Affinity For Plasminogen Research Articles

Structure-Function Relationships in Staphylokinase as Revealed by "Clustered Charge to Alanine" Mutagenesis

Eighteen mutants of recombinant staphylokinase (SakSTAR) in which clusters of two or three charged residues were converted to alanine ("clustered charge-to-alanine scan") were characterized. Fifteen of these mutants had specific plasminogen-activating activities of > 20% of that of wild-type SakSTAR, whereas three mutants, SakSTAR K11A D13A D14A (SakSTAR13), SakSTAR E46A K50A (SakSTAR48), and SakSTAR E65A D69A (SakSTAR67) had specific activities of 3%. SakSTAR13 had an intact affinity for plasminogen and a normal rate of active site exposure in equimolar mixtures with plasminogen. The plasmin-SakSTAR13 complex had a 14-fold reduced catalytic efficiency for plasminogen activation but was 5-fold more efficient for conversion of plasminogen-SakSTAR13 to plasmin-SakSTAR13. SakSTAR48 and SakSTAR67 had a 10-20-fold reduced affinity for plasminogen and a markedly reduced active site exposure; their complexes with plasmin had a more than 20-fold reduced catalytic efficiency toward plasminogen. Thus, plasminogen activation by catalytic amounts of SakSTAR is dependent on complex formation between plasmin(ogen) and SakSTAR, which is deficient with SakSTAR48 and SakSTAR67, but also on the induction of a functional active site configuration in the plasmin-SakSTAR complex, which is deficient with all three mutants. These findings support a mechanism for the activation of plasminogen by SakSTAR involving formation of an equimolar complex of SakSTAR with traces of plasmin, which converts plasminogen to plasmin and, more rapidly, inactive plasminogen-SakSTAR to plasmin-SakSTAR.

Interaction of Lp(a) with Plasminogen Binding Sites on Cells

Lp(a) competes with plasminogen for binding to cells but it is not known whether this competition is due to the ability of Lp(a) to interact directly with plasminogen receptors. In the present study, we demonstrate that Lp(a) can interact directly with plasminogen binding sites on monocytoid U937 cells and endothelial cells. The interaction of Lp(a) with these sites was time dependent, specific, saturable, divalent ion independent and temperature sensitive, characteristics of plasminogen binding to these sites. The affinity of plasminogen and Lp(a) for these sites also was similar (Kd = 1-3 microM), but Lp(a) bound to fewer sites (approximately 10-fold less). Both gangliosides and cell surface proteins with carboxy-terminal lysyl residues, including enolase, a candidate plasminogen receptor, inhibited Lp(a) binding to U937 cells. Additionally, Lp(a) interacted with low affinity lipoprotein binding sites on these cells which also recognized LDL and HDL. The ability of Lp(a) to interact with sites on cells that recognize plasminogen may contribute to the pathogenetic consequences of high levels of circulating Lp(a).

Substitution of Arginine 719 for Glutamic Acid in Human Plasminogen Substantially Reduces Its Affinity for Streptokinase

In isolation human plasminogen possesses no enzymatic activity, yet upon formation of an equimolar complex with the bacterial protein streptokinase, it acquires a plasminogen activator function. The region(s) of plasminogen and of streptokinase which mediate complex formation has (have) not been previously published. Here it is reported that a single-residue substitution (Arg719-->Glu) in the serine protease domain of full-length Glu-plasminogen substantially reduces its affinity for streptokinase. The plasminogen variant displays no other significant differences from the wild-type molecule with respect to activation by two-chain urokinase-type plasminogen activator, recognition by monoclonal antibodies, or ability to undergo conformational change. It is concluded that Arg719 in human plasminogen is an important determinant of the streptokinase binding site, although further sites are likely to contribute both to the affinity of plasminogen for streptokinase and to mechanisms by which the active site is formed within the complex.

