Urinary tissue type plasminogen activator and plasminogen activator inhibitor in renal transplant patients.
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Biopsy, Needle↗
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Heparin enhances active site-dependent binding of tissue-type plasminogen activator to endothelial cells.
Human umbilical vein endothelial cells (HUVEC) in culture express two classes of binding sites for tissue-type plasminogen activator (t-PA). The high-affinity binding site has been identified as PA inhibitor type 1 (PAI-1), which binds to the catalytic portion of the molecule, while the second site binds t-PA through an active-site independent domain. Because recombinant t-PA (rt-PA) is often administered concomitantly with heparin, we investigated the effects of heparin on rt-PA binding to HUVEC. Preincubation of HUVEC with heparin at 4 degrees C increased the binding of radiolabeled rt-PA in a time- and dose-dependent manner. One-half maximal increase in binding was observed within 10 minutes of heparin addition. When HUVEC were preincubated with optimal concentrations (5 U/mL) of heparin for 4 hours at 4 degrees C, a 2.5- +/- 0.2-fold increase in specific binding was observed (mean +/- SEM, n = 12, P less than .01). Other highly sulfated glycosaminoglycans and fucoidan (a sulfated polymer of fucose) stimulated rt-PA binding as well, whereas glycosaminoglycans with lower sulfate content than heparin did not. Several results suggested that heparin increased the binding of rt-PA to "cell-associated" PAI-1. First, only active-site-dependent binding was enhanced by heparin, whereas binding of active-site blocked rt-PA was not affected. Second, extracts from HUVEC preincubated with heparin contained increased amounts of rt-PA-PAI-1 complexes as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Third, antibodies to PAI-1 blocked the increased binding entirely. HUVEC preincubated with heparin also bound increased amounts of enzymatically active radiolabeled urokinase-type PAs. However, HUVEC preincubated with heparin did not express increased amounts of immunoreactive PAI-1. Therefore, heparin, at therapeutic concentrations, may enhance or stabilize the association of PAs with endothelial cell-associated PAI-1.
Binding Sites↗
Impaired fibrinolytic response to DDAVP in patients with von Willebrand's disease.
The fibrinolytic response after infusion of 1-deamino-8-d-arginine vasopressin (DDAVP) was examined in 4 patients with the severe form and 17 patients with the moderate form of Von Willebrand's disease (VWd) and compared to that of 9 normal subjects. Tissue plasminogen activator (t-PA) antigen (Ag) and activity and tissue plasminogen activator inhibitor (PAI) were measured before and after DDAVP. t-PA Ag was found to be significantly higher before DDAVP in patients than in controls. No release of t-PA Ag was observed in the 4 patients with the severe form of VWd but increased release was observed in patients with moderate forms. However, t-PA released into the circulation in these 17 patients had a lower functional activity as compared to that of normal subjects. PAI was significantly lower in patients before DDAVP than in normal subjects and the decrease in PAI after DDAVP was significantly less in patients than in controls. It is concluded from this study that patients with VWd have an abnormal fibrinolytic response after stimulation regardless of the severity of the disease. Furthermore, the results suggest either that patients with VWd have a double defect in VWF and tissue plasminogen activator or that the primary deficiency of VWF influences the synthesis and/or release of t-PA by endothelial cells.
Adolescent↗
Regulation of proteolytic activity in human bone marrow stromal cells by basic fibroblast growth factor, interleukin-1, and transforming growth factor beta.
Plasminogen activators (PAs) and/or plasmin may be involved in hematopoietic regulation. These enzymes release biologically relevant cytokines such as basic fibroblast growth factor (bFGF) from matrix and cell surfaces. In addition, transforming growth factor beta (TGF beta) and interleukin-1 beta (IL-1 beta) are converted from inactive to active forms by plasmin. Therefore, we studied the regulation of PAs and their specific inhibitors, PA inhibitor 1 (PAI-1) and PA inhibitor 2 (PAI-2), in human bone marrow stromal fibroblasts by IL-1 beta, bFGF, and TGF beta. All three cytokines stimulated PA secretion. IL-1 beta at 10(4) U/mL increased urokinase (u-PA) levels approximately 10-fold, bFGF at 0.2 ng/mL also increased production 10-fold, but increased predominantly tissue PA (t-PA) expression. TGF beta at 0.2 ng/mL increased u-PA production up to 300-fold. PAI-1 and PAI-2 are also regulated by these cytokines. IL-1 beta decreased PAI-1 levels by 50% and stimulated PAI-2 levels sixfold. bFGF had minimal effects on PAI-1 and TGF beta increased PAI-1 levels twofold. Neither of these agents had an effect on PAI-2 levels. Thus, three cytokines relevant to bone marrow physiology regulate PA and inhibitor production by human bone marrow stromal fibroblasts. In this manner PA and plasmin generation in specific microenvironments in the bone marrow may be one of the factors orchestrating the complex series of events, which results in an efficient exquisitely regulated hematopoietic process.
