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PubMed2002

Antibiotic A10255 (thioplabin) enhances fibrin binding and activation of plasminogen.

Three thiopeptide metabolites that enhance fibrin binding of plasminogen were isolated from a culture of Streptomyces sp. R1401. A combination of spectroscopic analyses revealed that these compounds were identical with the antibiotic A10255B, E and G. These agents enhanced fibrin binding of plasminogen and plasminogen/urokinase-mediated fibirinolysis at concentrations of 5 to approximately 20 microM. A10255B reversibily increased urokinase-catalyzed activation of plasminogen by lowering Km, while the agent did not enhance urokinase activity when substrates other than plasminogen were used, indicating that the agent affects plasminogen to increase its affinity to urokinase. A smaller but significant increase in activation was also observed when conformationally relaxed plasminogen derivatives such as Lys-plasminogen and mini-plasminogen were used. Two related thiopeptide antibiotics with a C-terminal amide had no effect on plasminogen activation, suggesting a role of the terminal carboxyl of the A10255 molecule in activity.

Anti-Bacterial Agents↗

PubMed2002

Soluble tissue actor interferes with angiostatin-mediated inhibition of endothelial cell proliferation by lysine-specific interaction with plasminogen kringle domains.

Experimental and clinical data suggest that tissue factor (TF), the major initiator of blood coagulation cascade, as well as proteases and components of the fibrinolytic system are involved in tumor growth at least in some solid tumors via effects on angiogenesis. Whereas the pro- and anti-angiogenic effects of the plasminogen/plasmin system and plasminogen kringle domains, respectively, are well characterized, the pathways responsible for the pro-angiogenic properties of TF remain poorly understood. To learn more about the biological significance of the recently described binding of plasminogen to the extracellular domain of TF, we examined the effects of soluble TF (sTF) on angiostatin-inhibited proliferation of endothelial cells. In solid phase binding assays, we found that sTF binds specifically to plasminogen, to the plasminogen kringle domains K1-3, K1-5, K4, as well as to mini-plasminogen. Inhibition of binding of plasminogen and its kringle domains to sTF by the lysine analog 6-aminohexanoic acid (AHA) suggests that lysine-binding sites are involved in plasminogen interaction with TF. Moreover, in the presence of sTF, the inhibitory effect of K1-5 on bFGF-mediated HUVEC proliferation was dose-dependently and saturably abolished. This suggests that TF can interfere with the antagonistic effect of K1-5 on endothelial cell proliferation. In contrast, sTF by itself had no effect on the endothelial cell proliferation. Whereas the interference of TF with K1-5-mediated effect was prevented by AHA, this lysine analog did not abolish the proliferation inhibition of K1-5. In conclusion, the binding of sTF to the plasminogen fragment K1-5 seems to antagonize the anti-angiogenic effects of this plasminogen fragment.

Angiostatins↗

PubMed1990

The role of fragment X polymers in the fibrin enhancement of tissue plasminogen activator-catalyzed plasmin formation.

When thrombin-mediated fibrin formation and tissue plasminogen activator (t-PA)-mediated fibrinolysis proceed in dynamic interaction, desA-(desB beta 1-42)-fragment X polymers are shown to be the predominant fibrin derivatives present during the rapid second phase of Glu1- and Lys78-plasminogen activation. To further investigate the effect of this intermediate, a method was developed for the production and purification of fibrinogen-derived desA-(desB beta 1-42)-fragment X, deprived of both COOH-terminal A alpha-chains, but still capable of thrombin-mediated polymerization. DesA-(desB beta 1-42)-fragment X polymer was compared to intact fibrin with regard to its stimulatory effect on Glu1-, Lys78-, and Val443-plasminogen activation, and its binding of Glu1- and Lys78-plasminogen. Pure fragment X polymer gave rise to a biphasic activation pattern like that of fibrin, demonstrating similar kinetics of rapid phase activation. The dissociation constant for the binding of plasminogen to the effector decreases by a factor of 14, and the stoichiometry increases by a factor of 2 upon plasmin-catalyzed cleavage of both native Glu1- to Lys78-plasminogen, and fibrin to fragment X polymer. We conclude that desA-fibrin protofibril formation is sufficient to initiate fibrin enhancement of t-PA-catalyzed plasminogen activation, and that optimal stimulation depends on further plasmin-mediated modification of the fibrin effector to desA-fragment X-related moieties. Optimal stimulation is dependent on the presence of the kringle 1-4 domains of plasminogen and probably results from altered and increased binding of both plasminogen and t-PA to the modified effector.

