Mechanism of the inhibitory effect of angiostatin on plasminogen activation by its physiologic activators

The influence of angiostatin K1-4.5, a fragment of the heavy chain of plasmin and a powerful inhibitor of angiogenesis, on kinetic parameters (k _Pg and K _Pg) of human Glu-plasminogen activation under the action of urokinase (uPA) not having affinity for fibrin and fibrin-specific tissue plasminogen activator (tPA) was investigated. Angiostatin does not affect on the k _Pg value, but increases the value of K _Pg plasminogen activation by urokinase. A decrease in the k _Pg value and an increase in the K _Pg value were found for fibrin-stimulated plasminogen activation by tPA with increasing concentrations of angiostatin. The obtained results show that angiostatin is a competitive inhibitor of the uPA activator activity, while it inhibits the activator activity of tPA with a mixed type. Such an influence of angiostatin on the kinetic constants of the plasminogen activation by urokinase suggests that angiostatin dose-dependent manner replaces plasminogen in the binary enzyme-substrate complex uPA-Pg. In the case of fibrin-stimulated plasminogen activation by tPA, both zymogen and tPA are bound to fibrin with the formation of the effective triple tPA-Pg-fibrin complex. Angiostatin replaces plasminogen both from the fibrin surface and from the enzyme-substrate tPA-Pg complex, which leads to a decrease in k _Pg and an increase in K _Pg of the plasminogen activation. Inhibition constants by angiostatin (K _i) of plasminogen-activator activities of uPA and tPA determined by the Dixon method were found to be 0.59 ± 0.04 and 0.12 ± 0.05 μM, respectively.     

Inhibitory effect of angiostatins on activity of the plasminogen/plasminogen activator system

Angiostatins, kringle-containing fragments of plasminogen, are potent inhibitors of angiogenesis. Effects of three angiostatin forms, K1–3, K1–4, and K1-4.5 (0–2 μM), on the rate of native Glu-plasminogen activation by its physiological activators in the absence or presence of soluble fibrin were investigated in vitro. Angiostatins did not affect the intrinsic amidolytic activities of plasmin and plasminogen activators of tissue type (tPA) and urokinase type (single-chain scuPA and two-chain tcuPA), but inhibited conversion of plasminogen to plasmin in a dose-dependent manner. All three angiostatins suppressed Glu-plasminogen activation by tcuPA independently of the presence of fibrin, and the inhibitory effect increased in the order: K1-3 < K1-4 < K1-4.5. The inhibitory effects of angiostatins on the scuPA activator activity were lower and further decreased in the presence of fibrin. Angiostatin K1-3 (up to 2 μM) had no effect, while 2 μM angiostatins K1-4 and K1-4.5 inhibited the fibrin-stimulated Glu-plasminogen activation by tPA by 50 and 100%, respectively. The difference in effects of the three angiostatins on the Glu-plasminogen activation by scuPA, tcuPA, and tPA in the absence or presence of fibrin is due to the differences in angiostatin structures, mechanisms of action, and fibrin-specificity of plasminogen activators, as well as due to the influence of fibrin on the Glu-plasminogen conformation. Angiostatins in vivo, which mimic plasminogen-binding activity, can inhibit plasminogen activation stimulated by various proteins (including fibrin) of extracellular matrix, thereby blocking cell migration and angiogenesis. The data of this work indicate that the inhibition of Glu-plasminogen activation under the action of physiological plasminogen activators by angiostatins can be implicated in the complex mechanism of their antiangiogenic and antitumor action.     

