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D Collen

Publications and source records attributed to D Collen.

At least 55 records · Page 3Linked to original sources
PubMed1991

Enhancement of clot lysis in vitro and in vivo with a bispecific monoclonal antibody directed against human fibrin and against urokinase-type plasminogen activator.

A hybrid hybridoma (FU1-74), secreting a bispecific monoclonal antibody (bs mAb), was obtained by fusion of a murine hybridoma secreting a monoclonal antibody (mAb) specific for human fibrin with a murine hybridoma secreting a mAb against urokinase-type plasminogen activator (u-PA). The bs mAb (MA-FU1-74), purified to homogeneity from mouse ascitic fluid, migrated as a single band with apparent Mr 150,000 on nonreduced SDS-PAGE and had an affinity for both human fibrin (Ka = 2 x 10(7) M-1) and for u-PA (Ka = 10(8) M-1) comparable to that of the mAbs obtained from the respective parental hybridomas. MA-FU1-74 did not influence the enzymatic activity of two-chain u-PA (tcu-PA) towards plasminogen or towards a chromogenic substrate. The complex of MA-FU1-74 with recombinant single chain u-PA (rscu-PA) or with tcu-PA (urokinase) enhanced the fibrinolytic potency of the plasminogen activator towards clotted human plasma 20-fold and 5-fold, respectively. In a hamster pulmonary embolism model, the rscu-PA/MA-FU1-74 complex had a 13- to 17-fold increased thrombolytic potency (percent lysis per mg/kg u-PA administered) relative to that of rscu-PA. The specific thrombolytic activity (percent lysis per microgram/ml steady state plasma level of u-PA antigen) of the complex was, however, not significantly different from that of rscu-PA. The complex of rscu-PA with the parental anti-u-PA mAb (MA-UK1-3) had only a 2-fold enhanced thrombolytic potency relative to that of rscu-PA and had a 5-fold decreased specific thrombolytic activity. The plasma clearance rates of the complexes of rscu-PA with both MA-FU1-74 and MA-UK1-3 were about 10-fold lower than that of rscu-PA. In a rabbit jugular vein thrombosis model, the rscu-PA/MA-FU1-74 complex had a 4-fold enhanced thrombolytic potency, an unchanged specific thrombolytic activity and 20-fold reduced plasma clearance. In both animal models, the rscu-PA/MA-FU1-74 complex did not cause more extensive systemic activation of the fibrinolytic system than rscu-PA. It is concluded that the bispecific anti-fibrin/anti-u-PA mAb MA-FU1-74 targets u-PA to the fibrin clot, resulting in a significantly enhanced thrombolytic potency of the plasminogen activator.

Amino Acid Sequence↗
PubMed1991

Effect of retinoic acid on the synthesis of tissue-type plasminogen activator and plasminogen activator inhibitor-1 in human endothelial cells.

The synthesis of plasminogen activators and inhibitors in endothelial cells is highly regulated by hormones, drugs and growth factors. The present study evaluates the effect of retinoic acid on the synthesis of tissue-type plasminogen activator (t-PA) and of plasminogen activator inhibitor-1 (PAI-1) by cultured human umbilical vein endothelial cells (HUVEC). Retinoic acid produced a time- and concentration-dependent increase in the secretion of t-PA-related antigen but not of PAI-1 related antigen into the culture medium. A maximal sevenfold increase of t-PA antigen after 24 h was observed with 10 microM and a half-maximal increase with 0.1 microM retinoic acid. Retinoic acid induced a time-dependent increase of the t-PA mRNA, with a maximum at 8 h and returning to normal at 24 h. The protein kinase inhibitor H7 decreased the t-PA antigen induced by both retinoic acid and phorbol 12-myristate 13-acetate. These results suggest that treatment of HUVEC with retinoic acid increases t-PA production by a pathway which, at some level, involves protein kinases. Thus, retinoic acid induces t-PA synthesis in the absence of altered PAI-1 synthesis, which may enhance the fibrinolytic potential of the endothelium.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1991

Characterization of a chimeric plasminogen activator consisting of a single-chain Fv fragment derived from a fibrin fragment D-dimer-specific antibody and a truncated single-chain urokinase.

