Partial purification and characterization of a new fast-acting plasmin inhibitor from human platelets. Evidence for non-identity with the known plasma proteinase inhibitors.

An inhibitor of the plasma proteinase plasmin (EC 3.4.21.7) was partially purified from washed and lysed human blood platelets by (NH4)2SO4 fractionation and affinity chromatrography on Sepharose-linked purified plasminogen. The material contained none of the known plasma proteinase inhibitors when studied by crossed-immunoelectrophoresis and electroimmunoassay, but inhibited a clot-lysis-time assay and an esterolytic assay that used the synthetic substrate S-2251 (D-Val-Leu-Lys-p-nitroanilide). The inhibitory activity had the same mobility as the alpha 2-plasma proteins on preparative agarose-gel electrophoresis. Titration of the inhibitor preparation by active-site-titrated plasmin demonstrated a dissociation constant of approx. 0.1 nM. The inhibition was complete within 1 min. The inhibitor increased the mobility in agarose-gel electrophoresis of purified activator-free plasmin or 125I-labelled plasmin, as demonstrated by crossed-immunoelectrophoresis against specific immunoglobulins against plasminogen or by radioautography. The results strongly suggest the presence in platelets of a plasmin inhibitor different from the known plasma proteinase inhibitors.     

A chromogenic assay for the detection of plasmin generated by plasminogen activator immobilized on nitrocellulose using a para-nitroanilide synthetic peptide substrate

A direct solid phase chromogenic assay has been developed for the detection of plasmin (EC 3.4.21.7), generated by the interaction of a nitrocellulose-bound plasminogen activator, using the plasmin specific tripeptide substrate, H-D-valyl-leucyl-lysine - p-nitroaniline. para-Nitroaniline released by the cleavage of the lysine - p-nitroaniline bound by plasmin was derivatized to its diazonium salt and subsequently coupled to N-1-napthylethylenediamine in situ to form a diazoamino of an intense red color at the site of the plasminogen activator. This method was used to assay for the streptococcal plasminogen activator, streptokinase, not only in crude bacterial supernatants, but also to detect streptokinase secreted by individual bacterial colonies. In addition, this solid phase assay was used to identify monoclonal antibodies specific for streptokinase which could inhibit the activation of human plasminogen by streptokinase. This method also permitted simultaneous immunological and biochemical identification of the plasminogen activator, thus permitting unequivocal comparative observations. This assay is quantitative and sensitive to nanogram amounts of activator comparable to those obtained with soluble assays. This method may also be applicable for the detection of other plasminogen activators, such as tissue plasminogen activator, urokinase, and staphylokinase, and also for the detection of immobilized proteases which can cleave other substrates derivatized with p-nitroaniline. The reagents used in this assay are inexpensive and easy to prepare.     

Single-chain urokinase-type plasminogen activator does not possess measurable intrinsic amidolytic or plasminogen activator activities.

The question whether single-chain urokinase-type plasminogen activator (Sc-uPA) possesses an enzymatic activity has been a subject of intense investigation for a number of years but still remains unresolved. Recent studies from several laboratories suggest that Sc-uPA or its plasmin-resistant mutants obtained by site-directed mutagenesis possess significant, albeit low, amidolytic and plasminogen activator activities, ranging from 0.1% to 1% of that observed for two-chain urokinase (Tc-uPA). In an effort to characterize these putative intrinsic activities, Sc-uPA was repeatedly treated with dansyl-Glu-Gly-Arg chloromethyl ketone (dansyl-EGRck) or diisopropyl fluorophosphate (DFP) (0.1-0.25 mM added thrice over a period of 24 h at 0 degrees C). This treatment exhaustively inactivated the Tc-uPA contaminant but did not affect Sc-uPA, as evidenced by the lack of significant incorporation of radiolabeled inhibitor in Sc-uPA and full activation of the inhibitor-treated Sc-uPA by plasmin. Assayed in the presence of excess DFP or dansyl-EGRck to ensure trapping of any Tc-uPA generated in the assay mixture, Sc-uPA (84 micrograms/mL, 10,500 latent units/mL) did not elicit any detectable cleavage of the chromogenic substrate S-2444 (detection limit 0.1 unit of Tc-uPA/mL). However, if the Tc-uPA inhibitors were removed prior to assay, a trace amount of amidolytic activity invariably reappeared in the Sc-uPA preparation. Incorporation experiments with [3H]DFP suggested that the appearance of this amidolytic activity was due to formation of Tc-uPA. Plasminogen activator assay of DFP- and dansyl-EGRck-treated Sc-uPA (0.45-2.25 microM), performed in the presence of these inhibitors and Trasylol (10 microM) to ensure entrapment of any Tc-uPA or plasmin generated in the reaction mixture, showed no significant cleavage of 125I-labeled plasminogen (detection limit 0.1 nM). However, if dansyl-EGRck and DFP were removed from the inhibitor-treated Sc-uPA and the assay was performed in the presence of Trasylol alone, there was significant cleavage of 125I-plasminogen due to contamination by Tc-uPA. Fibrin, a positive effector of plasminogen activation by Tc-uPA or Sc-uPA preparations in the absence of DFP and dansyl-EGRck, did not promote cleavage of plasminogen or S-2444 by Sc-uPA in the presence of the Tc-uPA inhibitors.(ABSTRACT TRUNCATED AT 400 WORDS)     

