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PubMed1989

A rapid and simple method for the separation of four molecular forms of human plasminogen.

At least four molecular forms of plasminogen are known. Two of those forms have glutamic acid at their amino-terminal end, and are designated as glu-plasminogen. The other two have lysine, methionine and/or valine as amino-terminal amino acid and are collectively designated as lys-plasminogen. Two subforms (I and II) each of glu- and lys-plasminogen exist. The I-forms are glycosylated at asn-288 and thr-345, whereas the II-forms are only glycosylated at thr-345. In a previous publication (Thromb Haemostas 1984; 52: 347-349) we have described the separation of the I- and II-forms of plasminogen in lysine-Sepharose in phosphate buffers. Now we have combined those findings with the differential affinity of glu- and lys-plasminogen for aminohexyl-Sepharose through their aminohexyl-sites, recently described by Christensen (Biochem J 1984; 223: 431-421). Acid/urea electrophoresis, end-group determination and carbohydrate analysis show that the combination of affinity chromatography on lysine-Sepharose in phosphate buffers, and on aminohexyl-Sepharose provides an efficient procedure to separate the four molecular forms of plasminogen.

Carbohydrates↗
PubMed1988

Behavior of plasminogen at the luminal surface of the normal and deendothelialized rabbit aorta in vivo and in vitro.

The behavior of purified rabbit plasminogen at the luminal surface of the uninjured and deendothelialized rabbit aorta has been studied in vivo and in vitro. After intravenous injection, 125I-plasminogen associated rapidly with the endothelium (approximately 0.1 pmol/cm2 at saturation) and passed through to accumulate in the subendothelium. At two to 15 hours after injection, 11 to 15 times more radioactivity was associated with the subendothelium than with the endothelium. Removal of the endothelium by balloon catheter led to a rapid adsorption of 125I-plasminogen by the luminal surface of the vessel; saturation (9.1 pmol/cm2) was attained at ten to 20 minutes after deendothelialization. Of the adsorbed plasminogen (radioactivity), only 2% to 4% was associated with the adherent platelet monolayer. Uptake of 125I-plasminogen by the deendothelialized vessel was not significantly inhibited by epsilon-aminohexanoic acid whether injected before or after the 125I-plasminogen. No evidence of plasmin activity at the aorta surface was found from either transmission electron microscopy studies or from amidolytic assays of plasminogen-saturated deendothelialized aorta samples before or after urokinase treatment. Balloon catheter treatment in vivo, however, generated significant antiplasmin activity of the deendothelialized aorta surface. We conclude that plasmin formed in vivo is probably inactivated by the antiplasmin activity that is associated with the subendothelium.

Animals↗
PubMed1987

[Plasminogen-binding centers of molecules of fibrinogen, fibrin and products of their proteolysis].

Using affinity chromatography, the binding of Lys-plasminogen to fibrinogen, fibrin and the consecutively formed products of their proteolysis was studied. The optimal conditions for this binding were elaborated, and the quantitative parameters of Lys-plasminogen binding to fibrinogen-Sepharose were determined. It was found that the interaction of Lys-plasminogen with fibrinogen- and fibrin-Sepharose is provided for by the lysine-binding sites of the proenzyme molecule. After partial hydrolysis of fibrinogen by plasmin, the amount of adsorbed plasminogen increases and the type of binding changes; part of the proenzyme molecules bind in the presence of 0.003 M 6-aminohexanoic acid, i.e., when lysine-binding sites appear to be blocked. A comparative study of plasminogen binding to fibrinogen fragments was carried out. The resistance of the complexes formed to the effect of 6-aminohexanoic acid and arginine competing for the binding sites was determined. The data obtained testify to the appearance of additional plasminogen-binding sites in the fibrinogen molecule during proteolysis. These sites are complementary for both lysine-and arginine-binding sites of the plasminogen molecule and are localized in the peripheral domains of the fibrinogen molecule.

Binding Sites↗
PubMed1987

Plasminogen activators and tumor development in the human colon: activity levels in normal mucosa, adenomatous polyps, and adenocarcinomas.