Amino Acid Sequence↗

PubMedDate not supplied

[Plasminogen depletion and incorporation in experimental venous thrombosis].

The objective of this piece of work is to study (using animals): venous thrombosis, the depletion and incorporation of plasminogen in thrombus of different stages of time, in order to devise better guide lines for the treatment of thrombosis than the conventional treatment. The studies relating to the plasminogen content of the thrombus show us that, on experimental venous thrombosis, as from the fifth day after which it occurred, the content of the plasminogen thrombus is reduced considerably. This data underlines the fact some venous thrombosis of over five days are resistant to conventional thrombolytic treatment. In relation to this we have to report that the mean lifespan according to our experiments of the plasminogen on beagle dogs is approximately sixteen hours. On the other hand the study of the addition of 125Lis-plasminogen, on experimental thrombosis of different stages of oldness show us that the above addition results in a 54% intake into the thrombus on three day, old thrombosis and 17% on five day old thrombosis. In conclusion we can suggest that the resistance to conventional thrombolytic treatment of old venous thrombosis (5-7 days old) is probably due to a reduction of efficiency of the thrombus plasminogen. At the same time the contribution of exogenous lis-plasminogen does not produce a sufficient enrichment of the thrombus in plasminogen. Therefore the therapeutic exogenous plasminogen addition is not advisable for relatively old thrombosis (less than or equal to 5 days old).

Animals↗

PubMed1989

Plasminogen interactions with immobilized fibrinogen.

This report describes the binding of plasminogen to fibrinogen adsorbed onto polystyrene wells. Binding was determined by enzyme linked immunosorbent assay. Both glu- and lys-plasminogen bound to immobilized fibrinogen in a dose-dependent fashion. However, more lys- than glu-plasminogen bound when equal concentrations of either were added to immobilized fibrinogen. Plasminogen binding was inhibited by epsilon aminocaproic acid indicating that binding was mediated via lysine-binding regions of plasminogen. Soluble fibrinogen added in excess of immobilized fibrinogen did not compete for plasminogen binding but fibrinogen fragments produced by plasmin digestion of fibrinogen did. Treatment of immobilized fibrinogen with thrombin caused a small but significant (p less than 0.01) increase in plasminogen binding. These studies demonstrate that immobilized fibrinogen binds both glu- and lys-plasminogen and that binding is mediated via lysine-binding regions. These interactions may facilitate plasminogen binding to fibrinogen adsorbed on to surfaces and to cells such as platelets which bind fibrinogen.

Aminocaproic Acid↗

PubMed1986

Investigation of a congenital abnormal plasminogen, Frankfurt I, and its relationship to thrombosis.

A new abnormal plasminogen, Frankfurt I, has been identified in the plasma of a 42 year-old male patient who has recurrent deep vein thrombosis. Clinical laboratory data showed normal hemostasis test results. Since plasma plasmin generation rates gave low values, the fibrinolytic system was analyzed for a possible fibrinolytic system defect. Functional and antigen plasminogen concentrations both in the plasma and with the isolated, purified plasminogen showed that only 49% of the antigen concentration had potential functional active sites. Also, a reduced antigen concentration was found in both the propositus, and his mother (46% active sites). Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the purified Frankfurt I plasminogen showed a normal native Glu-plasminogen band. Crossed-immunoelectrophoresis revealed a peak with normal size and shape, but displaced with respect to normal Glu-plasminogen toward the anode, i.e., was, as a whole, more negatively charged. Isoelectric focusing followed by zymography on a agarose-fibrin plate proved this observation, but did not indicate a separation of the normal from the abnormal plasminogen molecular species, also, fewer bands were found in the abnormal plasminogen isozyme pattern. Kinetic studies of Frankfurt I Glu-plasminogen and plasmin led to the conclusion that most of the functional abnormality is related to absence of active sites in half of the molecules. The plasmin generated was very unstable in the absence of stabilizing ligands and/or substrates. After reduction, the plasmin was completely converted to the typical two plasmin chains, A and B.

Adult↗

PubMed1987

Plasminogen activator in synovial fluid from patients with rheumatoid arthritis.

