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PubMed1994

Characterization of plasminogen activation by glycosylphosphatidylinositol-anchored urokinase.

The characteristics of plasminogen activation by glycosylphosphatidylinositol (GPI)-anchored urokinase were evaluated and compared with those reported previously for receptor-bound urokinase. When expressed in cultured bovine aortic endothelial cells, GPI anchoring of single-chain urokinase plasminogen activator (scu-PA) potentiated plasmin generation as compared with GPI-anchored scu-PA that had been released into solution from the cell surface by enzymatic cleavage of the GPI anchor ("released" scu-PA). The potentiation of plasmin generation by GPI-anchored scu-PA was inhibited in a dose-dependent manner by 6-aminohexanoic acid, a lysine analog, suggesting that the augmentation of plasmin generation by GPI-anchored scu-PA was dependent on simultaneous binding of plasminogen to the cell surface. GPI-anchored two-chain urokinase (tcu)-PA cleaved a peptide substrate at a rate equivalent to that of released urokinase. However, at a plasminogen concentration of 0.5 microM, GPI-anchored tcu-PA activated plasminogen less rapidly than did released urokinase. Modeling of kinetics of individual reactions revealed that cell-associated plasminogen activation by GPI-anchored tcu-PA was characterized by a Km of approximately 0.15 microM. This value of Km was 70-fold below that for activation of solution plasminogen by GPI-anchored urokinase. There was a concomitant decrease in Vmax for plasminogen activation by anchored tcu-PA. These alterations in kinetic parameters are similar to those reported previously for the activation of plasminogen by receptor-bound tcu-PA. In addition, GPI-anchored tcu-PA exhibited a modest resistance to plasminogen activator inhibitor 1 inactivation. The enzymatic characteristics of GPI-anchored urokinase reported here resemble closely those reported previously for receptor-bound urokinase. These data suggest that the urokinase receptor may regulate plasmin generation through a relatively nonspecific localization of urokinase to the cell surface rather than through any intrinsic property of the urokinase receptor.

Aminocaproic Acid↗
PubMed1987

Plasminogen activator production by human tumor cells: effect on tumor cell-extracellular matrix interactions.

Cell lines derived from 3 different types of human tumor (e.g., squamous carcinomas, melanomas and gliomas) were examined for production of plasminogen activator activity and for attachment and spreading on various extracellular matrix components in the presence or absence of plasminogen. All of the squamous carcinoma and melanoma lines produced high levels of plasminogen activator activity. In contrast, 4 of 6 glioma lines had undetectable activity. Cells from all 3 tumor types attached and spread on fibrinogen-coated or fibrin-coated plastic dishes in the absence of plasminogen. In the presence of exogenous plasminogen, the attachment and spreading of the cells which produced high levels of plasminogen activator activity was inhibited. The plasminogen activator-deficient cells were much less sensitive to exogenous plasminogen. In the presence of plasminogen, attachment and spreading on fibronectin-coated dishes was also partially inhibited. In contrast, plasminogen had no effect on the attachment and spreading of the cells on type-I or -IV collagen, laminin or thrombospondin. Previous studies have shown that tumor-cell adhesion to the extracellular matrix depends on the synthesis of receptors for extracellular matrix components or on the synthesis of extracellular matrix components themselves. The present study shows that, in addition, the production of enzymes which are capable of degrading these components also influences tumor-cell adhesion to extracellular matrix moieties.

Carcinoma, Squamous Cell↗
PubMed1994

Interactions of plasminogen and fibrinogen with model silica glass surfaces: adsorption from plasma and enzymatic activity studies.