Malignant changes are often accompanied by alterations in activity and composition of the plasminogen activators (PA). To study the relationship between PA expression and the development of colorectal cancer, we determined urokinase-type plasminogen activator (u-PA) and tissue-type plasminogen activator (t-PA) activity in normal mucosa (n = 80), adenomatous polyps (n = 76), and adenocarcinomas (n = 71) of the colon. Tissues obtained from surgical resection or polypectomy were analyzed for t-PA and u-PA activity in a specific enzymatic assay using plasminogen, a chromogenic substrate, and selective quenching with monospecific antibodies to both activators. The plasminogen activator activities were found to be changed in adenocarcinomas as compared to normal mucosa. The relative contribution of u-PA (expressed as percentage of u-PA) was raised from 6 to 50% for, respectively, normal mucosa and adenocarcinoma. This change could be attributed to a 3-fold decrease in t-PA activity and a 5-fold increase in u-PA activity in the carcinomas. Adenomatous polyps as a group showed percentages of u-PA [20.2 +/- 1.3 (SE)] which were intermediate as well as significantly different (P less than 0.001) from those of normal mucosa and adenocarcinomas. This observation was strengthened by a gradual rise in the relative contribution of u-PA in four resection specimens containing both adenomatous polyps and adenocarcinomas. Zymography showed the presence of minor quantities of PA-PA inhibitor complexes in the tissue extracts studied. The present study shows that the sequence of normal mucosa-adenomatous polyp-adenocarcinoma in the colon is associated with a parallel change in plasminogen activator activity. Thus, change in the regulation of plasminogen activator activity is an early event in the development of colorectal cancer.

Adenocarcinoma↗
PubMed1981

Immunochemical characterization of the kringle 4 fragment of human plasminogen.

Antisera were raised in rabbits against kringle 4, one of the five homologous domains of human plasminogen, and one which contains a site that binds lysine. A double antibody radioimmunoassay was developed for kringle 4 and used for cross-reactivity studies with plasminogen, kringle 1 + 2 + 3, kringle 5 + light chain, and the plasmin-antiplasmin complex, as competitors of either 125I-kringle 4 or 125I-Lys-plasminogen for binding to anti-kringle 4 antisera. Lys- and Glu-plasminogen caused 50% inhibition of anti-kringle 4 binding to 125I-kringle 4 at ratios of 4 and 20 mol/mol (competitor/kringle 4), respectively. The plasmin-antiplasmin complex behaved similarly to Glu-plasminogen. Kringle 1 + 2 + 3 inhibited at a ratio of about greater than 1000 mol/mol. Our results suggest that most of the surface antigenic sites of kringle 4 are exposed on Lys-plasminogen, Glu-plasminogen, and the plasmin-antiplasmin complex. There may be weak antigenic homology between kringle 4 and kringle 1 + 2 + 3, but there is little or no homology with kringle 5 + light chain and prothrombin fragments 1 and 2. Both 6-aminohexanoic acid and trans-4-(aminomethyl)cyclohexanecarboxylic acid can cause a maximum of 30% inhibition, with the latter being a much better inhibitor. Our results suggest that part of the antibody population is directed against a region of kringle 4 containing a lysine-binding site.

Cross Reactions↗
PubMed1995

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).

Binding Sites↗
PubMed1978

Specificity of response in hamster cells induced to produce plasminogen activator by the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate.

A phorbol ester promoting agent, 12-O-tetradecanoylphorbol-13-acetate, enhances plasminogen activator production in hamster cell lines that synthesize plasminogen activator immunologically identical with plasminogen activator of normal hamster lung cells. 12-O-Tetradecanoylphorbol-13-acetate exhibits a high degree of specificity in the type of plasminogen activator evoked, which is always of the lung form. No enhancement of plasminogen activator production was detected in hamster cell lines synthesizing plasminogen activator of different antigenic form, although in the presence of 12-O-tetradecanoylphorbol-13-acetate, cells of one such line initiated synthesis of plasminogen activator of the lung form.