The plasminogen activator in synovial fluid from patients with rheumatoid arthritis (RA) and osteoarthritis (OA) was analyzed on a molecular basis. The level of plasminogen activator in RA was found to be higher than in OA. The plaminogen activators of both RA and OA revealed 3 different molecular weights: 90,000, 55,000 and 33,000. RA demonstrated the 3 plasminogen activators in broadly comparable ratios, but OA had the 55,000 form dominantly. The 90,000 plasminogen activator was a tissue-type plasminogen activator, while the 55,000 and 33,000 plasminogen activators were of the urokinase-type. beta-Methasone suppressed the tissue-type plasminogen activator, and urinary trypsin inhibitor suppressed the urokinase-type plasminogen activators. When urinary trypsin inhibitor was injected clinically into the joint space of a patient with RA, the urokinase-type plasminogen inhibitor was suppressed as in the in vitro study, and the clinical signs and symptoms were markedly improved. Open trials of intraarticular injections of urinary trypsin inhibitor demonstrated improvement of the clinical signs and symptoms.

Adult↗

PubMed1983

Extrinsic plasminogen activator: a new principle in fibrinolysis.

Fibrinolysis in the blood seems to be regulated by specific molecular interactions between plasminogen activator, plasmin(ogen), fibrin and alpha 2-antiplasmin. Plasmin(ogen) contains structures, called lysine-binding sites, which mediate its interaction with fibrin and with alpha 2-antiplasmin. In plasma normally no systemic plasminogen activation by plasminogen activator occurs and plasmin, if formed, is efficiently neutralized by alpha 2-antiplasmin. When fibrin is formed in plasma a small amount of plasminogen is bound via its lysine-binding sites. Plasminogen activator present or released in the blood is strongly adsorbed to the fibrin and activates bound plasminogen in situ. The formed plasmin, which remains transiently complexed to fibrin, both by its lysine-binding site(s) and active center, is only slowly inactivated by alpha 2-antiplasmin, while plasmin which is released from digested fibrin is rapidly and irreversibly neutralized. The fibrinolytic process thus seems to be triggered by and confined to fibrin. An important consequence of this molecular model for fibrinolysis is that specific thrombolysis is only expected with the use of a specific activator, like the physiological extrinsic plasminogen activator, which confines the activation of plasminogen to the fibrin surface. Recent in vitro and in vivo studies have confirmed that the extrinsic plasminogen activator (tissue-type) might constitute a superior thrombolytic agent compared to urokinase or streptokinase.

Antifibrinolytic Agents↗

PubMed1981

Purification and characterization of the plasminogen activator secreted by human melanoma cells in culture.

The plasminogen activator secreted by a cultured human melanoma cell line was purified and compared with urokinase and with tissue plasminogen activator from human uterus. The purification procedure consisted of chromatography on zinc chelate-agarose, concanavalin A-agarose, and Sephadex G-150 in the presence of 0.01% (v/v) Tween 80. The purified material was obtained from the culture medium with a yield of 46% and a purification factor of 263. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed one main band with a molecular weight of about 72,000, and in the presence of reducing agents, two bands of 33,000 and 39,000. Addition of the protease inhibitor Aprotinin to the culture media and column buffers yielded a one-chain plasminogen activator with a molecular weight of about 72,000. One molecule of activator reacted with about one molecular of [3H]diisopropylfluorophosphate. The melanoma plasminogen activator and the uterine tissue plasminogen activator appeared to be very similar on dodecyl sulfate-polyacrylamide gel electrophoresis, amino acid analysis, and amidolytic properties. Both activators bound to fibrin clots, while urokinase did not. In immunodiffusion, as well as in quenching experiments of the fibrinolytic activities, the melanoma plasminogen activator appeared to be immunologically identical with the uterine tissue plasminogen activator, but unrelated to urokinase. All these findings indicate that the plasminogen activator secreted by human melanoma cells in culture is very similar to, or identical with, the plasminogen activator found in normal tissue, but different from urokinase.

Amino Acids↗

PubMed1993

On the mechanism of the activation of human plasminogen by recombinant staphylokinase.

