Comparison of the effects of fibrin binding on the biochemical properties of single-chain tissue-type plasminogen activator (t-PA) and single-chain chimeric plasminogen activator (t-PA/scu-PA).

The effect of the binding of the single-chain chimeric plasminogen activator t-PA/scu-PA, which contains amino acids 1 to 274 of tissue-type plasminogen activator (t-PA) and amino acids 138 to 411 of single-chain urokinase-type plasminogen activator (scu-PA), to fibrin on its biochemical properties was investigated in a purified system. In contrast to the binding of single-chain tissue-type plasminogen activator (sct-PA) on fibrin, which causes an increase in its intrinsic activity, t-PA/scu-PA enzyme activity is not elevated. In contrast to sct-PA which retains its single-chain form during fibrin-binding, t-PA/scu-PA is converted to its more active two-chain form. The activating process of t-PA/scu-PA is accelerated by increasing fibrin concentrations. With constant concentrations of fibrin monomer, the activation velocity also increases with time. Since this effect is inhibited by epsilon-aminocaproic acid and by a monoclonal antibody directed against the fibrin-binding site of t-PA, the activation process depends on the fibrin-binding of the molecule. The results point to the fact that t-PA/scu-PA is autocatalytically converted to its two-chain form during fibrin-binding. The conspicuous differences of the effect of the fibrin-binding on the biochemical properties of sct-PA and t-PA/scu-PA are caused obviously by small differences in the structures of the protease-domains and/or by different communications between the identical A-chains and the protease domains of the enzymes.     

Comparative thrombolytic properties of tissue-type plasminogen activator (t-PA), single-chain urokinase-type plasminogen activator (u-PA) and K1K2Pu (a t-PA/u-PA chimera) in a combined arterial and venous thrombosis model in the dog.

The chimeric molecule K1K2Pu, comprising the two kringle domains (K1 and K2) of tissue-type plasminogen activator (t-PA) and the COOH-terminal region with the serine protease domain (Pu) of urokinase-type plasminogen activator (u-PA), was previously shown to have a 5- to 10-fold reduced clearance rate with maintained specific thrombolytic activity, resulting in an increased thrombolytic potency in animal models of venous and arterial thrombosis. To document the thrombolytic potential of K1K2Pu, the thrombolytic potency and fibrin specificity were studied in a combined platelet-rich arterial eversion graft thrombosis and venous whole blood clot model in heparinized dogs (100 U/kg bolus and 50 U/kg per h infusion). Dose-response effects of bolus injections of K1K2Pu (0.032 to 0.25 mg/kg) were compared with those of recombinant t-PA (rt-PA) and of recombinant single chain u-PA (rscu-PA) (0.25 to 1.0 mg/kg each) in groups of five or six dogs, each given heparin with or without the thromboxane synthase inhibitor/prostaglandin endoperoxide receptor antagonist ridogrel. Heparin and ridogrel in the absence of a thrombolytic agent did not produce arterial reflow or venous clot lysis in five dogs. Addition of K1K2Pu, rt-PA or rscu-PA resulted in a dose-dependent induction of arterial reflow and of venous clot lysis in the absence of systemic fibrinolytic activation and fibrinogen breakdown. Consistent arterial reflow required 0.063 mg/kg of K1K2Pu and 0.5 mg/kg of rt-PA or of rscu-PA. The thrombolytic potency for venous clot lysis, expressed as percent lysis per mg compound administered per kg body weight, was (mean +/- SEM) 750 +/- 160 for K1K2Pu, 68 +/- 17 for rscu-PA (p less than 0.001 vs. K1K2Pu) and 110 +/- 29 for rt-PA (p less than 0.001 vs. K1K2Pu). The plasma clearance rates were significantly lower for K1K2Pu than for rscu-PA and rt-PA. In the absence of ridogrel, arterial reflow was significantly slower and was followed by cyclic reocclusion and reflow; however, venous clot lysis was unaffected. Template bleeding times were not significantly altered in the absence but were markedly prolonged in the presence of ridogrel. These results confirm and establish that, when given as a bolus injection, K1K2Pu has an approximately 10-fold higher thrombolytic potency for arterial and venous thrombolysis than does rt-PA or rscu-PA. Thrombolysis with K1K2Pu is obtained in the absence of systemic fibrinolytic activation and fibrinogen breakdown. These properties suggest that K1K2Pu offers potential for thrombolytic therapy by bolus administration in patients with thromboembolic disease.     

