Kinetic analysis of plasminogen activation by purified plasma kallikrein

Kinetics of plasminogen activation by purified activated plasma kallikrein have been studied in a purified system using Glu-plasminogen as a substrate. A synthetic paranitroanilide substrate was used for quantification of the formed plasmin. In that system kallikrein cleaved plasminogen with a Km value of 0.56 microM, a kcat of 1.6 X 10(-4) s-1 and a catalytic efficiency kcat/Km of 2.7 X 10(-4) s-1 microM-1. Addition of CNBr fibrinogen fragments resulted in an increase of Km to 1.18 microM, an increase of kcat to 5.1 X 10(-4) s-1 and an increase in the catalytic rate constant kcat/Km to 4.3 X 10(-4) s-1 microM-1. Addition of purified high molecular weight kininogen had no effect on the kinetics of plasminogen activation whether or not stimulating fibrinogen fragments were present. A stimulating effect of fibrinogen fragments could also be shown for the cleavage of the low molecular weight paranitroanilide substrate H-D-Pro-Phe-Arg-pNA by kallikrein; in that system the kcat for substrate cleavage by kallikrein increased from 200 s-1 to 280 s-1, while the Km value remained unchanged. From these data it can be concluded that based on enzyme kinetic studies plasminogen activator activity of purified plasma kallikrein is about 1/1000 of that of high molecular weight urokinase and is only slightly influenced by addition of stimulating fibrinogen fragments. Addition of high molecular weight kininogen does not affect plasminogen activator activity of purified plasma kallikrein.     

The effect of the carboxy-terminal lysine of urokinase on the catalysis of plasminogen activation.

When single-chain pro-UK is activated by plasmin or kallikrein, the Lys158-Ile159 bond is cleaved, leaving a C-terminal lysine on the A-chain (Lys-UK). Two-chain, high molecular weight urokinase (UK) purified from urine, however, has been shown to contain a phenylalanine residue as the C-terminal of the A-chain (Phe-UK). Since C-terminal lysine residues have a strong binding affinity for plasminogen that may promote its activation, we undertook kinetic studies comparing plasminogen activation by Lys- and Phe-UK. A two-stage method was employed in order to minimize factors known to interfere with plasminogen activation and plasmin determination. The Lys-UK was prepared by plasmin activation of pro-UK purified from human fetal kidney cell culture medium. The Phe-UK was prepared by carboxypeptidase B (CpB) treatment of Lys-UK. Removal of the C-terminal lysine of Lys-UK by CpB produced small but significant increases in the Michaelis constants for the activation of both Glu- and Lys-plasminogen. The apparent Michaelis constants for Glu-plasminogen activation by Lys- and Phe-UK were 3.7 microM +/- .36 microM and 5.9 microM +/- .70 microM, respectively and the Michaelis constants for Lys-plasminogen activation by Lys- and Phe-UK were 5.4 microM +/- .72 microM and 15.2 microM +/- 1.4 microM, respectively. The catalytic efficiency (kcat/Km) of Lys-UK was approximately 2-fold greater than that of Phe-UK for the activation of either Glu- or Lys-plasminogen. When the fibrinolytic activities of Lys- and Phe-UK were compared in a plasma milieu no significant differences were detected. In conclusion, the findings indicate that the C terminal lysine on the A-chain of UK significantly promotes the catalysis of plasminogen in a purified system. However, the higher catalytic efficiency of Lys-UK was not found to induce significant acceleration of clot lysis at pharmacological concentrations in plasma.     

A precise and rapid microtitre plate clot lysis assay: methodology, kinetic modeling and measurement of catalytic constants for plasminogen activation during fibrinolysis

