Endothelial-dependent fibrinolysis in subjects with the lupus anticoagulant and thrombosis

To investigate the hypothesis that diminished endothelial fibrinolytic activity contributes to the pathogenesis of thrombosis in patients with the lupus anticoagulant (LA), we assessed the ability of endothelium to release tissue-type plasminogen activator (t-PA) in response to standardized venous occlusion (VO) of the arm, and the extent of inhibition of t-PA, in 11 subjects with LA and a history of thrombosis and in 36 healthy normal subjects. The mean rise in plasma t-PA antigen after VO, the mean plasma free t-PA activity after VO, and the mean plasma t-PA inhibitor level prior to VO were not significantly different in subjects with LA and thrombosis and in normal subjects. Four subjects with LA and thrombosis (36%), and five of 36 healthy control subjects (14%) generated no detectable free t-PA activity after VO ("non-responders"); this difference was not statistically significant. All four "non-responders" with LA and thrombosis had normal t-PA antigen release after VO, indicating that the lack of detectable free t-PA activity after VO was due to increased inhibition of released t-PA. We conclude that abnormally reduced endothelial fibrinolytic activity is not present in the majority of subjects with LA and thrombosis. In the subset of subjects with LA and thrombosis who generate no detectable t-PA activity after VO, a stimulatory effect of LA on endothelial production of t-PA inhibitor cannot be excluded.     

Tissue-type plasminogen activator, type 1 plasminogen activator inhibitor and their complex in plasma with disseminated intravascular coagulation.

The levels of tissue-type plasminogen activator (t-PA), type 1 plasminogen activator inhibitor (PAI-1), and t-PA/PAI-1 complex antigens were analyzed in the plasma of disseminated intravascular coagulation (DIC) patients and healthy controls. Other fibrinolytic parameters such as the levels of plasminogen, alpha 2-antiplasmin (alpha 2-AP), plasmin/alpha 2-AP (PAP), and D-dimer were also estimated to clarify the fibrinolytic states in these plasmas. The antigens of t-PA, PAI-1, and t-PA/PAI-1 complex were found to increase from 8.5 +/- 4.3, 54.4 +/- 21.2, and 8.6 +/- 3.5 ng/ml in normal plasma to 36.4 +/- 25.1, 106.8 +/- 54.7, and 46.6 +/- 34.5 ng/ml in DIC plasma, respectively. The molar ratio of total t-PA to total PAI-1 was 1:6 and 1:3 in normal plasma and DIC plasma, respectively, indicating an enhanced fibrinolytic state in the DIC plasma. The DIC plasma revealed a significant consumption of plasminogen (62.1 +/- 27.8%), and alpha 2-AP (63.7 +/- 25.3%) and an increase in PAP (2.6 +/- 2.7 micrograms/ml) and D-dimer (3.9 +/- 10.7 micrograms/ml). These results suggest that the production and secretion of t-PA and PAI-1 from endothelial cells were enhanced in DIC, resulting in an increased t-PA/PAI-1 complex with dominant fibrinolytic activity.     

Induction of synthesis of plasminogen activator inhibitor type-1 by tissue-type plasminogen activator in human hepatic and endothelial cells

Plasminogen activator inhibitor type-1 (PAI-1) can modify fibrinolytic activity in vitro and in vivo. The present study was performed to determine whether pharmacologic concentrations of tissue-type plasminogen activator (t-PA) can initiate negative feedback by stimulating PAI-1 synthesis. In both human hepatoma cells (Hep G2) and human umbilical vein endothelial cells (HUVEC), t-PA increased the total concentrations and appearance of newly synthesized protein in conditioned media of free PAI-1 and PAI-1 complexed with t-PA in a dose and time dependent fashion judging from results after immunoprecipitation of metabolically labeled PAI-1. The t-PA effect was not attributable simply to release of stored or matrix-bound PAI-1. In HUVEC, Northern blot analyses indicated that t-PA increased steady-state levels of PAI-1 mRNA two-fold. In contrast PAI-1 mRNA expression was not increased in Hep G2 cells. Thus, mechanisms of stimulation appeared to differ in the two cell lines. The results obtained are consistent with the hypothesis that increased PAI-1 synthesis and secretion in response to t-PA may limit or attenuate fibrinolysis locally or systemically in vivo.     

