Fibrin-specific Plasminogen Activator Research Articles

Rationale for the bolus administration of fibrin-specific thrombolytic agents

Summary The bolus administration of fibrin-specific thrombolytic agents, mainly recombinant tissue-type plasminogen activator (rt-PA), is supported by a number of experimental and clinical pharmacology studies with these agents. Experimental animal studies have shown that the thrombolytic activity of rt-PA is sustained beyond its plasma clearance and that the thrombolytic effect of a rt-PA dose is inversely correlated with the duration of infusion while its haemorrhagic effect is directly correlated with the duration of infusion. Furthermore, experimental bleeding with rt-PA is delayed up to 1 h after the start of its infusion suggesting that bleeding after rt-PA is, at least in part, the result of the generation of a plasma proteolytic state. This is possibly due to the interaction in the circulation of rt-PA with the large fibrin degradation fragments (XL-FDP) produced by the thrombus lysis. Bolus administration of rt-PA is potentially less haemorrhagic than its prolonged infusion because the short half-life of this agent precludes its interaction with circulating XL-FDP. Most of the experimental studies on the bolus administration of fibrin specific plasminogen activators have been performed with rt-PA. Experimental studies have also compared the efficacy and safety of the bolus administration of rt-PA and plasminogen activators with prolonged half-life. Clinical pharmacology studies provide further support for the bolus administration of a fibrin specific plasminogen activator. These studies have shown that the thrombolytic activity of rt-PA is sustained over time and that bolus administration of rt-PA produces less thrombin generation as compared with a continuous infusion. A number of fibrin specific plasminogen activators have been recently administered as a bolus in patients with myocardial infarction, unstable angina and pulmonary embolism. Taken together, these clinical trials showed promising although non-definitive results. Recently planned clinical outcome trials will define the value of the bolus administration of fibrin specific plasminogen activators.

Preliminary crystallographic study on a low molecular weight form of bacterial plasminogen activator staphylokinase.

Staphylokinase, a 17 kDa protein, produced by certain strains of Staphylococcus aureus functions as a fibrin-specific plasminogen activator. During its interaction with plasminogen, staphylokinase is converted into a low molecular weight form by loss of ten amino-terminal residues. This low molecular weight form of recombinant staphylokinase has been crystallized using the hanging-drop vapor-diffusion technique with polyethylene glycol 4000 as precipitant. Crystals belong to the orthorhombic space group C222(1) with unit-cell dimensions a = 43.78, b = 59.86 and c = 103.25 A and one molecule in the asymmetric unit. These crystals diffract to about 2.4 A resolution.

Crystallization and preliminary X-ray diffraction studies of recombinant staphylokinase.

Staphylokinase, a fibrin-specific plasminogen activator, was highly expressed in Escherichia coli and purified by ion-exchange and gel-filtration chromatography. The purified recombinant staphylokinase was fully active and readily crystallized against 1.2 M sodium citrate in 100 mM Tris-HCl buffer at pH 8.0 using the hanging-drop method. Crystals of staphylokinase diffract to better than 2.2 A resolution. The crystal belongs to the tetragonal space group P4(1)2(1)2 or its enantiomorph with unit-cell parameters a = b = 67.5, c = 150.1 A. There are two molecules in the asymmetric unit. In this paper, we described the first crystallization of a kind of plasminogen activator and present the results of preliminary X-ray diffraction data from the native protein.

Limited Fibrin Specificity of Tissue-type Plasminogen Activator and Its Potential Link to Bleeding

Plasminogen activators initiate the fibrinolytic process by converting plasminogen to plasmin. Though plasminogen activators are effective in the treatment of thrombotic disorders, bleeding complications are associated with their use. The development of plasminogen activators with greater fibrin specificity was expected to reduce the incidence of bleeding complications; however, this has not occurred. In our rabbit model (a) bleeding from standardized ear incisions induced by tissue-type plasminogen activator (t-PA) is attenuated when fibrinogenolysis is reduced by the coadministration of alpha 2-antiplasmin and (b) when used in doses that produce equivalent thrombolysis, vampire bat plasminogen activator (b-PA), an agent that is more fibrin specific than t-PA, causes less bleeding than t-PA. In addition, we have found that the (DD)E complex formed as a result of degradation of crosslinked fibrin is a potent stimulator of t-PA-induced plasminogen activation but has no effect on b-PA. Fragment X, a high-molecular-weight clottable fibrinogen degradation product, accumulates after treatment with t-PA but not with t-PA given with alpha 2-antiplasmin or with b-PA. These findings suggest that there is a link between plasminogen activator-induced fibrinogenolysis and bleeding, and that the composition of fibrin within hemostatic plugs may influence susceptibility to lysis. Whether these results mean that fibrin-specific plasminogen activators like b-PA will have a better risk-to-benefit profile in humans requires rigorous testing in well-designed clinical trials. However, at the very least, our findings suggest that the development of plasminogen activators that are more fibrin specific than t-PA is a worth-while exercise.

