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D Collen

Publications and source records attributed to D Collen.

At least 451 records · Page 25Linked to original sources
PubMed1988

Mechanisms of plasminogen activation by mammalian plasminogen activators.

Plasminogen activators convert the proenzyme plasminogen to the active serine protease plasmin by hydrolysis of the Arg560-Val561 peptide bond. Physiological plasminogen activation is however regulated by several additional molecular interactions resulting in fibrin-specific clot lysis. Tissue-type plasminogen activator (t-PA) binds to fibrin and thereby acquires a high affinity for plasminogen, resulting in efficient plasmin generation at the fibrin surface. Single-chain urokinase-type plasminogen activator (scu-PA) activates plasminogen directly but with a catalytic efficiency which is about 20 times lower than that of urokinase. In plasma, however, it is inactive in the absence of fibrin. Chimeric plasminogen activators consisting of the NH2-terminal region of t-PA (containing the fibrin-binding domains) and the COOH-terminal region of scu-PA (containing the active site), combine the mechanisms of fibrin specificity of both plasminogen activators. Combination of t-PA and scu-PA infusion in animal models of thrombosis and in patients with coronary artery thrombosis results in a synergic effect on thrombolysis, allowing a reduction of the therapeutic dose and elimination of side effects on the hemostatic system.

Animals↗
PubMed1988

Rapid and sustained coronary artery recanalization with combined bolus injection of recombinant tissue-type plasminogen activator and monoclonal antiplatelet GPIIb/IIIa antibody in a canine preparation.

The effects of bolus injections of recombinant single-chain tissue-type plasminogen activator (rt-PA) and of F(ab')2 fragments of a murine monoclonal antibody (7E3) against the human platelet GPIIb/IIIa receptor [7E3-F(ab')2] on coronary arterial thrombolysis and reocclusion was studied in a canine preparation of coronary artery thrombosis superimposed on high-grade stenosis. Bolus intravenous injections of rt-PA at a dose of 0.45 mg/kg, repeated at 15 min intervals until reperfusion occurred (maximum of four injections) caused reperfusion in five of seven dogs within 100 min (33 +/- 15 min, mean +/- SD). Reperfusion was rapidly followed (generally within 10 min) by reocclusion and then by periods of cyclical reflow and reocclusion. A single intravenous injection of 7E3-F(ab')2 alone at 0.8 mg/kg caused reperfusion within 100 min in two of six dogs (19 and 37 min) without subsequent reocclusion. Single bolus injections of different amounts (0.1 to 0.8 mg/kg) of 7E3-F(ab')2 were then combined with bolus injections of 0.45 mg/kg of rt-PA. Stable reperfusion without reocclusion was accomplished with 0.8 or 0.6 mg/kg 7E3-F(ab')2 and a single injection of 0.45 mg/kg rt-PA within 6 +/- 3 min (n = 6, p less than .01) and 8 +/- 5 min (n = 5, p less than .02), respectively. None of these animals suffered reocclusion of the coronary artery. Lower doses (0.1 to 0.2 mg/kg) of 7E3-F(ab')2 did not significantly shorten the time to reperfusion and did not prevent reocclusion.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Coronary arterial thrombolysis with combined infusion of recombinant tissue-type plasminogen activator and urokinase in patients with acute myocardial infarction.