Fibrinolytic potential and antiphospholipid antibodies in systemic lupus erythematosus and other connective tissue disorders.

We studied the fibrinolytic response before and after venous occlusion (VO) in 30 patients with systemic lupus erythematosus (SLE), 25 with rheumatoid arthritis (RA) and 25 with different connective tissue disorders. Results were compared in patients with and without antiphospholipid antibodies (APA) and a history of either thrombosis or abortions. Before occlusion plasma levels of tissue-type plasminogen activator (t-PA) antigen and its inhibitor (PAI-1) were significantly higher in the patient group (p < 0.001). After occlusion, a low fibrinolytic activity on fibrin plates (p < 0.005) was observed in the same group. t-PA capacity and t-PA release were similar in relation to controls. The plasma PAI-1 activity was significantly elevated in each group of patients (p < 0.005) as compared to the control group. No significant differences with respect to t-PA and PAI-1 were observed in patients as to the presence or absence of thrombosis. There was also no correlation between the fibrinolytic changes and the presence of APA. It is concluded that an impairment of the fibrinolytic system, mainly related to increased PAI-1 levels, is present in most patients with connective tissue disorders, although these changes did not correlate with the presence of APA or the incidence of thrombosis.     

A specific immunologic assay for functional plasminogen activator inhibitor 1 in plasma--standardized measurements of the inhibitor and related parameters in patients with venous thromboembolic disease.

A new assay for functional plasminogen activator inhibitor 1 (PAI-1) in plasma was developed. The assay is based on the quantitative conversion of PAI-1 to urokinase-type plasminogen activator (u-PA)-PAI-1 complex the concentration of which is then determined by an ELISA employing monoclonal anti-PAI-1 as catching antibody and monoclonal anti-u-PA as detecting antibody. The assay exhibits high sensitivity, specificity, accuracy, and precision. The level of functional PAI-1, tissue-type plasminogen activator (t-PA) activity and t-PA-PAI-1 complex was measured in normal subjects and in patients with venous thromboembolism in a silent phase. Blood collection procedures and calibration of the respective assays were rigorously standardized. It was found that the patients had a decreased fibrinolytic capacity. This could be ascribed to high plasma levels of PAI-1. The release of t-PA during venous occlusion of an arm for 10 min expressed as the increase in t-PA + t-PA-PAI-1 complex exhibited great variation and no significant difference could be demonstrated between the patients with a thrombotic tendency and the normal subjects.     

Decrease of fibrinolytic activity in human endothelial cells by arsenite

Blackfoot disease (BFD) is an endemic peripheral vascular occlusive disease that occurred in the southwest coast of Taiwan. It is believed that arsenic in the drinking water from artesian wells plays an important role in the development of the disease. We have previously shown that BFD patients had significant lower tissue-type plasminogen activator (t-PA) antigen level and higher plasminogen activator inhibitor, Type 1 (PAI-1) antigen level than normal controls. The purpose of this study was to investigate the effects of arsenite on the fibrinolytic and anticoagulant activities of cultured macrovascular and microvascular endothelial cells. Incubation of human microvascular endothelial cells (HMEC-1), but not human umbilical vein endothelial cells (HUVECs), with arsenite caused a decrease of t-PA mRNA level, a rise of both PAI-1 mRNA level and PAI activity. Arsenite could also inhibit the thrombomodulin (TM) mRNA expression and reduce the TM antigen level in HMEC-1. In conclusion, arsenite had a greater effect on HMEC-1 as compared to HUVECs in lowering the fibrinolytic activity and may be responsible for the reduced capacity of fibrinolysis associated with BFD.     

Determinants of induction of increased synthesis of plasminogen activator inhibitor type-1 in human endothelial cells by t-PA.

