Abstract

Stimulation of platelets by the extracellular matrix protein collagen leads to activation of a tyrosine kinase-dependent mechanism resulting in secretion and aggregation. Tyrosine phosphorylation of the tyrosine kinase Syk and phospholipase Cgamma2 are early events in collagen-induced activation. We recently proposed that collagen-signaling in platelets involves a receptor or a receptor-associated protein containing an immunoreceptor tyrosine-based activation motif (ITAM) enabling interaction with Syk. In this report we show that collagen stimulation of platelets causes rapid tyrosine phosphorylation of the ITAM containing Fc receptor gamma-chain and that this is precipitated by the tandem Src homology 2 (SH2) domains of Syk expressed as a fusion protein. In addition we demonstrate an association between the Fc receptor gamma-chain with endogenous Syk in collagen-stimulated platelets. The Fc receptor gamma-chain undergoes tyrosine phosphorylation in platelets stimulated by a collagen-related peptide which does not bind the integrin alpha2beta1 and by the lectin wheat germ agglutinin. In contrast, cross-linking of the platelet low affinity receptor for immune complexes, FcgammaRIIA, or stimulation by thrombin does not induce phosphorylation of the Fc receptor gamma-chain. The present results provide a molecular basis for collagen activation of platelets which is independent of the integrin alpha2beta1 and involves phosphorylation of the Fc receptor gamma-chain, its association with Syk and subsequent phosphorylation of phospholipase Cgamma2. Collagen is the first example of a nonimmune receptor stimulus to signal through a pathway closely related to signaling by immune receptors.

Highlights

  • Stimulation of platelets by the extracellular matrix protein collagen leads to activation of a tyrosine kinasedependent mechanism resulting in secretion and aggregation

  • We recently proposed that collagensignaling in platelets involves a receptor or a receptorassociated protein containing an immunoreceptor tyrosine-based activation motif (ITAM) enabling interaction with Syk

  • In this report we show that collagen stimulation of platelets causes rapid tyrosine phosphorylation of the ITAM containing Fc receptor ␥-chain and that this is precipitated by the tandem Src homology 2 (SH2) domains of Syk expressed as a fusion protein

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Summary

EXPERIMENTAL PROCEDURES

Materials—Anti-FcR ␥-chain antiserum [14] was kindly provided by Dr J.-P. Kinet (Beth Israel Hospital, Boston, MA), the monoclonal antibody (mAb) 6F1 was a gift from Dr B. Coller (Mount Sinai School of Medicine, New York, NY), and the rabbit polyclonal antibody to the T cell ␨-chain was kindly donated by Dr D. A collagen-related peptide (CRP: GCP*(GPP*)10GCP*G; single amino acid code P* ϭ hydroxyproline) was synthesized and cross-linked as described previously [15]. Anti-phosphotyrosine mAb 4G10 was from Upstate Biotechnology (TCS Biologicals Ltd., Botolph Claydon, Bucks, UK), anti-Syk antibodies: Syk(LR) was from Santa Cruz Biotechnology Inc. (Santa Cruz, CA) and mAb 101 from Wako GmbH (Neuss, Germany), and anti-Fc␥RII mAb IV. was purchased from Medarex Inc.

The abbreviations used are
RESULTS
DISCUSSION
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