Determination of plasmin-alpha 2-antiplasmin complex in plasma samples by means of a radioimmunoassay

Immunization of a goat with partially reduced and S-carboxymethylated plasmin B-chain-alpha 2-antiplasmin complex resulted in a large population of antibodies with rather high specificity towards the complex. These antibodies do not react with plasminogen or plasmin in complex with other inhibitors than alpha 2-antiplasmin. However, they react fully with native alpha 2-antiplasmin, but a 200-fold higher concentration, as compared to plasmin-alpha 2-antiplasmin complex, is needed to obtain a similar displacement curve in a double-antibody radioimmunoassay. The results indicate a conformational change in the vicinity of the reactive site in alpha 2-antiplasmin, as a result of complex formation with plasmin. A method for determination of plasmin-alpha 2-antiplasmin complex in plasma has been elaborated using the described radioimmunoassay. About 1.5 mg plasmin-alpha 2-antiplasmin complex/l can be detected, which equals the condition when about 1% of the alpha 2-antiplasmin in plasma is in complex with plasmin. In normal individuals plasmin-alpha 2-antiplasmin complex could be detected only rarely. However, patient with acute processes, as evidenced by high fibrinogen levels, surgical patients postoperatively or patients with malignancy have often detectable levels.     

Determination of tissue plasminogen activator and its "fast" inhibitor in plasma

We describe efficient, accurate methods for specific determination of tissue plasminogen activator (t-PA, EC 3.4.21.31) and its "fast" inhibitor in plasma. In this coupled assay, a sample containing t-PA is incubated with plasminogen, a plasmin (EC 3.4.21.7) substrate of low Km and high Kcat, and fibrin as a stimulator. The inhibitor of t-PA is determined by incubating the sample with a known amount of t-PA in excess, then determining the residual t-PA. Both t-PA and t-PA inhibitor can be determined in many samples simultaneously within a few hours. These assays are modifications of procedures described by us (Clin Chim Acta 1983;127:279-88 and Thromb Res 1983;31:427-36). Their accuracy as assessed by analytical recovery of pure t-PA added to blood samples (91 +/- 4%) or of partly purified inhibitor added to plasma samples (102 +/- 10%) is satisfactory, as is their precision. For the t-PA assay the CV was 1.6% (within run) or 4.1% (between run). The corresponding values for the inhibitor assay were 4.5% (within run) or 8.4% (between run) if the inhibitor concentration exceeded 3 arb. units/mL.     

Measurement of different forms of tissue plasminogen activator in plasma

BACKGROUND: We evaluated assays to measure both total tissue plasminogen activator (tPA) and the three principle forms of tPA in plasma: active tPA, tPA complexed with plasminogen activator inhibitor type 1 (PAI-1), and tPA complexed with C1-inhibitor. METHODS: Active tPA was measured by use of an indirect amidolytic assay and immunofunctional assays. tPA/PAI-1, tPA/C1-inhibitor, and total tPA antigen were measured by use of microtiter plates coated with anti-tPA antibodies and, respectively, anti-PAI-1, anti-C1-inhibitor, and anti-tPA antibodies conjugated to peroxidase. RESULTS: The immunofunctional tPA assay detected 1 U/L (0.001 U/mL) tPA and recovered 108% +/- 12% of active tPA added to samples containing high (mean, 60 000 IU/L) PAI-1 activities vs a detection limit of 10 U/L (0.01 U/mL) and 13% +/- 25% recovery for the indirect amidolytic tPA activity assay. For measurement of tPA/PAI-1 complex, polyclonal anti-PAI-1 conjugates recovered 112% +/- 20% of the expected tPA/PAI-1 vs recovery of only 38% +/- 16% when monoclonal anti-PAI-1 conjugates were used. Of three methods tested, two total tPA antigen assays correlated well (r(2) = 0.85) and showed recoveries near 100%, whereas the third method showed lower correlations, higher intercepts, and falsely high recovery. A single anti-tPA capture antibody that performed the best in the individual assay evaluations was used to measure the different forms of tPA in 22 samples with a range of tPA and PAI-1 values. The sum of the molar concentrations of active tPA, tPA/PAI-1, and tPA/C1-inhibitor using the optimized methods was equal to 94% +/- 7% of measured total tPA. CONCLUSION: Optimized assays based on a single anti-tPA capture antibody can be used to accurately measure the major forms of tPA in plasma.     