Tissue-type plasminogen activator (t-PA) induces synthesis of a rapidly acting inhibitor, plasminogen activator inhibitor type-1 (PAI-1) in human umbilical vein endothelial cells (HUVEC) in culture. In vivo, an analogous process may induce negative feedback on the fibrinolytic system. To define specific determinants in the t-PA molecule contributing to the induction, PAI-1 synthesis was characterized in 35S-methionine labeled HUVEC in response to several molecular variants of t-PA. Catalytically active variants devoid of several specific structural domains in the A-chain retained the capacity to form complexes with PAI-1 and to induce increased concentrations of total PAI-1 (free and complexed) in conditioned media without depleting PAI-1 from the extracellular matrix. Surprisingly, a mutant t-PA with markedly reduced catalytic activity reflecting replacement of the active site serine with threonine (S478T) formed complexes with PAI-1 and induced increased PAI-1 synthesis as well. However, in contrast to wild-type t-PA and A-chain variants, it did not release 35S-methionine labeled PAI-1 from the extracellular matrix. Thus, its effects appeared to reflect increased secretion exclusively. Our results suggest that induction of PAI-1 synthesis in HUVEC by t-PA depends on its protease domain but that an active site serine is not a requirement.     

Hypofibrinolysis in patients with a history of idiopathic deep vein thrombosis and/or pulmonary embolism.

An impaired fibrinolytic activity after a venous occlusion test is the most common abnormality associated with thomboembolic disease. To better characterize the causes of abnormal responses we have measured different fibrinolytic parameters, before and after 10 and 20 min of venous occlusion, in 77 patients with a history of idiopathic deep vein thrombosis and/or pulmonary embolism and in 38 healthy volunteers. The patients had a lower mean fibrinolytic response to venous occlusion than the controls and higher antigen levels of tissue-type plasminogen activator (t-PA:Ag) and plasminogen activator inhibitor type 1 (PAI-1:Ag). Before venous occlusion, PAI-1 levels were at a molar excess over those of t-PA in all patients and controls. After 20 min of venous occlusion, the release of t-PA from the vascular endothelium resulted in a molar excess of t-PA over PAI-1 in the majority of controls (72%) but only in a minority of patients (39%). To identify patients with fibrinolytic abnormalities, reference intervals (RI) for fibrinolytic activity, t-PA:Ag and PAI-1:Ag were established in healthy controls. None of the patients had low levels of t-PA:Ag, but 17 (22%) had elevated PAI-1:Ag levels before venous occlusion and 12 (16%) exhibited low fibrinolytic activity after 20 min of venous occlusion. Ten of these were among the 17 subjects with high PAI-1:Ag levels before venous occlusion. Thus, the measurement of PAI-1:Ag levels before venous occlusion (i.e. in samples taken without any stimulation) is a sensitive (83%) and specific (89%) assay for the detection of patients with an impaired fibrinolytic response to venous occlusion.     

Heparin cofactor II inhibits thrombin-stimulated release of tissue plasminogen activator from cultured human endothelial cells in the presence of dermatan sulfate

To investigate the involvement of heparin cofactor II (HC II) in fibrinolytic system, endothelial cells from human umbilical vein were cultured in the presence of HC II or antithrombin III (AT III) combined with or without thrombin. Although AT III significantly inhibited thrombin-induced increase in tissue plasminogen activator antigen (t-PA:Ag) release, HC II did not exhibit such a suppressive effect. In contrast, in the presence of dermatan sulfate, HC II inhibited thrombin stimulation of t-PA:Ag release more strongly than AT III did. The release of plasminogen activator inhibitor-1 antigen (PAI-1:Ag) was also stimulated by thrombin; this stimulation was inhibited only by the combination of HC II and dermatan sulfate. Comparatively high concentrations of HC II significantly suppressed thrombin stimulation of t-PA:Ag and PAI-1:Ag release but did not cause an obvious change of both release in the absence of thrombin. Based on these results, it was suggested that HC II may inhibit an increase in fibrinolytic activity mediated by thrombin-stimulated endothelial cells in the liquid phase through a suppression of thrombin stimulation of t-PA:Ag release, when plasma is exposed to vascular smooth muscle cells or fibroblasts which synthesize a significant amount of dermatan sulfate.     

Impaired fibrinolysis and postoperative thromboembolism in orthopedic patients.