The mechanism of activation of human plasminogen by recombinant staphylokinase (STAR) was studied using the active site titrant p-nitrophenyl-p'-guanidinobenzoate (NPGB). NPGB prevented active site exposure in equimolar mixtures of plasminogen and STAR but reacted stoichiometrically with mixtures preincubated in the absence of titrant. Active site generation occurred progressively, with a marked initial lag phase followed by an exponential growth phase, and was associated with the conversion of single-chain plasminogen to two-chain plasmin. Incubation of mixtures of plasminogen and STAR with catalytic amounts (< 0.2% molar ratio) of preformed plasmin.STAR complex or of urokinase shortened the lag hase, whereas catalytic amounts (5% molar ratio) of the plasmin inhibitor alpha 2-antiplasmin delayed active site generation. The following kinetic model for the activation of plasminogen (P) by STAR (S) fits the experimental data, [formula: see text] and is described by [formula: see text] or [formula: see text] In this model, plasminogen and STAR produce an inactive complex (P.S), in which active plasmin.STAR (p.S) is generated in a rate limiting step, which is accelerated by plasminogen activators and delayed by plasmin inhibitors. At room temperature in a 0.1 M Veronal buffer, pH 8.3, containing 0.1 M arginine, the data are adequately fitted by the integrated equation with k1 = 4.0 x 10(-7) s-1 and k2 = 1.3 x 10(-2) microM-1 s-1. The k1 value could be explained by contamination of the plasminogen preparation with 3 ppm plasmin, converted by S to p.S. It is concluded that STAR activates plasminogen via a mechanism which differs in several essential aspects from that of streptokinase.

Amino Acid Sequence↗

PubMed1997

Regulation of plasminogen gene expression by interleukin-6.

Plasmin, the primary fibrinolytic enzyme, has a broad substrate spectrum and participates in other biological processes dependent upon proteolytic activity. Consequently, plasmin activity is tightly regulated by plasminogen activators and protease inhibitors. In this study, we examined whether regulation of plasminogen gene expression also might provide a new mechanism for controlling this system. We examined the effects of recombinant human interleukin-6 (rhIL-6), a pleiotropic cytokine, on plasminogen mRNA expression in primary murine hepatocytes and Hep3B human hepatoma cells. In primary hepatocytes, rhIL-6 and hydrocortisone separately increased plasminogen mRNA expression, but hydrocortisone did not markedly enhance the response to rhIL-6. Hep3B hepatoma cells exhibited more modest responses to rhIL-6. We used the polymerase chain reaction to amplify a 1,067-bp fragment of the human plasminogen promoter/5' flanking region. This fragment was cloned upstream of a luciferase reporter gene. Hep3B cells transiently transfected with this construct provided approximately 100-fold higher luciferase activity compared to cells transfected with control plasmids, and luciferase activity was increased approximately 4.5-fold when these cells were treated with rhIL-6. Furthermore, mice injected with rhIL-6 exhibited increases in hepatic plasminogen mRNA. Circulating plasminogen levels were significantly higher in the mice injected with rhIL-6 compared to mice injected with saline. Mice injected with lipopolysaccharide (an inducer of IL-6 in vivo) also showed increased hepatic plasminogen mRNA. Thus, plasminogen gene expression can be modulated by rhIL-6, suggesting a new mechanism for regulating biological systems that use plasmin.

Animals↗

PubMed1999

Urokinase regulates embryonic cardiac cushion cell migration without converting plasminogen.

Urokinase-type plasminogen activator (uPA) activation of plasminogen is an important mediator of cell migration in many cell types. In the developing avian heart, uPA has been implicated as a mediator of atrioventricular (AV) cushion cell migration; however, the role of the plasminogen/plasmin system has not been examined. The purpose of this study was to test the hypothesis that uPA conversion of plasminogen to plasmin mediates AV cushion cell migration in vitro. Stage 17/18 chicken atrioventricular tissue lysates converted plasminogen into plasmin through uPA activity but no tissue-type plasminogen activator activity was detected. Zymograms on living cultured AV explants also activated plasminogen producing plasmin that degraded extracellular protein. The migratory capacity of cushion cells was assessed in the presence or absence of various test reagents known to alter the plasminogen/plasmin system. Addition of either human or chicken plasminogen or aprotinin (an inhibitor of plasmin) had no effect on cell migration. However, an anti-catalytic uPA antibody that blocked AV uPA activity, significantly decreased cell migration at all concentrations tested. These results showed that uPA mediated a portion of cushion cell migration in vitro. Although AV segments activated plasminogen and degraded extracellular proteins, uPA's functional role in cushion cell migration did not involve the plasminogen/plasmin system.

Animals↗

PubMed1993

Actin stimulates plasmin generation by tissue and urokinase-type plasminogen activators.

