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Biomedical subjects

D Collen

Publications and source records attributed to D Collen.

At least 253 records · Page 14Linked to original sources
PubMed1995

Malondialdehyde-modified low density lipoproteins in patients with atherosclerotic disease.

The murine monoclonal antibody mAb-1H11 raised against malondialdehyde (MDA)-modified LDL, was used to detect cross-reacting material in human atheromatous tissue and in plasma. MDA-modified LDL levels in plasma were 0.19 +/- 0.02 mg/dl (mean +/- SEM) in 44 control subjects, 0.24 +/- 0.02 mg/dl in 15 patients with chronic stable angina pectoris (P = NS vs LDL cholesterol matched controls), 1.4 +/- 0.1 mg/dl in 60 patients with acute myocardial infarction (P < 0.001 vs controls), and 0.86 +/- 0.11 mg/dl in 22 patients with carotid atherosclerosis (P < 0.001 vs controls). Modified LDL, isolated from pooled LDL of 10 patients, showed a higher electrophoretic mobility on agarose gels, a higher content of thiobarbituric acid reactive substances, and a higher cholesterol/protein ratio than native LDL and had a similar reactivity (antigen/protein ratio) in the assay as the in vitro MDA-modified LDL used for calibration. Its apo B-100 moiety was not fragmented. Uptake of this modified LDL by macrophages resulted in foam cell generation. In conclusion, elevated plasma levels of atherogenic MDA-modified LDL may be a marker for unstable atherosclerotic cardiovascular disease.

Aged↗
PubMed1995

Clinical features and pathogenesis of intracerebral hemorrhage after rt-PA and heparin therapy for acute myocardial infarction: the Thrombolysis in Myocardial Infarction (TIMI) II Pilot and Randomized Clinical Trial combined experience.

Parenchymatous intracerebral hemorrhage (ICH) is a serious, infrequent complication of thrombolytic therapy for acute myocardial infarction. We studied the clinical and radiologic features, manner of presentation, associated factors, and temporal course in 23 patients with ICH associated with 150 mg or 100 mg recombinant tissue-type plasminogen activator (rt-PA) and heparin therapy for acute myocardial infarction in the Thrombolysis in Myocardial Infarction (TIMI) II Pilot and Randomized Clinical Trial. In TIMI II, 13 of the 23 ICH patients developed or maintained systolic blood pressure > or = 160 mm Hg or diastolic blood pressure > or = 90 mm Hg during the rt-PA infusion and before the onset of neurologic symptoms. Six patients (26%) had life-threatening ventricular arrhythmias, five before onset of neurologic symptoms. A decreased level of consciousness was the earliest neurologic abnormality in 15 (65%) and the most common initial physical finding (in 19, or 82%). Onset was usually gradual (70%), but time to maximal deficit was frequently (61%) within 6 hours of onset. The locations of the primary ICH sites were lobar in 16 (70%), thalamic in four (17%), and brainstem-cerebellum in three (13%), but the putamen was never the primary site. Multiple lobar hemorrhages occurred in six cases (26%). The timing and size of ICH was similar among patients treated with 150 mg rt-PA and 100 mg rt-PA. Brain CT demonstrated an arteriovenous malformation in one case. Four patients had hypofibrinogenemia, which was profound in three patients. Pathologic findings were available for five patients. Of these, three patients had cerebral amyloid angiopathy, and one had hemorrhagic transformation of an ischemic cerebral infarction found at autopsy. We conclude that ICH following rt-PA and heparin therapy for acute myocardial infarction presents as a distinctive clinical syndrome. Intracerebral bleeding after combined thrombolytic and antithrombotic therapy may be associated with cerebral amyloid angiopathy and other vascular lesions. Acute or persistent hypertension before or during rt-PA infusion, life-threatening ventricular arrhythmias, and hypofibrinogenemia, either alone or in combination, may play roles in some cases. Care should be exercised when considering thrombolytic therapy for patients with risk factors for ICH.