The adsorption of fibrinogen and plasminogen from plasma to silica glass, sulfonated silica glass, and lysine-derivatized silica glass has been investigated. The data indicate that the sulfonated material has a high affinity for both fibrinogen and plasminogen, but that the ratio of plasminogen to fibrinogen is greater on the lysine-derivatized surface. The adsorption data also suggest plasminogen as a possible contributor to the fibrinogen Vroman effect, whereby initially absorbed fibrinogen is displaced from the surface. The plasmin activity of plasminogen adsorbed to the lysine-derivatized silica glass and its sulfonated precursor was assessed by both a chromogenic substrate assay and a radioimmunoassay for the plasmin cleavage product of fibrinogen, the B beta 1-42 peptide. The data indicate that 1) the adsorbed plasminogen is not inherently plasmin-like; 2) the enzymatic activity associated with the bound plasminogen is significantly enhanced on both surfaces in the presence of activator; and 3) in the presence of activator, the plasmin activity per mole of bound plasminogen on the lysinized material is approximately a factor of two greater than on the sulfonated material based on the chromogenic substrate assay, and a factor of four greater based on the B beta 1-42 radioimmunoassay. The lysinized material thus exhibits several properties that are different from its sulfonated precursor. It adsorbs more plasminogen relative to fibrinogen after the Vroman peak, and this adsorbed plasminogen appears to be in a conformation that is more readily activated to plasmin. Once activated, the surface bound plasmin shows enhanced ability to cleave either a low molecular weight chromogenic substrate or a macromolecular substrate.(ABSTRACT TRUNCATED AT 250 WORDS)

Adsorption↗
PubMed1986

Involvement of both heparanase and plasminogen activator in lymphoma cell-mediated degradation of heparan sulfate in the subendothelial extracellular matrix.

The effect of plasminogen on the ability of highly metastatic ESb mouse lymphoma cells to degrade heparan sulfate (HS) in the subendothelial extracellular matrix (ECM) was studied. A metabolically sulfate-labeled ECM was incubated with the lymphoma cells, and labeled degradation products were analyzed by gel filtration on Sepharose 6B. Heparanase-mediated release of low-Mr (0.5 less than Kav less than 0.85) HS cleavage products was stimulated fourfold in the presence of plasminogen. Incubation of plasminogen alone with the ECM resulted in its conversion into plasmin, which released high-Mr (Kav less than 0.33) labeled proteoglycans from the ECM. Heating the ECM (80 degrees C, 1 hr) abolished its ability to convert plasminogen into plasmin, yet plasminogen stimulated, through its activation by the ESb plasminogen activator, heparanase-mediated release of low-Mr HS fragments. Heparin inhibited both the basal and plasminogen-stimulated degradation of HS side chains but not the total amount of labeled material released from the ECM. In contrast, aprotinin inhibited the plasminogen-stimulated release of high- as well as low-Mr material. In the absence of plasminogen, degradation of heated ECM by ESb cells was completely inhibited by aprotinin, but there was only a partial inhibition of the degradation of native ECM and no effect on the degradation of soluble HS proteoglycan. These results demonstrate that proteolytic activity and heparanase participate synergistically in the sequential degradation of ECM HS and that the ESb proteolytic activity is crucial for this degradation when the ECM-associated protease is inactivated. Plasminogen may serve as a source for the proteolytic activity that produces a more accessible substrate to the heparanase.

Animals↗
PubMed1996

Habutobin releases plasminogen activator (U-PA) from bovine pulmonary artery endothelial cells.

Habutobin is a thrombin-like enzyme, contained in the venom of Trimeresurus flavoviridis, which has the strongest toxic effect in cases of habu bite. The present study was undertaken to examine the effect of habutobin on the release of plasminogen activators using cultured endothelial cells of the bovine pulmonary artery. The chemical characteristics of the plasminogen activators released into the conditioned medium were determined by fibrin autography and immunological analysis. A chromogenic substrate (S-2251) microassay was employed for quantitative estimation of the plasminogen activator activity in the conditioned medium and euglobulin fraction derived from the conditioned medium. The levels of plasminogen activator inhibitor released into the conditioned medium were determined by reverse fibrin autography. Fibrin autography revealed that cultured bovine pulmonary artery endothelial cells spontaneously released tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA) into the conditioned medium with no stimulus. Exposure of confluent cultures to 50 nM habutobin, however, induced a time-dependent increase in the level of plasminogen activator activity in both the conditioned medium and euglobulin fraction, and the plasminogen activator activity in the euglobulin fraction at 24 hr was significantly higher than that in the control (P < 0.05). Reverse fibrin autography demonstrated that the lysis-resistant zone of the supernatant of the euglobulin fraction (habutobin exposure) was wider than that in the case of no stimulus. These findings suggest that habutobin induced a time-dependent increase in the levels of plasminogen activator and plasminogen activator inhibitor concomitantly.

