Thromb Haemost 2017; 117(06): 1101-1114
DOI: 10.1160/TH16-09-0711
Cellular Haemostasis and Platelets
Schattauer GmbH

Delayed-onset of procoagulant signalling revealed by kinetic analysis of COAT platelet formation

Lorenzo Alberio
1  UMR S949 INSERM, Établissement Français du Sang Grand Est, University of Strasbourg, Strasbourg, France
2  Division of Hematology and Central Hematology Laboratory, Centre Hospitalier Universitaire Vaudois, University of Lausanne, Lausanne, Switzerland
,
Catherine Ravanat
1  UMR S949 INSERM, Établissement Français du Sang Grand Est, University of Strasbourg, Strasbourg, France
,
Béatrice Hechler
1  UMR S949 INSERM, Établissement Français du Sang Grand Est, University of Strasbourg, Strasbourg, France
,
Pierre H. Mangin
1  UMR S949 INSERM, Établissement Français du Sang Grand Est, University of Strasbourg, Strasbourg, France
,
François Lanza
1  UMR S949 INSERM, Établissement Français du Sang Grand Est, University of Strasbourg, Strasbourg, France
,
Christian Gachet
1  UMR S949 INSERM, Établissement Français du Sang Grand Est, University of Strasbourg, Strasbourg, France
› Author Affiliations
Financial support: This study was performed during a six month sabbatical leave (from March to August 2011) granted from the INSELSpital, University Hospital of Bern, Switzerland to LA. Additional funding was provided by a research grant from the ISTH2007 Presidential Fund (LA) and an unrestricted grant from NovoNordisk (LA).
Further Information

Publication History

Received: 19 September 2016

Accepted after major revision: 19 March 2017

Publication Date:
28 November 2017 (online)

Summary

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.

Supplementary Material to this article is available online at www.thrombosis-online.com.