Plasma t-PA/PAI-1 complex and blood coagulability in patients with coronary artery disease.

The t-PA/PAI-1 complex is a good indicator of the release of fibrinolysis activators and inhibitors from the vascular wall, but its clinical significance in chronic ischemic heart disease is unclear. The plasma levels of tissue-type plasminogen activator (t-PA), plasminogen activator inhibitor-1 (PAI-1), and the t-PA/PAI-1 complex (including various coagulation factors) were assayed in 72 patients with coronary artery disease (CAD) and 29 control (C) subjects. The CAD patients were subdivided into 3 groups: single-vessel disease (G1, n = 30), double-vessel disease (G2, n = 20), and triple-vessel disease (G3, n = 22). The patients with triple-vessel disease had higher fibrinogen values (G3: 318 +/- 75 mg/dl, C: 263 +/- 56), factor VII activity (G3: 143 +/- 36%, C: 123 +/- 14), and t-PA antigen levels (G3: 4.7 +/- 0.8 ng/ml, C: 3.3 +/- 0.7) than controls. Patients with double- and triple-vessel disease also showed higher levels of factor VIII, vWF antigen, thrombin-antithrombin III complex (G1: 2.3 +/- 0.6 ng/ml, G2: 2.7 +/- 0.5, G3: 3.1 +/- 0.5, C: 2.0 +/- 0.5), and t-PA/PAI-1 complex (G1: 13.9 +/- 6.1 ng/ml, G2: 16.4 +/- 4.6, G3: 18.2 +/- 5.9, C: 10.7 +/- 4.9) than control subjects. The t-PA/PAI-1 complex levels were correlated significantly with the activities of factors VII and VIII and the thrombin-antithrombin III complex. These findings suggest that patients with CAD have greater blood coagulability than controls, and that this difference is related to the severity of the disease.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fibrinolytic response during exercise and epinephrine infusion in the same subjects.

To determine whether exercise-induced increases in tissue plasminogen activator (t-PA) were related to plasma epinephrine concentration during exercise, 14 healthy men (aged 24 to 62 years) were studied during epinephrine infusions (10, 25 and 50 ng/kg per min) and graded supine bicycle exercise, beginning at 33 W and increasing in 33-W increments until exhaustion. Plasma epinephrine, active and total t-PA, active plasminogen activator inhibitor type 1 (PAI-1) and t-PA/PAI-1 complex concentrations were measured at each exercise and infusion level. During epinephrine infusion, active and total t-PA levels increased linearly with the plasma epinephrine concentration (respective slopes [+/- SEM] of 0.062 +/- 0.003 and 0.076 +/- 0.003 pmol/ng epinephrine). During exercise, t-PA levels did not increase until plasma epinephrine levels increased, after which both active and total t-PA levels again increased linearly with the plasma epinephrine concentration, but at twice the rate observed with epinephrine infusion (0.131 +/- 0.005 and 0.147 +/- 0.005 pmol/ng, respectively). The t-PA level in blood was directly proportional to the plasma epinephrine concentration during both exercise and epinephrine infusion, suggesting that epinephrine release during exercise stimulates t-PA secretion. In these healthy subjects, active plasminogen activator inhibitor type 1 and t-PA/PAI-1 complex levels were low (41 +/- 11 and 21 +/- 5 pmol/liter, respectively) and did not change significantly during exercise or epinephrine infusion. It is concluded that approximately 50% of the increase in t-PA during exercise is due to stimulated release of t-PA by epinephrine.(ABSTRACT TRUNCATED AT 250 WORDS)     

复方丹参滴丸对冠心病患者纤溶系统t-PA、PAI-1含量与活性的调控

目的观察复方丹参滴丸对冠心病患者内源性纤溶活性及血管内皮功能的影响，探讨其作用机制。方法冠心病患者78例，随机分为对照组和复方丹参滴丸组。对照组采用常规治疗；复方丹参滴丸组在常规治疗基础上加用复方丹参滴丸每次10粒，每日3次。两组用药时间均为4周。比较治疗前后血浆组织型纤溶酶原激活剂（t-PA）、纤溶酶原激活剂抑制因子-1（PAI-1）活性及浓度。结果复方丹参滴丸治疗4周后，患者血浆PAI-1活性及浓度下降（P〈0．05），t—PA活性及浓度含量升高（P〈0．05），与治疗前比较差异有统计学意义；常规治疗组治疗前后t-PA和PAI-1活性差异无统计学意义。结论复方丹参滴丸可有效地调控改善冠心病患者内源性纤溶活性及血管内皮功能。     

Failure of peripheral arterial thrombolysis due to elevated plasminogen activator inhibitor type 1.

