Standardization of methods for measuring plasminogen activator inhibitor activity in human plasma

We have standardized the measurement of plasminogen activator inhibitor type 1 (PAI-1) activity in plasma. One-chain tissue-type plasminogen activator (t-PA; EC 3.4.21.31; final activity, 5 int. units/mL) was incubated with plasma (final dilutions 1:4 to 1:40) in phosphate buffer (pH 7.4, ionic strength = 0.15) for 15 min at 37 degrees C, followed by acidification and measurement of residual t-PA activity by an amidolytic method. The PAI-1 activity assay was 98% specific for PAI-1 activity in samples from both pregnancy and nonpregnancy, and varied linearly with added plasma volume when the percent inhibition of t-PA was between 8% and 50%. For the standardized method, analytical recovery was 93 +/- 5%, the detection limit was 1.6 arbitrary units per milliliter (1 arb. unit of PAI-1 activity = inhibition of 1 int. unit of t-PA activity), and total imprecision was 10.2 (SD 0.7) arb. units/mL (CV = 7%, n = 20). The average PAI-1 activity in 10 healthy individuals drawn between 0800 and 1000 hours was 23.9 +/- 15.4 arb. units/mL. Compared with the standardized assay, two of three previously described assays underestimated PAI-1 activity in plasma by 77% and 85%, respectively.     

Characterization and comparative evaluation of a structurally unique PAI-1 inhibitor exhibiting oral in-vivo efficacy.

Plasminogen activator inhibitor-1 (PAI-1) is the major physiological inhibitor of both tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA). Elevated levels of PAI-1 are associated with thrombosis and vascular disease, suggesting that high plasma PAI-1 may promote a hypercoagulable state by disrupting the natural balance between fibrinolysis and coagulation. In this study, we identify WAY-140312 as a structurally novel small molecule inactivator of PAI-1, compare its inhibitory activity with other previously identified small molecule inhibitors, and investigate the mechanism of inactivation of PAI-1 in the presence of both tPA and uPA. In an immunofunctional assay, WAY-140312 inhibited PAI-1 with an estimated inhibitory concentration (IC(50)) of 11.7 micro m, which was the lowest value obtained of the four different PAI-1 inactivators tested. Surface activity profiling indicated that the critical micelle concentration for WAY-140312 was 95.8 micro m, and that each inhibitor exhibited unique physical chemical properties. Using a sensitive direct activity assay, the IC(50) for WAY-140312 was similar when either tPA or uPA was used as the target protease. Immunoblot analysis demonstrated that WAY-140312 near the IC(50) inhibited the complex formation between either tPA or uPA and PAI-1. After oral administration, WAY-140312 exhibited 29% bioavailability with a plasma half-life of approximately 1 h. In an in-vivo model of vascular injury, a 10 mg kg(-1) oral dose of WAY-140312 was associated with improvement in arterial blood flow and reduction in venous thrombosis. Thus, WAY-140312 represents a structurally novel small molecule inhibitor of PAI-1, and is the first such molecule to exhibit efficacy in animal models of vascular disease following oral administration.     

Thrombin upregulates production of plasminogen activator inhibitor type 1 in human peritoneal mesothelial cells.