An Mr 57,000 single-chain chimeric plasminogen activator, K12G0S32, consisting of a variable region fragment (Fv) derived from the fibrin fragment D-dimer-specific monoclonal antibody MA-15C5 and of a 33-kDa (amino acids Ala132 to Leu411) recombinant single-chain urokinase-type plasminogen activator (rscu-PA-33k) was studied. K12G0S32, secreted by infected Spodoptera frugiperda insect cells at a rate of 1.5 micrograms/10(6) cells/48 h, was purified to homogeneity by ion-exchange chromatography and gel filtration. It was obtained essentially as a single-chain molecule with a Ka = 5.5 x 10(9) M-1 for immobilized fragment D-dimer, similar to that of MA-15C5. The specific activity of both its single-chain and two-chain forms on fibrin plates was 100,000 IU/mg of urokinase-type plasminogen activator (u-PA) equivalent. Activation of plasminogen by two-chain K12G0S32 obeyed Michaelis-Menten kinetics with Km = 2.9 +/- 0.6 microM and a k2 = 3.7 +/- 0.6 s-1 (mean +/- S.D.; n = 3), as compared to Km = 12 microM and k2 = 4.8 s-1 for rtcu-PA-32k (recombinant low Mr two-chain u-PA consisting of amino acids Leu144 to Leu411). Single-chain K12G0S32 induced a dose- and time-dependent lysis of a 125I-fibrin-labeled human plasma clot immersed in citrated human plasma; 50% lysis in 2 h was obtained with 0.70 +/- 0.07 micrograms/ml (mean +/- S.D.; n = 5), as compared with 8.8 +/- 0.1 micrograms/ml for rscu-PA-32k (recombinant low Mr single-chain u-PA consisting of amino acids Leu144 to Leu411) (mean +/- S.D.; n = 3). With two-chain K12G0S32, 50% clot lysis in 2 h required 0.25 +/- 0.03 micrograms/ml (mean +/- S.D.; n = 3), as compared with only 0.62 +/- 0.04 micrograms/ml (mean +/- S.D.; n = 2) for rtcu-PA-32k. These results indicate that low Mr single-chain u-PA can be targeted to a fibrin clot with a single-chain Fv fragment of a fibrin-specific antibody, resulting in a 13-fold increase of the fibrinolytic potency of the single-chain form and a 2.5-fold increase of the potency of the two-chain form.

Amides↗
PubMed1991

Comparative fibrinolytic properties of staphylokinase and streptokinase in animal models of venous thrombosis.

The thrombolytic and pharmacokinetic properties of staphylokinase were compared with those of streptokinase in hamsters with a pulmonary embolus produced from human plasma or from hamster plasma, and in rabbits with a jugular vein blood clot produced from rabbit blood. In both models, a continuous intravenous infusion of staphylokinase and streptokinase over 60 min in hamsters or over 4 h in rabbits, induced dose-dependent progressive clot lysis in the absence of significant systemic activation of the fibrinolytic system. The results of thrombolytic potency (clot lysis at 30 min after the end of the infusion, in percent, versus dose administered, in mg/kg) were fitted with an exponentially transformed sigmoidal function (formula: see text) and the maximal percent clot lysis (c), the maximal rate of lysis (z = 1/4 ac.eb) and the dose at which the maximal rate of lysis is achieved (b) were determined. In hamsters with a pulmonary embolus produced from human plasma, streptokinase had a somewhat higher thrombolytic potency than staphylokinase, as revealed by a higher z value (2,100 +/- 1,100% lysis per mg/kg streptokinase administered versus 1,100 +/- 330% lysis per mg/kg for staphylokinase). In hamsters with a pulmonary embolus produced from hamster plasma, staphylokinase had a somewhat higher thrombolytic potency than streptokinase (z = 1,600 +/- 440 versus 1,200 +/- 370% lysis per mg/kg). Staphylokinase had a higher thrombolytic potency than streptokinase in rabbits,as revealed by a higher z-value (950 +/- 350% lysis per mg/kg staphylokinase administered versus 330 +/- 39% lysis per mg/kg for streptokinase) and a lower b-value (0.035 +/- 0.010 mg/kg staphylokinase versus 0.091 +/- 0.008 mg/kg for streptokinase.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Characterization of a recombinant single-chain molecule comprising the variable domains of a monoclonal antibody specific for human fibrin fragment D-dimer.

