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The effect of isolated platelet plasma membranes on fibrinolysis
j M o l Cell C a r d i o l 19 ( S u p p l e m e n t IV) (1987)
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INTERACTION OF I~ATI~ET PROTEIN 4 . 1 WI~I THE CYTOSKELETON OF ACTIVATED I~AT!~ETS. ~. C. H o m e , C. A. O o l i s c h , a n d V. T. M a r c h e s i , Y a l e U n i v e r s i t y School of ~edicine, New H a v e n , c'r. Platelets, like other nonerythroid cells, contain an analog of protein 4.1, a component o f t h e e r y t h r o c y t e membrane s k e l e t o n . The g o a l s o f t h e s t u d y w e r e a) t o compare the structural and immunological properties of the platelet and erythrocyte proteins, a n d b) t o c h a r a c t e r i z e the interaction of the platelet protein with the cytoskeleton in resting and activated platelets. ~cstern blotting of platelets, using polyclonal IgG r a i s e d a g a i n s t i n t a c t e r y t h r o c y t e protein 4.1 or against synthetic peptides with sequences derived from specific sites within the erythrocyte protein detected a protein slightly smaller than the erythrocyte f o r m (M --~77kI)a). r The d i s t i n c t i v e positions of cysteine residues within the erythrocyte protein ~ere conserved in the platelet protein. Most o f t h e s e q u e n c e s d e t e c t e d b y W e s t e r n blotting were present in approximately equal levels in platclets and e rythrocytes. An e x c e p t i o n w a s a s e q u e n c e i n t h e s p e c t r i n / a c t i n - b i n d i n g domain of the erythrocyte p r o t e i n w h i c h was m a r k e d l y r e d u c e d i n p l a t e l e t s . Washed p l a t e l e t s were activated with thrombin (0.2-1U/ml) or with 12-0-tetradecanoyl phorbol 13-acetate (TPA), a n d lysed2+ w i t h T r i t o n X-100 in" t h e p r e s e n c e o f l e u p e p t i n a n d F~TA t o i n h i b i t Ca - a c t i v a t e d protease. The c y t o s k e l c t o n s w e r e i s o l a t e d and a s s a y e d f o r p r o t e i n 4.1 by ~cstern blotting. Activating platelets w i t h e i t h e r t h r o m b i n o r TPA i n c r e a s e d t h e amount o f c y t o s k e l e t o n - a s s o c i a t e d protein 4.1.
4 4 A PURE PRIMARY RESPONSEPATHWAY IN PLATELETS. G.D. MacFarlane, M.C. Herzberg, G.H.R. Rao, q. Zhao, K.D. Hauch. Departmentof Periodontics, University of Minnesota, Minneapolis, MN 55455 Certain viridans streptococci interact with platelets in v i t r o , resulting in activation, dense granule secretion, and aggregation into thrombi. Platelet response mechanisms to oral streptococci have not been well characterized. In contrast to other agonists, bacterial dilutions result in increased lag time to onset of aggregation, with extent nearly invariant, suggesting an independencefrom secondary wave responses. S. sanguis cells induced aggregation of platelets from patients with Hermansky-Pudlak syndrome. Inhibitors of secondary wave aggregation (apyrase, lmg/mL; CP/CPK, lmM:5OU/mL; crosslinked azidoATP, lmM; ASA, 100 pM; indomethacin, 10 pM) or target potential second messengers (PMA, O.l~g/mL; yohimbine, 20~M; stelazine, lOOpM) reduced the extent of aggregation and dense granule secretion of normal platelets in response to other agonists. These have l i t t l e effect on S. sanguisinduced aggregation. Prostaglandin inhibitors of primary aggregation TPGEI & PGI2, lOOnM) increased the lag time to onset, but had only minimal effects on the extent of aggregation. Therefore, the response pathway to S. sanguis is independent of secretion of dense granule products, may be independent of secondary wave responses, representing fewer steps between the receptor and the response.
45 THE EFFECT OF ISOLATED PLATELET F~_ASMAMEMBRANES ON FIBRINOLYSIS. W. P. FAY, and W. G. OWEN. HematologyResearch, Mayo Clinic, Rochester, Minnesota. Platelets have been found to both promote and inhibit fibrinolysis. We find that in a reconstituted clot lysis assay containing plasminogen (Pg), tissue plasminogen activator (t-PA), and fluorescein-labelled f i b r i n , isolated human platelet plasma membranes (PPM) cause a 90% reduction in the rate of fibrinolysis. However,analysis of the individual reactions comprising this system revealed opposing effects. Whenincubated with Pg (20 mcg/mL) and t-PA (200 ng/mL), PPM caused a 10-20 fold acceleration of plasmin formation. This degree of acceleration was 5-i0 fold greater than that observed with membranes prepared from washed human erythrocytes, but 25-fold less than that observed with soluble fibrin(ogen) fragments. Conversely, fibrin lysis by plasmin (0.5 mcg/mL) was reduced 80% by PPM, which had no effect on hydrolysis of the plasmin substrate, H-D-Nle-HHT-Lys-pNA. Erythrocyte membranes had no effect on plasmin-catalyzed fibrinolysis. These findings demonstrate that PPM have opposing effects on different components of the fibrinolytic system, and illustrate how platelets may enhance, inhibit, or have no effect on fibrinolysis as experimental conditions differ.
