Normal ADP-Induced Aggregation and Absence of Dissociation of the Membrane GP IIb-IIIa Complex of Intact Rat Platelets Pretreated with EDTA
Christian Gachet
INSERM U.311, Biologie et Pharmacologie des Interactions du Sang avec les Vaisseaux et les Biomatériaux, Centre Régional de Transfusion Sanguine, Strasbourg, France
,
Anita Stierlé
INSERM U.311, Biologie et Pharmacologie des Interactions du Sang avec les Vaisseaux et les Biomatériaux, Centre Régional de Transfusion Sanguine, Strasbourg, France
,
Philippe Ohlmann
INSERM U.311, Biologie et Pharmacologie des Interactions du Sang avec les Vaisseaux et les Biomatériaux, Centre Régional de Transfusion Sanguine, Strasbourg, France
,
François Lanza
INSERM U.311, Biologie et Pharmacologie des Interactions du Sang avec les Vaisseaux et les Biomatériaux, Centre Régional de Transfusion Sanguine, Strasbourg, France
,
Daniel Hanau
INSERM U.311, Biologie et Pharmacologie des Interactions du Sang avec les Vaisseaux et les Biomatériaux, Centre Régional de Transfusion Sanguine, Strasbourg, France
,
Jean-Pierre Cazenave
INSERM U.311, Biologie et Pharmacologie des Interactions du Sang avec les Vaisseaux et les Biomatériaux, Centre Régional de Transfusion Sanguine, Strasbourg, France
ADP-induced platelet aggregation requires the presence of external calcium and fibrinogen. When human platelets are incubated for 30 min at 37° C with 5mM EDTA and then resuspended in a calcium containing medium, they lose their ability to bind fibrinogen and to aggregate in response to ADP stimulation. Under these conditions, the effect of EDTA is irreversible and accompanied by dissociation of the glycoprotein (GP) IIb-IIIa complex into its free subunits, GP IIb and GP IIIa. We studied the effect of incubation of intact rat platelets with 5 mM EDTA at 37° C from 30 to 120 min. EDTA treated rat platelets showed normal aggregation in response to 5 εM ADP in the presence of added purified rat fibrinogen and bound 125I-labeled rat fibrinogen at the same rate and magnitude after stimulation with 5 εM ADP as untreated platelets. Control and EDTA treated rat platelets, labeled or not with 125I and solubilized in Triton X-100, had a similar pattern of immunoprecipitates after crossed immunoelectrophoresis (CIE) analysis. The rat GP IIb-IIIa arc was located by incorporation of an 125I-labeled polyclonal anti-human GP IIb-IIIa antibody. In contrast, in experiments using rat platelet lysates, we demonstrated that the rat GP IIb-IIIa is a Ca2+-dependent heterodimer as it was dissociated by EDTA. Using SDS-PAGE and two-dimensional SDS-PAGE, the rat GP IIb-IIIa complex was found to have characteristics similar to the human complex with the exception that the light chain of the rat GP IIb was undetectable after 125I surface labeling. Small structural particularities of rat GP IIb-IIIa might still explain the functional differences observed between intact rat and human platelets treated with EDTA.
References
1
Kinlough-Rathbone RL,
Mustard JF,
Packham MA,
Perry PW,
Reimers HJ,
Cazenave J-P.
Properties of washed human platelets. Thromb Haemostas 1977; 37: 291-308
3
Marguerie GA,
Plow EF,
Edgington TS.
Human platelets possess an inducible and saturable receptor specific for fibrinogen. J Biol Chem 1979; 254: 5357-463
7
Hagen I,
Nurden AT,
Bjerrum OJ,
Solum NO,
Caen JP.
Immunochemical evidence for protein abnormalities in platelets, from patients with Glanzmann's thrombasthenia and the Bernard-Soulier syndrome. J Clin Invest 1980; 65: 722-31
12
McEver RP,
Bennett EM,
Martin MN.
Identification of two structurally and functionally distinct sites on human platelets membrane glycoprotein IIb-IIIa using monoclonal antibodies. J Biol Chem 1983; 258: 5269-75
13
Jennings LK,
Phillips DR.
Purification of glycoprotein IIb and IIIa from human platelets plasma membranes and characterization of a calcium dependent glycoprotein IIb-IIIa complex. J Biol Chem 1982; 257: 10458-66
16
Cazenave J-P,
Hemmendinger S,
Beretz A,
Sutter-Bay A,
Launay J.
L'agrégation plaquettaire: outil d'investigation clinique et d'étude pharmacologique. Ann Biol Clin 1983; 41: 167-79
20
Phillips DR,
Poh Agin P.
Platelet plasma membrane glycoproteins. Evidence for the presence of non equivalent disulfide bonds using non-reduced two dimensional electrophoresis. J Biol Chem 1977; 252: 2121-6
26
Gachet C,
Stierlé A,
Cazenave J-P,
Ohlmann P,
Lanza F,
Bouloux C,
Maffrand JP.
The thienopyridine PCR 4099 selectively inhibits ADP-induced platelet aggregation and fibrinogen binding without modifying the membrane glycoprotein IIb-IIIa complex in rat and in man. Biochem Pharmacol 1990; 40: 229-38
27
Fitzgerald LA,
Charo IF,
Phillips DR.
Human and bovine endothelial cells synthesize membrane proteins similar to human platelet glycoproteins IIb and IIIa. J Biol Chem 1985; 260: 10893-6
29
Lam SCT,
Plow EF,
D'Souza SE,
Cheresh DA,
Frelinger AL,
Ginsberg MH.
Isolation and characterization of a platelet membrane protein related to the vitronectin receptor. J Biol Chem 1989; 264: 3742-9
31
Poncz M,
Newman PJ.
Analysis of rodent platelet glycoprotein IIb: evidence for evolutionarily conserved domains and alternative proteolytic processing. Blood 1990; 75: 1282-9
32
Harfenist EJ,
Packham MA,
Mustard JF.
Expression of fibrinogen on the surface of ADP-stimulated platelets: comparison of human and rabbit platelets. Thromb Haemostas 1988; 59: 319-22
34
Harfenist EJ,
Packham MA,
Mustard JF.
Effects of the cell adhesion peptide, Arg-Gly-Asp-Ser, on responses of washed platelets from humans, rabbits and rats. Blood 1988; 71: 132-6
