Submitted January 18, 2007
Accepted September 5, 2007
The factor VIII C1 domain contributes to platelet binding
Ting-Chang Hsu, Kathleen P. Pratt, and Arthur R. Thompson*
Department of Medicine, Division of Hematology, Puget Sound Blood Center & University of Washington, Seattle, WA, United States
* Corresponding author; email: arthomps{at}u.washington.edu.
Activated factor VIII (FVIIIa) forms a procoagulant complex with factor IXa on negatively charged membranes, including activated platelet surfaces. Membrane attachment involves the FVIII C2 domain; involvement of the adjacent C1 domain has not been established. Binding of recombinant FVIII C1C2 and C2 proteins to platelets was detected by flow cytometry using (1) anti-C2 monoclonal antibody ESH8 followed by a phycoerythrin-labeled secondary antibody; (2) biotinylated C1C2 detected by phycoerythrin-labeled streptavidin, and (3) C1C2 and C2 site-specifically labeled with fluorescein. Highest binding and lowest background were obtained using fluorescein-conjugated proteins. Over 90% of activated platelets bound C1C2, compared to ~50% for equimolar C2. Estimates using fluorescent microbeads indicated ~7,000 C1C2 binding sites per platelet, ~1,400 for C2 and ~3,000 for fluorescein-labeled FVIIIa. Unlike C2 and FVIII(a), C1C2 bound to ~700 sites/platelet before activation. C1C2 binding to activated platelets appeared independent of von Willebrand factor and was competed effectively by FVIII(a), but only partially by excess C2. Fluorescein-labeled FVIIIa was competed much more effectively by C1C2 than C2 for binding to activated platelets. Two monoclonal antibodies that inhibit C2 binding to membranes competed platelet binding of C2 more effectively than C1C2. Thus, the C1 domain of FVIII contributes to platelet binding affinity.