Comparison of fibrin-mediated stimulation of plasminogen activation by tissue-type plasminogen activator (t-PA) and fibrin-dependent enhancement of amidolytic activity of t-PA.

Studies in the past 10 years have shown that there are two different, but related pathways for the acceleration of tissue-type plasminogen activator (t-PA) catalysis: (1) fibrin-dependent enhancement of t-PA amidolytic activity by fibrin binding; (2) fibrin-mediated stimulation of plasminogen activation by t-PA via the formation of a ternary complex of fibrin, t-PA and plasminogen. The common characteristic of both phenomena is the affinity of t-PA for fibrin, which is realized by the same enzyme binding site. However, a comparison of the kinetic data, the participating fibrin structures and the differences between single-chain and two-chain t-PA (sct-PA and tct-PA, respectively) shows that both phenomena have different causes. Fibrin-mediated stimulation of plasminogen activation involves both sct-PA and tct-PA and different fibrinogen derivatives such as fibrin, fibrinogen cyanogen bromide fragment FCB-2, fibrin alpha-chain and poly-lysine. This mechanism is described by a marked apparent decrease in the KM value. In contrast, fibrin-dependent enhancement of t-PA activity against low molecular weight peptides is exclusive to sct-PA and is characterized by an increase in the kcat value and, depending on the nature of the substrate, by an increase in kcat and a decrease in KM. Thus, sct-PA activity modulation depends strictly on the correct three-dimensional folding of fibrin and is not mediated by fibrinogen fragment FCB-2 or isolated fibrin chains.(ABSTRACT TRUNCATED AT 250 WORDS)     

Coronary thrombolysis with tissue-type plasminogen activator (t-PA): emerging strategies

Fundamental observations and the conceptual framework underlying coronary thrombolysis have a history dating back to 1789. Recent enthusiasm for it is predicated on the recently established safety of cardiac catheterization in critically ill patients, the high incidence of coronary thrombosis underlying acute transmural myocardial infarction and demonstrable benefit conferred to the heart and the patient when thrombolysis is initiated early after the onset of ischemia. Clot-selective activators of the fibrinolytic system offer promise for safe induction of coronary thrombolysis without marked predisposition to bleeding. One such activator, tissue-type plasminogen activator (t-PA), has been synthesized by recombinant deoxyribonucleic acid (DNA) technology, amenable to large scale production of pharmaceutical agents and hence widespread availability. Initial clinical trials conducted with t-PA have demonstrated opening rates of completely occluded, infarct-related coronary arteries of approximately 75% without marked depletion of fibrinogen. The focus of research in progress includes: noninvasive delineation of recanalization and estimation of the extent of myocardium salvaged by initial recanalization, development of alternative routes of administration of thrombolytic agents potentially exploitable by paramedical personnel and, perhaps, high risk patients themselves, and definitive elucidation of the extent to which benefits conferred by thrombolysis can be enhanced with adjunctive pharmacologic interventions as well as early angioplasty or surgery.     

The release of plasminogen activators (t-PA and u-PA) and plasminogen activator inhibitor (PAI-1) after venous stasis.

The aim of the present study was to compare plasma levels of urokinase-type plasminogen activator (u-PA), before and after 20 min of venous stasis, with those of tissue-type plasminogen activator (t-PA), type 1 plasminogen activator inhibitor (PAI-1) and t-PA/PAI-1 complexes, to determine whether both plasminogen activators and their inhibitor respond similarly to the same stimulus. We studied 36 patients with recurrent venous thrombosis in whom no coagulation defects predisposing them to thrombosis had been detected (mean age 38.2 years, range 15-70 years). Twenty healthy individuals (mean age 34.3 years, range 20-60 years) served as a control group. t-PA, PAI-1 and u-PA activity and antigen, as well as the t-PA/PAI-1 complex antigen, were measured before and after venous stasis. Post-stasis fibrinolytic parameters were corrected for the haemoconcentration which occurred during the venous occlusion test. Pathologically high PAI-1 levels (antigen and activity) were found in four out of 36 patients who were excluded from study. Functional and antigenic u-PA increased significantly after venous stasis when analysed as the absolute differences between paired samples (P less than 0.01). This increase in u-PA did not correlate with changes in t-PA or PAI-1 (r = 0.28 and r = 0.36 respectively). This leads us to suggest that different mechanisms relating to clearance and/or release from diverse sources might be involved in elevations of u-PA in response to a local endothelial stimulus. We conclude that venous stasis might not be the elective choice when evaluating 'bad responders' predisposed to thrombosis.     