Plasminogen Adsorption to Sulfonated and Lysine Derivatized Model Silica Glass Materials

Silica glass and silylated silica glass have been used extensively as model surfaces for studying the interactions of biological systems with specific chemical functions. In this work, the adsorption of plasminogen to silylated silica glass materials has been investigated with the objective of providing data for the development of a profibrinolytic or clot lysing surface for blood-contacting applications. The adsorption of plasminogen from Tris buffer to silica glass, lysine modified silica glass, and its sulfonated silica glass precursor has been investigated. The isotherms (25°C) suggest high affinity, Langmuir type adsorption. Adsorption capacities are in the order sulfonated silica > lysinized silica > unmodified silica. Estimates of the apparent affinity constants based on the Langmuir equation indicate that the affinity of plasminogen is greater for the lysinized surface than for either the silica glass or sulfonated silica glass. Experiments on the adsorption of plasminogen to the sulfonated and lysinized silica glass in the presence of ϵ-amino caproic acid (EACA), known to interfere with plasminogen binding to lysine, were also carried out. Adsorption plateau values for the sulfonated surface with and without EACA in the buffer were the same. For the lysinized surface, adsorption was reduced in the presence of EACA. Desorption experiments against Tris buffer showed that for the lysinized and sulfonated surfaces, 70-80% of the protein was irreversibly bound, with slightly higher reversibility on the lysinized surface. It is concluded that the mechanisms of adsorption of plasminogen to the lysinized surface and sulfonated surface are different, and that the lysine binding sites of plasminogen may be utilized in its interactions with the lysinized surface.

Carboxypeptidase U : A novel plasma carboxypeptidase with affinity for plasminogen

Carboxypeptidase U : A novel plasma carboxypeptidase with affinity for plasminogen

The Kinetics of Plasminogen Activation by Thrombin-Cleaved Pro-Urokinase and Promotion of its Activity by Fibrin Fragment E-2 and by Tissue Plasminogen Activator

Thrombin hydrolyzes the Arg156-Phe157 bond in pro-urokinase (pro-UK), two residues from the activation site, generating a two-chain form (thromb-UK) believed to have little activity and that is resistant to plasmin activation. The kinetic constants for thromb-UK against synthetic substrate (S2444) were found to be essentially identical to pro-UK. Against native plasminogen, thromb-UK had a lower Michaelis constant (KM) and a higher (2-fold) catalytic efficiency. However, this difference with pro-UK was nullified by carboxypeptidase B (CpB) treatment of thromb-UK to remove the C-terminal arginine on the A-chain. Plasminogen activation by thromb-UK was substantially promoted by fibrin fragment E-2 but not by other fibrin derivatives, a phenomenon previously observed with pro-UK. Similarly, clot lysis by thromb-UK was promoted by tissue plasminogen activator because their combined effect was synergistic. Fibrinogenolysis in plasma occurred at 80-fold the concentration of thromb-UK as pro-UK, reflecting the 90-fold greater plasmin resistance of thromb-UK. Addition of a CpB inhibitor to the plasma enhanced fibrinogenolysis by thromb-UK and pro-UK by approximately 16%, consistent with the promotion of both forms by certain C-terminal lysines. In conclusion, CpB-thromb-UK corresponds functionally to a plasmin resistant form of pro-UK, indicating that the catalytic site of the single-chain pro-UK is unaffected by thrombin cleavage. The effect of CpB indicates that the C-terminal Arg of thromb-UK slightly enhances its affinity for plasminogen. Thromb-UK has potential plasminogen-activating activity at surfaces where C-terminal lysines, functionally comparable to fragment E-2, are found.

The kinetics of plasminogen activation by thrombin-cleaved pro- urokinase and promotion of its activity by fibrin fragment E-2 and by tissue plasminogen activator