Comparison of plasminogen activator inhibitor activity and antigen in plasma samples

A double antibody radioimmunoassay for determination of plasminogen activator inhibitor (PA-inhibitor) in plasma samples has been developed. The reliability of the method as assessed by determining the specificity, the accuracy, the detectability and the variability seems sufficient for use in clinical practice. The correlation between PA-inhibitor antigen as measured with this method and PA-inhibitor activity as measured with a spectrophotometric assay in 111 patients with thrombotic diseases was very good (r = 0.89). As calculated from the regression line or from the mean activity and mean antigen values a specific activity of about 800,000-900,000 arb U/mg was obtained for PA-inhibitor in these samples. In 15 healthy individuals a similar figure was obtained. The results suggest that PA-inhibitor in most plasma samples is fully active or close to fully active. PA-inhibitor activity and PA-inhibitor antigen have also been measured after venous occlusion. The data suggest that small amounts of PA-inhibitor is released on venous occlusion, but at the same time an inactivation takes place, most likely due to the formation of enzymatically inactive complex with simultaneously released t-PA.     

The regulation by fibrinogen and fibrin of tissue plasminogen activator kinetics and inhibition by plasminogen activator inhibitor 1

BACKGROUND: Tissue plasminogen activator (tPA) is unusual in the coagulation and fibrinolysis cascades in that it is produced as an active single-chain enzyme (sctPA) rather than a zymogen. Two chain tPA (tctPA) is produced by plasmin but there are conflicting reports in the literature on the behaviour of sc- and tctPA and little work on inhibition by the specific inhibitor plasminogen activator inhibitor-1 (PAI-1) under physiological conditions. OBJECTIVES: To perform a systematic study on the kinetics of sctPA and tctPA as plasminogen activators and targets for PAI-1. METHODS: Detailed kinetic studies were performed in solution and in the presence of template stimulators, fibrinogen and fibrin, including native fibrin and partially digested fibrin. Numerical simulation techniques were utilized to cope with the challenges of investigating kinetics of activation and inhibition in the presence of fibrin(ogen). RESULTS: Enzyme efficiency (k(cat)/K(m)) was higher for tctPA than sctPA in solution with chromogenic substrate (3-fold) and plasminogen (7-fold) but in the presence of templates, such as fibrinogen and native or cleaved fibrin, the difference disappeared. sctPA was more susceptible to PAI-1 in buffer solution and in the presence of fibrinogen; however, in the presence of fibrin, PAI-1 inhibited more slowly and there was no difference between sc and tctPA. CONCLUSIONS: Fibrinogen and fibrin modulate the activity of tPA differently in regard to their activation of plasminogen and inhibition by PAI-1. Fibrinogen and fibrin stimulate tPA activity against plasminogen but fibrin protects tPA from PAI-1 to promote fibrinolysis.     

Lipoprotein(a), tissue plasminogen activator and plasminogen activator inhibitor 1 levels in hyperlipidaemic patients in Kuwait

Plasma levels of lipoprotein(a) [Lp(a)], tissue plasminogen activator (tPA) and plasminogen activator inhibitor type 1 (PAI-1) were assessed in addition to anthropometry and levels of glucose, total cholesterol, triglycerides, high-density lipoprotein (HDL), low-density lipoprotein (LDL) and apo A1 and B in 73 patients (36 men and 37 women) with primary hyperlipidaemia (group NDHL) in Kuwait. Lp(a) levels (212 mg L^?1, 8–600 mg L^?1, median and range) were similar to those obtained in a matched group of 32 non-insulin-dependent diabetes mellitus (NIDDM) patients with hyperlipidaemia (218 mg L^?1, 50–610 mg L^?1) and slightly higher, although not significantly so (P = 0.06), than levels seen in 68 healthy normolipidaemic control subjects (182 mg L^?1, 70–488 mg L^?1). tPA levels (8.4 ng mL^?1, 3.8–18.4 ng mL^?1, median and range) in group NDHL were lower than in the diabetic group (11.4 ng mL^?1, 5.2–14.2 ng mL^?1) but higher than in the healthy control subjects (7.4 ng mL^?1, 2.8–12.6 ng mL^?1). PAI-1 levels in group NDHL (40.4 ng mL^?1, 8.6–55 ng mL^?1, median and range) were higher than in the control subjects (32.5 ng mL^?1, 14.6–46.4 ng mL^?1) but lower than in diabetic patients (43.8 ng mL^?1, 15.6–55 ng mL^?1). Hyperlipidaemia phenotype (hypercholesterolaemia or hypertriglyceridaemia) did not influence tPA and PAI-1 levels, but Lp(a) levels were significantly lower with hypertriglyceridaemia. Gender, cigarette smoking and racial origin (Kuwaitis, other Arabs or South Asians) did not affect Lp(a), tPA and PAI-1 levels, but tPA levels were higher in postmenopausal subjects. Low-density lipoprotein (LDL) levels (whether in total cholesterol or as apo B) correlated significantly (P < 0.05) with Lp(a) levels. tPA levels were correlated with age and the plasma levels of glucose and uric acid (P < 0.05); this correlation with glucose may explain the high levels associated with diabetes, whereas the age association might account not only for the differences observed between group NDHL and the younger control group but also for the higher levels in the postmenopausal women. PAI-1 levels correlated with tPA and triglyceride (TG) levels in the groups of subjects (normo- and hyperlipidaemic). In the normolipidaemic control group, the significant associations of tPA and PAI-1 were with body mass, expressed as the body mass index or the waist–hip ratio. These results suggest that different factors influence the plasma levels of the prothrombotic factors Lp(a), tPA and PAI-1 in healthy control subjects and in patients with hyperlipidaemia. In the latter, hyperlipidaemia phenotype, age, glycaemic status and uric acid levels are important determinants of the levels of these prothrombotic variables, whereas in the healthy, young control population, body mass was the single important association with tPA and PAI–1.     

