人t-PA溶栓突变体的研究进展

人t-PA在机体循环中的纤溶系统中起重要作用,是一种内源性溶血栓因子,t-PA蛋白分子可直接用于溶栓治疗,但天然的t-PA分子在体内半衰期短,极最被清除,因而限制其广泛应用,根据它的结构特点而改造的一系列t-PA变体分子将成为新一代溶栓药物,在溶栓治疗中广泛应用。     

Comparison of recombinant tissue-type plasminogen activator (rt-PA) expressed in mouse C127 cells and human vascular plasminogen activator (HV-PA)

Tissue-type plasminogen activator produced by recombinant DNA technology (rt-PA) has now been recognized as a promising clot-selective thrombolytic agent. We have compared the properties of rt-PA expressed in mouse C127 cells with those of naturally occurring human vascular plasminogen activator (HV-PA). The molecular weight of HV-PA and rt-PA was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to be approx. 66,000. HV-PA and rt-PA were labile and rapidly lost their activities at pH values below 5.5. The optimum pH of HV-PA and rt-PA for plasminogen activation was around 8.5. HV-PA and rt-PA appeared to be very similar in amidolytic properties, amino-acid composition and carbohydrate composition. Moreover, the N-terminal amino-acid sequence of HV-PA was in good agreement with that of rt-PA. The purified preparations of HV-PA and rt-PA had specific activities of about 250,000 and 600,000 IU/mg, respectively. Both activators bound to fibrin clots to similar degree. In immunodiffusion as well as in the quenching experiments of the fibrinolytic activities, rt-PA appeared to be immunodiffusion as well as in the quenching experiments of the fibrinolytic activities, rt-PA appeared to be immunologically indistinguishable from HV-PA. All these findings indicate that rt-PA expressed in mouse C127 cells is identical with naturally occurring HV-PA in physical and chemical properties.     

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.     

Protein engineering of novel plasminogen activators with increased thrombolytic potency in rabbits relative to activase.

Human tissue-type plasminogen activator (t-PA) is a glycoprotein used currently in thrombolytic therapy for patients with acute myocardial infarction. Due to its rapid rate of clearance from the circulation, continuous intravenous administration of approximately 100 mg over 3 h is recommended. We have previously characterized novel thrombolytic variant forms of t-PA which offer the potential of administration by bolus injection and reduced dosage due to their slower rates of clearance, relative to t-PA. This study was undertaken to quantitatively compare the pharmacokinetics, thrombolytic activity, and hemostatic effects of two of these variant forms, called delta FE1X and delta FE3X plasminogen activator (PA), with commercially available recombinant t-PA (Activase). These evaluations were performed in rabbits after bolus intravenous injection of the proteins. Following injection of 0.25 mg of protein/kg of body weight, the rates of clearance for delta FE3X and delta FE1X PA antigen were decreased approximately 9- and 18-fold, respectively, relative to Activase. Plasma plasminogen activator activity was also measured and the rates of clearance of delta FE3X and delta FE1X PA activity were similarly decreased by approximately 9- and 22-fold, respectively, relative to Activase. To quantitate thrombolytic activity we used the rabbit jugular vein thrombosis model and demonstrated that approximately 50% thrombolysis was achieved with delta FE1X and delta FE3X PA at approximately an 8.6- and 3-fold lower dose than Activase, respectively. No major differences in fibrinogen and alpha 2-antiplasmin depletion were observed among the agents at doses required to produce 50% thrombolysis, indicating similarities in fibrin specificities among these agents. These results demonstrate a reciprocal relationship between thrombolysis and rate of clearance for these thrombolytic proteins. The 8.6-fold increase in potency of delta FE1X PA relative to Activase supports the future clinical testing of this novel engineered protein as a thrombolytic agent.     

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.     

