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Action of thrombin of platelets. Characterization of total and membrane proteins. Platelet-fibrinogen interaction
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ABSTRACTS OF 12TH INTNAT’L CONGRESS
Vol. 65, Suppl. 1
P274 PLATELET RELEASE REACTION: VlSUALJZATlON OF SECRETED FIBRINOGEN BY THE MONOCLONAL ANTIBODY (D4) DIRECTLY COUPLED TO COLLOIDAL GOLD J. Suttnar’, N. Belitser>, Y. Vekhch’, M. Anishukl, T. Pozdnyakoval 0. Gorkunl and J.E. Dyr’ L Institute of Hematology and Blood Transfusion, Prague, CS I Institute of Biochemistry, Kiev, Ukraine Gel filtered human platelets were stimulated with thrombio (0.05 NlH u/ml) in a protein-free medium containing 1 mM CaCl’. After 5 mitt of stimulation, the conversion of secreted fibrinogen to fibrin was stopped by the addition of 20-fold excess of hirudin and the samples were incubated for 20 min with the anti-fibrinogen monoclooal antibody (D4) directly coupled to 15 nm colloidal gold particles (D4-Au). Secreted fibrinogen was localized as being bound to discrete sites on the individual platelet surfaces and filling intercellular spaces of platelet microaggregates. The results obtained also showed that fibrin fibers preformed before the addition of D4-Au, either at platelet plasma membranes or in the lumina of secretory vacuoles, did not bind D4-Au, whereas dispersed and/or aggregated fibrinogen and fibrinogen-containing extracellular structures were heavily stained by the gold. Labeled contents of internal vacuoles and channels indicated that these compartments were “open” during incubation with D4-Au, being thus available for labeling.
P275 ACTION OF THROMBJN OF PLATELETS. CHARACTERIZATION OF TOTAL AND MEMBRANE PROTEINS. PLATELET-FIBRINOGEN INTERACTION V. Vila, V. Martfnex-Sales, E. R&anon Research Center, “La Fe” Universitary Hospital, Valencia, E Platelet protein obtained by cryofracture and solubilixation with SDS or by lysis with triton X-100 were charadericed by SDS-PAGE, immunotransfer and autoradiography. Twenty main platelet proteins, with molecular wheight of 390,000 to 20,000, were identified, and the major proteins of the outer membrane were shown to be: Fg, lb, Ifb, 138 Kd and Hla. The different solubilities of the proteins with the agents used made it possible to see that the proteins identified as actin, lb and 138 Kd partially dissolve when Triton X-l 00 at 1% is used. When platelets are activated with thrombin at concentrations ranging from 0.05 to 1 U/ml, slight variations in the electrophoretic pattern of the membrane proteins are observed, and the electrophoretic band identifided immunologically as albumin is the one that shows the most significant release. Electron microscopy reveals morphological alterations when platelets are activated with low concentrations of thrombin (0.05-0.5 U/ml), with no significant variations in the cytoplasmic lactate dehydrogenase content. Fibrinogen binding to platelets activated with thrombin increases linearly as the enzyme concentrations increases, reaching its maximum at a thrombin concentration of 0.2-0.3 U/ml. It is of function of the time course of tbrombio action on platelets, which reaches a plateau after 15 minutes.
ABSTRACTS OF 12TH INTNAT’L CONGRESS
Vol. 65, Suppl. 1
P274 PLATELET RELEASE REACTION: VlSUALJZATlON OF SECRETED FIBRINOGEN BY THE MONOCLONAL ANTIBODY (D4) DIRECTLY COUPLED TO COLLOIDAL GOLD J. Suttnar’, N. Belitser>, Y. Vekhch’, M. Anishukl, T. Pozdnyakoval 0. Gorkunl and J.E. Dyr’ L Institute of Hematology and Blood Transfusion, Prague, CS I Institute of Biochemistry, Kiev, Ukraine Gel filtered human platelets were stimulated with thrombio (0.05 NlH u/ml) in a protein-free medium containing 1 mM CaCl’. After 5 mitt of stimulation, the conversion of secreted fibrinogen to fibrin was stopped by the addition of 20-fold excess of hirudin and the samples were incubated for 20 min with the anti-fibrinogen monoclooal antibody (D4) directly coupled to 15 nm colloidal gold particles (D4-Au). Secreted fibrinogen was localized as being bound to discrete sites on the individual platelet surfaces and filling intercellular spaces of platelet microaggregates. The results obtained also showed that fibrin fibers preformed before the addition of D4-Au, either at platelet plasma membranes or in the lumina of secretory vacuoles, did not bind D4-Au, whereas dispersed and/or aggregated fibrinogen and fibrinogen-containing extracellular structures were heavily stained by the gold. Labeled contents of internal vacuoles and channels indicated that these compartments were “open” during incubation with D4-Au, being thus available for labeling.
P275 ACTION OF THROMBJN OF PLATELETS. CHARACTERIZATION OF TOTAL AND MEMBRANE PROTEINS. PLATELET-FIBRINOGEN INTERACTION V. Vila, V. Martfnex-Sales, E. R&anon Research Center, “La Fe” Universitary Hospital, Valencia, E Platelet protein obtained by cryofracture and solubilixation with SDS or by lysis with triton X-100 were charadericed by SDS-PAGE, immunotransfer and autoradiography. Twenty main platelet proteins, with molecular wheight of 390,000 to 20,000, were identified, and the major proteins of the outer membrane were shown to be: Fg, lb, Ifb, 138 Kd and Hla. The different solubilities of the proteins with the agents used made it possible to see that the proteins identified as actin, lb and 138 Kd partially dissolve when Triton X-l 00 at 1% is used. When platelets are activated with thrombin at concentrations ranging from 0.05 to 1 U/ml, slight variations in the electrophoretic pattern of the membrane proteins are observed, and the electrophoretic band identifided immunologically as albumin is the one that shows the most significant release. Electron microscopy reveals morphological alterations when platelets are activated with low concentrations of thrombin (0.05-0.5 U/ml), with no significant variations in the cytoplasmic lactate dehydrogenase content. Fibrinogen binding to platelets activated with thrombin increases linearly as the enzyme concentrations increases, reaching its maximum at a thrombin concentration of 0.2-0.3 U/ml. It is of function of the time course of tbrombio action on platelets, which reaches a plateau after 15 minutes.