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PubMed1998

Synoviocytes from osteoarthritis and rheumatoid arthritis produce plasminogen activators and plasminogen activator inhibitor-1 and display u-PA receptors on their surface.

The production of plasminogen activators and their inhibitors was studied in vitro in osteoarthritic (OA) and rheumatoid arthritic (RA) synovial fibroblasts (SF), obtained from RA and OA patients undergoing joint surgery. Subcultured SF were cultivated for 2, 4, 6, 8, 10 and 13 days and the medium assayed for the presence of both plasminogen activators (PAs) and plasminogen activator inhibitor-1 (PAI-1). The presence of urokinase-Plasminogen Activator (u-PA) receptors (u-PAR) on the surface of synovial cells was investigated by radio-ligand binding assay and cross-linking and by transmission electron microscopy (TEM) of a gold-u-PA complex. Our results showed a low production of tissue-type-Plasminogen Activator (t-PA) in both OA and RA SF, but relatively high levels of u-PA, until confluence, both in OA and in RA. SF were also able to produce plasminogen activator inhibitor in large amounts, in particular in RA since the very beginning of the culture. Receptors for u-PA were evident on both RA and OA SF. Our data show that SF in vitro produce mainly u-PA, the most important plasminogen activator involved in tissue modifications. The demonstration of u-PA receptors on the surface of OA and RA SF represents a step forward in the understanding of the possible role of fibrinolytic and tissue destructive proteinase cascade in joint inflammation.

Arthritis, Rheumatoid↗

PubMed1990

Kinetic studies on novel plasminogen activators. Demonstration of fibrin enhancement for hybrid enzymes comprising the A-chain of plasmin (Lys-78) and B-chain of tissue-type plasminogen activator (Ile-276) or urokinase (Ile-159).

The activation of plasminogen by two novel hybrid enzymes, constructed from the A-chain of plasmin and the B-chains of tissue-type plasminogen activator (t-PA) or urokinase, was compared with the activation by the parent enzymes. Basal kinetic constants for 'Lys-plasminogen' (human plasminogen with N-terminal lysine) and 'Glu-plasminogen' (human plasminogen with N-terminal glutamic acid) activation were similar to those of the parent activators. The Km for plasminogen turnover for both hybrid enzymes was considerably decreased in the presence of both soluble fibrin and a mimic, a CNBr digest of fibrinogen. These enhancements and the related apparent negative co-operativity are similar to the behaviour of t-PA itself. The results are discussed with regard to the molecular features involved in the mechanism of fibrin stimulation.

Aminocaproic Acid↗

PubMed2000

A truncated plasminogen activator inhibitor-1 protein induces and inhibits angiostatin (kringles 1-3), a plasminogen cleavage product.

Plasminogen activator inhibitor-1 (PAI-1) is a serpin protease inhibitor that binds plasminogen activators (uPA and tPA) at a reactive center loop located at the carboxyl-terminal amino acid residues 320-351. The loop is stretched across the top of the active PAI-1 protein maintaining the molecule in a rigid conformation. In the latent PAI-1 conformation, the reactive center loop is inserted into one of the beta sheets, thus making the reactive center loop unavailable for interaction with the plasminogen activators. We truncated porcine PAI-1 at the amino and carboxyl termini to eliminate the reactive center loop, part of a heparin binding site, and a vitronectin binding site. The region we maintained corresponds to amino acids 80-265 of mature human PAI-1 containing binding sites for vitronectin, heparin (partial), uPA, tPA, fibrin, thrombin, and the helix F region. The interaction of "inactive" PAI-1, rPAI-1(23), with plasminogen and uPA induces the formation of a proteolytic protein with angiostatin properties. Increasing amounts of rPAI-1(23) inhibit the proteolytic angiostatin fragment. Endothelial cells exposed to exogenous rPAI-1(23) exhibit reduced proliferation, reduced tube formation, and 47% apoptotic cells within 48 h. Transfected endothelial cells secreting rPAI-1(23) have a 30% reduction in proliferation, vastly reduced tube formation, and a 50% reduction in cell migration in the presence of VEGF. These two studies show that rPAI-1(23) interactions with uPA and plasminogen can inhibit plasmin by two mechanisms. In one mechanism, rPAI-1(23) cleaves plasmin to form a proteolytic angiostatin-like protein. In a second mechanism, rPAI-1(23) can bind uPA and/or plasminogen to reduce the number of uPA and plasminogen interactions, hence reducing the amount of plasmin that is produced.

