title: The protein kinase C inhibitor RO318220 potentiates thrombin-stimulated platelet-supported prothrombinase activity.
abstract: Prothrombinase activity was tested on thrombin- and SFLLRN-activated platelets treated with RO318220, a potent inhibitor of protein kinase C. RO318220 completely inhibited platelet dense and alpha-granule secretion at a concentration of 20 microM but had no effect on prothrombinase activity in the presence of excess factor Va (20 nM). This indicates that protein kinase C activity and agonist-initiated secretion are not necessary for the development of a procoagulant surface. Treatment with 75 to 150 microM RO318220 potentiated platelet-supported thrombin generation up to 280% of control platelets with no change in Kd appFXa. Treated with increasing concentrations of RO318220, an increasing proportion of thrombin-stimulated platelets bound annexin V with decreasing binding sites per platelet. A lower mean forward scatter (FSC-H) of platelets treated with RO318220 suggested platelet vesiculation as a result of RO318220 treatment; however, 100 microM calpeptin pretreatment eliminated the decrease in FSC-H without affecting either the increase in platelets positive for annexin V binding, the decrease in binding sites per platelet, or the 3-fold increase in prothrombinase activity. Thus, RO318220 appears to increase prothrombinase activity by increasing platelet responsiveness to thrombin rather than by inducing platelet vesiculation. This suggests that RO318220 inhibits a signaling molecule within a negative regulatory pathway that governs platelet procoagulant surface changes.
authors.affiliation: Sol Sherry Thrombosis Research Center, Department of Biochemistry, Temple University School of Medicine, Philadelphia, PA, USA. flon@astro.temple.edu
authors.forename: Fredda S
authors.initials: FS
authors.lastname: London
mesh_terms: D017304:Annexin A5 / Q000493:pharmacokinetics; D001792:Blood Platelets / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D015943:Factor Va / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D007211:Indoles / Q000494:pharmacology*; D010743:Phospholipids / Q000378:metabolism; D010974:Platelet Aggregation / Q000187:drug effects / Q000502:physiology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors*; D018179:Receptors, Thrombin / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D013917:Thrombin / Q000096:biosynthesis*; D013925:Thromboplastin / Q000378:metabolism*; D017186:Titrimetry
chemical_list: D017304:Annexin A5; D004791:Enzyme Inhibitors; D007211:Indoles; D010743:Phospholipids; D018179:Receptors, Thrombin; D015943:Factor Va; D013925:Thromboplastin; D011493:Protein Kinase C; D013917:Thrombin; C112908:protease-activated receptor 4; C064758:Ro 31-8220