in rabbits by injecting bovine serum albumin (BSA) as an antigen. Measurements of the enzyme activities in the fibrinolytic system confirmed that a rapid and strong increase of plasminogen activator (PA) was induced during anaphylaxis. The euglobulin fibrinolytic activity (EFA) as estimated by the plasminogen-rich fibrin plate method rose significantly, peaking at 15 min after the BSA injection (when the arterial pressure was minimum). However, EFA was not detected by the plasminogen-poor fibrin plate method. The tissue-type PA (t-PA) activity using the natural substrate plasminogen increased significantly with a peak at 15 min. The amidolytic activity also simultaneously increased significantly using the t-PA substrate, H-D-Ile-Pro-Arg-pNA. The plasminogen activator inhibitor (PAI) activity remained at baseline levels until 30 min, but rose fourfold at 90 min. The main plasma fibrinolytic enzyme which increased in anaphylaxis was proved by zymography to be t-PA with a molecular weight (MW) of 69,000.     

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.     

Structure and function of human tissue-type plasminogen activator (t-PA)

Full-length tissue-type plasminogen activator (t-PA) cDNA served to construct deletion mutants within the N-terminal "heavy" (H)-chain of the t-PA molecule. The H-chain cDNA consists of an array of structural domains homologous to domains present on other plasma proteins ("finger," "epidermal growth factor," "kringles"). These structural domains have been located on an exon or a set of exons. The endpoints of the deletions nearly coincide with exon-intron junctions of the chromosomal t-PA gene. Recombinant t-PA deletion mutant proteins were obtained after transient expression in mouse Ltk- cells, transfected with SV40-pBR322-derived t-PA cDNA plasmids. It is demonstrated that the serine protease moiety of t-PA and its substrate specificity for plasminogen is entirely contained within the C-terminal "light" (L)-chain of the protein. The presence of cDNA, encoding the t-PA signal peptide preceding the remaining portion of t-PA, suffices to achieve secretion of (mutant) t-PA into the medium. The stimulatory effect of fibrin on the plasminogen activator activity of t-PA was shown to be mediated by the kringle K2 domain and, to a lesser extent, by the finger domain. The other domains on the H-chain, kringle K1, and the epidermal growth-factor-like domain, do not contribute to this property of t-PA. These findings correlate well with the fibrin-binding properties of the rt-PA deletion-mutant proteins, indicating that stimulation of the activity is based on aligning of the substrate plasminogen and its enzyme t-PA on the fibrin matrix. The primary target for endothelial plasminogen activator inhibitor (PAI) is located within the L-chain of t-PA. Deleting specific segments of t-PA H-chain cDNA and subsequent transient expression in mouse Ltk- cells of t-PA deletion-mutant proteins did not affect the formation of a stable complex between mutant t-PA and PAI.     

叶下珠有效部位的溶栓作用及其对PAI-1和tPA活性的影响

采用改良的Charlton和Tomihisa等方法评价叶下珠植物(Phyllanthus urinaria)含corilagin的水溶性有效部位(代号PUW)对电刺激大鼠颈动脉血栓的溶栓作用;应用发色底物方法测定PUW在体内外对血浆tPA、血浆或血小板释放的PAl-1活性的影响。结果显示,5mg／kg的PUW,其再通率为50％,再栓率为60％;10mg／kg PUW的血管再通率为60％,其再栓率为33．3％,低于2万U／kg尿激酶42．9%的再栓率。再通后1h内,血管开放状态表现为,2万U／kg尿激酶组的血管开放状态与5mg／kg PUW组的相似;10mg／kg PUW组,其持续再通率高于2万U／kg的尿激酶组。PUW在体外或静脉注射均明显降低血浆PAI—1活性,同时提高血浆tPA的活性;PUW静注还明显抑制血小板释放的PA1-1活性。本实验结果提示,PUW静脉注射显著提高闭塞颈动脉的再通率,同时降低再通后颈动脉的再栓率;抑制PAI—1活性,同时提高tPA的活性可能是PUW具有较好溶栓作用的分子机制。     

Plasminogen activation by tissue plasminogen activator on fibrin clots with different surface structures

Transformation of fibrinogen into fibrin with consequent formation of the fibrin clot trimeric structure is one of the final steps in the blood coagulation system. The plasminogen activation by the tissue plasminogen activator (t-PA) is one of the fibrinolysis system key reactions. The effect of different factors on transformation of plasminogen into plasmin is capable to change essentially the equilibrium between coagulation and fibrinolytic sections of haemostasis system. We have studied the plasminogen activation by tissue plasminogen activator on fibrin clots surface formed on the interface between two phases and in presence of one phase. The t-PA plasminogen activation rate on fibrin clots both with film and without it the latter has been analyzed. These data allow to assume that the changes of fibrin clot structure depend on its formations, as well as are capable to influence essentially on plasminogen activation process by means of its tissue activating agent.     