Adult↗
PubMed1995

Thrombolytic agents in development.

The quest continues for thrombolytic agents with a higher thrombolytic potency, specific thrombolytic activity and/or a better fibrin selectivity. Several lines of research towards improvement of thrombolytic agents are being explored, including the construction of mutants and variants of plasminogen activators (PAs), chimaeric PAs, conjugates of PAs with monoclonal antibodies, and PAs from animal or bacterial origin. Some of these new thrombolytic agents have shown promise in animal models of venous or arterial thrombosis and in pilot clinical studies. Such molecules include numerous mutants of tissue-type PA (t-PA) with prolonged in vivo half-life and/or resistance to protease inhibitors, and chimaeric PAs consisting of different regions of t-PA and of urokinase-type PA (u-PA). Several molecular forms of the thrombolytic substance in the saliva of the vampire bat have been characterised and cloned. Vampire bat PA exhibits 85% homology to human t-PA but lacks kringle 2 and the plasmin-sensitive cleavage site. A thrombolytic enzyme of 203 amino acids is present in the venom of a southern copperhead snake. This polypeptide, termed fibrolase, is now produced by recombinant technology. Fibrolase does not activate plasminogen or protein C, but directly degrades the alpha and beta chains of fibrin and fibrinogen. Recombinant staphylokinase is not an enzyme, but it forms a 1:1 stoichiometric complex with plasminogen, which becomes active after conversion of plasminogen to plasmin. It is a potent and highly fibrin specific thrombolytic agent in animals and patients.

Amino Acid Sequence↗
PubMed1995

Differential inhibition with antifibrinolytic agents of staphylokinase and streptokinase induced clot lysis.

The inhibitory effects of antifibrinolytic amino acids on clot lysis induced with recombinant staphylokinase (SakSTAR) or with streptokinase (SK) were evaluated in a human plasma milieu in vitro and in a hamster pulmonary embolism model in vivo. Addition of tranexamic acid to a system composed of 60 microliters 125I-fibrin-labeled plasma clots submerged in 0.5 ml human plasma, caused dose-dependent inhibition of lysis; complete lysis in 120 min required 30 nM SakSTAR or 100 nM SK and was reduced to 50% with 0.015 mM or with 0.07 mM tranexamic acid, respectively. Aprotinin also produced dose-dependent inhibition; lysis with SakSTAR or with SK was reduced to 50% of the control value with 8 KIU/ml or with 10 KIU/ml aprotinin, respectively. Thus, in human plasma in vitro the antifibrinolytic potency of tranexamic acid was 5-fold higher towards SakSTAR than towards SK, whereas that of aprotinin was comparable towards both agents. In hamsters with pulmonary embolism given 0.063 mg/kg SakSTAR or 0.20 mg/kg SK over 30 min, the antifibrinolytic potency of tranexamic acid, administered as a single bolus injection or as a bolus injection followed by continuous infusion, was 8- to 10-fold higher towards SakSTAR than toward SK (50% reduction of clot lysis with SakSTAR at 12.5 mg/kg, as compared to 100-150 mg/kg with SK). In contrast, aprotinin was equipotent towards SakSTAR and SK (50% reduction of clot lysis with 2,000 to 2,700 KIU/kg).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Staphylokinase: fibrinolytic properties and current experience in patients with occlusive arterial thrombosis.