To determine whether tissue-type plasminogen activator (t-PA) and urokinase (UK) act synergistically to achieve coronary thrombolysis, incremental doses of both drugs were infused intravenously over 60 min. In 146 consecutive patients treated 3.0 +/- 1.0 hr from symptom onset, coronary angiography was performed 90 min after the start of the infusion and at 7 days. The groups of patients treated by different dose regimen were as follows: group I, 14 patients treated with t-PA 25 mg and UK 0.5 million U; group II, 20 patients given t-PA 25 mg and UK 1.0 million U; group III, 24 patients given t-PA 1.0 mg/kg and UK 0.5 million U; group IV, 33 patients treated with t-PA 1.0 mg/kg and UK 1.0 million U; and group V, 55 patients given t-PA 1.0 mg/kg and UK 2.0 million U. In groups I and II, patency of the infarct-related vessel at 90 min was only 36% and 42%, respectively. With 1 mg/kg t-PA and increasing doses of UK (groups III to V), patency ranged from 72% to 75% (overall 73%). Repeat catheterization at 7 days demonstrated reocclusion in groups III to V in 10 of 110 (9%). The patency and reocclusion rates in groups III to V were not significantly different from those in our previous study of 386 patients treated with t-PA alone (150 mg over 6 to 8 hr). In that study the patency rate of the infarct-related vessel at 90 min was 75% (p = .66) and reocclusion occurred in 15% (p = .11).(ABSTRACT TRUNCATED AT 250 WORDS)

Angioplasty, Balloon↗
PubMed1988

Prevention of coronary artery reocclusion and reduction in late coronary artery stenosis after thrombolytic therapy in patients with acute myocardial infarction. A randomized study of maintenance infusion of recombinant human tissue-type plasminogen activator.

Sixty-eight patients with acute "transmural" myocardial infarction presenting within 6 hours (range, 1.3-5.8 hours) of onset of chest pain were given intravenous recombinant tissue-type plasminogen activator (rt-PA) at a dosage of 1 mg/kg during 90 minutes. Coronary angiography at 90 minutes revealed a patent infarct-related coronary artery in 52 patients (76%). These patients were randomized either to treatment by continuous infusion of heparin alone (27 patients) or to treatment by heparin and a maintenance infusion of rt-PA at a dosage of 0.8 mg/kg during 4 hours (25 patients). Coronary angiography was repeated 60 minutes after the start of the maintenance infusion and again after 8-14 days. Acute symptomatic reocclusion of the infarct-related artery occurred during the 1-hour observation period in five (19%) patients treated with heparin alone but in none of the patients treated with rt-PA (p = 0.05). The measured residual stenosis of the patent infarct-related coronary artery was similar in the heparin-treated and the rt-PA-treated groups at 90 minutes infusion: 66 +/- 14% versus 68 +/- 13% diameter stenosis, respectively (mean +/- SD) and 1.1 +/- 1.1 mm2 versus 0.82 +/- 0.7 mm2 area (p = 0.35). At 8-14 days after infusion, residual stenosis was unchanged in the heparin-treated group, but it improved to 55 +/- 17% (p = 0.001) and 1.6 +/- 1.2 mm2 (p = 0.003) in the rt-PA-treated group. At 90 minutes of infusion, residual intraluminal thrombus was observed in 29 of the 52 patients (56%) with a comparably measured distribution in the two groups (p = 0.43). At 150 minutes, however, the extent of intraluminal thrombus was significantly reduced in the rt-PA-treated group as compared with the heparin-treated group (p = 0.03). In-hospital ischemic events (symptomatic reocclusion, unstable angina, or cardiovascular death) occurred in 12 patients of the heparin-treated group but only in three patients of the rt-PA-treated group (p = 0.03). Fibrinogen levels decreased to 65 +/- 21% of baseline at 90 minutes of rt-PA infusion. During the rt-PA maintenance infusion, fibrinogen fell slightly from 63 +/- 26 to 57 +/- 28% (p = 0.18). This study shows that after successful reperfusion with 1 mg/kg rt-PA during 90 minutes, a maintenance infusion of 0.8 mg/kg rt-PA during 4 hours prevents acute symptomatic coronary artery reocclusion, and it reduces the frequency of ischemic events and the severity of residual coronary artery stenosis at hospital discharge.

Aged↗
PubMed1988

Monoclonal antibody against the platelet glycoprotein (GP) IIb/IIIa receptor prevents coronary artery reocclusion after reperfusion with recombinant tissue-type plasminogen activator in dogs.

