title: Delayed-onset of procoagulant signalling revealed by kinetic analysis of COAT platelet formation.
abstract: The combined action of collagen and thrombin induces the formation of COAT platelets, which are characterised by a coat of procoagulant and adhesive molecules on their surface. Although recent work has started to highlight their clinical relevance, the exact mechanisms regulating the formation of procoagulant COAT platelets remain unclear. Therefore, we employed flow cytometry in order to visualise in real time surface and intracellular events following simultaneous platelet activation with convulxin and thrombin. After a rapid initial response pattern characterised by the homogenous activation of the fibrinogen receptor glycoprotein IIb/IIIa in all platelets, starting with a delay of about 2 minutes an increasing fraction transforms to procoagulant COAT platelets. Their surface is characterised by progressive loss of PAC-1 binding, expression of negative phospholipids and retention of α-granule von Willebrand factor. Intracellular events in procoagulant COAT platelets are a marked increase of free calcium into the low micromolar range, concomitantly with early depolarisation of the mitochondrial membrane and activation of caspase-3, while non-COAT platelets keep the intracellular free calcium in the nanomolar range and maintain an intact mitochondrial membrane. We show for the first time that the flow-cytometrically distinct fractions of COAT and non-COAT platelets differentially phosphorylate two signalling proteins, PKCα and p38MAPK, which may be involved in the regulation of the different calcium fluxes observed in COAT versus non-COAT platelets. This study demonstrates the utility of concomitant cellular and signalling evaluation using flow cytometry in order to further dissect the mechanisms underlying the dichotomous platelet response observed after collagen/thrombin stimulation.
authors.affiliation: Lorenzo Alberio, MD, Service et Laboratoire central d'hématologie, Centre Hospitalier Universitaire Vaudois, Rue du Bugnon 46, 1011 Lausanne, Switzerland, Tel.: +41 21 314 34 32, Fax: +41 21 314 43 23, E-mail: lorenzo.alberio@chuv.ch.
authors.forename: Lorenzo
authors.initials: L
authors.lastname: Alberio
authors.forename: Catherine
authors.initials: C
authors.lastname: Ravanat
authors.forename: Béatrice
authors.initials: B
authors.lastname: Hechler
authors.forename: Pierre H
authors.initials: PH
authors.lastname: Mangin
authors.forename: François
authors.initials: F
authors.lastname: Lanza
authors.forename: Christian
authors.initials: C
authors.lastname: Gachet
mesh_terms: D001777:Blood Coagulation; D001792:Blood Platelets / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D002469:Cell Separation; D002478:Cells, Cultured; D003094:Collagen / Q000378:metabolism*; D003435:Crotalid Venoms / Q000378:metabolism; D054641:Dual Specificity Phosphatase 2 / Q000378:metabolism; D005434:Flow Cytometry; D006801:Humans; D037181:Lectins, C-Type / Q000378:metabolism*; D008928:Mitochondria / Q000378:metabolism*; D015539:Platelet Activation; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000378:metabolism; D011485:Protein Binding; D011493:Protein Kinase C / Q000378:metabolism; D015398:Signal Transduction; D013917:Thrombin / Q000378:metabolism*; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism; D014841:von Willebrand Factor / Q000378:metabolism
chemical_list: D003435:Crotalid Venoms; D037181:Lectins, C-Type; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D014841:von Willebrand Factor; C028890:convulxin; D003094:Collagen; D011493:Protein Kinase C; D048051:p38 Mitogen-Activated Protein Kinases; C517032:DUSP2 protein, human; D054641:Dual Specificity Phosphatase 2; D013917:Thrombin; D002118:Calcium
keywords: COAT; Platelets; apoptosis; calcium; flow cytometry; intracellular signalling; phosphorylation; procoagulant; surface