Staphylokinase is a profibrinolytic agent that forms a 1:1 stoichiometric complex with plasminogen which, following conversion to plasmin, activates other plasminogen molecules to plasmin. The plasmin, staphylokinase complex, unlike the plasmin, streptokinase complex, is rapidly inhibited by alpha 2-antiplasmin. In a plasma milieu, staphylokinase is able to dissolve fibrin clots without associated fibrinogen degradation. This fibrin-specificity of staphylokinase is the result of reduced inhibition by alpha 2-antiplasmin of plasmin, staphylokinase complex bound to fibrin, recycling of staphylokinase from the plasmin, staphylokinase complex following inhibition by alpha 2-antiplasmin, and prevention of the conversion of plasminogen, staphylokinase to plasmin, staphylokinase by alpha 2-antiplasmin. In several experimental animal models, staphylokinase appears to be equipotent to streptokinase for the dissolution of whole blood or plasma clots, but significantly more potent for the dissolution of platelet-rich or retracted thrombi. The feasibility of fibrin-specific coronary thrombolysis with an intravenous infusion over 30 min of 10 mg recombinant staphylokinase was demonstrated in two small pilot studies in patients with acute myocardial infarction with angiographically confirmed total occlusion of the infarct-related coronary artery. However, neutralizing antibodies against staphylokinase were demonstrable from the third week on in all patients. Definition of the therapeutic benefit of recombinant staphylokinase will require more detailed dose-finding studies followed by randomized efficacy studies against other thrombolytic agents. An interim analysis after 50 patients of a randomized trial of recombinant tissue-type plasminogen activator versus staphylokinase in patients with acute myocardial infarction revealed similar rates of coronary patency at 90 minutes but a significantly higher fibrin specificity of the latter compound.

Animals↗
PubMed1995

Interactions of staphylokinase with human platelets.

The interactions of recombinant staphylokinase (SakSTAR) with human platelets were investigated in a buffer milieu, in a human plasma milieu in vitro, and in plasma from patients with acute myocardial infarction (AMI) treated with SakSTAR. In a buffer milieu, the activation rate of plasminogen by SakSTAR or streptokinase (SK) was not significantly altered by addition of platelets. Specific binding of SakSTAR or SK to either resting or thrombin-activated platelets was very low. ADP-induced or collagen-induced platelet aggregation in platelet-rich plasma (PRP) was 94 +/- 2.7% or 101 +/- 1.7% of control in the presence of 0.1 to 20 microM SakSTAR, with corresponding values of 95 +/- 2.8% or 90 +/- 4.6% of control in the presence of 0.1 to 4 microM SK. No effects were observed on platelet disaggregation. ATP secretion following collagen-induced platelet aggregation was 4.3 +/- 0.26 microM for SakSTAR (at concentrations of 0.1 to 20 microM) and 4.4 +/- 0.35 microM for SK (at concentrations of 0.1 to 4 microM), as compared to 3.4 +/- 0.70 microM in the absence of plasminogen activator. Fifty % lysis in 2 h (C50) of 60 microliters 125I-fibrin labeled platelet-poor plasma (PPP) clots prepared from normal plasma or from plasma of patients with Glanzmann thrombasthenia and immersed in 0.5 ml normal plasma, was obtained with 12 or 16 nM SakSTAR and with 49 or 40 nM SK, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1995

Comparison of a low-molecular-weight heparin (nadroparin calcium) and unfractionated heparin as adjunct to coronary thrombolysis with alteplase and aspirin in dogs.

