- Email: [email protected]
221 Proteolysis of plasminogen by polymorpho nuclear leukocyte
64 POSTER PRESENTATIONS P-XI Pericellular Proteolysis: Growth Factors a n d C y t o k i n e s
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HIGH LOCAL CONCENTRATION O F P A I - 2 -- L E S S INFLAMMATORY SYMPTOMS l, 2,3Kinnby B, 2 Matsson L and 3~stedt B Departments of Ipedodontics and 2pefiodontology, School of Dentistr3, Lund University, and 3Research Laboratory of the Department of
ACTIVATION OF METALLOPROTEINASES FROM METASTATIC CARCINOMATOUS LINE BY PHOSPHORYLATED UROKINASE-PLASMINOGEN
Obstetrics and Gynecology, Lurid University Hospital, Sweden. The clinical manifestations of gingival inflammation in reaction to bacterial plaque are aggravated during pregnancy. In vitro studies suggest a hormonal influence on PAI-2 (1), of importance for tissue proteolysis. A disturbance of the fibrinolytic system could thus help to explain "pregnancy gingivitis". The aim of this study was therefore to investigate the plasminogen activators and their inhibitors in gingival crevicular fluid (GCF) in pregnant and non-pregnant states. GCF was sampled in 14 women in pregnant and post-pregnant states. The gingival condition was assessed by an ordinal scale, grading swelling and redness (the gingival index -- GI) and the amount of bacterial plaque by the plaque index (PI). The ratio of the number of sites with gingivitis to the number of sites with plaque was calculated (G/P-ratio). Based on differences in G/P-ratio between pregnancy and post-pregnancy, subgrouping was done into a high-reacting (HR) and a low-reacting (LR) group. Levels of t-PA, u-PA, PAI-I and PAl-2 in GCF were determined with ELISAs and 17B-estradiol and progesterone in serum with RIAs. For each individual the differences (A) between pregnancy and post-pregnancy in hormone levels as well as PAs and PAIs were calculated. A significant correlation between Aprogesterone and APAI-2 was noted: the higher Aprogesterone, the lower APAI-2 (r2=0.27; P : 0 . 0 4 8 ) The women in the HR group showed loweror unchanged PAI-2 in GCF during pregnancy (mean -5nM), while those in the LR group showed a great increase (mean +25riM). The lower inhibitory capacity in terms of low concentrations of P A I - 2 during pregnancy in women with more pronounced inflammatory reaction indicates that the fibrinolytic system may be involved in pathogenesis of pregnancy gingivitis and implies that PAl-2 serves as an inhibitor of importance for tissue proteolysis. The present finding contributes to the explanation of pregnancy gingivitis and adds to the understanding of the significance of PAI-2 in tissue proteolysis I. Kinnby B, Astedt B, Cassl6n B. Fibrinolysis 1995; 9:152-1 56
1Takahashi
K, 2 K o h S and 3 K w a a n
CELLS OF A TYROSINEACTIVATOR
HC
1Department of Physiology, Shimane Medical College, Izumo 693, Japan, 2Department of Internal Medicine, Shinshu University, Matsumoto 390, Japan, and 3Hematology/Oncology, School of Medicine, Northwestern University, Chicago, II 60611, USA. Urokinase-plasminogen activator (uPA) associated with surface receptors of various types of tumor cells is generally considered to involve in cellular invasion by braking down proteins of extracellular matrix. Other proteaees such as collagenases or gelatinases are also important in the destruction of the surrounding architecture. To answer the question whether uPA is able to activate the latent matrix metalloproteinases (MMP) associated with a metastatic carcinomatous cell line, Detroit 562, affinity purified MMPs on a column of gelatin-Sepharose from the conditioned medium or membrane fractions were used to test either by cellular phosphorylated uPA (p-uPA)or urinary uPA. In comparison with this, the latter was labeled with biotin and phosphorylated by the incubation with materials co-precipitated with antipp60 c-src antibody or with p43 v'abl. They were evaluated by blots ol SDSPAGE gels by use of avidin-conjugated peroxidase or anti-mouse IgG conjugated with peroxidase following by enhanced chemiluminescence. MMPs were examined by gelatin-containing zymography and immunoblots using antibodies against MMPs. Both the plasminogen and uPA or p-uPA were immobilized on disks of nitrocellulose filters and incubated with the MMP. Activation of plasminogen was examined by use of a synthetic substrate, S-2251, and that of MMPs was by determinations at 420 nm of hydrolysates of a synthetic substrate (PZ-PLGPR). MMP-2 and MMP-9 were identified to be associated with membrane fractions and they were found to be activated solely by the p-uPA. Addition of plasminogen did not enhance the activation of MMPs. It was also able to activate plasminogen at a higher rate than that of unphosphorylated one. Inhibitors (PAl-1 and PAl-2) did not interfere the p-uPA activity. Thus, it suggests that inhibitor resistant p-uPA and MMPs may involve in a concomitant functional network of proteases on cell surfaces enabling an efficient hydrolysis of matrix proteins.