Bone Marrow↗
[Decreased fibrinolytic capacity in coronary patients by increased plasminogen activator inhibitor activity].
The fibrinolytic capacity of the blood mainly depends on the amount of tissue-plasminogen activator (t-PA) antigen and plasminogen activator inhibitor (PAI). In this study the fibrinolytic response to a venous occlusion test (VOT) was measured in 109 patients with angiographically documented coronary artery disease (CAD) and in 20 healthy volunteers at comparable age (controls). CAD-patients had higher plasma plasminogen activator inhibitor capacity before (24.4 +/- 11.0 vs. 15.4 +/- 5.2 arbitrary units [AU/ml]; p less than 0.0002) and after VOT (19.6 +/- 13.2 vs. 10.9 +/- 5.3 AU/ml; p less than 0.0001) compared with controls. Furthermore they showed significant lower plasma t-PA activity after VOT (3.0 +/- 6.8 vs. 6.6 +/- 10.6 AU/ml; p less than 0.0001). However there were no difference between both groups in plasma t-PA antigen levels after VOT (17.3 +/- 12.1 vs. 18.7 +/- 14.4 ng/ml). In 10% of patients the decrease in fibrinolytic activity resulted from a lower t-PA release ("lower" was defined as mean minus one standard deviation of the control group). 40% showed elevated plasma PAI capacity before VOT ("elevated" was defined as mean plus two standard deviations of the control group). Both caused significantly reduced post occlusion plasma t-PA activity and prolonged Euglobulin clot lysis time (p less than 0.003). A positive correlation was found between PAI capacity and serum triglyceride levels. Reduced fibrinolytic activity in 109 patients with coronary heart disease based either on a decrease in t-PA antigen release or a increased in PAI capacity in comparison with healthy controls. The mechanism of these findings is not yet well-known.(ABSTRACT TRUNCATED AT 250 WORDS)
Adult↗
Urokinase-type plasminogen activator in human aqueous humor.
In the anterior segment of the eye, fibrin clots must be rapidly resorbed to prevent further fibrosis and scarring. The aqueous humor of patients undergoing cataract surgery was analyzed for the presence of components of the fibrinolytic cascade. In 30 patients, aqueous humor and plasma were compared for their content of urokinase-type plasminogen activator (uPA), tissue-type plasminogen activator (tPA), plasminogen activators inhibitors (PAIs), plasminogen, and total proteins. With gel electrophoresis and zymographic assays of serial dilutions of plasma and aqueous humor, all these components were found to be present at lower concentrations in aqueous humor than in plasma. For total proteins, the aqueous/plasma ratio was approximately 0.003, and for plasminogen it was 0.001. Interestingly, the aqueous/plasma ratio for uPA was not as low and varied from 0.01 to 0.03. A significant proportion of the uPA in aqueous humor was present in the two-chain active form. In addition to uPA, aqueous humor contained lower levels of tPA, but no detectable levels of reactive plasminogen activators inhibitors (PAIs). The presence of a relatively high concentration of active uPA shows that the proteolytic balance of the aqueous humor in the anterior chamber of the eye is shifted toward fibrinolysis.
Aqueous Humor↗
The inhibition of tissue type plasminogen activator by plasminogen activator inhibitor-1. The effects of fibrinogen, heparin, vitronectin, and lipoprotein(a).