A recombinant single-chain molecule, scFv-K12G0, containing the variable domains of the monoclonal antibody MA-15C5, specific for fragment D-dimer of human cross-linked fibrin, was constructed and expressed in Spodoptera frugiperda, Sf9, insect cells. The Arg108 carboxyl-terminal amino acid of the variable domain of the light-chain of the antibody was connected through a synthetic Ala-Gly-Gln-Gly-Ser-Ser-Val peptide linker with the Gln1 amino-terminal amino acid of the variable domain of its heavy chain. scFv-K12G0 was secreted by the infected Sf9 cells at a rate of 10 micrograms/10(6) cells within 48 h, resulting in conditioned medium with a maximal concentration of 15 mg of scFv-K12G0/liter. The molecule, purified to homogeneity by ion exchange chromatography and gel filtration, migrated as a single Mr band on reduced sodium dodecyl sulfate-gel electrophoresis. It bound to immobilized fragment D-dimer with an affinity constant of 4.0 x 10(9) M-1 (2.0 x 10(10) M-1 for intact MA-15C5). Clearing of scFv-K12G0 from the circulation in rabbits occurred with an initial half-life (t1/2 alpha) of 10 min and a clearance of 5.1 ml min-1, as compared to 90 min and 210 ml min-1 for intact MA-15C5. Nephrectomy resulted in a prolongation of t1/2 alpha to 110 min, suggesting that the rapid clearance of scFv-K12G0 occurs primarily via the kidney, presumably by glomerular filtration. The results indicate that the single-chain recombinant molecule scFv-K12G0 is secreted in functionally intact form and suggest that it may be useful for targeting of radioisotopes or plasminogen activators to blood clots in vivo.

Amino Acid Sequence↗
PubMed1991

Hemorrhagic events during therapy with recombinant tissue-type plasminogen activator, heparin, and aspirin for acute myocardial infarction. Results of the Thrombolysis in Myocardial Infarction (TIMI), Phase II Trial.

OBJECTIVES: To assess the effects of invasive procedures, hemostatic and clinical variables, the timing of beta-blocker therapy, and the doses of recombinant plasminogen activator (rt-PA) on hemorrhagic events. DESIGN: A multicenter, randomized, controlled trial. SETTING: Hospitals participating in the Thrombolysis in Myocardial Infarction, Phase II trial (TIMI II). INTERVENTIONS: Patients received rt-PA, heparin, and aspirin. The total dose of rt-PA was 150 mg for the first 520 patients and 100 mg for the remaining 2819 patients. Patients were randomly assigned to an invasive strategy (coronary arteriography with percutaneous angioplasty [if feasible] done routinely 18 to 48 hours after the start of thrombolytic therapy) or to a conservative strategy (coronary arteriography done for recurrent spontaneous or exercise-induced ischemia). Eligible patients were also randomly assigned to either immediate intravenous or deferred beta-blocker therapy. MEASUREMENTS: Patients were monitored for hemorrhagic events during hospitalization. MAIN RESULTS: In patients on the 100-mg rt-PA regimen, major and minor hemorrhagic events were more common among those assigned to the invasive than among those assigned to the conservative strategy (18.5% versus 12.8%, P less than 0.001). Major or minor hemorrhagic events were associated with the extent of fibrinogen breakdown, peak rt-PA levels, thrombocytopenia, prolongation of the activated partial thromboplastin time (APTT) to more than 90 seconds, weight of 70 kg or less, female gender, and physical signs of cardiac decompensation. Immediate intravenous beta-blocker therapy had no important effect on hemorrhagic events when compared with delayed beta-blocker therapy. Intracranial hemorrhages were more frequent among patients treated with the 150-mg rt-PA dose than with the 100-mg rt-PA dose (2.1% versus 0.5%, P less than 0.001). The extent of the plasmin-mediated hemostatic defect was also greater in patients receiving the 150-mg dose. CONCLUSIONS: Increased morbidity due to hemorrhagic complications is associated with an invasive management strategy in patients with acute myocardial infarction. Our findings show the complex interaction of several factors in the occurrence of hemorrhagic events during thrombolytic therapy.

Aspirin↗
PubMed1991

Effect of chemical conjugation of recombinant single-chain urokinase-type plasminogen activator with monoclonal antiplatelet antibodies on platelet aggregation and on plasma clot lysis in vitro and in vivo.