Staphylokinase--a specific plasminogen activator

Staphylokinase is a 135 amino acid protein produced by certain strains of Staphylococcus aureus. It belongs to fibrin-specific plasminogen activator. Staphylokinase converts plasminogen--the inactive proenzyme--to the plasmin, which dissolves the fibrin of a blood clots. This review will focus on the biochemical and thrombolytic properties of staphylokinase and its derivatives, which would make use of treatment in acute myocardial infarction and other cardiovascular diseases.     

Fatal ischaemic brain oedema after early thrombolysis with tissue plasminogen activator in acute stroke.

Two patients with acute major, disabling cerebral infarction with presumed middle cerebral artery occlusion were treated with the clot specific thrombolytic agent tissue plasminogen activator roughly three and a half hours after the onset of symptoms. Both patients had a normal computed tomography (CT) scan before treatment. No appreciable systemic bleeding complications occurred, apart from bruising. One patient had bleeding into the subarachnoid space from a microscopic angioma, which was found at necropsy. Haematological monitoring of the two patients showed pronounced fibrinogenolysis and alpha 2 antiplasmin consumption in one. One patient showed transient improvement during the infusion. In both cases extensive infarction, partly haemorrhagic in one, with massive concomitant oedema was found on repeated CT. Both patients deteriorated and eventually died as a consequence of transtentorial herniation. In the one patient who came to necropsy a moderate, probably pre-existing smooth stenosis of the ipsilateral carotid artery was found, all cerebral vessels being patent. It is concluded that thrombolytic treatment with a clot specific agent such as tissue plasminogen activator started three to four hours after a major ischaemic stroke may be hazardous, not because of haemorrhagic transformation of the original ischaemia but because early reperfusion may promote massive, potentially fatal cerebral oedema.     

Characterization of a modified human tissue plasminogen activator comprising a kringle-2 and a protease domain

To study structure/function relationships of tissue plasminogen activator (t-PA) activity, one of the simplest modified t-PA structures to activate plasminogen in a fibrin-dependent manner was obtained by constructing an expression vector that deleted amino acid residues 4-175 from the full-length sequence of t-PA. The expression plasmid was introduced into a Syrian hamster cell line, and stable recombinant transformants, producing high levels of the modified plasminogen activator, were isolated. The resulting molecule, mt-PA-6, comprising the second kringle and serine protease domains of t-PA, produced a doublet of plasminogen activator activity having molecular masses of 40 and 42 kDa. The one-chain mt-PA-6 produced by cultured Syrian hamster cells was purified in high yield by affinity and size exclusion chromatography. The purified mt-PA-6 displayed the same two types of microheterogeneity observed for t-PA. NH2-terminal amino acid sequencing demonstrated that one-chain mt-PA-6 existed in both a GAR and a des-GAR form. Purified mt-PA-6 also existed in two glycosylation forms that accounted for the 40- and 42-kDa doublet of activity produced by the cultured Syrian hamster cells. Separation of these two forms by hydrophobic interaction chromatography and subsequent tryptic peptide mapping demonstrated that both forms contained N-linked glycosylation at Asn448; in addition, some mt-PA-6 molecules were also glycosylated at Asn184. Plasmin treatment of one-chain mt-PA-6 converted it to a two-chain molecule by cleavage of the Arg275-Ile276 bond. This two-chain mt-PA-6, like t-PA, had increased amidolytic activity. The fibrinolytic specific activities of the one- and two-chain forms of mt-PA-6 were similar and twice that of t-PA. The plasminogen activator activity of one-chain mt-PA-6 was enhanced greater than 80-fold by CNBr fragments of fibrinogen, and the one-chain enzyme lysed human clots in vitro in a dose-dependent manner. The ability to produce and purify a structurally simple plasminogen activator with desirable fibrinolytic properties may aid in the development of a superior thrombolytic agent for the treatment of acute myocardial infarction.     