Inactivation of the plasminogen activator from HeLa cells by peptides of arginine chloromethyl ketone.

The binding specificities of human urinary urokinase (EC 3.4.99.26) and HeLa cell plasminogen activator were studied using peptidyl chloromethyl ketone inhibitors. A 125I-labeled fibrin assay has been developed to yield kinetic information. Reagents of the sequence X-Gly-ArgCH2Cl were the most effective. The susceptibility of the HeLa cell plasminogen activator differed from that of urokinase in several respects indicating the utility of this type of inhibitor in distinguishing between proteases of this specificity.     

Activation of pro-urokinase and plasminogen on human sarcoma cells: a proteolytic system with surface-bound reactants

Human HT-1080 fibrosarcoma cells produce urokinase-type plasminogen activator (u-PA) and type 1 plasminogen activator inhibitor (PAI-1). We found that after incubation of monolayer cultures with purified native human plasminogen in serum-containing medium, bound plasmin activity could be eluted from the cells with tranexamic acid, an analogue of lysine. The bound plasmin was the result of plasminogen activation on the cell surface; plasmin activity was not taken up onto cells after deliberate addition of plasmin to the serum-containing medium. The cell surface plasmin formation was inhibited by an anticatalytic monoclonal antibody to u-PA, indicating that this enzyme was responsible for the activation. Preincubation of the cells with diisopropyl fluorophosphate-inhibited u-PA led to a decrease in surface-bound plasmin, indicating that a large part, if not all, of the cell surface plasminogen activation was catalyzed by surface-bound u-PA. In the absence of plasminogen, most of the cell surface u-PA was present in its single-chain proenzyme form, while addition of plasminogen led to formation of cell-bound two-chain u-PA. The latter reaction was catalyzed by cell-bound plasmin. Cell-bound u-PA was accessible to inhibition by endogenous PAI-1 and by added PAI-2, while the cell-bound plasmin was inaccessible to serum inhibitors, but accessible to added aprotinin and an anticatalytic monoclonal antibody. A model for cell surface plasminogen activation is proposed in which plasminogen binding to cells from serum medium is followed by plasminogen activation by trace amounts of bound active u-PA, to form bound plasmin, which in turn serves to produce more active u-PA from bound pro-u-PA. This exponential process is subject to regulation by endogenous PAI-1 and limited to the pericellular space.     

Increased natural killer cell activity in experimental American trypanosomiasis

When purified murine plasminogen was added to cultures of mouse spleen B cells, active plasmin progressively appeared in the supernatants. This reaction, resulting from the specific cleavage of the plasminogen by lymphocyte plasminogen activator (LPA), was measured in a fibrinolysis assay using 125I-fibrinogen. T cells were totally ineffective; under certain conditions, they could even antagonize the B cell action. Of the various B populations studied, i.e., obtained from spleen, lymph nodes, or blood of various mouse strains, all expressed the same property of plasminogen activation, which concerned mainly medium-sized B cells. Since only slight activities were detected in extracellular or intracellular compartments, a membrane-associated proteolytic enzyme may be responsible for plasminogen activation. Submitted to a series of group-specific antiproteases, the lymphocyte plasminogen activator essentially behaved as a serine-protease, with sensitivity to diisopropyl fluorophosphate, phenyl methyl sulfonyl fluoride, and nitro phenyl guanidino benzoate. The fast renewal of the enzyme in the membrane was also demonstrated by different techniques, using modifiers of cell physiology, like cycloheximide and dexamethasone, or following the reexpression of the enzyme by the cell kinetically.     

