Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: UNIPROT:P00750 (PLA)
 16,800 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The Kunitz-type trypsin and tissue plasminogen activator (t-PA)-inhibitor from Erythrina caffra seeds was cleaved by trypsin at low pH to yield a disulphide linked two-chain molecule with reduced hydrophobicity. This change was used to separate cleaved from native inhibitor by phenyl-Sepharose chromatography. The inhibitor was not cleaved by t-PA. Trypsin, but not t-PA, catalysed resynthesis of the cleaved bond. Although the cleaved protein retained inhibitory activity for both trypsin and t-PA, 6-10 times higher concentrations were required for equivalent inhibition. Removal of the active site arginine (Arg63) from the cleaved inhibitor by digestion with carboxypeptidase B resulted in a further loss of inhibitory activity towards both proteases. The activity of the inhibitor could also be decreased by modification of one susceptible arginine residue with peptidyl arginine deiminase. These results suggest that the trypsin-reactive site of the Erythrina inhibitor is also involved in the interaction between the inhibitor and t-PA.
 ...
PMID:The Erythrina protease inhibitor: interactions with tissue plasminogen activator. 177 16

A novel triple-kringle plasminogen activator protein, PK1 delta FE1X, has been produced which is a genetic chimera between the fibrin binding kringle 1 domain of plasminogen and the two kringles and serine protease domains of naturally occurring wild-type tissue plasminogen activator (wt t-PA). This chimera also contains a modification to prevent high mannose type N-linked glycosylation on kringle 1 of t-PA. PK1 delta FE1X is biochemically and fibrinolytically similar to wt t-PA in vitro but retains the decreased plasma clearance rate characteristic of other t-PA variants which lack fibronectin finger-like and epidermal growth factor domains. The serine protease domain of PK1 delta FE1X exhibits the amidolytic activity characteristic of wt t-PA. In an indirect coupled plasminogen activator assay, the specific activity of PK1 delta FE1X is approximately 1.4 times greater than that of wt t-PA. In a fibrin film-binding assay, greater binding to untreated fibrin is observed with wt t-PA than with PK1 delta FE1X. However, following limited plasmin digestion of the fibrin film, PK1 delta FE1X binding increases to the level observed with wt t-PA. The incremental binding to plasmin-digested fibrin observed with PK1 delta FE1X is eliminated if plasmin digestion of the fibrin film is followed by carboxypeptidase B treatment. This result suggests that plasminogen kringle 1 binds plasmin-digested fibrin even after recombination with a heterologous protein. The fibrinolytic activity of PK1 delta FE1X in human plasma clot lysis assays was similar to that of wt t-PA at activator concentrations of approximately 1 microgram/ml. At substantially lower concentrations, approximately 0.1 microgram/ml, PK1 delta FE1X was only slightly less active than wt t-PA. Pharmacokinetic analysis showed that wt t-PA activity is cleared approximately 15 times as rapidly as PK1 delta FE1X following intravenous bolus injection. In a rabbit jugular vein clot lysis model, intravenous bolus injection of 0.06 mg/kg of PK1 delta FE1X showed greater thrombolytic potency than a similar administration of 0.5 mg/kg of wt t-PA. Thus it appears that in vitro exon shuffling techniques can be used to generate novel fibrinolytic agents which biochemically and pharmacologically represent the combination of individual domains of naturally occurring proteins.
 ...
PMID:Replacement of finger and growth factor domains of tissue plasminogen activator with plasminogen kringle 1. Biochemical and pharmacological characterization of a novel chimera containing a high affinity fibrin-binding domain linked to a heterologous protein. 184 87