Analysis of factors influencing the blood levels and activities of tissue-type plasminogen activator (t-PA)

This study aimed to correlate plasmatic tissue-type plasminogen activator (t-PA) levels and activity with parameters of artery blood flow and vessel walls, nail fold microcirculation, hemorheology, serum glucose, and lipids. Thirty healthy volunteers (female/male 12/18) aged 40-60 (average 46) were included in the study. In citrate venous blood, the following parameters were determined: carotid mean velocity, carotid intimal-medial-thickness (IMT), capillary circulation parameters, hemorheology index, serum glucose, and lipids. Analysis of data showed that t-PA concentration was positively and significantly correlated with total cholesterol, triglycerides, and serum glucose (P<0.05, P<0.05, and P<0.01), but t-PA activity showed no correlation with them; among the hemorheology factors investigated, t-PA concentration showed the strongest positive correlation with both whole blood viscosity and reduced blood viscosity at high and low shear rate separately (P<0.01), t-PA activity showed no correlation with any hemorheology factors; t-PA concentration showed no correlation with any investigated nail fold capillary parameters, whereas t-PA activity was significantly and negatively associated with capillary loop number (P<0.05); t-PA concentration and activity was not associated with values of carotid maximum intimal-medial-thickness (mIMT) and mean velocity or systolic, diastolic blood pressure (P>0.05). But subjects with mIMT 1.0 mm showed higher t-PA levels compared with those with mIMT < 1.0 mm (P<0.05) and decreased carotid mean velocity (P<0.01). These findings suggest that multiple vascular disease risk factors would influence the t-PA level; t-PA concentration does not parallelize with t-PA activity.     

A one-step enzyme immunoassay for the determination of total tissue-type plasminogen activator (t-PA) antigen in plasma.

A reproducible and sensitive one-step enzyme immunoassay (EIA) was developed to determine total tissue-type plasminogen activator (t-PA) antigen in plasma. The EIA comprises two monoclonal catching antibodies and a polyclonal (goat) tagging antibody conjugated with horseradish peroxidase. There is an equal reactivity towards the several physiological t-PA forms, i.e., single-chain t-PA, two-chain t-PA and t-PA in complex with its naturally occurring inhibitor plasminogen activator inhibitor-type 1 (t-PA/PAI-1 complex). Additionally, the EIA does not discriminate between human melanoma t-PA and recombinant t-PA (Activase). The assay has a lower detection limit of approximately 0.5 ng t-PA per ml plasma, with a time-to-result of only 3.5 h.     

Distribution of tissue-type plasminogen activator (activity and antigen) in rat tissues

Tissue-type plasminogen activator (PA) activity and antigen was measured in nine different tissues from healthy rats (brain, lung, heart, liver, spleen, kidney, adrenal, aorta and skeletal muscle). After extraction in a KSCN buffer (for kidney) or in an acid acetate buffer (for all other tissues), total PA activity was determined by an improved spectrophotometric procedure, and tissue-type PA (tPA) activity was determined by quenching with anti-rat tPA Ig; tPA antigen was determined by an ELISA procedure. tPA was the major PA (greater than 90%) in all tissues, except kidney and liver (65%) and spleen (40%). Lung yielded the highest tPA activity (1400 U/g), followed by kidney, brain, heart and adrenal (150-300 U/g), and then by liver, aorta, spleen and muscle (15-30 U/g). In agreement with fibrin autographic studies, which demonstrated the presence of tPA-PAI complexes in the tissue extracts, the tPA antigen/activity ratio was generally greater than one. Free PA inhibitor activity could not be demonstrated in any tissue.     