Effect of thrombolysis on myocardial injury: recombinant tissue plasminogen activator vs. alfimeprase

Plasmin-dependent thrombolytic agents are potentially prothrombotic and proinflammatory. Alfimeprase, a zinc-containing metalloproteinase, degrades fibrin directly and achieves thrombolysis independent of plasmin formation. This study examines the hypothesis that thrombolysis in the absence of plasmin generation results in improved myocardial salvage on reperfusion. The thrombolytic effects of recombinant tissue plasminogen activator [rt-PA; 0.022 mg/kg, 1/10 of which was administered as a loading dose; the rest (9/10) was infused over 60 min by intracoronary (ic) administration] or alfimeprase (0.5 mg/kg over 1 min ic) were evaluated in a canine model of arterial thrombosis involving electrolytic injury of the left circumflex (LCX) coronary artery. Both agents induced thrombolysis, with onset of reperfusion being more rapid after alfimeprase compared with rt-PA (1.5 +/- 0.6 vs. 10.1 +/- 2.1 min). In the absence of adjunctive therapy, time to reocclusion after alfimeprase was 3.2 +/- 0.5 min compared with 77.5 +/- 31.9 min with rt-PA. The glycoprotein IIb/IIIa platelet receptor antagonist CRL-42796 prolonged reperfusion time after thrombolysis with alfimeprase or rt-PA. The effect of each lytic agent on myocardial infarct size was examined in a separate group of dogs subjected to 60 min of LCX coronary artery ligation and 4 h of reperfusion. Myocardial infarct size, expressed as percentage of the risk region, was larger (32.16 +/- 3.95%) after rt-PA compared with alfimeprase (19.85 +/- 3.61%) or that of the saline control group (18.46 +/- 3.34%). rt-PA in contrast to alfimeprase, a direct-acting fibrinolytic agent, is associated with an increase in myocyte reperfusion injury.     

Construction and expression of hybrid plasminogen activators prepared from tissue-type plasminogen activator and urokinase-type plasminogen activator genes

Recent data from several studies have suggested that the non-protease domains in tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA) determine their biological specificities, including binding to fibrin clots and survival in the circulatory system (Van Zonneveld, A.-J., Veerman, H., and Pannekoek, H. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 4670-4674; Rijken, D. C., and Emeis, J. J. (1986) Biochem. J. 238, 643-646). Structural manipulations (e.g. deletions, additions, or substitutions) in these domains can thus be utilized to maximize the desired biological effects. Using recombinant DNA technology, we constructed a number of hybrid molecules from the t-PA and u-PA genes. In hybrid A, the epidermal growth factor and finger domains of t-PA (residues 1-91) were replaced by the epidermal growth factor and kringle of u-PA (residues 1-131). In hybrids B and C, the u-PA kringle (residues 50-131) was inserted either before (residue 92) or after (residue 261) the double-kringle region of t-PA. All these hybrid PAs containing three kringles were expressed in mouse fibroblast cells (C-127). The hybrid proteins were synthesized in predominantly a single-chain form with molecular weights of 70,000-80,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were enzymatically active as assayed by the fibrin-agar plate method. In vitro studies on the binding of hybrid PAs to fibrin showed that hybrid B, like t-PA, possesses affinity toward fibrin, while hybrid A shows lower binding. This suggests that the finger domain, which is not present in hybrid A, plays a role in conferring fibrin affinity to the hybrid PAs. The enzymatic activities of the hybrids were compared with that of recombinant t-PA (rt-PA) expressed in the same vector/host system and found to be similar in activity toward a chromogenic peptide substrate. In addition, plasminogen activation with all the hybrid-PAs, as with rt-PA, was stimulated by fibrin, with the order of activity being rt-PA greater than or equal to hybrid B greater than hybrid C greater than hybrid A. This study shows the feasibility of shuffling functional domain(s) of known specificity in plasminogen activators which may lead to the design of a superior thrombolytic agent.     