Animals↗
PubMed2004

Plasminogen fragmentation and increased production of extracellular matrix-degrading proteinases are associated with serous epithelial ovarian cancer progression.

OBJECTIVE: Elevated levels of proteases are linked to the malignant phenotype in a wide variety of solid tumors. Therefore, the expression of plasminogen, matrix metalloproteinases (MMP-2 and MMP-9), and of the serine protease urokinase-type plasminogen activator (uPA) in serous epithelial carcinoma of the ovary were investigated. METHODS: Plasminogen antigen was analyzed in tissue extracts and in the urine of patients with normal (n = 12), benign (n = 6), borderline (n = 9), and invasive serous tumors (n = 22) by Western immunoblotting using rabbit polyclonal plasminogen and murine monoclonal angiostatin antibodies. In the same tissue extracts, semiquantitative estimates of MMP-2, MMP-9, total MMP activity, and uPA activity were determined using semiquantitative gelatin zymography in the presence or absence of human plasminogen. RESULTS: Bands corresponding to Glu-plasminogen (approximately 92 kDa) and Lys-plasminogen (approximately 86 kDa) were detected in all ovarian tissues and in corresponding urine samples. Densitometric analysis of combined Glu- or Lys-plasminogen levels showed significantly decreased levels in malignant compared to normal tissue. In Grade 3 cancers, there was no evidence of Glu-plasminogen or angiostatin. MMP activity was significantly elevated in both borderline and in Grade 3 ovarian cancer tissues. Increased tissue uPA activity on zymograms was detected only in Grade 3 ovarian cancer tissue. CONCLUSION: These data suggest that proteolytic activity of the plasminogen activation cascade increases in serous epithelial ovarian carcinoma.

Blotting, Western↗
PubMedDate not supplied

Limited fibrin specificity of tissue-type plasminogen activator and its potential link to bleeding.

Plasminogen activators initiate the fibrinolytic process by converting plasminogen to plasmin. Though plasminogen activators are effective in the treatment of thrombotic disorders, bleeding complications are associated with their use. The development of plasminogen activators with greater fibrin specificity was expected to reduce the incidence of bleeding complications; however, this has not occurred. In our rabbit model (a) bleeding from standardized ear incisions induced by tissue-type plasminogen activator (t-PA) is attenuated when fibrinogenolysis is reduced by the coadministration of alpha 2-antiplasmin and (b) when used in doses that produce equivalent thrombolysis, vampire bat plasminogen activator (b-PA), an agent that is more fibrin specific than t-PA, causes less bleeding than t-PA. In addition, we have found that the (DD)E complex formed as a result of degradation of crosslinked fibrin is a potent stimulator of t-PA-induced plasminogen activation but has no effect on b-PA. Fragment X, a high-molecular-weight clottable fibrinogen degradation product, accumulates after treatment with t-PA but not with t-PA given with alpha 2-antiplasmin or with b-PA. These findings suggest that there is a link between plasminogen activator-induced fibrinogenolysis and bleeding, and that the composition of fibrin within hemostatic plugs may influence susceptibility to lysis. Whether these results mean that fibrin-specific plasminogen activators like b-PA will have a better risk-to-benefit profile in humans requires rigorous testing in well-designed clinical trials. However, at the very least, our findings suggest that the development of plasminogen activators that are more fibrin specific than t-PA is a worth-while exercise.

Animals↗
PubMed1996

Regulation of plasminogen receptor expression on monocytoid cells by beta1-integrin-dependent cellular adherence to extracellular matrix proteins.

