Factor VIII lacking the C2 domain retains cofactor activity in vitro.
Factor (F) VIII consists of a heavy chain (A1A2B domains) and light chain (A3C1C2 domains). The activated form of FVIII, FVIIIa, functions as a cofactor for FIXa in catalyzing the membrane-dependent activation of FX. Whereas the FVIII C2 domain is believed to anchor FVIIIa to the phospholipid surface, recent x-ray crystal structures of FVIII suggest that the C1 domain may also contribute to this function. We constructed a FVIII variant lacking the C2 domain (designated DeltaC2) to characterize the contributions of the C1 domain to function. Binding affinity of the DeltaC2 variant to phospholipid vesicles as measured by energy transfer was reduced approximately 14-fold. However, the activity of DeltaC2 as measured by FXa generation and one-stage clotting assays retained 76 and 36%, respectively, of the WT FVIII value. Modest reductions ( approximately 4-fold) were observed in the functional affinity of DeltaC2 FVIII for FIXa and rates of thrombin activation. On the other hand, deletion of C2 resulted in significant reductions in FVIIIa stability ( approximately 3.6-fold). Thrombin generation assays showed peak thrombin and endogenous thrombin potential were reduced as much as approximately 60-fold. These effects likely result from a combination of the intermolecular functional defects plus reduced protein stability. Together, these results indicate that FVIII domains other than C2, likely C1, make significant contributions to membrane-binding and membrane-dependent function.
Department of Biochemistry and Biophysics, University of Rochester School of Medicine, Rochester, New York 14642, USA.
Hironao
H
Wakabayashi
Amy E
AE
Griffiths
Philip J
PJ
Fay
D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D015949:Factor IXa / Q000378:metabolism; D005169:Factor VIII / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D010743:Phospholipids / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000235:genetics / Q000502:physiology; D013917:Thrombin / Q000378:metabolism
D010743:Phospholipids; C078147:F8 protein, human; D005169:Factor VIII; D015949:Factor IXa; D013917:Thrombin