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The factor VIII C1 domain contributes to platelet binding.

ABSTRACT: 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. More than 90% of activated platelets bound C1C2, compared with approximately 50% for equimolar C2. Estimates using fluorescent microbeads indicated approximately 7,000 C1C2-binding sites per platelet, approximately 1,400 for C2, and approximately 3,000 for fluorescein-labeled FVIIIa. Unlike C2 or FVIII(a), C1C2 bound to approximately 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.

SUBMITTER: Hsu TC

PROVIDER: S-EPMC2200805 | biostudies-other | 2008 Jan

REPOSITORIES: biostudies-other

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The factor VIII C1 domain contributes to platelet binding.

Hsu Ting-Chang TC Pratt Kathleen P KP Thompson Arthur R AR

Blood 20071004 1

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 phycoerythr ...[more]

PMID: 17916745

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Project description:Essentials Inhibitor formation remains a challenging complication of hemophilia A care. The Bethesda assay is the primary method used for determining bleeding risk and management. Antibodies that block factor VIII binding to von Willebrand factor can increase FVIII clearance. Antibodies that increase clearance contribute to antibody pathogenicity. SUMMARY:Background The development of neutralizing anti-factor VIII (FVIII) antibodies remains a challenging complication of modern hemophilia A care. In vitro assays are the primary method used for quantifying inhibitor titers, predicting bleeding risk, and determining bleeding management. However, other mechanisms of inhibition are not accounted for in these assays, which may result in discrepancies between the inhibitor titer and clinical bleeding symptoms. Objectives To evaluate FVIII clearance in vivo as a potential mechanism for antibody pathogenicity and to determine whether increased FVIII dosing regimens correct the associated bleeding phenotype. Methods FVIII-/- or FVIII-/- /von Willebrand factor (VWF)-/- mice were infused with anti-FVIII mAbs directed against the FVIII C1, C2 or A2 domains, followed by infusion of FVIII. Blood loss via the tail snip bleeding model, FVIII activity and FVIII antigen levels were subsequently measured. Results Pathogenic anti-C1 mAbs that compete with VWF for FVIII binding increased the clearance of FVIII-mAb complexes in FVIII-/- mice but not in FVIII-/- /VWF-/- mice. Additionally, pathogenic anti-C2 mAbs that inhibit FVIII binding to VWF increased FVIII clearance in FVIII-/- mice. Anti-C1, anti-C2 and anti-A2 mAbs that do not inhibit VWF binding did not accelerate FVIII clearance. Infusion of increased doses of FVIII in the presence of anti-C1 mAbs partially corrected blood loss in FVIII-/- mice. Conclusions A subset of antibodies that inhibit VWF binding to FVIII increase the clearance of FVIII-mAb complexes, which contributes to antibody pathogenicity. This may explain differences in the bleeding phenotype observed despite factor replacement in some patients with hemophilia A and low-titer inhibitors.

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The factor VIII C1 domain contributes to platelet binding.

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The factor VIII C1 domain contributes to platelet binding.

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