Tumor invasion through the human amniotic membrane: requirement for a proteinase cascade

To understand the role of proteinases in tumor invasion, the effects of inhibitors of metallo-, serine-, and cysteine-proteinases on this process were studied using 125I-iododeoxyuridine-labeled B16/BL6 cells grown on human amnion basement membrane. Cellular invasion was quantitated by measuring the radioactivity associated with the amniotic membrane after the B16/BL6 cells on the basement membrane were removed by lysis followed by scraping. The results obtained with proteinase inhibitors showed that inhibitors of collagenase and plasmin prevented invasion of the amnion. Tissue invasion was also blocked by antiurokinase antibodies. On the contrary, cysteine-proteinase inhibitors and anti-tissue plasminogen activator antiserum were ineffective. Mersalyl, a compound known to activate collagenase, stimulated invasion under conditions where plasmin formation or activity were inhibited. Evidence for the role of a plasminogen activator-plasmin-collagenase activation cascade in B16 invasion is provided.     

Plasminogen activation with single-chain urokinase-type plasminogen activator (scu-PA). Studies with active site mutagenized plasminogen (Ser740----Ala) and plasmin-resistant scu-PA (Lys158----Glu)

The mechanism of the activation of plasminogen by single-chain urokinase-type plasminogen activator (single-chain u-PA, scu-PA) was studied using rscu-PA-Glu158, a recombinant plasmin-resistant mutant of human scu-PA obtained by site-specific mutagenesis of Lys158 to Glu, and rPlg-Ala740, a recombinant human plasminogen in which the catalytic site is destroyed by mutagenesis of the active-site Ser740 to Ala. Conversion of 125I-labeled single-chain plasminogen to two-chain plasmin was quantitated on reduced sodium dodecyl sulfate-gel electrophoresis combined with autoradiography and radioisotope counting of gels bands. The efficiencies of both rscu-PA-Glu158 and rscu-PA for the activation of rPlg-Ala740 and of natural plasminogen were comparable and were 250-500-fold lower than that of recombinant two-chain u-PA (rtcu-PA) for rscu-PA-Glu158 and 100-200-fold lower for rscu-PA. Pretreatment of rscu-PA-Glu158 or rscu-PA with excess alpha 2-antiplasmin, which efficiently neutralizes all contaminating rtcu-PA, did not significantly reduce the catalytic efficiency of these single-chain moieties, indicating that they have a low but significant intrinsic plasminogen activating potential. The low intrinsic catalytic efficiency of rscu-PA for the conversion of plasminogen to plasmin may be sufficient to generate trace amounts of plasmin, which may regulate plasminogen activation by converting poorly active rscu-PA to very active rtcu-PA.     

Binding and activation of plasminogen at the surface of Staphylococcus aureus. Increase in affinity after conversion to the Lys form of the ligand.

Untreated Staphylococcus aureus cells, strain Cowan I, specifically bound 125I-Glu-plasminogen. The binding was inhibited by both unlabeled Glu-plasminogen and Glu-plasmin. The Lys form of plasminogen, which lacks the 8-kDa amino-terminal activation peptide, was approximately 100-fold more effective than the Glu form in competing with the binding of 125I-labeled Glu-plasminogen. This suggests an increase in binding affinity upon removal of the activation peptide. Fibronectin, fibrinogen and IgG, plasma components known to bind to the staphylococcal surface, did not significantly interfere with the binding. The competing activity in plasma was abolished by specifically absorbing plasminogen from the plasma sample. L-Lysine and a fragment of plasminogen containing three of the first five protein attachment domains present in the molecule (kringle structures) also competed with plasminogen for binding suggesting that the lysine-binding sites of plasminogen were involved in its interaction with staphylococci. Scatchard analysis revealed high- and low-affinity binding sites. Kd and the number of high-affinity binding sites were 1.7 nM and 780 binding sites/bacterial cell, respectively. 125I-Glu-plasminogen bound to staphylococcal surface was converted to plasmin by tissue-type plasminogen activator. The conversion took place also in the presence of plasma. If the conversion was carried out in the absence of low-molecular-mass plasmin inhibitors such as aprotinin, the bound Glu-plasmin was further converted to Lys-plasmin. The surface-bound plasmin was enzymically active, as judged by digestion of the synthetic substrate, S-2251. The plasminogen conversion shown by the present experiments not only leads to the surface-bound plasmin but seems to considerably increase the affinity of plasmin for its binding site. This may represent a physiologically relevant method for a bacterial cell to retain surface-bound active plasmin which is also protected from its soluble plasma inhibitors. This novel mechanism for staphylococci to adopt surface-bound proteolytic activity, without the interference of plasma components, may have some role in the tissue penetration and invasion of microbes during infection.     