OBJECTIVE: To determine the influence of thrombin, which is generated intraperitoneally during peritoneal dialysis, on the synthesis of fibrinolytic system components in human peritoneal mesothelial cells (HMC). METHODS: Confluently grown HMC, isolated from the omental tissue, were used in the experiments. Conditioned media were obtained by incubating cells with serum-free M199 containing the appropriate concentration of the test compound. Tissue type plasminogen activator (tPA) and plasminogen activator inhibitor type 1 (PAI-1) antigen concentrations were measured by ELISA. Northern blot analysis was conducted for mRNA expression experiments. To test thrombin specificity, we used the thrombin inhibitor hirudin. The protein kinase C (PKC) inhibitor Ro 31-8220 was inserted to examine whether the effect of thrombin depends on PKC activity. RESULTS: Thrombin increased PAI-1 antigen in the conditioned media of HMC in a time- and concentration-dependent manner. After 24 hours incubation, PAI-1 levels increased from 350+/-30 ng/10(5) cells in control conditions to 620+/-30 ng/10(5) cells in HMC exposed to 5 U/mL thrombin (n = 8, p < 0.05). In contrast, there was no effect of thrombin on tPA antigen levels. An increase of PAI-1 mRNA expression was also observed by Northern blot hybridization. Hirudin (10 U/mL) inhibited the thrombin-induced increase in PAI-1 synthesis. In addition, a complete inhibition of the stimulating effect of thrombin on PAI-1 synthesis was obtained by blocking PKC activity with Ro 31-8220 (3 micromol/L). CONCLUSIONS: Thrombin increases PAI-1 synthesis in HMC via a PKC-dependent mechanism.Thereby the synthesis of tPA is not affected. Thus, thrombin may not only promote fibrin formation in the peritoneal cavity, but may also inhibit fibrin degradation by release of free PAI-1 from HMC.     

The glycocalyx protects erythrocyte-bound tissue-type plasminogen activator from enzymatic inhibition

Coupling tissue-type plasminogen activator (tPA) to carrier red blood cells (RBC) prolongs its intravascular life span and permits its use for thromboprophylaxis. Here, we studied the susceptibility of RBC/tPA to PA inhibitors including plasminogen activator inhibitor-1 (PAI-1) that constrain its activity and may reduce the duration of its effect. Despite lesser spatial and diffusional limitations, soluble tPA was far less effective than RBC/tPA in dissolving clots formed in vitro from blood of wild-type (WT) mice (40 versus 80% lysis at equal doses of tPA). Furthermore, after i.v. injection, soluble tPA lost activity faster in transgenic mice expressing a high level of PAI-1 than in WT mice, whereas the activity of RBC/tPA was unaffected. PAI-1 inactivated soluble tPA at equimolar ratios in vitro, but it had no effect on the amidolytic or fibrinolytic activity of RBC/tPA. RBC/tPA was also more resistant than soluble tPA to in vitro inhibition by other serpins (alpha2-macroglobulin and alpha1-antitrypsin) and pathologically high levels of glucose. However, coupling to RBC did not protect a truncated tPA mutant, Retavase, from plasma inhibitors. Chemical removal of the RBC glycocalyx negated tPA protection from inhibitors: tPA coupled to glycocalyx-stripped RBC bound twice as much 125I-PAI-1 as did tPA coupled to naive RBC, and susceptibility of the bound tPA to inhibition by PAI-1 was restored. Thus, the RBC glycocalyx protects RBC-coupled tPA against inhibition. Resistance to high levels of inhibitors in vivo contributes to the potential utility of RBC/tPA for thromboprophylaxis.     

Vitamin C affects thrombosis/ fibrinolysis system and reactive hyperemia in patients with type 2 diabetes and coronary artery disease

OBJECTIVE: To examine the effect of vitamin C on forearm vasodilatory response to reactive hyperemia and on plasma level of plasminogen activator inhibitor 1 (PAI-1), von Willebrand factor (vWF), tissue plasminogen activator (tPA), antithrombin III (ATIII), proteins C and S, and factors V (fV) and VII (fVII) in patients with both type 2 diabetes and CAD. RESEARCH DESIGN AND METHODS: A total of 39 patients with type 2 diabetes and CAD were divided into two groups and received vitamin C (2 g/day) or no antioxidant for 4 weeks. Forearm blood flow was determined using venous occlusion gauge-strain plethysmography at baseline and after treatment. Forearm vasodilatory response to reactive hyperemia (RH%) or nitrate (NTG%) was defined as the percent change of flow from baseline to the maximum flow during reactive hyperemia or after administration of nitrate, respectively. Biochemical markers were determined by enzyme-linked immunosorbent assay (ELISA) or other standard methods. RESULTS: RH% was significantly increased after treatment with vitamin C (from 62.4 +/- 7.2 to 83.1 +/- 9.3%, P = 0.024) but remained unaffected in the control group. Vitamin C decreased plasma levels of fV (from 143 +/- 5.4 to 123 +/- 6.03%, P = 0.038), vWF (from 133.5 +/- 14.5 to 109.5 +/- 11.4%, P = 0.016), and tPA (from 12.3 +/- 0.99 to 8.40 +/- 0.60 ng/ml, P = 0.001), whereas these levels remained unaffected in the control group. The changes in RH%, vWF, and tPA were significantly greater (P = 0.028, 0.036, and 0.007, respectively) in the vitamin C-treated group than in the control group. Levels of ATIII, proteins S and C, fVII, and PAI-1 remained unchanged in all groups. CONCLUSIONS: Short-term treatment with high doses of vitamin C improved RH% and decreased plasma levels of tPA and vWF in patients with type 2 diabetes and CAD.     