The experiments reported here were carried out to define in greater detail actin's stimulation of plasmin generation by t-PA. Actin did not alter t-PA's hydrolysis of a synthetic substrate, and thus is unlikely to have a direct effect upon t-PA's proteolytic activity. When studied in a single-stage assay, actin accelerated t-PA-mediated plasmin generation from both Glu-plasminogen and Lys-plasminogen, indicating the central role of ternary complex formation. Although actin does not appear to bind two-chain urokinase (tcu-PA), it stimulates tcu-PA's cleavage of Glu-plasminogen. This finding suggests that actin alters the conformation of Glu-plasminogen to an open form. The failure of actin to increased plasmin generation by tcu-PA acting on Lys-plasminogen, which is in an open configuration, is consistent with this interpretation. Immunoglobin G, which shares with actin the property of binding to Glu-plasminogen after nicking by plasmin, did not stimulate tcu-PA's cleavage of Glu-plasminogen, indicating the uniqueness of actin's effects and suggesting interactions between actin and plasminogen at multiple binding sites. Unlike fibrin and heparin, whose stimulation of t-PA is related to polymer length actin is able to stimulate t-PA when presented in either a monomeric or polymeric form. Denaturation of actin by exposure to urea and guanidine increased its ability to stimulate plasmin generation by t-PA. Because actin's structure is maintained by a noncovalently bound adenine nucleotide (ATP or ADP), exposure to ATP/ADPases found in plasma and on cell membranes might also result in its denaturation. Actin treated with an enzyme functionally similar to such ecto-ATP/ADPases, potato apyrase, was more potent than native actin in stimulating plasmin generation by t-PA. The effects of apyrase were blocked by the addition of the plasma actin-binding proteins, gelsolin and the vitamin D-binding protein (DBP). Thus, denaturation of actin may occur in under physiologic conditions, with potential biological consequences. Actin thus appears to be unique with regard to its interactions with the fibrinolytic system and plasma actin-binding proteins may serve to protect the host from the effects of denatured actin.

Actins↗

PubMed1992

Generation of cell surface-bound plasmin by cell-associated urokinase-type or secreted tissue-type plasminogen activator: a key event in melanoma cell invasiveness in vitro.

Recently, we have shown that plasminogen activators (PAs) of both types, urokinase-type (uPA) as well as tissue-type (tPA), are involved in the in vitro invasiveness of human melanoma cells. The present study is focused on the generation and importance of cell surface-bound plasmin in this process. The human melanoma cell lines MelJuso and MeWo expressed plasminogen binding sites on the cell surface. Plasminogen binding was saturable and not species-specific, since human and bovine plasminogen bound to the cells with comparable efficiency. The activation of the proenzyme plasminogen bound on MelJuso cells, which expressed surface-associated uPA activity, occurred almost synchronously with binding to the cell surface. Removal of cell-associated uPA considerably reduced plasmin generation on these cells. In contrast, plasminogen activation on MeWo cells, which secreted tPA into the culture supernatant and which were devoid of surface-associated PA activity, was by far less effective. The efficiency of the activation process could be increased by addition of exogenous tPA. With both cell lines, plasmin generation on the cell surface was suppressed by inhibitory monoclonal antibodies specific for the respective PA type. Selective inhibition of cell surface-associated plasmin by preincubating the cells with an inhibitory monoclonal antibody or with aprotinin, as well as removal of plasmin from the cell surface, led to a significant decrease in cellular invasiveness of both cell lines into various biological substrates such as fibrin gel, the basement membrane extract Matrigel, or intact extracellular matrix. Both cell lines were able to penetrate an intact cell layer of the human keratinocyte line HaCaT, a process, which also proved to be dependent on cell-associated plasmin. In conclusion, these data provide evidence that plasminogen activation associated with the surface of human melanoma cells is catalyzed much more efficiently by cell-associated uPA (MelJuso) than by secreted tPA (MeWo). Cell-associated plasmin, which is protected from inactivation by serum inhibitors, represents the essential component of the proteolytic cascade of plasminogen activation during in vitro invasiveness of human melanoma cells.

Animals↗

PubMed2005

Stimulation of cell surface plasminogen activation by membrane-bound melanotransferrin: a key phenomenon for cell invasion.

The activation of plasminogen at the cell surface is a crucial step in cell migration and invasion. In the present study, the effect of membrane-bound melanotransferrin (mMTf), also known as human melanoma antigen p97, on cell surface plasminogen binding and activation was investigated by using Chinese Hamster Ovary (CHO) cells transfected with full-length melanotransferrin (MTf) cDNA and SK-MeL-28 melanoma cells. The expression of mMTf in CHO increased cell surface plasminogen binding by about 2-fold. In addition, application of the monoclonal antibody L235 against MTf as well as truncated, soluble MTf (sMTf) abolished plasminogen binding to MTf-transfected and SK-MeL-28 cells, indicating that mMTf is a potential cell surface plasminogen receptor. Moreover, mMTf expression in CHO cells stimulates plasminogen activation at the cell surface by about 2.5-fold. In addition to the induced binding and activation of plasminogen, cell motility, migration and invasion were about 3-fold higher in CHO cells expressing mMTf. Both monoclonal antibody L235 and truncated sMTf inhibited mMTf-stimulated CHO cell motility, migration and invasion. Overall, our results indicate a key role for mMTf in cell surface plasminogen binding and in activation processes involved during cell migration and invasion.