Plasminogen binding sites function to arm cell surfaces with the proteolytic activity of plasmin, critical for degradation of extracellular matrices. We have assessed the effects of adhesion of the representative monocytoid cell lines, THP-1 and U937, to purified extracellular matrix proteins on their expression of plasminogen receptors. After adhesion to immobilized fibronectin, adherent and nonadherent subpopulations of cells were separated. Plasminogen binding to the nonadherent population of cells increased 3-fold, whereas binding to the adherent population decreased by 60%. These changes were due to differences in the plasminogen binding capacities of the cells, while the affinities of the cells for plasminogen were unchanged. The up-regulation of receptor expression in the nonadherent cell population was: 1) induced rapidly and reversibly, 2) independent of new protein synthesis, 3) required an interaction between adherent and nonadherent cell populations, and 4) associated with an enhanced ability of the cells to promote plasminogen activation and to degrade fibronectin. Other immobilized adhesive proteins, laminin and vitronectin, also supported up-regulation of plasminogen receptors in the nonadherent cells. Carboxypeptidase B treatment eliminated the increment in the plasminogen binding capacity of the nonadherent cells, suggesting that the increase in binding was due to exposure of new carboxyl-terminal lysyl residues on the cell surfaces. Furthermore, both the adherence of the cells and up-regulation of plasminogen binding sites was abolished by beta1-integrin monoclonal antibodies. These results suggest that proteins found in extracellular matrices have the capacity to modulate the expression of plasminogen binding sites, thus regulating local proteolysis and cell migration.

Carboxypeptidase B↗
PubMed2003

Inhibition of plasminogen activation by lipoprotein(a): critical domains in apolipoprotein(a) and mechanism of inhibition on fibrin and degraded fibrin surfaces.

Similarity between the apolipoprotein(a) (apo(a)) moiety of lipoprotein(a) (Lp(a)) and plasminogen suggests a potentially important link between atherosclerosis and thrombosis. Lp(a) may interfere with tissue plasminogen activator (tPA)-mediated plasminogen activation in fibrinolysis, thereby generating a hypercoagulable state in vivo. A fluorescence-based system was employed to study the effect of apo(a) on plasminogen activation in the presence of native fibrin and degraded fibrin cofactors and in the absence of positive feedback reactions catalyzed by plasmin. Human Lp(a) and a physiologically relevant, 17-kringle recombinant apo(a) species exhibited strong inhibition with both cofactors. A variant lacking the protease domain also exhibited strong inhibition, indicating that the apo(a)-plasminogen binding interaction mediated by the apo(a) protease domain does not ultimately inhibit plasminogen activation. A variant in which the strong lysine-binding site in kringle IV type 10 had been abolished exhibited substantially reduced inhibition whereas another lacking the kringle V domain showed no inhibition. Amino-terminal truncation mutants of apo(a) also revealed that additional sequences within kringle IV types 1-4 are required for maximal inhibition. To investigate the inhibition mechanism, the concentrations of plasminogen, cofactor, and a 12-kringle recombinant apo(a) species were systematically varied. Kinetics for both cofactors conformed to a single, equilibrium template model in which apo(a) can interact with all three fibrinolytic components and predicts the formation of ternary (cofactor, tPA, and plasminogen) and quaternary (cofactor, tPA, plasminogen, and apo(a)) catalytic complexes. The latter complex exhibits a reduced turnover number, thereby accounting for inhibition of plasminogen activation in the presence of apo(a)/Lp(a).

Amino Acid Substitution↗
PubMed1997

Factor XIIa activation of plasminogen is enhanced by contact activating surfaces and Zn2+.

The native form of plasminogen is Glu-plasminogen, which by plasmin cleavage gives Lys-plasminogen. Lys-plasminogen is a considerably better substrate compared with Glu-plasminogen for plasminogenolytic enzymes. The contact activation of the intrinsic pathway of coagulation consisting of factor XII, prekallikrein and high Mr kininogen has been implicated to play a role in the intrinsic fibrinolysis. Here activation of Glu- and Lys-plasminogen by factor XIIa in the absence of prekallikrein/kallikrein and high Mr kininogen was studied in a purified system by the generation of amidolytic activity towards pyroGlu-Phe-Lys-pNA (S-2403), a chromogenic substrate of plasmin. A slow activation rate of both Glu- and Lys-plasminogen by factor XIIa was enhanced approximately 60-fold in the presence of Zn2+ and a negatively charged surface. 6-Aminohexanoic acid further enhanced the activation of Glu-plasminogen but inhibited the activation of Lys-plasminogen. The presence of a specific factor XIIa inhibitor completely prevented the generation of plasmin amidolytic activity indicating that activation was mediated by proteolytical cleavage, although this could not be proven by Western-blotting. Physiological concentration of factor XIIa was as more efficient than soluble u-PA to lyse fibrin as a result of activation of Glu-plasminogen. This did not require the presence of Zn2+ or sulfatide.