Angiostatins↗

PubMed1995

Tissue plasminogen activator (tPA) inhibits plasmin degradation of fibrin. A mechanism that slows tPA-mediated fibrinolysis but does not require alpha 2-antiplasmin or leakage of intrinsic plasminogen.

Thrombolysis is dramatically slower when high concentrations of lytic agent are used. This paradoxical observation, first described as "plasminogen steal," was originally believed to be due to depletion of extrinsic plasminogen and consequent leaching of clot-bound plasminogen. We report that administration of increasing concentrations of recombinant human tissue plasminogen activator (tPA) to fibrin gels resulted in lysis rates that displayed a maximum, with significantly slower rates found at higher tPA, regardless of whether plasminogen was supplied extrinsically or intrinsically. A similar maximum in lysis rates was observed in a system lacking an extrinsic phase when plasminogen was added to fibrin suspensions preincubated with increasing tPA. Thus, intrinsic plasminogen leakage and alpha 2-antiplasmin were not required for the decreased lysis at high tPA. No maximum was observed for increasing concentrations of urokinase. Using fibrin suspensions or gels preincubated with tPA before addition of plasmin, we report that tPA, but not urokinase, caused a dose-dependent inhibition of the fibronolytic action of plasmin. With respect to optimal dosage schemes and the design of novel lytic agents, these findings indicate that (a) there exists a biochemical mechanism against minimizing reperfusion time with increasing tPA dosages and (b) the fibrin affinity of tPA may cause reduced fibrinolysis by plasmin.

Dose-Response Relationship, Drug↗

PubMed2005

The plasminogen activation system in skeletal muscle regeneration: antagonistic roles of urokinase-type plasminogen activator (uPA) and its inhibitor (PAI-1).

The plasminogen activation (PA) system is an extensively used mechanism for the generation of proteolytic activity in the extracellular matrix, where it contributes to tissue remodeling in a wide range of physiopathological processes. Despite the limited information available at present on plasminogen activators, their inhibitors and cognate receptors in skeletal muscle, increasing evidence is accumulating on their important roles in the homeostasis of muscle fibers and their surrounding extracellular matrix. The development of mice deficient for the individual components of the PA system has provided an incisive approach to test the proposed muscle functions in vivo. Skeletal muscle regeneration induced by injury has been analyzed in urokinase-type plasminogen activator (uPA)-, tissue-type plasminogen activator (tPA)-, plasminogen (Plg)- and plasminogen activator inhibitor-1 (PAI-1)-deficient mice and has demonstrated profound effects of these molecules on the fibrotic state and the inflammatory response, which contribute to muscle repair. In particular, the opposite roles of uPA and its inhibitor PAI-1 in this process are highlighted. Delineating the mechanisms by which the different plasminogen activation system components regulate tissue repair will be of potential therapeutic value for severe muscle disorders.

Animals↗

PubMed1995

Effect of individual caseins on plasminogen activation by bovine urokinase-type and tissue-type plasminogen activators.

The effect was examined of individual caseins on the rate of plasminogen activation by bovine urokinase-type and tissue-type plasminogen activators. All individual caseins (alpha-CN, beta-CN, and kappa-CN) enhanced the activity of both types of plasminogen activators. Optimal concentrations for alpha-CN and beta-CN were 5 and 25 micrograms/ml, respectively. The enhancement of enzymatic activity declined when concentrations of alpha-CN and beta-CN were higher. In contrast, increasing concentrations of kappa-CN from 0 to 200 micrograms/ml resulted in corresponding increases in activity of both types of plasminogen activators. On a weight basis, alpha-CN was the most effective enhancer of plasminogen activator activity. Indirect evidence obtained with experiments utilizing alpha-CN immobilized on agarose suggested that the effect is related to extensive binding of plasminogen and both types of plasminogen activators to casein.

Amino Acid Sequence↗

PubMed1992

Depression of tissue-type plasminogen activator and enhancement of urokinase-type plasminogen activator as an expression of local inflammation.