Monocytes and monocytoid cell lines previously have been shown to express receptors for plasminogen and urokinase (u-PA). In the present study, the monocytoid cell lines, U937 and THP-1, are shown to bind tissue plasminogen activator (t-PA) in a specific, saturable, and reversible manner. These cells bound t-PA with low affinity (kd = 0.67 to 0.97 mumol/L) and high capacity (0.71 to 3.3 x 10(6) receptors/cell). Human peripheral blood monocytes bound t-PA with a kd (0.9 mumol/L) similar to that of the monocytoid cells but with a lower capacity (0.17 x 10(6) sites/cell). These binding parameters also were similar to the low-affinity interaction of t-PA with endothelial cells as measured with the cells in suspension (kd = 0.73 mumol/L and 1.1 x 10(6) sites/cell). Lysine analogues and active or diisopropylfluorophosphate-inactivated u-PA inhibited t-PA binding to monocytes, monocytoid cells, and endothelial cells with similar IC50 (concentration producing 50% inhibition) values, suggesting that the same recognition specificity mediates t-PA binding to all of these cell types. The existence of a high-affinity binding site for t-PA on monocytoid cells was also explored in detail. Unlike endothelial cells where plasminogen activator inhibitor-1 has been implicated in mediating a high-affinity interaction of t-PA with the cells, no evidence for a role of this inhibitor in ligand binding to the monocytoid cells was found. Furthermore, using both high and low 125I-t-PA concentrations, competition analyses with lysine analogues or u-PA, or treatment of the cells with carboxypeptidase B, failed to indicate the presence of distinguishable classes of t-PA binding sites. In sum, low-affinity receptors for t-PA are expressed at high density on monocytes and monocytoid cells, identifying a new element in the fibrinolytic arsenal of these cells.
 ...
PMID:Binding of tissue plasminogen activator to human monocytes and monocytoid cells. 193 47

The binding of recombinant tissue-type plasminogen activator (rt-PA) to fibrin increases upon digestion of fibrin with plasmin. Optimal binding is observed following a limited plasmin digestion of fibrin, coinciding with the generation of fibrin fragment X polymers. We studied the involvement of the separate domains of the amino-terminal "heavy" (H) chain of rt-PA in this augmentation of fibrin binding. The fibrin-binding characteristics of a set of rt-PA deletion mutants, lacking either one or more of the structural domains of the H chain, were determined on intact fibrin matrices and on fibrin matrices that were subjected to limited digestion with plasmin. The augmented fibrin binding of rt-PA is partially abolished when the plasmin-degraded fibrin matrices are subsequently treated with carboxypeptidase B, demonstrating that this increased binding is dependent on the generation of carboxyl-terminal lysine residues in the fibrin matrix. Evidence is provided that this increase of fibrin binding is mediated by the kringle 2 (K2) domain that contains a lysine-binding site. Further increase of the fibrin binding of rt-PA is independent of the presence of carboxyl-terminal lysines. It is shown that the latter increase is not mediated by the K2 domain. Based on our data, we propose that the increase in fibrin binding, unrelated to the presence of carboxyl-terminal lysine residues, is mediated by the finger (F) domain, provided that this domain is correctly exposed in the remainder of the protein.
 ...
PMID:Identification of the domains of tissue-type plasminogen activator involved in the augmented binding to fibrin after limited digestion with plasmin. 252 27

Sequencing of two internal peptides from the putative human endothelial cell tissue plasminogen activator (t-PA) receptor identified an analog of the calcium- and phospholipid-binding protein, annexin II (Ann-II). The polymerase chain reaction-derived, full-length cDNA revealed complete sequence identity with the heavy chain of Ann-II, and ligand-precipitated receptor protein immunoreacted specifically with a monoclonal antibody to Ann-II. Transfected 293 cells bound plasminogen (Kd = 114 nM; Bmax = 347,000) as well as t-PA (Kd = 48 nM; Bmax = 380,000). Antisense oligonucleotides directed against endothelial cell Ann-II mRNA inhibited binding of both t-PA and plasminogen by 49% and 38%, respectively. The K307T mutant of Ann-II expressed on 293 cells failed to bind plasminogen, while the K328I mutant bound this ligand in a manner equivalent to the wild-type. Binding of plasminogen to both the wild-type and the K328I mutant was blocked by pretreatment of 293 cells with carboxypeptidase B. These data suggest a novel mechanism whereby a plasmin-like serine protease may cleave Ann-II at Lys307-Arg308, exposing a new carboxyl-terminal lysine residue (Lys307) for binding and efficient activation of plasminogen.
 ...
PMID:An endothelial cell receptor for plasminogen/tissue plasminogen activator. I. Identity with annexin II. 806 40