Trinitrobenzoylated poly(D-lysine) as a stimulator of interactions between plasminogen, plasmin, and tissue-type plasminogen activator

Trinitrobenzyl alkylation of poly(D-lysine) provides a novel powerful stimulator of tissue-type plasminogen activator. Its stimulatory effect on plasminogen activation is far greater than that of the original poly(D-lysine), and even surpasses that of fibrin. Its effect on plasmin-catalysed modification of both tissue-type plasminogen activator (t-PA) and native (Glu-1-) plasminogen are also investigated. Cleavage of one-chain t-PA to its two-chain form is monitored by measuring the increase in amidolytic activity which accompanies this transformation. Presupposing apparent first-order reaction kinetics, a theory is developed by which the rate constant, kcat/Km = 1.0 X 10(6) M-1 X s-1 of plasmin cleavage of one-chain t-PA can be calculated. Plasmin-catalysed transformation of 125I-labelled Glu-1- to Lys-77-plasminogen is quantified following separation by polyacrylamide gel electrophoresis at pH 3.2. A rate constant, kcat/Km = 4.4 X 10(3) M-1 X s-1 is obtained for the reaction between plasmin and Glu-1-plasminogen in the presence of 1 mM trans-4-(aminomethyl)cyclohexane-1-carboxylic acid. Both of the above plasmin-catalysed reactions are strongly enhanced by trinitrobenzoylated poly(D-lysine). The mechanism of action of this stimulator is elucidated by studying its binding to both activator and plasmin(ogen), and by direct comparison of the results with measurements of plasminogen activation kinetics in the presence of the stimulator. Binding studies are performed exploiting the observation that an insoluble yellow complex is formed between plasminogen and modified poly(D-lysine). Protein-polymer interactions are also studied with solubilised components in an aqueous two-phase partition system containing dextran and poly(ethylene glycol). The rate enhancement of plasminogen activation is found to be closely correlated to the association of plasminogen to the stimulator. It is proposed that the stimulator effects of this simple polymer on the enzymatic activities of both plasminogen activator and plasmin are brought about by association of the proteinase and its substrate to a common matrix. Similarities between the action of the artificial and the natural stimulator (fibrin) are stressed. These properties of trinitrobenzoylated poly(D-lysine) makes it useful as a model for the study of the regulatory mechanism of the fibrinolytic process at the molecular level.     

Inhibition of a plasminogen activator from oncogenic virus-transformed mouse cells by rabbit antibodies against the enzyme

Antisera were raised in rabbits against an electrophoretically pure 48 000 dalton plasminogen activator from mouse cells transformed by an oncogenic virus. The IgG fraction of the antisera inhibited 48 000 dalton mouse plasminogen activators from a variety of sources (neoplastic and nonneoplastic), a 29 00) dalton plasminogen activator from mouse urine and a 48 000 dalton plasminogen activator from rat urine. No inhibition was observed of a 75 000 dalton plasminogen activator extracted from mouse lung, of mouse plasmin or of plasminogen activators from human urine and from oncogenic-virus transformed chicken cells. The IgG antibodies were stronger and more specific inhibitors of the 48 000 dalton mouse plasminogen activator than any previously tested compounds.     

Monoclonal antibody to human 66,000 molecular weight plasminogen activator from melanoma cells. Specific enzyme inhibition and one-step affinity purification

Hybridomas producing a monoclonal IgG1 antibody to a human plasminogen-activating enzyme with an apparent mol. wt. of 66,000 (66 K, HPA66) from human melanoma cells were obtained by fusion of NSI-Ag 4/1 mouse myeloma cells with spleen cells from a mouse immunized with a partially purified preparation of the enzyme. Screening for clones of hybridomas producing antibodies to HPA66 was performed with the impure enzyme preparation. A preliminary screening included enzyme-linked immunosorbent assay and SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by immunoblotting; the final identification was based on inhibition of the enzymatic activity of HPA66 which was complete at high antibody concentrations. No inhibition of three other human and murine plasminogen activators or of plasmin was observed. Employing a one-step affinity procedure with the antibody coupled to Sepharose, HPA66 was purified approximately 200-fold from conditioned medium from the melanoma cells with a yield of 79%. The purified HPA66 was homogeneous as evaluated by SDS-PAGE. Electrophoresis under reducing conditions indicated that it consisted of one polypeptide chain. The binding constant between the antibody and 125I-labelled HPA66 was approximately 2.5 x 10(9) l/mol. The antibody did not bind to a variety of other plasminogen activators, including 52-K and 36-K human enzymes and 48-K and 75-K murine enzymes. Previously, a monoclonal antibody against another enzyme was derived by the sole use of enzyme inhibition for screening. The present study represents a modification of this procedure that can be used when antibody-unrelated inhibitors of the enzyme are present in hybridoma culture fluid.     