Inflammatory processes are accompanied by extravascular deposition and breakdown of fibrin. We measured fibrinolytic parameters in synovial fluid (SF) and in plasma of 36 patients with rheumatoid arthritis (RA). As a control, SF of 13 patients with blunt knee trauma, and plasma of 17 healthy volunteers were studied. In RA patients, extravascular t-PA mediated plasminogen activation was depressed: mean SF tissue-type plasminogen activator (t-PA:Ag) concentration (2.1 +/- 1.6 ng/ml) was four-fold lower, and plasminogen activator inhibitor (PAI) activity (284 +/- 212%) four-fold higher than the plasma values of the same patients or of healthy donors. In contrast, u-PA related plasminogen activation was strongly enhanced: urokinase-type plasminogen activator (u-PA) antigen (23.1 +/- 12.4 ng/ml) was more than four-fold higher, single-chain u-PA (scu-PA) (5.3 +/- 1.9 ng/ml) three-fold higher than in plasma of the same patients or of healthy donors, and active two-chain u-PA (tcu-PA) was detected in 14 of the 36 SF samples of RA patients. All of these changes in extravascular fibrinolytic parameters correspond with those induced by inflammatory mediators in cell cultures. In joint effusions of patients with a blunt knee trauma, the effects were intermediate: u-PA related parameters showed moderate changes in the same direction as in arthritis; t-PA antigen was also decreased. The only exception was that PAI was not increased. We conclude that the findings in traumatic effusions reflect transient effects as a reaction to trauma. In joint inflammation, the depressed t-PA mediated plasminogen activation, although more than compensated by the enhanced u-PA mediated plasminogen activation, results in protraction of fibrin removal. Besides, the enhanced u-PA activation might lead to proteolytic damage of the cartilage.

Arthritis, Rheumatoid↗

PubMed1990

Plasminogen activation at low temperatures in plasma samples containing therapeutic concentrations of tissue-type plasminogen activator or other thrombolytic agents.

It is known that in vitro plasminogen activation in blood samples taken during thrombolytic therapy with tissue-type plasminogen activator (t-PA) may lead to artefactually low fibrinogen and alpha 2-antiplasmin values. To mimic this phenomenon, pooled normal plasma was supplemented with 2.5 micrograms/ml t-PA and incubated at various temperatures. The rates of fibrinogen degradation and alpha 2-antiplasmin consumption were most pronounced at 37 degrees C, were less pronounced at 25 degrees C, but surprisingly, did not further decrease at 10 degrees C, 0 degrees C or -8 degrees C. In contrast, when plasma was supplemented with 160 IU/ml urokinase or 30 IU/ml streptokinase, the rates of fibrinogen degradation and alpha 2-antiplasmin consumption gradually decreased with incubation temperature and were negligible at 10 degrees C and lower temperatures. The rate of plasminogen activation also decreased gradually with temperature in mixtures of purified fibrinogen, plasminogen, alpha 2-antiplasmin and t-PA. These results imply that, in a plasma milieu, additional factors with a stimulatory activity are involved in t-PA-induced plasminogen activation at around 0 degrees C. The abnormally high reaction rate at low temperatures explains in vitro plasminogen activation observed during the processing of t-PA-containing blood samples. In contrast to the activation of plasminogen by t-PA, the slow inhibition of t-PA (2.5 micrograms/ml) by proteinase inhibitors in plasma could be minimized to a negligible level by keeping the plasma samples at 0 degrees C. This makes it possible to reliably monitor t-PA activity during thrombolytic therapy.

Chromogenic Compounds↗

PubMed1999

Increased expression of plasminogen activator and plasminogen activator inhibitor during liver fibrogenesis of rats: role of stellate cells.