Localized thrombosis was produced in the left anterior descending (LAD) coronary artery of open chest dogs by constricting a segment so as to produce greater than 90% stenosis (reducing blood flow to 40 +/- 10% of baseline), and placing a thrombus in the segment immediately proximal to the stenosis by inducing endothelial cell injury and instilling a mixture of blood and thrombin. Intravenous infusion of recombinant tissue-type plasminogen activator (rt-PA) at a rate of 15-30 micrograms/kg per min for 30 or 60 min in eight dogs induced coronary artery reperfusion within 23 +/- 7 min (mean +/- SD), but reocclusion occurred despite heparin anticoagulation in all but one of these dogs within 7 +/- 5 min. Intravenous injection of 0.8 mg/kg of the F(ab')2 fragment of a monoclonal antibody (7E3) directed against the platelet GPIIb/IIIa receptor, prevented reocclusion in 10/10 dogs during an observation period of 2 h (P less than 0.001 vs. rt-PA alone). The antibody abolished ADP-induced platelet aggregation and markedly prolonged the bleeding time. Intravenous aspirin or dipyridamole prevented reocclusion for 1 h or more in only 2/7 and 1/6 dogs, respectively. We conclude that the monoclonal antibody is very effective in preventing reocclusion after successful thrombolysis of occluded coronary arteries with rt-PA.

Animals↗
PubMed1988

Plasminogen receptors, urokinase receptors, and their modulation on human endothelial cells.

Endothelial cells are centrally involved in regulation of fibrinolysis, and receptors for plasminogen and urokinase provide a mechanism by which cells can regulate their fibrinolytic function. Therefore, the existence and characteristics of receptors for these fibrinolytic components on cultured human umbilical vein endothelial cells were examined. We verified the presence of plasminogen receptors on these cells (Kd = 2.1 +/- 1.3 mumol/L, and 1.8 +/- 1.3 x 10(7) binding sites/cell). These binding parameters and other characteristics indicate that these receptors are closely related to the plasminogen receptors on many circulating and adherent cells. Specific binding sites that interact with two-chain urokinase of mol wt 55,000 with a dissociation constant of 2.1 +/- 1.7 nmol/L, with 2.9 +/- 2.9 x 10(5) sites/cell were also identified. Single-chain urokinase of mol wt 55,000, but not the two-chain degradation product of mol wt 33,000 bound to the cells, implicating the amino-terminal aspects of the ligand in receptor recognition. When endothelial cells were stimulated with thrombin, an agent that modulates their fibrinolytic potential, both receptor types were modestly affected; urokinase binding increased 17%, whereas plasminogen binding decreased 19%. The presence and modulation of plasminogen and urokinase receptors provide a potentially important additional mechanism by which endothelial cells may regulate fibrinolysis.

Aminocaproic Acid↗
PubMed1988

Fibrin-specific thrombolytic agents.

The mammalian fibrinolytic system comprises a proenzyme, plasminogen, which can be converted to the active enzyme plasmin, which will degrade fibrin. Plasminogen activation is mediated by plasminogen activators which are classified as either tissue-type plasminogen activator (t-PA) or urokinase-type plasminogen activator (u-PA). t-PA and single-chain u-PA (scu-PA) induce clot-specific thrombolysis, however via entirely different mechanisms. t-PA is relatively inactive in the absence of fibrin, but fibrin strikingly enhances the activation rate of plasminogen by t-PA. This is explained by an increased activity of fibrin-bound t-PA for plasminogen and not by alteration of the catalytic efficiency of the enzyme. scu-PA has a high affinity for plasminogen but, however, does not activate plasminogen in plasma in the absence of a fibrin clot, due to competitive inhibition. Fibrin-specific thrombolysis appears to be due to the fact that fibrin reverses the competitive inhibition, but this does not seem to occur via specific binding of scu-PA to fibrin. The thrombolytic efficacy and fibrin-specificity of natural and recombinant t-PA has been demonstrated in animal models of pulmonary embolism, venous thrombosis and coronary artery thrombosis. In all these studies thrombolysis and relative fibrinogen sparing effect of t-PA was recently confirmed in several multicenter clinical trials in patients with acute myocardial infarction. Specific thrombolysis by scu-PA has also been demonstrated in animal models of pulmonary embolism, venous thrombosis and coronary artery thrombosis.