To reduce the risk of intracerebral hemorrhage during thrombolytic therapy, a lower dose of tissue plasminogen activator (t-PA) or urokinase is used for acute peripheral arterial thrombi versus coronary thrombi. We hypothesized that elevated plasminogen activator inhibitor-1 (PAI-1) activity could neutralize infused t-PA or urokinase, resulting in lysis failure. Active PAI-1, active t-PA and total t-PA antigen were measured in 20 patients receiving t-PA, and active PAI-1 was measured in four patients receiving urokinase for acute peripheral arterial thrombosis. The 18 patients that successfully lysed their thrombi all had low active PAI-1 levels (10 +/- 19 pmol/l) during infusion of thrombolytic therapy, while six patients that failed to lyse their thrombi had high active PAI-1 levels (1533 +/- 1384 pmol/l, P = 0.00007) during infusion. Active t-PA levels during t-PA infusion were higher in the group that lysed their thrombi (536 +/- 423 pmol/l versus 42 +/- 45 pmol/l, P = 0.04) even though total t-PA levels were lower (1240 +/- 493 pmol/l versus 1956 +/- 709 pmol/l, P = 0.03). In the patients that failed to lysed their thrombi, > 95% of infused t-PA was neutralized by PAI-1. We conclude that elevated PAI-1 during acute peripheral arterial thrombolysis is associated with an increased risk of lysis failure due to reduced levels of circulating active t-PA or urokinase.     

体外治疗性超声对大鼠纤溶系统的影响

目的　研究体外治疗性超声对大鼠血浆组织型纤溶酶原激活因子 (t-PA)、纤溶酶原激活物抑制因子 -1(PAI -1)的影响。方法　采用酶联免疫吸附法测定超声治疗前后大鼠血浆t-PA、PAI -1的改变。结果　超声治疗后ETUS组t-PA(0 .5 4± 0 .19)IU/ml,PAI -1(0 .44± 0 .18)AU/ml,对照组t -PA(0 .5 1± 0 .2 5 )IU/ml,PAI -1(0 .43± 0 .19)AU/ml,二者比较无明显差异 (P >0 .0 5 )。结论　治疗剂量超声对大鼠纤溶系统没有影响     

Plasminogen activator in normal subjects after exercise and venous occlusion: t-PA circulates as complexes with C1-inhibitor and PAI-1

Exercise to exhaustion was associated with the appearance in plasma of plasminogen activator (PA) in several mol wt forms, as identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with zymography. A number of active bands, all immunologically identified as tissue-type PA (t-PA), were observed. The major form had an apparent mol wt of approximately 60,000 and is due to free t-PA. The other strong bands had apparent mol wts of approximately 110,000 and 180,000. The 110,000 band, also present in pre-exercise samples, represents t-PA complexed with its major inhibitor (PAI-1), and the 180,000 band is due to t-PA complexed with C1 inhibitor. The released forms of t-PA were cleared rapidly after cessation of exercise at exhaustion. Urokinase-type PA (u-PA) activity was also identified in pre- and postexercise samples at an apparent mol wt of approximately 50,000. This is consistent with its being free u-PA; no complexed forms of u-PA were observed. Qualitatively similar changes in plasma PA were observed after venous occlusion. Small quantities of plasmin were generated after strenuous exercise, as observed by detection of plasmin- alpha 2-antiplasmin complex by two-dimensional immunoelectrophoresis in three of five subjects. This complex was cleared rapidly after cessation of exercise. Plasmin-alpha 2-antiplasmin complex was not detected in any of the subjects after venous occlusion.     