Animals↗
PubMed1994

The specific roles of finger and kringle 2 domains of tissue-type plasminogen activator during in vitro fibrinolysis.

The specific roles of the finger (F) and kringle 2 (K2) domains of tissue-type plasminogen activator (t-PA) were quantified with regard to fibrin binding and kinetic parameters for plasminogen activation by employing domain-deletion variants. On an intact fibrin clot, active site-blocked 125I-t-PA has a dissociation constant (Kd) of 0.36 +/- 0.08 microM and a single binding site per fibrin monomer (n = 1.1 +/- 0.1). Deletion of the K2 domain results in a 3-fold increase of the Kd (1.1 +/- 0.2 microM) and a 2-fold increase of the binding sites per fibrin monomer (n = 2.0 +/- 0.3). Deletion of the F domain results in nonsaturable binding with high Kd (3.2 +/- 0.6 microM). These results indicate that the high affinity binding of t-PA to intact fibrin is the resultant of the cooperation of two low affinity binding sites assembled on intact t-PA. Furthermore, fibrin clot lysis experiments were performed, using polymerized fibrin and plasminogen. Enzymatic activity of t-PA (variants) was assessed by following the decrease in turbidity of the polymerized fibrin. Intact recombinant t-PA exhibited a Michaelis constant for plasminogen activation (Km) of 37 +/- 2 nM. Deletion of either the K2 or F domain results in an increase of the Km for plasminogen of 4- and 16-fold, respectively. We interpret these kinetic parameters in terms of the ternary complex model: binding of t-PA to fibrin, mediated simultaneously by both the F and K2 domain, is essential for a correct orientation of the enzyme on the fibrin polymer to yield the optimal "Km-driven" stimulation of fibrin on the activation of plasminogen. The different potencies of various deletion mutants in a plasma clot are explained by decreased affinity of the enzymes both for fibrin and for the substrate plasminogen.

Binding Sites↗
PubMed1994

Plasminogen mutants activated by thrombin. Potential thrombus-selective thrombolytic agents.

Plasmin, the enzyme responsible for degradation of fibrin in blood clots and thus thrombolysis, is normally formed when its zymogen plasminogen is activated by cleavage of the Arg561-Val562 bond by specific plasminogen activators. We have altered the activation characteristics of plasminogen by substituting the P3, P2, and P1' cleavage site residues with sequences from thrombin-cleavable proteins to produce a novel thrombolytic agent which instead is activated by the blood clotting system. Plasminogen variants with thrombin cleavage sites from fibrinogen, the thrombin receptor, factor XIII, and factor XI were cleaved by thrombin with times to 50% cleavage of 28 h, 2.5 h, 5.7 min, and 3 min, respectively. In vitro clot lysis studies have shown that a variant in which the P3-P1' residues of plasminogen were substituted by the P7-P1' residues (Thr363-Ile370) from factor XI (T51) was sufficiently rapidly cleaved by thrombin to be activated by the endogenous thrombin produced by the coagulation cascade, resulting in rapid clot dissolution. Thrombin-activatable plasminogen therefore has the capacity to short circuit the physiological hemostatic mechanisms and produce fibrinolytic activity localized to the site of thrombin formation, that is, at the thrombus itself. The novel activation mechanism combined with the natural long circulating half-life of plasminogen gives this type of thrombolytic agent the potential for thrombus-selective plasmin generation and an extended duration of action.

Amino Acid Sequence↗
PubMed1996

Isolated familial plasminogen deficiency may not be a risk factor for thrombosis.