Aminocaproic Acid↗
PubMed1975

Structural relationship between "glutamic acid" and "lysine" forms of human plasminogen and their interaction with the NH2-terminal activation peptide as studied by affinity chromatography.

Urokinase digestion of maleinated plasminogen results in cleavage of the single peptide bond Arg-68-Met-69, which is one of the bonds normally cleaved during the first step of the activation procedure. The inactive intermediate compound formed in this way was subjected to NH2-terminal amino acid sequence analysis, which clearly demonstrates the structural relationship between the forms of plasminogen with different NH2-terminal amino acids. It is thus shown that lysine-78 and valine-79 in the "glutamic acid" plasminogen actually are the NH2-terminal amino acids in "lysine" and "valine" plasminogen respectively. The forms with glutamic acid in NH2-terminal position are called plasminogen A, while all other forms lacking the NH2-terminal part of the molecule and which can be activated in a single step are called plasminogen B. By affinity chromatographic studies of the NH2-terminal activation peptide on insolubilized plasminogen B, it was demonstrated that this peptide has specific affinity for plasminogen B. It was also shown that this noncovalent interaction is broken by 6-aminohexanoic acid in two concentration. The tryptic heptapeptide (Ala-Phe-Gln-Tyr-His-Ser-Lys) which occupies the positions number 45 to 51 in the NH2-terminal activation peptide (as well as in the intact plasminogen molecule) is importance for the conformational state of the plasminogen molecule.

Amino Acid Sequence↗
PubMed1984

Novel properties of human monocyte plasminogen activator.

Human peripheral monocytes stimulated by either muramyl dipeptide [N-acetyl-muramoyl-L-alanyl-D-isoglutamine], bacterial lipopolysaccharide or lymphokine-containing supernatants of human lymphocytes, could be shown to produce and secrete appreciable activities of a 52 000-Mr plasminogen activator. This enzyme was suppressed in control and stimulated cultures by dexamethasone (0.1 microM). Monocyte plasminogen activator could only be assayed under conditions of low ionic strength and had no detectable activity at 0.15 M NaCl. Intracellular enzyme was present as a proenzyme, requiring activation by preincubation with plasminogen containing traces of plasmin, before its activity could be seen on sodium dodecyl sulphate/polyacrylamide gel electrophoresis by a fibrin overlay method. Secreted enzyme was in the active form. Further incubation of lysate or supernatant plasminogen activator with plasminogen did not produce any active enzyme species of Mr 36 000, unlike incubations of urokinase with plasminogen. Moreover, comparisons with other plasminogen activators of Mr 52 000 from transformed cell lines showed that the monocyte activator was unique in its resistance to monocyte minactivin, a specific inactivator of urokinase-type plasminogen activators, and in its sensitivity to human alpha 2-macroglobulin. It was therefore concluded that human monocyte plasminogen activator, although sharing an Mr of 52 000 in common with other such activators, is not identical to the high Mr form of urokinase or the plasminogen activators of transformed cells. On present evidence it is the least likely of these enzymes to be active extracellularly under normal physiological conditions.

Cells, Cultured↗
PubMed1984

Influence of cyanogen-bromide-digested fibrinogen on the kinetics of plasminogen activation by urokinase.

The catalytic efficiency (kcat/Km) of high-molecular-mass urokinase for the activation of Glu-plasminogen is increased about 10-fold in the presence of CNBr-digested fibrinogen. This stimulation is similar to that observed with 6-aminohexanoic acid, and yields kinetic parameters comparable to those for the activation of Lys-plasminogen by urokinase. The increase of the activation rate of Glu-plasminogen by urokinase in the presence of CNBr-Fg can thus be explained by a conformational change in the plasminogen molecule similar to that observed upon conversion of Glu-plasminogen to Lys-plasminogen and upon binding of 6-aminohexanoic acid to Glu-plasminogen. Stabilization of the Michaelis complex between urokinase and plasminogen by formation of a cyclic ternary complex with CNBr-Fg, which has been invoked to explain the dramatic stimulatory effect of CNBr-Fg on the activation of plasminogen by tissue-type plasminogen activator, does not appear to play a significant role in the increased activation rate.