全蝎纯化液对实验性动脉血栓形成t-PA、PAI-1的影响

目的观察全蝎纯化液对新西兰白兔颈总动脉血栓形成组织型纤溶酶原激活物(t-PA)、纤溶酶原活化物抑制剂(PAI-1)、血小板计数(Pt)的变化。方法将50只健康新西兰白兔随机分成5组:空白组、模型组、全蝎纯化液大剂量组(20mg/kg)、全蝎纯化液中剂量组(10mg/kg)和全蝎纯化液小剂量组(5mg/kg)。耳缘静脉注射药物(空白组、模型组注射同等体积的生理盐水)后,采用H2O2损伤新西兰白兔颈总动脉制备血栓模型,造模2h后,采血并取血栓,酶联免疫吸附法(ELISA法)检测血浆t-PA、PAI-1的含量。结果与空白组比较,模型组t-PA明显降低,PAI-1明显升高,差异有统计学意义(P<0.01);与模型组相比,全蝎纯化液各剂量组能明显抑制PAI-1活性,增加t-PA活性,差异均有统计学意义(P<0.01),而各组的血小板计数差异无统计学意义(P>0.05)。结论全蝎纯化液各剂量组可明显促进血浆t-PA的分泌,抑制血浆PAI-1活性,提示全蝎纯化液抗血栓作用机制可能与其促纤溶作用有关。     

The release of plasminogen activators (t-PA and u-PA) and plasminogen activator inhibitor (PAI-1) after venous stasis.

The aim of the present study was to compare plasma levels of urokinase-type plasminogen activator (u-PA), before and after 20 min of venous stasis, with those of tissue-type plasminogen activator (t-PA), type 1 plasminogen activator inhibitor (PAI-1) and t-PA/PAI-1 complexes, to determine whether both plasminogen activators and their inhibitor respond similarly to the same stimulus. We studied 36 patients with recurrent venous thrombosis in whom no coagulation defects predisposing them to thrombosis had been detected (mean age 38.2 years, range 15-70 years). Twenty healthy individuals (mean age 34.3 years, range 20-60 years) served as a control group. t-PA, PAI-1 and u-PA activity and antigen, as well as the t-PA/PAI-1 complex antigen, were measured before and after venous stasis. Post-stasis fibrinolytic parameters were corrected for the haemoconcentration which occurred during the venous occlusion test. Pathologically high PAI-1 levels (antigen and activity) were found in four out of 36 patients who were excluded from study. Functional and antigenic u-PA increased significantly after venous stasis when analysed as the absolute differences between paired samples (P less than 0.01). This increase in u-PA did not correlate with changes in t-PA or PAI-1 (r = 0.28 and r = 0.36 respectively). This leads us to suggest that different mechanisms relating to clearance and/or release from diverse sources might be involved in elevations of u-PA in response to a local endothelial stimulus. We conclude that venous stasis might not be the elective choice when evaluating 'bad responders' predisposed to thrombosis.     

The specific activity of plasminogen activator inhibitor-1 in disseminated intravascular coagulation with acute promyelocytic leukemia.

In disseminated intravascular coagulation (DIC) with acute promyelocytic leukemia (APL) in the absence of severe infection, marked fibrinolysis was noted in comparison with normal levels of antithrombin III, which is a major inhibitor of the coagulation system. Increased plasminogen activator inhibitor-1 (PAI-1) antigen levels in plasma from patients with septicemia decreased the ratio of the plasma clot lysis rate induced by an anti-alpha 2-plasmin inhibitor monoclonal antibody to the tissue-type plasminogen activator (t-PA) concentration. This decrease was not as prominent in plasma from patients with DIC, especially those with APL. To explore the character of PAI-1 in these plasmas, we measured the specific activity of PAI-1 by determining the ratio of active PAI-1 antigen to t-PA-unbound PAI-1 antigen. To calculate the amount of active PAI-1 antigen, the amount of t-PA/PAI-1 complex before and after the addition of a fixed amount of t-PA to the sample was measured by a sandwich solid-phase enzyme-linked immunosorbent assay using anti-PAI-1 and anti-t-PA monoclonal antibodies. The assay to measure total PAI-1 antigen used three monoclonal anti-PAI-1 antibodies and had similar sensitivities to free active, latent, vitronectin-bound and t-PA-bound PAI-1. The specific activity of PAI-1 decreased in patients with DIC (43.7% +/- 30.6%) and in DIC cases with APL (10.3% +/- 6.0%) in comparison to patients with septicemia (83.7% +/- 20.2%) or normal controls (85.8% +/- 27.3%). In DIC associated with APL, degraded forms of PAI-1 were detected in plasma by immunoblotting. These results suggest that a decrease in the specific activity of PAI-1 and an increase in secondary fibrinolysis result in a hyperfibrinolytic state in DIC patients with APL.     

