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BioMed Research International
Volume 2017, Article ID 9795271, 10 pages
Research Article

The Phosphatase Inhibitor Calyculin-A Impairs Clot Retraction, Platelet Activation, and Thrombin Generation

1Department of Laboratory Medicine, Faculty of Medicine, University of Debrecen, 98 Nagyerdei krt., Debrecen 4032, Hungary
2Department of Physiology, Faculty of Medicine, University of Debrecen, 98 Nagyerdei krt., Debrecen 4032, Hungary
3Department of Medical Chemistry, Faculty of Medicine, University of Debrecen, 98 Nagyerdei krt., Debrecen 4032, Hungary
4Department of Hematology, Inselspital, University of Bern, Murtenstrasse 40, 3008 Bern, Switzerland

Correspondence should be addressed to János Kappelmayer; uh.bedinu.dem@reyamleppak

Received 2 February 2017; Revised 10 April 2017; Accepted 24 April 2017; Published 7 June 2017

Academic Editor: Marlien Pieters

Copyright © 2017 Renáta Hudák et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


The aim of this study was to investigate the effect of the serine/threonine protein phosphatase inhibitor, calyculin-A (CLA), on clot formation and on the procoagulant activity of human platelets. Platelet-rich plasma (PRP) samples were preincubated with buffer or CLA and subsequently platelets were activated by the protease-activated receptor 1 (PAR-1) activator, thrombin receptor activating peptide (TRAP). Clot retraction was detected by observing clot morphology up to 1 hour, phosphatidylserine- (PS-) expression was studied by flow cytometry, and thrombin generation was measured by a fluorimetric assay. For the intracellular Ca2+ assay, platelets were loaded with calcium-indicator dyes and the measurements were carried out using a ratiometric method with real-time confocal microscopy. CLA preincubation inhibited clot retraction, PS-expression, and thrombin formation. TRAP activation elicited Ca2+ response and PS-expression in a subset of platelets. The activated PRP displayed significantly faster and enhanced thrombin generation compared to nonactivated samples. CLA pretreatment abrogated PS-exposure and clot retraction also in TRAP-activated samples. As a consequence of the inhibitory effect on calcium elevation and PS-expression, CLA significantly downregulated thrombin generation in PRP. Our results show that CLA pretreatment may be a useful tool to investigate platelet activation mechanisms that contribute to clot formation and thrombin generation.