Plasminogen activator inhibitor-1 (PAI-1) regulates fibrinolysis by inhibiting tissue type plasminogen activator (t-PA). Fibrinogen, heparin, and vitronectin enhance the rate of inhibition of t-PA by PAI-1. Kinetic studies indicate that both fibrinogen and heparin increase the second-order inhibition constant by a maximum of approximately 4-fold, whereas vitronectin increases the rate constant by a maximum of approximately 6-fold. The dissociation constants of fibrinogen, heparin, and vitronectin for the inhibition reaction were 200 nM, 20 nM, and 600 pM, respectively. In addition, PAI-1 inhibition of t-PA may be regulated by the presence of lipoprotein(a) (Lp(a)). Previous studies demonstrated that Lp(a) competes with plasminogen for the active site of fibrinogen- and heparin-bound t-PA. Kinetic studies described here demonstrate that Lp(a) prevents the inhibition of t-PA by PAI-1 in the presence of fibrinogen and heparin, but has no effect on the reaction in the presence of vitronectin or in the absence of either fibrinogen or heparin. The data suggest that fibrinogen and heparin may enhance the rate of inhibition through an interaction with t-PA, and that vitronectin may enhance the inhibition through an interaction with PAI-1. In addition, these experiments indicate that Lp(a) may regulate fibrinolysis by competing with PAI-1 and plasminogen for fibrinogen- and heparin-bound t-PA. These data suggest that PAI-1 inhibition of t-PA in vivo is primarily mediated via interaction with fibrinogen, heparin, vitronectin, and Lp(a), and therefore, the functional levels of PAI-1 activity in the vasculature may be regulated by the presence of these components.
Fibrinogen↗
Vitronectin governs the interaction between plasminogen activator inhibitor 1 and tissue-type plasminogen activator.
The "serpin" plasminogen activator inhibitor 1 (PAI-1) is the fast acting inhibitor of plasminogen activators (tissue-type (t-PA) and urokinase type-PA) and is an essential regulatory protein of the fibrinolytic system. Its P1-P1' reactive center (R346 M347) acts as a "bait" for tight binding to t-PA/urokinase-type PA. In vivo, PAI-1 is encountered in complex with vitronectin, an interaction known to stabilize its activity but not to affect the second-order association rate constant (k1) between PAI-1 and t-PA. Nevertheless, by using PAI-1 reactive site variants (R346M, M347S, and R346M M347S), we show that the binding of vitronectin to the PAI-1 mutant proteins improves plasminogen activator inhibition. In the absence of vitronectin the PAI-1 R346M mutants are virtually inactive toward t-PA (k1 less than 1 x 10(3) M-1 s-1). In contrast, in the presence of vitronectin the rate of association increases about 1,000-fold (k1 of 6-8 x 10(5) M-1 s-1). This inhibition coincides with the formation of serpin-typical, sodium dodecyl sulfide-stable t-PA.PAI-1 R346M (R346M M347S) complexes. As evidenced by amino acid sequence analysis, the newly created M346-M/S347 peptide bond is susceptible to attack by t-PA, similar to the wild-type R346-M347 peptide bond, indicating that in the presence of vitronectin M346 functions as an efficient P1 residue. In addition, we show that the inhibition of t-PA and urokinase-type PA by PAI-1 mutant proteins is accelerated by the presence of the nonprotease A chains of the plasminogen activators.
Base Sequence↗
The interaction of plasminogen activator inhibitor 1 with plasminogen activators (tissue-type and urokinase-type) and fibrin: localization of interaction sites and physiologic relevance.
Plasminogen activator inhibitor 1 (PAI-1), an essential regulatory protein of the fibrinolytic system, harbors interaction sites for plasminogen activators (tissue-type [t-PA] and urokinase-type [u-PA]) and for fibrin. In this study, anti-PAI-1 monoclonal antibodies (MoAbs) were used to identify interaction sites of PAI-1 with these components. The binding sites of 18 different MoAbs were established and are located on five distinct "linear" areas of PAI-1. MoAbs, binding to two distinct areas of PAI-1, are able to prevent the inhibition of t-PA by PAI-1. In addition, two interaction sites for fibrin were identified on PAI-1. The area located between amino acids 110 and 145 of PAI-1 contains a binding site for both components and its significance is discussed in the context of the t-PA inhibition by fibrin-bound PAI-1. Subsequently, the MoAbs were used to assess the role of platelet-PAI-1 in clot-lysis. An in vitro clot-lysis system was used to demonstrate that clot-lysis resistance is dependent on the presence of activated platelets and that PAI-1 is a major determinant for lysis-resistance. We propose that, upon activation of platelets, PAI-1 is fixed within the clot by binding to fibrin and retains its full capacity to inhibit t-PA and u-PA.