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ELUCIDATION OF THE INHIBITORY PRO PER'P/OF "rwo UK MONOCLONALS IN EX'IRACELLULAR PRO'lEO LVSIS USING U93 7 CELL S. THE b IFFERENCE OF THE INHIB ITORY PRO PERTV TOWARDS ENDOGENOUS AND EXOGENOUS UPA GIo,~ttaV. Ishb'aki, $hiaem H ayashi Thrombosis Chernical Institute, Tokyo, dspaa
PROTEOLYSIS OF PLASMINOGEN BY POLYMORPHO NUCLEAR LEUKOCYTE Duboscq C., Genand V., Kordich L Laboratorio de Hemostasia y Tmmbosis - Facultad de Ciencias Exactas y Naturales - Universidad de Buenos Aires - Argentina
It h ~ been suggested by previous inv'estig~ors that other rneohat~isrn rn~ye~ist to iniciate u PA sctivation of pla.~minogen reJtherth sa solely s.t:tributing it t:hrough the intrinsic property'of uPAR. We atternpted to elucidate this phenomen~byu~ing 2 UK Me.be When e×ogenous uPA is s~:lded to PMk.-stimulated U937 cel Is, there is ~33~; increase of tots] uPA [ endogenous s.nd exogenous] bound ~,s determined b'y ELISA by using a broad ba..~ed &finit y UK ~ 5E8D3 produced in our IoJ3orator,/. T,~o other UK monoolonsJs were used to block the endogenous cellbound uPA on U937 adhesive cells seeded on "1"24wells. Blocking wss done overnight at 4 C. Plssminogen-dch fibrin oel were overlaved directlyon the s.dhesi're ¢ell:s and incubated over night.at 37 C. "lhere ,#~ a eomplet • inhibitiorl of fibrinolysis e:~'~ibited by 1OA7A4 UK I ~ ~nd partied inhibition by 2F4B9 UK Ms.b. However non4blooked cell levers show ed oomplete fibHnolvsis. Another set ,of cells of the ssme batch, ~,ere in cuba.t:ed with e >0ogenous u PA over n ight, the n blocked with ~ ~nd subsequently the plssrnir~ogen-rich fibrin gel wss overisved on top of the cell laver, inct4bated overnight, ~d.fibrinol',/sis ~,~ observed in ell condition s, blocked ~ d non -blocked cell lave re. The two Wpes of UK I ~ were net unsble to inhibit the f ibrinol'.ctie sctivityof ecogenousu PA is s~ spected to be envolve d in a te replete ra echa~is rn ~here i n u PA bin ds to t h • plasrninogen prose n t in the cell surf~e stitnulating f ibri nolysis. This further Sulaports findings that the simultaneous presence of u PA an d plasminog en on the c • II surfsce results in th • generati on of pls.srnin, speJ't fro rn the sctivation o~used by o e II-bound u PA
Elastase like proteins (ELP) of human granulocytes degrade plasminogen producing several fragments, one of them called mini- plasminogen. Polymorphonuclear lcokocytes (PMN) were isolated from human peripheral blood by Boyun method. Two volume of PMN (10 8 cell/rnl) preincubated with cytochalasin B (10 paX,l)was mixed with one volume of: a) PLG (1 mg/ml) (Assay A) b) PLG (0.2 mg/ml) (Assay B) c) Normal hun~an plasma (Assay C) d) PLG (l mg/ml) and(xt antitrypsina (AT) (6 mg/ml) (Assay D). Mixlure were stimulated by FMLP (33 gM), incubated at 37°C during 12 he. and the reaction was stopped with phenilmethylsulfonyl fluoride. Then the supernatant was store at -20°C.Degranulation was measured by elastase and mieloperoxidase activity (% Degranulation= 55:t:5%). Plasminogen and its proteolytic fragments were investigated by SDS-PAGE and Western blot using a peliclonal anti-human plasminogcn antibody. Western blot of A showed three bands between 45 Kd and 70 Kd, similar to fragments obtained by limited proteolysis in vitro with leukocyte elastase. Western blot of assay B and C showed neither an intact plasminogen's band nor any kind of proteulytic fragments. Assay D presented only one band corresponding to intact plasminogen showing that Qqantitrypsin was enough to inhibit all proteolytic activity. These results show that the plasmatic AT was not able to inhibit plasminogen's proteolysis when PMN degranulation was stimulated in presence of human plasma.
