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Updated: Jun 10, 2026

Characterizing Modulators of Protease-Activated Receptors with a Calcium Mobilization Assay Using a Plate Reader
Published on: May 24, 2024
Protein phosphatase inhibitor calyculin-A modulates activation markers in TRAP-stimulated human platelets
Zsuzsa Simon1, Andrea Kiss, Ferenc Erdödi
1Department of Clinical Biochemistry and Molecular Pathology, Hungarian Academy of Sciences, Research Center for Molecular Medicine, Medical and Health Science Center, University of Debrecen, Hungary.
Abstract:
Platelet activation is accompanied with the phosphorylation of a number of proteins on serine (Ser) and threonine (Thr) residues. The phosphorylation level of these proteins is dependent upon the protein kinase/phosphatase activity ratio. The aim of this study was to investigate the consequences of inhibiting protein phosphatase 1 (PP1) and 2A (PP2A) on platelet functions. Protein phosphatases were inhibited by preincubation of platelet rich plasma (PRP) samples with calyculin-A (CLA). Subsequently, platelets were activated by thrombin-receptor activating peptide (TRAP) and platelet aggregation, platelet-derived microparticle (PMP) formation, surface expressions of P-selectin (CD62), lysosome-associated membrane protein (CD63), glycoprotein Ib and IIb were examined. Phosphatase activity was determined by using phosphorylated 20 kDa myosin light chain (P-MLC20) as substrate. In CLA-treated platelets substantial decrease of P-MLC20 phosphatase activity was observed. CLA significantly suppressed TRAP-induced surface expression of P-selectin and CD63 in a concentration-dependent manner as compared to non-treated samples and moderately decreased platelet aggregation. In TRAP-activated samples, 50 nM of CLA pretreatment completely abolished the level of PMPs and the prevention of GPIb downregulation was also observed; however, no difference was found in GPIIb expression. In conclusion, PP1 and PP2A-catalyzed dephosphorylation processes have crucial roles in PMP formation and in the regulation of alpha-granule and lysosome secretion in human platelets.
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