Thrombin hydrolyzes the Arg156-Phe157 bond in pro-urokinase (pro-UK), two residues from the activation site, generating a two-chain form (thromb-UK) believed to have little activity and that is resistant to plasmin activation. The kinetic constants for thromb-UK against synthetic substrate (S2444) were found to be essentially identical to pro-UK. Against native plasminogen, thromb-UK had a lower Michaelis constant (KM) and a higher (2-fold) catalytic efficiency. However, this difference with pro-UK was nullified by carboxypeptidase B (CpB) treatment of thromb-UK to remove the C-terminal arginine on the A- chain. Plasminogen activation by thromb-UK was substantially promoted by fibrin fragment E-2 but not by other fibrin derivatives, a phenomenon previously observed with pro-UK. Similarly, clot lysis by thromb-UK was promoted by tissue plasminogen activator because their combined effect was synergistic. Fibrinogenolysis in plasma occurred at 80-fold the concentration of thromb-UK as pro-UK, reflecting the 90- fold greater plasmin resistance of thromb-UK. Addition of a CpB inhibitor to the plasma enhanced fibrinogenolysis by thromb-UK and pro- UK by approximately 16%, consistent with the promotion of both forms by certain C-terminal lysines. In conclusion, CpB-thromb-UK corresponds functionally to a plasmin resistant form of pro-UK, indicating that the catalytic site of the single-chain pro-UK is unaffected by thrombin cleavage. The effect of CpB indicates that the C-terminal Arg of thromb- UK slightly enhances its affinity for plasminogen. Thromb-UK has potential plasminogen-activating activity at surfaces where C-terminal lysines, functionally comparable to fragment E-2, are found.

Isolation, molecular cloning, and partial characterization of a novel carboxypeptidase B from human plasma.

A novel plasminogen-binding protein has been isolated from human plasma utilizing plasminogen-Sepharose affinity chromatography. This protein copurified with alpha 2 antiplasmin when the plasminogen affinity column was eluted with high concentrations of epsilon-aminocaproic acid (greater than 20 mM). Analysis by sodium dodecyl sulfate suggests this protein has an apparent Mr of 60,000. The amino-terminal amino acid sequence showed no similarity to other protein sequences. Based on the amino-terminal amino acid sequence, oligonucleotide probes were designed for polymerase chain reaction primers, and an approximately 1,800 base pair cDNA was isolated that encodes this Mr 60,000 protein. The deduced amino acid sequence reveals a primary translation product of 423 amino acids that is very similar to carboxypeptidase A and B and consists of a 22-amino acid signal peptide, a 92-amino acid activation peptide, and a 309-amino acid catalytic domain. This protein shows 44 and 40% similarity to rat procarboxypeptidase B and human mast cell procarboxypeptidase A, respectively. The residues critical for catalysis and zinc and substrate binding of carboxypeptidase A and B are conserved in the Mr 60,000 plasminogen-binding protein. The presence of aspartic acid at position 257 of the catalytic domain suggests that this protein is a basic carboxypeptidase. When activated by trypsin, it hydrolyzes carboxypeptidase B substrates, hippuryl-Arg and hippuryl-Lys, but not carboxypeptidase A substrates, and it is inhibited by the specific carboxypeptidase B inhibitor (DL-5-guanidinoethyl)mercaptosuccinic acid. We propose that the Mr 60,000 plasminogen-binding protein isolated here is a novel human plasma carboxypeptidase B and that it be designated pCPB.

The effect of the carboxy-terminal lysine of urokinase on the catalysis of plasminogen activation

When single-chain pro-UK is activated by plasmin or kallikrein, the Lys 158-Ile 159 bond is cleaved, leaving a C-terminal lysine on the A-chain (Lys-UK). Two-chain, high molecular weight urokinase (UK) purified from urine, however, has been shown to contain a phenylalanine residue as the C-terminal of the A-chain (Phe-UK). Since C-terminal lysine residues have a strong binding affinity for plasminogen that may promote its activation, we undertook kinetic studies comparing plasminogen activation by Lys-and Phe-UK. A two-stage method was employed in order to minimize factors known to interfere with plasminogen activation and plasmin determination. The Lys-UK was prepared by plasmin activation of pro-UK purified from human fetal kidney cell culture medium. The Phe-UK was prepared by carboxypeptidase B (CpB) treatment of Lys-UK. Removal of the C-terminal lysine of Lys-UK by CpB produced small but significant increases in the Michaelis constants for the activation of both Glu- and Lys-plasminogen. The apparent Michaelis constants for Glu-plasminogen activation by Lys- and Phe-UK were 3.7 μM ± .36 μM and 5.9 μM ± .70 μM, respectively and the Michaelis constants for Lys-plasminogen activation by Lys- and Phe-UK were 5.4 μM ± .72 μM and 15.2 μM ± 1.4 μM, respectively. The catalytic efficiency ( k cat / K m ) of Lys-UK was approximately 2-fold greater than that of Phe-UK for the activation of either Glu- or Lys-plasminogen. When the fibrinolytic activities of Lys- and Phe-UK were compared in a plasma milieu, no significant differences were detected. In conclusion, the findings indicate that the C-terminal lysine on the A-chain of UK significantly promotes the catalysis of plasminogen in a purified system. However, the higher catalytic efficiency of Lys-UK was not found to induce significant acceleration of clot lysis at pharmacological concentrations in plasma.

