title: Platelet microparticle formation and thrombin generation under high shear are effectively suppressed by a monoclonal antibody against GPIba.
abstract: We studied the inhibition of platelet microparticle (MP) formation and thrombin generation under high shear forces. We hypothesized that an inhibitor of the GPIb a -von Willebrand factor (vWF) interaction would be more effective in suppressing MP formation and thrombin generation than GPIIb/IIIa inhibitors. Platelet-rich plasma (PRP) anticoagulated with PPACK (D-Phe-Pro-Arg chloromethyl ketone) was exposed in a cone-and-plate viscometer (shear: 5,000 s(-1) for 5 min) in the presence of antagonists to GPIb a (the monoclonal antibody [Mab] Ib-23) or to GPIIb/IIIa (abciximab, tirofiban, eptifibatide) at their IC90 determined in platelet aggregometry with ristocetin or ADP, respectively. We used double labeling (CD41-PE and annexin-V-FITC) for flow cytometric detection of MP and their aminophospholipid exposure. Thrombin generation was measured using PRP prepared from ACD anticoagulated blood. About 40% of the thrombin generation was found to be mediated by the MP fraction of the PRP. Blockade of GPIb a with Mab Ib-23 reduced MP formation and thrombin generation by 50%, and was more effective than any GPIIb/IIIa antagonist. The combination of Mab Ib-23 with one of the GPIIb/IIIa inhibitors further reduced the MP formation to ~ 30%. The antibody also partially inhibited thrombin induced platelet aggregation. Epitope mapping suggested that Mab Ib-23 binds between the amino acids 201 and 268 of GPIb a , explaining the interference with vWF and thrombin interaction. In contrast to the commonly used GPIIb/IIIa antagonists, the blockade of GPIb a with Mab Ib-23 effectively reduces the prothrombotic MP generation and thrombin formation at shear rates typically found in arterial stenoses.
authors.affiliation: Department of Medicine, Laboratory for Thrombosis Research, Kantonsspital Baden, Switzerland.
authors.forename: Luca
authors.initials: L
authors.lastname: Pontiggia
authors.forename: Beat
authors.initials: B
authors.lastname: Steiner
authors.forename: Hans
authors.initials: H
authors.lastname: Ulrichts
authors.forename: Hans
authors.initials: H
authors.lastname: Deckmyn
authors.forename: Marc
authors.initials: M
authors.lastname: Forestier
authors.forename: Jürg H
authors.initials: JH
authors.lastname: Beer
mesh_terms: D000077284:Abciximab; D017304:Annexin A5 / Q000378:metabolism; D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000494:pharmacology; D001666:Binding Sites, Antibody; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D018604:Epitope Mapping; D000077542:Eptifibatide; D005434:Flow Cytometry; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000494:pharmacology; D066298:In Vitro Techniques; D010455:Peptides / Q000494:pharmacology; D010718:Phosphatidylserines / Q000378:metabolism; D010974:Platelet Aggregation / Q000187:drug effects; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology; D010979:Platelet Function Tests; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000037:antagonists & inhibitors / Q000378:metabolism*; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000276:immunology / Q000378:metabolism*; D013314:Stress, Mechanical; D013917:Thrombin / Q000378:metabolism*; D000077466:Tirofiban; D014443:Tyrosine / Q000031:analogs & derivatives / Q000494:pharmacology; D014841:von Willebrand Factor / Q000378:metabolism
chemical_list: D017304:Annexin A5; D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D010455:Peptides; D010718:Phosphatidylserines; D010975:Platelet Aggregation Inhibitors; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D019038:Platelet Glycoprotein GPIb-IX Complex; D014841:von Willebrand Factor; D014443:Tyrosine; D013917:Thrombin; D000077466:Tirofiban; D000077542:Eptifibatide; D000077284:Abciximab