BACKGROUND: Low-molecular-weight heparins may have a higher benefit to risk ratio than unfractionated heparin in preventing perioperative thrombosis. The antithrombotic effects of low-molecular-weight heparins, given as adjunctive therapy to alteplase and aspirin, have not previously been compared with those of unfractionated heparin in experimental models of coronary artery thrombosis. METHODS: Occlusive coronary thrombosis was induced in 5 groups of 10 dogs by placing a copper coil into the left anterior descending coronary artery. After 1 h of occlusion, intravenous alteplase (0.1 mg/kg bolus followed by 0.01 mg/kg/min for 30 min), and aspirin (bolus of 5 mg/kg) were administered in combination with one of the following study treatments given intravenously for 2 h: placebo (group 1); unfractionated heparin (200 IU/kg bolus followed by 100 IU/kg/h, group II); the low-molecular weight heparin, nadroparin calcium, in three different doses (100 IU/kg bolus followed by 50 IU/kg/h, group III; 200 IU/kg bolus followed by 100 IU/kg/h, group IV; and 300 IU/kg followed by 150 IU/kg/h, group V). Coronary patency was assessed with angiography at 10 min intervals and hemostasis parameters were measured at baseline, after 1 h of occlusion, and 30 and 120 min after commencing drug administration. RESULTS: Optimal reperfusion [Thrombolysis in Myocardial Infarction (TIMI) flow grade 3 without reocclusion] was more frequently observed in groups II (6/10), IV (8/10) and V (9/10) than in groups I (1/10) and III (3/10) (P < 0.05). Groups II and IV had similar patency rates (P = NS) and were therefore assumed to represent equivalent antithrombotic doses. Both nadroparin calcium and unfractionated heparin effectively prevented new thrombin generation as shown by repeated measurements of thrombin-antithrombin III complex levels in plasma. At equivalent antithrombotic doses, nadroparin calcium (group IV) was associated with significantly lower steady state values than standard heparin (group II) for activated partial thromboplastin time (41.3 +/- 48.9 versus 134.7 +/- 61.6 s), anti-Xa levels (2.4 +/- 0.5 vs 3.4 +/- 0.9 U/ml) and anti-IIa levels (0.8 +/- 0.1 versus 2.1 +/- 0.7 U/ml). CONCLUSION: Both nadroparin calcium and unfractionated heparin significantly enhance alteplase-induced thrombolysis in aspirin-treated dogs. At equivalent antithrombotic doses, nadroparin calcium was associated with less prolongation of the activated partial thromboplastin time and lower steady-state anti-Xa and anti-IIa activities.

Animals↗
PubMed1995

Biological effects of combined inactivation of plasminogen activator and plasminogen activator inhibitor-1 gene function in mice.

Mice with combined homozygous deficiency of tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA) (T-U-), of t-PA and plasminogen activator inhibitor-1 (PAI-1) (T-P-), of u-PA and PAI-1 (U-P-) or of t-PA, u-PA, and PAI-1 (T-U-P-) were generated by inbreeding of mice with the respective deficiencies. Homologous recombination at the t-PA, u-PA and PAI-1 locus was verified by Southern blot analysis of genomic tail tip DNA, and confirmed by measurement of antigen levels in plasma or urine. T-P- and U-P- mice were apparently healthy and fertile. T-U- mice showed extensive fibrin deposition with calcification in the liver, whereas T-U-P- mice were significantly (p < 0.001) less affected. Spontaneous in vivo clot lysis measured 4 h after injection of a 125I-fibrin-labeled clot prepared from plasma of wild-type (WT) mice into the jugular vein, was (mean +/- SEM of n experiments) 2 +/- 1% (n = 8) for T-P-, 49 +/- 6% (n = 9) for U-P-, 1 +/- 1% (n = 4) for T-U- and 3 +/- 3% (n = 3) for T-U-P- mice, as compared to 32 +/- 4% (n = 10) for WT, 1 +/- 0% (n = 7) for T-, 30 +/- 5% (n = 5) for U- and 58 +/- 10% (n = 6) for P- mice. Plasminogen-dependent lysis of 125I-fibrin-labeled matrix and of 3H-proline-labeled subendothelial matrix (mean +/- SEM; n = 4 to 6) was lower with thioglycollate-stimulated macrophages obtained from U-P- mice (22 +/- 7% and 5 +/- 1%, respectively), as compared to WT mice (57 +/- 14% and 18 +/- 5%, respectively) and T-P- mice (87 +/- 6% and 27 +/- 4%, respectively). A similar decrease was previously observed with U- mice, but not with T- or P- mice. Thus, the phenotype of mice with combined deficiency of t-PA and PAI-1 or of u-PA and PAI-1 is similar to the phenotype observed in mice with single deficiency of the plasminogen activator. Additional deletion of PAI-1 does not affect viability, fertility, macrophage function or thrombolytic potential of the single deficient mice. Additional deletion of PAI in mice with combined deficiency of t-PA and u-PA does not restore the deficient in vivo fibrinolytic capacity, but significantly reduces the thrombotic phenotype, as revealed by fewer, smaller and less calcified fibrin deposits in the liver.

