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Efficient tyrosine phosphorylation of proteins after activation of platelets with thrombin depends on intact glycoprotein Ib
Authors:Maribel Diaz-Ricart  Berta Fuste  Eva Estebanell  Raul Tonda  Miguel Lozano  Gines Escolar
Affiliation:1. Servicio de Hemoterapia y Hemostasia, Hospital Clinic, IDIBAPS, Barcelona, Spainmdiaz@clinic.ub.es;3. Servicio de Hemoterapia y Hemostasia, Hospital Clinic, IDIBAPS, Barcelona, Spain
Abstract:The role of platelet glycoprotein Ib as a thrombin receptor has been often a subject of controversy. We have investigated the role of the thrombin receptors, GPIb and protease-activated receptor (PAR)-1. Tyrosine phosphorylation in whole platelet lysates and in cytoskeletal extracts was evaluated after activation with thrombin and with the thrombin receptor-activating peptide (TRAP). Different experimental approaches were applied including: (i) congenital deficiency of platelet GPIb (Bernard Soulier syndrome, BSS), (ii) antibody to GPIb (AP1), (iii) selective protease cleavage (metalloprotease), and (iv) antibody to (PAR)-1. After activation of control platelets with thrombin or TRAP, multiple proteins became tyrosine phosphorylated in platelet lysates and some of them associated with the cytoskeletal fraction. These effects were absent in BSS platelets. Presence of AP1 or metalloprotease treatment showed an inhibitory effect when platelets were activated with a low concentration of thrombin or TRAP. Blockade of PAR-1 with a specific antibody, SPAN 12, inhibited platelet response to both agonists. This study reinforces the hypothesis that GPIb is the high-affinity receptor for thrombin. The signaling mechanisms occurring through tyrosine phosphorylation of proteins triggered by thrombin seem to be dependent on intact GPIb. Moreover, our results indicate that both receptors, GPIb and PAR-1, are necessary to achieve a full platelet response to thrombin.
Keywords:Glycoprotein Ib  PAR-1  tyrosine phosphorylation  platelets
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