The role of carbohydrate side chains of plasminogen in its activation by staphylokinase

Kinetic parameters (k(Pg) and K(Pg)) were determined for activation of Glu-plasminogen (Glu-Pg) and Lys-plasminogen (Lys-Pg) type I (with N-linked carbohydrate chain at Asn-289) and type II (with unsubstituted Asn-289) by plasmin-staphylokinase (Pm-STA) complex. The K(Pg) values for Glu-Pg I and Lys-Pg I (17.1 and 11.2 microM, respectively) were higher than those for Glu-Pg II and Lys-Pg II (14.9 and 5.4 microM, respectively), while only minor differences in the k(Pg) values were observed between plasminogens type I and type II. Soluble fibrin significantly increased the k(Pg)/K(Pg) values for activation of all four plasminogens due to a decrease in the K(Pg) values but did not alter the k(Pg) values. However, the activation of plasminogens type I was stimulated by fibrin lesser degree than that of plasminogens type II. These findings indicate that N-glycosylation of kringle 3 of plasminogen decreases the stability of Pm-STA-Pg ternary enzyme-substrate complex in solution as well as interferes with its formation and rearrangement on the fibrin surface.     

Protonation state of a single histidine residue contributes significantly to the kinetics of the reaction of plasminogen activator inhibitor-1 with tissue-type plasminogen activator

Stopped-flow fluorometry was used to study the kinetics of the reactive center loop insertion occurring during the reaction of N-((2-(iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-3-diazole (NBD) P9 plasminogen activator inhibitor-1 (PAI-1) with tissue-(tPA) and urokinase (uPA)-type plasminogen activators and human pancreatic elastase at pH 5.5-8.5. The limiting rate constants of reactive center loop insertion (k(lim)) and concentrations of proteinase at half-saturation (K(0.5)) for tPA and uPA and the specificity constants (k(lim)/K(0.5)) for elastase were determined. The pH dependences of k(lim)/K(0.5) reflected inactivation of each enzyme due to protonation of His57 of the catalytic triad. However, the specificity of the inhibitory reaction with tPA and uPA was notably higher than that for the substrate reaction catalyzed by elastase. pH dependences of k(lim) and K(0.5) obtained for tPA revealed an additional ionizable group (pKa, 6.0-6.2) affecting the reaction. Protonation of this group resulted in a significant increase in both k(lim) and K(0.5) and a 4.6-fold decrease in the specificity of the reaction of tPA with NBD P9 PAI-1. Binding of monoclonal antibody MA-55F4C12 to PAI-1 induced a decrease in k(lim) and K(0.5) at any pH but did not affect either the pKa of the group or an observed decrease in k(lim)/K(0.5) due to protonation of the group. In contrast to tPA, the k(lim) and K(0.5) for the reactions of uPA with NBD P9 PAI-1 or its complex with the monoclonal antibody were independent of pH in the 6.5-8.5 range. Since slightly acidic pH is a feature of a number of malignant tumors, alterations in PAI-1/tPA kinetics could play a role in the cancerogenesis. Changes in the protonation state of His(188), which is placed closely to the S1 site and is unique for tPA, has been proposed to contribute to the observed pH dependences of k(lim) and K(0.5).     

一种新的纤溶酶原激活反应动力学研究方法

 在溶栓研究中 ,纤溶酶原激活剂 (plasminogen activator,PA)激活纤溶酶原 (plasminogen,Plg)反应的动力学常数的测定占有重要地位 .在前人的研究中 ,虽然进行了这项实验 ,但是并未给出一个方便、快捷的测定方法 .所以建立一种更准确 ,更适合一般的实验室条件的 PA分子激活 Plg反应动力学常数的测定方法是必要的 .在对该反应进行数学分析的基础上 ,得到由可测量表示的纤溶酶 (plasmin,Plm)生成速度 (v(Plm) )的计算公式 ,由 v(Plm)及相应已知量可进一步推导出 Km、kcat的表达式 ,最终测得相应动力学常数 .用这种方法测定的由甲醇酵母 (pichia pastoris)表达的人单链尿激酶型纤溶酶原激活剂 (human single chain urokinase- PA,scu- PA)激活 Plg的反应的 Km=0 .648μmol·L-1,kcat=0 .0 62 6s-1,与文献报导相符 (Km=0 .4～ 1 .1 μmol· L-1,kcat=0 .0 2～ 0 .0 93) s-1,说明此方法是可靠的 .又因该法只需相应底物及酶标仪且为连续测定 ,所以十分简便 .     