Imbalance of tissue-type plasminogen activator (t-PA) and its specific inhibitor (PAI-1) in patients with rheumatoid arthritis associated with disease activity

Summary The relationship between plasma fibrinogen, D-dimer (DD), t-PA and PAI-1 and their correlation with disease activity (DA) were studied in 45 patients with rheumatoid arthritis (RA) (group B) to further understand the implication of fibrinolysis in the pathophysiology of RA. The control group constituted 24 healthy subjects (group A). A Stoke index (SI) of DA was assigned to each patient. Patients were divided into two groups: C, minimal-mild DA (SI 1–7); D, moderate-severe DA (SI 8–17). Fibrinogen was elevated in RA correlating positively with SI and CRP. Hypercoagulability counteracted by reactive fibrinolysis was inferred from a 10-fold increase of DD in group B as compared to group A. The relatively lower levels of DD in group D compared to group C and their negative correlation with SI (r_s=–0.45, –0.49, p=0.0006) indicate the tendency of fibrinolysis to decrease with the increase of DA. Significant elevation of t-PA and PAI-1 were found in group B compared to group A. While t-PA progressively decreased with the increase of DA (r_s=–0.45, p=0.0019), a positive relation of PAI-1 to DA was observed (r_s=0.42, p=0.0042). A 2-fold increase of PAI-1/t-PA molar ratio in group D compared to groups A and C as well as its positive correlation with SI (r_s=0.63, p=0.0001) indicate the displacement of balance between t-PA and PAI-1 in favour of the inhibitor with the increase of DA in RA. The involvement of inflammatory mediators in PAI-1/t-PA imbalance was proposed from the relation of fibrinolytic abnormalities with the activity of systemic inflammatory process.     

Interrelationship between coagulant activity and tissue-type plasminogen activator (t-PA) system in acute ischaemic heart disease. Possible role of the endothelium.

Patients with unstable angina pectoris (UAP; n = 20) and acute myocardial infarction (AMI; n = 34) were studied in the acute phase of ischaemic heart disease. We found significantly higher levels of thrombin-antithrombin-III (TAT) complexes, lower levels of systemic tissue plasminogen activator (t-PA) activity, and higher levels of plasminogen activator inhibitor (PAI) activity in the AMI patients compared to the UAP patients. In contrast to these specific changes, general acute phase reactants such as C-reactive protein, fibrinogen and von Willebrand factor did not differ significantly between the two groups. Studies of the relationship between coagulation (TAT-complexes) and fibrinolysis data revealed a significant positive correlation between plasma antigen concentrations of TAT-complexes and t-PA (P less than 0.02), and between TAT-complexes and PAI-I (P less than 0.002). These observations indicate a common pathophysiological mechanism underlying the changes in coagulation and fibrinolysis, suggesting that coagulation activity and t-PA-related fibrinolysis are interrelated processes in vivo, and probably take place at the level of the endothelial cell.     

Serum antibodies to distinct epitopes of the tissue-type plasminogen activator (t-PA) in patients with systemic lupus erythematosus

Defective fibrinolysis due to decreased tissue-type plasminogen activator (t-PA) activity is a well-established finding in patients with systemic lupus erythematosus (SLE). The possibility that this decrease in t-PA activity may be related to the presence of autoantibodies directed against t-PA, and the possible role of these autoantibodies in the patho-physiology of fibrinolysis in SLE, were investigated. Serum samples from 115 SLE patients and 63 normal volunteers were analyzed for the presence of such antibodies. The search for antibodies to t-PA was performed by means of several systems, allowing for the identification of epitopes presented in different conformational physical states of t-PA: free or associated to its inhibitor (PAI-1) in plasma, specifically bound to fibrin surface, or passively adsorbed to solid supports. Antibodies in variable amounts were detected by all systems used; however, t-PA activity was not inhibited by the IgG fraction of the positive sera in a fibrin-agar fibrinolysis system. Moreover, the demonstration of serum anti-t-PA antibodies was not associated with clinical or laboratory abnormalities related to vasoocclusive episodes. These results indicate that, as in the case of other autoantibodies, their detection in serum does not imply their direct participation in the pathophysiology of thrombosis in SLE. © 1995 Wiley-Liss, Inc.     