Humans↗
PubMed1988

Pharmacokinetics and thrombolytic properties of deletion mutants of human tissue-type plasminogen activator in rabbits.

The following mutants of human tissue-type plasminogen activator (t-PA) were constructed by deletion mutagenesis of t-PA cDNA, expressed in Chinese hamster ovary cells and purified to homogeneity: (a) t-PA-delta FE:t-PA lacking both the fibronectin fingerlike (F) domain and the epidermal growth factor (E) domain, (b) t-PA-delta FE1X:t-PA-delta FE with the glycosylated 117Asn mutagenized to Gln, and (c) t-PA-delta FE3X:t-PA-delta FE with the three known glycosylated Asn residues replaced by Gln. The mutant and natural t-PA (Mel-t-PA obtained from melanoma cell culture) were infused intravenously for four hours into rabbits with jugular vein thrombosis at doses ranging between 0.12 and 0.75 mg/kg. Fifty percent thrombolysis, determined by interpolation, was obtained with 0.4 mg/kg Mel-t-PA, 0.37 mg/kg t-PA-delta FE, 0.2 mg/kg t-PA-delta FE1X, and 0.40 mg/kg t-PA-delta FE3X. These infusion rates resulted in plateau levels of t-PA antigen in plasma of 0.055, 2.1, 0.6, and 0.5 micrograms/mL, respectively. At 50% lysis, the residual fibrinogen 30 minutes after the end of the infusion was 100%, 81%, 100% and 85% of baseline, and the residual alpha 2-antiplasmin was 82%, 55%, 85%, and 90%, respectively. These results indicate that t-PA-delta FE1X and t-PA-delta FE3X have a specific thrombolytic activity and fibrin specificity comparable to that of Mel-t-PA. t-PA-delta FE has a comparable specific thrombolytic activity but a lower fibrin specificity than Mel-t-PA. After the end of the infusion, t-PA-related antigen disappeared from plasma with an initial t1/2 of four minutes for Mel-t-PA, 25 minutes for t-PA-delta FE, 42 minutes for t-PA-delta FE1X, and 14 minutes for t-PA-delta FE3X. It is concluded that t-PA can be modified by deletion mutagenesis to yield variants with a markedly longer half-life in the blood. Some of these variants have a specific thrombolytic activity and fibrin specificity similar to that of natural t-PA. These variants may be useful to identify the structures in t-PA responsible for its clearance, specific thrombolytic activity, and fibrin specificity in vivo.

Animals↗
PubMed1988

Fibrin-specific thrombolytic agents.

The fibrinolytic system comprises a proenzyme, plasminogen, which can be converted to the active enzyme plasmin, which will degrade fibrin. Plasminogen activation is mediated by plasminogen activators which are classified as either tissue-type plasminogen activator (t-PA) or urokinase-type plasminogen activator (u-PA). Inhibition of the fibrinolytic system may occur at the level of the activators or at the level of generated plasmin. Plasmin has a low substrate specificity and when circulating freely in the blood will degrade several proteins including fibrinogen, Factor V and Factor VIII. Plasma does, however, contain a fast-acting plasmin inhibitor, alpha 2-antiplasmin, which will inhibit free plasmin extremely rapidly (t1/2: 0.1 s) but which reacts much slower (10(3)-fold) with plasmin bound to fibrin. A "systemic fibrinolytic state" may, however, occur by extensive activation of plasminogen (conc. in plasma 2 microM) and depletion of alpha 2-antiplasmin (conc. in plasma 1 microM). Clot-specific thrombolysis therefore requires plasminogen activation restricted to the vicinity of the fibrin. Two physiological plasminogen activators, t-PA and single-chain u-PA (scu-PA) induce clot-specific thrombolysis, however via entirely different mechanisms. t-PA is relatively inactive in the absence of fibrin, but fibrin strikingly enhances the activation rate of plasminogen by t-PA. This is explained by an increased affinity of fibrin-bound t-PA for plasminogen and not by alteration of the catalytic rate constant of the enzyme. The high affinity of t-PA for plasminogen in the presence of fibrin thus allows efficient activation on the fibrin clot while no significant plasminogen activation by t-PA occurs in plasma.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