Characterization of plasminogen binding to human capillary and arterial endothelial cells

Phenotypic diversity of endothelial cells that line the various vascular spaces has been well established. However, it is not known if biochemical differences also exist, particularly in the numbers of receptors for plasma proteins. Equilibrium binding techniques were used to assess potential differences in the binding of 125I-labelled plasminogen to cultured human umbilical arterial endothelial cells and capillary endothelium, as compared with umbilical venous cells. The kinetic behaviour of plasminogen binding to all three types of cells was similar, with optimal binding occurring between 20 and 30 min of incubation. Binding of plasminogen to arterial, capillary, and venous cells was concentration dependent and reversible upon addition to excess unlabelled plasminogen. Scatchard analyses showed that artery, capillary, and venous endothelial cells all possess low affinity sites for plasminogen with Kd values of 0.30 +/- 0.07, 0.40 +/- 0.06, and 0.40 +/- 0.08 microM, respectively. Vein cells also possess an additional higher affinity binding site with a Kd of 0.07 +/- 0.01 microM, exhibiting a 6-fold greater affinity for plasminogen than the lower affinity sites on capillary and arterial endothelial cells. Assuming a stoichiometry of 1:1 for binding, the data indicate that arterial and capillary endothelial cells contain approximately 4.2 (+/- 0.9) x 10(6) and 4.1 (+/- 0.6) x 10(6) plasminogen receptors per cell. Venous cells contain both low and high density binding sites with 6.2 (+/- 0.8) x 10(6) and 12.4 (+/- 2.4) x 10(6) sites per endothelial cell. The presence of a higher affinity site on vein cells, but not on artery or capillary cells, may signal functional differences relating to fibrinolytic activity on the surface of these cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of cell-surface lysines in plasminogen binding to cells: identification of .alpha.-enolase as a candidate plasminogen receptor

Plasminogen binding to cell surfaces results in enhanced plasminogen activation, localization of the proteolytic activity of plasmin on cell surfaces, and protection of plasmin from alpha 2-antiplasmin. We sought to characterize candidate plasminogen binding sites on nucleated cells, using the U937 monocytoid cell as a model, specifically focusing on the role of cell-surface proteins with appropriately placed lysine residues as candidate plasminogen receptors. Lysine derivatives with free alpha-carboxyl groups and peptides with carboxy-terminal lysyl residues were effective inhibitors of plasminogen binding to the cells. One of the peptides, representing the carboxy-terminal 19 amino acids of alpha 2-antiplasmin, was approximately 5-fold more effective than others with carboxy-terminal lysines. Thus, in addition to a carboxy-terminal lysyl residue, other structural features of the cell-surface proteins may influence their affinity for plasminogen. Affinity chromatography has been used to isolate candidate plasminogen receptors from U937 cells. A major protein of Mr 54,000 was recovered and identified as alpha-enolase by immunochemical and functional criteria. alpha-Enolase was present on the cell surface and was capable of binding plasminogen in ligand blotting analyses. Plasminogen binding activity of a molecular weight similar to alpha-enolase also was present in a variety of other cell types. Carboxypeptidase B treatment of alpha-enolase abolished its ability to bind plasminogen, consistent with the presence of a C-terminal lysyl residue. Thus, cell-surface proteins with carboxy-terminal lysyl residues appear to function as plasminogen binding sites, and alpha-enolase has been identified as a prominent representative of this class of receptors.

Thrombolytic therapy

Thrombolytic therapy

Purification and properties of a single-chain urokinase-type plasminogen activator form produced by subcultured human umbilical vein endothelial cells.