Antibodies, Monoclonal↗
Characterization of monoclonal antibodies against human tissue plasminogen activator (tPA): quantitation of free tPA in human cell cultures by an ELISA.
Seven murine monoclonal antibodies produced against tissue plasminogen activator (tPA) were evaluated by means of enzyme-linked immunosorbent assays (ELISAs), and their effects on the enzymatic activities of tPA towards a synthetic substrate (S-2288) and plasminogen were investigated. One of the antibodies, TPA1-70, strongly inhibited the enzymatic activity of tPA in a fibrin agarose plate assay, while it did not affect the enzymatic activity towards the synthetic substrate or plasminogen. The antibody is directed to an epitope on the B-chain of tPA, which is necessary for the formation of a ternary complex of tPA, fibrin and plasminogen, but probably not to the active site. Another antibody, TPA2-14, partially inhibited the enzymatic activities of tPA towards the synthetic substrate and plasminogen, but it was not able to bind to the inactive tPA complexed with plasminogen activator inhibitor-1 (PAI-1). The antibody is directed to an epitope on the second kringle region, which is probably one of the PAI-1 binding sites. This property of the antibody enabled us to develop an ELISA for selective quantitation of free tPA in culture media conditioned with several human cell lines. The results indicate that tPA in these media exists either partially or almost entirely in a complex with PAI-1.
Animals↗
On the target specificity of plasminogen activator inhibitor 1: the role of heparin, vitronectin, and the reactive site.
Plasminogen activator inhibitor 1 (PAI-1) is the fast-acting inhibitor of both tissue-type and urokinase-type plasminogen activators (t-PA, u-PA) and is an essential regulatory protein of the fibrinolytic system. In the presence of either the protein vitronectin or the glycosaminoglycan heparin, PAI-1 is also an efficient inhibitor of thrombin. To assess whether these cofactors turn PAI-1 into a general protease inhibitor or whether their influence is restricted to thrombin, the second-order association rate constants between PAI-1 and the human plasma proteases t-PA, u-PA, plasmin, thrombin, Factor Xa (FXa), and Factor XIIa (FXIIa) in the absence and in the presence of either vitronectin or heparin are determined. In addition, the role of the PAI-1 reactive site P3 to P3' residues for the specificity of inhibition was studied by using PAI-1 reactive site mutants. Our results show that: (1) Heparin exclusively increases the rate of inhibition of thrombin by PAI-1, whereas in the presence of heparin the rate of inhibition of the other proteases is not altered; (2) Vitronectin is an obligatory cofactor for the inhibition of thrombin by PAI-1. In addition, vitronectin moderately increases the rate of inhibition by PAI-1 of u-PA and of plasmin, but does not alter the rate of inhibition of t-PA, FXa, or FXIIa; (3) Apart from the important role of the P1 residue, no consensus can be presented on the nature of other residues within the P3 to P3' region with regard to target protease specificity.
Antithrombin III↗
A kinetic model of the circulatory regulation of tissue plasminogen activator.
A computer simulation was developed to study the regulation of active tissue plasminogen activator (t-PA) levels in plasma by kinetically modeling t-PA secretion, t-PA inhibition by plasminogen activator inhibitor type 1 (PAI-1), and hepatic clearance of t-PA, PAI-1 and t-PA/PAI-1 complex throughout a simplified human circulatory system. The model indicates that as the active PAI-1 concentration increases, the percent of t-PA in the active form decreases exponentially. Further, the reaction between t-PA and PAI-1 substantially reduces the half-lives of both active factors. By adjusting the t-PA and PAI-1 secretion rates to provide the best fit between simulated and measured circadian variations in t-PA, PAI-1 and complex, the model predicts that the diurnal rhythm in active t-PA levels is principally due to changes in the rate of PAI-1 secretion and not to variations in the t-PA secretion rate. In conclusion, the model predicts that PAI-1 is an important regulator of the concentration, half-life and circadian variation of active t-PA.