The streptokinase–human plasminogen activator complex. Composition and identity of a subcomponent with activator activity

Reactions between purified plasminogen and streptokinase, labelled with ¹³¹I and ¹²⁵I respectively, were investigated by polyacrylamide-gel discontinuous electrophoresis. A molecular complex consisting of both ¹³¹I-labelled plasminogen and ¹²⁵I-labelled streptokinase migrated between plasminogen and streptokinase. This complex contained bovine plasminogen activator activity. The relative quantities of ¹³¹I-labelled plasminogen and ¹²⁵I-labelled streptokinase in this complex were markedly affected by reaction conditions. A fragment that retained 50% or more of the parent activator activity was released from the complex after exposure to mercaptoethanol. This subcomponent had an estimated molecular weight of 70000, and contained both ¹³¹I-labelled plasminogen and ¹²⁵I-labelled streptokinase.     

Plasminogen activator-anti-human fibrinogen conjugate

A covalent conjugate between the plasminogen activator urokinase and polyclonal rabbit anti-human fibrinogen has been formed using the heterobifunctional coupling reagent N-succinimidyl 3-(2-pyridyldithio) propionate. The resultant urokinase-anti-human fibrinogen conjugate was separated from unreacted material by gel filtration. The conjugate exhibited amidase activity against the small chromogenic substrate pyroglutamyl-glycyl-arginine-p-nitroanilide as well as plasminogen activator activity in an assay employing plasminogen and the plasmin substrate D-valyl-leucyl-lysine-p-nitroanilide. Retention of antibody specificity for fibrinogen was demonstrated using an enzyme linked immunoassay procedure. The conjugate was found to have greater stability in human plasma than unconjugated urokinase.     

The Receptor for Urokinase-Type Plasminogen Activator of a Human Keratinocyte Line (HaCaT)

It is assumed that plasmin participates in pericellular proteolysis in the epidermis. Plasmin is generated by keratinocyte-associated plasminogen activators from the proenzyme plasminogen; plasminogen activation can proceed at the keratinocyte surface. The resultant plasmin interferes with cell to matrix adhesion and does possibly contribute to keratinocyte migration during reepithelialization. Here we describe the receptor for urokinase-type plasminogen activator (uPA-R) in the human keratinocyte cell line HaCaT, which serves to direct plasminogen activation to the cell surface; we relate the receptor to the uPA-R previously described in human myclo-/monocytes. Binding of uPA to the receptor accelerated plasminogen activation by a factor of ≈10, compared to uPA in solution. Receptor-bound uPA was susceptible to inhibition by the plasminogen activator inhibitors 1 and 2. uPA and uPA-R antigen, as well as uPA activity, were localized to the leading front of expanding sheets of HaCaT cells. Exposure of HaCaT cells to plasminogen was followed by detachment of the cells. Detachment was prevented by an anti-catalytic anti-uPA antibody, by the plasmin-specific inhibitor aprotinin, and by the lysine analogue tranexamic acid, the latter of which prevents plasmin(ogen) binding to the cell surface. Our findings support the hypothesis that uPA-mediated plasminogen activation is characteristic of mobile rather than sessile keratinocytes. Moreover, the uPA-R seems to focalize plasminogen activation to the surface of cells at the site of keratinocyte migration.     