[Comparative randomized study of the effectiveness of intravenous recombinant tissue-type plasminogen activator and intravenous streptokinase in patients with acute myocardial infarct. Report of the European Cooperative Study Group for Recombinant Tissue-Type Plasminogen Activator].

In a single-blind randomised trial in patients with acute myocardial infarction of less than 6 h duration, the frequency of coronary patency was found to be higher after intravenous administration of recombinant human tissue-type plasminogen activator (rt-PA) than after intravenous streptokinase. 64 patients were allocated to 0.75 mg rt-PA/kg over 90 min, and the infarct-related coronary artery was patent in 70% of 61 assessable coronary angiograms taken 75-90 min after the start of infusion; 65 patients were allocated to 1,500,000 IU streptokinase over 60 min, and the infarct-related vessel was patent in 55% of 62 assessable angiograms. The 95% confidence interval of the difference ranges from +30 to -2% (p = 0.054). Bleeding episodes and other complications were less common in the rt-PA patients than in the streptokinase group. Hospital mortality was identical in the 2 treatment groups. At the end of the rt-PA infusion the circulating fibrinogen level was 61 +/- 35% of the starting value, as measured by a coagulation-rate assay, and 69 +/- 25% as measured by sodium sulphite precipitation. After streptokinase infusion, corresponding fibrinogen levels were 12 +/- 18% and 20 +/- 11%. In the rt-PA group only 4.5% of the fibrinogen was measured as incoagulable fibrinogen degradation products, compared with 30% in the streptokinase group. Activation of the systemic fibrinolytic system was far less pronounced with rt-PA than with streptokinase.

Adult↗
PubMed1988

[Randomized double-blind study comparing tissue-type plasminogen activator with placebo in acute myocardial infarct].

In a double-blind randomised trial 129 patients with first myocardial infarction of less than 6 h duration were allocated to treatment with human recombinant tissue-type plasminogen activator (rt-PA) given intravenously over 90 min, or to placebo infusion. Coronary angiography at the end of this infusion showed that the infarct-related vessel was patent in 61% of 62 assessable coronary angiograms in the rt-PA-treated group compared with 21% in the control group. Treatment with rt-PA was not accompanied by any major complications. In the rt-PA group the circulating fibrinogen level at the end of the catheterisation was 52 +/- 29% (mean +/- SD) of the starting value.

Adult↗
PubMed1988

Measurement of plasminogen activator inhibitor 1 in biologic fluids with a murine monoclonal antibody-based enzyme-linked immunosorbent assay.