In the preceding paper (Hajjar, K. A., Jacovina, A. T., and Chacko, J. (1994) J. Biol. Chem. 269, 21191-21197), we identified a M(r) = 40,000 endothelial cell receptor for tissue plasminogen activator (t-PA) and plasminogen (PLG) as the calcium- and phospholipid-binding protein, annexin II (Ann-II). Here, we examined the effect of Ann-II on t-PA-dependent plasminogen activation in a purified system. Purified native Ann-II bound t-PA, plasminogen, and plasmin with high affinity (Kd = 25 nM, 161 nM, and 75 nM, respectively). At fixed plasminogen concentrations, preincubation with purified native Ann-II was associated with an approximately 21-fold increase in the rate of Glu-PLG activation and an approximately 14-fold increase in activation of Lys-PLG. Three irrelevant proteins had no effect on plasmin formation, while fibrinogen increased the rate of Glu-PLG activation by approximately 4-fold. Annexin-II-mediated enhancement of t-PA-dependent plasminogen activation was 90-95% inhibited by epsilon-aminocaproic acid or by pretreatment of Ann-II with carboxypeptidase B, indicating a carboxyl-terminal lysine-dependent interaction. Kinetic analyses revealed that Ann-II conferred an approximately 60-fold increase in catalytic efficiency upon t-PA-dependent activation of either Glu-PLG or Lys-PLG. Thus, Ann-II-mediated assembly of plasminogen and t-PA may promote and localize constitutive plasmin generation on the surface of the blood vessel wall.
 ...
PMID:An endothelial cell receptor for plasminogen/tissue plasminogen activator (t-PA). II. Annexin II-mediated enhancement of t-PA-dependent plasminogen activation. 806 41

It is well established that tissue-type plasminogen activator (t-PA) binds to the D region of fibrin(ogen) and that two distinct CNBr fragments of fibrinogen (FCB), FCB-2 and FCB-5, comprising parts of this region, stimulate plasminogen activation by t-PA. In the present work, ligand-binding studies were performed to characterize the interactions between t-PA and the corresponding fibrin regions using a well defined model of a fibrin surface and both FCB-2 and FCB-5 in liquid and solid phase. Binding isotherms showed a characteristic Langmuir adsorption saturation profile. The dissociation constants determined for the binding of t-PA to immobilized FCB-2 (Kd = 0.70 +/- 0.10 nM) and FCB-5 (Kd = 0.47 +/- 0.08 nM) were of the same order of magnitude as the Kd for fibrin binding (Kd = 1 +/- 0.2 nM). The specificity of the binding was demonstrated by the ability of soluble FCB-2 and FCB-5 to inhibit t-PA binding to solid-phase fibrin (Ki = 3.3 microM and 6.4 microM, respectively). The binding of t-PA to fibrin and to immobilized FCB-2 was partially inhibited by the lysine analogue 6-aminohexanoic acid (Ki = 123 +/- 47 microM and 364 microM, respectively) but was not modified by carboxypeptidase B, thus indicating involvement of internal lysine residues. Removal of lysine residues by treatment with, successively, plasmin and carboxypeptidase B, produced only a partial inhibition of t-PA binding, thus confirming the existence of both a lysine-dependent and a lysine-independent mechanism of binding of t-PA to both fibrin and FCB-2. In contrast, the binding of t-PA to FCB-5 was not significantly affected by 6-aminohexanoic acid. Altogether, these data indicate that the mechanism of binding of t-PA to fibrin involves mainly a lysine-independent interaction with the D region which is contributed by sequences present in FCB-5 and FCB-2; contribution to binding by a lysine-dependent interaction was detected only in FCB-2 and is probably of minor relevance as suggested by the limited effect of 6-aminohexanoic acid.
 ...
PMID:Study of tissue-type plasminogen activator binding sites on fibrin using distinct fragments of fibrinogen. 811 48