A rapid and precise turbidimetric clot lysis assay employing a microtitre plate reader and personal computer is described in detail. The use of such widely available instrumentation, the convenience and rapid throughput suggest the assay could be developed as a reference method with which to measure the potency of tissue plasminogen activator (t-PA) in conjunction with the WHO reference preparation. The method has been used to investigate molecular parameters involved in fibrinolysis. Aggregation status of the fibrin does not appear to influence the mechanism of plasminogen activation and clot lysis by plasmin. High ratios of plasminogen to fibrin resulted in a change in clot turbidity and in a change in the lysis profile of turbidity versus time. This is probably the result of plasminogen binding to fibrin and consequent restriction of the access of plasmin to its sites of cleavage in the fibrin. A simple model is proposed, and equations have been derived, for the kinetics of lysis which adequately describe the mechanism and which are confirmed by experimental data. This model results in estimates of the Km and kcat for the activation of plasminogen by t-PA during clot lysis of approximately 150 nM and 0.1 s-1, respectively, in excellent agreement with published values. The assay should therefore prove useful in quantitative evaluations of the molecular phenomena occurring during fibrinolysis. The more rapid activation of lys-plasminogen than glu-plasminogen by t-PA was confirmed. However, evidence was obtained that the lys-form binds more tightly to fibrin by the same factor. This observation suggested that the appropriate substrate in the kinetic model is fibrin-bound plasminogen.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biological and thrombolytic properties of proenzyme and active forms of human urokinase--IV. Variability in fibrinolytic response of plasma of several mammalian species

The fibrinolytic and fibrinogenolytic properties of recombinant pro-urokinase (Rec-pro-UK) and recombinant urokinase (Rec-UK) were compared with those of natural urokinase (Nat-UK) and of tissue-type plasminogen activator (t-PA) in an in vitro system consisting of 125I-labeled autologous plasma clots immersed in plasma of humans, five primate species, dogs, rabbits and pigs. With each of the four plasminogen activators, a dose-dependent clot lysis was observed, the degree of which differed, however, very markedly from one species to the other. At a concentration of 100 IU/ml of urokinase extensive plasma clot lysis was obtained in plasma of man, Macaca mulatta, Macaca fascicularis and Macaca radiata, while the plasma clots of Papio cynocephalus, Papio anubis and rabbit, dog and pig were much more resistant to lysis. No significant differences in the extent of lysis were observed between Rec-pro-UK and Rec-UK nor between Rec-UK and Nat-UK. Comparable degrees of lysis were obtained with t-PA at 3- to 5-fold lower concentrations. Lysis with Rec-UK or Nat-UK was always associated with extensive activation of the fibrinolytic system in plasma, evidenced by fibrinogen breakdown and plasminogen activation and alpha 2-antiplasmin consumption. With t-PA, extensive clot lysis was obtained in the absence of fibrinolytic activation in the plasma. With Rec-pro-UK the response was intermediate; at high concentrations (200 IU/ml) extensive lysis in the reactive species was associated with fibrinogen consumption, while at intermediate concentrations (50-100 IU/ml) significant clot lysis was obtained in the reactive species in the absence of marked activation of the fibrinolytic system in the plasma.     

Effects of kringles derived from human plasminogen on fibrinolysis in vitro

Plasminogen kringle 1+2+3 (K1-3) containing lysine-binding sites inhibited the reaction of plasmin with alpha 2-plasmin inhibitor (alpha 2PI), in a rate assay using a synthetic chromogenic substrate, S-2251. However, K1-3 did not inhibit the reaction to any degree between alpha 2PI and mini-plasmin which lacked the kringle 1 to 4 portion of plasmin. These results suggest that K1-3 blocked the binding of alpha 2PI to the lysine-binding site of plasmin. In the urokinase (UK)-induced fibrinolysis, K1-3 shortened the human plasma clot lysis time at low concentration (0.5-6 microM), and prolonged the lysis time at a high concentration (20 microM). Similar results were obtained in the lysis time of a fibrin clot consisting of plasminogen, fibrinogen and alpha 2PI isolated from human plasma. The kringle 4 (K4) of human plasminogen did not accelerate human plasma clot lysis at any concentration (1.2-24.1 microM). Furthermore, in the tissue plasminogen activator (TPA)-induced fibrinolysis, K1-3 also shortened both the lysis time of human plasma clot and fibrin clot as observed in UK-induced fibrinolysis, but K4 did not. The above findings indicate that the reaction of alpha 2PI with the lysine-binding site of plasmin is involved in the inhibition of plasmin activity by alpha 2PI, and in the presence of an inhibitor of this reaction, the balance of coagulofibrinolytic activity in plasma will be shifted towards the fibrinolytic side.     