纤溶酶原激活物及其抑制剂-1的血浆含量测定在急性肺血栓栓塞症中的意义

目的探讨在急性肺血栓栓塞症(APE)发病中的组织型纤溶酶原激活物(tPA)及其抑制剂-1(PAI-1)的血浆含量、作用及其该病诊断中的意义。方法,对44例APE患者和56例健康正常对照者应用酶联免疫吸附双抗体夹心法(ELISA法)定量测定血浆tPA和PAI-1抗原水平。结果与正常对照组(tPA含量为11．05ng／ml和PAI-1含量为61．31ng／m1)相比,APE组的IPA含量(33．88ng／ml)和PAI-1含量(111．50ng／ml)较高,两组间差异有显著性。在急性肺血栓栓塞症的疾病诊断中,tPA和PAI-1的血浆含量合理诊断截断点分别为21．7ng／ml和79．4ng／ml.结论急性肺血栓栓塞症的发病是由于PAI-1抗原产生和释放增多,而非tPA抗原释放或产生不足所致．tPA和PAI-1抗原血浆含量测定在APE的疾病诊断中具有要意义。     

Simple and practical sandwich-type enzyme immunoassay for human oxidatively modified low density lipoprotein using antioxidized phosphatidylcholine monoclonal antibody and antihuman apolipoprotein-B antibody

OBJECTIVES: To develop a simple and practical enzyme immunoassay (EIA) for oxidatively modified low density lipoprotein (Ox-LDL) in human blood, a biological marker of atherogenesis. DESIGN AND METHODS: A sandwich EIA suitable for the measurement of human Ox-LDL was developed using the mouse monoclonal antibody FOH1a/DLH3. This antibody, specific for oxidized phosphatidylcholine, was used as the capture antibody, and a horseradish peroxidase (HRP)-labeled goat anti-human apolipoprotein-B (Apo-B) IgG was used for detection. Copper-oxidized human LDL, prepared under controlled conditions, was used as a standard and the results of the EIA were expressed in arbitrary units (U/mL). RESULTS: This EIA meets all the requirements for use in routine clinical assays in terms of sensitivity (detection limit: 1 U/mL), reproducibility (total CV: 2.3-7.7%), accuracy (recovery: 90.6-103.8%), simplicity and rapidity (<4 h). Clinical performance of the assay was assessed by measurement of the Ox-LDL in the plasma of normal subjects (10.8 +/- 2.8 U/mL, mean +/- SD) and patients with coronary heart disease (CHD) (19.7 +/- 10.2). The present EIA had a sensitivity of 79% and a specificity of 75% for CHD. CONCLUSIONS: We have developed a new assay, suitable for the measurement of Ox-LDL in human blood, which meets the requirements for routine clinical assay.     

An amidolytic assay for determination of alpha 1-antitrypsin in serum and in cerebrospinal fluid

An amidolytic assay using the chromogenic substrate tosyl-glycyl-prolyl-lysine-4-nitranilide acetate (Chromozym PL) has been studied for the determination of alpha 1-antitrypsin in serum and in cerebrospinal fluid (CSF). It was found that binding of trypsin to alpha 2-macroglobulin has to be taken into consideration when alpha 1-antitrypsin is determined by an amidolytic assay. In serum this accounts for 7.4 +/- 3.2% and in CSF 2.8 +/- 1.8% of the total antitrypsin capacity. The reference range of alpha 1-antitrypsin in sera was found to be x = 60.3 +/- 20.8 kIU/l, and in CSF mean = 186 +/- 99 IU/l. Within-run precision showed a C.V. of 1.36% in sera, and a C.V. of 1.86% in CSF. There was a good correlation with immunochemical methods: r = 0.946 in 65 sera (p less than 0.001) and r = 0.986 in 55 samples of CSF (p less than 0.001).     