BACKGROUND/AIMS: Plasminogen activators and plasminogen activator inhibitors are important regulators of the balance between the proteolytic and antiproteolytic activities that determine extracellular matrix turnover. We examined the expression of plasminogen activator-plasmin system components in experimental liver fibrosis of rats. METHODS: Liver fibrosis was produced in rats by injecting carbon tetrachloride for 6 to 12 weeks. Gene expression for plasminogen activator inhibitor-1 (PAI-1), urokinase and tissue plasminogen activators (uPA and tPA), urokinase plasminogen activator receptor (uPAR), and transforming growth factor-beta1 (TGF-beta1) was examined by Northern analysis. Western analysis was performed to detect protein expression of PAI-1, uPA and uPAR. An immunohistochemical study was performed to detect the localization of PAI-1. Additionally, primary cultured liver cells were examined by Northern and Western analyses for this protein with or without prior incubation with TGF-beta1. RESULTS: At 6 weeks, when fibrosis had occurred, uPA and uPAR mRNAs had increased 2.8-fold and 1.8-fold, respectively; PAI-1 and tPA mRNA levels were unchanged. At the cirrhotic stage (9 to 12 weeks), mRNA levels for PAI-1, uPA, uPAR and tPA were all increased. Western analysis also showed increased uPA and uPAR expressions in fibrotic liver, and increased PAI-1, uPA and uPAR expressions in cirrhotic liver. PAI-1 protein was also demonstrated immunohistochemically along sinusoids, vessels, and bile duct cells of normal and fibrotic liver. In liver cell cultures, Kupffer cells, hepatocytes, and especially stellate cells, expressed PAI-1. Expression was enhanced in stellate cells cultured from fibrotic or cirrhotic liver or stimulated in vitro with TGF-beta1. CONCLUSION: Though increased uPA and uPAR may act on matrix degradation in fibrotic liver, increased PAI-1 together with uPA, uPAR and tPA are associated with overall inhibition of matrix degradation in cirrhotic liver. Hepatic stellate cells are an important source of PAI-1 during liver fibrosis.

Animals↗

PubMed1996

Suppression of plasminogen activator inhibitor-1 release from human cerebral endothelium by plasminogen activators. A factor potentially predisposing to intracranial bleeding.

BACKGROUND: Intracranial bleeding is the most catastrophic potential complication of treatment with thrombolytic agents. To identify potential factors that may contribute to this problem, we characterized elaboration by human brain endothelial cells of plasminogen activator inhibitor-1 (PAI-1) and measured PAI-1 mRNA levels. METHODS AND RESULTS: When human cerebral microvascular endothelial cells (HCMEC), pial arterial endothelial cells, and middle meningeal arterial endothelial cells were exposed to 10 to 1000 ng/mL recombinant tissue-type plasminogen activator (RTPA), urokinase-type plasminogen activator (UPA), or streptokinase/ plasminogen (37 U streptokinase plus 2 mumol/L plasminogen) for 24 hours, they exhibited concentration-dependent decreases in elaboration of PAI-1 of 65 +/- 3%, 48 +/- 3%, and 59 +/- 8%. UPA and streptokinase/plasminogen elicited decreases of 33 +/- 8% and 35 +/- 4%, respectively, that were specific with respect to the protease agonists as to total protein synthesis and cell type; ie, neither human umbilical vein endothelial cells nor cerebral pericytes exhibited inhibition of PAI-1 elaboration. No decrease in HCMEC PAI-1 elaboration was induced by coagulation factor XB (10 nmol/L). A 2.7 +/- 0.5-fold increase was induced by alpha-thrombin (10 nmol/L). PAI-1 secretion from HCMEC decreased within 4 hours of exposure to 100 ng/mL RTPA. In HCMEC exposed to RTPA for 8 hours, PAI-1 mRNA decreased from 176 +/- 20 to 43 +/- 2.2 pg/microgram RNA. CONCLUSIONS: These results indicate that brain endothelial cells exposed to RTPA exhibit paradoxically diminished elaboration of PAI-1. This property may render brain vasculature vulnerable to attack by serine proteases, thereby predisposing to injury and initiating an underlying subsequent intracerebral hemorrhage in patients given plasminogen activators for treatment of coronary thrombosis.

Aprotinin↗

PubMed1994

Influence of exogenous growth factors on the expression of plasminogen activators and plasminogen activator inhibitors by cells isolated from normal and healing rabbit ligaments.

