Role of fibronectin in the migration of fibroblasts into plasma clots

The adhesion and migration of human diploid fibroblasts on plasma clots were measured. The role of plasma fibronectin was examined by depleting plasma of fibronectin before clotting. Fibronectin was not essential for cell adhesion and spreading, although rates were slightly slower on depleted clots. Rates of migration on the surface of clots were unaffected by fibronectin depletion. In contrast, fibronectin was an absolute requirement for migration of cells into plasma clots. Cells migrated rapidly into control clots but completely failed to penetrate the surface of fibronectin-depleted clots. The effect of depletion could only be reversed by adding fibronectin to depleted plasma before clotting. Adsorption of fibronectin after clotting failed to reverse the effect of depletion, suggesting that fibronectin had to be cross-linked by transglutaminase during the clotting process.     

Quantitative characterization of the binding of plasminogen to intact fibrin clots, lysine-sepharose, and fibrin cleaved by plasmin

The binding of human Glu- and Lys-plasminogens to intact fibrin clots, to lysine-Sepharose, and to fibrin cleaved by plasmin was quantitatively characterized. On intact fibrin clots, there was one strong binding site for Glu-plasminogen with a dissociation constant, Kd, of 25 microM and one strong binding site for Lys-plasminogen with a Kd of 7.9 microM. In both cases, the number of plasminogen binding sites per fibrin monomer was 1. Also, a much weaker binding site for Glu-plasminogen was observed with a Kd of about 350 microM. Limited digestion of fibrin by plasmin created additional binding sites for plasminogen with Kd values similar to the binding of plasminogen to lysine-Sepharose. This was predictable given the observations that plasminogen binds to lysine-Sepharose and can be eluted with epsilon-aminocaproic acid [Deutsch, D.G., & Mertz, E.T. (1970) Science (Washington, D.C.) 170, 1095-1096] and that plasmin preferentially cleaves fibrin at the carboxy side of lysyl residues [Weinstein, M.J., & Doolittle, R.F. (1972) Biochim. Biophys. Acta 258, 577-590], because the structures of the lysyl moiety in lysine-Sepharose and of epsilon-aminocaproic acid are identical with the structure of a COOH-terminal lysyl residue created by plasmin cleavage of fibrin. The Kd for the binding of Glu-plasminogen to lysine-Sepharose was 43 microM and for fibrin partially cleaved by plasmin 48 microM. The Kd for the binding of Lys-plasminogen to lysine-Sepharose was 30 microM. With fibrin partially cleaved by plasmin, there were two types of binding sites for Lys-plasminogen, one with a Kd of 7.6 microM and the other with a Kd of 44 microM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of plasminogen activators in peritoneal adhesion formation

Intra-abdominal adhesion formation is a major complication of serosal repair following surgery, ischaemia or infection, leading to conditions such as intestinal obstruction and infertility. It has been proposed that the persistence of fibrin, due to impaired plasminogen activator activity, results in the formation of adhesions between damaged serosal surfaces. This study aimed to assess the role of fibrinolysis in adhesion formation using mice deficient in either of the plasminogen activator proteases, tissue-type plasminogen activator (tPA) or urokinase-type plasminogen activator (uPA). We hypothesize that, following serosal injury, mice with decreased peritoneal fibrinolytic activity will be more susceptible to adhesion formation. Adhesion formation was induced in tPA- and uPA-deficient and wild-type mice following either surgical trauma to the serosa with haemorrhage and acute or chronic intraperitoneal inflammation. Adhesion formation was assessed from 1 to 4 weeks post-injury. Mice deficient in tPA were more susceptible to adhesion formation following both a surgical insult and a chronic inflammatory episode compared with uPA-deficient and wild-type mice. In addition, the time of maximal adhesion formation varied depending on the nature of the initial insult. It is proposed that the persistence of fibrin due to decreased tPA activity following surgery or chronic inflammation plays a major role in peritoneal adhesion formation.     

Fluorescence polarization assay of plasmin, plasminogen, and plasminogen activator

A chromatographic method involving medium-pressure liquid chromatography on alumina impregnated with silver nitrate is described for the separation of a series of closely related C₂₇ sterol precursors of cholesterol differing only in the number and location of olefinic double bonds. The features of the described system are compared with those of previously described thin-layer, gas-liquid, gravity column, and high-pressure liquid chromatographic methods.     