Life-threatening anaphylactoid shock caused by recombinant tissue plasminogen activator

Recombinant tissue plasminogen activator (rt-PA) is currently the only approved agent for treating acute ischemic stroke. However, rt-PA may cause fatal symptomatic intracranial hemorrhage and other adverse effects like bleeding complications and allergic reactions. Patients taking angiotensin-converting enzyme (ACE) inhibitors have increased risk of allergic reactions. This report is about a patient with a history of ACE inhibitor intake who experienced life-threatening anaphylactoid shock during rt-PA administration. The relationship between rt-PA and ACE inhibitor was also discussed.     

Modulation of sympathetic activity by tissue plasminogen activator is independent of plasminogen and urokinase

Sympathetic neurons synthesize, transport, and release tissue-type plasminogen activators (t-PAs) and urinary-type plasminogen activators (u-PAs). We reported that t-PA enhances sympathetic neurotransmission and exacerbates reperfusion arrhythmias. We have now assessed the role of u-PA and plasminogen. Neurogenic contractile responses to electrical field stimulation (EFS) were determined in vasa deferentia (VD) from mice lacking t-PA (t-PA(-/-)), plasminogen activator inhibitor-1 (PAI-1(-/-)), plasminogen (plgn(-/-)), u-PA (u-PA(-/-)), and wild-type (WT) controls. Similar levels of t-PA were present in VD and cardiac synaptosomes of WT, PAI-1(-/-), plgn(-/-), and u-PA(-/-) mice, whereas t-PA was undetectable in t-PA(-/-) tissues. EFS responses were potentiated and attenuated in VD from PAI-1(-/-) and t-PA(-/-) mice, respectively, but indistinguishable from WT responses in VD from plgn(-/-) and u-PA(-/-) mice. Moreover, t-PA inhibition with t-PA(stop) decreased EFS response in WT mice, whereas u-PA(stop) did not. VD responses to ATP, norepinephrine, and K(+) in t-PA(-/-), PAI-1(-/-), plgn(-/-), and u-PA(-/-) mice were similar to those in WT, whereas t-PA(stop) did not modify VD responses to norepinephrine in WT, t-PA(-/-), and PAI-1(-/-) mice, indicating a prejunctional site of action for t-PA-induced potentiation of sympathetic neurotransmission. Indeed, K(+)-induced norepinephrine exocytosis from cardiac synaptosomes was potentiated in PAI-1(-/-), attenuated in t-PA(-/-) and not different from WT in u-PA(-/-) and plgn(-/-) mice. Likewise, ATP exocytosis was decreased in t-PA(-/-) and attenuated by t-PA(stop) in WT mice. Thus, t-PA-induced enhancement of sympathetic neurotransmission is a prejunctional event associated with increased transmitter exocytosis and independent of u-PA and plasminogen availability. This novel t-PA action may be a potential therapeutic target in hyperadrenergic states.     