Absence of synergism between tissue-type plasminogen activator (t-PA), single-chain urokinase-type plasminogen activator (scu-PA) and urokinase on clot lysis in a plasma milieu in vitro

A potential synergic effect of tissue-type plasminogen activator (t-PA), single-chain urokinase-type plasminogen activator (scu-PA) or urokinase on clot lysis was investigated in a whole human plasma system in vitro. The system consisted of a human plasma clot labeled with 125I-fibrinogen, immersed in citrated whole human plasma, to which the thrombolytic agents were added. Clot lysis was quantitated by measurement of released 125I, and activation of the fibrinolytic system in the surrounding plasma by measurements of fibrinogen and alpha 2-antiplasmin. t-PA, scu-PA and urokinase induced a dose-dependent and time-dependent clot lysis; 50 percent lysis after 2 h was obtained with 5 nM t-PA, 20 nM scu-PA and 12 nM urokinase. At these concentrations no significant activation of the fibrinolytic system in the plasma was observed with t-PA and scu-PA, whereas urokinase caused significant alpha 2-antiplasmin consumption and concomitant fibrinogen degradation. The shape of the dose-response curves was different; t-PA and urokinase showed a log linear dose-response whereas that of scu-PA was sigmoidal. Combinations of t-PA and scu-PA, of t-PA and urokinase or of scu-PA and urokinase at thrombolytic doses of each showed no synergism for thrombolysis. Fifty percent clot lysis in 2 h was obtained at total concentrations of the combined agents of 5 to 15 nM with molar ratios ranging from 1:4 to 4:1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetic analyses of the activation of Glu-plasminogen by urokinase in the presence of fibrin, fibrinogen or its degradation products

The kinetics of the activation of Glu-plasminogen (Glu-plg) and Lys-plasminogen (Lys-plg) by urokinase (UK) were studied in purified systems. The activation of plasminogen by UK in the purified systems followed Michaelis-Menten kinetics with a Michaelis constant (Km) of 1.45 microM and a catalytic rate constant (kcat) of 0.93/sec for Glu-plg as compared to 0.25 microM (Km) and 0.82/sec (kcat) for Lys-plg. In the presence of fibrin and fibrinogen or its plasmin degradation products (fragment D and fragment E), Km for Glu-plg hardly changed, whereas kcat for Glu-plg increased. Effect on increase in kcat was in the order of fibrin greater than fibrinogen greater than D greater than E. Fibrin, fibrinogen, D and E did not influence the activation of Lys-plg by UK. These results indicate that Glu-plg bound to fibrin, fibrinogen, D or E becomes easily activatable by UK. The activation of Lys-plg, however, is not influenced in the presence of fibrin, fibrinogen, D or E.     

Effects of tranexamic acid, CIS-AMCHA, and 6-aminohexanoic acid on the activation rate of plasminogen by urokinase in the presence of clot

When human plasma was mixed with 50 units of urokinase and clotted with thrombin in the presence of S-2251, the hydrolysis of S-2251 in the clot was higher than that in the plasma. The presence of 10 μM of tranexamic acid resulted in decrease in the activation rate of plasminogen in the clot but not in the plasma. Further increase in its concentration resulted in marked increase in plasminogen activation in both clot and plasma. The addition of 6-aminohexanoic acid or cis-AMCHA showed changes in the rate of plasminogen activation similar to that shown in the presence of tranexamic acid. Fifty μM of tranexamic acid totally inhibited urokinase induced clot lysis, while 1 mM of cis-AMCHA and 6-aminohexanoic acid totally inhibited clot lysis. The presence of 1 mM of these ω-aminoacids remarkably increased the activation of plasminogen by urokinase, but no clot lysis was observed.     

Stimulation of the plasma fibrinolytic activity in rats by the prostacyclin analogue CG 4203

In anesthetized rats the intravenous infusion (15-120 min) of the prostacyclin analogue CG 4203 (0.215-2.15 micrograms.kg-1.min-1) resulted in a time and dose dependent shortening of the ex vivo euglobulin clot lysis time (ECLT). This effect that appeared to be significant already in the non-hypotensive dose range of CG 4203, was still existing at 2 hours after cessation of the infusion. The phosphodiesterase inhibitor theophylline (4.64 mg.kg-1 i.v.) potentiated the ECLT shortening effect of CG 4203. Even the highest dose of CG 4203 did not change the plasma fibrinogen levels. In contrast to low molecular weight urokinase (100 PU/ml) CG 4203 (10 microM) did not shorten the in vitro lysis of preformed euglobulin clots from untreated rats nor did it reduce the 125J-fibrin content of human thrombi in the Chandler loop system. From these results it is concluded that intravenously infused CG 4203 increases the plasma fibrinolytic activity in rats by a c-AMP dependent mechanism, probably by release of plasminogen activator. Direct urokinase like activation of plasminogen does not occur with CG 4203. The relevance of this activity is discussed with respect to the CG 4203 treatment of occlusive vascular diseases.     

