Sensitive and Specific Enzyme-Linked Immunosorbent Assay for Urokinase-Type Plasminogen Activator in Human Plasma

Grøndahl-Hansen, J.; Agerlin, Niels; Nielsen, Lene; Danø, Keld

doi:10.1055/s-0038-1644425

Cited by 24 publications

(28 citation statements)

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“…The plasma concentration ofuPA is in the order of -1 nM (69,70), close to the dissociation constant for uPA/uPAR interaction ( 18). Therefore, ligand-bound uPAR may also be present in the ascitic fluid or plasma of ovarian carcinoma patients or in the plasma of healthy volunteers.…”

Section: Resultsmentioning

confidence: 99%

A ligand-free, soluble urokinase receptor is present in the ascitic fluid from patients with ovarian cancer.

Pedersen¹,

Schmitt²,

Rønne³

et al. 1993

J. Clin. Invest.

107

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We have identified a soluble form of the human urokinase plasminogen activator (uPA) receptor (uPAR) in the ascitic fluids from patients with ovarian cancer. After purification of uPAR from the ascitic fluids by ligand-affinity chromatography (prouPA Sepharose), the uPAR was initially identified by crosslinking to a radiolabeled amino-terminal fragment of human uPA. The uPAR purified from the ascitic fluid has no bound ligand (uPA), as similar amounts can be purified by ligand-affinity chromatography as by immuno-affinity chromatography. uPAR from ascitic fluids partitions in the water phase after a temperature-dependent phase separation of a detergent extract. It therefore lacks at least the lipid moiety of the glycophospholipid anchor present in cellular-bound uPARs. It is highly glycosylated and the deglycosylated form has the same electrophoretic mobility as previously characterized cellular uPAR from other sources. The immunoreactivity of the purified uPAR from the ascitic fluid is indistinguishable from that of characterized uPAR, demonstrated by Western blotting with three different anti-uPAR monoclonal antibodies. The uPAR was found in 11 of 11 ascitic fluids from patients with ovarian cancer and in elevated amounts in the plasma from 2 of 3 patients. The concentration of soluble uPAR in the ascitic fluid was estimated to range between 1 and 10 ng/ml. Human soluble uPAR, derived from the tumor cells, was also found in the ascitic fluid and serum from nude mice xenografted intraperitoneally with three different human ovarian carcinomas. (J. Clin. Invest. 1993.92:2160-2167.) Key words: ovarian cancer. urokinase plasminogen activator receptor * ascitic fluid * plasma.soluble receptor

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Section: Resultsmentioning

confidence: 99%

A ligand-free, soluble urokinase receptor is present in the ascitic fluid from patients with ovarian cancer.

Pedersen¹,

Schmitt²,

Rønne³

et al. 1993

J. Clin. Invest.

107

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“…The amounts of u-PA in normal plasma, measured by both OS and AD u-PA ELISA (Table 1), were similar to previously reported values. [18][19][20][21] Each ELISA indicated patients with the QPD had larger increases in u-PA in their platelets than their plasmas because many patients had normal plasma u-PA levels (Table 1). u-PA ELISA confirmed For personal use only.…”

Section: Resultsmentioning

confidence: 99%

Platelets from patients with the Quebec platelet disorder contain and secrete abnormal amounts of urokinase-type plasminogen activator

