1. Platelet type III collagen binding protein (TIIICBP) presents high biochemical and functional similarities with kindlin-3
- Author
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Françoise Fauvel-Lafève, Valérie Labas, Samia Mourah, Joëlle Vinh, Monique Lemesle, Chantal Legrand, Ibtissem Djaafri, Pascal Maurice, Brigitte Arbeille, Hemostase, Endothelium, Angiogenese (UMR_S_553), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Diderot - Paris 7 (UPD7), Physiologie de la reproduction et des comportements [Nouzilly] (PRC), Centre National de la Recherche Scientifique (CNRS)-Université de Tours-Institut Français du Cheval et de l'Equitation [Saumur]-Institut National de la Recherche Agronomique (INRA), Unité de Spectrométrie de Masse Biologique et Protéomique, ESPCI ParisTech-Centre National de la Recherche Scientifique (CNRS), Plateforme des Microscopies, Université de Tours, Institut Universitaire d'Hématologie (IUH), Université Paris Diderot - Paris 7 (UPD7), Université Paris Diderot - Paris 7 (UPD7)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut National de la Recherche Agronomique (INRA)-Institut Français du Cheval et de l'Equitation [Saumur]-Université de Tours-Centre National de la Recherche Scientifique (CNRS), Spectrométrie de Masse Biologique et Protéomique (USR3149 / FRE2032) (SMBP), Ecole Superieure de Physique et de Chimie Industrielles de la Ville de Paris (ESPCI Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Centre National de la Recherche Scientifique (CNRS), Neurobiologie et diversité cellulaire (NDC), Université de Tours (UT), Hématologie -Immunologie -Cibles thérapeutiques, Centre National de la Recherche Scientifique (CNRS)-Ecole Superieure de Physique et de Chimie Industrielles de la Ville de Paris (ESPCI Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL), and Institut National de la Recherche Agronomique (INRA)-Institut Français du Cheval et de l'Equitation [Saumur]-Université de Tours (UT)-Centre National de la Recherche Scientifique (CNRS)
- Subjects
MESH: Neoplasm Proteins ,MESH: Platelet Adhesiveness ,Platelet Aggregation ,MESH: Sequence Homology, Amino Acid ,[SDV]Life Sciences [q-bio] ,MESH: Amino Acid Sequence ,Biochemistry ,Collagen receptor ,MESH: Thrombin ,0302 clinical medicine ,Peptide sequence ,MESH: Blood Platelets ,MESH: Platelet Aggregation ,MESH: Collagen Type III ,0303 health sciences ,biology ,Chemistry ,Thrombin ,Convulxin ,General Medicine ,Neoplasm Proteins ,3. Good health ,Adenosine Diphosphate ,030220 oncology & carcinogenesis ,MESH: Crotalid Venoms ,MESH: Membrane Proteins ,GPVI ,Blood Platelets ,Receptors, Collagen ,Molecular Sequence Data ,Integrin ,Platelet Membrane Glycoproteins ,Antibodies ,03 medical and health sciences ,Platelet Adhesiveness ,[SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN] ,Crotalid Venoms ,MESH: Platelet Activation ,Humans ,Immunoprecipitation ,Lectins, C-Type ,Amino Acid Sequence ,Platelet activation ,030304 developmental biology ,MESH: Molecular Sequence Data ,MESH: Humans ,MESH: Adenosine Diphosphate ,Sequence Homology, Amino Acid ,MESH: Immunoprecipitation ,MESH: Antibodies ,Binding protein ,MESH: Platelet Membrane Glycoproteins ,Membrane Proteins ,Platelet Activation ,MESH: Receptors, Collagen ,Molecular biology ,Collagen Type III ,Membrane protein ,biology.protein ,MESH: Lectins, C-Type - Abstract
International audience; Type III collagen binding protein (TIIICBP) was previously described as a platelet membrane protein that recognizes the KOGEOGPK peptide sequence within type III collagen. In order to better characterize this protein, we performed different approaches including mass spectrometry sequencing and functional experiments. This study leads to identify high biochemical and functional similarities between TIIICBP and kindlin-3, a member of a family of focal adhesion proteins. Indeed, mass spectrometry surveys indicated that TIIICBP contains several peptides identical to kindlin-3, covering 41% of the amino acid sequence. Polyclonal antibodies raised against a kindlin-3 specific N-terminal sequence, recognized and immunoprecipitated TIIICBP from platelet lysates. Electron microscopy and flow cytometry experiments showed that kindlin-3, as well as TIIICBP, were present associated to platelet membrane and a trans-location of cytosolic kindlin-3 to the platelet membrane was observed after platelet activation. Similarly to anti-TIIICBP antibodies and the KOGEOGPK peptide, anti-kindlin-3 antibodies inhibited platelet interactions with type III collagen under flow conditions and slowed down platelet aggregation induced by glycoprotein VI agonists; e.g. collagen-related peptides and convulxin. In addition, the anti-kindlin-3 antibody inhibited platelet aggregation induced by low e but not high e doses of ADP or thrombin which depends on a IIb b 3 integrin function. In conclusion, our results show that the peptides identified by mass spectrometry from purified TIIICBP correspond to the kindlin-3 protein and demonstrate biochemical and functional similarities between TIIICBP and kindlin-3, strengthening a key role for TIIICBP/kindlin-3 in platelet interactions with collagen by cooperating with glycoprotein VI activation and integrin clustering in focal adhesion complexes.
- Published
- 2012