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INTERACTION OF I~ATI~ET PROTEIN 4 . 1 WI~I THE CYTOSKELETON OF ACTIVATED I~AT!~ETS. ~. C. H o m e , C. A. O o l i s c h , a n d V. T. M a r c h e s i , Y a l e U n i v e r s i t y School of ~edicine, New H a v e n , c'r. Platelets, like other nonerythroid cells, contain an analog of protein 4.1, a component o f t h e e r y t h r o c y t e membrane s k e l e t o n . The g o a l s o f t h e s t u d y w e r e a) t o compare the structural and immunological properties of the platelet and erythrocyte proteins, a n d b) t o c h a r a c t e r i z e the interaction of the platelet protein with the cytoskeleton in resting and activated platelets. ~cstern blotting of platelets, using polyclonal IgG r a i s e d a g a i n s t i n t a c t e r y t h r o c y t e protein 4.1 or against synthetic peptides with sequences derived from specific sites within the erythrocyte protein detected a protein slightly smaller than the erythrocyte f o r m (M --~77kI)a). r The d i s t i n c t i v e positions of cysteine residues within the erythrocyte protein ~ere conserved in the platelet protein. Most o f t h e s e q u e n c e s d e t e c t e d b y W e s t e r n blotting were present in approximately equal levels in platclets and e rythrocytes. An e x c e p t i o n w a s a s e q u e n c e i n t h e s p e c t r i n / a c t i n - b i n d i n g domain of the erythrocyte p r o t e i n w h i c h was m a r k e d l y r e d u c e d i n p l a t e l e t s . Washed p l a t e l e t s were activated with thrombin (0.2-1U/ml) or with 12-0-tetradecanoyl phorbol 13-acetate (TPA), a n d lysed2+ w i t h T r i t o n X-100 in" t h e p r e s e n c e o f l e u p e p t i n a n d F~TA t o i n h i b i t Ca - a c t i v a t e d protease. The c y t o s k e l c t o n s w e r e i s o l a t e d and a s s a y e d f o r p r o t e i n 4.1 by ~cstern blotting. Activating platelets w i t h e i t h e r t h r o m b i n o r TPA i n c r e a s e d t h e amount o f c y t o s k e l e t o n - a s s o c i a t e d protein 4.1.
4 4 A PURE PRIMARY RESPONSEPATHWAY IN PLATELETS. G.D. MacFarlane, M.C. Herzberg, G.H.R. Rao, q. Zhao, K.D. Hauch. Departmentof Periodontics, University of Minnesota, Minneapolis, MN 55455 Certain viridans streptococci interact with platelets in v i t r o , resulting in activation, dense granule secretion, and aggregation into thrombi. Platelet response mechanisms to oral streptococci have not been well characterized. In contrast to other agonists, bacterial dilutions result in increased lag time to onset of aggregation, with extent nearly invariant, suggesting an independencefrom secondary wave responses. S. sanguis cells induced aggregation of platelets from patients with Hermansky-Pudlak syndrome. Inhibitors of secondary wave aggregation (apyrase, lmg/mL; CP/CPK, lmM:5OU/mL; crosslinked azidoATP, lmM; ASA, 100 pM; indomethacin, 10 pM) or target potential second messengers (PMA, O.l~g/mL; yohimbine, 20~M; stelazine, lOOpM) reduced the extent of aggregation and dense granule secretion of normal platelets in response to other agonists. These have l i t t l e effect on S. sanguisinduced aggregation. Prostaglandin inhibitors of primary aggregation TPGEI & PGI2, lOOnM) increased the lag time to onset, but had only minimal effects on the extent of aggregation. Therefore, the response pathway to S. sanguis is independent of secretion of dense granule products, may be independent of secondary wave responses, representing fewer steps between the receptor and the response.
45 THE EFFECT OF ISOLATED PLATELET F~_ASMAMEMBRANES ON FIBRINOLYSIS. W. P. FAY, and W. G. OWEN. HematologyResearch, Mayo Clinic, Rochester, Minnesota. Platelets have been found to both promote and inhibit fibrinolysis. We find that in a reconstituted clot lysis assay containing plasminogen (Pg), tissue plasminogen activator (t-PA), and fluorescein-labelled f i b r i n , isolated human platelet plasma membranes (PPM) cause a 90% reduction in the rate of fibrinolysis. However,analysis of the individual reactions comprising this system revealed opposing effects. Whenincubated with Pg (20 mcg/mL) and t-PA (200 ng/mL), PPM caused a 10-20 fold acceleration of plasmin formation. This degree of acceleration was 5-i0 fold greater than that observed with membranes prepared from washed human erythrocytes, but 25-fold less than that observed with soluble fibrin(ogen) fragments. Conversely, fibrin lysis by plasmin (0.5 mcg/mL) was reduced 80% by PPM, which had no effect on hydrolysis of the plasmin substrate, H-D-Nle-HHT-Lys-pNA. Erythrocyte membranes had no effect on plasmin-catalyzed fibrinolysis. These findings demonstrate that PPM have opposing effects on different components of the fibrinolytic system, and illustrate how platelets may enhance, inhibit, or have no effect on fibrinolysis as experimental conditions differ.
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