The incidence of deep vein thrombosis (DVT) and pulmonary embolism was studied prospectively in patients undergoing elective total hip replacement. 96 patients were randomly allocated to receive either low molecular weight heparin (LMWH) or unfractionated heparin (UFH). All patients had bilateral phlebography and pulmonary perfusion/ventilation scintigraphy 10-12 days after surgery. The following fibrinolytic variables were analysed in plasma and related to thromboembolism: tissue plasminogen activator (t-PA) activity, t-PA antigen (t-PA Ag), plasminogen activator inhibitor (PAI-1) activity and PAI-1 antigen (PAI-1 Ag). No significant difference was found, regarding the fibrinolytic response to surgery, between patients treated with LMWH and UFH. The level of PAI-1 activity was significantly increased before operation in patients developing DVT as compared to non-DVT patients (p less than 0.03). Immediately after surgery and in the morning the first postoperative day the levels of PAI-1 activity, PAI-1 Ag and t-PA Ag were positively correlated to thromboembolism. PAI-1 activity was the only preoperative fibrinolytic variable correlated to thromboembolism.     

Stimulation of tissue-type plasminogen activator synthesis by retinoids in cultured human endothelial cells and rat tissues in vivo

Tissue-type plasminogen activator (t-PA) and its inhibitor, plasminogen activator inhibitor 1 (PAI-1), play an important role in regulating the fibrinolytic capacity of plasma. Both t-PA and PAI-1 are synthesized by the endothelium. We report that retinoic acid (vitamin A acid) and other retinoids rather specifically stimulate the production of t-PA by cultured human umbilical vein endothelial cells. Effective retinoids induced a dose-dependent (range: 0.01-50 microM) increase in the production of t-PA of maximally about six-fold, while simultaneously causing no or only a small increase (less than two-fold) of PAI-1. The effects on t-PA synthesis were apparent by 4-8 h, and reached maximal values after about 24-48 h of incubation with retinoid. The retinoid effect on t-PA production was accompanied by increased t-PA mRNA levels, without any parallel change in PAI-1 or GAPDH mRNA concentrations. The study also shows that modifications at the carboxyl group of retinoic acid are associated with a decrease in stimulatory potency. The stimulatory pathway appears to be identical for all retinoids but distinct from a pathway by which another strong inducer, sodium butyrate, induces t-PA synthesis in endothelial cells. The induction of t-PA by retinoids might involve protein kinase C (PKC) as judged by an experiment using a specific PKC inhibitor. The effect of retinoids on the fibrinolytic system in vivo was assessed by feeding rats with a vitamin A deficient diet or a diet with excess of vitamin A or other retinoids.(ABSTRACT TRUNCATED AT 250 WORDS)     

Impaired fibrinolysis in obstructive jaundice--evidence from clinical and experimental studies

Microvascular thrombosis is considered an important pathogenetic factor in renal failure associated with obstructive jaundice but the mechanisms leading to fibrin deposition are still unknown. The plasma levels of plasminogen activator inhibitor (PAI) in 29 patients with obstructive jaundice were found significantly increased as compared to 20 nonjaundiced patients. Fibrin autography of plasma supplemented with tissue plasminogen activator (t-PA) revealed that in icteric samples most of the added activator migrated with an apparent Mr of 100 kDa, corresponding to t-PA-PAI complex, whereas in control samples virtually all t-PA migrated as free enzyme. PAI activity detected in icteric samples is similar to the endothelial type PAI since it is neutralized by a monoclonal antibody against PAI-1. Venous stasis in jaundiced patients was neither associated with an increase in blood fibrinolytic activity nor with a decrease in PAI activity. Immunologic assay showed that t-PA release was impaired in 3 out of 4 patients. In controls, venous occlusion induced an increase in both fibrinolytic activity and t-PA antigen and a reduction in PAI activity. Bile duct recanalization in jaundiced patients subjected to surgery was accompanied by a decrease in plasma PAI activity which paralleled the decrease in serum bilirubin levels. In nonjaundiced patients, surgical treatment did not cause significant changes in either parameter. Rabbits made icteric by bile duct ligation showed an early and progressive increase in plasma PAI activity indicating that obstructive jaundice itself causes the elevation of circulating PAI. it is concluded that obstructive jaundice is associated with a severe impairment of fibrinolysis which might contribute to microvascular thrombosis and renal failure.     