Activation of the fibrinolytic system and utilization of the coagulation inhibitors in sepsis: comparison with severe sepsis and septic shock

OBJECTIVES: To determine whether the fibrinolytic system is activated and coagulation inhibitors are utilized in sepsis, to compare the findings detected in sepsis with those found in severe sepsis and septic shock, and to compare the role played by different infectious pathogens on fibrinolysis and coagulation inhibitors. DESIGN AND SETTING: Prospective study comparing patients with sepsis, severe sepsis, and septic shock and healthy volunteers in the general intensive care unit of a tertiary university hospital. PATIENTS: Eighty-two consecutive septic patients (47 with sepsis, 18 with severe sepsis, and 17 with septic shock), and 14 healthy volunteers (controls). MEASUREMENTS AND RESULTS: After blood sampling we measured activation markers of fibrinolysis [plasmin/alpha(2)-antiplasmin complexes (PAP), complexes of tissue plasminogen activator/plasminogen activator inhibitor (tPA/PAI), fibrin(ogen) degradation products (FDPs), D-dimmers fibrin degradation products (D-d)], the utilization marker of antithrombin III (ATIII) thrombin/antithrombin complexes (TAT), several factors of fibrinolysis [plasminogen, tissue plasminogen activator (tPA), plasminogen activator inhibitor 1 (PAI-1), alpha(2)-antiplasmin], and the natural coagulation inhibitors [ATIII, protein C (PrC), protein S (PrS)]. In sepsis, PAP, FDPs, D-d, and TAT were increased to 439.8+/-32.35 microg/l, 57% positive, 49% positive, and 3.46+/-0.27 microg/l, respectively, compared with control subjects (205.57+/-28.58 microg/l, 0% positive, 7% positive, and 1.61+/-0.1 microg/l, respectively). These markers further increased in severe sepsis and septic shock. With the exception of a decrease in ATIII and an increase in tPA and PAI-1, coagulation inhibitors and factors of fibrinolysis were not changed in sepsis. In severe sepsis and mainly in septic shock, coagulation inhibitors (ATIII, PrC) and plasminogen were markedly decreased, whereas tPA and PAI-1 were further increased. All changes were independent of the causative infectious pathogen. CONCLUSIONS: Fibrinolysis is strongly activated and ATIII is utilized in sepsis. These findings are further enhanced in severe sepsis and septic shock. In sepsis only ATIII is decreased. In contrast, in severe sepsis and mainly in septic shock plasminogen and the main coagulation inhibitors (i.e., ATIII, PrC) are depleted, indicating exhaustion of fibrinolysis and coagulation inhibitors. Finally, Gram-positive, Gram-negative and other micro-organisms produce identical impairment.     

Cardiac fibrinolytic capacity is markedly increased after brief periods of local myocardial ischemia, but declines following successive periods in anesthetized pigs

BACKGROUND: Fibrinolysis in blood is mainly reflected by the activities of tissue plasminogen activator (tPA) and of plasminogen activator inhibitor-1 (PAI-1). The effect of myocardial ischemia on their activities in the coronary circulation is, however, not established. OBJECTIVES: With an improved experimental model, we therefore examined the effect of a brief period of myocardial ischemia on their activities. Furthermore, the consequences of repeated periods of ischemia, mimicking the situations in patients with unstable angina, were investigated. METHODS: In six anesthetized pigs, we occluded the distal left anterior descending coronary artery (LAD) four times for 10 min with 40 min intervals and determined the activities of tPA and PAI-1 in arterial and coronary venous blood. By simultaneously recording LAD flow, we could estimate cardiac release of these factors at baseline conditions and during reperfusion. RESULTS: Neither net cardiac release of PAI-1 nor alterations in plasma PAI-1 levels were demonstrated during the experiment. However, a significant net release of tPA activity of 10.4 +/- 3.2 IU mL(-1) (P < 0.005) was recorded during baseline conditions. During reperfusion following the first period of ischemia, the cardiac release of tPA activity increased to a peak of 103 +/- 30-fold baseline release, but declined progressively after repeated periods of ischemia. After the fourth period, tPA release did not exceed an estimated baseline accumulation during ischemia and early reperfusion. CONCLUSIONS: In this porcine model, a substantial local increase in fibrinolytic capacity was observed after brief periods of ischemia, but declined subsequently by repeated periods of ischemia.     