Animals↗
PubMed1995

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)

Clinical Trials as Topic↗
PubMed1995

Fibrinolytic agents: mechanisms of activity and pharmacology.

Fibrinolytic (thrombolytic) agents activate the fibrinolytic system by conversion of the inactive proenzyme, plasminogen into the active enzyme plasmin, that degrades fibrin. Agents available for clinical use are: the physiologic tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA)--either in a single chain (scu-PA, prourokinase) or a two-chain (tcu-PA, urokinase) form, and the bacterial activator plasminogen streptokinase or its anisoylated complex with plasminogen (APSAC). Despite their widespread use, mainly in patients with acute myocardial infarction, all these agents suffer from a number of significant limitations, including resistance to reperfusion, the occurrence of acute coronary reocclusion and bleeding complications. Several lines of research towards improvement of thrombolytic agents are being explored, including the construction of mutants and variants of plasminogen activators, chimeric plasminogen activators, or plasminogen activators from animal (e.g. vampire bat) or bacterial (e.g. staphylokinase) origin. Pilot studies in patients with acute myocardial infarction have been performed with a few selected agents. Definition of their relative therapeutic benefit, or lack thereof, will require more detailed dose-finding studies, followed by randomized clinical trials against presently available thrombolytic agents.

Animals↗
PubMed1995

Gene targeting and gene transfer studies of the biological role of the plasminogen/plasmin system.

A possible role of the plasminogen/plasmin or fibrinolytic system in several biological processes has been implied from correlations between fibrinolytic activity and (patho)physiological phenomena. However, such indirect evidence does not allow to definitively establish the biological relevance of this system. Two recently developed technologies, gene targeting and gene transfer, have allowed to more definitively characterize the in vivo role of gene products. The consequences of gain or loss of function of fibrinolytic system components on reproduction, development, health, survival and on hemostasis, thrombosis, neointima formation, tissue remodeling, brain function, malignancy and neovascularization is summarized below. In addition, the possible use of transgenic mice to study gene regulation or to generate monoclonal antibodies against conserved epitopes in the targeted proteins is discussed.

Animals↗
PubMed1995

Immunoassay of murine t-PA, u-PA and PAI-1 using monoclonal antibodies raised in gene-inactivated mice.

Three enzyme-linked immunosorbent assays for the quantitation of murine tissue-type plasminogen activator (t-PA), urokinase-type plasminogen activator (u-PA) and plasminogen activator inhibitor 1 (PAI-1), were developed using monoclonal antibodies raised against the autologous proteins in gene-inactivated mice. Dose-response was linear for t-PA and PAI-1 between 5 and 0.1 ng/ml and for u-PA between 50 and 1 ng/ml, with intra-assay, inter-assay and inter-dilution coefficients of variation of 6 to 14%. Assay recoveries of proteins (5 to 100 ng/ml) added to plasma were 73 to 95% for t-PA and PAI-1. Linear correlations (r = 0.65, r = 0.91 and r = 0.92, for t-PA, u-PA and PAI-1 respectively) were found between antigen and activity in plasma, urine and tissue extracts. Levels of t-PA and PAI-1 antigen in murine plasma were 2.5 +/- 1.0 ng/ml (mean +/- SD, n=9) and 1.9 +/- 0.6 ng/ml (mean +/- SD, n = 8), respectively, in wild-type mice and undetectable in gene-inactivated mice. Bradykinin injection in mice provoked a 12-fold increase (p < 0.0002) of t-PA and endotoxin injection an 80-fold increase (p < 0.005) of PAI-1 levels. u-PA antigen levels in urine from wild-type mice ranged between 0.2 and 8.2 micrograms/ml (1.8 +/- 1.9 micrograms/ml, mean +/- SD, n = 17) and were undetectable in gene-inactivated mice. Thus, these assays may be useful for studies on the role of these proteins in tissue remodeling, atherosclerosis, embryogenesis, etc., in established mouse models. Gene-inactivated mice may constitute a general approach for the generation of monoclonal antibodies against the deficient translation products and for the development of specific immunoassays for murine proteins.