Salvianolic acid B modulates hemostasis properties of human umbilical vein endothelial cells

Salviae miltiorrhizae (SM), a clinical, commonly used herb, can activate blood circulation and resolve stasis. We have investigated the effects of salvianolic acid B (Sal B), a pure compound extracted from the dried SM roots, on fibrinolytic (tissue-type plasminogen activator and plasminogen activator inhibitor, t-PA and PAI) and anticoagulant (thrombomodulin,TM) properties of cultured human umbilical vein endothelial cells (HUVECs). When HUVECs were treated with Sal B, a dose- (0.0125–0.5 mg/ml) and a time-dependent decrease in PAI activity were observed. PAI type 1 (PAI-1) antigen and PAI-1 mRNA expression significantly decreased compared to control values in the conditioned media of HUVECs pretreated with Sal B for 12 h. Moreover, TM activity reached a maximum stimulation of 1.25-fold over control levels in the pretreatment of Sal B for 12 h and t-PA and TM specific mRNA expression also increased (1.7- and 1.8-fold, respectively). In conclusion, Sal B increased the fibrinolytic and anticoagulant potential of cultured HUVECs by up-regulating the expression of t-PA and TM and by down-regulating the expression of PAI-1. These data suggest that Sal B is clinically effective because of its ability to change the gene expression profile of endothelial cells thereby preventing vascular events.     

Quantitation of tissue-type plasminogen activator in human endothelial cell cultures by use of an enzyme immunoassay

Endothelial cells from human umbilical cord were cultured to study plasminogen activator synthesis and secretion. Since simultaneous production of plasminogen activator inhibitor(s) prevented detection of plasminogen activators by use of fibrinolytic assays, an enzyme immunoassay for tissue-type plasminogen activator (t-PA) was developed. In this assay, t-PA in test samples was adsorbed onto microtiter plates coated with rabbit antibody against t-PA and then quantitated by successive incubation with goat antibody against t-PA and enzyme labeled rabbit antibody against goat IgG. The sensitivity of the assay was found to be 1 ng t-PA/ml. In the absence of serum, arterial and venous endothelial cells continuously produce t-PA antigen during a 24 h period, reaching a level of 5.1 +/- 2.5 ng t-PA/ml (n = 8). In serum containing (20%) medium, 9.3 +/- 6.0 ng t-PA/ml (n = 17) was produced during this period (0.1 - 0.2 ml medium per cm2 confluent cells). It is concluded that the enzyme immunoassay is a useful method for quantitating t-PA secretion by endothelial cells in the presence of proteinase inhibitors.     

Hyperfibrinolysis in alcoholic cirrhosis: relative plasminogen activator inhibitor type 1 deficiency

BACKGROUND: Over activity of the fibrinolytic system (hyperfibrinolysis) occurs in cirrhosis and has been shown to correlate with the risk of variceal hemorrhage. We have developed a model for assessing acute tissue plasminogen activator (t-PA) release in vivo in man. The aims of the study were to assess the contribution of basal and stimulated t-PA release to hyperfibrinolysis in patients with alcoholic cirrhosis. METHODS: Bilateral forearm blood flow and plasma fibrinolytic variables were measured in 8 patients with biopsy proven alcohol induced cirrhosis, ascites and portal hypertension, and 8 age and sex matched healthy controls during infusion of bradykinin (100-900 pmol/min; endothelium-dependent vasodilator that releases t-PA) followed by sodium nitroprusside (SNP 2-8 microg/min; a control endothelium-independent vasodilator). RESULTS: Baseline plasma t-PA antigen concentrations were higher in patients (14+/-2 vs 9+/-1 ng/mL; p<0.05) whereas plasma plasminogen activator inhibitor type-1 (PAI-1) antigen concentrations were similar (59+/-16 vs 55+/-11 ng/mL; p=NS). This resulted in an increased t-PA activity (3+/-1 vs 0+/-0 IU/mL; p<0.05) and reduced PAI-1 activity (9+/-2 vs 21+/-2 AU/mL; p<0.05) indicating a relative deficiency of PAI-1 in patients with cirrhosis. Bradykinin and SNP caused dose-dependent vasodilatation (p<0.001 for both) that did not differ between the two groups. Bradykinin caused a similar release of t-PA antigen (p<0.05 for both) in both patients and controls (24+/-17 vs 23+/-7 ng/100 mL/min; p=ns) without affecting PAI-1 concentrations. Local t-PA activity was increased in patients following acute stimulated t-PA release (5+/-1 vs 3+/-1 IU/mL; p<0.05). SNP caused no significant change in fibrinolytic parameters. CONCLUSION: Patients with alcoholic cirrhosis have a higher basal plasma t-PA activity because of a failure to increase plasma concentrations of its inhibitor, PAI-1. Furthermore, despite releasing normal amounts of t-PA acutely, higher t-PA activity remained due to the relative deficiency of PAI-1. This suggests that the pathogenesis of hyperfibrinolysis in alcoholic cirrhosis is the result of a relative PAI-1 deficiency and enhanced basal t-PA release.     