Effects of losartan on urinary secretion of extracellular matrix and their modulators in type 2 diabetes mellitus patients with microalbuminuria

PURPOSE: Angiotensin II receptor Type 1 antagonists postpone the development of nephropathy in type 2 diabetes mellitus (DM). We hypothesize that Losartan may ameliorate renal function in diabetic patients through the regulation on the generation of transforming growth factor (TGF)-beta and fibrinolytic regulators. METHODS: Twenty-two type 2 DM patients with microalbuminuria were treated with 50-100 mg/day of Losartan for 6 months. Urinary secretion of TGF-, plasminogen activator inhibitor-1 (PAI-1), tissue and urokinase plasminogen activators (tPA and uPA) fibronectin, collagen IV and plasma levels of TGF-beta, PAI-1, tPA and uPA of the patients before and after the treatment were analyzed using enzyme-linked immunoabosorbance assay. RESULTS: Losartan effectively reduced arterial blood pressure and urinary albumin excretion. The levels of TGF-beta in urine, but not in plasma, were reduced after 2, 4 and 6 months of the treatment (-32% to -48%, P < 0.05 or 0.01). Urinary or plasma levels of PAI-1, tPA or uPA, and urinary secretion of fibronectin or collagen IV were not significantly altered by Losartan treatment. Urinary levels of collagen IV positively correlated with uPA, and that of fibronectin negatively correlated with PAI-1 in the patients (P < 0.01). Urinary TGF-beta negatively correlated uPA in urine of the patients (P < 0.01). CONCLUSION: Losartan reduced urinary excretion of TGF-beta and albumin in type 2 DM patients with microalbuminuria. Fibrinolytic regulators and TGF-beta are implicated in the regulation of ECM turnover in kidneys of the patients with diabetic nephropathy.     

Plasminogen and plasmin activity in patients with coronary artery disease

OBJECTIVE: While coronary artery disease (CAD) is associated with disturbances of the plasma fibrinolytic system, the nature of these disturbances is not fully defined. Fibrinolysis is regulated by plasmin, whose production is mediated by plasminogen activator conversion of plasminogen (Plg) to plasmin. The cascade is modulated by feedback loops that include Plg activator inhibitor 1 (PAI-1). Molecular interactions with Plg kringle domains play an important role in regulating plasmin production and its modulation of fibrinolysis. We hypothesized that interactions of tissue plasminogen activator (tPA) with Plg kringle domains regulates plasmin levels in patients with stable CAD. METHODS: Plasma was collected from patients (n = 33) with an angiographically significant CAD and controls (n = 18) with angiographically established normal or minimally diseased arteries. Plasmin activity, tPA activity, and plasma levels of Plg, PAI-1, uPA, and tPA were determined. RESULTS: CAD patients had 1.7-fold greater plasmin activity (P = 0.02) that correlated with 1.5-fold higher tPA activity when compared to controls. Epitope mapping of Plg domains showed Plg differences in epitope exposure between the two groups. Plasma from CAD patients had 50% less (P < 0.001) detectable kringle 4 and 48% less (P = 0.007) detectable kringles 1-3. CONCLUSIONS: Based on detectable differences in Plg, we conclude that in patients with stable CAD, Plg complexed with tPA exists in a conformation that enables increased tPA activity and Plg conversion to plasmin.     