The murine monoclonal antiplatelet antibodies MA-TSPI-1 (directed against human thrombospondin) and MA-PMI-2, MA-PMI-1, and MA-LIBS-1 (directed against ligand-induced binding sites [LIBS] on human platelet glycoprotein IIb/IIIa) were conjugated with recombinant single-chain urokinase-type plasminogen activator (rscu-PA) using the cross-linking reagent N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP). The conjugates (rscu-PA/MA-TSPI-1, rscu-PA/MA-PMI-2, rscu-PA/MA-PMI-1, and rscu-PA/MA-LIBS-1), purified by immunoadsorption and gel filtration, were obtained with recoveries of 34% to 45%, with an average stoichiometry of 1.6 to 1.8 IgG molecules per rscu-PA molecule, and with unaltered specific activities and affinities. Preincubation of human platelet-rich plasma with rscu-PA/MA-PMI-2, rscu-PA/MA-PMI-1, or unconjugated rscu-PA resulted in partial inhibition of ADP-induced aggregation; 25% inhibition was obtained with 63 micrograms/mL rscu-PA and with 6 micrograms u-PA/mL rscu-PA/MA-PMI-2 or 1.2 micrograms u-PA/mL rscu-PA/MA-PMI-1. In an in vitro system composed of a 125I-fibrin-labeled platelet-rich human plasma clot immersed in normal human plasma, the conjugates had threefold to greater than 15-fold less fibrinolytic potency than unconjugated rscu-PA. The thrombolytic potency of rscu-PA/MA-PMI-1 and rscu-PA/MA-LIBS-1 was compared with that of rscu-PA and that of a control conjugate rscu-PA/MA-1C8 in a pulmonary embolism model in the hamster, using clots prepared from platelet-poor or platelet-rich human plasma. Lysis was measured 30 minutes after the end of a 60-minute intravenous infusion of the thrombolytic agents. rscu-PA, rscu-PA/MA-PMI-1, rscu-PA/MA-LIBS-1, as well as rscu-PA/MA-1C8 had comparable thrombolytic potencies (percent lysis per dose administered) towards platelet-poor human plasma clots. In contrast, the thrombolytic potency of rscu-PA/MA-PMI-1 and of rscu-PA/MA-LIBS-1 towards platelet-rich clots was 2.3- to 3-fold higher than that of rscu-PA (P less than .005) and fivefold to sevenfold higher than that of the control conjugate (P less than .01).

Adenosine Diphosphate↗
PubMed1991

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.

Animals↗
PubMed1991

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.

Antifibrinolytic Agents↗
PubMed1991

Molecular forms of plasminogen activator inhibitor-1 (PAI-1) and tissue-type plasminogen activator (t-PA) in human plasma.

Molecular forms of plasminogen activator inhibitor-1 (PAI-1) and tissue-type plasminogen activator (t-PA), identified by gel filtration and specific immunoassays, were studied in plasma from subjects with normal and elevated PAI-1 levels before and after in vitro or in vivo addition of t-PA. In normal plasma, PAI-1 occurs in three molecular forms, a Mr greater than 700 KDa inactive form of heterogeneous composition, an active 450 KDa form containing PAI-1/vitronectin complex and an inactive peak at Mr 50 KDa containing free PAI-1. Stimulation of platelets results in a significant increase of the 50 KDA form and a slight increase of the 450 KDa form. Patients with increased PAI activity levels have an increase of both the 450 KDa and the 50 KDa forms, whereas patients with thrombotic thrombocytopenic purpura have an increased 50 KDa form. In normal plasma, collected in the presence or absence of D-Phe-Pro-Arg-CH2Cl, t-PA occurs primarily as a Mr greater than 700 KDa form containing t-PA/PAI-1 complex. Addition of high concentrations of t-PA (70 ng/ml) to plasma in vitro or t-PA injection in vivo, results in t-PA inhibitor complexes, including t-PA/ alpha 2 antiplasmin. It is concluded that in subjects with increased PAI-1 levels in plasma, PAI-1 may occur as high molecular weight complexes with vitronectin of which 450 KDa was the most important part and as a 50 KDa inactive form; t-PA circulates primarily in complex with inhibitors. Thus, some of the molecular interactions between PAI-1, t-PA and vitronectin, previously demonstrated in purified systems in vitro, also occur in plasma.

Amino Acid Sequence↗
PubMed1991

Role of alpha 2-antiplasmin in fibrin-specific clot lysis with single-chain urokinase-type plasminogen activator in human plasma.