The gender-specific role of polymorphisms from the fibrinolytic, renin-angiotensin, and bradykinin systems in determining plasma t-PA and PAI-1 levels

Tissue plasminogen activator (t-PA) and plasminogen activator inhibitor 1 (PAI-1) directly influence thrombus formation and degradation and thus risk for arterial thrombosis. We report here results from a genetic analysis of plasma t-PA and PAI-1 levels in a large population-based sample from the PREVEND study in Groningen, the Netherlands (n = 2,527). We measured polymorphisms from genes of the fibrinolytic system, the renin-angiotensin system (RAS), and the bradykinin system. We found that males had higher levels of natural-log transformed t-PA, and PAI-1 (P < 0.01) compared to females. When stratifying females by menopausal status, PAI-1 levels were only significantly different between pre-menopausal females and males (p < 0.001). Furthermore, we found that age, body mass index, and waist-to-hip ratio were significant predictors of t-PA and PAI-1 in both females and males, and that the regression relationships between these factors and plasma t-PA and PAI-1 were dependent on gender. In addition, we found that the PAI-1 4G/5G polymorphism was a significant predictor of PAI-1 levels in both females and males, that the angiotensin II type I receptor A1166C was a significant predictor of t-PA and PAI-1 levels in females, and that the bradykinin receptor B2 58CT polymorphism was a significant predictor of t-PA levels in females. In conclusion, this large population-based study showed that t-PA and PAI-1 levels are determined by several demographic and genetic factors involved in the fibrinolytic, RAS and bradykinin system. In addition, the results support the idea that the biology of t-PA and PAI-1 is different between females and males.     

Fluvastatin inhibits basal and stimulated plasminogen activator inhibitor 1, but induces tissue type plasminogen activator in cultured human endothelial cells

The effects of fluvastatin, a synthetic hydroxymethylglutaryl coenzyme A (HMG-CoA) inhibitor, on the biosynthesis of tissue plasminogen activator (t-PA) and of its major physiological inhibitor (plasminogen activator inhibitor type 1, PAI-1) were investigated in cultured human umbilical vein endothelial cells (HUVEC). Fluvastatin (0.1 to 2.5 microM), concentration-dependently reduced the release of PAI-1 antigen by unstimulated HUVEC, subsequent to a reduction in PAI-1 steady-state mRNA levels and de novo protein synthesis. In contrast, it increased t-PA secretion. The drug also reduced PAI-1 antigen secreted in response to 10 microg/ml bacterial lipopolysaccharide (LPS), 100 U/ml tumour necrosis factor alpha (TNFalpha) or 0.1 microM phorbol myristate acetate (PMA). Mevalonate (100 microM), a precursor of isoprenoids, added to cells simultaneously with fluvastatin, suppressed the effect of the drug on PAI-1 both in unstimulated and stimulated cells as well as on t-PA antigen. Among intermediates of the isoprenoid pathway, all-transgeranylgeraniol (5 microM) but not farnesol (10 microM) prevented the effect of 2.5 microM fluvastatin on PAI-1 antigen, which suggests that the former intermediate of the isoprenoid synthesis is responsible for the observed effects.     