Tissue-type plasminogen activator and its inhibitor (PAI-1) in plasma in cases of non-insulin-dependent diabetes mellitus (NIDDM)

Parameters of fibrinolysis, including plasminogen, alpha 2 plasmin-inhibitor (alpha 2 PI), tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) antigens, and fibrinogen were assayed in 53 patients (28 women and 27 men; mean age: 64 years, age range: 32-87 years) with non-insulin-dependent diabetes mellitus (NIDDM). The control group was similarly aged (mean age: 60.4 years, age range: 38-81). The levels of t-PA and t-PA/PAI-1 ratio of the diabetic group (mean +/- SD; 9.8 +/- 4.3 ng/ml, 0.94 +/- 0.47, respectively) were significantly higher than that of the control group (5.5 +/- 2.5 ng/ml, 0.51 +/- 0.23, respectively). The increased levels of t-PA antigen and t-PA/PAI-1 ratio in diabetics mean that free t-PA has been released. However, there was no significant difference in the level of PAI-1 between the diabetic group (12.9 +/- 6.4 ng/ml) and the control group (12.1 +/- 5.6 ng/ml). Levels of fibrinogen, plasminogen and alpha 2 PI in plasma were not different in the two groups. Duration of the disease, levels of glycosylated hemoglobin, differences in treatment and presense of diabetic nephropathy or retinopathy did not affect the fibrinolytic parameters. The levels of fibrinogen was higher in those with nephropathy than in the diabetics without nephropathy and retinopathy (p less than 0.05). There were no significant differences in the levels of t-PA, t-PA/PAI-1 ratio and PAI-1 between younger (less than 65 years) and older (65 years or more) subjects, in either the control or diabetic groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mean transit times and the sites of synthesis and catabolism of tissue plasminogen activator and plasminogen activator inhibitor type 1 in young subjects.

Using an invasive technique, we studied the mean transit time, the net quantitative turnover rate, and the sites of synthesis and catabolism of tissue plasminogen activator (t-PA) and plasminogen activator inhibitor type 1 (PAI-1) in healthy young volunteers in the fasting, steady state. Blood was sampled simultaneously from a large hepatic vein, an artery and the inferior caval vein, while measuring the splanchnic plasma flow rate and the plasma volume. We found that the catabolism of active t-PA and t-PA antigen took place in the splanchnic circulation with net rates of 7.2 and 6.3 pmol/min, respectively. The extraction fraction and the mean transit time in the splanchnic circulation were, respectively, 0.63 and 5.6 min for active t-PA and 0.17 and 21 min for t-PA antigen. Active PAI-1 was synthesized in the splanchnic circulation at a rate of 890 IU/min and had a mean transit time of about 9.8 min. No net extraction of PAI-1 antigen took place in the splanchnic circulation. In conclusion, we demonstrated that active t-PA and t-PA antigen are catabolized and active PAI-1 produced in the splanchnic circulation in young healthy subjects during steady state. Furthermore, our data show that active t-PA was also eliminated outside the splanchnic region with a catabolism rate of about 8.4 pmol/min. No net complex formation could be demonstrated in the peripheral circulation. We therefore suggest that active t-PA is eliminated by a re-uptake in the endothelium in the peripheral vessels or in the lung circulation.     