Animals↗

PubMed2005

The plasminogen activator/plasmin system is essential for development of the joint inflammatory phase of collagen type II-induced arthritis.

The plasminogen activator (PA) system has been proposed to have important roles in rheumatoid arthritis. Here we have used the autoimmune collagen type II (CII)-induced arthritis (CIA) model and mice deficient for urokinase-type PA (uPA) or plasminogen to investigate the role of the PA system for development of arthritis. Our data revealed that uPA-deficient mice have a lower severity and incidence of CIA than wild-type mice. Furthermore, although >80% of wild-type control mice developed CIA, we found that none of the 50 plasminogen-deficient littermates that were tested developed CIA within a 40-day period. Antibody generation after CII immunization as well as the binding of labeled anti-CII antibodies to the surface of cartilage were similar in wild-type and plasminogen-deficient mice. No sign of inflammation was seen when plasminogen-deficient mice were injected with a mixture of monoclonal antibodies against CII. However, after daily injections of human plasminogen, these mice developed arthritis within 5 days. Our finding that infiltration of inflammatory cells into the synovial joints was impaired in plasminogen-deficient mice suggests that uPA and plasminogen are important mediators of joint inflammation. Active plasmin is therefore essential for the induction of pathological inflammatory joint destruction in CIA.

Animals↗

PubMed1992

Requirement of zymogen modification for activation of porcine plasminogen.

In physiological salt solutions, porcine plasminogen is refractory to activation by urokinase or trypsin and to proteolysis at Lys77 by plasmin or trypsin. Plasminogen becomes a substrate for urokinase (at Arg560), plasmin (at Lys77), and trypsin (at both bonds) if chloride ion is removed or if 6-aminohexanoate (2.5 mmol/L) is added. Irrespective of salts, activation of des(1-77)plasminogen is as efficient as activation of des(kringle1-4)plasminogen and is inhibited 50% by 2.5 mmol/L 6-aminohexanoate. In solutions lacking chloride or containing 6-aminohexanoate, plasminogen, des(1-77)plasminogen, and des(kringle1-4)plasminogen show no tendency to saturate urokinase in physiologically relevant concentrations (10 mumol/L). The findings are interpreted as indicating that plasminogen requires modification, either by proteolysis or by ligands, for activation.

Amino Acid Sequence↗

PubMed2002

Plasminogen activation is stimulated by prion protein and regulated in a copper-dependent manner.

Prion diseases are associated with the conversion of the normal prion protein, PrP(C), to the infectious disease form PrP(Sc). Discrimination between these isoforms would significantly enhance diagnosis of these diseases, and it has recently been reported that PrP(Sc) is specifically recognized by the serine protease zymogen plasminogen (Fischer et al. (2000) Nature 408, 479). Here we have tested the hypothesis that PrP is a regulator of the plasminogen activation system. The effect of recombinant PrP, either containing copper (holo-PrP) or devoid of it (apo-PrP), on plasminogen activation by both uPA and tPA was determined. PrP had no effect on plasminogen activation by uPA. By contrast, the activity of tPA was stimulated by up to 280-fold. This was observed only with the apo-PrP isoforms. The copper-binding octapeptide repeat region of PrP was involved in the effects, as a mutant lacking this region failed to stimulate plasminogen activation, although a synthetic peptide corresponding to this region was unable to stimulate tPA activity. Competition experiments demonstrated that, in addition to plasminogen binding, the stimulation required a high-affinity interaction between tPA and PrP (K(d) < 2.5 nM). Kinetic analysis revealed a template mechanism for the stimulation, suggesting independent binding sites for tPA and plasminogen. Lack of copper-binding may be an early event in the conversion of PrP(C) to PrP(Sc), and our data therefore suggest that tPA-catalyzed plasminogen activation may provide the basis for a sensitive detection system for the early stages of prion diseases and also play a role in the pathogenesis of these diseases.

Animals↗