Molecular basis of specific inhibition of urokinase plasminogen activator by amiloride

The urokinase plasminogen activator (uPA) and tissue plasminogen activator (tPA) are very similar serine proteases with the same physiological function, the activation of plasminogen. An increased amount or activity of uPA but not tPA has been detected in human cancers. The PAs are weak proteolytic enzymes, but they activate plasminogen to plasmin, a strong proteolytic enzyme largely responsible for the malignant properties of cancers. It has been shown recently that the administration of uPA inhibitors can reduce tumor size. Inhibitors of uPA could therefore be used as anti-cancer and anti-angiogenesis agents. It has been found that amiloride competitively inhibits the catalytic activity of uPA but not tPA. Modification of this chemical could therefore produce a new class of uPA specific inhibitors and a new class of anti-cancer agents. The X-ray structure of the uPA complex with amiloride is not known. There are structural differences in the specificity pocket of uPA and tPA. However, the potential energy of binding amiloride is lower outside this cavity in the case of tPA. A region responsible for binding amiloride to tPA has been proposed as the loop B93-B101, reached in negatively charged amino acids present in tPA but not uPA.     

Effects of extracellular DNA on plasminogen activation and fibrinolysis

The increased levels of extracellular DNA found in a number of disorders involving dysregulation of the fibrinolytic system may affect interactions between fibrinolytic enzymes and inhibitors. Double-stranded (ds) DNA and oligonucleotides bind tissue-(tPA) and urokinase (uPA)-type plasminogen activators, plasmin, and plasminogen with submicromolar affinity. The binding of enzymes to DNA was detected by EMSA, steady-state, and stopped-flow fluorimetry. The interaction of dsDNA/oligonucleotides with tPA and uPA includes a fast bimolecular step, followed by two monomolecular steps, likely indicating slow conformational changes in the enzyme. DNA (0.1-5.0 μg/ml), but not RNA, potentiates the activation of Glu- and Lys-plasminogen by tPA and uPA by 480- and 70-fold and 10.7- and 17-fold, respectively, via a template mechanism similar to that known for fibrin. However, unlike fibrin, dsDNA/oligonucleotides moderately affect the reaction between plasmin and α(2)-antiplasmin and accelerate the inactivation of tPA and two chain uPA by plasminogen activator inhibitor-1 (PAI-1), which is potentiated by vitronectin. dsDNA (0.1-1.0 μg/ml) does not affect the rate of fibrinolysis by plasmin but increases by 4-5-fold the rate of fibrinolysis by Glu-plasminogen/plasminogen activator. The presence of α(2)-antiplasmin abolishes the potentiation of fibrinolysis by dsDNA. At higher concentrations (1.0-20 μg/ml), dsDNA competes for plasmin with fibrin and decreases the rate of fibrinolysis. dsDNA/oligonucleotides incorporated into a fibrin film also inhibit fibrinolysis. Thus, extracellular DNA at physiological concentrations may potentiate fibrinolysis by stimulating fibrin-independent plasminogen activation. Conversely, DNA could inhibit fibrinolysis by increasing the susceptibility of fibrinolytic enzymes to serpins.     

Urokinase and tissue type plasminogen activators in human keratinocyte culture

Using immunocytochemical and biochemical techniques, we have demonstrated that cultured human epidermal keratinocytes contain both urokinase and tissue type plasminogen activators. In subconfluent colonies the distribution of the two enzymes differed. Tissue type plasminogen activator (tPA) was distributed evenly throughout the colony, while, as we have demonstrated previously, urokinase type plasminogen activator (uPA) was preferentially localized at the migrating edges of the colony. Using zymographic analyses, both tPA and uPA activities were detected in cell extracts. Depending on the procedure used to prepare cell extracts, tPA was detected either as free enzyme or in complex with PA inhibitor type 1. PA inhibitor type 1 was deposited onto the extracellular matrix of the keratinocyte cultures and formed a complex with cell-associated tPA when cells and matrix were extracted together. The most differentiated keratinocytes in the culture, which were spontaneously shed from the culture surface, also contained both tPA and uPA. However, these spontaneously shed cells had a higher ratio of tPA:uPA than did the less differentiated cells from the same culture. In conjunction with our previous studies, these results demonstrate the complex nature of the plasminogen activator system, including enzymes and inhibitors, that is present in human keratinocytes. In addition, our data suggest that the relative amounts of uPA and tPA in epidermal cells vary with differentiation state.     