Cyanogen Bromide↗
PubMed1986

A monoclonal antibody directed against the high-affinity lysine-binding site (LBS) of human plasminogen. Role of LBS in the regulation of fibrinolysis.

One of thirty murine monoclonal antibodies, raised by immunization with human plasmin-alpha 2-antiplasmin complex, was found to be directed against the high-affinity lysine-binding site in plasminogen. Indeed, this antibody (MA-HAL) reacted with plasminogen and with a fragment of plasminogen composed of the first three triple-loop structures (LBS I) and was displaced by 6-aminohexanoic acid (50% displacement at 25 microM). In competitive radioimmunoassays the binding of radiolabeled plasminogen to MA-HAL was reduced to 50% with 2.3 microM alpha 2-antiplasmin or 1.3 microM histidine-rich glycoprotein, which corresponds to the known dissociation constants between these ligands and the high-affinity lysine-binding site of plasminogen. MA-HAL did not influence the activation of plasminogen by tissue-type plasminogen activator in the absence of CNBr-digested fibrinogen, but abolished the effect of CNBr-digested fibrinogen on the Michaelis constant of the reaction. MA-HAL reduced the reaction rate between plasmin and alpha 2-antiplasmin by a factor 20 and abolished the binding of plasminogen to fibrin. These results indicate that MA-HAL specifically binds to and masks the high-affinity lysine-binding site of plasminogen. It therefore is a useful tool for the investigation of the role of this structure in the regulation of fibrinolysis, both at the level of fibrin-stimulated activation of plasminogen and of the inhibition of generated plasmin.

Animals↗
PubMed1990

Receptors for human plasminogen on gram-negative bacteria.

A total of 188 strains representing 11 species of gram-negative bacteria were examined for the ability to interact with human plasminogen. Highly purified human plasminogen was labeled with 125I, and its uptake by different bacterial strains was measured. All 14 strains of Haemophilus influenzae and all 13 strains of Branhamella catarrhalis tested were positive with respect to plasminogen uptake. Also, eight species belonging to the family Enterobacteriaceae were tested, and of those, Proteus mirabilis demonstrated the most substantial uptake, with 28 of 39 strains taking up more than 10% of the plasminogen. Ten strains of Pseudomonas aeruginosa were also tested, of which seven showed uptake values higher than 10%. With H. influenzae and B. catarrhalis strains, Scatchard analysis indicated a two-phase receptor interaction, one more-avid receptor with a Kd of 6 to 8 nM and 2,000 to 2,500 sites per bacterium and a second receptor with a Kd of 50 to 80 nM and 9,000 sites per bacterium. With Pseudomonas aeruginosa strains, a single receptor interaction was detected with a Kd of 60 nM and the number of sites was estimated as 8,000 per bacterium. Scatchard analysis with strains of P. mirabilis indicated binding of a less-specific nature. However, plasminogen uptake by this species could be reduced by 50% by the addition of 2 mM unlabeled plasminogen. This estimate of Kd, as well as uptake studies with plasminogen fragments, suggests different properties of this receptor. With all receptor types, the addition of plasmin-aprotinin complex inhibited plasminogen uptake, which demonstrates that both forms of the molecule react with the same receptors. Plasminogen uptake could be eliminated by the addition of lysine or epsilon-aminocaproic acid, which suggests that the lysine-binding sites of the plasminogen molecule are involved in the receptor-ligand interaction.

Aprotinin↗
PubMed1996

Borrelia burgdorferi induces secretion of pro-urokinase-type plasminogen activator by human monocytes.