In this investigation, we demonstrate that cells from normal and healing rabbit ligaments are selective in their responsiveness to various growth factors. The cells analyzed included fibroblasts isolated from the synovium, the anterior cruciate ligament, and the medial collateral ligament (midsubstance and epiligament). Fibroblasts isolated from scar tissue of medial collateral ligament that had been allowed to heal for 3 weeks also were analyzed. The addition of insulin-like growth factor-2 or transforming growth factor-beta 1 was observed to alter, in a dose-dependent manner, the expression of plasminogen activator and plasminogen activator inhibitor by connective tissue cells. However, the response to these growth factors was cell specific. Fibroblasts isolated from the midsubstance, epiligament, and scar tissue of the medial collateral ligament were responsive to these growth factors; fibroblasts isolated from the anterior cruciate ligament and synovium did not have a detectable response. The cells from the normal and healing medial collateral ligament responded to both growth factors by increasing plasminogen activator inhibitor activity. This was observed at both the protein and RNA level. In contrast, the addition of insulin-like growth factor-1 or acidic or basic fibroblast growth factor to cells derived from normal or healing ligament did not result in any detectable alteration of plasminogen activator or plasminogen activator inhibitor activity. These results are similar to those observed with an explant system and indicate that cells isolated from ligament tissue maintain their responsiveness to these growth factors in the absence of matrix. As the major effect of insulin-like growth factor-2 and transforming growth factor-beta 1 on the cells tested was to increase plasminogen activator inhibitor activity, such an alteration should diminish the activity of plasminogen activator, an enzyme capable of directly and indirectly proteolyzing matrix molecules, and thus contribute to a more anabolic environment.

Animals↗

PubMed1992

Allosteric regulation of tPA-mediated plasminogen activation by a modifier mechanism: evidence for a binding site for plasminogen on the tPA A-chain.

We studied the mechanism responsible for nonlinear double reciprocal plots for tissue type plasminogen activator (tPA)-mediated plasminogen activation reported previously by several groups. We found nonlinear Eadie-Scatchard plots for Glu-plasminogen activation by recombinant single-chain tPA confirming a non-Michaelis-Menten behavior of tPA. In order to characterize this mechanism, enzyme kinetic studies with truncated substrates (Lys- and miniplasminogen) and modified or truncated enzymes (two-chain tPA and tPA B-chain) were performed. Thereby it could be excluded that product-mediated modifications of the enzyme or the substrate are responsible for the nonlinear plots. Linear plots, i.e., Michaelis-Menten kinetics, were only found when tPA B-chain was used as a plasminogen activator, indicating that the tPA A-chain should be responsible for the non-Michaelis-Menten behavior. Binding studies of plasminogen to immobilized tPA A-chain in fact demonstrated a saturable binding of Glu- and miniplasminogen to the A-chain of tPA with a KD approximately 0.1 microM and one binding site per molecule of tPA A-chain. These data suggested a modifier mechanism responsible for the nonlinear plots whereby the substrate plasminogen itself could function as a modifier. When such a mechanism was included into a model for tPA-mediated plasminogen activation, the experimentally obtained data could be fitted into such a model by nonlinear regression analysis with resulting p-values of less than 0.001.

Allosteric Regulation↗

PubMed1990

Specific binding of plasminogen to vitronectin. Evidence for a modulatory role of vitronectin on fibrin(ogen)-induced plasmin formation by tissue plasminogen activator.

Vitronectin immobilized onto polystyrene microtiter wells was demonstrated to specifically bind plasminogen in a concentration-dependent manner, yielding an estimated KD = 0.4 microM. Heparin only moderately interfered with the vitronectin-plasminogen interaction, whereas high concentrations of 6-amino-hexanoic acid inhibited binding. Utilizing a ligand-blotting procedure in which plasminogen was reacted with proteolytic fragments of vitronectin, transblotted onto nitrocellulose, the plasminogen-binding site of vitronectin was localized to the heparin-binding domain of the adhesive protein. Moreover, vitronectin was found to inhibit in a dose-dependent fashion the fibrin(ogen)-induced activation of plasminogen by tissue plasminogen activator. These results provide the first evidence for a novel vitronectin-mediated control of plasminogen activation potentially relevant for directional clot-lysis and plasmin-dependent proteolysis in extracellular matrices.

Electrophoresis, Polyacrylamide Gel↗

PubMed1988

Plasminogen activation by tissue plasminogen activator in the presence of platelets.