Animals↗
PubMed1994

Characterization of the murine plasma fibrinolytic system.

The main components of the murine plasma fibrinolytic system, including fibrinogen, plasminogen, alpha 2-antiplasmin, tissue-type plasminogen activator and plasminogen activator inhibitor-1, were purified to homogeneity and their interactions were quantitated and compared with those of the human counterparts. Initial activation rates of murine and human plasminogen by autologous tissue-type plasminogen activator were comparable (catalytic efficiencies, k2/Km, of 0.4 and 0.6 mM-1 s-1, respectively), but murine plasminogen appeared to be resistant to activation by human tissue-type plasminogen activator (k2/Km = 0.01 mM-1 s-1). Plasminogen activation by tissue-type plasminogen activator was stimulated 100- and 160-fold in autologous murine and human systems, respectively, with saturating concentrations of 0.45 and 0.32 microM, respectively, of CNBr-digested fibrinogen. Nearly quantitative binding (85-90%) of tissue-type plasminogen activator to fibrin was observed both in autologous and heterologous systems. Murine and human plasmin were very rapidly inhibited by autologous and heterologous alpha 2-antiplasmin (second-order inhibition rate constants, k1,app, of 2.1-2.3 x 10(7) M-1 s-1) and murine and human tissue-type plasminogen activator were very rapidly inhibited by autologous or heterologous plasminogen activator inhibitor-1 (k1,app of 1.8-4.9 x 10(7) M-1 s-1). Two-chain murine tissue-type plasminogen activator (added at a concentration of 1 microgram/ml) was inhibited in normal or plasminogen activator inhibitor-1-deficient murine plasma with half-lives of 6.5 min and 4.2 min, respectively, as compared to 80 min for human tissue-type plasminogen activator, suggesting that murine plasma contains proteinase inhibitors other than plasminogen activator inhibitor-1 which efficiently inhibit autologous tissue-type plasminogen activator. Clot lysis experiments in autologous plasma revealed that the murine plasma fibrinolytic system is more resistant to activation than the human system (20-30% clot lysis in 2 h with 100 nM tissue-type plasminogen activator in the murine system, as compared to 50% clot lysis in 2 h with 3.5 nM tissue-type plasminogen activator in the human system). Several mechanisms appear to be involved in this relative resistance observed in the murine system, including resistance of murine plasminogen to quantitative activation and short plasma half-life of murine tissue-type plasminogen activator. Thus, although these quantitative interactions between purified components of the murine fibrinolytic system appear to be comparable to those between the human counterparts, murine plasma clots are > 30-fold more resistant to lysis with autologous tissue-type plasminogen activator than human plasma clots.

Amino Acid Sequence↗
PubMed1994

Characterization of the binding of urokinase-type plasminogen activator (u-PA) to plasminogen, to plasminogen-activator inhibitor-1 and to the u-PA receptor.

