Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.68 (tissue plasminogen activator)
 11,311 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Apolipoprotein(a) [apo(a)] is a glycoprotein with Mr approximately equal to 280,000 that is disulfide linked to apolipoprotein B in lipoprotein(a) particles. Elevated plasma levels of lipoprotein(a) are correlated with atherosclerosis. Partial amino acid sequence of apo(a) shows that it has striking homology to plasminogen. Plasminogen is a plasma serine protease zymogen that consists of five homologous and tandemly repeated domains called kringles and a trypsin-like protease domain. The amino-terminal sequence obtained for apo(a) is homologous to the beginning of kringle 4 but not the amino terminus of plasminogen. Apo(a) was subjected to limited proteolysis by trypsin or V8 protease, and fragments generated were isolated and sequenced. Sequences obtained from several of these fragments are highly (77-100%) homologous to plasminogen residues 391-421, which reside within kringle 4. Analysis of these internal apo(a) sequences revealed that apo(a) may contain at least two kringle 4-like domains. A sequence obtained from another tryptic fragment also shows homology to the end of kringle 4 and the beginning of kringle 5. Sequence data obtained from two tryptic fragments show homology with the protease domain of plasminogen. One of these sequences is homologous to the sequences surrounding the activation site of plasminogen. Plasminogen is activated by the cleavage of a specific arginine residue by urokinase and tissue plasminogen activator; however, the corresponding site in apo(a) is a serine that would not be cleaved by tissue plasminogen activator or urokinase. Using a plasmin-specific assay, no proteolytic activity could be demonstrated for lipoprotein(a) particles. These results suggest that apo(a) contains kringle-like domains and an inactive protease domain.
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PMID:Partial amino acid sequence of apolipoprotein(a) shows that it is homologous to plasminogen. 347 6

The clearance of tissue-type plasminogen activator (t-PA) was studied, in rats, by use of a functional assay for t-PA. Half-lives in the circulation were about one minute both for human (melanoma cell-derived) t-PA and for the rat's own t-PA. The clearance of t-PA required an intact liver blood flow. In isolated liver perfusion experiments the hepatic extraction of t-PA did not require any plasma proteins, including fast-acting t-PA inhibitor. Competition experiments, using monosaccharides, suggested that known hepatic glycoprotein receptors were not involved in hepatic t-PA extraction.
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PMID:Hepatic clearance of tissue-type plasminogen activator in rats. 393 64

Thrombospondin (TSP), a multifunctional alpha-granule glycoprotein of human platelets binds fibrinogen, fibronectin, heparin, histidine-rich glycoprotein (HRGP), and plasminogen (Plg), and thus, may play an important role in regulating thrombotic influences at vessel surfaces. In this study we have demonstrated that purified human platelet TSP formed a trimolecular complex with human Plg and HRGP. Complex formation was detected by a specific binding enzyme-linked immunosorbent assay (ELISA) which demonstrated simultaneous binding of fluid-phase Plg and HRGP to TSP adsorbed to microtitration wells. While neither ligand inhibited complex formation of the other with TSP, 10 mM epsilon-amino-n-caproic acid selectively blocked incorporation of Plg into the complex, suggesting that TSP contains independent binding sites for Plg and HRGP. Comparable extent of trimolecular complex formation was also detected when TSP monomer was substituted for whole TSP in the ELISA. HRGP covalently cross-linked to Sepharose 4B simultaneously bound both 125I-TSP and 131I-Plg, confirming trimolecular complex formation. Rocket immunoelectrophoresis of mixtures of the purified radiolabeled proteins into anti-Plg containing agarose also confirmed trimolecular complex formation. The TSP-HRGP-Plg complex bound a similar amount of heparin as the TSP-HRGP complex, demonstrating that the HRGP within the trimolecular complex maintained functional capability. Similarly, using a fluorometric plasmin substrate, the trimolecular complex was shown to be an effective substrate for tissue plasminogen activator. Significant amounts of plasmin were generated from the TSP-HRGP-Plg complex (equivalent to that from the TSP-Plg complex), but the rate of plasmin generation from the trimolecular complex was greater than from the bimolecular complex, suggesting an important interaction of HRGP with Plg when both are complexed to TSP. The macromolecular assembly of these three proteins on cellular surfaces, such as the platelet, may serve important regulatory functions, both prothrombotic at sites of active fibrin deposition and proteolytic in nonfibrin-containing microenvironments.
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PMID:Platelet thrombospondin forms a trimolecular complex with plasminogen and histidine-rich glycoprotein. 400 52

