title: The majority of circulating platelet-derived microparticles fail to bind annexin V, lack phospholipid-dependent procoagulant activity and demonstrate greater expression of glycoprotein Ib.
abstract: It has been widely accepted that microparticles expose phosphatidylserine which in turn binds annexin V. It was the objective of this study to compare the antigenic characteristics and phospholipid-dependent procoagulant activity of annexin V positive and -negative subpopulations of platelet-derived microparticles. Annexin V positive and -negative microparticles were identified and characterised using flow cytometry and procoagulant activity was measured by a phospholipid-dependent assay (XACT). In unstimulated platelet-poor plasma, 80% of platelet-derived microparticles failed to bind annexin V. Varying the assay constituents (buffer, calcium and annexin V concentration) did not alter annexin V binding. The proportion of microparticles that bound annexin V was dependent upon the agonist, with physiological agonists such as collagen resulting in fewer annexin V binding microparticles than non-physiological agonists such as ionophore. CD42b (glycoprotein Ib) expression was significantly decreased and CD62p and CD63 expression were significantly increased in annexin V positive compared to annexin V negative subpopulations. There was no significant difference in CD41, CD61, CD42a and CD40L expression between annexin V positive and -negative subpopulations. A significant correlation between annexin V binding and XACT was found (p=0.033). Annexin V inhibited greater than 95% of phospholipid activity, suggesting that annexin V binding was a true reflection of procoagulant activity. The majority of platelet-derived microparticles in unstimulated plasma failed to bind annexin V and showed significantly increased levels of CD42b compared to annexin V positive events. Phospholipid-dependent procoagulant activity is limited to the annexin V positive subpopulation and is agonist-dependent. The significance of annexin V negative microparticles is unclear, however, it is possible that they possess other activities aside from procoagulant phospholipid activity.
authors.affiliation: Department of Haematology and Haematological Stem Cell Transplantation, St Vincent's Hospital, Sydney, New South Wales, Australia. dconnor@stvincents.com.au
authors.forename: David Ewan
authors.initials: DE
authors.lastname: Connor
authors.forename: Thomas
authors.initials: T
authors.lastname: Exner
authors.forename: David Dang Fung
authors.initials: DD
authors.lastname: Ma
authors.forename: Joanne Emily
authors.initials: JE
authors.lastname: Joseph
mesh_terms: D017304:Annexin A5 / Q000378:metabolism*; D015703:Antigens, CD / Q000096:biosynthesis; D001777:Blood Coagulation / Q000187:drug effects; D001780:Blood Coagulation Tests; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D055252:Cell-Derived Microparticles / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D002478:Cells, Cultured; D003094:Collagen / Q000494:pharmacology; D005434:Flow Cytometry; D006801:Humans; D007476:Ionophores / Q000494:pharmacology; D010718:Phosphatidylserines / Q000378:metabolism; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding
chemical_list: D017304:Annexin A5; D015703:Antigens, CD; D007476:Ionophores; D010718:Phosphatidylserines; D019038:Platelet Glycoprotein GPIb-IX Complex; D003094:Collagen