Biological control of tissue plasminogen activator-mediated fibrinolysis

Fibrinolysis, the body's ability to degrade fibrin, is an integrated part of hemostasis. Overactivity in the fibrinolytic system causes bleeding and underactivity causes thrombosis. Tissue plasminogen activator (tPA), plasminogen activator inhibitor type 1 (PAI-1), alpha 2-antiplasmin (alpha 2-AP) and plasminogen are definitely involved in fibrinolysis because: (1) these components can be assigned a fibrinolytic role in purified systems, i.e. in vitro, and (2) abnormal structural variants and abnormal levels of these components give rise to bleeding or to thrombosis. The biological control of tPA-mediated fibrinolysis is both cellular and humoral. The cellular regulation compasses synthesis of tPA and PAI-1 and release/uptake of these components. The humoral regulation involves: (1) the reaction between tPA and PAI-1; (2) the fibrin-stimulated plasminogen activation; (3) the reaction between plasmin and alpha 2-AP and (4) plasmin degradation of fibrin. The highly developed biological control of tPA-mediated fibrinolysis is indicative of its physiological importance.     

The contribution of the exosite residues of plasminogen activator inhibitor-1 to proteinase inhibition

The binding of plasminogen activator inhibitor-1 (PAI-1) to serine proteinases, such as tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA), is mediated by the exosite interactions between the surface-exposed variable region-1, or 37-loop, of the proteinase and the distal reactive center loop (RCL) of PAI-1. Although the contribution of such interactions to the inhibitory activity of PAI-1 has been established, the specific mechanistic steps affected by interactions at the distal RCL remain unknown. We have used protein engineering, stopped-flow fluorimetry, and rapid acid quenching techniques to elucidate the role of exosite interactions in the neutralization of tPA, uPA, and beta-trypsin by PAI-1. Alanine substitutions at the distal P4' (Glu-350) and P5' (Glu-351) residues of PAI-1 reduced the rates of Michaelis complex formation (k(a)) and overall inhibition (k(app)) with tPA by 13.4- and 4.7-fold, respectively, whereas the rate of loop insertion or final acyl-enzyme formation (k(lim)) increased by 3.3-fold. The effects of double mutations on k(a), k(lim), and k(app) were small with uPA and nonexistent with beta-trypsin. We provide the first kinetic evidence that the removal of exosite interactions significantly alters the formation of the noncovalent Michaelis complex, facilitating the release of the primed side of the distal loop from the active-site pocket of tPA and the subsequent insertion of the cleaved reactive center loop into beta-sheet A. Moreover, mutational analysis indicates that the P5' residue contributes more to the mechanism of tPA inhibition, notably by promoting the formation of a final Michaelis complex.     

Interaction of heparin with plasminogen activators and plasminogen: effects on the activation of plasminogen

The amidolytic plasmin activity of a mixture of tissue plasminogen activator (tPA) and plasminogen is enhanced by heparin at therapeutic concentrations. Heparin also increases the activity in mixtures of urokinase-type plasminogen activator (uPA) and plasminogen but has no effect on streptokinase or plasmin. Direct analyses of plasminogen activation by polyacrylamide gel electrophoresis demonstrate that heparin increases the activation of plasminogen by both tPA and uPA. Binding studies show that heparin binds to various components of the fibrinolytic system, with tight binding demonstrable with tPA, uPA, and Lys-plasminogen. The stimulation of tPA activity by fibrin, however, is diminished by heparin. The ability of heparin to promote plasmin generation is destroyed by incubation of the heparin with heparinase, whereas incubation with chondroitinase ABC or AC has no effect. Also, stimulation of plasmin formation is not observed with dextran sulfate or chondroitin sulfate A, B, or C. Analyses of heparin fractions after separation on columns of antithrombin III-Sepharose suggest that both the high-affinity and the low-affinity fractions, which have dramatically different anticoagulant activity, have similar activity toward the fibrinolytic components.     