Platelets were found to provide a surface for activation of plasminogen by the tissue-type plasminogen activator (t-PA) at an optimum concentration and to potentiate the generation of plasmin by the amidolytic method, fibrin lysis time and fibrin plate method. The effect of platelets on amidolytic activity on S-2251 was due to the potentiating effect of plasminogen activation by t-PA, because it was observed only in the presence of both plasminogen and t-PA. Plasmin generation was also evidenced in the SDS-PAGE profile of the supernatant from a mixture containing t-PA and plasminogen with platelets. These findings suggests that the potentiating activity of platelets on plasminogen activation by t-PA in circulation is one of the causes of fibrinogenolysis during fibrinolytic therapy with a high dose of t-PA. Platelets from patients with various diseases showed different potentiating activity on plasminogen activation by t-PA. The assay of this ability of platelets may be a new tool for evaluating their role in the blood fibrinolytic process.

Blood Platelets↗

PubMed1991

The effects of polysaccharides on plasminogen activation by single chain-and two chain-tissue plasminogen activator.

We examined the effects of polysaccharides on t-PA mediated plasminogen activation using single-chain tissue plasminogen activator (sct-PA) and two-chain tissue plasminogen activator (tct-PA). Unfractionated heparin, low molecular weight heparin (LMW heparin) and dextran sulfates enhanced the activation rate of plasminogen by sct-PA about three-fold to six-fold. Chondroitin sulfate C did not enhance the activation. The activation of plasminogen by tct-PA was slightly enhanced by unfractionated heparin, but not by other polysaccharides. Conversion of sct-PA to tct-PA was not stimulated by polysaccharides. SDS-PAGE showed no enhancement of the conversion from sct-PA to tct-PA by plasmin in the presence of polysaccharides. However, the enhancement of sct-PA mediated activation of plasminogen by unfractionated heparin, LMW heparin and dextran sulfates in the presence of aprotinin was shown with SDS-PAGE. It was suggested that unfractionated heparin, LMW heparin and dextran sulfates form complex with sct-PA and plasminogen, and stimulate the conversion of sct-PA to tct-PA.

Animals↗

PubMed1984

Human endothelial cells produce a plasminogen activator inhibitor and a tissue-type plasminogen activator-inhibitor complex.

Serum-free conditioned media and cell extracts from cultured human umbilical vein endothelial cells were analyzed for plasminogen activator by SDS-polyacrylamide gel electrophoresis and enzymography on fibrin-indicator gels. Active bands of free and complexed tissue-type plasminogen activator (t-PA) or urokinase-type plasminogen activator (u-PA) were identified by the incorporation of specific antibodies against, respectively, t-PA or u-PA in the indicator gel. The endothelial cells predominantly released a high-molecular-weight t-PA (95 000-135 000). This t-PA form was converted to Mr-72 000 t-PA by 1.5 M NH4OH/39 mM SDS. A component with high affinity for both t-PA and u-PA could be demonstrated in serum-free conditioned medium and endothelial cell extract. The complex between this component and Mr-72 000 t-PA comigrated with high-molecular-weight t-PA. From the increase in Mr of t-PA or u-PA upon complex formation, the Mr of the endothelial cell component was estimated to be 50 000-70 000. The reaction between t-PA or u-PA and the plasminogen activator-binding component was blocked by 5 mM p-aminobenzamidine, while the complexes, once formed, could be cleaved by 1.5 M NH4OH/39 mM SDS. These observations indicated that the active center of plasminogen activator was involved in the complex formation. It was further noted that serum-free conditioned medium or endothelial cell extract inhibited plasminogen activator activity when assayed by the fibrin-plate method. Evidence is provided that the plasminogen activator-binding component was different from a number of the known plasma serine proteinase inhibitors, the placenta inhibitor and the fibroblast surface protein, proteinase-nexin. We conclude that cultured endothelial cells produce a rapid inhibitor of u-PA and t-PA as well as a t-PA-inhibitor complex.

Amyloid beta-Protein Precursor↗

PubMed1991

Single-chain urokinase-type plasminogen activator does not possess measurable intrinsic amidolytic or plasminogen activator activities.

