title: Regulation of platelet glycoprotein IIb/IIIa (integrin alpha IIB beta 3) function via the thrombin receptor.
abstract: Binding sites on glycoprotein (GP) IIb/IIIa exposed by 0.5 unit/ml alpha-thrombin are insensitive to prostaglandin I2 (PGI2), in contrast with sites exposed by ADP or platelet-activating factor. Here we show that the thrombin receptor agonist peptide (TRAP) (SFLLRN; 15 microM) opens almost the same number of GPIIb/IIIa molecules as 0.5 unit/ml alpha-thrombin (64840 +/- 8920 compared with 81050 +/- 6030 molecules of fibronectin bound/platelet), but these sites rapidly close on addition of PGI2. To investigate whether alpha-thrombin and TRAP initiate different signalling pathways, we measured phospholipase C (PLC)-mediated control of GPIIb/IIIa and its sensitivity to cyclic AMP. Optimal concentrations of alpha-thrombin and TRAP activated PLC maximally, but TRAP induced only about 50% protein kinase C PKC) activation after 10 min stimulation compared with alpha-thrombin. These concentrations also suppressed PGI2-induced cyclic AMP accumulation, with alpha-thrombin inducing complete inhibition and TRAP about 10% less. Direct activation of PKC by phorbol 12-myristate 13-acetate confirmed earlier observations that PGI2-induced cyclic AMP accumulation is partly inhibited via PKC. Applying different concentration of alpha-thrombin, TRAP or a combination of alpha-thrombin and the thrombin receptor inhibitory peptide (TRIP) (Mpr-F-Cha-Cha-RKPNDK-NH2; 800 microM) (Mpr, 3-mercaptopropionic acid; Cha, cyclohexylalanine), we show that the different means of stimulating the thrombin receptor all suppressed PGI2-induced cyclic AMP accumulation via (i) activation of PKC and (ii) activation of the heterotrimeric G-protein, Gi. We conclude that complete inhibition of cyclic AMP accumulation requires activation of both PKC and Gi, as observed with 0.5 unit/ml alpha-thrombin. Although TRAP almost fully exposes GPIIb/IIIa, its activation of PKC is incomplete, enabling PGI2 to raise cyclic AMP concentration from 1.4 +/- 0.7 to 4.1 +/- 1.3 nmol/10(11) platelets (P < 0.005) which is sufficient to close exposed GPIIb/IIIa molecules.
authors.affiliation: Department of Haematology, University Hospital Utrecht, The Netherlands.
authors.forename: A N
authors.initials: AN
authors.lastname: Giesberts
authors.forename: G
authors.initials: G
authors.lastname: van Willigen
authors.forename: E G
authors.initials: EG
authors.lastname: Lapetina
authors.forename: J W
authors.initials: JW
authors.lastname: Akkerman
mesh_terms: D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000262:Adenylyl Cyclases / Q000378:metabolism; D000595:Amino Acid Sequence; D000242:Cyclic AMP / Q000378:metabolism; D004789:Enzyme Activation; D006801:Humans; D016023:Integrins / Q000378:metabolism*; D008969:Molecular Sequence Data; D010455:Peptides / Q000494:pharmacology; D037342:Pertussis Toxin; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D011493:Protein Kinase C / Q000378:metabolism; D016024:Receptors, Cytoadhesin / Q000378:metabolism*; D018179:Receptors, Thrombin / Q000819:agonists / Q000378:metabolism*; D010738:Type C Phospholipases / Q000378:metabolism; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
chemical_list: D016023:Integrins; D010455:Peptides; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D016024:Receptors, Cytoadhesin; D018179:Receptors, Thrombin; D010566:Virulence Factors, Bordetella; D000246:Adenosine Diphosphate Ribose; D000242:Cyclic AMP; D037342:Pertussis Toxin; D011493:Protein Kinase C; D010738:Type C Phospholipases; D000262:Adenylyl Cyclases