A Comprehensive Proteomics and Genomics Analysis Reveals Novel Transmembrane Proteins in Human Platelets and Mouse Megakaryocytes Including G6b-B, a Novel Immunoreceptor Tyrosine-based Inhibitory Motif Protein

Abstract

The platelet surface is poorly characterized due to the low abundance of many membrane proteins and the lack of specialist tools for their investigation. In this study we identified novel human platelet and mouse megakaryocyte membrane proteins using specialist proteomics and genomics approaches. Three separate methods were used to enrich platelet surface proteins prior to identification by liquid chromatography and tandem mass spectrometry: lectin affinity chromatography, biotin/NeutrAvidin affinity chromatography, and free flow electrophoresis. Many known, abundant platelet surface transmembrane proteins and several novel proteins were identified using each receptor enrichment strategy. In total, two or more unique peptides were identified for 46, 68, and 22 surface membrane, intracellular membrane, and membrane proteins of unknown subcellular localization, respectively. The majority of these were single transmembrane proteins. To complement the proteomics studies, we analyzed the transcriptome of a highly purified preparation of mature primary mouse megakaryocytes using serial analysis of gene expression in view of the increasing impor-

G6b-B Inhibits Constitutive and Agonist-induced Signaling by Glycoprotein VI and CLEC-2

“…series of experiments were undertaken to investigate the possible regulation of constitutive and agonist-induced signaling through the GPVI-FcR ␥-chain complex and CLEC-2 by the platelet immunoglobulin receptor G6b-B, which contains two ITIMs in its cytosolic tail (9,10). xpression of G6b-B significantly inhibited constitutive signaling by both receptors by more than 60% (Fig.…”

“…The second ITIM in G6b-B is located ϳ20 amino acids downstream of the first ITIM and has a slightly different sequence to the above, TXYXXV. G6b-B is the third ITIM-containing protein to be identified on platelets (10,13). The other two platelet ITIM receptors are platelet/ endothelial cell adhesion molecule-1 (PECAM-1) and TREMlike transcript-1 (14 -16).…”

“…G6b-B is the only one of these variants to contain both a transmembrane region and two immunoreceptor tyrosine-based inhibitory motifs (ITIMs) that support binding to the two SH2 domaincontaining protein tyrosine phosphatases, SHP1 and SHP2 (9,10). ITIMs are defined by the consensus sequence (L/I/V/S)-X-Y-X-X-(L/V) and are commonly found in pairs separated by 15 to 30 amino acid residues (11,12).…”

Vascular proteomics

“…These studies have been extended and novel platelet signaling proteins and phophorylation events have been identified [145][146][147][148][149]. Using LC-MS/MS, lectin affinity chromatography and free flow electrophoresis, has permitted the identification of novel transmembrane proteins in human platelets [150]. The study of activated platelets is very attractive in relation with the atherosclerotic process since activated platelets adhere to the atherosclerotic lesions and could be a source of plaque marker proteins as they release proteins after activation.…”

The Tetraspanin CD9 Localizes to Platelet-Platelet Contacts and Regulates Thrombus Stability

“…They are believed to modulate integrin function, and indeed, both antibody-blocking and knockout studies yield platelets with incomplete GPIIb-IIIa functioning. Megakaryocytes have been shown to contain the transcripts for the tetraspanins NET5 (TSPAN9) and TSPAN33, and these may also be expressed at the protein level in platelets (Senis 2007). …”