Thrombomodulin (TM) expressed on endothelial cells binds thrombin and initiates anticoagulant pathways. Soluble functional proteolytic fragments of TM are also present in circulating plasma. Recently, it was reported that TM accelerated thrombin-dependent plasma procarboxypeptidase B (pro-pCPB) activation in a purified system and suggested that TM may inhibit fibrinolysis in crude plasma. The aim of present study was to evaluate any functional role of soluble TM fragments in plasma or purified TM added into plasma to the regulation of coagulation and fibrinolysis. Addition of rabbit TM (1-200 ng/ml) to plasma resulted in a concentration-dependent prolongation of urokinase (UK)- or tissue plasminogen activator (t-PA)-induced clot lysis time. The concentration of TM required for the inhibition of fibrinolysis was lower than that required for the inhibition of coagulation. Addition of anti-rabbit TM IgG or anti-human TM IgG into plasma reduced UK- or t-PA-induced clot lysis time without affecting clotting times, indicating that exogenous TM or soluble TM fragments in normal human plasma participated in regulation of fibrinolysis. Moreover, the TM-dependent inhibition of fibrinolysis was observed only in the presence of thrombin and blocked by addition of carboxypeptidase B inhibitors, but not mediated by protein C activation or direct inhibition of UK, t-PA or plasmin. Analysis of various substrates and inhibitors indicated that TM accelerated thrombin-dependent pro-pCPB activation in plasma. The present results indicate that TM, including soluble TM fragments in plasma, inhibit fibrinolysis via activation of pro-pCPB in plasma.
 ...
PMID:Thrombomodulin in human plasma contributes to inhibit fibrinolysis through acceleration of thrombin-dependent activation of plasma procarboxypeptidase B. 949 93

A complex of d-dimer noncovalently associated with fragment E ((DD)E), a degradation product of cross-linked fibrin that binds tissue plasminogen activator (t-PA) and plasminogen (Pg) with affinities similar to those of fibrin, compromises the fibrin specificity of t-PA by stimulating systemic Pg activation. In this study, we examined the effect of thrombin-activable fibrinolysis inhibitor (TAFI), a latent carboxypeptidase B (CPB)-like enzyme, on the stimulatory activity of (DD)E. Incubation of (DD)E with activated TAFI (TAFIa) or CPB (a) produces a 96% reduction in the capacity of (DD)E to stimulate t-PA-mediated activation of Glu- or Lys-Pg by reducing k(cat) and increasing K(m) for the reaction; (b) induces the release of 8 mol of lysine/mol of (DD)E, although most of the stimulatory activity is lost after release of only 4 mol of lysine/mol (DD)E; and (c) reduces the affinity of (DD)E for Glu-Pg, Lys-Pg, and t-PA by 2-, 4-, and 160-fold, respectively. Because TAFIa- or CPB-exposed (DD)E produces little stimulation of Glu-Pg activation by t-PA, (DD)E is not degraded into fragment E and d-dimer, the latter of which has been reported to impair fibrin polymerization. These data suggest a novel role for TAFIa. By attenuating systemic Pg activation by (DD)E, TAFIa renders t-PA more fibrin-specific.
 ...
PMID:Thrombin-activable fibrinolysis inhibitor attenuates (DD)E-mediated stimulation of plasminogen activation by reducing the affinity of (DD)E for tissue plasminogen activator. A potential mechanism for enhancing the fibrin specificity of tissue plasminogen activator. 1097 Aug 91

The binding of human plasminogen and plasmin to the promastigote form of Leishmania mexicana was investigated. L. mexicana was capable to bind both molecules, the binding being inhibited by epsilon-aminocaproic acid. Scatchard plot analysis revealed a dissociation constant (Kd) value of 2.4+/-0.8 microM and 0.9+/-0.1 x 10(4) binding sites per cell for plasminogen and a Kd value of 1.2+/-0.4 microM and 1.6+/-0.2 x 10(5) binding sites per cell for plasmin. C-terminal lysine residues are involved in plasminogen binding to cells, since carboxypeptidase B treatment reduced this binding by 34%. Ligand blotting analysis showed a group of proteins, with molecular masses between 105 and 115 kDa, capable to interact with plasminogen. Zymogram analysis showed that the protease activity acquired by L. mexicana, due to the interaction with either plasminogen or plasmin, comprises an important fraction of the total protease activity at pH 7.7. Plasminogen activation by tissue-type plasminogen activator (t-PA) was enhanced by the presence of L. mexicana promastigotes. These results raise the question whether the interaction of L. mexicana with components of the fibrinolytic system is involved in the virulence of the parasite.
 ...
PMID:Interaction of Leishmania mexicana promastigotes with the plasminogen-plasmin system. 1107 Dec 75


1 2 3 Next >>