Specific fibrinolytic properties of different molecular forms of pro-urokinase from a monkey kidney cell culture

The specific fibrinolytic properties of both high molecular weight (55 kd) and low molecular weight (30 kd) pro-urokinase from a monkey kidney cell culture were evaluated in a plasma clot lysis system and compared with those of human urokinase. The system was composed of a radiolabelled plasma clot immersed in plasma containing the fibrinolytic agent. On unit base, 55 kd pro-urokinase was approximately 1.5 times more effective in lysing the clot than 30 kd pro-urokinase and equally effective as urokinase. In contrast to urokinase, both pro-urokinase forms induced clot lysis without degrading fibrinogen in the surrounding plasma. However, a considerable activation of the fibrinolytic system in the plasma occurred as a large amount of ₂-antiplasmin was consumed, indicating that pro-urokinase was not fully fibrin-specific. Quenching antibodies against tissue-type plasminogen activator (t-PA) added to the plasma clot lysis system retarded but did not prevent pro-urokinase-induced clot lysis. This indicated that not only was t-PA in plasma involved in the activation of pro-urokinase (probably via plasmin), but that an additional mechanism also existed.     

Analysis of the activated partial thromboplastin time test using mathematical modeling

Activated partial thromboplastin time (APTT) is a laboratory test for the diagnosis of blood coagulation disorders. The test consists of two stages: The first one is the preincubation of a plasma sample with negatively charged materials (kaolin, ellagic acid etc.) to activate factors XII and XI; the second stage begins after the addition of calcium ions that triggers a chain of calcium-dependent enzymatic reactions resulting in fibrinogen clotting. Mathematical modeling was used for the analysis of the APTT test. The process of coagulation was described by a set of coupled differential equations that were solved by the numerical method. It was found that as little as 2.3 x 10(-9) microM of factor XIIa (1/10000 of its plasma concentration) is enough to cause the complete activation of factor XII and prekallikrein (PK) during the first 20 s of the preincubation phase. By the end of this phase, kallikrein (K) is completely inhibited, residual activity of factor XIIa is 54%, and factor XI is activated by 26%. Once a clot is formed, factor II is activated by 4%, factor X by 5%, factor IX by 90%, and factor XI by 39%. Calculated clotting time using protein concentrations found in the blood of healthy people was 40.5 s. The most pronounced prolongation of APTT is caused by a decrease in factor X concentration.     

Kinetic analyses of the enhancement of the activities of t-PA induced by the presence of monoclonal antibody (C9-5)

Nine monoclonal antibodies (Mab) against two chain tissue plasminogen activator (t-PA(W] were obtained. The effects of these Mabs on the enzymatic activities of one chain t-PA (t-PA(TD] and two chain t-PA (t-PA(W] were examined by incubating t-PA and Mabs with S-2288, or with plasminogen (plg) and S-2251 in the presence or absence of fibrin. One of Mabs called C9-5 significantly enhanced the activities of t-PA, which were kinetically analyzed. The kinetic analyses of the hydrolysis of S-2288 by t-PA showed increase in kcat from 5.17/sec to 7.75/sec in the presence of 75 micrograms/ml of C9-5 without change in Km. When Glu-plg was activated by t-PA(TD) in the absence of fibrin, Km decreased from 2 microM to 0.17 microM in the presence of C9-5 without change in Vmax. The addition of fibrin resulted in further decrease in Km from 0.133 microM to 0.077 microM. When Lys-plg was activated by t-PA(TD) in the absence of fibrin, Km decreased from 0.408 microM to 0.185 microM in the presence of C9-5, and kcat increased from 0.131 sec-1 to 0.465 sec-1. The presence of fibrin further increased kcat of the activation of Lys-plg. Similar results were obtained when plasminogen was activated by t-PA(W). Mab, C9-5, was shown to bind to B-chain of t-PA by immunoblotting. These results suggest that a dimolecular complex of t-PA and C9-5 has a higher affinity to plasminogen in the presence or absence of fibrin.     

