The inhibition of high and low molecular weight urokinase in plasma

Murano, Genesio; Aronson, David L.; Williams, L. David; Brown, Larry N.

doi:10.1182/blood.v55.3.430.430

Cited by 45 publications

(27 citation statements)

References 21 publications

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“…The FCS inhibition was stronger with the 52-kDa form of active u-PA than with the 33-kDa form (c.f. Murano et al, 1980) but clearly the amino-terminal region of u-PA (the portion lacking in 33-kDa u-PA) was not required for inhibition by serum. Pro-u-PA was considerably less affected ( Figure 2), showing that the active site of u-PA was involved.…”

Section: Resultsmentioning

confidence: 87%

“…Our results obtained with the RC2A myelomonocytic leukemia cell line show first the partition of active u-PA between bovine serum a2M and cell-surface receptors. Slow inactivation of active u-PA in the medium by serum a2M (Murano et al, 1980;Waller et al, 1983;Straight et al, 1985;Sottrup-Jensen et al, 1989) leads to loss of measurable u-PA activity in the medium, but it is not able to significantly reduce the amount of active u-PA that rapidly binds to the cell surface. This outcome is evidently deter-…”

Section: Discussionmentioning

confidence: 99%

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Alpha 2-macroglobulin restricts plasminogen activation to the surface of RC2A leukemia cells.

et al. 1991

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Human RC2A myelomonocytic leukemia cells are able to activate the prourokinase (pro-u-PA) they secrete so that active u-PA is present both in serum-free conditioned medium from these cells, as well as on the cell surface. When the cells are grown in serum-containing medium, no u-PA activity can be found in the medium but active u-PA is found bound to the cell surface where it can generate bound plasmin. This distribution of u-PA activity was shown to be, first, the net result of slow inactivation of free active u-PA by serum inhibitor(s) and simultaneous rapid uptake of u-PA onto the cell surface. Binding to cells was at least six times faster than inactivation by 10% serum. The principal serum inhibitor of u-PA was identified as alpha 2-macroglobulin (alpha 2M), and prior inactivation of u-PA by purified human alpha 2M was also shown to prevent uptake of u-PA activity onto cells. Second, although endogenous u-PA could form covalent complexes with purified alpha 2M in the culture medium of RC2A cells, covalent alpha 2M complexes were not formed by u-PA on the cell surface; the u-PA taken up in this compartment was protected against alpha 2M inhibition. u-PA anchored to plastic surfaces via monoclonal antibodies to the amino-terminal region of u-PA was also protected against alpha 2M, suggesting that the protection of cell surface u-PA results from a steric effect. These results provide evidence as to how the active u-PA produced by leukemia cells can contribute to proteolytic activity on their cell surface in the presence of serum inhibitors.

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Section: Resultsmentioning

confidence: 87%

Section: Discussionmentioning

confidence: 99%

Alpha 2-macroglobulin restricts plasminogen activation to the surface of RC2A leukemia cells.

et al. 1991

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“…The purity of the enzymes was verified by sodium dodecyl sulfate polyacrylamide gel electro phoresis (SDS-PAGE) and inhibitor studies. 3,5,6 Digestion of the BM components was performed at 25°C using matched enzymatic activities. Urokinase failed to degrade laminin significantly, or any of the other components.…”

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confidence: 99%

Degradation of Glycoprotein and Collagenous Components of the Basement Membrane: Studies with Urokinase-Type Plasminogen Activator, α-Thrombin, and Plasmin

Goldfarb¹,

Murano²,

Brundage³

et al. 1986

Semin Thromb Hemost

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Penetration of the basement membrane takes place at several stages of tumor invasion and meta stasis and appears to involve a variety of degradative proteolytic enzymes directly or indirectly under the regulation of malignant cells. 1 ' 2 ' 4 We have therefore evaluated the role of the neutral serine proteases: urokinase (plasminogen activator), α-thrombin, and plasmin in the degradation of intact basement membrane (BM) and BM components. 3 ' 5 ' 7 Homogeneously purified enzymes were incubated with isolated components of the BM and with whole amnion BM. The components comprised acidextracted type IV collagen, pepsin fragments of type IV collagen, type V collagen, laminin and fibronectin.The purity of the enzymes was verified by sodium dodecyl sulfate polyacrylamide gel electro phoresis (SDS-PAGE) and inhibitor studies. 3,5,6 Digestion of the BM components was performed at 25°C using matched enzymatic activities. Urokinase failed to degrade laminin significantly, or any of the other components. Under the same native condi tions, however, plasmin and α-thrombin cleaved laminin and fibronectin into multiple, specific frag ments but did not produce a major cleavage of acid-extracted type IV collagen, pepsinized type IV collagen, or type V collagen. α-Thrombin selectively degraded only the 400,000 M r β-chain of laminin, whereas plasmin degraded both chains. Utilization of a 14 C-labeled laminin revealed that degrada tion by the serine proteases was concentration and time dependent. In addition to metastatic tumor cells, macrophages and polymorphonuclear leukocytes contained significant laminin-degrading proteolytic activity. This activity was enhanced by the addition of plasminogen. Type V collagen was cleaved by α-thrombin and plasmin at 35°C but not at temperatures below 33°C. Electron microscopy demonstrated preservation of the lamina densa fol lowing treatment of whole-amnion BM with any of the neutral serine proteases employed. Immunohistologic studies revealed that laminin, but not type IV collagen, was removed from the whole BM by plasmin treatment.The results suggest that these BM components are poor substrates for urokinase-type plasminogen activator, and plasmin alone cannot degrade the whole BM. 3,5,8,9 The conversion of plasminogen to plasmin, generated through the catalytic activity of plasminogen activator appears to play a significant role in the degradation of the noncollagenous com ponents of the BM in tumor invasion. The plas minogen activator-mediated generation of plasmin also appears to play an important role in activating latent type IV collagenase and in enhancing the enzymatic activity of a heparan sulfate-degrading heparanase (endoglycosidase) derived from invasive tumor cells. 1,2