The 4G/5G PAI-1 polymorphism influences the endothelial response to IL-1 and the modulatory effect of pravastatin

BACKGROUND: Increased plasminogen activator inhibitor (PAI-1) levels lead to impaired fibrinolytic function associated with higher cardiovascular risk. PAI-1 expression may be regulated by different inflammatory cytokines such as interleukin-1alpha (IL-1). Several polymorphisms have been described in the PAI-1 gene. AIM: We examined the influence of the 4G/5G polymorphism in the promoter region on IL-1alpha-induced PAI-1 expression by human umbilical vein endothelial cells (HUVEC) in presence or absence of pravastatin. METHODS AND RESULTS: Genotyped HUVEC were incubated with IL-1alpha (500 U mL(-1)) in presence or absence of pravastatin (1-10 microm). PAI-1 expression was analyzed by real time polymerase chain reaction (PCR), and PAI-1 antigen measured in supernatants by ELISA. IL-1alpha increased PAI-1 secretion in a genotype-dependent manner, and higher values were observed for 4G/4G compared with both 4G/5G and 5G/5G cultures (P < 0.05). Preincubation of HUVEC with 10 microm pravastatin significantly reduced IL-1-induced PAI-1 expression in 4G/4G HUVEC compared with untreated cultures (177.5% +/- 24.5% vs. 257.9% +/- 39.0%, P < 0.05). Pravastatin also attenuated the amount of secreted PAI-1 by 4G/4G HUVEC after IL-1 stimulation (5020.6 +/- 165.7 ng mL(-1) vs. 4261.1 +/- 309.8 ng mL(-1), P < 0.05). This effect was prevented by coincubation with mevalonate, indicating a dependence on HMG-CoA reductase inhibition. CONCLUSIONS: The endothelial 4G/5G PAI-1 genotype influences the PAI-1 response to IL-1alpha and the modulatory effect of pravastatin. As increased PAI-1 levels have been linked to cardiovascular disease the observed endothelial modulation by pravastatin may have potential clinical implications.     

Increased plasminogen activator inhibitor-1 activity in offspring of type 2 diabetic patients: lack of association with plasma insulin levels

OBJECTIVE: To determine whether a dysregulation of the fibrinolytic system exists in normal glucose tolerant offspring of type 2 diabetic patients. RESEARCH DESIGN AND METHODS: In this cross-sectional study, 32 offspring of type 2 diabetic patients and 26 subjects with no family history of diabetes were studied. With respect to the metabolic parameters, plasma fasting and 2-h postload (75 g glucose) glucose and insulin levels, total cholesterol, triglycerides, and HDL cholesterol concentrations were determined. To evaluate the status of hemostatic factors, fibrinogen, tissue plasminogen activator (tPA) antigen level, plasminogen activator inhibitor-1 (PAI-1) antigen level, and PAI-1 activity were assessed. The statistical analyses included the Mann-Whitney U test to check the significance of differences between variables in the two groups and Spearman's rank correlation tests to check the interrelationships between the hemostatic and metabolic parameters in the offspring group. RESULTS: All subjects had normal glucose tolerance according to the American Diabetes Association criteria. Plasma fasting and postload insulin concentrations were significantly higher in offspring compared with control group (P<0.00001 and P<0.01, respectively). Plasma fasting and postload glucose, fibrinogen, tPA antigen, total cholesterol, and BMI were comparable between the groups. The offspring had significantly higher waist-to-hip ratio (WHR) (P = 0.03), higher triglycerides (P = 0.01), and lower HDL cholesterol (P<0.01) compared with the control group. PAI-1 antigen level and PAI-1 activity were higher in the offspring (P = 0.05 and P = 0.04, respectively). In the offspring group, PAI-1 activity was correlated with plasma PAI-1 antigen level (r = 0.40, P = 0.02), fibrinogen (r = 0.45, P = 0.01), and HDL cholesterol (r = -0.36, P = 0.04). However, tPA antigen level, fasting and postload plasma glucose and insulin, total cholesterol, triglycerides, WHR, and BMI did not correlate with PAI-1 activity. CONCLUSIONS: These data suggest that normal glucose tolerant offspring of type 2 diabetic subjects have elevated PAI-1 activity indicating to hypofibrinolysis in this group. The elevated PAI-1 activity has no association with plasma insulin concentration.     

