Protein phosphatase inhibitor calyculin-A modulates activation markers in TRAP-stimulated human platelets.
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.
Simon
Zsuzsa
Z
Department of Clinical Biochemistry and Molecular Pathology, Hungarian Academy of Sciences, Research Center for Molecular Medicine, Medical and Health Science Center, University of Debrecen, Hungary.
Kiss
Andrea
A
Erdödi
Ferenc
F
Setiadi
Hendra
H
Beke Debreceni
Ildikó
I
Nagy
Béla
B
Kappelmayer
János
J
D001792:Blood Platelets / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism / Q000502:physiology; D015536:Down-Regulation; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006801:Humans; D008387:Marine Toxins; D010080:Oxazoles / Q000494:pharmacology*; D019007:P-Selectin / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000097:blood; D010446:Peptide Fragments / Q000494:pharmacology; D015539:Platelet Activation / Q000187:drug effects* / Q000502:physiology; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000037:antagonists & inhibitors / Q000378:metabolism; D054645:Protein Phosphatase 1 / Q000037:antagonists & inhibitors* / Q000097:blood; D054648:Protein Phosphatase 2 / Q000037:antagonists & inhibitors* / Q000097:blood; D017452:Receptors, IgG / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000097:blood
D004791:Enzyme Inhibitors; C489017:FCGR1A protein, human; D008387:Marine Toxins; D010080:Oxazoles; D019007:P-Selectin; D010446:Peptide Fragments; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D017452:Receptors, IgG; C086309:thrombin receptor peptide SFLLRNP; C059041:calyculin A; D054645:Protein Phosphatase 1; D054648:Protein Phosphatase 2