Protein phosphatase inhibitor calyculin A modulates activation markers in TRAP-stimulated human platelets

Zsuzsa Simon, A. Kiss, F. Erdődi, Hendra Setiadi, Ildik Beke Debreceni, B. Nagy, J. Kappelmayer

Research output: Contribution to journalArticle

2 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)555-562
Number of pages8
JournalPlatelets
Volume21
Issue number7
DOIs
Publication statusPublished - Nov 2010

Fingerprint

thrombin receptor peptide SFLLRNP
Phosphoprotein Phosphatases
Blood Platelets
Protein Phosphatase 1
Protein Phosphatase 2
P-Selectin
Platelet Aggregation
Phosphoric Monoester Hydrolases
Phosphorylation
Lysosome-Associated Membrane Glycoproteins
Platelet Glycoprotein GPIb-IX Complex
Myosin Light Chains
Platelet-Rich Plasma
Platelet Activation
Threonine
Lysosomes
Protein Kinases
Serine
calyculin A
Proteins

Keywords

  • Calyculin-A
  • microparticles
  • P-selectin
  • Ser/Thr specific protein phosphatase

ASJC Scopus subject areas

  • Hematology

Cite this

Protein phosphatase inhibitor calyculin A modulates activation markers in TRAP-stimulated human platelets. / Simon, Zsuzsa; Kiss, A.; Erdődi, F.; Setiadi, Hendra; Beke Debreceni, Ildik; Nagy, B.; Kappelmayer, J.

In: Platelets, Vol. 21, No. 7, 11.2010, p. 555-562.

Research output: Contribution to journalArticle

@article{5a8dcd0446fb4fee85f9833e21826557,
title = "Protein phosphatase inhibitor calyculin A modulates activation markers in TRAP-stimulated human platelets",
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.",
keywords = "Calyculin-A, microparticles, P-selectin, Ser/Thr specific protein phosphatase",
author = "Zsuzsa Simon and A. Kiss and F. Erdődi and Hendra Setiadi and {Beke Debreceni}, Ildik and B. Nagy and J. Kappelmayer",
year = "2010",
month = "11",
doi = "10.3109/09537104.2010.499156",
language = "English",
volume = "21",
pages = "555--562",
journal = "Platelets",
issn = "0953-7104",
publisher = "Informa Healthcare",
number = "7",

}

TY - JOUR

T1 - Protein phosphatase inhibitor calyculin A modulates activation markers in TRAP-stimulated human platelets

AU - Simon, Zsuzsa

AU - Kiss, A.

AU - Erdődi, F.

AU - Setiadi, Hendra

AU - Beke Debreceni, Ildik

AU - Nagy, B.

AU - Kappelmayer, J.

PY - 2010/11

Y1 - 2010/11

N2 - 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.

AB - 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.

KW - Calyculin-A

KW - microparticles

KW - P-selectin

KW - Ser/Thr specific protein phosphatase

UR - http://www.scopus.com/inward/record.url?scp=77958017209&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=77958017209&partnerID=8YFLogxK

U2 - 10.3109/09537104.2010.499156

DO - 10.3109/09537104.2010.499156

M3 - Article

C2 - 20670106

AN - SCOPUS:77958017209

VL - 21

SP - 555

EP - 562

JO - Platelets

JF - Platelets

SN - 0953-7104

IS - 7

ER -