Specific interaction of tissue-type plasminogen activator (t-PA) with annexin II on the membrane of pancreatic cancer cells activates plasminogen and promotes invasion in vitro

BACKGROUND: Overexpression of tissue plasminogen activator (t-PA) in pancreatic cancer cells promotes invasion and proliferation in vitro and tumour growth and angiogenesis in vivo. AIMS: To understand the mechanisms by which t-PA favours cancer progression, we analysed the surface membrane proteins responsible for binding specifically t-PA and studied the contribution of this interaction to the t-PA promoted invasion of pancreatic cancer cells. METHODS: The ability of t-PA to activate plasmin and a fluorogenic plasmin substrate was used to analyse the nature of the binding of active t-PA to cell surfaces. Specific binding was determined in two pancreatic cancer cell lines (SK-PC-1 and PANC-1), and complex formation analysed by co-immunoprecipitation experiments and co-immunolocalisation in tumours. The functional role of the interaction was studied in Matrigel invasion assays. RESULTS: t-PA bound to PANC-1 and SK-PC-1 cells in a specific and saturable manner while maintaining its activity. This binding was competitively inhibited by specific peptides interfering with the interaction of t-PA with annexin II. The t-PA/annexin II interaction on pancreatic cancer cells was also supported by co-immunoprecipitation assays using anti-t-PA antibodies and, reciprocally, with antiannexin II antibodies. In addition, confocal microscopy showed t-PA and annexin II colocalisation in tumour tissues. Finally, disruption of the t-PA/annexin II interaction by a specific hexapeptide significantly decreased the invasive capacity of SK-PC-1 cells in vitro. CONCLUSION: t-PA specifically binds to annexin II on the extracellular membrane of pancreatic cancer cells where it activates local plasmin production and tumour cell invasion. These findings may be clinically relevant for future therapeutic strategies based on specific drugs that counteract the activity of t-PA or its receptor annexin II, or their interaction at the surface level.     

Acute reduction of lipoprotein(a) by tissue-type plasminogen activator.

BACKGROUND. Lipoprotein(a) [Lp(a)] is a low density lipoprotein-like particle whose apolipoprotein B (apo B) moiety is disulfide-linked to apo(a), a plasminogen-like inhibitor of fibrinolysis in vitro. We hypothesized that plasma concentrations of Lp(a) are acutely affected by intravenous tissue-type plasminogen activator (t-PA). METHODS AND RESULTS. Patients with unstable angina were randomized to receive either intravenous t-PA (n = 15) or placebo (n = 11). Two-way ANOVA using repeated measures revealed a significant effect of t-PA on concentrations of Lp(a) (p = 0.026). There was a 48% fall in Lp(a) from baseline concentrations in the t-PA group at 12 hours (p = 0.031) but not at 72 hours. Lp(a) in the placebo group was unchanged. CONCLUSIONS. We conclude that t-PA produces a sharp and substantial but reversible reduction in plasma Lp(a). These data suggest that Lp(a) concentration is not as static in vivo as had been believed and might be acutely modifiable through some mechanism that induces its removal from the freely circulating state.     

Comparative thrombolytic properties of bolus injections and continuous infusions of a chimeric (t-PA/u-PA) plasminogen activator in a hamster pulmonary embolism model

The recombinant chimeric plasminogen activator, rt-PA-delta FE/scu-PA- e, consisting of amino acids 1 to 3 and 87 to 274 of tissue-type plasminogen activator (t-PA) and amino acids 138 to 411 of single-chain urokinase-type plasminogen activator (scu-PA), has a markedly increased thrombolytic potency following its continuous intravenous infusion in animal models of venous thrombosis (Collen et al, Circulation, in press). In the present study, the thrombolytic potencies of intravenous bolus injections of rt-PA-delta FE/scu-PA-e, of recombinant t-PA (rt- PA), and of recombinant scu-PA (rscu-PA), given alone or in combination, were compared with those of intravenous infusions in a hamster pulmonary embolism model. Dose-dependent clot lysis was obtained in the absence of systemic activation of the fibrinolytic system and fibrinogen breakdown. In bolus injection experiments, the maximal rate of clot lysis, expressed in percent clot lysis per milligrams per kilogram compound administered, was 120 +/- 10 for rt- PA, 54 +/- 8 for rscu-PA, and 2,100 +/- 500 for rt-PA-delta FE/scu-PA-e (P less than .01 v rt-PA or rscu-PA). Comparative results with continuous infusion over 1 hour were 270 +/- 64, 99 +/- 18, and 1,500 +/- 250 (P less than .01 v rt-PA or rscu-PA) percent lysis per mg/kg compound infused for rt-PA, rscu-PA, and rt-PA-delta FE/scu-PA-e, respectively. Thus, rt-PA and rscu-PA are more potent when administered as an infusion than as a bolus, whereas rt-PA-delta FE/scu-PA-e is at least as potent when administered as a bolus. Combined bolus injections of rt-PA and rscu-PA had a 2.2-fold synergistic effect on clot lysis, but no synergism was observed with combined bolus injections or with combined infusions of rt-PA and rt-PA-delta FE/scu-PA-e, or of rscu-PA and rt-PA-delta FE/scu-PA-e. The present study thus shows that rt-PA- delta FE/scu-PA-e is much more potent for clot lysis than rt-PA or rscu- PA when administered as a bolus injection, but no synergistic interaction is observed between the chimera and either rt-PA or rscu-PA.     

