title: Reversible inhibition of the platelet procoagulant response through manipulation of the Gardos channel.
abstract: The platelet procoagulant response requires a sustained elevation of the intracellular Ca2+ concentration, [Ca2+]i, causing exposure of phosphatidylserine (PS) at the outer surface of the plasma membrane. An increased [Ca2+]i also activates Ca2+-dependent K+ channels. Here, we investigated the contribution of the efflux of K+ ions on the platelet procoagulant response in collagen-thrombin-activated platelets using selective K+ channel blockers. The Gardos channel blockers clotrimazol, charybdotoxin, and quinine caused a similar decrease in prothrombinase activity as well as in the number of PS-exposing platelets detected by fluorescence-conjugated annexin A5. Apamin and iberiotoxin, inhibitors of other K+ channels, were without effect. Only clotrimazol showed a significant inhibition of the collagen-plus-thrombin-induced intracellular calcium response. Clotrimazol and charybdotoxin did not inhibit aggregation and release under the conditions used. Inhibition by Gardos channel blockers was reversed by valinomycin, a selective K+ ionophore. The impaired procoagulant response of platelets from a patient with Scott syndrome was partially restored by pretreatment with valinomycin, suggesting a possible defect of the Gardos channel in this syndrome. Collectively, these results provide evidence for the involvement of efflux of K+ ions through Ca2+-activated K+ channels in the procoagulant response of platelets, opening potential strategies for therapeutic interventions.
authors.affiliation: Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University, PO Box 616, 6200 MD Maastricht, the Netherlands.
authors.forename: Jef L
authors.initials: JL
authors.lastname: Wolfs
authors.forename: Simone J
authors.initials: SJ
authors.lastname: Wielders
authors.forename: Paul
authors.initials: P
authors.lastname: Comfurius
authors.forename: Theo
authors.initials: T
authors.lastname: Lindhout
authors.forename: John C
authors.initials: JC
authors.lastname: Giddings
authors.forename: Robert F
authors.initials: RF
authors.lastname: Zwaal
authors.forename: Edouard M
authors.initials: EM
authors.lastname: Bevers
mesh_terms: D017304:Annexin A5 / Q000737:chemistry; D001030:Apamin / Q000494:pharmacology; D001779:Blood Coagulation Factors / Q000378:metabolism*; D001792:Blood Platelets / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D018999:Charybdotoxin / Q000494:pharmacology; D003022:Clotrimazole / Q000494:pharmacology; D006801:Humans; D007476:Ionophores / Q000494:pharmacology; D010455:Peptides / Q000494:pharmacology; D010718:Phosphatidylserines / Q000737:chemistry; D011188:Potassium / Q000737:chemistry; D013925:Thromboplastin / Q000378:metabolism; D014634:Valinomycin / Q000494:pharmacology
chemical_list: D017304:Annexin A5; D001779:Blood Coagulation Factors; D007476:Ionophores; D010455:Peptides; D010718:Phosphatidylserines; D018999:Charybdotoxin; D014634:Valinomycin; D001030:Apamin; C064719:iberiotoxin; D013925:Thromboplastin; D003022:Clotrimazole; D011188:Potassium; D002118:Calcium