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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.

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.

We have investigated whether urokinase-type plasminogen activator (u-PA) is present in the mouse in vivo as the proenzyme or as the active enzyme. u-PA in extracts of various murine tissues was of a one-polypeptide chain form with an electrophoretic mobility indistinguishable from purified proenzyme (pro-u-PA), as demonstrated by SDS-polyacrylamide gel electrophoresis under recucing conditions followed by immunoblotting. No 2-chain u-PA was detected in any of the extracts (detection limit 10% of that of one-chain u-PA). In bladder urine more than half of the u-PA was of the one-chain form. Together with previous immunocytochemical studies of the normal murine tissues and studies of the Lewis lung carcinoma, the present results indicate that in these tissues the one-chain proenzyme is the predominant form of u-PA in intracellular stores and for the first time demonstrates that at least in some cases the one-chain form constitutes a sizeable fraction of the u-AP in extracellular fluids in the intact organism.

Both the urokinase-type and tissue-type plasminogen activator can convert their 5̃4 kDa type-1 inhibitor (PAI-1) to an inactive form with a lower apparent molecular mass. We have determined the amino-terminal amino acid sequences of human native and converted PAI-1, and isolated PAI-1 cDNA and determined the nucleotide sequence in regions corresponding to the amino-terminus and the cleavage site. The data show that the conversion of the inhibitor consists of cleavage of an Arg-Met bond 33 residues from the carboxy-terminus, thus localizing the reactive center of the inhibitor to that position. In addition, a heterogeneity was found at the amino-terminus, with a Ser-Ala-Val-His-His form and a two-residue shorter form (Val-His-His-) occurring in approximately equal quantities.

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 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.

Both the urokinase‐type and tissue‐type plasminogen activator can convert their 5̃4 kDa type‐1 inhibitor (PAI‐1) to an inactive form with a lower apparent molecular mass. We have determined the amino‐terminal amino acid sequences of human native and converted PAI‐1, and isolated PAI‐1 cDNA and determined the nucleotide sequence in regions corresponding to the amino‐terminus and the cleavage site. The data show that the conversion of the inhibitor consists of cleavage of an Arg‐Met bond 33 residues from the carboxy‐terminus, thus localizing the reactive center of the inhibitor to that position. In addition, a heterogeneity was found at the amino‐terminus, with a Ser‐Ala‐Val‐His‐His form and a two‐residue shorter form (Val‐His‐His‐) occurring in approximately equal quantities.

We have investigated whether urokinase‐type plasminogen activator (u‐PA) is present in the mouse in vivo as the proenzyme or as the active enzyme. u‐PA in extracts of various murine tissues was of a one‐polypeptide chain form with an electrophoretic mobility indistinguishable from purified proenzyme (pro‐u‐PA), as demonstrated by SDS‐polyacrylamide gel electrophoresis under recucing conditions followed by immunoblotting. No 2‐chain u‐PA was detected in any of the extracts (detection limit 10% of that of one‐chain u‐PA). In bladder urine more than half of the u‐PA was of the one‐chain form. Together with previous immunocytochemical studies of the normal murine tissues and studies of the Lewis lung carcinoma, the present results indicate that in these tissues the one‐chain proenzyme is the predominant form of u‐PA in intracellular stores and for the first time demonstrates that at least in some cases the one‐chain form constitutes a sizeable fraction of the u‐AP in extracellular fluids in the intact organism.

In a retrospective study, uPA and PAI-I were assayed by enzyme-linked immunosorbent assay (ELISA) in detergent extracts prepared from 400 primary breast tumours. The patients were followed for a mediati of 9 years and for all patients relevant clinical findings were recorded.We found a correlation between uPA/PAI-1 and classical prognostic factors such as number of lymph nodes involved, grade, tumour size, hormone receptor status and histology, but no correlation according to menopause or age.Disease-free (DFS) and over-all survival (OS) were analyzed using a Cox's proportional hazard model. As cut-off point the median uPA and PAI-I values were used. Breast cancer patients with high content of PAI-I (11.0ng/mg protein) have an increased risk of relapse and death (RR: 1.74 (1.27–2.39), while high uPA (4.3ng/mg protein) had no impact.Despite PAI-l's correlation to other prognosticators, PAI-I retained its independent significance in the Cox-model in contrast to uPA.