Molecular Basis of Fibrinolysis, as Relevant for Thrombolytic Therapy

The fibrinolytic system comprises an inactive proenzyme, plasminogen, that is converted by plasminogen activators to the active enzyme, plasmin, that degrades fibrin. Two physiological plasminogen activators have been identified: tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA). Plasminogen activation for clot lysis is regulated by specific molecular interactions between tissue-type plasminogen activator (t-PA), plasminogen and fibrin, whereby the lysine-binding sites of the plasminogen molecule play a crucial role by mediating its binding to fibrin, and by controlling the inhibition rate of plasmin by alpha 2-antiplasmin. The recognition that thrombosis within the infarct related coronary artery plays a major role in the pathogenesis of acute myocardial infarction and the observation that early administration of thrombolytic agents results in recanalization of occluded coronary arteries, have provided the basis for the development of thrombolytic therapy in acute myocardial infarction. The elucidation of the biochemical mechanism of fibrin-specific plasminogen activation has fueled the hope that specific and efficacious thrombolytic agents might become available. Comparative studies between the non-fibrin-selective streptokinase and fibrin-selective recombinant t-PA (rt-PA) have shown a difference in efficacy for early coronary artery recanalization, whereas the GUSTO trial has established that clinical benefit in patients with acute myocardial infarction is indeed correlated with the rapidity and frequency of sustained recanalization and that effective thrombolysis requires adequate anticoagulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Staphylokinase, a fibrin-specific plasminogen activator with therapeutic potential?

Staphylokinase, a fibrin-specific plasminogen activator with therapeutic potential?

Fragmentation pattern of fibrinogen during thrombolysis in acute myocardial infarction with fibrin-specific plasminogen activators — Detection of high-molecular weight fibrinogen degradation products

In thrombolysis in acute myocardial infarction, the fibrin-specific plasminogen activators are infused in non-physiologically high doses. Only limited fibrin-specific in nature, this may give rise to extensive fibrinogen degradation. In a series of 65 patients — 24 rt-PA, 41 pro-urokinase — we investigated, whether fibrinogen degradation follows a similar fragmentation pattern as known from thrombolysis with streptokinase. During thrombolysis, fibrinogen levels were stable (3.2 ± 1.7 before and 3.1 ± 1.3 g/l at 2 hours), whereas plasminogen and α 2-antiplasmin levels decreased from 96.1 ± 23.8 to 59.3 ± 25.6 and 93.4 ± 26.9 to 31.9 ± 32.4%, respectively. Simultaneously low molecular weight fibrinogen — and fibrin degradation products increased from 0.55 ± 0.42 to 3.31 ± 5.69 mg/l and from 0.61 ± 0.83 to 6.58 ± 6.78 mg/I respectively. Moreover high molecular weight fibrin degradation products (250–300,000 D) were detected in the plasmas of 14 pts (8 pro-urokinase, 6 rt-PA) with means of SDS-PAGE (Polyacrylamide-gradient 6–12%, 250 V, 30 mA, silver staining). Furthermore generation of high molecular weight degradation products was correlated to consumption of the plasminogen and α 2-antiplasmin. Thus the fibrinogen fragmentation pattern of all plasminogen activators is similar and is correlated to the extent of systemic lytic state.

Alpha 2-antiplasmin supplementation inhibits tissue plasminogen activator-induced fibrinogenolysis and bleeding with little effect on thrombolysis.

Tissue plasminogen activator (t-PA) causes fibrinogen proteolysis when alpha 2-antiplasmin levels fall, and this may contribute to t-PA-induced hemorrhage. Because clot-bound plasmin is protected from alpha 2-antiplasmin inhibition, we tested the possibility that alpha 2-antiplasmin supplementation would block t-PA-induced fibrinogenolysis and bleeding without affecting thrombolysis. When added to human or rabbit plasma, alpha 2-antiplasmin inhibits t-PA-induced fibrinogenolysis, but hat little effect on the lysis of 125I-fibrin clots. To examine its effect in vivo, rabbits with preformed 125I-labeled-jugular vein thrombi were randomized to receive t-PA, t-PA and alpha 2-antiplasmin, or saline. alpha 2-Antiplasmin infusion produced a modest decrease in t-PA-induced thrombolysis (from 40.2% to 30.1%, P = 0.12), but reduced fibrinogen consumption from 87% to 27% (P = 0.0001), and decreased blood loss from standardized ear incisions from 5,594 to 656 microliter (P < 0.0001). We hypothesize that alpha 2-antiplasmin limits t-PA-induced hemorrhage by inhibiting fibrinogenolysis and subsequent fragment X formation because (a) SDS-PAGE and immunoblot analysis indicate less fragment X formation in alpha 2-antiplasmin treated animals, and (b) when added to a solution of fibrinogen and plasminogen clotted with thrombin in the presence of t-PA, fragment X shortens the lysis time in a concentration-dependent fashion. These findings suggest that fragment X incorporation into hemostatic plugs contributes to t-PA-induced bleeding. By blocking t-PA-mediated fibrinogenolysis, alpha 2-antiplasmin supplementation may improve the safety of fibrin-specific plasminogen activators.