Thrombolysis with tissue plasminogen activator in patients with acute myocardial infarction

Thrombolytic agents are being employed clinically in increasing numbers of patients in the attempt to eliminate occlusive coronary thrombi in patients with evolving myocardial infarction. When administered by the intracoronary route, streptokinase lyses is successful in coronary thrombi in more than two-thirds of patients, but when administered intravenously is successful in only one-third. Since streptokinase is a nonselective plasminogen activator, it induces fibrinogenolysis when administered selectively or systematically with an attendant marked reduction in plasma fibrinogen levels and significant bleeding complications. In contrast, the action of tissue plasminogen activator (t-Pa) is relatively selective for fibrinolysis (as opposed to fibrinogenolysis). It induces coronary thrombolysis in at least 60% of patients when administered either into a coronary ostium or a peripheral vein without producing substantial reductions in circulating fibrinogen. Bleeding complications are modest and usually related to high administered doses and concomitant heparinization, and occur primarily at sites of vascular access. Thus, t-Pa appears to be a promising agent for thrombolytic treatment of patients with evolving acute myocardial infarction.     

Release of plasminogen activator by pentosan polysulfate

In isolated perfused organs (pig ear, rabbit ear, rat lung) pentosan polysulphate caused an increase in the release of plasminogen activator. The activator was released in a dose-dependent manner, the release being repeatedly induced as demonstrated with the rabbit ear. An increase in activator activity was also found in experimental animals (mini-pig, rat, rabbit). In the isolated perfused organ and the whole animal, the activator released proved to be tissue-type plasminogen activator. For the release mechanism displacement of mural plasminogen activator by pentosan polysulphate seems to be of importance. The release of tissue-type activator plays a decisive role for the regulation of the temporarily insufficient fibrinolytic system, for the thrombolytic process and for the antithrombotic action of pentosan polysulphate.     

p22 is a novel plasminogen fragment with antiangiogenic activity.

Tumor or tumor-associated cells cleave circulating plasminogen into three or four kringle-containing antiangiogenic fragments, collectively referred to as angiostatin. Angiostatin blocks tumor growth and metastasis by preventing the growth of endothelial cells that are critical for tumor vascularization. Here, we show that cancer and normal cells convert plasminogen into a novel 22 kDa fragment (p22). Production of this plasminogen fragment in a cell-free system has allowed characterization of the structure and activity of the protein. p22 consists of amino acid residues 78-180 of plasminogen and therefore embodies the first plasminogen kringle (residues 84-162) as well as additional N- and C-terminal residues. Circular dichroism and intrinsic fluorescence spectrum analysis have defined structural differences between p22 and recombinant plasminogen kringle 1 (rK1), therefore suggesting a unique conformation for kringle 1 within p22. Proliferation of capillary endothelial cells but not cells of other lineages was selectively inhibited by p22 in vitro. In addition, p22 prevented vascular growth of chick chorioallantoic membranes (CAMs) in vivo. Furthermore, administration of p22 at low dose suppressed the growth of murine Lewis lung carcinoma (LLC) metastatic foci in vivo. This is the first identification of a single kringle-containing antiangiogenic plasminogen fragment produced under physiological conditions.     

Longistatin, a novel plasminogen activator from vector ticks, is resistant to plasminogen activator inhibitor-1

Thrombo-occlusive diseases are major causes of morbidity and mortality, and tissue-type plasminogen activator (t-PA) is recommended for the treatment of the maladies. However, both t-PA and u-PA are rapidly inactivated by plasminogen activator inhibitor-1 (PAI-1). Here, we show that longistatin, a novel plasminogen activator isolated from the ixodid tick, Haemaphysalis longicornis is resistant to PAI-1. Longistatin was relatively less susceptible to the inhibitory effect of SDS-treated platelet lysate than physiologic PAs. Platelet lysate inhibited t-PA and tcu-PA with the IC₅₀ of 7.7 and 9.1 μg/ml, respectively, whereas for longistatin inhibition IC₅₀ was 20.1 μg/ml (p < 0.01). Similarly, activated PAI-1 (20 nM) inhibited only 21.47% activity of longistatin but almost completely inhibited t-PA (99.17%) and tcu-PA (96.84%). Interestingly, longistatin retained 76.73% initial activity even after 3 h of incubation with 20 nM of PAI-1. IC₅₀ of PAI-1 during longistatin inhibition was 88.3 nM while it was 3.9 and 3.2 nM in t-PA and tcu-PA inhibition, respectively. Longistatin completely hydrolyzed fibrin clot by activating plasminogen efficiently in the presence of 20 nM of PAI-1. Importantly, unlike t-PA, longistatin did not form complex with PAI-1. Collectively, our results suggest that longistatin is resistant to PAI-1 and maybe an interesting tool for the development of a PAI-1 resistant effective thrombolytic agent.     