Despite many reports of individuals with congenital plasminogen deficiency and thrombosis, there is still uncertainty whether heterozygous deficiency represents a real thrombophilic risk factor or simply a coincidental finding. We have addressed this issue by testing for plasminogen deficiency in a cohort of 9611 blood donors. Out of 66 donors with reduced plasminogen activity on two occasions 28 were shown to have a familial deficiency state (including 3 with dysplasminogenaemia). Our observed prevalence rate for familial plasminogen deficiency, calculated at 2.9/1000 (95% CI = 1.9-4.2 per 1000), was not significantly different from that calculated from published reports of congenital plasminogen deficiency in thrombotic cohorts (5.4/1000). Furthermore, with only two exceptions, all 80 donors and relatives with familial deficiency were asymptomatic with regard to thrombosis-including a 29 year old donor with suspected compound heterozygous hypoplasminogenaemia. These findings add further weight to the argument that familial heterozygous plasminogen deficiency, at least in isolation, does not constitute a significant thrombotic risk factor. However, it remains uncertain whether plasminogen deficiency, when combined with other thrombophilic conditions, may become more clinically important.

Adult↗
PubMed1997

Studies on the effect of human lys-plasminogen in a rat model of global cerebral ischemia.

Human lys-plasminogen and the corresponding formulation buffer were tested in a rat model of global cerebral ischemia (clamping of both carotid arteries, withdrawal of 5 ml blood for 30 min). The two main parameters, tested in different experimental set-ups, were 1. brain edema (water content) 23.5 h after reperfusion and 2. assessment of neurological deficits 24, 48 and 72 h after reperfusion. In some groups of animals of the first set-up, brains were examined histologically for microvascular fibrin deposits. In a separate group of animals the fibrinolytic plasma activity of rats treated with 500 CU/kg lys-plasminogen was studied. Concerning brain water content lys-plasminogen completely antagonized the formation of brain edema when given with 500 caseolytic Units (CU)/kg i.v. with blood reperfusion and was still effective when given 30 min later. 200 CU/kg i.v. given with blood reperfusion as well as 500 CU/kg i.v. given 60 min after blood reperfusion proved ineffective. In none of the brains investigated microvascular fibrin deposits were found. In experiments with assessment of neurological deficits, animals treated with 500 CU/kg lys-plasminogen i.v. showed almost no disabilities (like sham operated animals) when compared to ischemic (positive) controls which were rather severely handicapped. The formulation buffer of lys-plasminogen, tested in an equivalent volume, was without any effect in both set-ups. No fibrinolytic activity was found in plasma samples of rats up to 240 min after treatment with 500 CU/kg lys-plasminogen i.v. It is concluded from these experiments that human lys-plasminogen has a protective effect in rats against the sequelae of global cerebral ischemia which is not related to the well-known fibrinolytic potential but might be a separate quality.

Animals↗
PubMed1996

Difference in expression of the plasminogen activation system in synovial tissue of patients with rheumatoid arthritis and osteoarthritis.

Proteolytic joint destruction in inflammatory and non-inflammatory arthropathy is believed to be mediated, at least in part, by the plasminogen activation (PA) system. To further investigate possible involvement of the PA system, we quantified immunoreactive urokinase-type plasminogen activator (u-PA), tissue-type plasminogen activator (t-PA), both plasminogen activator inhibitors (PAI-1 and PAI-2) and u-PA-receptor (u-PAR) in synovial tissue extracts of 14 patients with rheumatoid arthritis (RA) and 12 with osteoarthritis (OA). u-PA, PAI-1, PAI-2 and u-PAR concentrations were significantly higher in RA than in OA patients. t-PA antigen levels were significantly lower in RA than in OA synovial tissue extracts. Immunohistochemistry was performed to compare the distribution and staining intensity of these components in samples of RA and OA synovial tissue. Intense immunostaining of u-PA, u-PAR, PAI-1 and, to a lesser degree, PAI-2 was observed predominantly in the synovial lining of RA patients. In OA patients, u-PA, PAI-1, PAI-2 and u-PAR were barely detectable. t-PA immunostaining was restricted to the endothelial side of vascular walls in both groups. We conclude that the observed increase of u-PA, u-PAR and PAI expression, distributed mainly in the synovial lining area of proliferative and invasively growing synovial tissue in RA patients, supports a pathogenic role for the PA system in destructive arthritis. Depressed t-PA-mediated plasminogen activation might contribute to delayed intra-articular fibrin removal.