Differential effect of platelets on plasminogen activation by tissue plasminogen activator, urokinase, and streptokinase

In this report, we have examined the effects of platelets on plasminogen activation by different activators. Platelets enhance activation of plasminogen by both 1- and 2-chain tissue plasminogen activator (t-PA). The primary effect of platelets is to lower the Km with a corresponding 5-8-fold increase in the kcat/Km. The effect is saturable with respect to the platelet concentration. Platelets enhance activation of both glu- and lys-plasminogen by t-PA. Platelets have no effect on plasminogen activation by streptokinase, and high and low molecular weight urokinase. Thus, there are marked differences in the effects of platelets on plasminogen activation depending on the plasminogen activator. These differences are likely to reflect differences in the interaction between platelets and the plasminogen activators.     

Inhibition of activated haceman factor (factor XIIa) by an inhibitor of the plasminogen activation (PA inhibitor)

Purified preparations of an inhibitor which prevents one or more reactions leading to the activation of plasminogen (PA inhibitor) but with no effect on plasmin have been tested for their effects on factors XII, XIIa, IXa and Xa. The PA inhibitor was found to be a fast reacting inhibitor of factor XIIa but had no effects on factors XII, XIa, IXa or Xa. The PA inhibitor also blocked the kaolin induced generation of fibrinolytic activity initiated by factor XIIa and a crude euglobulin fraction prepared from factor XII deficient plasma. These data indicate that the PA inhibitor of human plasma readily inactivates factor XIIa as assayed in two biological systems.     

A comparison of the abilities of plasma kallikrein, beta-Factor XIIa, Factor XIa and urokinase to activate plasminogen

In order to compare the relative potencies of plasma kallikrein, beta-Factor XIIa, Factor XIa and urokinase as plasminogen activators, plasminogen activation by these proteins was studied using a radiolabeled fibrin plate assay. Urokinase was approximately 20,000 times more active than kallikrein or Factor XIa and 300,000 times more active than beta-Factor XIIa. Kallikrein and Factor XIa were approximately equal in plasminogen activator activity and were 20 times more potent than beta-Factor XIIa.     

Correlation between progressive adsorption of plasminogen to blood clots and their sensitivity to lysis

The binding of plasminogen to preformed human plasma clots immersed in citrated human plasma was measured and correlated with the sensitivity of these clots to lysis with recombinant tissue-type plasminogen activator (rt-PA), recombinant single-chain urokinase-type plasminogen activator (rscu-PA) or two chain urokinase-type plasminogen activator (tcu-PA, urokinase). When 0.15 ml plasma clots were compressed mechanically to about 1% of their original weight, and immersed in 0.15 ml plasma, 131I-labeled native plasminogen (Glu-plasminogen) adsorbed progressively from the plasma milieu onto the clot; binding was 3 +/- 1% (n = 10) after 1 h, 7 +/- 1% after 12 h and 12 +/- 1% after 48 h. This was associated with an increased sensitivity of the clot to lysis; 50% clot lysis in 4 h was obtained with 65 +/- 5 ng/ml (n = 3) rt-PA before and 30 +/- 5 ng/ml (n = 3) after 48 h preincubation in plasma (p less than 0.01), with corresponding values of 660 +/- 55 ng/ml (n = 3) and 280 +/- 25 ng/ml (n = 3) for rscu-PA, (p less than 0.01), and 800 +/- 85 ng/ml (n = 3) and 270 +/- 35 ng/ml (n = 3) for urokinase (p less than 0.01). Additional binding of plasminogen and increased sensitivity to lysis were reduced or abolished when the clot was preincubated in plasminogen-depleted or in t-PA-depleted plasma, or when 20 mM 6-aminohexanoic acid or 2,000 KIU/ml aprotinin were added.(ABSTRACT TRUNCATED AT 250 WORDS)     

Assembly of high molecular weight kininogen and activation of prekallikrein on cell matrix

