Protein Tyrosine Phosphorylation in Human Platelets during Shear Stress-Induced Platelet Aggregation (SIPA) Is Regulated by Glycoprotein (GP) Ib/IX as well as GP IIb/IIIa and Requires Intact Cytoskeleton and Endogenous ADP

Atsushi Oda; Kenji Yokoyama; Mitsuru Murata; Michihide Tokuhira; Kosei Nakamura; Makoto Handa; Kiyoaki Watanabe; Yasuo Ikeda

doi:10.1055/s-0038-1649806

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 1995; 74(02): 736-742
DOI: 10.1055/s-0038-1649806

Original Article

Platelets

Schattauer GmbH Stuttgart

Protein Tyrosine Phosphorylation in Human Platelets during Shear Stress-Induced Platelet Aggregation (SIPA) Is Regulated by Glycoprotein (GP) Ib/IX as well as GP IIb/IIIa and Requires Intact Cytoskeleton and Endogenous ADP

Atsushi Oda

¹The Division of Hematology, Department of Internal Medicine, Keio University, Tokyo, Japan

,

Kenji Yokoyama

¹The Division of Hematology, Department of Internal Medicine, Keio University, Tokyo, Japan

,

Mitsuru Murata

¹The Division of Hematology, Department of Internal Medicine, Keio University, Tokyo, Japan

,

Michihide Tokuhira

¹The Division of Hematology, Department of Internal Medicine, Keio University, Tokyo, Japan

,

Kosei Nakamura

¹The Division of Hematology, Department of Internal Medicine, Keio University, Tokyo, Japan

,

Makoto Handa

²The Blood Center, School of Medicine, Keio University, Tokyo, Japan

,

Kiyoaki Watanabe

³The Department of Laboratory Medicine, School of Medicine, Keio University, Tokyo, Japan

,

Yasuo Ikeda

¹The Division of Hematology, Department of Internal Medicine, Keio University, Tokyo, Japan

› Author Affiliations

Abstract

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Summary

Shear stress-induced platelet aggregation (SIPA) may be essential in thrombus formation in pathologically stenotic arteries. Intracellular events during SIPA are, however, poorly understood. Washed platelets were exposed to shear stress (108 dyne/cm²) in the presence of von Willebrand factor (vWf, 10 μg/ml) and 1 mM CaCl₂ for various time intervals, and then lyzed in SDS. Platelet proteins were separated by 10% SDS-PAGE and tyrosine phosphorylated proteins were detected by immunoblotting with an anti-phosphotyrosine monoclonal antibody. Increased tyrosine phosphorylation of proteins of 130, 100, 85, 74, 70, 64, 58, and 40 kDa was observed within 30 s after the beginning of exposure of platelets to high shear force and the degree of tyrosine phosphorylation continued to increase up to approximately 2 min after the exposure. A monoclonal antibody (MoAb) against vWf-binding domain of glycoprotein (GP) Ibα (GUR83-35), anti-vWf MoAb that inhibits binding of vWf to GPIbα (NMC-4), or a MoAb against GP IIb/IIIa complex (AP-2) inhibited SIPA as well as tyrosine phosphorylation of these proteins. Apyrase (an ADP scavenger, 2 U/ml), EDTA (5 mM), or RGDS peptide (200 μg/ml) also had inhibitory effects on both SIPA and tyrosine phosphorylation. However, Cytochalasin D (2 μM) or staurosporin (1 μM) did not affect SIPA, while they inhibited SIPA-associated tyrosine phosphorylation of those proteins. SIPA-associated tyrosine phosphorylation is a novel post-aggregatory pathway in signal transduction, which is dependent on the binding of vWf to GP Ib/IX and GP IIb/IIIa, endogenous ADP, and intact cytoskeleton.

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References

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