Type 1 plasminogen activator inhibitor binds to fibrin via vitronectin

Type 1 plasminogen activator inhibitor (PAI-1), the primary inhibitor of tissue-type plasminogen activator (t-PA), circulates as a complex with the abundant plasma glycoprotein, vitronectin. This interaction stabilizes the inhibitor in its active conformation In this report, the effects of vitronectin on the interactions of PAI-1 with fibrin clots were studied. Confocal microscopic imaging of platelet-poor plasma clots reveals that essentially all fibrin-associated PAI-1 colocalizes with fibrin-bound vitronectin. Moreover, formation of platelet-poor plasma clots in the presence of polyclonal antibodies specific for vitronectin attenuated the inhibitory effects of PAI-1 on t-PA-mediated fibrinolysis. Addition of vitronectin during clot formation markedly potentiates PAI-1-mediated inhibition of lysis of (125)I-labeled fibrin clots by t-PA. This effect is dependent on direct binding interactions of vitronectin with fibrin. There is no significant effect of fibrin-associated vitronectin on fibrinolysis in the absence of PAI-1. The binding of PAI-1 to fibrin clots formed in the absence of vitronectin was characterized by a low affinity (K(d) approximately 3.5 micrometer) and rapid loss of PAI-1 inhibitory activity over time. In contrast, a high affinity and stabilization of PAI-1 activity characterized the cooperative binding of PAI-1 to fibrin formed in the presence of vitronectin. These findings indicate that plasma PAI-1.vitronectin complexes can be localized to the surface of fibrin clots; by this localization, they may modulate fibrinolysis and clot reorganization.     

Effects of clot contraction on clot degradation: A mathematical and experimental approach

Thrombosis, resulting in occlusive blood clots, blocks blood flow to downstream organs and causes life-threatening conditions such as heart attacks and strokes. The administration of tissue plasminogen activator (t-PA), which drives the enzymatic degradation (fibrinolysis) of these blood clots, is a treatment for thrombotic conditions, but the use of these therapeutics is often limited due to the time-dependent nature of treatment and their limited success. We have shown that clot contraction, which is altered in prothrombotic conditions, influences the efficacy of fibrinolysis. Clot contraction results in the volume shrinkage of blood clots, with the redistribution and densification of fibrin and platelets on the exterior of the clot and red blood cells in the interior. Understanding how these key structural changes influence fibrinolysis can lead to improved diagnostics and patient care. We used a combination of mathematical modeling and experimental methodologies to characterize the process of exogenous delivery of t-PA (external fibrinolysis). A three-dimensional (3D) stochastic, multiscale model of external fibrinolysis was used to determine how the structural changes that occur during the process of clot contraction influence the mechanism(s) of fibrinolysis. Experiments were performed based on modeling predictions using pooled human plasma and the external delivery of t-PA to initiate lysis. Analysis of fibrinolysis simulations and experiments indicate that fibrin densification makes the most significant contribution to the rate of fibrinolysis compared with the distribution of components and degree of compaction (p < 0.0001). This result suggests the possibility of a certain fibrin density threshold above which t-PA effective diffusion is limited. From a clinical perspective, this information can be used to improve on current therapeutics by optimizing timing and delivery of lysis agents.     

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.     

The Effect of Tranexamic Acid on the Fibrinolytic System During Anaphylaxis in Rabbits; The Importance of the Fibrinolytic System During Anaphylaxis

Abstract

We previously reported (Shimaya et al. (1992) Enzyme, 46, 204) that a rapid and strong increase of plasminogen activator (PA) was induced during anaphylaxis, and that the main plasma fibrinolytic enzyme which increased in the anaphylaxis group was shown to be tissue-type plasminogen activator (t-PA). Anaphylaxis was induced in rabbits by giving BSA after t-AMCHA injection. 44% of those rabbits died within 3 h after BSA injection. In the dead group, the euglobulin fibrinolytic activity (EFA) could not be detected by the plasminogen-rich fibrin plate method and the t-PA activity, using the natural substrate plasminogen, did not rise significantly reaching a peak at 10–15 min. However, the EFA and t-PA activity increased significantly in the surviving group. A significant prolongation of the activated partial thromboplastin time (AFTT) and the prothrombin time (PT) was observed during anaphylaxis in both groups. These findings suggest that increased PA activity during anaphylaxis is an important defense mechanism against the rapid increase in the blood coagulation system.     