The role of plasma alpha 2-antiplasmin (alpha 2-AP) in the fibrin-specificity of clot lysis by recombinant single-chain urokinase-type plasminogen activator (rscu-PA) and in the conversion of rscu-PA to its two-chain derivative (rtcu-PA, urokinase) was investigated in an in vitro human plasma clot lysis system. Fifty % lysis in 2 h of a 0.1 ml 125I-fibrin labeled human plasma clot immersed in 0.5 ml normal human plasma was obtained with 1.4 +/- 0.15 micrograms/ml rscu-PA (mean +/- SD, n = 8). This was associated with degradation of 23 +/- 7% of fibrinogen and generation of 0.20 +/- 0.09 micrograms/ml rtcu-PA. In alpha 2-AP-depleted plasma 50% clot lysis in 2 h required 2-fold less rscu-PA which was associated with 3-fold more extensive fibrinogen degradation and 2-fold more rtcu-PA generation. Fifty % lysis in 2 h, of a 0.1 ml alpha 2-AP-depleted plasma clot, submersed in 0.5 ml normal plasma, was obtained with 0.80 +/- 0.05 micrograms/ml rscu-PA (n = 3, p less than 0.001 vs normal clot) and was associated with 17 +/- 6% fibrinogen breakdown (p = 0.22 vs normal clot) and 0.08 +/- 0.02 micrograms/ml rtcu-PA generation (p less than 0.05 vs normal clot). In alpha 2-AP-depleted plasma the equipotent rscu-PA concentration was 4-fold lower than in normal plasma and was associated with 3-fold more fibrinogen degradation and a similar extent of rtcu-PA generation.(ABSTRACT TRUNCATED AT 250 WORDS)

Fibrinogen↗
PubMed1991

On the reversible interaction of plasminogen activator inhibitor-1 with tissue-type plasminogen activator and with urokinase-type plasminogen activator.

The reaction between plasminogen activators and plasminogen activator inhibitor-1 is characterized by an initial rapid formation of an inactive reversible complex. The second-order association rate constant (k1) of complex formation of recombinant two-chain tissue-type plasminogen activator (rt-PA) or recombinant two-chain urokinase-type plasminogen activator (rtcu-PA) by recombinant plasminogen activator inhibitor-1 (rPAI-1) is 2.9 +/- 0.4 x 10(7) M-1 s-1 (mean +/- S.D., n = 30) and 2.0 +/- 0.6 x 10(7) M-1 s-1 (n = 12), respectively. Different molecular forms of tissue- or urokinase-type plasminogen activator which do not form covalent complexes with rPAI-1, including rt-PA-Ala478 (rt-PA with the active-site Ser478 mutagenized to Ala) and anhydro-urokinase (rtcu-PA with the active-site Ser356 converted to dehydroalanine) reduced k1 in a concentration-dependent manner, compatible with 1:1 stoichiometric complex formation between rPAI-1 and these ligands. The apparent dissociation constant (KD) of the complex between rPAI-1 and rt-PA-Ala478, determined as the concentration of rt-PA-Ala478 which reduced k1 to 50% of its control value, was 3-5 nM. Corresponding concentrations of active-site-blocked two-chain rt-PA were 150-250-fold higher. The concentration of anhydro-urokinase which reduced k1 to 50% was 4-6 nM, whereas that of active-site-blocked rtcu-PA was 100-250-fold higher. Recombinant single-chain urokinase-type plasminogen activator had an apparent KD of about 2 microM. These results suggest that inhibition of rt-PA or rtcu-PA by rPAI-1 proceeds via a reversible high affinity interaction which does not require a functional active site but which is markedly reduced following inactivation of the enzymes with active-site titrants.

Animals↗
PubMed1991

Correlation between baseline plasminogen activator inhibitor levels and clinical outcome during therapy with tissue plasminogen activator for acute myocardial infarction.