Arthritis, Rheumatoid↗
PubMed2000

Angiostatin generation by human tumor cell lines: involvement of plasminogen activators.

Angiostatin is a tumor-derived angiogenesis inhibitor consisting of an internal fragment of plasminogen. Little is known about the production of angiostatin by human tumors. In this study, we examined the in vitro angiostatin-generating capacities of a panel of human tumor cell lines (total n = 75) and the proteolytic molecule(s) involved. Angiostatin formation was determined by assessing the level of plasminogen digestion in conditioned medium by Western-blot analysis. We found that the capacity to produce angiostatin is a common feature of many cell lines, depending on the tumor type. All 6 bladder-carcinoma and 6 out of 7 prostate-carcinoma cell lines showed intermediate to potent angiostatin-generating activity. In contrast, only 2 out of 7 colon-carcinoma and 2 out of 9 renal-cell carcinoma cell lines were able to generate angiostatin at intermediate levels. Out of 25 melanoma cell lines, only one line failed to generate angiostatin. In the other cell-line groups (cervix, breast and ovary), angiostatin formation varied. Remarkably, angiostatin bands were not of equal size in all plasminogen digests. Since reported data have indicated that plasminogen activators (uPA and tPA) were able to excise the angiostatin fragment from the plasminogen parent molecule via plasmin generation, we determined levels of uPA and tPA and PAI-1 antigen in the conditioned media, and correlated the results with angiostatin-generating capacity. Whereas prostate- and bladder-carcinoma lines capable of generating high levels of angiostatin showed high uPA levels, angiostatin generation in melanoma cell lines was correlated with tPA levels. Generally, angiostatin non-producers did not express uPA or tPA. In 6 out of 75 cell lines, however, we found angiostatin generation combined with low or absent levels of plasminogen activator, suggesting the involvement of alternative proteolytic pathways in the generation of angiostatin.

Angiostatins↗
PubMed1993

Extracellular matrix produced by cultured corneal and aortic endothelial cells contains active tissue-type and urokinase-type plasminogen activators.

Incubation of plasminogen with the subendothelial extracellular matrix (ECM) synthesized by cultured bovine corneal and aortic endothelial cells resulted in generation of fibrinolytic activity, indicated by proteolysis of 125I-fibrin in a time- and dose-dependent manner. Both tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA) were identified in the ECM by fibrin zymography, immunoblotting, and inhibition of plasminogen activation by anti-u-PA and anti-t-PA antibodies. Most of the ECM-resident plasminogen activator (PA) activity did not originate from intracellular PA release occurring when the endothelial cells were lyzed and the ECM exposed, since a comparable amount of PA was associated with the ECM when the cells were lyzed with Triton X-100 or removed intact by treatment with 2 M urea. Active u-PA and t-PA were released from ECM by treatment with heparanase (endo-beta-D-glucuronidase), indicating that some of the ECM-resident PA activity is sequestered by heparan sulfate side chains. These results indicate that both u-PA and t-PA produced by endothelial cells are firmly sequestered in an active form by the subendothelial ECM. It is suggested that ECM-resident plasminogen activators participate in sequential matrix degradation during cell invasion and tumor metastasis. PA activity may also function in release of ECM-bound growth factors (i.e., basic fibroblast growth factor) and activation of proenzymes (i.e., prothrombin), resulting in modulation of the ECM growth-promoting and thrombogenic properties.

Absorption↗
PubMed1988

Placental-type plasminogen activator inhibitor in preeclampsia.

The present cross-sectional study in patients with preeclampsia and gestational hypertension and in gestational age-matched controls was undertaken to investigate further the fibrinolytic system in these conditions. In preeclampsia we observed increased levels of total plasminogen activator inhibitor (p less than 0.001) but low levels of placental-type plasminogen activator inhibitor (p less than 0.05) compared with controls. The levels of placental-type plasminogen activator inhibitor were even more reduced (p less than 0.002) in pregnancies with a poor fetal outcome. It is concluded that placental-type plasminogen activator inhibitor does not contribute to the increased levels of total plasminogen activator inhibitor activity in preeclampsia. Placental-type plasminogen activator inhibitor levels correlated significantly with birth weight and placenta weight and may therefore reflect placental function.

