title: Sustained integrin ligation involves extracellular free sulfhydryls and enzymatically catalyzed disulfide exchange.
abstract: Studies have suggested a pivotal role for free sulfhydryls in platelet integrin function, and enzyme-mediated reduction of disulfide bonds on platelets has been implicated. The platelet fibrinogen receptor alpha(IIb)beta(3) is the best-studied platelet integrin and serves as a model system for studying the structure-function relation in this family of adhesion receptors. The demonstration of free sulfhydryls on the exofacial domain of purified alpha(IIb)beta(3), specifically in its activated conformation, prompted us to explore the potential for activation-dependent, enzymatically catalyzed thiol expression on intact platelets and the possible role of surface-associated protein disulfide isomerase (PDI) in alpha(IIb)beta(3) ligation. Using the membrane-impermeant sulfhydryl blocker para-chloromercuriphenyl sulfonate, the inhibitor of disulfide exchange bacitracin, and the monoclonal anti-PDI antibody RL90, we examined fibrinogen binding to alpha(IIb)beta(3) as well as ligation-induced allosteric changes in the conformation of alpha(IIb)beta(3). We sought to distinguish the possible involvement of disulfide exchange in agonist-induced platelet stimulation from its role in integrin ligation. Analysis of the role of free thiols in platelet aggregation suggested a thiol-independent initial ligation followed by a thiol-dependent stabilization of binding. Flow cytometric analysis showed that sustained binding of fibrinogen, as well as expression of ligand-induced binding site epitopes and ligand-bound conformation, depended on free thiols and disulfide exchange. Expression of P-selectin was minimally affected, even with complete inhibition of alpha(IIb)beta(3) function. These data indicate that although agonist-induced platelet stimulation is independent of ecto-sulfhydryls, engagement of integrin alpha(IIb)beta(3) on the intact platelet depends totally on their enzymatically catalyzed surface expression.
authors.affiliation: Coagulation Laboratory, Institute of Hematology, Rabin Medical Center-Beilinson Campus, Petah-Tiqva, Israel. jlahav@netvision.net.il
authors.forename: Judith
authors.initials: J
authors.lastname: Lahav
authors.forename: Kerstin
authors.initials: K
authors.lastname: Jurk
authors.forename: Oded
authors.initials: O
authors.lastname: Hess
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Barnes
authors.forename: Richard W
authors.initials: RW
authors.lastname: Farndale
authors.forename: Jacob
authors.initials: J
authors.lastname: Luboshitz
authors.forename: Beate E
authors.initials: BE
authors.lastname: Kehrel
mesh_terms: D000911:Antibodies, Monoclonal / Q000494:pharmacology; D001792:Blood Platelets / Q000187:drug effects / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005340:Fibrinogen / Q000378:metabolism* / Q000494:pharmacology; D018922:HL-60 Cells; D006801:Humans; D007700:Kinetics; D010974:Platelet Aggregation / Q000187:drug effects; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000187:drug effects / Q000378:metabolism*; D019704:Protein Disulfide-Isomerases / Q000037:antagonists & inhibitors / Q000097:blood*; D011994:Recombinant Proteins / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000494:pharmacology*; D020245:p-Chloromercuribenzoic Acid / Q000494:pharmacology
chemical_list: D000911:Antibodies, Monoclonal; D004791:Enzyme Inhibitors; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; D020245:p-Chloromercuribenzoic Acid; D005340:Fibrinogen; D019704:Protein Disulfide-Isomerases