Role of the streptokinase alpha-domain in the interactions of streptokinase with plasminogen and plasmin

The role of the streptokinase (SK) alpha-domain in plasminogen (Pg) and plasmin (Pm) interactions was investigated in quantitative binding studies employing active site fluorescein-labeled [Glu]Pg, [Lys]Pg, and [Lys]Pm, and the SK truncation mutants, SK-(55-414), SK-(70-414), and SK-(152-414). Lysine binding site (LBS)-dependent and -independent binding were resolved from the effects of the lysine analog, 6-aminohexanoic acid. The mutants bound indistinguishably, consistent with unfolding of the alpha-domain on deletion of SK-(1-54). The affinity of SK for [Glu]Pg was LBS-independent, and although [Lys]Pg affinity was enhanced 13-fold by LBS interactions, the LBS-independent free energy contributions were indistinguishable. alpha-Domain truncation reduced the affinity of SK for [Glu]Pg 2-7-fold and [Lys]Pg 相似文献   

Role of plasminogen/plasmin in functional activity of blood cells

The article deals with the data concerning structural peculiarities of plasminogen/plasmin molecule, which define the specificity of intermolecular interactions and provide the variety of its biological functions. The main principles of the modern classification of plasminogen receptors and factors, which modulate their expression, have been presented. We have considered the mechanisms regulating both plasmin formation and activity on the surface of cells, fibrin and proteins of extracellular matrix. The data of previous investigators and our own results, concerning the influence of plasminogen/plasmin on platelet aggregation induced by different agonists, have been summarized. The participation of plasminogen/plasmin in atherogenesis and angiogenesis mediated by endotheliocyte receptors has been discussed. Special attention was given to plasminogen/plasmin proinflammatory function, which is realized by regulatory processes of activation, secretion, migration and apoptosis of monocytes and macrophages.     

Purification of plasminogen activators from human seminal plasma.

Two plasminogen activators (1 and 2) were isolated from human seminal plasma by hiigh-speed centrifugation, Sephadex-gel filtration and ion-exchange chromatography. The activators were shown to be homogeneous by polyacrylamide-disc -gel electrophoresis at pH 8.3 and 4.5, and by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The molecular weights of activators 1 and 2 were estimated as 69 000 and 74 000. Their amino acid compositions are very similar, both being high in aspartic acid, glutamic acid, serine, glycine and leucine, and low in methionine, tryptophan, tyrosine, isoleucine and histidine. Activators 1 and 2 each possess 16 cysteine residues. Both activators have isoelectric points of approx. 7.0, are stable over a wide pH range at temperatures up to 60 degrees C, but lose activity at higher temperatures, particularly under very basic or acidic conditions. They are not inhibited by EDTA, Mg2+ and Ca2+ at 10 mM concentrations, but their activity decreases on addition of 10 mM-cysteine or Fe2+ and 6-aminohexanoate or sera from pregnant women. The precipitin band formed between urokinase and its antiserum is continuous with the precipitin bands formed between the seminal plasminogen activators and the urokinase antiserum. Antisera to urokinase inhibit both the activity of urokinase and the seminal plasminogen activators.     

Identification of a reversible inhibitor of plasminogen activators in blood plasma

Inhibition of tissue-type plasminogen activator (t-PA) by pooled plasma could be ascribed for only 60% to the endothelial cell type PA inhibitor. The residual inhibition is ascribed to a so-far undescribed plasma component present at 0.2 nmol/l. This component shows reversible binding to t-PA with an apparent K_i of 10 pmol/l (does not hinder t-PA binding to fibrin); also reacts with urokinase, but not with DIP-t-PA; is stable at 37°C and does not occur in media of endothelial cells, hepatocytes and fibroblasts. This PA binding component in plasma adds to the regulation of plasminogen activator activities.

Fibrinolysis Tissue-type plasminogen activator Urokinase Blood plasma Endothelial cell type plasminogen activator inhibitor Protease inhibitor     

Electrochemical assay of plasminogen activators in plasma using polyion-sensitive membrane electrode detection

Two relatively simple electrochemical assay methods suitable for the measurement of plasminogen activators (including urokinase (u-PA), streptokinase (SK), and tissue plasminogen activator (t-PA)) in plasma samples are described. In one approach, the initial rate of decrease in the potentiometric response of a polycation-sensitive membrane electrode toward protamine is monitored after addition of a preincubated reaction mixture containing the sample and exogenous plasminogen. The plasmin formed from plasminogen by the activators catalyzes the decomposition of the arginine-rich protamine substrate, yielding smaller polycationic fragments that are not sensed by the electrode. Alternately, the sample, plasminogen, and protamine can be incubated together, and the remaining protamine in this reaction mixture can be measured at a fixed point in time by placing the electrode into the mixture and recording the electromotive force response. Working curves found with both methods for plasma samples spiked with varying levels of the activators cover the expected therapeutic activity ranges found in the plasma of patients treated with these "clot-busting" drugs.     