Binding parameters [association-rate (kass) and dissociation-rate (kdiss) constants, and affinity constants (KA = kass/kdiss)] for the interaction between urokinase-type plasminogen activator (u-PA) and its substrate plasminogen, its inhibitor plasminogen activator inhibitor-1 (PAI-1) and its receptor (u-PAR), were determined by real-time biospecific interaction analysis (BIA). The KA values for the binding of [S741A]recombinant plasminogen (plasminogen with N-terminal Glu and with the active site Ser741 mutagenized to Ala) or of active site-blocked plasmin (D-ValPheLysCH2-plasmin) to the 54-kDa or 32-kDa molecular forms of recombinant single-chain u-PA (rscu-PA) ranged between 0.57 x 10(6) M-1 and 1.7 x 10(6) M-1, compared to 14-22 x 10(6) M-1 for binding to the corresponding active site-blocked recombinant two-chain u-PA (rtcu-PA) moieties. KA values for binding of these plasmin(ogen) moieties to [Ser356deHAla]rtcu-PA (rtcu-PA with the active site Ser356 converted to dehydroAla) were 81 x 10(6) M-1 and 670 x 10(6) M-1, respectively. Binding of active site-blocked LMM-plasmin (a low-molecular-mass plasmin derivative lacking kringles 1-4) and of the plasmin B chain to [Ser356deHAla]rtcu-PA occurred with KA values of 3.7 x 10(6) M-1 and 0.33 x 10(6) M-1, compared to 670 x 10(6) M-1 for the binding of intact D-ValPheLysCH2-plasmin to [Ser356deHAla]rtcu-PA. The KA values for binding of latent PAI-1 to 54-kDa or 32-kDa molecular forms of rscu-PA and rtcu-PA were in the range 0.34-2.1 x 10(6) M-1. Reactivated PAI-1 bound to 54-kDa and 32-kDa rtcu-PA moieties with KA values of 26 x 10(6) M-1 and 28 x 10(6) M-1, compared to 0.77 x 10(6) M-1 and 3.2 x 10(6) M-1 for binding to the corresponding single-chain u-PA species, and 450 x 10(6) M-1 for binding to [Ser356deHAla]rtcu-PA. KA values for binding of plasmin(ogen) to the covalent rtcu-PA/PAI-1 complex were similar or somewhat higher than those for binding to uncomplexed rtcu-PA. Single-chain and two-chain 54-kDa u-PA moieties bound with a 1:1 stoichiometry and with very high affinity to u-PAR (KA of 4.6-8.5 x 10(9) M-1), whereas no significant binding of 32-kDa u-PA moieties was observed (KA < or = 0.2 x 10(6) M-1).(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1994

Characterization of the interaction between plasminogen and staphylokinase.

Binding parameters [association (ka) and dissociation (kd) rate constants, and affinity constants (Ka = ka/kd)] for the interaction between recombinant staphylokinase (SakSTAR) and plasmin(ogen) were determined by real-time biospecific interaction analysis. The Ka value for binding of SakSTAR to native human Glu-plasminogen was 0.93 x 10(8) M-1 as compared to 2.0 x 10(8) M-1 and 1.6 x 10(8) M-1, respectively, for the binding to [S741A]recombinant plasminogen or Lys-[S741A]recombinant plasminogen (intact or proteolytically degraded plasminogen with the active site Ser741 replaced by alanine). Binding of SakSTAR to active plasmin or to active-site blocked plasmin occurred with Ka values of 4.0 x 10(8) M-1 and 8.4 x 10(8) M-1, respectively, whereas active-site blocked LMM-plasmin (a plasmin derivative lacking kringles 1-4) and the plasmin B-chain bound with Ka values of 1.0 x 10(8) M-1 and 0.49 x 10(8) M-1, respectively. Lysine-binding site I (a plasminogen derivative consisting of kringles 1-3) and lysine-binding site II (a plasminogen derivative consisting of kringle 4) bound with much lower affinity (Ka values of 1.2 x 10(5) M-1 and 2.9 x 10(5) M-1, respectively). The binding of these plasminogen derivatives to streptokinase occurred with similar relative Ka values. The Ka values for binding of the plasmin-SakSTAR complex to streptokinase and binding of the plasmin-streptokinase complex to SakSTAR, were, respectively, 44-fold and 30-fold lower than the values for free plasmin. The Ka for binding of plasminogen to the inactive mutants [M26R]Sak42D or [M26A]Sak42D (site-specific mutagenesis of Met26 to arginine or alanine) were 10-20-fold lower than that of native staphylokinase. These results indicate that: (a) the affinity of staphylokinase for Glu-plasminogen and Lys-plasminogen is comparable; (b) the active site in the plasmin molecule is not required for binding; (c) kringle structures 1-4 of plasminogen do not contribute significantly to plasminogen binding of staphylokinase; (d) Met26 in staphylokinase is important for its high-affinity binding to plasminogen; (e) the binding sites on plasmin for staphylokinase and streptokinase overlap at least partially.