The function of fibrinolysis is to dissolve fibrin clots. The agent of fibrinolysis is plasmin, a glycoprotein with gram molecular weight (GMW) of 90,000. Under natural conditions, plasminogen is converted to plasmin by tissue plasminogen activator (TPA). Activation occurs on the fibrin surface, thus confining proteolytic activity to the appropriate site. Tissue plasminogen activator, produced by monoclonal methods, has recently been made available for limited therapeutic use. Currently streptokinase and urokinase are widely used therapeutically to activate plasminogen. These agents cause plasmin to be formed which is free in the circulation as well as bound to fibrin, resulting in proteolysis of circulating plasminogen and clotting factors. Fibrinolytic therapy has proven to be more beneficial than anticoagulation alone for deep vein thrombi and for pulmonary emboli. During therapy, laboratory studies demonstrate reduced concentrations of plasminogen, fibrinogen, and of alpha-2 plasmin inhibitor, and prolongation of activated partial thromboplastin time and thrombin time. Laboratory findings must be correlated with the clinical course. Demonstration of circulating plasmin-antiplasmin complex may be a useful indicator of active fibrinolysis.
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PMID:Fibrinolysis--a review. 623 87

Cultured bovine aortic endothelial cells are associated with an unusually stable fibrinolytic inhibitor (Loskutoff, D.J., van Mourik, J.A., Erickson, L.A., and Lawrence, D. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 2956-2960). This inhibitor was purified to apparent homogeneity from medium conditioned by these cells by a combination of concanavalin A affinity chromatography and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It is a single-chain glycoprotein of apparent Mr 50,000 +/- 2,500 and isoelectric point of 4.5-5.0, and inhibits the ability of both urokinase and tissue-type plasminogen activator to cleave and active plasminogen. This inhibition of plasminogen activator activity is associated with the formation of an enzyme-inhibitor complex which can be detected after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The purified inhibitor retains full activity after incubation in the presence of 0.1% sodium dodecyl sulfate, or at pH 2.7, two treatments which rapidly destroy the activity of protease nexin, another cellular inhibitor of fibrinolysis. The inhibitor purified from cloned endothelial cells cultured in the presence of L-[3,4,5-3H]leucine represented 2.5-12% of the total radiolabeled protein released by the cells in a 24-h period. These results indicate that cultured bovine aortic endothelial cells synthesize and secrete a protein which inhibits plasminogen activators and is distinct from protease nexin. It is a major endothelial cell product, and, as such, probably plays an important role in regulating the fibrinolytic system of these cells.
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PMID:Purification of an inhibitor of plasminogen activator (antiactivator) synthesized by endothelial cells. 643 6

Thrombospondin (TSP), a multifunctional alpha-granule glycoprotein of platelets, binds fibrinogen, fibronectin, heparin, and histidine-rich glycoprotein and thus may play an important role in regulating thrombotic influences at vessel surfaces. In this study we have demonstrated that purified human platelet TSP formed a complex with purified human plasminogen (Plg). Complex formation was detected by rocket immunoelectrophoresis of mixtures of the purified radiolabeled proteins. Significant complex formation of fluid-phase Plg with adsorbed TSP was also demonstrated by enzyme-linked immunosorbent assay (ELISA). The complex formation was specific, saturable, and inhibited by excess fluid-phase TSP, with an apparent KD of approximately 35 nM. In both ELISA and rocket immunoelectrophoresis systems, complex formation was inhibited by 10 mM epsilon-amino-n-caproic acid, implying that there is a role for the lysine binding sites of Plg in mediating the interaction. TSP also formed a complex with plasmin as detected by ELISA but did not directly inhibit plasmin activity measured with a synthetic fluorometric substrate or with a 125I-fibrin plate assay. TSP, when incubated with Plg before addition to 125I-fibrin plates significantly inhibited the generation of plasmin activity by tissue plasminogen activator (TPA) in a manner that was calcium dependent. A kinetic study of Plg activation by TPA in the presence of TSP demonstrated that Michaelis-Menten kinetics were followed and that TSP acted as a noncompetitive inhibitor. These studies support the hypothesis that TSP, acting as a multifunctional regulator in focal areas of active hemostasis, could serve as a prothrombotic influence, leading to increased deposition of fibrin.
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PMID:Complex formation of platelet thrombospondin with plasminogen. Modulation of activation by tissue activator. 643 54

The effects of gynaecological surgery on the fibrinolytic and inhibitor mechanisms were followed up for 24 h post-operatively in patients receiving a single dose of ketorolac infusion (n = 18) as compared with those not receiving ketorolac infusion (n = 11). A pre-operative state of lower mean t-PA activity and higher PAI-1 levels with increased platelet activation than that reported in normal subjects were observed in both groups of patients. Increased t-PA activity upon anaesthetic induction together with a decreased level at 24 h post-operation was seen in both groups. However, fibrinolytic 'shut-down' was not evident as significant increase in D-dimer levels was observed post-operatively, suggesting an enhanced lytic state concurrent with an enhanced activation of coagulation and diminished platelet activation although beta-TG remained above the normal level; plasmin from this enhanced lytic state affects platelet adhesion and cleaves platelet glycoprotein Ib thus inhibit release reaction. Ketorolac infusion elicited a significant response in PAI-1 activity within 24 h post-operation and this was not seen in the non-ketorolac group in spite of the rising trend by 24 h post-operation which did not achieve statistical significance. There were no statistical significant differences in blood loss and duration of surgery between the two groups of patients. Overall, both groups of patients showed similar haemostatic changes post-operatively for 24 h, a longer duration of post-operative study would have revealed any subtle changes in the molecular markers of thrombosis which was not the objective of this study.
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PMID:The effects of gynaecological surgery on coagulation activation, fibrinolysis and fibrinolytic inhibitor in patients with and without ketorolac infusion. 750 76