Molecular mechanisms of fibrinolysis and their application to fibrin-specific thrombolytic therapy

The fibrinolytic system comprises a proenzyme, plasminogen, which can be converted to the active enzyme, plasmin, which degrades fibrin. Plasminogen activation is mediated by plasminogen activators, which are classified as either tissue-type plasminogen activators (t-PA) or urokinase-type plasminogen activators (u-PA). Inhibition of the fibrinolytic system may occur at the level of the activators or at the level of generated plasmin. Plasmin has a low substrate specificity, and when circulating freely in the blood it degrades several proteins including fibrinogen, factor V, and factor VIII. Plasma does, however, contain a fast-acting plasmin inhibitor, alpha 2-antiplasmin, which inhibits free plasmin extremely rapidly but which reacts much slower with plasmin bound to fibrin. A "systemic fibrinolytic state" may, however, occur by extensive activation of plasminogen and depletion of alpha 2-antiplasmin. Clot-specific thrombolysis therefore requires plasminogen activation restricted to the vicinity of the fibrin. Two physiological plasminogen activators, t-PA and single-chain u-PA (scu-PA) induce clot-specific thrombolysis, via entirely different mechanisms, however. t-PA is relatively inactive in the absence of fibrin, but fibrin strikingly enhances the activation rate of plasminogen by t-PA. This is explained by an increased affinity of fibrin-bound t-PA for plasminogen and not by alteration of the catalytic rate constant of the enzyme. The high affinity of t-PA for plasminogen in the presence of fibrin thus allows efficient activation on the fibrin clot, while no significant plasminogen activation by t-PA occurs in plasma. scu-PA has a high affinity for plasminogen (Km = 0.3 microM) but a low catalytic rate constant (kcat = 0.02 sec-1). However, scu-PA does not activate plasminogen in plasma in the absence of a fibrin clot, owing to the presence of (a) competitive inhibitor(s). Fibrin-specific thrombolysis appears to be due to the fact that fibrin reverses the competitive inhibition. The thrombolytic efficacy and fibrin specificity of natural and recombinant t-PA has been demonstrated in animal models of pulmonary embolism, venous thrombosis, and coronary artery thrombosis. In all these studies intravenous infusion of t-PA at sufficiently high rates caused efficient thrombolysis in the absence of systemic fibrinolytic activation. The efficacy and relative fibrinogen-sparing effect of t-PA was recently confirmed in three multicenter clinical trials in patients with acute myocardial infarction.(ABSTRACT TRUNCATED AT 400 WORDS)     

Functional properties of the recombinant kringle-2 domain of tissue plasminogen activator produced in Escherichia coli

The kringle-2 domain (residues 176-262) of tissue-type plasminogen activator (t-PA) was cloned and expressed in Escherichia coli. The recombinant peptide, which concentrated in cytoplasmic inclusion bodies, was isolated, solubilized, chemically refolded, and purified by affinity chromatography on lysine-Sepharose to apparent homogeneity. [35S]Cysteine-methionine-labeled polypeptide was used to study the interactions of kringle-2 with lysine, fibrin, and plasminogen activator inhibitor-1. The kringle-2 domain bound to lysine-Sepharose and to preformed fibrin with a Kd = 104 +/- 6.2 microM (0.86 +/- 0.012 binding site) and a Kd = 4.2 +/- 1.05 microM (0.80 +/- 0.081 binding site), respectively. Competition experiments and direct binding studies showed that the kringle-2 domain is required for the formation of the ternary t-PA-plasminogen-intact fibrin complex and that the association between the t-PA kringle-2 domain and fibrin does not require plasmin degradation of fibrin and exposure of new COOH-terminal lysine residues. We also observed that kringle-2 forms a complex with highly purified guanidine-activated plasminogen activator inhibitor-1, dissociable by 0.2 M epsilon-aminocaproic acid. The kringle-2 polypeptide significantly inhibited tissue plasminogen activator/plasminogen activator inhibitor-1 interaction. The kringle-2 domain bound to plasminogen activator inhibitor-1 in a specific and saturable manner with a Kd = 0.51 +/- 0.055 microM (0.35 +/- 0.026 binding site). Therefore, the t-PA kringle-2 domain is important for the interaction of t-PA not only with fibrin, but also with plasminogen activator inhibitor-1 and thus represents a key structure in the regulation of fibrinolysis.     