An enzyme-linked immunosorbent assay for plasminogen activator inhibitor-1 (PAI-1) in biologic fluids was developed on the basis of two murine monoclonal antibodies raised against PAI-1 purified from HT-1080 fibrosarcoma cells. The lower limit of sensitivity of the assay in plasma is 2 ng/mL. The assay is 12 times less sensitive toward the PAI-1/human tissue-type plasminogen activator (t-PA) complex as compared with free PAI-1. The intraassay, interassay, and interdilution coefficients of variation are 5.2%, 8.0%, and 7.1%, respectively. The level of PAI-1 in platelet-poor plasma of healthy subjects is 18 +/- 10 ng/mL (mean +/- SD, n = 45). In platelet-rich plasma after freezing and thawing, 92% of PAI-1 antigen is released from platelets, whereas only 8% is found in the corresponding platelet-poor plasma. In platelet-poor plasma from healthy subjects, a linear correlation (r = 0.80) was found between PAI activity and PAI-1 antigen. In plasma approximately two thirds of the PAI-1 antigen was functionally active, whereas only 5% of the PAI-1 antigen released from platelets was active. During pregnancy a progressive increase of PAI-1 antigen levels up to three- to sixfold the control value was observed. In plasma of patients with recurrent deep vein thrombosis, PAI-1 levels were 44 +/- 20 ng/mL (mean +/- SD, n = 7), during a clinically silent phase. Four of these patients had a level above 38 ng/mL (mean +/- 2 SD of normal). The present assay, based on stable and reproducible reagents, allows the specific determination of PAI-1 antigen in biologic fluids. It may facilitate interlaboratory comparisons and be useful for further investigations of the role of PAI-1 in clinical conditions associated with impaired fibrinolysis and/or a tendency to thrombosis and investigations of the role of PAI-1 in platelets.

Animals↗
PubMed1987

Fibrinolytic response and fibrin fragment D-dimer levels in patients with deep vein thrombosis.

An enzyme-linked immunosorbent assay for fragment D-dimer was developed with the use of two monoclonal antibodies directed against specific non-overlapping antigenic determinants, present in fragment D-dimer of crosslinked fibrin but not in fragment D of non crosslinked fibrin or of fibrinogen. The lower limit of sensitivity of the assay when applied to human plasma, is 25 ng/ml. Concentration of fragment D-dimer in plasma from healthy individuals was 177 +/- 83 ng/ml (mean +/- SD). In plasma of 11 out of 12 patients with phlebographically confirmed acute deep vein thrombosis, fragment D-dimer levels were significantly increased. Fragment D-dimer was not increased in 9 out of 10 patients with recurrent idiopathic deep vein thrombosis during clinically silent episodes. Total t-PA antigen and free t-PA antigen concentrations were measured using previously developed ELISAs. Nine of the 12 patients with acute deep vein thrombosis showed a significant increase of total t-PA antigen (from 8.6 +/- 6.9 ng/ml to 21 +/- 16 ng/ml) after venous occlusion but in 3 of these free t-PA remained undetectable. Five of the 10 patients with recurrent deep vein thrombosis responded to venous occlusion with a significant increase of total t-PA antigen (from 6.7 +/- 3.2 ng/ml to 14 +/- 7.9 ng/ml) but, in all patients, free t-PA antigen remained undetectable. It is concluded that the combined assays of total and free t-PA antigen and of fragment D-dimer may be useful for the evaluation of the dynamics of the fibrinolytic system in physiological and pathological conditions.

Aged↗
PubMed1987

Fibrinolytic response to venous occlusion and fibrin fragment D-dimer levels in normal and complicated pregnancy.

The fibrinolytic response to venous occlusion was assessed in 29 women with normal or complicated pregnancy, by measurements of total t-PA and free t-PA with specific ELISAs. The release of t-PA from the vessel wall was 11 +/- 9 ng/ml in non-pregnant women (mean +/- SD, n = 6) but was markedly reduced throughout pregnancy. Following venous occlusion, free t-PA increased by 12 +/- 11 ng/ml in non-pregnant women but remained below the detection limit of 2 ng/ml towards the end of pregnancy. A markedly reduced t-PA release with absence of free t-PA was also observed during late pregnancy in patients with insulin-dependent diabetes mellitus, intra-uterine growth retardation and pre-eclampsia. Plasma levels of fragment D-dimer of cross-linked fibrin were measured with a specific ELISA in 79 pregnant women. D-dimer levels were 129 +/- 36 ng/ml (mean +/- SD, n = 8) in non-pregnant women and increased to 400 +/- 170 ng/ml (n = 25) and 440 +/- 220 ng/ml (n = 22) during the second and third trimester of pregnancy respectively. Significantly higher levels than observed in uncomplicated third trimester pregnancies were found in 3 out of 6 diabetic and in 2 out of 7 pre-eclamptic women. It is concluded that the t-PA release after venous occlusion is significantly reduced during pregnancy. In addition, released t-PA is rapidly inhibited. The levels of fragment D-dimer increase during pregnancy, suggesting that, notwithstanding the marked impairment of the fibrinolytic response to venous occlusion, the fibrinolytic system remains functionally active.