Cardiopulmonary bypass (CPB) induces a bleeding defect which leads to enhanced blood loss. A double-blind study was carried out comparing aprotinin with placebo in patients undergoing re-operation for heart valve replacement. The results confirm that aprotinin is effective at reducing such loss. In the placebo treated group, significant increases were observed, during CPB, in the plasma concentrations of fibrinolytic activity, tissue plasminogen activator antigen, D-dimer, and beta-thromboglobulin. Platelet counts fell within 5-10 min of the patients going onto CPB, but this could be accounted for by the dilutional effect of the extracorporeal circuit. Inhibition of responsiveness of platelets, as judged by aggregometry, was significant only at the end of bypass when collagen was the agonist and after protamine reversal when ristocetin was the agonist. CPB did not enhance the release, into the circulation, of glycocalicin (a proteolytic fragment of glycoprotein Ib). In the aprotinin-treated group, the formation of fibrin degradation products as measured by D-dimer was inhibited. However, aprotinin did not influence the change in platelet count, suppress beta-thromboglobulin release from platelets, prevent the inhibition of platelet function or influence the concentration of plasma glycocalicin during the study period. These observations confirm that CPB leads to a fibrinolytic state and less responsive platelets. This study also indicates that aprotinin-induced reduction in blood loss is associated with inhibition of plasmin-mediated fibrin digestion and that the mechanism by which aprotinin reduces blood loss is not via protection of platelets during CPB.
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PMID:Aprotinin reduces cardiopulmonary bypass-induced blood loss and inhibits fibrinolysis without influencing platelets. 751 Sep 90

We examined the efficacy of the monoclonal antibody (MoAb) 7E3 F(ab')2 fragment, an inhibitor of the platelet glycoprotein (GP)IIb/IIIa receptor, to prevent coronary artery rethrombosis after successful thrombolysis with rt-PA. The circumflex coronary artery of anesthetized dogs was instrumented with a flow probe, an electrode, and a stenosis. After recovery from the surgical procedure, the animals were reanesthetized on post-operative day 9, and vessel wall injury was induced with current applied to the intimal surface of the circumflex coronary artery. The resulting occlusive thrombus was aged for 30 min, and recombinant tissue plasminogen activator (rt-PA) was administered. The animals were allocated to receive either placebo or a single dose of 7E3 [0.8 mg/kg intravenous (i.v.) bolus] as the sole adjunctive agent. Ex vivo platelet function and coronary artery blood flow velocity were recorded on each of 5 consecutive days. Reocclusion and mortality were reduced significantly in animals treated with 7E3 as compared with the placebo-treated group. Significant inhibition of ex vivo platelet aggregation persisted for 48 h after a single injection of 7E3. The MoAb 7E3 F(ab')2 fragment is effective as the sole adjunctive agent with rt-PA for prevention of rethrombosis. The present study is unique in that it examined the efficacy of GPIIb/IIIa inhibition in an experimental model for an extended time, demonstrating the duration of antiplatelet therapy required to prevent rethrombosis after thrombolysis.
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PMID:Prevention of rethrombosis after coronary thrombolysis in a chronic canine model. I. Adjunctive therapy with monoclonal antibody 7E3 F(ab')2 fragment. 751 47

Plasminogen activator inhibitor type 1 (PAI-1) is the rapid physiologic inhibitor of tissue-type plasminogen activator and urokinase-type plasminogen activator (uPA). In plasma and the extracellular matrix, PAI-1 is associated with the adhesive glycoprotein vitronectin. In order to characterize the PAI-1 structural domain responsible for binding to vitronectin, the segment of the PAI-1 cDNA encoding amino acids 13-147 (nucleotides 248-650) was randomly mutagenized and subcloned into a bacterial expression vector containing the mature PAI-1 coding sequence. Recombinant PAI-1 mutants were expressed in Escherichia coli and bacterial lysates assayed in duplicate for uPA inhibitory activity and vitronectin binding. Of 190 clones screened, six consistently demonstrated decreased vitronectin binding relative to uPA inhibitory activity. DNA sequence analysis of four of these clones identified 10 unique missense mutations, all located between base pairs 298 and 641, with each clone containing between one and four substitutions. Each substitution was expressed independently by site-directed mutagenesis and again analyzed for uPA inhibitory activity and vitronectin binding. Five point mutations that selectively disrupt vitronectin binding were identified. All 5 residues are located on the exterior of the PAI-1 structure. These findings appear to define a complex binding surface that bridges alpha-helices C and E to beta-strand 1A and includes amino acids 55, 109, 110, 116, and 123. These results suggest that vitronectin binding may stabilize the active conformation of PAI-1 by restricting the movement of beta-sheet A and thereby preventing insertion of the reactive center loop.
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PMID:Localization of vitronectin binding domain in plasminogen activator inhibitor-1. 751 53


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