The question whether single-chain urokinase-type plasminogen activator (Sc-uPA) possesses an enzymatic activity has been a subject of intense investigation for a number of years but still remains unresolved. Recent studies from several laboratories suggest that Sc-uPA or its plasmin-resistant mutants obtained by site-directed mutagenesis possess significant, albeit low, amidolytic and plasminogen activator activities, ranging from 0.1% to 1% of that observed for two-chain urokinase (Tc-uPA). In an effort to characterize these putative intrinsic activities, Sc-uPA was repeatedly treated with dansyl-Glu-Gly-Arg chloromethyl ketone (dansyl-EGRck) or diisopropyl fluorophosphate (DFP) (0.1-0.25 mM added thrice over a period of 24 h at 0 degrees C). This treatment exhaustively inactivated the Tc-uPA contaminant but did not affect Sc-uPA, as evidenced by the lack of significant incorporation of radiolabeled inhibitor in Sc-uPA and full activation of the inhibitor-treated Sc-uPA by plasmin. Assayed in the presence of excess DFP or dansyl-EGRck to ensure trapping of any Tc-uPA generated in the assay mixture, Sc-uPA (84 micrograms/mL, 10,500 latent units/mL) did not elicit any detectable cleavage of the chromogenic substrate S-2444 (detection limit 0.1 unit of Tc-uPA/mL). However, if the Tc-uPA inhibitors were removed prior to assay, a trace amount of amidolytic activity invariably reappeared in the Sc-uPA preparation. Incorporation experiments with [3H]DFP suggested that the appearance of this amidolytic activity was due to formation of Tc-uPA. Plasminogen activator assay of DFP- and dansyl-EGRck-treated Sc-uPA (0.45-2.25 microM), performed in the presence of these inhibitors and Trasylol (10 microM) to ensure entrapment of any Tc-uPA or plasmin generated in the reaction mixture, showed no significant cleavage of 125I-labeled plasminogen (detection limit 0.1 nM). However, if dansyl-EGRck and DFP were removed from the inhibitor-treated Sc-uPA and the assay was performed in the presence of Trasylol alone, there was significant cleavage of 125I-plasminogen due to contamination by Tc-uPA. Fibrin, a positive effector of plasminogen activation by Tc-uPA or Sc-uPA preparations in the absence of DFP and dansyl-EGRck, did not promote cleavage of plasminogen or S-2444 by Sc-uPA in the presence of the Tc-uPA inhibitors.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Chloromethyl Ketones↗

PubMed2003

Phospholipid-associated annexin A2-S100A10 heterotetramer and its subunits: characterization of the interaction with tissue plasminogen activator, plasminogen, and plasmin.

Annexin A2 (p36) is a highly alpha-helical molecule that consists of two opposing sides, a convex side that contains the phospholipid-binding sites and a concave side, which faces the extracellular milieu and contains multiple ligand-binding sites. The amino-terminal region of annexin A2 extends along the concave side of the protein and contains the binding site for the S100A10 (p11) subunit. The interaction of these subunits results in the formation of the heterotetrameric form of the protein, annexin A2-S100A10 heterotetramer (AIIt). To simulate the orientation of AIIt on the plasma membrane we bound AIIt to a phospholipid bilayer that was immobilized on a BIAcore biosensor chip. Surface plasmon resonance was used to observe in real time the molecular interactions between phospholipid-associated AIIt or its annexin A2 subunit and the ligands, tissue-type plasminogen activator (t-PA), plasminogen, and plasmin. AIIt bound t-PA (Kd = 0.68 microm), plasminogen (Kd = 0.11 microm), and plasmin (Kd = 75 nm) with moderate affinity. Contrary to previous reports, the phospholipid-associated annexin A2 subunit failed to bind t-PA or plasminogen but bound plasmin (Kd = 0.78 microm). The S100A10 subunit bound t-PA (Kd = 0.45 microm), plasminogen (Kd = 1.81 microm), and plasmin (Kd = 0.36 microm). Removal of the carboxyl-terminal lysines from the S100A10 subunit attenuated t-PA and plasminogen binding to AIIt. These results show that the carboxyl-terminal lysines of S100A10 form t-PA and plasminogen-binding sites. In contrast, annexin A2 and S100A10 contain distinct binding sites for plasmin.

Annexin A2↗