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HIGH LOCAL CONCENTRATION O F P A I - 2 -- L E S S INFLAMMATORY SYMPTOMS l, 2,3Kinnby B, 2 Matsson L and 3~stedt B Departments of Ipedodontics and 2pefiodontology, School of Dentistr3, Lund University, and 3Research Laboratory of the Department of
ACTIVATION OF METALLOPROTEINASES FROM METASTATIC CARCINOMATOUS LINE BY PHOSPHORYLATED UROKINASE-PLASMINOGEN
Obstetrics and Gynecology, Lurid University Hospital, Sweden. The clinical manifestations of gingival inflammation in reaction to bacterial plaque are aggravated during pregnancy. In vitro studies suggest a hormonal influence on PAI-2 (1), of importance for tissue proteolysis. A disturbance of the fibrinolytic system could thus help to explain "pregnancy gingivitis". The aim of this study was therefore to investigate the plasminogen activators and their inhibitors in gingival crevicular fluid (GCF) in pregnant and non-pregnant states. GCF was sampled in 14 women in pregnant and post-pregnant states. The gingival condition was assessed by an ordinal scale, grading swelling and redness (the gingival index -- GI) and the amount of bacterial plaque by the plaque index (PI). The ratio of the number of sites with gingivitis to the number of sites with plaque was calculated (G/P-ratio). Based on differences in G/P-ratio between pregnancy and post-pregnancy, subgrouping was done into a high-reacting (HR) and a low-reacting (LR) group. Levels of t-PA, u-PA, PAI-I and PAl-2 in GCF were determined with ELISAs and 17B-estradiol and progesterone in serum with RIAs. For each individual the differences (A) between pregnancy and post-pregnancy in hormone levels as well as PAs and PAIs were calculated. A significant correlation between Aprogesterone and APAI-2 was noted: the higher Aprogesterone, the lower APAI-2 (r2=0.27; P : 0 . 0 4 8 ) The women in the HR group showed loweror unchanged PAI-2 in GCF during pregnancy (mean -5nM), while those in the LR group showed a great increase (mean +25riM). The lower inhibitory capacity in terms of low concentrations of P A I - 2 during pregnancy in women with more pronounced inflammatory reaction indicates that the fibrinolytic system may be involved in pathogenesis of pregnancy gingivitis and implies that PAl-2 serves as an inhibitor of importance for tissue proteolysis. The present finding contributes to the explanation of pregnancy gingivitis and adds to the understanding of the significance of PAI-2 in tissue proteolysis I. Kinnby B, Astedt B, Cassl6n B. Fibrinolysis 1995; 9:152-1 56
1Takahashi
K, 2 K o h S and 3 K w a a n
CELLS OF A TYROSINEACTIVATOR
HC
1Department of Physiology, Shimane Medical College, Izumo 693, Japan, 2Department of Internal Medicine, Shinshu University, Matsumoto 390, Japan, and 3Hematology/Oncology, School of Medicine, Northwestern University, Chicago, II 60611, USA. Urokinase-plasminogen activator (uPA) associated with surface receptors of various types of tumor cells is generally considered to involve in cellular invasion by braking down proteins of extracellular matrix. Other proteaees such as collagenases or gelatinases are also important in the destruction of the surrounding architecture. To answer the question whether uPA is able to activate the latent matrix metalloproteinases (MMP) associated with a metastatic carcinomatous cell line, Detroit 562, affinity purified MMPs on a column of gelatin-Sepharose from the conditioned medium or membrane fractions were used to test either by cellular phosphorylated uPA (p-uPA)or urinary uPA. In comparison with this, the latter was labeled with biotin and phosphorylated by the incubation with materials co-precipitated with antipp60 c-src antibody or with p43 v'abl. They were evaluated by blots ol SDSPAGE gels by use of avidin-conjugated peroxidase or anti-mouse IgG conjugated with peroxidase following by enhanced chemiluminescence. MMPs were examined by gelatin-containing zymography and immunoblots using antibodies against MMPs. Both the plasminogen and uPA or p-uPA were immobilized on disks of nitrocellulose filters and incubated with the MMP. Activation of plasminogen was examined by use of a synthetic substrate, S-2251, and that of MMPs was by determinations at 420 nm of hydrolysates of a synthetic substrate (PZ-PLGPR). MMP-2 and MMP-9 were identified to be associated with membrane fractions and they were found to be activated solely by the p-uPA. Addition of plasminogen did not enhance the activation of MMPs. It was also able to activate plasminogen at a higher rate than that of unphosphorylated one. Inhibitors (PAl-1 and PAl-2) did not interfere the p-uPA activity. Thus, it suggests that inhibitor resistant p-uPA and MMPs may involve in a concomitant functional network of proteases on cell surfaces enabling an efficient hydrolysis of matrix proteins.