Investigations determined if extracellular matrix of endothelial cells (EC) is a platform for HK assembly and PK activation. In buffers containing bovine serum albumin, biotin-HK binding to ECV304 cells or their matrix requires > or = 50 microM added Zn2+. Ortho-phenanthroline or a HK domain 5 peptide blocks HK binding. Binding to umbilical vein EC or matrix, but not ECV304 cells or matrix, is mediated by cytokeratin 1. Biotin-HK binds to ECV304 cells or matrix with a Kd of 15.8 or 9.0 nM and a Bmax of 2.6 x 10(7) or 2.4 x 10(7) sites/cell, respectively. PK activation on ECV304 cells or matrix is blocked by antipain or SBTI and corn trypsin inhibitor partially inhibits kallikrein formation. PK activation occurs on ECV304 cells or matrix prepared without serum or in human factor XII deficient serum, indicating that the PK activator is not factor XIIa. EC matrix promotes plasminogen activation after the assembly of HK, PK and pro-urokinase. These studies indicate that matrix of various EC has the ability to assemble HK allowing for PK activation and subsequent activities.     

Identification and characterization of the plasma kallikrein-kinin system inhibitor, haemaphysalin, from hard tick, Haemaphysalis longicornis

The plasma kallikrein-kinin system inhibitor, haemaphysalin, from the hard tick, Haemaphysalis longicornis, was identified. It was found that haemaphysalin inhibited activation of the plasma kallikrein-kinin system by interfering with reciprocal activation between factor XII and prekallikrein. It did not, however, inhibit amidolytic activities of factor XIIa and kallikrein. Direct binding assay indicated that factor XII/XIIa and high molecular weight kininogen (HK) are the target molecules of haemaphysalin, and that Zn2+ ions are involved in the interactions of haemaphysalin with these target molecules. This suggests that haemaphysalin interacts with target molecules by recognizing their conformational changes induced by Zn2+ ions. Furthermore, haemaphysalin interacted with the fibronectin type II domain and domain D5, the cell binding domains of factor XII and HK, respectively. This finding suggests that haemaphysalin interferes with the association of factor XII and the prekallikrein-HK complex with a biologic activating surface by binding to these cell-binding domains, leading to inhibition of the reciprocal activation between factor XII and prekallikrein.     

Euglobulin clot lysis induced by tissue-type plasminogen activator is reduced in subjects with increased levels of lipoprotein (a).

Several reports have evaluated the in vitro effect of lipoprotein(a) [Lp(a)] levels on the fibrinolytic system, suggesting that high Lp(a) levels may inhibit fibrinolysis by competing for plasminogen binding in different systems. We have studied plasminogen activation induced by tissue-type plasminogen activator (t-PA), as well as other fibrinolytic parameters, in 25 subjects with Lp(a) levels greater than 30 mg/dl and the results were compared with those found in 23 subjects with Lp(a) less than 30 mg/dl. Both groups were similar in age, sex distribution, living habits and lipid pattern. Plasminogen activation, when measured by t-PA-induced euglobulin clot lysis, was significantly decreased in the group with elevated Lp(a) levels (lysis time, 16.7 +/- 3.3 min) compared with the group with low Lp(a) levels (11.8 +/- 2.0 min), although 8 of the 25 subjects with high Lp(a) levels showed plasminogen activation within the range of the control group. A positive significant correlation between Lp(a) levels and t-PA-induced euglobulin clot lysis time was found. No statistical differences were demonstrated between groups for the other fibrinolytic parameters studied. Addition of purified Lp(a) to the euglobulin fraction or to plasma resulted in a decrease in euglobulin clot lysis. The present study shows that t-PA induced plasminogen activation is decreased in individuals with high circulating levels of Lp(a) supporting the hypothesis that Lp(a) may interfere with the physiological functions of plasminogen.     

Studies on the influence of reactants and buffer environment on clot lysis induced by human plasminogen activators

Clot lysis induced by tissue plasminogen activator and urokinase has been studied and the influences of pH, ionic environment and reactant concentrations have been determined. Both pH and ionic strength strongly affect the rate of clot lysis and distinctly different dependency profiles are obtained for tissue plasminogen activator and urokinase. Variations in concentration of plasminogen also profoundly affect the rate of clot lysis, maxima being obtained at different plasminogen concentrations depending on the concentration of fibrinogen. For urokinase, these maxima occurred at about a tenfold higher concentration of plasminogen than for the tissue plasminogen activator. The lysis times are directly dependent on the concentration of fibrinogen. Variation in thrombin concentration did not significantly affect the lysis times. Suitable conditions for the assays of tissue plasminogen activator and urokinase are suggested.