Age dependence of tissue plasminogen activator concentrations in plasma, as studied by an improved enzyme-linked immunosorbent assay

A procedure for improving the specificity of enzyme-linked immunosorbent assays (ELISA) was devised, based on addition of antigen-specific or non-immune immunoglobulins to the citrated plasma sample and defining the difference in assay response between these two mixtures as the antigen-specific part of the response. When applied to measurement of tissue plasminogen activator (t-PA; EC 3.4.21.31) antigen in plasma, this procedure resulted in elimination of the overestimates obtained in a large proportion (10-20%) of patients' samples when assayed according to the conventional ELISA technique. Basal t-PA concentrations in plasma were found to be highly age-dependent, normal values being about 3 micrograms/L for adults near 30 years of age and about 10 micrograms/L for those over 60. Patients with gallbladder stone disease had increased mass concentrations of t-PA in plasma, even when corrected for the age effect; patients with multi-infarct dementia did not.     

Acute endothelial tissue plasminogen activator release in pregnancy

Summary. Objective: Pregnancy is associated with marked changes in vascular physiology and an increased risk of thrombosis. The aim of the study was to assess the effect of pregnancy on the acute release of tissue plasminogen activator (t‐PA) from the endothelium. Methods and results: Ten primigravida pregnant women were recruited in the third trimester of pregnancy (week 36 ± 1) and compared with 20 age‐matched non‐pregnant women (day 9.8 ± 0.3 of menstrual cycle). Blood flow and plasma fibrinolytic factors were measured in both forearms by venous occlusion plethysmography and blood sampling, respectively, during unilateral brachial artery infusions of bradykinin (100–1000 pmol min^?1). Pregnant women had higher plasma plasminogen activator inhibitor type 1 (PAI‐1) antigen concentrations (77.1 ± 12.4 vs. 21.5 ± 9.8 ng mL^?1; P = 0.004) that resulted in lower basal t‐PA/PAI‐1 ratios (0.2 ± 0.1 vs. 0.6 ± 0.1; P = 0.02) and plasma t‐PA activity concentrations (0.17 ± 0.02 vs. 0.58 ± 0.06 IU mL^?1; P < 0.0004). In both groups, bradykinin caused dose‐dependent increases in blood flow and local release of plasma t‐PA antigen and activity (P < 0.005 for all). Both the plasma t‐PA/PAI‐1 ratios and the net release of active t‐PA were markedly reduced in pregnant women (P < 0.05 for both). Area under the curve for net active t‐PA release was reduced by 36%. Conclusions: Pregnancy is associated with major perturbations of endogenous fibrinolytic capacity with an overwhelming increase in plasma PAI‐1 concentrations and an inadequate release of active t‐PA. These prothrombotic effects may, in part, explain the increased risk of arterial and venous thrombosis in pregnant women.     

Optimum conditions for the stabilization and measurement of tissue plasminogen activator activity in human plasma

Current studies show a more than 50-fold variation in the estimated level of tissue-type plasminogen activator (t-PA) activity in normal resting blood samples that result from major differences in the methods used to sample, preserve, and assay t-PA activity in blood. In this study we developed optimized methods for stabilizing and measuring t-PA activity in plasma by using a coupled plasminogen-chromogenic substrate (amidolytic) assay. To maximize the recovery of t-PA activity, blood should be acidified within 60 seconds after being drawn by adding 2 parts whole blood to 1 part 0.5 mol/L sodium acetate, pH 4.2. This method prevents hemolysis and eliminates 70% of the alpha 2-plasmin inhibitor. Optimum conditions for measuring t-PA activity are pH 8.0 to 8.3 (37 degrees C), ionic strength 0.02 to 0.04, 0.5 mumol/L plasminogen, 80 micrograms/ml CNBr-cleaved fibrinogen, and a chromogenic substrate concentration of 0.65 mmol/L D-valyl-leucyl-lysyl-p-nitroanilide, 0.25 mmol/L D-valyl-phenylalanyl-lysyl-p-nitroanilide, or 0.2 mmol/L D-norleucyl-hexahydrotyrosyl-lysyl-p-nitroanilide. The final assay is linear with respect to added one-chain t-PA, two-chain t-PA, and acidified plasma. There was no difference in t-PA activity measured with ethylenediaminetetraacetic acid anticoagulant versus that measured with citrate anticoagulant after correction for dilution effects (average resting t-PA activity in plasma from 20 healthy individuals = 1.59 IU/ml). We conclude that assay conditions can have major effects on the measurement of t-PA activity in plasma and that suboptimal conditions may result in a significant underestimation of t-PA activity.     