The interaction of streptokinase.plasminogen activator complex, tissue-type plasminogen activator, urokinase and their acylated derivatives with fibrin and cyanogen bromide digest of fibrinogen. Relationship to fibrinolytic potency in vitro.

The effects of purified soluble fibrin and of fibrinogen fragments (fibrin mimic) on the activation of Lys-plasminogen (i.e. plasminogen residues 77-790) to plasmin by streptokinase.plasminogen activator complex and by tissue-type plasminogen activator were studied. Dissociation constants of both activators were estimated to lie in the range 90-160 nM (fibrin) and 16-60 nM (CNBr-cleavage fragments of fibrinogen). The kinetic mechanism for both types of activator comprised non-essential enzyme activation via a Rapid Equilibrium Ordered Bireactant sequence. In order to relate the fibrin affinity of plasminogen activators to their fibrinolytic potency, the rate of lysis of supported human plasma clots formed in the presence of unmodified or active-centre-acylated precursors of plasminogen activators was studied as a function of the concentration of enzyme derivative. The concentrations of unmodified enzyme giving 50% lysis/h in this assay were 0.9, 2.0 and 11.0 nM for tissue-type plasminogen activator, streptokinase.plasmin(ogen) and urokinase respectively. However, the potencies of active-centre-acylated derivatives of these enzymes suggested that acylated-tissue plasminogen activator and streptokinase.plasminogen complexes of comparable hydrolytic stability were of comparable potency. Both types of acyl-enzyme were significantly more potent than acyl-urokinases.     

Kinetics of the inhibition of plasminogen activators by the plasminogen-activator inhibitor. Evidence for ''second-site'' interactions.

The reactions between plasminogen-activator inhibitor (PAI) and different plasminogen activators were studied in the presence of chromogenic peptide substrates for the enzymes. Our findings suggest that the rate constants for the reactions of PAI with single-chain tissue plasminogen activator (tPA), two-chain tPA, high-Mr urokinase and low-Mr urokinase are high and quite similar (1.6 X 10(7)-3.9 X 10(7) M-1.s-1). A free active site in the enzymes seems to be necessary for their reaction with PAI. Amino acids with antifibrinolytic properties did not interfere with the reactions. However, di-isopropyl phosphorofluoridate-inactivated tPA inhibited the reaction between PAI and all plasminogen activators in a similar way. These findings clearly demonstrated that a 'second-site' interaction, in addition to that between the enzyme active site and the inhibitor 'bait' peptide bond, is of importance for the high reaction rate. The reaction rate between PAI and single-chain tPA in the presence of an activator substrate (D-Ile-Pro-Arg p-nitroanilide) was decreased in the presence of fibrin. Fibrin caused a decrease in the Km for the single-chain tPA-substrate reaction. As a consequence, the 'free' concentration of single-chain tPA in the system decreased in the presence of fibrin, affecting the reaction rate between PAI and single-chain tPA. The phenomenon might be of physiological relevance, in the sense that single-chain tPA bound to fibrin in the presence of plasminogen would be protected against inactivation by PAI.     

Lysine 156 promotes the anomalous proenzyme activity of tPA: X-ray crystal structure of single-chain human tPA.

Tissue type plasminogen activator (tPA) is the physiological initiator of fibrinolysis, activating plasminogen via highly specific proteolysis; plasmin then degrades fibrin with relatively broad specificity. Unlike other chymotrypsin family serine proteinases, tPA is proteolytically active in a single-chain form. This form is also preferred for therapeutic administration of tPA in cases of acute myocardial infarction. The proteolytic cleavage which activates most other chymotrypsin family serine proteinases increases the catalytic efficiency of tPA only 5- to 10-fold. The X-ray crystal structure of the catalytic domain of recombinant human single-chain tPA shows that Lys156 forms a salt bridge with Asp194, promoting an active conformation in the single-chain form. Comparisons with the structures of other serine proteinases that also possess Lys156, such as trypsin, factor Xa and human urokinase plasminogen activator (uPA), identify a set of secondary interactions which are required for Lys156 to fulfil this activating role. These findings help explain the anomalous single-chain activity of tPA and may suggest strategies for design of new therapeutic plasminogen activators.     