Single-chain Mr 54,000 u-PA (scu-PA) was isolated, in the presence of aprotinin, from 3-liter batches of 60-h serum-free conditioned media obtained from subcultured (4-6th passage) human umbilical vein endothelial cells (HUVECs, approximately 1.8 x 10(9) cells). In the presence of heparin and endothelial cell growth factor, subcultured human umbilical vein endothelial cells produced u-PA proteins consisting of about 85-90% Mr 54,000 scu-PA and 10-15% two-chain Mr 54,000. The major scu-PA form was purified to homogeneity by ion-exchange chromatography on CM-Sephadex C-50, immunoadsorption on purified anti-u-PA IgG-Sepharose and affinity chromatography on p-amino-benzamidine-Agarose. Typically, about 8-10 micrograms of purified scu-PA protein (antigen/protein ratio = 1) was isolated from 3-liter batches of heparin-containing serum-free conditioned media with a yield of about 41% of the total starting u-PA antigen. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of this purified u-PA protein showed a single Ag-stained band (nonreduced and reduced), with an estimated molecular weight of about 54,000, which exhibited very low fibrinolytic activity. Purified HUVEC-derived scu-PA did not incorporate 3H-labeled diisopropyl fluorophosphate. This protein did, however, exhibit very low amidolytic activity (approximately 5,000 IU/mg) on the u-PA-specific synthetic substrate pyroglu-Gly-Arg-p-nitroanilide, very low plasminogen-dependent fibrinolytic activity on 125I-labeled fibrin coated plates, and directly activated 125I-labeled plasminogen following Michaelis-Menten kinetics with high affinity, Km = 0.72 microM and low turnover number, kcat = 0.0005 s-1. Treatment with plasmin rapidly converted the HUVEC-derived scu-PA to the active two-chain Mr 54,000 u-PA form (approximately 90,000 IU/mg). Binding to fibrin clots, using antigen quantitation, indicated about 20, 10, and 90% binding for equimolar amounts of HUVEC-derived scu-PA, two-chain u-PA, and tissue plasminogen activator standards, respectively. These results indicate that subcultured HUVECs synthesize and secrete their u-PA protein as a single-chain molecule with low intrinsic amidolytic and fibrinolytic activity, high affinity for plasminogen and no specific affinity for fibrin. The role of scu-PA in endothelial cell-mediated vascular function has yet to be clearly defined.

Variants of human tissue-type plasminogen activator that lack specific structural domains of the heavy chain.

The heavy chain of tissue plasminogen activator (t-PA) consists of four domains [finger, epidermal-growth-factor (EGF)-like, kringle 1 and kringle 2] that are homologous to similar domains present in other proteins. To assess the contribution of each of the domains to the biological properties of the enzyme, site-directed mutagenesis was used to generate a set of mutants lacking sequences corresponding to the axons encoding the individual structural domains. The mutant proteins were assayed for their ability to hydrolyze artificial and natural substrates in the presence and absence of fibrin, to bind to lysine-Sepharose and to be inhibited by plasminogen activator inhibitor-1. All the deletion mutants exhibit levels of basal enzymatic activity very similar to that of wild-type t-PA assayed in the absence of fibrin. A mutant protein lacking the finger domain has a 2-fold higher affinity for plasminogen than wild-type t-PA, while the mutant that lacks both finger and EGF-like domains is less active at low concentrations of plasminogen. Mutants lacking both kringles neither bind to lysine-Sepharose nor are stimulated by fibrin. However, mutants containing only one kringle (either kringle 1 or kringle 2) behave indistinguishably from one another and from the wild-type protein. We conclude that kringle 1 and kringle 2 are equivalent in their ability to mediate stimulation of catalytic activity by fibrin.

Mechanisms of plasminogen activation by mammalian plasminogen activators.