A prospective, randomized trial comparing combination half-dose tissue-type plasminogen activator and streptokinase with full-dose tissue-type plasminogen activator. Kentucky Acute Myocardial Infarction Trial (KAMIT) Group

BACKGROUND. The potential benefits of combination thrombolytic agents in the treatment of myocardial infarction remain uncertain. In a small pilot study, we demonstrated that combining half-dose tissue-type plasminogen activator (t-PA) with streptokinase (SK) achieved a high rate of infarct vessel patency and a low rate of reocclusion at half the cost of full-dose t-PA. METHODS AND RESULTS. We designed a prospective trial in which 216 patients were randomized within 6 hours of myocardial infarction to receive either the combination of half-dose (50 mg) t-PA with streptokinase (1.5 MU) during 1 hour or to the conventional dose of t-PA (100 mg) during 3 hours. Acute patency was determined by angiography at 90 minutes, and angioplasty was reserved for failed thrombolysis. Heparin and aspirin regimens were maintained until follow-up catheterization at day 7. Acute patency was significantly greater after t-PA/SK (79%) than with t-PA alone (64%, p less than 0.05). After angioplasty for failed thrombolysis, acute patency increased to 96% in both groups. Marked depletion of serum fibrinogen levels occurred after t-PA/SK compared with t-PA alone at 4 hours (37 +/- 36 versus 199 +/- 66 mg/dl, p less than 0.0001) and persisted 24 hours after therapy (153 +/- 66 versus 252 +/- 75 mg/dl, p less than 0.0001). Reocclusion (3% versus 10%, p = 0.06), reinfarction (0% versus 4%, p less than 0.05), and need for emergency bypass surgery (1% versus 6%, p = 0.05) tended to be less in the t-PA/SK group. Greater myocardial salvage was apparent in the t-PA/SK group as assessed by infarct zone function at day 7 (-1.9 SD/chord versus -2.3 SD/chord after t-PA alone, p less than 0.05). In-hospital mortality (6% versus 4%) and serious bleeding (12% versus 11%) were similar between the two groups. CONCLUSIONS. These results suggest that a less expensive regimen of half-dose t-PA with SK yields superior 90-minute patency and left ventricular function and a trend toward reduced reocclusion compared with the conventional dose of t-PA.     

Biochemical and physiologic principles of tissue-type plasminogen activator

After wound healing the protective fibrin clot is removed by the fibrinolytic system. In addition fibrinolysis is one of the most important counter-reactions of blood coagulation. Fibrinolysis is controlled by activation and inhibition processes. Tissue type plasminogen activator (t-PA) and Pro-urokinase (single chain urokinase; scu-PA) hold a key position in physiological plasminogen activation. Plasmin itself is a rather unspecific protease capable of degrading a great variety of proteins besides fibrin. In vivo however--except for certain pathological situations--the fibrinolytic process is restricted to its actual target the fibrin clot. This surprising situation in terms of structure function interrelation is physiologically managed by N-terminal modules in the protein structure of the essential factors providing fibrin affinity. Free plasmin will be immediately inactivated by alpha 2-antiplasmin. Therefore fibrin plays a central role as cofactor in the fibrinolytic system in determining initiation and localization of the fibrinolytic process. Because of the superior properties of t-PA and scu-PA with respect to fibrin specificity both activators must be regarded as the future thrombolytic agents for therapy.