Constriction↗
PubMed1987

Activation with plasmin of tow-chain urokinase-type plasminogen activator derived from single-chain urokinase-type plasminogen activator by treatment with thrombin.

Thrombin converts single-chain urokinase-type plasminogen activator (scu-PA) to an inactive two-chain derivative (thrombin-derived tcu-PA) by hydrolysis of the Arg-156--Phe-157 peptide bond. In the present study, we show that inactive thrombin-derived tcu-PA (specific activity 1000 IU/mg) can be converted with plasmin to active two-chain urokinase-type plasminogen activator (specific activity 43,000 IU/mg) by hydrolysis of the Lys-158--Ile-159 peptide bond. This conversion follows Michaelis-Menten kinetics with a Michaelis constant Km of 37 microM and a catalytic rate constant k2 of 0.013 s-1. The catalytic efficiency (k2/Km) for the activation of thrombin-derived tcu-PA by plasmin is about 500-fold lower than that for the conversion of intact scu-PA to tcu-PA. tcu-PA, generated by plasmin treatment of thrombin-derived tcu-PA, has similar properties to tcu-PA obtained by digestion of intact scu-PA with plasmin (plasmin-derived tcu-PA); its plasminogen activating potential and fibrinolytic activity in an in vitro plasma clot lysis system appear to be unaltered. These observations confirm that the structure of the NH2-terminal region of the B chain of u-PA is an important determinant for its enzymatic activity, whereas that of the COOH-terminal region of the A chain is not.

Amino Acids↗
PubMed1987

Immunological identity of heparin-dependent plasma and urinary protein C inhibitor and plasminogen activator inhibitor-3.

Purified plasma and urinary protein C inhibitors (PCI) formed heparin-dependent complexes with activated protein C (APC) which were detected by immunoblotting after nondenaturing gel electrophoresis. Bands representing APC.PCI complexes were also seen on immunoblots after incubation of plasma with APC and heparin. The same immunoblot pattern of complexes was detected by three different methods: method A, monoclonal antibody to plasminogen activator inhibitor-3 (PAI-3, urinary urokinase inhibitor) + 125I-labeled anti-mouse IgG; method B, polyclonal antibodies to PCI + 125I-labeled purified plasma PCI; and method C, monoclonal antibody to protein C + 125I-protein C. Plasma depleted of PAI-3 by immunoadsorption with insolubilized monoclonal antibody to PAI-3 showed no detectable antigen or complexes with APC as visualized by methods A or B. This PAI-3-depleted plasma had less than 10% of the heparin-dependent inhibitory activity of normal plasma toward APC. Purified plasma PCI was fully reactive in an enzyme-linked immunoabsorbent assay for PAI-3, and plasma and urinary PCI inhibited urokinase activity in a heparin-dependent manner. These data indicate that heparin-dependent plasma and urinary PCI and PAI-3 are immunologically and functionally very similar if not identical. This observation identifies a new interrelation between the protein C anticoagulant and the fibrinolytic systems. In addition, plasma contains a heparin-independent inhibitor of APC which is not immunologically related to plasma PCI or to PAI-3.

Antibodies, Monoclonal↗