title: Novel α2β1 integrin inhibitors reveal that integrin binding to collagen under shear stress conditions does not require receptor preactivation.
abstract: The interaction between α2β1 integrin (GPIa/IIa, VLA-2) and vascular collagen is one of the initiating events in thrombus formation. Here, we describe two structurally similar sulfonamide derivatives, BTT-3033 and BTT-3034, and show that, under static conditions, they have an almost identical effect on α2-expressing CHO cell adhesion to collagen I, but only BTT-3033 blocks platelet attachment under flow (90 dynes/cm(2)). Differential scanning fluorimetry showed that both molecules bind to the α2I domain of the recombinant α2 subunit. To further study integrin binding mechanism(s) of the two sulfonamides, we created an α2 Y285F mutant containing a substitution near the metal ion-dependent adhesion site motif in the α2I domain. The action of BTT-3033, unlike that of BTT-3034, was dependent on Tyr-285. In static conditions BTT-3034, but not BTT-3033, inhibited collagen binding by an α2 variant carrying a conformationally activating E318W mutation. Conversely, in under flow conditions (90 dynes/cm(2)) BTT-3033, but not BTT-3034, inhibited collagen binding by an α2 variant expressing E336A loss-of-function mutation. Thus, the binding sites for BTT-3033 and BTT-3034 are differentially available in distinct integrin conformations. Therefore, these sulfonamides can be used to study the biological role of different functional stages of α2β1. Furthermore, only the inhibitor that recognized the non-activated conformation of α2β1 integrin under shear stress conditions effectively blocked platelet adhesion, suggesting that the initial interaction between integrin and collagen takes place prior to receptor activation.
authors.affiliation: BioTie Therapies, Turku FI-20520, Finland.
authors.forename: Liisa
authors.initials: L
authors.lastname: Nissinen
authors.forename: Jarkko
authors.initials: J
authors.lastname: Koivunen
authors.forename: Jarmo
authors.initials: J
authors.lastname: Käpylä
authors.forename: Maria
authors.initials: M
authors.lastname: Salmela
authors.forename: Jonna
authors.initials: J
authors.lastname: Nieminen
authors.forename: Johanna
authors.initials: J
authors.lastname: Jokinen
authors.forename: Kalle
authors.initials: K
authors.lastname: Sipilä
authors.forename: Marjo
authors.initials: M
authors.lastname: Pihlavisto
authors.forename: Olli T
authors.initials: OT
authors.lastname: Pentikäinen
authors.forename: Anne
authors.initials: A
authors.lastname: Marjamäki
authors.forename: Jyrki
authors.initials: J
authors.lastname: Heino
mesh_terms: D000818:Animals; D001792:Blood Platelets / Q000737:chemistry / Q000166:cytology / Q000378:metabolism; D002448:Cell Adhesion / Q000187:drug effects; D002460:Cell Line; D024042:Collagen Type I / Q000378:metabolism*; D006801:Humans; D038982:Integrin alpha2beta1 / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D015394:Molecular Structure; D010980:Platelet Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D013314:Stress, Mechanical; D013449:Sulfonamides / Q000378:metabolism* / Q000494:pharmacology
chemical_list: D024042:Collagen Type I; D038982:Integrin alpha2beta1; D010980:Platelet Membrane Glycoproteins; D013449:Sulfonamides; C098341:platelet membrane glycoprotein VI