Behaviour of tissue plasminogen activator, plasminogen activator inhibitor 1 and their complex in various disease states.

Plasma levels of tissue plasminogen activator (t-PA) antigen, plasminogen activator inhibitor 1 (PAI-1) antigen and t-PA/PAI-1 complex were measured in plasmas from 18 healthy subjects and 75 patients with various diseases (28 patients with haematological malignancies, 20 with thrombotic diseases, five with infectious diseases, four with liver diseases, ten with bleeding disorders and eight miscellaneous conditions). In addition, we studied ten patients with bleeding disorders after DDAVP infusion and 18 healthy subjects after venous occlusion. Plasma levels of t-PA antigen, PAI-1 antigen and t-PA/PAI-1 complex were increased in the patients compared with the healthy subjects. t-PA/PAI-1 complex levels correlated well with t-PA antigen levels and molar concentrations of t-PA antigen were similar to those of the t-PA/PAI-1 complex. Venous occlusion induced an increase in both t-PA antigen and PAI-1 antigen and the molar concentration of the t-PA/PAI-1 complex was equivalent to that of t-PA antigen. Following DDAVP infusion, the levels of t-PA antigen and t-PA/PAI-1 complex increased but PAI-1 antigen levels decreased, and the increase of t-PA antigen was greater than that of t-PA/PAI-1 complex. These findings indicate that PAI-1 antigen exceeds t-PA antigen in healthy subjects and in patients with various diseases. We conclude that part of the t-PA/PAI-1 complex is rapidly cleared from the circulation and that free t-PA increases after DDAVP infusion.     

Inhibition of clot lysis and decreased binding of tissue-type plasminogen activator as a consequence of clot retraction.

Tissue-type plasminogen activator (t-PA) is less active in vivo and in vitro against clots that are enriched in platelets, even at therapeutic concentrations. The release of radioactivity from 125I-fibrin-labeled clots was decreased by 47% 6 hours after the addition of t-PA 400 U/mL when formed in platelet-rich versus platelet-poor plasma. This difference was not due to the release of plasminogen activator inhibitor-1 (PAI-1) by platelets. Thus, the fibrinolytic activity of t-PA in the supernatant was similar in the two preparations and fibrin autography demonstrated only a minor degree of t-PA-PAI-1 complex formation. Furthermore, a similar platelet-dependent reduction in clot lysis was seen with a t-PA mutant resistant to inhibition by PAI-1. The reduction in t-PA activity correlated with a decrease in t-PA binding to platelet-enriched clot (60% +/- 3% v platelet-poor clot, n = 5). This reduction in binding was also shown using t-PA treated with the chloromethylketone, D-Phe-Pro-Arg-CH2Cl (PPACK) (36% +/- 13%, n = 3), and with S478A, a mutant t-PA in which the active site serine at position 478 has been substituted by alanine (43% +/- 6%, n = 3). In contrast, fixed platelets and platelet supernatants had no effect on the binding or lytic activity of t-PA. Pretreatment with cytochalasin D 1 mumol/L, which inhibits clot retraction, also abolished the platelet-induced inhibition of lysis and t-PA binding by platelets. These data suggest that platelets inhibit clot lysis at therapeutic concentrations of t-PA as a consequence of clot retraction and decreased access of fibrinolytic proteins.     

Inhibition of tissue plasminogen activator activity by aspirin in vivo and its relationship to levels of tissue plasminogen activator inhibitor antigen, plasminogen activator and their complexes