Pericardial infusion of tissue plasminogen activator in fibropurulent pericarditis

A 61-year-old man developed a loculated fibropurulent pericarditis, a rare complication of bacteremia. This occurred as a complication of a Staphylococcal aureus bacteremia from a head and neck abscess following self-extraction of a tooth. Despite surgical intervention and placement of 2 pericardial drains, a refractory, inadequately drained infected pericardial effusion persisted. Although there is limited experience with thrombolytic therapy to dissolve a fibrin clot in the pericardium, break down loculated adhesions, and facilitate free drainage of infected material, lysis is well described in the management of exudative pleural effusions. After infusion of 30 mg of tissue plasminogen activator in 100 cc normal saline through the pericardial drain of the patient, a large amount of infected serosanginous material subsequently drained during the next 2 days. The patient became afebrile and culture negative, remained hemodynamically stable, and had resolution of his pericarditis and pericardial effusion on electrocardiogram and echocardiogram, respectively.     

Gender-specific correlations of plasminogen activator inhibitor-1 and tissue plasminogen activator levels with cardiovascular disease-related traits

Summary.  Background:  The purpose of this study was to examine the correlations between plasma levels of plasminogen activator inhibitor-1 (PAI-1) and tissue plasminogen activator (t-PA) and cardiovascular disease-related traits in a general population and whether these correlations differed between females and males. Methods:  Plasma PAI-1 and t-PA antigen levels and C-reactive protein (CRP), HDL-cholesterol, triglycerides, total cholesterol, systolic blood pressure, diastolic blood pressure, urinary albumin excretion, and glucose were measured in the population-based PREVEND study in Groningen, the Netherlands ( n  = 2527). Results:  Except for CRP and total cholesterol levels, all traits were significantly different between gender ( P  < 0.001). PAI-1 levels were correlated with all measured cardiovascular disease-related traits ( P  < 0.01) in both females and males. Except for urinary albumin excretion, similar results, albeit less significant, were found for t-PA levels. Age-adjusted correlations between PAI-1 and CRP, triglycerides, total cholesterol, systolic blood pressure, and diastolic blood pressure differed significantly between females and males ( P  < 0.01). Many of the gender differences were predominantly present between premenopausal females and males. Conclusion:  PAI-1 and t-PA levels were correlated with cardiovascular disease-related traits in subjects obtained from the general population and several of these correlations differed across gender. The correlations found in the present study suggest the presence of coordinated patterns of cardiovascular risk factors and indicate which traits might influence PAI-1 and t-PA levels and thereby provide a framework and potential tool for therapeutic intervention to reduce thromboembolic events in the general population.     

有氧运动训练对高蛋氨酸饮食大鼠血浆组织纤溶酶原激活物/纤维蛋白溶酶原激活因子抑制物-1和前列腺素I2/血栓烷A2系统的影响

目的探讨有氧运动训练对高蛋氨酸饮食大鼠血浆组织纤溶酶原激活物/纤维蛋白溶酶原激活因子抑制物-1（t-PA/PAI-1）及前列腺素I2/血栓烷A2（PGI2/TXA2）系统的影响。 方法将雄性Wistar大鼠随机分为对照组、高蛋氨酸组及运动干预组。对照组喂饲普通饲料,高蛋氨酸组及运动干预组大鼠喂饲含3％蛋氨酸的特制饲料,同时运动干预组大鼠每日进行90 min无负重游泳运动,每周训练6 d。于实验进行8周后采用高效液相色谱法测定大鼠血浆同型半胱氨酸（Hcy）含量,选用发色底物法测定t-PA及PAI-1含量,选用放射免疫非平衡法测定6-酮-前列腺素F1(6-keto-PGF1)及TXB2含量。 结果高蛋氨酸组大鼠血浆Hcy含量显著高于对照组,t-PA/PAI-1及6-keto-PGF1α／TXB2比值均较对照组显著降低(P<0.05);与高蛋氨酸组比较,运动干预组血浆Hcy含量明显降低（P<0.05）,t-PA /PAI-1及6-keto-PGF1α／TXB2比值显著升高(P<0.05),且上述各项指标与对照组间差异均无统计学意义（P&rt;0.05）。 结论高蛋氨酸饮食可诱发大鼠高半胱氨酸血症,导致血浆t-PA/PAI-1和PGI2/TXA2系统失衡;适宜有氧运动训练可降低高蛋氨酸饮食大鼠血浆Hcy水平,纠正t-PA/PAI-1及PGI2/TXA2系统失衡,从而预防高半胱氨酸血症发生。     

Intravenous tissue plasminogen activator and stroke in the elderly

Objective

Since publication in 1995 of the National Institute of Neurological Disorders and Stroke (NINDS) trial of intravenous tissue plasminogen activator (IV tPA) for acute ischemic stroke, the benefit and frequency of use of IV tPA in the elderly have remained uncertain.