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“…8 The enzymatic activity of the urokinase preparations were assayed by multiple methods, including plasminogen-dependent fibrinolysis of iodinated fibrin and fluorometric determinations on a fluorogenic substrate: CBZ-Gly-Gly-Argamino methyl coumarin. 5,7,9 Purity of the enzymes employed was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. [5][6][7] HMW urokinase mediated angiogenesis in eight of ten experiments, whereas LMW urokinase mediated neovascularization in six of ten experiments.…”

mentioning

confidence: 99%

“…5,7,9 Purity of the enzymes employed was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. [5][6][7] HMW urokinase mediated angiogenesis in eight of ten experiments, whereas LMW urokinase mediated neovascularization in six of ten experiments. In control experiments it was observed that 3 H diisopropylfluorophosphate ( 3 H-DFP) labeled preparations of pure HMW and LMW urokinase failed to mediate neovascularization.…”

mentioning

confidence: 99%

Plasminogen Activators (Urokinase) Mediate Neovascularization: Possible Role in Tumor Angiogenesis

Goldfarb¹,

Ziche²,

Murano³

et al. 1986

Semin Thromb Hemost

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Most malignant solid tumors induce the growth of new capillaries from the vascular bed by tumor angiogenesis, and dormant tumors can grow only if new capillaries are induced and penetrate into the tumor. 2 Since many human tumors produce a plasminogen activator (PA) immunochemically and biochemically identical to urokinase (the PA produced by the kidney and excreted in urine), highly purified preparations of both high and low M r urokinase have been examined for their potential to mediate neovascularization. This was of interest since PA activity has been correlated with various aspects of malignant transformation, tumor invasion, and tumor metastasis. 3 " 6 We report that homogeneously purified high molecular weight (HMW) and low molecular weight (LMW) urokinase can cause angiogenesis when angiogenic capacity was evaluated in the rabbit cornea assay. 8 The enzymatic activity of the urokinase preparations were assayed by multiple methods, including plasminogen-dependent fibrinolysis of iodinated fibrin and fluorometric determinations on a fluorogenic substrate: CBZ-Gly-Gly-Argamino methyl coumarin. 5,7,9 Purity of the enzymes employed was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. 5-7 HMW urokinase mediated angiogenesis in eight of ten experiments, whereas LMW urokinase mediated neovascularization in six of ten experiments. In control experiments it was observed that 3 H diisopropylfluorophosphate ( 3 H-DFP) labeled preparations of pure HMW and LMW urokinase failed to mediate neovascularization. It was demonstrated that 3 H-DFP-labeled urokinase preparations were inhibited in their enzymatic activity by greater than 98%. In addition, SDS-PAGE autoradiography demonstrated that 3 H-DFP was incorporated into both HMW and LMW urokinase and showed complete coincidence with Coomassie blue stained counterpart lanes in the same gels.It is therefore concluded that LMW and HMW urokinase directly, or indirectly, mediated neovascularization in the rabbit cornea and that the active site of the enzyme is required for this activity. Preliminary experiments have also suggested that tissue plasminogen activator also mediated neovascularization.It therefore appears that an important regulatory neutral serine protease that contributes to both normal degradative thrombolysis and pathologic tumor invasion and metastasis may contribute to neovascularization. During the course of our studies, an additional group of investigators simultaneously observed that LMW urokinase causes vascularization of the cornea. 1 REFERENCES 1. Berman M, S Winthrop, D Ausprunk, et al: Plasminogen activator (urokinase) causes vascularization of the cornea.

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The inhibition of high and low molecular weight urokinase in plasma

Cited by 45 publications

References 21 publications

Alpha 2-macroglobulin restricts plasminogen activation to the surface of RC2A leukemia cells.

Alpha 2-macroglobulin restricts plasminogen activation to the surface of RC2A leukemia cells.

Degradation of Glycoprotein and Collagenous Components of the Basement Membrane: Studies with Urokinase-Type Plasminogen Activator, α-Thrombin, and Plasmin

Plasminogen Activators (Urokinase) Mediate Neovascularization: Possible Role in Tumor Angiogenesis

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