What data support our current thrombolytic management of patients with acute myocardial infarction?

T HE DECADE of the eighties is characterized by major advances in the management of patients with evolving myocardial infarction. Large-scale, placebo-controlled studies have shown that intravenous thrombolytic therapy with streptokinase (SK), anisoylated plasminogen-streptokinase activator complex (APSAC), or the relatively fibrin-specific agent recombinant-tissue type plasminogen activator (rtPA), reduces short-term mortality in selected populations by 18% to 50%.le4 This difference is maintained for at least 15 months.@ The most recently reported trials, Gruppo Italian0 per lo Studio della Streptochinasi nell’ Infarto Miocardio (GISSI)-2 and International Study of Infarct Survival (ISIS)-3, failed to show statistically significant differences in 30 to 35 day mortality when comparing SK, APSAC, and rtPA.‘s8 Skepticism has surrounded these results because of dissent over the adequacy of the heparinization regimen. However, they surprised many who had speculated that treatment with rtPA, which in the Thrombolysis in Myocardial Infarction (TIMI)-I trial appeared to lyse coronary thrombi faster than streptokinase,9 would translate into improved survival rates. The numerous reperfusion, patency, and mortality trials conducted to date have firmly established thrombolysis as the cornerstone therapy for evolving myocardial infarction in eligible patients. They have provided a wealth of information on the pathophysiology of acute coronary occlusion, the thrombus/vessel wall interactions, and the advantages and disadvantages of relative fibrin specificity. They also gave new insights into clinical prognostic factors. The observation that coronary patency may be a better prognostic indicator of shortand longterm outcome than residual left ventricular (LV) function as assessed by ejection fraction,1°-13 led to the formulation of the “open artery” hypothesis. This generated a flurry of clinical research activity, as exemplified by the TIM1 and Thrombolysis and Angioplasty in Myocardial Infarction (TAMI) study groups, focusing on pharmacological and/or mechanical means of reestablishing or maintaining patency of the culprit coronary artery. With thrombolytic therapy for acute myocardial infarction (AMI) now widely accepted, physicians are constantly confronted with the fundamental guideline: “Primum non nocere.” While lytic therapy has reduced in-hospital mortality to 10% or less, compared with 12% to 15% in the prethrombolytic era, intracerebral hemorrhage has made its inroad in the clinical picture, with a reported incidence of 0.5% to 1.5%, depending on the agent used and the aggressiveness of the infusion regimen. As discussed by Top01 elsewhere in this symposium,14 the decision to administer thrombolysis, the choice of the thrombolytic agent, and the choice of any adjunctive mechanical or pharmacological treatment must be based on the expected net clinical benefit to the patient. Large studies

Bolus Thrombolysis in Venous Thromboembolism

Thrombolytic therapy is rarely used in venous thromboembolism because of the fear of hemorrhagic complications. Preliminary clinical experiences with recombinant tissue-type plasminogen activator (rt-PA) in patients with deep vein thrombosis have shown that even this fibrin-specific plasminogen activator causes an unacceptable rate of hemorrhagic complications. Theoretical considerations and the available experimental and clinical data suggest that infusion of rt-PA over a short period of time would result in a more favorable risk-benefit ratio. Shortening the period of rt-PA infusion results in higher peak plasma levels, thus allowing a higher concentration of the plasminogen activator on the surface and inside the occluding thrombus. In addition, a bolus infusion can prevent or minimize the interaction between rt-PA and the hemostatic system, reducing the likelihood of a systemic lytic state, of a platelet function defect, and, possibly, of bleeding side effects. In venous thromboembolism animal models, the efficacy of bolus rt-PA can be further increased by the adjunctive administration of an effective antithrombotic treatment. This is because the accretion of new fibrin on the thrombi counteracts the lysis of preformed fibrin and influences negatively the final thrombus size. Effective adjunctive antithrombotic treatment includes either high doses of heparin, producing an unclottable activated partial thromboplastin time (aPTT), or doses of recombinant hirudin, doubling the aPTT. When used as an alternative to rt-PA, bolus doses of a hybrid plasminogen activator with prolonged half-life efficiently reduce thrombus size by lysing preformed and newly formed fibrin. Preliminary clinical experience in patients with pulmonary embolism seems to confirm that rt-PA infused as a bolus is at least as effective as, and probably more effective than, rt-PA infused over a longer period.