Methods

We obtained data from the NINDS trial to summarize outcomes for randomized subjects older than 80 years. We used data from the Cardiovascular Health Study, a cohort study of 5888 elderly participants from 4 US communities followed longitudinally for stroke since 1989 to estimate the use of and hospital outcome after IV tPA in older adults following publication of the trial in 1995.

Results

In the NINDS trial, 44 subjects older than 80 years were randomized, and their 3-month functional outcomes were not significantly improved with IV tPA. Of 25 randomized to IV tPA, 4 experienced symptomatic intracranial hemorrhages within 36 hours of treatment. Compared with younger patients, older patients were 2.87 times more likely to experience a symptomatic intracranial hemorrhage within 36 hours of IV tPA (95% confidence interval, 1.04-7.93). Of 227 Cardiovascular Health Study participants hospitalized for ischemic stroke between 1995 and 2002, seven, whose mean age was 84 years, were treated with IV tPA (3.1%; 95% confidence interval 1.2-6.2). Two had symptomatic intracranial hemorrhages, 3 failed to improve, and 2 of the 7 had good outcomes.

Conclusions

These data highlight the need to clarify the risk-benefit profile of IV tPA in ischemic stroke victims who are older than 80 years.     

Tissue plasminogen activator promotes platelet disaggregation in plasma.

We studied the disaggregation of human platelets by tissue-type plasminogen activator (t-PA). When added to a suspension of human platelets induced to aggregate in plasma with adenosine 5'-diphosphate, t-PA promoted disaggregation of platelets over several minutes. Addition of fresh plasma or purified human fibrinogen to disaggregated platelets facilitated (reversible) aggregation and subsequent disaggregation. Aspirin treatment of platelets markedly potentiated the ability of t-PA to induce disaggregation. Disaggregation was inhibited by alpha-2-antiplasmin. Comparative analysis of the rate of proteolysis of platelet-bound fibrinogen with that of ambient plasma fibrinogen suggested that fibrinogenolysis of cohesive fibrinogen occurred more rapidly than fibrinogenolysis of ambient fibrinogen. These data demonstrate that t-PA facilitates platelet disaggregation in plasma through kinetically selective proteolysis of cohesive fibrinogen by plasmin, and suggest that thrombolytic mechanisms may serve both to remove platelets from platelet-fibrin thrombi and to disperse circulating platelet aggregates.     

Anaphylactoid reaction to recombinant tissue plasminogen activator.

Anaphylactoid reaction to recombinant tissue plasminogen activator for the thrombolytic treatment of acute ischemic stroke is an uncommon complication. An increased risk of anaphylaxis may be found in patients concomitantly being treated with angiotensin-converting enzyme inhibitors, as illustrated by this case report describing a patient who experienced an urticaric rash, hypotension, tachycardia, orolingual angioedema, and airway obstruction following intravenous administration of alteplase. Possible pharmacologic interactions resulting in excessive serum bradykinin and subsequent systemic hypersensitivity responses are discussed.     

Proactivator and activator levels of plasminogen in plasma as measured by caseinolysis.

A procedure is presented for the caseinolytic assay of plasminogen proactivator and activator levels in blood. The validity of the method was established and normal plasma proactivator levels were determined in 10 subjects. No detectible activator activity was found in these subjects. Venous occulsion produced a small rise in activator activity. Hageman-deficient and Fitzgerald trait plasmas did not produce activator activity after exposure to kaolin. The generation of activator activity in the Fitzgerald plasma was partly restored by the addition of purified high molecular weight kininogen, but the Hageman-deficient plasma was not corrected. The caseinolytic assay avoids some of the drawbacks which are inherent in methods which depend on fibrin dissolution for the detection of enhanced fibrinolytic states, provides a reproducible baseline, and may be sufficiently sensitive to detect changes of clinical interest.