Adsorption↗
PubMed1994

Conversion of plasminogen activator inhibitor-1 from inhibitor to substrate by point mutations in the reactive-site loop.

Plasminogen activator inhibitor-1 (PAI-1), the main physiological inhibitor of tissue-type plasminogen activator (t-PA), may occur in three interconvertible conformations: active, latent, and substrate. To delineate specific domains in the PAI-1 molecule responsible for its conformational flexibility and associated functional diversity, four mutants of PAI-1 (with the amino acids at positions P12, P10, P8, and P6, respectively, substituted with proline) were expressed in Escherichia coli, purified, and characterized. Wild-type PAI-1 (wtPAI-1) had a specific activity of 21 +/- 10% (mean +/- S.D., n = 3) of the theoretical maximum value. PAI-1-P12 (Ala-->Pro at P12), PAI-1-P10 (Ser-->Pro at P10), and PAI-1-P8 (Thr-->Pro at P8) had specific activities of 0.06 +/- 0.03% (n = 3), 2.6 +/- 1.0% (n = 4), and 2.7 +/- 1.1% (n = 3), respectively (p < 0.03 versus wtPAI-1). PAI-1-P6 (Val-->Pro at P6) has a specific activity of 12 +/- 3.3% (n = 3) of the theoretical maximum value (p = not significant versus wtPAI-1). SDS-polyacrylamide gel electrophoresis of mixtures of wtPAI-1 or PAI-1-P6 with a 2-fold molar excess of t-PA yielded a mixture of a covalent 110-kDa t-PA.PAI-1 complex (15-25%), nonreactive 45-kDa material (44-67%), and a 41-kDa band (18-31%) representing cleaved PAI-1. PAI-1-P12, PAI-1-P10, and PAI-1-P8 behaved as substrates, yielding predominantly the 41-kDa cleavage product (85-91%) and a small amount (9-15%) of non-reactive material. NH2-terminal amino acid sequencing revealed that cleavage occurred at the P1-P1' bond (Arg346-Met347). Incubation of PAI-1-P12, PAI-1-P10, or PAI-1-P8 with a 2-fold molar excess of urokinase-type plasminogen activator, plasmin, or thrombin also primarily generated a 41-kDa cleavage product (62-89%). Incubation of wtPAI-1 and PAI-1-P6 at 37 degrees C resulted in a loss of inhibitory activity, whereas the substrate behavior of PAI-1-P12, PAI-1-P10, and PAI-1-P8 remained unaltered. Treatment of the three substrate-like mutants with guanidinium Cl did not induce inhibitory activity. In conclusion, point mutations at positions P12, P10, and P8 yield PAI-1 variants with stable substrate properties, which may facilitate more detailed structure/function studies.

Amino Acid Sequence↗
PubMed1994

Physiological consequences of loss of plasminogen activator gene function in mice.

Indirect evidence suggests a crucial role for the fibrinolytic system and its physiological triggers, tissue-type (t-PA) and urokinase-type (u-PA) plasminogen activator, in many proteolytic processes. Inactivation of the t-PA gene impairs clot lysis and inactivation of the u-PA gene results in occasional fibrin deposition. Mice with combined t-PA and u-PA deficiency suffer extensive spontaneous fibrin deposition, with its associated effects on growth, fertility and survival.

Animals↗