title: Platelet type III collagen binding protein (TIIICBP) presents high biochemical and functional similarities with kindlin-3.
abstract: 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 translocation 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 - but not high - doses of ADP or thrombin which depends on α(IIb)β(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.
authors.affiliation: INSERM, U553, Hôpital Saint-Louis, 1 Avenue Claude Vellefaux, 75475 Paris cedex 10, France.
authors.forename: Ibtissem
authors.initials: I
authors.lastname: Djaafri
authors.forename: Pascal
authors.initials: P
authors.lastname: Maurice
authors.forename: Valérie
authors.initials: V
authors.lastname: Labas
authors.forename: Joëlle
authors.initials: J
authors.lastname: Vinh
authors.forename: Monique
authors.initials: M
authors.lastname: Lemesle
authors.forename: Brigitte
authors.initials: B
authors.lastname: Arbeille
authors.forename: Chantal
authors.initials: C
authors.lastname: Legrand
authors.forename: Samia
authors.initials: S
authors.lastname: Mourah
authors.forename: Françoise
authors.initials: F
authors.lastname: Fauvel-Lafeve
mesh_terms: D000244:Adenosine Diphosphate / Q000494:pharmacology; D000595:Amino Acid Sequence; D000906:Antibodies / Q000378:metabolism; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D024061:Collagen Type III / Q000378:metabolism*; D003435:Crotalid Venoms / Q000494:pharmacology; D006801:Humans; D047468:Immunoprecipitation; D037181:Lectins, C-Type; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000737:chemistry / Q000378:metabolism*; D015539:Platelet Activation / Q000187:drug effects; D010973:Platelet Adhesiveness / Q000187:drug effects; D010974:Platelet Aggregation / Q000187:drug effects; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism; D038981:Receptors, Collagen / Q000737:chemistry / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013917:Thrombin / Q000494:pharmacology
chemical_list: D000906:Antibodies; D024061:Collagen Type III; D003435:Crotalid Venoms; C477322:FERMT3 protein, human; D037181:Lectins, C-Type; D008565:Membrane Proteins; D009363:Neoplasm Proteins; D010980:Platelet Membrane Glycoproteins; D038981:Receptors, Collagen; C098341:platelet membrane glycoprotein VI; C519344:platelet receptor for type III collagen, human; C028890:convulxin; D000244:Adenosine Diphosphate; D013917:Thrombin