Adult↗
PubMed1994

Inhibition of tissue plasminogen activators and urokinase by human saliva.

The fibrinolytic activity found in body fluids is mediated through the generation of plasmin from plasminogen by plasminogen activator. Mixed fresh human saliva was found to have plasminogen activator inhibitory activity that has not yet been characterized. In the present study, mixed fresh human saliva was found to contain an inhibitor for tissue plasminogen activator and for urokinase. The plasminogen activator inhibitory activity was mainly found in the mixed fresh human saliva cellular pellet containing buccal epithelial cells and not in the supernatant salivary fluid. Saliva collected by Stenson's duct cannulation does not have any PA-inhibitor activity, although saliva collected from submandibular glands contains small amounts of such activity. The salivary plasminogen activator inhibitor is unstable, and its activity is lost during storage.

Adult↗
PubMed1985

A new function for activated protein C: activated protein C prevents inhibition of plasminogen activators by releasate from mononuclear leukocytes--platelet suspensions stimulated by phorbol diester.

Mononuclear leukocytes release an inhibitor of plasminogen activators. Mononuclear leukocyte mixtures (400 to 1,000/mm3) lysed fibrin (8.3 microM) clots in the presence of plasminogen (0.58 microM). Anti-urokinase IgG (0.16 microM) inhibited this fibrinolysis. 2-Deoxyglucose (5 mM) and oligomycin (2.3 microM) also inhibited fibrinolysis. Incubation of mononuclear leukocytes (3,200/microliter) with phorbol-12 myristate 13-acetate (20 nM) for ten minutes at 37 degrees C aggregated the monocyte and platelet components and inhibited fibrinolysis. The releasate from these stimulated cells in dilutions ranging from undiluted to 1:16 inhibited urokinase (1.6 pM) and tissue plasminogen activator (1.4 pM). This releasate did not inhibit plasmin (2.5 nM). Incubation of this releasate with activated protein C (33 nM to 333 nM) for ten minutes at 37 degrees C before addition of either urokinase, or tissue plasminogen activator and plasminogen completely prevented this inhibition. Thrombin, factor Xa, DIP-activated protein C had no affect on this inhibition. We conclude that activated protein C facilitates fibrinolysis by preventing inhibition of plasminogen activators. This may be a mechanism by which activated protein C increases fibrinolytic activity in vivo.

Blood Platelets↗
PubMed1995

Characterization of a plasminogen activator produced by Acanthamoeba castellanii.

Serine proteases play an important role in a diverse array of biological processes, including embryogenesis, metastasis, angiogenesis, thrombolysis and tissue invasion by certain parasites. The latter observation prompted us to explore the possibility that the tissue-invasive ocular parasite Acanthamoeba castellanii elaborates one or more serine proteases. Acanthamoeba sp. are pathogenic free-living amoebae that can produce an invasive, blinding inflammatory disease of the cornea, termed Acanthamoeba keratitis. The present study reports the preliminary purification and characterization of a novel plasminogen activator from an ocular isolate of A. castellanii. The parasite-derived enzyme has a molecular mass of approx. 40 kDa and produces a single band of lysis on fibrinogen-agarose zymographs. Activity of the enzyme is completely inhibited by treatment with diisopropylfluorophosphate, indicating that it is a serine protease. The parasite-derived serine protease is not inhibited by amiloride which is a strong inhibitor of urokinase-type plasminogen activator. Additionally, the enzyme is not inhibited by plasminogen activator inhibitor-1 which is the primary physiological inhibitor of both urokinase and tissue-type plasminogen activator. It does not cross-react with antibodies specific for human urokinase or tissue-type plasminogen activator. The parasite-derived enzyme activates plasminogen from several mammalian species, including human, cow and pig. Thus, it is possible that this parasite-derived serine protease contributes to the pathogenesis of Acanthamoeba keratitis.

Acanthamoeba↗