Molecular forms of plasminogen activator inhibitor-1 (PAI-1) and tissue-type plasminogen activator (t-PA) in human plasma

Molecular forms of plasminogen activator inhibitor-1 (PAI-1) and tissue-type plasminogen activator (t-PA), identified by gel filtration and specific immunoassays, were studied in plasma from subjects with normal and elevated PAI-1 levels before and after in vitro or in vivo addition of t-PA. In normal plasma, PAI-1 occurs in three molecular forms, a Mr greater than 700 KDa inactive form of heterogeneous composition, an active 450 KDa form containing PAI-1/vitronectin complex and an inactive peak at Mr 50 KDa containing free PAI-1. Stimulation of platelets results in a significant increase of the 50 KDA form and a slight increase of the 450 KDa form. Patients with increased PAI activity levels have an increase of both the 450 KDa and the 50 KDa forms, whereas patients with thrombotic thrombocytopenic purpura have an increased 50 KDa form. In normal plasma, collected in the presence or absence of D-Phe-Pro-Arg-CH2Cl, t-PA occurs primarily as a Mr greater than 700 KDa form containing t-PA/PAI-1 complex. Addition of high concentrations of t-PA (70 ng/ml) to plasma in vitro or t-PA injection in vivo, results in t-PA inhibitor complexes, including t-PA/ alpha 2 antiplasmin. It is concluded that in subjects with increased PAI-1 levels in plasma, PAI-1 may occur as high molecular weight complexes with vitronectin of which 450 KDa was the most important part and as a 50 KDa inactive form; t-PA circulates primarily in complex with inhibitors. Thus, some of the molecular interactions between PAI-1, t-PA and vitronectin, previously demonstrated in purified systems in vitro, also occur in plasma.     

Hepatic synthesis and clearance of components of the fibrinolytic system in healthy volunteers and in patients with different stages of liver cirrhosis

We determined plasma levels of tissue-type plasminogen activator (t-PA) antigen, urokinase-type plasminogen activator (u-PA) antigen, and activity of the fast acting inhibitor of plasminogen activator (PAI-1) in patients with different stages of liver cirrhosis (Child A, B, and C) and in age and sex-matched healthy controls to investigate the contribution of the liver to the metabolism of these main components of the fibrinolytic system. For control purposes routine clotting parameters were also determined. In patients with the most severe form of liver cirrhosis (Child C) t-PA antigen levels were significantly elevated as compared to patients with Child A or Child B (p less than 0.05) or to controls (p less than 0.01). Furthermore, Child C patients exhibited significantly decreased PAI-1 plasma levels (p less than 0.05) as compared to controls. We were not able to demonstrate, however, any significant correlation between liver function and u-PA plasma levels. Furthermore, t-PA antigen and albumin plasma levels were negatively correlated (r = 0.48; p = 0.0015) and t-PA antigen and bilirubin were positively correlated (r = 0.46; p = 0.0022) thus indicating that the liver is mainly involved in the clearance of t-PA antigen. PAI-1 activity, however, seems to depend partially on synthesis by the liver as demonstrated by a positive correlation between PAI-1 and albumin (r = 0.33; p = 0.037). These physiologic liver functions are both progressively attenuated in severe liver damage and an increase of t-PA plasma levels and a decrease of PAI-1 might contribute to the higher fibrinolytic tendency observed in those patients.     

Heparin induced increase of t-PA antigen plasma levels in patients with unstable angina: No evidence for clinical benefit of heparinization during the initial phase of treatment

Patients with unstable coronary artery disease were randomly treated either with a combination therapy consisting of nitrates and calcium-channel blockers without or with addition of clinical grade heparin administered subcutaneously; in order to evaluate the effect of heparin treatment on the fibrinolytic system, tissue plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) plasma levels were related to the clinical course of the disease. In heparinized patients thrombin time was prolonged more than 3-fold the normal range indicating effective heparin treatment. Heparinization led to a significant increase in t-PA antigen plasma levels (p <0.0001) within approximately four hours while PAI-1 activites remained unaltered. However, the measurable increase of the anticoagulant and pro-fibrinolytic activities of heparin did not result in a short-term benefit for the heparinized patients because the number of further ischemic attacks per patient during the observation period of three days was not different between the two study groups.