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.

Carbohydrate Structures of Human Tissue Plasminogen Activator Expressed in Chinese Hamster Ovary Cells

Recombinant human tissue plasminogen activator (rt-PA), produced by expression in Chinese hamster ovary cells, is a fibrin-specific plasminogen activator which has been approved for clinical use in the treatment of myocardial infarction. In this study, the structures of the Asn-linked oligosaccharides of Chinese hamster ovary-expressed rt-PA have been elucidated. High mannose and hybrid oligosaccharides were released from the protein by endoglycosidase H digestion, whereas N-acetyllactosamine-type ("complex") oligosaccharides were released by peptide:N-glycosidase F digestion. The oligosaccharides were fractionated by gel permeation chromatography and anion exchange high performance liquid chromatography (HPLC), and their structures were analyzed by composition and methylation analysis, high pH anion exchange chromatography, fast atom bombardment-mass spectrometry (FAB-MS), and 500-MHz 1H NMR spectroscopy. High mannose oligosaccharides were found to account for 38% of the total carbohydrate content of rt-PA and consisted of Man5GlcNAc2, Man6GlcNAc2, and Man7GlcNAc2 in the ratio 1.8:1.7:1. Two hybrid oligosaccharides were identified and accounted for 3% of the carbohydrate of rt-PA. The N-acetyllactosamine-type oligosaccharides were found to comprise diantennary (34% of total carbohydrate), 2,4-branched triantennary (11%), 2,6-branched triantennary (9%), and tetraantennary (5%) structures. Sialylation of these oligosaccharides was by alpha (2----3) linkages to galactose. Most (greater than 90%) of the N-acetyllactosamine-type structures contained fucose alpha (1----6) linked to the Asn-linked N-acetylglucosamine residue. The distribution of oligosaccharide structures at individual glycosylation sites (Asn residues 117, 184, and 448) was also determined. rt-PA exists as two variants that differ by the presence (type I) or absence (type II) of carbohydrate at Asn-184. Tryptic glycopeptides were isolated by reversed phase high performance liquid chromatography and treated with peptide:N-glycosidase F. The oligosaccharides released from each glycosylation site were analyzed by high pH anion exchange chromatography. By this analysis, Asn-117 was demonstrated to carry exclusively high mannose oligosaccharides. When glycosylated, Asn-184 carried diantennary, 2,4-branched triantennary, 2,6-branched triantennary, and tetraantennary N- acetyllactosamine oligosaccharides in the ratio 9.0:4.5:1.4:1. Asn- 448 carried the same types of oligosaccharides, but in the ratio 7.5:1.6:2.1:1. The distributions of Asn-linked oligosaccharides at positions 117 and 448 were found not to be affected by the presence or absence of carbohydrate at position 184. The relevance of the

Effect of Retraction on the Lysis of Human Clots with Fibrin Specific and Non-Fibrin Specific Plasminogen Activators

The effect of the serum content of human clots on their sensitivity to lysis with plasminogen activators was studied in a system composed of 125I-fibrin labeled clots immersed in buffer or in citrated plasma. The effect was studied with plasma clots before or after mechanical compression and with whole blood clots before or after retraction, using either the fibrin specific plasminogen activators recombinant tissue-type plasminogen activator (rt-PA) or recombinant single chain urokinase-type plasminogen activator (rscu-PA), and the non-fibrin specific activators recombinant two chain urokinase-type plasminogen activator (rtcu-PA), or streptokinase (SK). In a buffer milieu, all plasminogen activators had a similar fibrinolytic potency towards serum-rich clots (non-compressed plasma clots or non-retracted blood clots): 50% clot lysis in 4 h required 50 to 85 ng plasminogen activator per ml. Serum-poor clots (compressed plasma clots or retracted blood clots) were resistant to lysis in a buffer milieu but became sensitive to lysis following preincubation in plasma for 48 h. These findings indicate that plasma proteins entrapped in clots contribute significantly to their sensitivity to lysis and suggest that the amount of bound or entrapped plasminogen may be a limiting factor. In a plasma milieu, all plasminogen activators lysed serum-rich plasma or blood clots, albeit at higher concentrations (3 to 40 times higher than in the buffer milieu) and with different efficiencies: 50% clot lysis in 4 h required approximately 600 ng/ml of rtcu-PA but 1,500 to 2,000 ng/ml of rscu-PA.(ABSTRACT TRUNCATED AT 250 WORDS)