title: Quantitative determination of the binding of beta2-glycoprotein I and prothrombin to phosphatidylserine-exposing blood platelets.
abstract: The plasma protein beta2GPI (beta2-glycoprotein I) has been proposed to mediate phagocytosis of apoptotic cells and to play a role in the antiphospholipid syndrome. This suggestion is based mainly on the presumption that beta2GPI has an appreciable interaction with PS (phosphatidylserine)-exposing cell membranes. However, quantitative data on the binding of beta2GPI to PS-exposing cells under physiologically relevant conditions are scarce and conflicting. Therefore we evaluated the binding of beta2GPI to PS-expressing blood platelets. Flow cytometry showed that binding of beta2GPI is negligible at physiological ionic strength, in contrast with significant binding occurring at low ionic strength. Binding parameters of beta2GPI and (for comparison) prothrombin were quantified by ellipsometric measurement of protein depletion from the supernatant following incubation with platelets. At low ionic strength (20 mM NaCl, no CaCl2), a dissociation constant (K(d)) of 0.2 microM was found for beta2GPI, with 7.4x10(5) binding sites per platelet. Under physiologically relevant conditions (120 mM NaCl and 3 mM CaCl2), binding of beta2GPI was not detectable (extrapolated K(d)>80 microM). Prothrombin binding (at 3 mM CaCl2) was much less affected by ionic strength: K(d) values of 0.5 and 1.4 muM were observed at 20 and 120 mM NaCl respectively. The low affinity and the presence of many lipid-binding proteins in plasma that can compete with the binding of beta2GPI suggest that only a small fraction (<5%) of the binding sites on PS-exposing blood cells are likely to be occupied by beta2GPI. These findings are discussed in relation to the alleged (patho-)physiological functions of beta2GPI.
authors.affiliation: Cardiovascular Research Institute Maastricht, Maastricht University, P.O. Box 616, 6200 MD Maastricht, The Netherlands. em.bevers@bioch.unimaas.nl
authors.forename: Edouard M
authors.initials: EM
authors.lastname: Bevers
authors.forename: Marie P
authors.initials: MP
authors.lastname: Janssen
authors.forename: Paul
authors.initials: P
authors.lastname: Comfurius
authors.forename: Krishnakumar
authors.initials: K
authors.lastname: Balasubramanian
authors.forename: Alan J
authors.initials: AJ
authors.lastname: Schroit
authors.forename: Robert F A
authors.initials: RF
authors.lastname: Zwaal
authors.forename: George M
authors.initials: GM
authors.lastname: Willems
mesh_terms: D001665:Binding Sites; D001792:Blood Platelets / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods; D006023:Glycoproteins / Q000378:metabolism*; D006801:Humans; D015759:Ionomycin / Q000494:pharmacology; D009994:Osmolar Concentration; D010718:Phosphatidylserines / Q000378:metabolism*; D011485:Protein Binding; D011516:Prothrombin / Q000378:metabolism*; D053482:beta 2-Glycoprotein I
chemical_list: D006023:Glycoproteins; D010718:Phosphatidylserines; D053482:beta 2-Glycoprotein I; D015759:Ionomycin; D011516:Prothrombin