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.     

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.     

Mutational and immunochemical analysis of plasminogen activator inhibitor 1

We have undertaken a structural and functional analysis of recombinant plasminogen activator inhibitor type 1 (PAI-1) produced in Escherichia coli using site-directed mutagenesis and immunochemistry. Expression of recombinant PAI-1 yielded an inhibitor that was functionally indistinguishable from PAI-1 made in human endothelial cells. Mutations in both the reactive center P1 and P1' residues (Arg-Met) and a putative secondary binding site for plasminogen activators on PAI-1 have been engineered to assess their functional effects. The inhibition of a panel of serine proteases, including plasminogen activators, trypsin, elastase, and thrombin, has been studied. Substitution of the P1 arginine residue with lysine or the P1' residue with either valine or serine had no detectable effect on the rate of inhibition of plasminogen activators. However, replacement of both P1 and P1' by Met-Ser produced a variant with no detectable plasminogen activator inhibitor activity. Mutations introduced into either Asp102 or Lys104 in the second site did not affect the rate of inhibition of plasminogen activators. Complementary immunochemical experiments using antibodies directed against the same two regions of the PAI-1 protein confirm that the reactive center is the primary determinant of inhibitory activity and that the putative second site is not a necessary functional region.     

Role of the pleiotropic effects of plasminogen deficiency in infection experiments with plasminogen-deficient mice

Plasminogen-deficient mice hold great promise as tools for analyzing the contribution of plasminogen activators produced by infectious agents to pathogenesis. However, the pathology caused by congenital plasminogen deficiency complicates the interpretation of infection experiments conducted with these animals. This pathology, the most prominent features of which are poor weight gain, wasting after about 60 days of age, and shortened lifespan, results from the inability of the mice to clear small fibrin thrombi. This article describes strategies for distinguishing the contribution of this pathology from the direct effects of depriving infectious agents of plasminogen. These strategies depend on the use of mouse genotypes in which the correlation of plasminogen deficiency with fibrin-dependent pathology is broken. Mice with plasminogen activator deficiencies are unable to generate plasmin and develop pathologies identical to those seen in plasminogen-deficient mice. However, unlike plasminogen-deficient mice, they do make plasminogen available to the infectious agent. Fibrinogen-deficient mice also deficient for plasminogen do not develop the pathology typical of plasminogen deficiency. These mice allow examination of plasminogen deficiency in the absence of fibrin-dependent pathology. Use of fibrinogen-deficient mice is complicated by the possibility that fibrin may be the key substrate of plasmin generated by the infectious agent.     

Enhanced expression of an antibody-targeted plasminogen activator in Escherichia coli by fusion to decorsin

To improve the thrombolytic specificity of plasminogen activators, an antibody-targeted plasminogen activator was constructed consisting of a single-chain variable fragment of a monoclonal antibody SZ-51 raised specifically against human P-selectins on activated platelets and a low molecular weight single-chain urokinase. After fusion to the 3' end of the gene coding for decorsin, originally isolated from the leech Macrobdella decora, expression of the antibody-targeted plasminogen activator gene in E. coli strain Rosetta (DE3) pLysS was greatly enhanced.