Characterizing the role of tissue-type plasminogen activator in a mouse model of Group A streptococcal infection

Plasmin(ogen) acquisition is critical for invasive disease initiation by Streptococcus pyogenes (GAS). Host urokinase plasminogen activator (uPA) plays a role in mediating plasminogen activation for GAS dissemination, however the contribution of tissue-type plasminogen activator (tPA) to GAS virulence is unknown. Using novel tPA-deficient ALBPLG1 mice, our study revealed no difference in mouse survival, bacterial dissemination or the pathology of GAS infection in the absence of tPA in AlbPLG1/tPA^?/? mice compared to AlbPLG1 mice. This study suggests that tPA has a limited role in this humanized model of GAS infection, further highlighting the importance of its counterpart uPA in GAS disease.     

A high affinity interaction of plasminogen with fibrin is not essential for efficient activation by tissue-type plasminogen activator

Fibrin (Fn) enhances plasminogen (Pg) activation by tissue-type plasminogen activator (tPA) by serving as a template onto which Pg and tPA assemble. To explore the contribution of the Pg/Fn interaction to Fn cofactor activity, Pg variants were generated and their affinities for Fn were determined using surface plasmon resonance (SPR). Glu-Pg, Lys-Pg (des(1-77)), and Mini-Pg (lacking kringles 1-4) bound Fn with K(d) values of 3.1, 0.21, and 24.5 μm, respectively, whereas Micro-Pg (lacking all kringles) did not bind. The kinetics of activation of the Pg variants by tPA were then examined in the absence or presence of Fn. Whereas Fn had no effect on Micro-Pg activation, the catalytic efficiencies of Glu-Pg, Lys-Pg, and Mini-Pg activation in the presence of Fn were 300- to 600-fold higher than in its absence. The retention of Fn cofactor activity with Mini-Pg, which has low affinity for Fn, suggests that Mini-Pg binds the tPA-Fn complex more tightly than tPA alone. To explore this possibility, SPR was used to examine the interaction of Mini-Pg with Fn in the absence or presence of tPA. There was 50% more Mini-Pg binding to Fn in the presence of tPA than in its absence, suggesting that formation of the tPA-Fn complex exposes a cryptic site that binds Mini-Pg. Thus, our data (a) indicate that high affinity binding of Pg to Fn is not essential for Fn cofactor activity, and (b) suggest that kringle 5 localizes and stabilizes Pg within the tPA-Fn complex and contributes to its efficient activation.     

Effect of Specific Cleavage of Immunoglobulin G by Plasmin on the Binding and Activation of Plasminogen

A method of ELISA for measuring the binding of different samples of immunoglobulin (IgG) and its fragments to human plasminogen (Pg) has been developed. Instead of plasminogen, the heavy chain of plasminogen (Pg-H) containing five ligand-binding kringle domains, immobilized on the surface of the plate, was used in this method as a detector. It was found that IgG treated with plasmin (IgG_Pm-t) binds to the immobilized Pg-H 2.84 times more strongly than intact IgG. Both IgG samples showed a weak nonspecific binding to the immobilized light chain of plasminogen (Pg-L). It was shown that 0.2 M L-lysine inhibits the binding of IgG_Pm-t and does not affect the nonspecific binding of intact IgG to the immobilized Pg-H, indicating the involvement of lysine-binding regions of Pg-H in binding to IgG_Pm-t. A preliminary treatment of IgG samples with carboxypeptidase В (CPB) inhibited the binding of IgG_Pm-t and did not affect the nonspecific binding of intact IgG to the immobilized Pg-H, which indicates a key role of the С-terminal lysine of IgG_Pm-t in the specific binding to the lysine-binding sites of Pg. The study of the effects of intact IgG and IgG_Pm-t on the rate of activation of Glu- and Lys-forms of Pg (Glu-Pg and Lys-Pg) by a tissue activator of Pg (tPA) and urokinase (uPA) in buffer showed that intact IgG completely inhibited the activation of Glu-Pg and Lys-Pg with both tPA and uPA. Presumably, the inhibitory effect of intact IgG is due to steric hindrances that it creates for protein–protein interactions of the activators with the zymogen. IgG_Pm-t accelerated the generation of plasmin from Pg. In this case, the stimulatory effect of IgG_Pm-t on the activation of Glu-Pg under the action of tPA was ～25% higher than on the activation of Lys-Pg, which is explained by more significant conformational changes in the Glu-Pg molecule compared with the Lys-Pg molecule after their binding to IgG_Pm-t. The results suggest that the specific cleavage of IgG by plasmin may be one of the ways by which the plasminogen/plasmin system is involved in various physiological and pathological processes.     