The observation that aspirin inhibits the increment in tissue plasminogen activator (t-PA) activity induced by venous occlusion of the forearm became controversial with the publication of several nonconfirmatory studies. The current study was performed to confirm the original observation and determine the mechanism by which aspirin suppresses the incremental t-PA activity induced by venous occlusion. Aspirin (650 mg/d X 2) caused no change in resting levels of t-PA antigen (t-PA:Ag) or activity, plasminogen activator inhibitor 1 antigen (PAI-1:Ag), or activity or t-PA-PAI-1 complexes. In contrast, aspirin reduced the increments induced by venous occlusion as follows: t-PA:Ag by 45% (P = .001); t-PA activity (euglobulin lysis time, ELT) by 43% (P = .006); and t-PA activity (alpha 2-plasmin inhibitor-plasmin complexes, PIPC) by 41% (P = .003). The inhibition of incremental t-PA activity measured as ELT or PIPC was linearly correlated with the inhibition of incremental t-PA:Ag (respectively, r = .75, P less than .02; r = .67, P less than .05). Aspirin had no effect on the increment in PAI-1:Ag induced by venous occlusion, but similar to the effect on t- PA:Ag, aspirin induced a 51% inhibition of the increment in t-PA-PAI-1 complex formation. Aspirin did not alter the ability of alpha 2-plasmin inhibitor to bind plasmin, nor the ability of plasma to support the fibrin-catalyzed generation of plasmin by t-PA, nor the subsequent formation of PIPC. Aspirin inhibits the t-PA activity induced by venous occlusion primarily by inhibiting the release of t-PA antigen.     

A lifelong bleeding disorder associated with a deficiency of plasminogen activator inhibitor type 1

A 36-year-old patient was investigated for a lifelong history of epistaxis and delayed bleeding after minor surgeries. Deficiencies or abnormalities of the coagulation system, of platelet function, or of factor XIII and alpha-2-antiplasmin were excluded. Consistently, however, over a period of 7 years, a high basal euglobulin fibrinolytic activity was observed that was characterized by a high tissue-type plasminogen activator (t-PA) activity, normal t-PA antigen, and undetectable plasminogen activator inhibitor type-1 (PAI-1) antigen and activity. The high specific activity of t-PA (640,000 IU/mg) and the minimal amounts of t-PA/PAI-1 complexes detected by fibrin zymography suggest that in this patient all t-PA was active. This is in striking contrast to normal plasma, where the majority of t-PA is complexed to PAI-1. Thus, in this patient, a severe deficiency of PAI-1 is associated with a delayed type bleeding tendency. Our observation underscores the importance of plasma PAI-1 for the stabilization of the hemostatic plug.     

Formation, inhibition and clearance of plasmin in vivo

A 5 microg/kg bolus of tissue plasminogen activator (t-PA) was infused into 11 healthy subjects followed by measurement of t-PA activity and antigen, PAI-1 activity and antigen, t-PA/PAI-1 complex, plasmin/antiplasmin (PAP) complex and D-dimer over 4 h. Infusion of t-PA resulted in a rise in PAP levels in all subjects from a baseline of 2.4 +/- 1.1 nmol/l to a peak of 5.1 +/- 2.3 nmol/l, but had no effect on plasminogen, antiplasmin or D-dimer levels. Using a kinetic model of plasminogen activation in vivo, the second-order rate constant for t-PA binding to plasminogen was estimated to be 3,100 +/- 1,300 mol(-1)l x s(-1), 200-500 times slower than t-PA accelerated by fibrin. The half-life of PAP was 4.5 +/- 1.6 h. In healthy subjects, the PAP level in plasma represents the average rate of plasminogen activation over the last 2-8 h.     

益气活血复方对动脉粥样硬化家兔PAI-1及t—PA影响的实验研究

目的 观察益气活血复方对动脉粥样硬化家兔组织型纤溶酶原激活物(t-PA)、组织型纤溶酶原激活物抑制剂-1(PAI-1)及超微结构的影响.方法 40只雄性新西兰大耳白兔随机分为正常组、模型组、中药对照组、西药对照组、中药治疗组,每组8只.适应性喂养1周后,模型组、中药对照组、西药对照组、中药治疗组行颈总动脉球囊损伤术.术后正常组给予普通饲料,其他组给予高脂饲料喂养4周.实验第4周末开始分别予与益气活血复方、血脂康胶囊和辛伐他汀片,持续给药4周.在治疗前、后分别取血检测PAI-1、t-PA.8周末处死动物,取家兔左右颈总动脉,用透射电镜观察颈总动脉的超微结构.结果 4周末与正常组比较,模型组PAI-1水平持续升高,t-PA略有升高.8周末中药治疗组与模型组相比较,PAI-1水平明显下降(P<0.01),t-PA水平明显升高(P<0.01).各给药组组间PAI-1、t-PA水平无统计学意义(P>0.05);电镜水平证实益气活血中药能够改善颈内动脉的超微结构.结论 益气活血复方能促进动脉粥样硬化家兔纤溶酶原激活物的分泌,减少纤溶酶原激活物抑制物的分泌,从而增强纤溶活性,减少动脉粥样硬化的发生.     