Effects of thiol reagents on glucose transport in thymocytes

We have investigated the factors governing the plasminogen-dependent fibrinolysis catalyzed by the serine proteinase, plasminogen activator (EC 3.4.21.-), under physiologic conditions. We found that live rabbit fibroblasts digested much less fibrin than predicted by cell-free assay of the secreted plasminogen activator. The reduced catalytic activity of plasminogen activator expressed by cells growing on fibrin was regulated by the salt concentration of culture medium. The plasminogen activators of cells from several mammalian species were inhibited by physiologic salt concentrations (0.15 M NaCl) in cell-free assays. CaCl2 and KCl, but not D-glucose, were also effective inhibitors. The catalytic activity of purified human urokinase and of plasmin was unaffected by increased ionic strength. Plasminogen activators secreted both spontaneously and in response to stimulation by the tumor promoter, 12-O-tetradecanoyl-phorbol-13-acetate, were inhibited by 0.15 M NaCl. Physiologic salt concentration appeared to function by interacting with plasminogen activator, or plasminogen, and a third component, possibly a reversible inhibitor. One consequence of this regulation of plasminogen activator under physiologic conditions is the limitation of plasminogen-dependent fibrin degradation by living cells.     

Sulfated Glycosaminoglycans Enhance Tumor Cell Invasionin Vitroby Stimulating Plasminogen Activation

Metastasizing tumor cells invade host tissues by degrading extracellular matrix constituents. We report here that the highly sulfated glycosaminoglycans, heparin and heparan sulfate, as well as the sulfated polysaccharide, fucoidan, significantly enhanced tumor cell invasionin vitrointo fibrin, the basement membrane extract, Matrigel, or through a basement membrane-like extracellular matrix. The enhancement of tumor cell invasion was due to a stimulation of the proteolytic cascade of plasminogen activation since the effect required plasminogen activation and was abolished by inhibitors of urokinase-type plasminogen activator (uPA) or plasmin. Sulfated polysaccharides enhanced five reactions of tumor-cell initiated plasminogen activation in a dose-dependent manner. They amplified plasminogen activation in culture supernatants up to 70-fold by stimulating (i) pro-uPA activation by plasmin and (ii) plasminogen activation by uPA. (iii) In addition, sulfated polysaccharides partially protected plasmin from inactivation by α₂-antiplasmin. Sulfated polysaccharides also stimulated tumor-cell associated plasminogen activation, e.g., (iv) cell surface pro-uPA activation by plasmin and (v) plasminogen activation by cell surface uPA. These results suggest that sulfated glycosaminoglycans liberated by tumor-cell mediated extracellular matrix degradationin vivomight amplify pericellular plasminogen activation and locally enhance tumor cell invasion in a positive feedback manner.     

Stimulation of tPA-dependent provisional extracellular fibrin matrix degradation by human recombinant soluble melanotransferrin

Tissue-type plasminogen activator (tPA) and its substrate plasminogen (Plg) are key components in the fibrinolytic system. We have recently demonstrated, that truncated human recombinant soluble melanotransferrin (sMTf) could stimulate the activation of Plg by urokinase plasminogen activator and inhibit angiogenesis. Since various angiogenesis inhibitors were shown to stimulate tPA-mediated plasminogen activation, we examined the effects of sMTf on tPA-dependent fibrinolysis. This study demonstrated that sMTf enhanced tPA-activation of Plg by 6-fold. sMTf also increased the release of [125I]-fibrin fragments by tPA-activated plasmin. Moreover, we observed that the interaction of sMTf with Plg provoked a change in the fibrin clot structure by cleaving the fibrin alpha and beta chains. Overall, the present study shows that sMTf modulates tPA-dependent fibrinolysis by modifying the clot structure. These results also suggest that sMTf properties could involve enhanced dissolution of the provisional extracellular fibrin matrix.     

Inactive proenzyme to tissue-type plasminogen activator from human melanoma cells, identified after affinity purification with a monoclonal antibody

The human 66 000 mol. wt. plasminogen activator (HPA66; tissue-type plasminogen activator) has been purified from melanoma cells by a one-step affinity method with a monoclonal antibody. HPA66 purified in this way consists mainly of a one-polypeptide chain form with small amounts (15%) of a form containing two polypeptide chains held together by one or more disulphide bridges. The one-chain form was converted to the two-chain form by catalytic amounts of plasmin. During the conversion, the enzyme activity of HPA66, as measured by an [125I]plasminogen conversion assay and with a chromogenic substrate, increased linearly with the percentage of the two-chain form. A linear regression analysis showed that all enzyme activity could be accounted for by the two-chain form, while the one-chain form had no measurable enzyme activity (detection limit approximately 5% of the activity of the two-chain form). Together with previous findings of inactive proenzymes to murine and human approximately 50 000 mol. wt. (urokinase-type) plasminogen activators, these findings indicate that plasminogen activators are generally formed from inactive one-chain proenzymes which are converted to active two-chain enzymes by limited proteolysis, thus demonstrating a third step in a cascade reaction leading to extracellular proteolysis.