Kahr

Zheng

Sheth

et al. 2001

Blood

113

142

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The Quebec platelet disorder (QPD) is an autosomal dominant platelet disorder associated with delayed bleeding and ␣-granule protein degradation. The degradation of ␣-granule, but not plasma, fibrinogen in patients with the QPD led to the investigation of their platelets for a protease defect. Unlike normal platelets, QPD platelets contained large amounts of fibrinolytic serine proteases that had properties of plasminogen activators. Western blot analysis, zymography, and immunodepletion experiments indicated this was because QPD platelets contained large amounts of urokinase-type plasminogen activator (u-PA) within a secretory compartment. u-PA antigen was not increased in all QPD plasmas, whereas it was increased more than 100-fold in QPD platelets (P < .00009), which contained increased u-PA messenger RNA. Although QPD platelets contained 2-fold more plasminogen activator inhibitor 1 (PAI-1) (P < .0008) and 100-fold greater u-PA-PAI-1 complexes (P < .0002) than normal platelets, they contained excess u-PA activity, predominantly in the form of two chain (tcu-PA), which required additional PAI-1 for full inhibition. There was associated proteolysis of plasminogen in QPD platelets, to forms that comigrated with plasmin. When similar amounts of tcu-PA were incubated with normal platelet secretory proteins, many ␣-granule proteins were proteolyzed to forms that resembled degraded QPD platelet proteins. These data implicate u-PA in the pathogenesis of ␣-granule protein degradation in the QPD. Although patients with the QPD have normal to increased u-PA levels in their plasma, without evidence of systemic fibrinogenolysis, their increased platelet u-PA could contribute to bleeding by accelerating fibrinolysis within the hemostatic plug. QPD is the only inherited bleeding disorder in humans known to be associated with increased u-PA. IntroductionCongenital platelet disorders are usually associated with defective primary hemostasis. [1][2][3] The Quebec platelet disorder (QPD) is an autosomal dominant platelet disorder that has unusual clinical features: it is associated with moderate to severe delayed bleeding, that typically begins 12 to 24 hours after surgery or trauma, and its hemorrhagic manifestations can be controlled with fibrinolytic inhibitors but not with platelet transfusions. 1,[4][5][6] This disorder was initially designated as factor V Quebec because of the abnormalities found in platelet factor V of these patients. 7 Two families from Quebec have been identified with this condition, which is now known to be associated with other platelet abnormalities that include reduced to low-normal platelet counts, proteolytic degradation of soluble and membrane proteins stored in platelet ␣-granules, an apparent quantitative deficiency of the ␣-granule protein multimerin, and defective aggregation with epinephrine. 1,[4][5][6]8 Although patients with the QPD have elevated levels of fibrinogen degradation products (FDPs) in their serum (because of platelet fibrinogen degradation), their plasma contains normal amo...

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“…Clones #377 and #394 were purchased from American Diagnostica Inc. (Greenwich, Connecticut). A polyclonal IgG anti-uPA preparation (pAb1) was prepared from rabbits immunized with uPA (purified from human urine) and absorbed with human plasma proteins (Grøndahl-Hansen et al, 1988;Kristensen et al, 1984). This preparation was divided into two parts; one part was passed through a column with immobilized bovine serum albumin (pAb1), and the other part was passed through a column with immobilized uPA and was used as a negative control antibody preparation (pAbl Ϫ ).…”

Section: Antibodiesmentioning

confidence: 99%

Urokinase Plasminogen Activator Is Localized in Stromal Cells in Ductal Breast Cancer

Nielsen¹,

Sehested²,

Duun

et al. 2001

Laboratory Investigation

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SUMMARY:Urokinase plasminogen activator (uPA) regulates a proteolytic cascade that facilitates cancer invasion through degradation of the extracellular matrix, and high levels of uPA in human breast cancer tissue correlate with poor prognosis. We previously found that, in ductal breast cancer, uPA mRNA is highly expressed by myofibroblasts surrounding invasively growing cancer cells. However, the localization of uPA protein has not been settled in the published literature. Because uPA is a secreted molecule, it could conceivably be localized differently from its mRNA. We have studied the localization of uPA immunoreactivity in detail. Twenty-five cases of invasive ductal carcinoma were analyzed with three different uPA antibody preparations, all of which gave an essentially identical stromal staining pattern. Using double immunofluorescence, we identified uPA immunoreactivity in myofibroblasts and macrophages in all cases examined. Additionally, in approximately half of the tumors, we saw uPA staining of endothelial cells. In 3 of the 25 cases, a small subpopulation of the cancer cells was uPA-positive. We conclude that uPA immunoreactivity is almost exclusively associated with stromal cells, which thus play a major role in generation of proteolytic activity in ductal breast cancer. (Lab Invest 2001, 81:1485-1501.

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Sensitive and Specific Enzyme-Linked Immunosorbent Assay for Urokinase-Type Plasminogen Activator in Human Plasma

Cited by 24 publications

References 0 publications

A ligand-free, soluble urokinase receptor is present in the ascitic fluid from patients with ovarian cancer.

A ligand-free, soluble urokinase receptor is present in the ascitic fluid from patients with ovarian cancer.

Platelets from patients with the Quebec platelet disorder contain and secrete abnormal amounts of urokinase-type plasminogen activator

Urokinase Plasminogen Activator Is Localized in Stromal Cells in Ductal Breast Cancer

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