Circadian fluctuations of tissue plasminogen activator antigen and plasminogen activator inhibitor-1 antigens in vasospastic angina.

To elucidate the circadian variation of fibrinolytic components in vasospastic angina, plasma levels of tissue plasminogen activator antigen (t-PA), free plasminogen activator inhibitor antigen (free PAI-1), t-PA/PAI-1 complex, and total PAI-1 were measured in venous plasma samples. Samples were taken every 6 hours (6:00 AM, noon, 6:00 PM, and midnight) for 24 hours in 14 patients with vasospastic angina, in 9 patients with exertional angina, and in 19 normal subjects. Twenty-four-hour Holter monitoring (Holter monitor, Del Mar Avionics, Irvine, Calif.) was also carried out in all subjects. All of the fibrinolytic components showed circadian variation, with a peak level at 6:00 AM in every study group except for the t-PA/PAI-1 complex in the group of patients with exertional angina. The values for all or the fibrinolytic components at each sampling time were higher in patients with coronary artery disease than in normal subjects. In particular, the mean value of free PAI-1 at 6:00 AM in patients with vasospastic angina was significantly higher than that in normal subjects and that in patients with exertional angina. This value of free PAI-1 in patients with vasospastic angina was closely associated with the duration of ischemic attacks. These results suggested that the circadian fluctuation of fibrinolytic components may be an important factor that leads to coronary thrombosis at the time of coronary spasm, especially in the early morning.     

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.     

Dependence of fibrinolytic activity on the concentration of free rather than total tissue-type plasminogen activator in plasma after pharmacologic administration

To identify factors responsible for the decline of plasma tissue-type plasminogen activator (t-PA)-specific activity that we have observed after infusions of the activator and to define the potential usefulness of selected variants of t-PA in obviating them in patients with infarction, serial plasma samples from patients (n = 4) and rabbits (n = 15) given t-PA were assayed for total t-PA antigen, t-PA activity, and free as opposed to type-1 plasminogen activator inhibitor (PAI-1)--complexed t-PA. In patients, attenuation of t-PA specific activity after infusions was evident with concentrations of total t-PA antigen that were as much as sevenfold greater than pretreatment values (62 compared with 9 ng/ml). Attenuation of t-PA activity corresponded with the disappearance of free t-PA from plasma and was associated with persistence of complexes of t-PA with PAI-1. In normal rabbits (n = 4) given wild-type t-PA by bolus injection, PAI-1 activity was 4 +/- 1 arbitrary units/ml. Attenuation of t-PA activity was not evident until 60 minutes after injection at a time when total plasma t-PA antigen concentration was as low as 13 +/- 8 ng/ml. Under these conditions, plasma t-PA was composed predominantly of free t-PA. In rabbits (n = 5) given lipopolysaccharide to increase plasma PAI-1 activity to 193 +/- 84 arbitrary units/ml, the specific activity of t-PA was attenuated as early as 15 minutes after injection at a time when total t-PA antigen concentration was as high as 164 +/- 79 ng/ml. As was the case with samples from patients, attenuation was associated with the disappearance of free t-PA and the persistence of complexes of t-PA with PAI-1. A genetically engineered variant of t-PA with comparable specific activity and a comparable rate constant of association with PAI-1 but designed to persist in the circulation manifested prolonged clearance from plasma of normal rabbits (n = 3) (t1/2 = 24.6 +/- 1.6 minutes compared with an alpha phase t1/2 of 1.9 minutes for wild-type t-PA). The variant lacked the epidermal growth factor and kringle one domains and contained a duplicated kringle two domain.(ABSTRACT TRUNCATED AT 400 WORDS)