Plasminogen activators convert the proenzyme plasminogen to the active serine protease plasmin by hydrolysis of the Arg560-Val561 peptide bond. Physiological plasminogen activation is however regulated by several additional molecular interactions resulting in fibrin-specific clot lysis. Tissue-type plasminogen activator (t-PA) binds to fibrin and thereby acquires a high affinity for plasminogen, resulting in efficient plasmin generation at the fibrin surface. Single-chain urokinase-type plasminogen activator (scu-PA) activates plasminogen directly but with a catalytic efficiency which is about 20 times lower than that of urokinase. In plasma, however, it is inactive in the absence of fibrin. Chimeric plasminogen activators consisting of the NH2-terminal region of t-PA (containing the fibrin-binding domains) and the COOH-terminal region of scu-PA (containing the active site), combine the mechanisms of fibrin specificity of both plasminogen activators. Combination of t-PA and scu-PA infusion in animal models of thrombosis and in patients with coronary artery thrombosis results in a synergic effect on thrombolysis, allowing a reduction of the therapeutic dose and elimination of side effects on the hemostatic system.

Recombinant human tissue-type plasminogen activator therapy in acute thromboembolic stroke.

Systemic fibrinolytic therapy for acute stroke is no longer recommended because of resulting systemic fibrinolysis and the risk of intracerebral hemorrhage. Human tissue-type plasminogen activator (TPA) is a native enzyme that converts plasminogen to plasmin with subsequent clot lysis. The affinity for plasminogen is increased several-fold when the substrate is bound to fibrin. At appropriate dosage, "clot-specific" thrombolysis may be achieved at the surface of the thrombus without creating systemic fibrinolysis. The authors designed a study to evaluate the effect of intravenous TPA administered 2 hours after acute thromboembolic stroke in rats. This time course was chosen to simulate an analogous clinical situation. Middle cerebral artery embolic stroke was caused by intracarotid injection of 0.025 cc of human blood clot in 16 rats. Regional cerebral blood flow, measured by the hydrogen clearance technique, and electroencephalographic (EEG) recordings were obtained every 30 minutes for 5 hours after thromboembolism. Eight rats received a 1-hour infusion of intravenous TPA (1.5 mg/kg) 2 hours after injection of emboli. Ipsilateral blood flow increased significantly within 30 minutes after intravenous TPA and reached preembolic levels within 60 minutes. Blood flow did not improve in the eight control rats throughout the experiment. Power spectral analysis of the EEG recordings showed improvement in the treated group compared to the control group. Postmortem angiography revealed proximal middle cerebral artery occlusion in control animals and patent middle cerebral arteries in TPA-treated animals. Serum fibrinogen and fibrin split products were unchanged in both groups, indicating the absence of systemic fibrinolysis. There were no intracerebral hemorrhages. It is concluded that, in this rat model, TPA increases blood flow with subsequent improvement in the EEG recording after thromboembolic stroke without evidence of systemic fibrinolysis. Intravenous TPA may be useful in the treatment of acute stroke in man.

Molecular mechanisms of fibrinolysis and their application to fibrin-specific thrombolytic therapy.

The fibrinolytic system comprises a proenzyme, plasminogen, which can be converted to the active enzyme, plasmin, which degrades fibrin. Plasminogen activation is mediated by plasminogen activators, which are classified as either tissue-type plasminogen activators (t-PA) or urokinase-type plasminogen activators (u-PA). Inhibition of the fibrinolytic system may occur at the level of the activators or at the level of generated plasmin. Plasmin has a low substrate specificity, and when circulating freely in the blood it degrades several proteins including fibrinogen, factor V, and factor VIII. Plasma does, however, contain a fast-acting plasmin inhibitor, alpha 2-antiplasmin, which inhibits free plasmin extremely rapidly but which reacts much slower with plasmin bound to fibrin. A "systemic fibrinolytic state" may, however, occur by extensive activation of plasminogen and depletion of alpha 2-antiplasmin. Clot-specific thrombolysis therefore requires plasminogen activation restricted to the vicinity of the fibrin. Two physiological plasminogen activators, t-PA and single-chain u-PA (scu-PA) induce clot-specific thrombolysis, via entirely different mechanisms, however. t-PA is relatively inactive in the absence of fibrin, but fibrin strikingly enhances the activation rate of plasminogen by t-PA. This is explained by an increased affinity of fibrin-bound t-PA for plasminogen and not by alteration of the catalytic rate constant of the enzyme. The high affinity of t-PA for plasminogen in the presence of fibrin thus allows efficient activation on the fibrin clot, while no significant plasminogen activation by t-PA occurs in plasma. scu-PA has a high affinity for plasminogen (Km = 0.3 microM) but a low catalytic rate constant (kcat = 0.02 sec-1). However, scu-PA does not activate plasminogen in plasma in the absence of a fibrin clot, owing to the presence of (a) competitive inhibitor(s). Fibrin-specific thrombolysis appears to be due to the fact that fibrin reverses the competitive inhibition. The thrombolytic efficacy and fibrin specificity of natural and recombinant t-PA has been demonstrated in animal models of pulmonary embolism, venous thrombosis, and coronary artery thrombosis. In all these studies intravenous infusion of t-PA at sufficiently high rates caused efficient thrombolysis in the absence of systemic fibrinolytic activation. The efficacy and relative fibrinogen-sparing effect of t-PA was recently confirmed in three multicenter clinical trials in patients with acute myocardial infarction.(ABSTRACT TRUNCATED AT 400 WORDS)