Borrelia burgdorferi is transmitted by infected ticks and causes Lyme disease. To infect distant organ sites, B. burgdorferi spirochetes must disseminate from the site of the tick bite. During dissemination from the dermal tissue, they breach tissue barriers, probably by proteolysis. The previous findings that spirochetes bind serum-derived plasminogen and that plasmin favors spirochetal invasiveness and infectivity suggested a role for plasmin in the pathogenicity of B. burgdorferi. Binding of plasminogen to spirochetes and activation into plasmin is favored in a microenvironment that is rich in plasminogen and plasminogen activators. Plasminogen is abundant in plasma and interstitial fluids, and it is increased in inflammatory exudates. Since B. burgdorferi does not express endogenous plasminogen activators, the conversion of spirochete-bound plasminogen depends on host-derived plasminogen activators. In this report, we show that both intact B. burgdorferi organisms and its recombinant outer surface lipoprotein A induce human monocytes to express and secrete urokinase-type plasminogen activator in its zymogen form (pro-uPA). Moreover, we demonstrate that the presence of B. burgdorferi accelerates the interaction between (pro-)uPA and plasmin(ogen), leading to spirochete-bound plasmin. In a pro-uPA-serum mixture, spirochete-bound plasmin activity is generated. Taken together, the data suggest that B. burgdorferi may induce pro-uPA in a monocyte-containing inflammatory site and that the spirochetal surface provides an appropriate milieu for subsequent interactions between (pro-)uPA and plasmin(ogen), which result in spirochete-bound plasmin even in the presence of inhibitors for plasminogen activators and plasmin.

Borrelia burgdorferi Group↗
PubMed1988

Characterization of commercially available plasminogen preparations in vitro: purity and reactivity.

Three different commercially available plasminogen preparations (Immuno-, Kabi-, and Behring-plasminogens) were examined regarding purity and reactivity to different activators (high molecular weight [HMW] or low molecular weight [LMW] two-chain urokinase type plasminogen activator [tcu-PA], single chain urokinase type plasminogen activator [scu-PA], tissue type plasminogen activator [t-PA], and streptokinase [SK]). The Immuno-preparation was a Lys-plasminogen, commercially available for therapeutical use, whereas the research reagents for KabiVitrum and Behringwerke were Glu-plasminogen. Activity data provided by the manufacturers correlated well with our findings. Also a good correlation of reactivity to activators measured with a chromogenic substrate and on fibrin plates could be observed. The Immuno-plasminogen showed a minimum contamination with plasmin which has to be taken into consideration for the interpretation for its apparently higher activation by plasminogen activators compared to the plasmin-free plasminogens. Further in vitro and in vivo research has to be performed to find out criteria for a practicable scheme of administration of fibrinolytic agents for therapeutical thrombolysis.

Electrophoresis, Polyacrylamide Gel↗
PubMed1982

Thrombolysis with human extrinsic (tissue-type) plasminogen activator in dogs with femoral vein thrombosis.

Extrinsic (tissue-type) plasminogen activator (plasminogen activator) was isolated either as a single-chain or as a two-chain molecule from the culture medium of a human melanoma cell line. The thrombolytic activity of both molecular forms of activator was investigated in beagle dogs with an experimental femoral vein thrombosis and compared with that of urokinase. The 125I-fibrinogen-labeled thrombus was formed in an isolated 4-cm segment of the vein, aged for 30 min, and the thrombolytic substances were infused over a 4-h period. The degree of thrombolysis was measured 2 h later as the difference between the injected and recovered 125I. In six control animals with a saline infusion the extent of thrombolysis was 16.3 +/- 3.8% (mean +/- SEM), in five dogs receiving 100,000 IU urokinase, 17.4 +/- 3.7% (P less than 0.4) and in four dogs with 1,000,000 IU urokinase 40.6 +/- 4.8% (P less than 0.001). Infusion of 100.000 IU single-chain plasminogen activator in five dogs resulted in 3.5 +/- 7.8% lysis (P less than 0.05) and of 100,000 IU two-chain plasminogen activator in five dogs in 60.1 +/- 10.8% (P less than 0.001). Infusion of 300,000 IU one-chain plasminogen activator yielded 57.5% lysis and of the same amount of two-chain plasminogen activator 72.9%. Significant activation of plasminogen, consumption of alpha 2-antiplasmin, and fibrinogen breakdown in plasma was only observed in animals receiving the high doses of urokinase but not in the saline, plasminogen activator, or the low-dose urokinase groups. It is thus concluded that in this thrombosis model human extrinsic plasminogen activator has a higher specific thrombolytic effect that urokinase. Plasminogen activator also appears to induce thrombolysis without systemic fibrinolytic activation and fibrinogen breakdown.

Animals↗