Adult↗
[Changes of ovarian plasminogen activator and inhibitor during gonadotropin-induced ovulation in rhesus monkeys].
Changes of plasminogen activator (PA) and its inhibitor (PAI-1) activity and antigen have been investigated during PMSG/hCG induced ovulation in rhesus monkeys. It has been demonstrated that the ovarian tissue type PA (tPA) activity, which reaches maximum prior to ovulation and declines thereafter, is closely related to follicular rupture; significant increases in urokinase type PA (uPA) only occurs in granulosa cells after ovulation. Since the secretory activity of ovarian PAI-1 reaches its peak level 12-24 h earlier than tPA the rapid decrease in PAI-1 activity in the approach of ovulation is correlated with the elevation of tPA activity. It is, therefore, suggested that a counterbalance of tPA and PAI-1 activity within the ovary may play an important role in the ovulation mechanism, whereas uPA may be involved in the regulation of corpus luteum formation.
Animals↗
[Dynamic changes in fibrinolytic system in patients with acute myocardial infarction].
Dynamic change of serum tissue-type plasminogen, activator (t-PA) and PAI activity was studied in 22 cases with acute myocardial infarction (AMI) 11 cases with unstable angina (UA) and 24 healthy persons. The group of AMI was divided into two subgroups, one with severe complications, AMI-A subgroup and the other without complication, AMI-B subgroup. The results revealed: t-PA activity was decreased in AMI group, especially in AMI-A subgroup; PAI activity was markedly increased in AMI-A subgroup in first three days after onset; there was a marked decrease of active type in AMI-A subgroup. The study confirms that the function of fibrinolytic system is abnormal in patients with AMI and UA. The change of activity of t-PA and PAI is closely related with the severity of AMI. It is important to augment the activity of the fibrinolytic system in the treatment of AMI.
Adult↗
Occurrence of components of fibrinolysis pathways in situ in neoplastic and nonneoplastic human breast tissue.
The occurrence and distribution of components of fibrinolysis pathways were determined using immunohistochemical techniques applied to 10 cases of primary carcinoma of the breast, normal breast tissue obtained from two patients undergoing reductive mammoplasty, and three cases of benign breast tumors. Tumor cells stained for urokinase- and tissue-type plasminogen activators, plasminogen activation inhibitor-1, plasminogen, and plasmin-antiplasmin complex neoantigen. The tumor connective tissue stained for fibrinogen and its D fragment plasmin digestion product. By contrast, only occasional nonneoplastic duct epithelial cells stained for urokinase- and tissue-type plasminogen activators and there was little or no staining for the other antigens tested. These results are consistent with the existence of local amplification of expression of enzymatically active plasminogen activators, and particularly of urokinase-type plasminogen activator, in situ in primary breast cancer tissue. These features distinguish malignant from benign breast tissue and may modulate neoplastic progression through an effect on tumor cell proliferation, invasion, and metastatic dissemination.
Breast↗
Endothelial cell haemostatic function after heart transplantation.
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Antithrombin III↗
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.
Adult↗
[A new advance in theory of blood coagulation and fibrinolysis and its practical application].
New trends in tests for coagulation and fibrinolysis and advances in diagnosis for the hypercoagulable state and utilization of immunological techniques such as various polyclonal and monoclonal antibodies are reported. We discussed (1) the new markers for hypercoagulable states, (2) differential diagnosis of disseminated intravascular coagulation (DIC) and abnormalities of coagulation in liver cirrhosis (LC), and (3) new markers for fibrinolysis and vascular function. Thrombin-antithrombin III complex (TAT) levels were higher in thrombotic diseases than in healthy controls. Therefore, TAT should be a good marker for hypercoagulation as fibrinopeptide A (FPA) and soluble fibrin monomer complex (SFMC). Measurement of TAT, plasma-alpha 2 plasmin inhibitor complex (PIC), and D dimer were useful for differential diagnosis of DIC and liver cirrhosis. t-PA-PAI complex correlated well with t-PA, but not with fibrinolytic parameters such as PIC. The t-PA-PAI complex may be a good marker for the function of the vascular endothelium.
Antithrombin III↗