Activation of gelatinases by fibrin is PA/plasmin system-dependent in human glomerular endothelial cells

Evidence suggests that fibrin deposit is related to severity of glomerulonephropathy. Fibrin is considered to play an active role beyond a haemostatic plug or temporary matrix in response to injury. We have reported that fibrin induced specific morphological changes and up-regulated intercellular adhesion molecule-1 expression of glomerular endothelial cells (GECs). Changes of gelatinases activity have been implicated playing a prominent role in glomerular diseases involving matrix turnover. This study examined whether overlying fibrin influences the expression of gelatinase A and B in cultured human GECs and mechanism underlying the activation. No gelatinase activity was detectable in supernatant of cultured GECs; however, physiological concentration of fibrin (0.5–2.0 mg/ml) induced a dramatic expression of activated MMP-2 and MMP-9 at both mRNA and protein level in a dose and time dependent manner. Increased mRNA level of membrane-type 1 matrix metalloproteinases (MT1-MMPs) was also found. Interestingly, we observed that fibrin also induced the expression of tissue type plasminogen activator (tPA), urokinase type plasminogen activator (uPA) and plasminogen activator inhibitor-1 by casein zymographic and reverse zymographic analysis. Fibrin plate assay revealed the net activity was PA predominant. Serine protease inhibitor aprotinin blocked the conversion of pro-gelatinase A and B to their active forms. The results demonstrate that overlying fibrin increased the secretion of gelatinase A and B from GECs. PA/plasmin proteolytic pathways contributed to the activation of gelatinases.     

Tissue plasminogen activator binds to human vascular smooth muscle cells by a novel mechanism. Evidence for a reciprocal linkage between inhibition of catalytic activity and cellular binding.

Human vascular smooth muscle cells (VSMC) bind tissue plasminogen activator (tPA) specifically, saturably, and with relatively high affinity (K(d) 25 nM), and this binding potentiates the activation of cell-associated plasminogen (Ellis, V., and Whawell, S. A. (1997) Blood 90, 2312-2322). We have observed that this binding can be efficiently competed by DFP-inactivated tPA and S478A-tPA but not by tPA inactivated with H-D-Phe-Pro-Arg-chloromethyl ketone (PPACK). VSMC-bound tPA also exhibited a markedly reduced inhibition by PPACK, displaying biphasic kinetics with second-order rate constants of 7. 5 x 10(3) M(-1) s(-1) and 0.48 x 10(3) M(-1) s(-1), compared with 7. 2 x 10(3) M(-1) s(-1) in the solution phase. By contrast, tPA binding to fibrin was competed equally well by all forms of tPA, and its inhibition was unaltered. These effects were shown to extend to the physiological tPA inhibitor, plasminogen activator inhibitor 1. tPA.plasminogen activator inhibitor 1 complex did not compete tPA binding to VSMC, and the inhibition of bound tPA was reduced by 30-fold. The behavior of the various forms of tPA bound to VSMC correlated with conformational changes in tPA detected by CD spectroscopy. These data suggest that tPA binds to its specific high affinity site on VSMC by a novel mechanism involving the serine protease domain of tPA and distinct from its binding to fibrin. Furthermore, reciprocally linked conformational changes in tPA appear to have functionally significant effects on both the interaction of tPA with its VSMC binding site and the susceptibility of bound tPA to inhibition.