title: Platelet binding sites for factor VIII in relation to fibrin and phosphatidylserine.
abstract: Thrombin-stimulated platelets expose very little phosphatidylserine (PS) but express binding sites for factor VIII (fVIII), casting doubt on the role of exposed PS as the determinant of binding sites. We previously reported that fVIII binding sites are increased three- to sixfold when soluble fibrin (SF) binds the αIIbβ3 integrin. This study focuses on the hypothesis that platelet-bound SF is the major source of fVIII binding sites. Less than 10% of fVIII was displaced from thrombin-stimulated platelets by lactadherin, a PS-binding protein, and an fVIII mutant defective in PS-dependent binding retained platelet affinity. Therefore, PS is not the determinant of most binding sites. FVIII bound immobilized SF and paralleled platelet binding in affinity, dependence on separation from von Willebrand factor, and mediation by the C2 domain. SF also enhanced activity of fVIII in the factor Xase complex by two- to fourfold. Monoclonal antibody (mAb) ESH8, against the fVIII C2 domain, inhibited binding of fVIII to SF and platelets but not to PS-containing vesicles. Similarly, mAb ESH4 against the C2 domain, inhibited >90% of platelet-dependent fVIII activity vs 35% of vesicle-supported activity. These results imply that platelet-bound SF is a component of functional fVIII binding sites.
authors.affiliation: Departments of Medicine and Research, VA Boston Healthcare System, Boston, MA; Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA;
authors.forename: Gary E
authors.initials: GE
authors.lastname: Gilbert
authors.affiliation: Departments of Medicine and Research, VA Boston Healthcare System, Boston, MA;
authors.forename: Valerie A
authors.initials: VA
authors.lastname: Novakovic
authors.affiliation: Departments of Medicine and Research, VA Boston Healthcare System, Boston, MA; Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA;
authors.forename: Jialan
authors.initials: J
authors.lastname: Shi
authors.affiliation: Protein Chemistry Laboratory, Department of Molecular Biology, University of Aarhus, Aarhus, Denmark; and.
authors.forename: Jan
authors.initials: J
authors.lastname: Rasmussen
authors.affiliation: Departments of Pediatrics and Pathology, University of Michigan, Ann Arbor, MI.
authors.forename: Steven W
authors.initials: SW
authors.lastname: Pipe
mesh_terms: D001665:Binding Sites / Q000502:physiology; D001792:Blood Platelets / Q000378:metabolism*; D002478:Cells, Cultured; D005169:Factor VIII / Q000378:metabolism*; D005337:Fibrin / Q000378:metabolism*; D006801:Humans; D010718:Phosphatidylserines / Q000378:metabolism*; D015539:Platelet Activation / Q000502:physiology*; D011485:Protein Binding / Q000502:physiology
chemical_list: D010718:Phosphatidylserines; D005169:Factor VIII; D005337:Fibrin