The phosphatase inhibitor calyculin-a impairs clot retraction, platelet activation, and thrombin generation

Renáta Hudák, János Vincze, L. Csernoch, Ildikó Beke Debreceni, Tamás Oláh, F. Erdődi, Kenneth J. Clemetson, J. Kappelmayer

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

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.

Original languageEnglish
Article number9795271
JournalBioMed Research International
Volume2017
DOIs
Publication statusPublished - Jan 1 2017

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Clot Retraction
thrombin receptor peptide SFLLRNP
Platelet Activation
Platelets
Phosphoric Monoester Hydrolases
Thrombin
Phosphatidylserines
Chemical activation
Platelet-Rich Plasma
Blood Platelets
Plasmas
PAR-1 Receptor
Calcium
Assays
Phosphoprotein Phosphatases
Human Activities
Confocal Microscopy
Flow cytometry
calyculin A
Confocal microscopy

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Immunology and Microbiology(all)

Cite this

The phosphatase inhibitor calyculin-a impairs clot retraction, platelet activation, and thrombin generation. / Hudák, Renáta; Vincze, János; Csernoch, L.; Beke Debreceni, Ildikó; Oláh, Tamás; Erdődi, F.; Clemetson, Kenneth J.; Kappelmayer, J.

In: BioMed Research International, Vol. 2017, 9795271, 01.01.2017.

Research output: Contribution to journalArticle

Hudák, Renáta ; Vincze, János ; Csernoch, L. ; Beke Debreceni, Ildikó ; Oláh, Tamás ; Erdődi, F. ; Clemetson, Kenneth J. ; Kappelmayer, J. / The phosphatase inhibitor calyculin-a impairs clot retraction, platelet activation, and thrombin generation. In: BioMed Research International. 2017 ; Vol. 2017.
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