Evaluation of PAI-039 [{1-benzyl-5-[4-(trifluoromethoxy)phenyl]-1H-indol-3-yl}(oxo)acetic acid], a novel plasminogen activator inhibitor-1 inhibitor, in a canine model of coronary artery thrombosis

We tested a novel, orally active inhibitor of plasminogen activator inhibitor-1 (PAI-1) in a canine model of electrolytic injury. Dogs received by oral gavage either vehicle (control) or the PAI-1 inhibitor PAI-039 [{1-benzyl-5-[4-(trifluoromethoxy)phenyl]-1H-indol-3-yl}(oxo)acetic acid] (1, 3, and 10 mg/kg) and were subjected to electrolytic injury of the coronary artery. PAI-039 caused prolongation in time to coronary occlusion (control, 31.7 +/- 6.3 min; 3 mg/kg PAI-039, 66.0 +/- 6.4 min; 10 mg/kg, 56.7 +/- 7.4 min; n = 5-6; p < 0.05) and a reduced thrombus weight (control, 7.6 +/- 1.5 mg; 10 mg/kg PAI-039, 3.6 +/- 1.0 mg; p < 0.05). Although occlusive thrombosis was observed across all groups based upon the absence of measurable blood flow, a high incidence (>60%) of spontaneous reperfusion occurred only in those groups receiving PAI-039. Spontaneous reperfusion in the 10 mg/kg PAI-039 group accounted for total blood flow (area under the curve of coronary blood flow) of 99.6 +/- 11.7 ml after initial thrombotic occlusion (p < 0.05 compared with control). Plasma PAI-1 activity was reduced in all drug-treated groups (percentage of reduction in activity p < 0.05; 10 mg/kg PAI-039), whereas ADP-, 9,11-dideoxy-11alpha,9alpha-epoxymethanoprostaglandin F(2alpha) (U46619)-, and collagen-induced platelet aggregation, as well as template bleeding and prothrombin time, remained unaffected by PAI-039. Ex vivo clot lysis analysis revealed normal clot formation but accelerated clot lysis in PAI-039-treated groups. The pharmacokinetic profile of PAI-039 indicated an oral bioavailability of 43 +/- 15.3% and a plasma half-life of 6.2 +/- 1.3 h. In conclusion, PAI-039 is an orally active prothrombolytic drug that inhibits PAI-1 and accelerates fibrinolysis while maintaining normal coagulation in a model of coronary occlusion.     

Regulation of local availability of active tissue-type plasminogen activator in vivo in man.

Free, biologically active tissue-type plasminogen activator (tPA) is the main initiator of intravascular fibrinolysis, but little is known about the regulation of active tPA on the organ level. The aim was to investigate if the local availability of active tPA on the organ level depends on the local release rate of tPA or the arterial input of tPA and plasminogen activator inhibitor type 1 (PAI-1). Also, we wanted to evaluate if plasma levels predict capacity for endothelial release of fibrinolytic proteins. Invasive perfused-forearm studies were performed in 96 healthy subjects. Local release rates of fibrinolytic proteins were assessed at baseline and during endothelial stimulation. Stimulation by methacholine and desmopressin induced a 6- and 12-fold increase in total tPA release rates, respectively. With increasing local release rates of tPA a gradually closer correlation emerged between the total tPA secretion and the forearm output of active tPA (from r = 0.102, ns to r = 0.85, P < 0.0001). Forearm availability of active tPA was not related to arterial input of either tPA or PAI-1. Release rates and plasma levels of tPA were not correlated. Baseline release rates of active tPA increased to noon. The major determinant for the local availability of active tPA is the capacity of the endothelium to release tPA rather than the arterial input of PAI-1 or tPA. Despite a molar excess of PAI-1, the majority of tPA released during stimulation does not undergo local inactivation. The capacity to release tPA locally cannot be predicted from its plasma concentration.     