Baseline plasminogen activator inhibitor (PAI) levels were examined for their influence on the responses to thrombolysis with recombinant tissue plasminogen activator (rt-PA) administered for acute myocardial infarction during the Thrombolysis and Myocardial Infarction (TAMI)-I study. Baseline PAI activity was 19 +/- 21 IU/ml (normal less than 5 IU/ml) and baseline PAI-1 antigen 54 +/- 53 ng/ml (normal 27 +/- 16 ng/ml), confirming previous findings of elevated PAI levels during acute myocardial infarction. Among clinical outcomes, lower PAI-1 antigen levels correlated weakly with greater patency at the 90 min angiogram. Thus, high baseline plasma PAI-1 levels may be detrimental to reperfusion with t-PA. There was no correlation with other major in-hospital clinical outcomes including reocclusion at the 7-10 day angiogram, survival to discharge, or bleeding. During the follow up period of 2.0 +/- 0.4 years, no relationship between baseline PAI levels and post-discharge reinfarction was observed.

Angioplasty, Balloon, Coronary↗
PubMed1991

Fibrinolysis and coagulation in patients with infectious disease and sepsis.

Sepsis is often associated with hemostatic dysfunction. This study aimed to relate changes in fibrinolysis and coagulation parameters to sepsis and sepsis outcome. Urokinase-type plasminogen activator (u-PA) antigen, tissue-type plasminogen activator (t-PA) antigen and activity, plasminogen activator inhibitor (PAI) type 1 antigen, PAI activity, antithrombin (AT) III activity, and protein C activity were measured in 24 patients suffering from sepsis or septic shock and the results were compared with those observed in 30 non-sepsis patients with severe infectious disease. The u-PA level was markedly increased in plasma of sepsis patients as compared to non-sepsis patients (11.5 +/- 9.4 versus 1.6 +/- 1.5 ng/ml, p less than 0.0001). PAI-1 antigen and t-PA activity showed a significant increase in sepsis patients (320 +/- 390 ng/ml versus 120 +/- 200 ng/ml, and 3.0 +/- 3.6 IU/ml versus 1.0 +/- 0.7 IU/ml, respectively, p less than 0.01). AT III was decreased in sepsis patients (58 +/- 28% in sepsis versus 79 +/- 26% in severe infectious disease, p less than 0.01) as was protein C (30 +/- 18% versus 58 +/- 27%, p less than 0.001). No significant difference was found for t-PA antigen nor for PAI activity. Nonsurvivors of sepsis were distinguished mainly by a high u-PA antigen level and increased t-PA activity. It is concluded that plasma u-PA antigen showed the strongest significant difference, among the parameters evaluated, between sepsis and severe infection. u-PA antigen may be of prognostic value in patients admitted to the medical intensive care unit for severe infectious disease.

Adult↗
PubMed1991

Requirement of heparin for arterial and venous thrombolysis with recombinant tissue-type plasminogen activator.

The effect of concomitant intravenous (IV) heparin (200 U/kg bolus, followed by 100 U/kg/h) on the efficacy of arterial and venous thrombolysis with IV recombinant tissue-type plasminogen activator (rt-PA; 0.5 mg/kg over 1 hour) was investigated in a combined femoral arterial and venous thrombosis model in the dog. The arterial model consisted of a high-grade stenosis, endothelial damage, and a thrombotic occlusion, and the venous model consisted of a 125I-fibrin-labeled blood clot. After a dose-finding pilot study in four dogs, a randomized, prospective, and blind study was performed in 20 animals pretreated with 2.8 mg/kg IV acetyl salicylic acid (ASA). The combination of rt-PA and heparin (group I, n = 10) induced early (less than 30 minutes) arterial reperfusion in seven dogs, late (greater than 30 minutes) reflow in two dogs, and persistent occlusion in one dog. rt-PA alone (group II, n = 10) was associated with early reperfusion in one dog, late reflow in three dogs, and persistent occlusion in six dogs (P = .018). Reocclusion occurred in five of nine reperfused dogs of group I and in one of four reperfused dogs of group II (P = not significant). Venous clot lysis amounted to 81% +/- 4% (mean +/- SEM) for group I and to 49% +/- 7% for group II (P less than .001). Template bleeding times increased moderately, but significantly, in group I (from 2.2 +/- 0.2 minutes at baseline to 7.0 +/- 1.4 minutes at 30 minutes, P = .006), but only marginally in group II (from 2.2 +/- 0.2 minutes to 3.6 +/- 0.7 minutes, P = .09). No systemic fibrinogen depletion was observed. Thus, the concommitant use of heparin with rt-PA accelerates arterial reperfusion and enhances venous thrombolysis in dogs pretreated with ASA. These results, obtained in a randomized prospective study design, add to a growing body of experimental and clinical evidence, indicating that thrombolytic therapy with rt-PA requires concomitant adjunctive IV heparin for optimal efficacy, even in the face of treatment with ASA.