Disorders in the release of plasminogen activators

Impairment of the release of plasminogen activator has been looked for in patients with a predisposition to vascular disease or venous thrombosis. In normal people the fibrinolytic activity of the blood rises sharply after strenuous physical exercise or after the administration of certain drugs, among which DDAVP. These measures fail to elicit a normal response in many of these patients. In most cases this turned out to be due to a high level of a circulating plasminogen activator inhibitor which suppresses the rise in fibrinolytic activity. Release of activator can only be demonstrated reliably by the assay of t-PA-antigen. An impaired release appears to be very rare and in the experience of the author it occurs with some regularity only in patients with terminal renal insufficiency.     

Incorporation of fragment X into fibrin clots renders them more susceptible to lysis by plasmin

Bleeding, the most serious complication of thrombolytic therapy with tissue-type plasminogen activator (t-PA), is thought to result from lysis of fibrin in hemostatic plugs and from the systemic lytic state caused by unopposed plasmin. One mechanism by which systemic plasmin can impair hemostasis is by partially degrading fibrinogen to fragment X, a product that retains clottability but forms clots with reduced tensile strength that stimulate plasminogen activation by t-PA more than fibrin clots. The purpose of this study was to elucidate potential mechanisms by which fragment X accelerates t-PA-mediated fibrinolysis. In the presence of t-PA, clots containing fragment X were degraded faster than fibrin clots and exhibited higher rates of plasminogen activation. Although treatment with carboxypeptidase B, an enzyme that reduces plasminogen binding to fibrin, prolonged the lysis times of fragment X and fibrin clots, clots containing fragment X still were degraded more rapidly. Furthermore, plasmin or trypsin also degraded clots containing fragment X more rapidly than fibrin clots, suggesting that this effect is largely independent of plasminogen activation. Fragment X-derived degradation products were not preferentially released by plasmin from clots composed of equal concentrations of fibrinogen and fragment X, indicating that fragment X does not constitute a preferential site for proteolysis. These data suggest that structural changes resulting from incorporation of fragment X into clots promote their lysis. Thus, attenuation of thrombolytic therapy-induced fragment X formation may reduce the risk of bleeding.     

A sensitive fluorometric assay for plasminogen, plasmin, and streptokinase

A rapid, convenient, and highly sensitive fluorometric assay for plasmin (P), plasminogen (Pg), and streptokinase (SK) as the activator complex (SK·P) is described. These assays are based on the measurement of the fluorescence of β-naphthol (βN) released from α-N-methyl α-N-tosyl-l-lysine β-naphthol ester (MTLNE) by the P present or generated during the assay. The rate of βN release may be followed by direct recording or determined subsequently, following termination of the enzyme reaction at a fixed digestion time, by the addition of p-nitrophenyl-p′-guanidinobenzoate. The latter method may be readily automated. The K_m and V values for the hydrolysis of MTLNE by P or SK·P are equivalent. The Pg activator activity of P was shown to be very small (less than 0.2% of that of SK·P).     

Structure and function of plasminogen/plasmin system

The main physiological function of plasmin is blood clot fibrinolysis and restoration of normal blood flow. To date, however, it became apparent that in addition to thrombolysis, the plasminogen/plasmin system plays an important physiological and pathological role in a number of other essential processes: degradation of the extracellular matrix, embryogenesis, cell migration, tissue remodeling, wound healing, angiogenesis, inflammation, and tumor cell migration. This review focuses on structural features of plasminogen, regulation of its activation by physiological plasminogen activators, inhibitors of plasmin, and plasminogen activators, and the role of plasminogen binding to fibrin, cellular receptors, and extracellular ligands in various functions performed by plasmin thus formed.     

Follicular plasminogen and plasminogen activator and the effect of plasmin on ovarian follicle wall.

Plasminogen, plasminogen activator, protease inhibitors, and a proteolytic activity are shown to be present in bovine follicular fluid. Much of the proteolytic activity appears to be due to plasmin. In addition, plasminogen activator activity can be demonstrated in follicle wall homogenates. Evidence that plasmin decreases the tensile strength of follicle wall preparations is also reported. The potential for the involvement of these substances in ovulation is discussed.     

Identity of kininogenase and plasminogen activators in human granulocytes

It was shown that the plasminogen activator inhibitor, ZGlyGlyArgCH2Cl, inactivates the kininogenase and plasminogen activator activities in the whole human granulocyte lysate and human granulocyte proteinase fractions isolated by isoelectrofocusing from the granulocyte lysate (pH 3-10). The kinetics of irreversible inhibition of the ZGlyGlyArgpNA-amidase activity in granulocyte proteinase fractions (pI 10.75, 8.9 and 8.3) by ZGlyGlyArgCH2Cl was measured. These data confirm the earlier obtained results on the trypsin-like nature of the human granulocyte plasminogen activator and its identity to this cell kininogenase.