title: Platelet receptor interplay regulates collagen-induced thrombus formation in flowing human blood.
abstract: The platelet glycoproteins (GPs) Ib, integrin alpha(2)beta(1), and GPVI are considered central to thrombus formation. Recently, their relative importance has been re-evaluated based on data from murine knockout models. To examine their relationship during human thrombus formation on collagen type I fibers at high shear (1000 s(-1)), we tested a novel antibody against GPVI, an immunoglobulin single-chain variable fragment, 10B12, together with specific antagonists for GPIb alpha (12G1 Fab(2)) and alpha(2)beta(1) (6F1 mAb or GFOGER-GPP peptide). GPVI was found to be crucial for aggregate formation, Ca(2+) signaling, and phosphatidylserine (PS) exposure, but not for primary adhesion, even with more than 97% receptor blockade. Inhibiting alpha(2)beta(1) revealed its involvement in regulating Ca(2+) signaling, PS exposure, and aggregate size. Both GPIb alpha and alpha(2)beta(1) contributed to primary adhesion, showing overlapping function. The coinhibition of receptors revealed synergism in thrombus formation: the coinhibition of adenosine diphosphate (ADP) receptors with collagen receptors further decreased adhesion and aggregation, and, crucially, the complete eradication of thrombus formation required the coinhibition of GPVI with either GPIb alpha or alpha(2)beta(1). In summary, human platelet deposition on collagen depends on the concerted interplay of several receptors: GPIb in synergy with alpha(2)beta(1) mediating primary adhesion, reinforced by activation through GPVI, which further regulates the thrombus formation.
authors.affiliation: Department of Biochemistry, University of Cambridge, Main Bldg, Downing Site, Tennis Court Rd, CB2 1QW Cambridge, United Kingdom. prms2@mole.bio.cam.ac.uk
authors.forename: Pia R-M
authors.initials: PR
authors.lastname: Siljander
authors.forename: Imke C A
authors.initials: IC
authors.lastname: Munnix
authors.forename: Peter A
authors.initials: PA
authors.lastname: Smethurst
authors.forename: Hans
authors.initials: H
authors.lastname: Deckmyn
authors.forename: Theo
authors.initials: T
authors.lastname: Lindhout
authors.forename: Willem H
authors.initials: WH
authors.lastname: Ouwehand
authors.forename: Richard W
authors.initials: RW
authors.lastname: Farndale
authors.forename: Johan W M
authors.initials: JW
authors.lastname: Heemskerk
mesh_terms: D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000991:Antithrombins / Q000494:pharmacology; D001777:Blood Coagulation / Q000502:physiology*; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology; D024042:Collagen Type I / Q000494:pharmacology; D006801:Humans; D038982:Integrin alpha2beta1 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010973:Platelet Adhesiveness / Q000502:physiology; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000037:antagonists & inhibitors / Q000378:metabolism; D010980:Platelet Membrane Glycoproteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011673:Pulsatile Flow; D058919:Purinergic P2 Receptor Antagonists; D018048:Receptors, Purinergic P2 / Q000378:metabolism; D013927:Thrombosis / Q000378:metabolism*
chemical_list: D000590:Amino Acid Chloromethyl Ketones; D000991:Antithrombins; D024042:Collagen Type I; D038982:Integrin alpha2beta1; D019038:Platelet Glycoprotein GPIb-IX Complex; D010980:Platelet Membrane Glycoproteins; D058919:Purinergic P2 Receptor Antagonists; D018048:Receptors, Purinergic P2; C032193:glycoprotein receptor GPIb-IX; C098341:platelet membrane glycoprotein VI; C040308:phenylalanyl-prolyl-arginine-chloromethyl ketone