220
221
ELUCIDATION OF THE INHIBITORY PRO PER'P/OF "rwo UK MONOCLONALS IN EX'IRACELLULAR PRO'lEO LVSIS USING U93 7 CELL S. THE b IFFERENCE OF THE INHIB ITORY PRO PERTV TOWARDS ENDOGENOUS AND EXOGENOUS UPA GIo,~ttaV. Ishb'aki, $hiaem H ayashi Thrombosis Chernical Institute, Tokyo, dspaa
PROTEOLYSIS OF PLASMINOGEN BY POLYMORPHO NUCLEAR LEUKOCYTE Duboscq C., Genand V., Kordich L Laboratorio de Hemostasia y Tmmbosis - Facultad de Ciencias Exactas y Naturales - Universidad de Buenos Aires - Argentina
It h ~ been suggested by previous inv'estig~ors that other rneohat~isrn rn~ye~ist to iniciate u PA sctivation of pla.~minogen reJtherth sa solely s.t:tributing it t:hrough the intrinsic property'of uPAR. We atternpted to elucidate this phenomen~byu~ing 2 UK Me.be When e×ogenous uPA is s~:lded to PMk.-stimulated U937 cel Is, there is ~33~; increase of tots] uPA [ endogenous s.nd exogenous] bound ~,s determined b'y ELISA by using a broad ba..~ed &finit y UK ~ 5E8D3 produced in our IoJ3orator,/. T,~o other UK monoolonsJs were used to block the endogenous cellbound uPA on U937 adhesive cells seeded on "1"24wells. Blocking wss done overnight at 4 C. Plssminogen-dch fibrin oel were overlaved directlyon the s.dhesi're ¢ell:s and incubated over night.at 37 C. "lhere ,#~ a eomplet • inhibitiorl of fibrinolysis e:~'~ibited by 1OA7A4 UK I ~ ~nd partied inhibition by 2F4B9 UK Ms.b. However non4blooked cell levers show ed oomplete fibHnolvsis. Another set ,of cells of the ssme batch, ~,ere in cuba.t:ed with e >0ogenous u PA over n ight, the n blocked with ~ ~nd subsequently the plssrnir~ogen-rich fibrin gel wss overisved on top of the cell laver, inct4bated overnight, ~d.fibrinol',/sis ~,~ observed in ell condition s, blocked ~ d non -blocked cell lave re. The two Wpes of UK I ~ were net unsble to inhibit the f ibrinol'.ctie sctivityof e
Elastase like proteins (ELP) of human granulocytes degrade plasminogen producing several fragments, one of them called mini- plasminogen. Polymorphonuclear lcokocytes (PMN) were isolated from human peripheral blood by Boyun method. Two volume of PMN (10 8 cell/rnl) preincubated with cytochalasin B (10 paX,l)was mixed with one volume of: a) PLG (1 mg/ml) (Assay A) b) PLG (0.2 mg/ml) (Assay B) c) Normal hun~an plasma (Assay C) d) PLG (l mg/ml) and(xt antitrypsina (AT) (6 mg/ml) (Assay D). Mixlure were stimulated by FMLP (33 gM), incubated at 37°C during 12 he. and the reaction was stopped with phenilmethylsulfonyl fluoride. Then the supernatant was store at -20°C.Degranulation was measured by elastase and mieloperoxidase activity (% Degranulation= 55:t:5%). Plasminogen and its proteolytic fragments were investigated by SDS-PAGE and Western blot using a peliclonal anti-human plasminogcn antibody. Western blot of A showed three bands between 45 Kd and 70 Kd, similar to fragments obtained by limited proteolysis in vitro with leukocyte elastase. Western blot of assay B and C showed neither an intact plasminogen's band nor any kind of proteulytic fragments. Assay D presented only one band corresponding to intact plasminogen showing that Qqantitrypsin was enough to inhibit all proteolytic activity. These results show that the plasmatic AT was not able to inhibit plasminogen's proteolysis when PMN degranulation was stimulated in presence of human plasma.