Animals↗
PubMed1991

Thrombolytic and pharmacokinetic properties of human tissue-type plasminogen activator variants, obtained by deletion and/or duplication of structural/functional domains, in a hamster pulmonary embolism model.

A pulmonary embolism model in hamsters was used for the quantitative evaluation of the thrombolytic and pharmacokinetic properties of variants of tissue-type plasminogen activator (t-PA). A 25 microliters 125I-fibrin labeled human plasma clot was made in vitro and injected into the jugular vein of heparinized hamsters. The extent of thrombolysis within 90 min was determined as the difference between the radioactivity injected in the jugular vein and that recovered in the heart and lungs. Recombinant t-PA (home-made rt-PA or Activase) infused intravenously over 60 min caused dose-dependent progressive thrombolysis. The results of thrombolytic potency (clot lysis in percent versus dose administered in mg/kg) and of specific thrombolytic activity (clot lysis in percent versus steady state plasma level in microgram/ml) were fitted with an exponentially transformed sigmoidal function y = 100 c/(1 + e-a(ax-eh] and the maximal percent lysis (c), the dose or plasma level at which maximal rate of lysis is achieved (b) and the maximal rate of lysis (z = 1/4 ac.eb) were determined. With rt-PA, these parameters were c = 72 +/- 6% (mean +/- SEM), b = 0.19 +/- 0.08 mg/kg, z = 68 +/- 25% lysis per mg/kg, with corresponding values of 87 +/- 5%, 0.07 +/- 0.03 mg/kg and 150 +/- 38% lysis per mg/kg for Activase (p = NS). Deletion of the finger and growth factor domains in rt-PA (rt-PA-delta FE) was not associated with marked alteration of the thrombolytic potency (c = 90 +/- 30%, b = 0.34 +/- 0.35 mg/kg, and z = 54 +/- 14% per mg/kg), but was associated with a significant reduction of the specific thrombolytic activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Synthesis and secretion of plasminogen activator inhibitor 1 by human endothelial cells in vitro. Effect of active site mutagenized tissue-type plasminogen activator.

The effects of recombinant tissue-type plasminogen activator (rt-PA) and of an inactive mutant of rt-PA, obtained by mutagenesis of the active site Ser478 to Ala (rt-PA-Ala478), on the synthesis and secretion of plasminogen activator inhibitor-1 (PAI-1) by human umbilical vein endothelial cells (HUVEC) in culture were studied. Under base-line conditions, PAI-1 antigen secretion was 4.3 +/- 1.0 micrograms (mean +/- S.D., n = 8) per 10(6) cells in 24 h. This PAI-1 had a low specific activity (6,000 +/- 1,600 units/mg) and Mr of 50,000, which was not altered by addition of rt-PA. In HUVEC cultured with 2 micrograms/ml rt-PA-Ala478, PAI-1 antigen secretion was 2.1 +/- 0.8 micrograms (n = 5) per 10(6) cells in 24 h with a specific activity of 120,000 +/- 42,000 units/mg and Mr of 50,000. Addition of rt-PA to this conditioned medium resulted in generation of three main components: 16% migrated as an Mr 106,000 rt-PA.PAI-1 complex, 16% as an Mr 81,000 degraded rt-PA.PAI-1 complex and the remainder as an Mr 45,000 degradation product of PAI-1. HUVEC cultured with 2 micrograms/ml rt-PA secreted 3.9 +/- 0.6 micrograms (n = 8) PAI-1 antigen per 10(6) cells within 24 h, of which 20-50% occurred as intact or degraded complexes with t-PA (Mr 106,000 and 81,000) and the rest as an inactive Mr 45,000 degradation product of PAI-1. PAI-1 mRNA levels, determined by Northern blot analysis and expressed relative to beta-actin mRNA levels, were very similar for HUVEC cultured in the absence or the presence of rt-PA or rt-PA-Ala478. It is concluded that PAI-1 is secreted by HUVEC in culture in fully active form which spontaneously inactivates. PAI-1 can be stabilized by addition of rt-PA-Ala478 to the culture medium, resulting in a 20-fold increase in specific activity. Interaction of rt-PA with active PAI-1 produces both t-PA.PAI-1 complex and an inactive degradation product of PAI-1.

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