Research Article The Phosphatase Inhibitor Calyculin-A Impairs Clot Retraction, Platelet Activation, and Thrombin Generation Renáta Hudák, 1 János Vincze, 2 László Csernoch, 2 Ildikó Beke Debreceni, 1 Tamás Oláh, 2 Ferenc Erdydi, 3 Kenneth J. Clemetson, 4 and János Kappelmayer 1 1 Department of Laboratory Medicine, Faculty of Medicine, University of Debrecen, 98 Nagyerdei krt., Debrecen 4032, Hungary 2 Department of Physiology, Faculty of Medicine, University of Debrecen, 98 Nagyerdei krt., Debrecen 4032, Hungary 3 Department of Medical Chemistry, Faculty of Medicine, University of Debrecen, 98 Nagyerdei krt., Debrecen 4032, Hungary 4 Department of Hematology, Inselspital, University of Bern, Murtenstrasse 40, 3008 Bern, Switzerland Correspondence should be addressed to J´ anos Kappelmayer; kappelmayer@med.unideb.hu Received 2 February 2017; Revised 10 April 2017; Accepted 24 April 2017; Published 7 June 2017 Academic Editor: Marlien Pieters Copyright © 2017 Ren´ ata Hud´ ak et al. Tis 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. Te aim of this study was to investigate the efect 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 bufer 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 fow cytometry, and thrombin generation was measured by a fuorimetric assay. For the intracellular Ca 2+ 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 Ca 2+ response and PS-expression in a subset of platelets. Te activated PRP displayed signifcantly 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 efect on calcium elevation and PS-expression, CLA signifcantly 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. 1. Introduction Platelets play a crucial role in the pathogenesis of atheroscle- rotic diseases including acute coronary syndrome or ischemic stroke that are leading causes of death and disability world- wide. Tese events are triggered by disruption of the endothe- lium and plaque rupture or during interventions on coro- naries, when platelets are tethered to surface-bound von Willebrand Factor (vWF), which initiates platelet activation and allows them to adhere to subendothelial components. Protease-activated receptor 1 (PAR-1), the primary platelet thrombin receptor, is G-protein-coupled. Te acti- vator of this receptor is the strongest platelet agonist and an important contributor to atherothrombotic processes. Modulation of the PAR-1 receptor is the target for novel and promising antiplatelet drugs [1, 2]. PAR-1 receptor activation via thrombin or relevant thrombin receptor activating peptides (TRAPs) results in a series of signaling events terminating in platelet shape change and granule secretion via the G 12/13 proteins and intracellular calcium release via the G q mediated inositol triphosphate (IP 3 ) pathway. Intracellular IP3 receptors can be directly activated by pharmacological agents like thiomersal that has been used previously as a calcium mobilizer and cell function- modulating agent [3]. Serine/threonine protein phosphatases (PP) are present in platelets predominantly as type 1 protein phosphatase (PP1) and type 2A protein phosphatase (PP2A) subtypes. Calyculin-A (CLA), a naturally occurring phosphatase inhibitor, present in marine sponges, in nanomolar concen- tration, primarily inhibits PP2A and indirectly PP1 [4, 5]. Hindawi BioMed Research International Volume 2017, Article ID 9795271, 10 pages https://doi.org/10.1155/2017/9795271