d-dimer predicts major bleeding,cardiovascular events and all-cause mortality during warfarin treatment

ObjectivesPrevious studies have shown that biomarkers in blood plasma can predict bleeding complications during anticoagulant treatment as well as thromboembolic events and may improve existing risk stratification schemes in patients on or considered for oral anticoagulant treatment. The aim of this study was to investigate if levels of d-dimer, tissue plasminogen activator (tPA) and its complex with plasminogen inhibitor type 1 (tPA/PAI-1 complex) can predict major bleedings, cardiovascular events and all-cause mortality in patients with warfarin treatment.Design and methodsIn a longitudinal cohort study, 719 patients on oral anticoagulant treatment were followed for a total of 3001 treatment years. Major bleeding, stroke, arterial emboli, myocardial infarction and death were recorded and classified. Blood samples collected at baseline were analyzed for d-dimer, tPA, and tPA/PAI-1 complex.ResultsIn multivariate Cox regression analysis, high levels of d-dimer were associated with major bleeding (HR 1.27 per SD; 95% CI: 1.01–1.60), cardiovascular events (HR 1.23 per SD; 95% CI: 1.05–1.45) and all-cause mortality (HR 1.25 per SD; 95% CI: 1.06–1.47). Neither tPA nor the tPA/PAI-1 complex was associated with major bleeding, cardiovascular events or all-cause mortality.ConclusionWe conclude that high levels of d-dimer predict major bleeding, cardiovascular events and all-cause mortality during warfarin treatment.     

117例急性白血病患者纤溶抑制物研究

目的 探讨急性白血病(AL)患者凝血酶激活的纤溶抑制物(TAFI)、纤溶酶原激活剂抑制物(PAI)、α2抗纤溶酶(α2:-PI)等纤溶抑制物的变化及其临床意义.方法 采用ELISA法测定117例AL患者和50名正常对照的PAI-1抗原(PAI-1:Ag)含量、TAFI抗原含量;采用发色底物法测定PAI活性、α2-PI活性、TAFI活性.结果 ①AL患者的α2-PI活性水平明显低于对照组,其中急性淋巴细胞白血病患者[(96.8±21.2)%]较对照组[(129.1±13.1)%]下降更明显;②急性髓系白血病患者PAI-1:Ag含量[(37.8±9.2)μg/L]高于对照组[(33.8±4.9)μg/L];③急性早幼粒细胞白血病患者PAI-1:Ag含量[(37.8±9.0)μg/L]高于对照组,TAFI活性水平[(13.3±4.8)mg/L]低于对照组[(16.9±2.6)mg/L],急性单核细胞白血病患者PAI-1:Ag含量[(39.9±11.6)μg/L]高于对照组;④复发/难治组的PAI-1:Ag含量[(39.6±11.6)μg/L]高于对照组;⑤明显出血组TAFI活性水平[(13.2±5.3)mg/L]低于对照组及无出血组[(17.0±4.6)mg/L];⑥TAFI活性与出血程度呈显著负相关,r=-0.276(P《0.05).结论 α2-PI及TAFI活性降低是AL出血的原因之一,且TAFI活性与卅血程度呈显著负相关.     

缺血性脑卒中患者血纤溶活性变化规律的研究

目的 研究脑梗死组和非脑梗死组患者急性期,亚急性期和恢复期组织型纤溶酶原激活物（tPA）,纤溶酶原激活物抑制物-1（PAI-1）的变化规律.方法 135例脑梗死组患者于发病96h内,病后14d、3月、6月、9月分别采血,77例非脑梗死组患者先采血一次,然后分别在第一次采血后14d、3月、6月、9月采血.结果 脑梗死急性期tPA含量明显升高,PAI-1的含量明显升高.恢复期脑梗死即发病后3月,6月,9月tPA的含量比急性期明显降低,且脑梗死后时间越长tPA含量越低,恢复期脑梗死PAI-1的含量比急性期明显降低.结论 在缺血性卒中的患者,高tPA含量提示纤溶活性的降低.高PAI-1代表纤溶抑制增强.