The molecular form of α2-antiplasmin with affinity for plasminogen is selectively bound to fibrin by factor XIII

Two molecular forms of α 2-antiplasmin (PB = plasminogen-binding; NPB = non-PB) exist. We have studied the extent of binding of these two forms to fibrin by factor XIII. A modified crossed immunoelectrophoresis technique which separately determines both forms showed that, compared with plasma, the PB-form is reduced by 31 ± 11% (SD) in serum of twelve individuals and the NPB form by only 6%. Laurell assay showed a reduction of 18 ± 9% (n = 12) in total α 2-antiplasmin antiqen (PB + NPB) in serum; the immediate olasmin inhibition test (mainly recording the PS-form) revealed 35 ± 6% (n = 12) reduction in inhibition. Coagulation of blood, platelet-rich and platelet-poor plasma yielded comparable results. No decrease in α 2-antiplasmin or change in its composition was observed when factor XIII-deficient plasma was clotted. Fibrin binding was not significantly different from normal in either plasminogen-depleted or plasminogen-enriched plasma. It is concluded that the PB-form of α 2-antiplasmin becomes selectively bound to fibrin.

Molecular exchange between blood and in vitro thrombi.

Uptake of radioiodinated Lys-plasminogen (125I-PLG), albumin (125I-ALB), and tritiated water (3H-H2O) by in vitro thrombi and interchange of these molecules with blood in an in vitro perfusion system were investigated. The radioisotopes were taken up by thrombi either by incorporation during formation in radiolabelled blood or by perfusion of preformed nonradioactive thrombi in radiolabelled blood. Release of the radioisotopes into a perfusion medium of nonradiolabelled blood was then monitored over a 120-min period. The small molecules of 3H-H2O were rapidly released from the thrombi and achieved equilibrium with the perfusion medium by 120 min in that the specific radioactivity (cpm/mg) of the thrombi equalled that of the medium. The larger molecules of 125I-PLG and 125I-ALB were more slowly released and did not reach equilibrium with the perfusion medium over the period studied. Release of 125I-ALB was intermediate between that of 3H-H2O and 125I-PLG. The greater retention of 125I-PLG by the thrombi was consistent with the high binding affinity of plasminogen for fibrin. The demonstrated movement of these radioisotopes between medium and thrombus suggests that thrombi exist in blood in a dynamic state of flux, exhibiting a fluid exchange of molecules between the interstitial compartment of the thrombus and blood.

Importance, localization and functional properties of the cell-associated form of plasminogen activator in mouse peritoneal macrophages

In inflammatory macrophages, plasminogen activator exists in two active forms, a soluble form released into the extracellular medium and a cell-associated form. This communication describes some properties of the cellular form of plasminogen activator, in intact macrophages and in cell lysates. Cellular plasminogen activator is a membrane protein, associated with the outer face of the plasma membrane; in intact macrophages, it participates in the activation of exogenous plasminogen and, thus, has to be considered as an entoenzyme. A plasminogen activator activity can be detected in cell lysates (macrophage monoloyers lysed in 0.1% Triton X-100) only when plasmin production is followed by the use of small synthetic substrates because a soluble inhibitor, released during extraction, blocks plasmin fibrinolytic activity. In these lysates, plasminogen activator molecules exist as high molecular weight unstable complexes exhibiting a high affinity for plasminogen.