Involvement of finger domain and kringle 2 domain of tissue-type plasminogen activator in fibrin binding and stimulation of activity by fibrin.

Human tissue-type plasminogen activator (t-PA) catalyses the conversion of inactive plasminogen into active plasmin, the main fibrinolytic enzyme. This process is confined to the fibrin surface by specific binding of t-PA to fibrin and stimulation of its activity by fibrin. Tissue-type plasminogen activator contains five domains designated finger, growth factor, kringle 1, kringle 2 and protease. The involvement of the domains in fibrin specificity was investigated with a set of variant proteins lacking one or more domains. Variant proteins were produced by expression in Chinese hamster ovary cells of plasmids containing part of the coding sequence for the activator. It was found that kringle 2 domain only is involved in stimulation of activity by fibrin. In the absence of plasminogen and at low concentration of fibrin, binding of t-PA is mainly due to the finger domain, while at high fibrin concentrations also kringle 2 is involved in fibrin binding. In the presence of plasminogen, fibrin binding of the kringle 2 region of t-PA also becomes important at low fibrin concentrations.     

Characterization of a recombinant fusion protein of the finger domain of tissue-type plasminogen activator with a truncated single chain urokinase-type plasminogen activator

Human recombinant single chain urokinase-type plasminogen activator (recombinant scu-PA) and a hybrid between human tissue-type plasminogen activator (t-PA) and scu-PA, obtained by ligation of cDNA fragments encoding the NH2-terminal region (amino acids 1-67) of t-PA and the COOH-terminal region (amino acids 136-411) of scu-PA, were expressed in a mammalian cell system. The proteins were purified from conditioned culture media containing 2% fetal calf serum by chromatography on zinc chelate-Sepharose, immunoadsorption chromatography on an insolubilized murine monoclonal antibody directed against urokinase, benzamidine-Sepharose chromatography, and Ultrogel AcA 44 gel filtration. Between 180 and 230 micrograms of the purified proteins were obtained per liter of conditioned medium, with a yield of approximately 18% and a purification factor of 720-1900. On sodium dodecyl sulfate gel electrophoresis under reducing conditions, the proteins migrated as single bands with approximate Mr 50,000 for recombinant scu-PA and Mr 43,000 for the t-PA/scu-PA hybrid. Following conversion to urokinase with plasmin, the proteins had a specific amidolytic activity comparable to that of natural scu-PA. Both proteins activated plasminogen directly with Km = 0.53 and 1.4 microM and k2 = 0.0034 and 0.0027 s-1, respectively. Both proteins did not bind specifically to fibrin and had a comparable degree of fibrin selectivity as measured in a system composed of a whole human 125I-fibrin-labeled plasma clot suspended in human plasma. It is concluded that this chimeric protein, consisting of the NH2-terminal "finger-like" domain of t-PA and the COOH-terminal region of scu-PA, has very similar enzymatic properties as compared to scu-PA, but has not acquired the fibrin affinity of t-PA.     

猪心组织型纤溶酶原激活剂的纯化鉴定

本文介绍了一种简便易行的纯化组织型纤溶酶原激活剂(t-PA)的方法。新鲜猪心组织经丙酮脱脂脱水,制成干粉,再经醋酸钾缓冲液提取。提取液经阳离子交换柱、肝素柱亲和层析及凝胶柱层析分离纯化。经SDS凝胶电泳分析证明纯化的t-PA只在分子量为67000道尔顿位置出现一条染色带。同时把该蛋白带转移到纤维蛋白板上,也在同一位置出现溶解带。     

Plasminogen activators and their potential in therapy

Plasminogen activators (PAs) are proteases that convert plasminogen to plasmin. Plasmin, in turn, is a protease that can lyse a fibrin clot and, therefore, PAs have a primary role in fibrinolysis. Two PAs, urokinase (UK) and streptokinase (SK), have been available for therapeutic use for years. Unfortunately, both can cause systemic fibrinogenolysis and other side effects which have limited their use. Interest has focused on a different enzyme, tissue plasminogen activator (t-PA), which will cause specific clot lysis without systemic problems. The gene for t-PA has been cloned and many biotechnology firms are preparing to produce t-PA for therapeutic use. The properties and potential for therapy of t-PA are reviewed and compared to new forms of other activators, such as pro-urokinase. How the interactions of PAs and inhibitors may affect the use of PAs is also discussed.