Abstract
Factor VIII (FVIII) is activated by proteolytic cleavages with thrombin and factor Xa (FXa) in the intrinsic blood coagulation pathway. The anti-C2 monoclonal antibody ESH8, which recognizes residues 2248-2285 and does not inhibit FVIII binding to von Willebrand factor or phospholipid, inhibited FVIII activation by FXa in a clotting assay. Furthermore, analysis by SDS-polyacrylamide gel electrophoresis showed that ESH8 inhibited FXa cleavage in the presence or absence of phospholipid. The light chain (LCh) fragments (both 80 and 72 kDa) and the recombinant C2 domain dose-dependently bound to immobilized anhydro-FXa, a catalytically inactive derivative of FXa in which dehydroalanine replaces the active-site serine. The affinity (K(d)) values for the 80- and 72-kDa LCh fragments and the C2 domain were 55, 51, and 560 nM, respectively. The heavy chain of FVIII did not bind to anhydro-FXa. Similarly, competitive assays using overlapping synthetic peptides corresponding to ESH8 epitopes (residues 2248-2285) demonstrated that a peptide designated EP-2 (residues 2253-2270; TSMYVKEFLISSSQDGHQ) inhibited the binding of the C2 domain or the 72-kDa LCh to anhydro-FXa by more than 95 and 84%, respectively. Our results provide the first evidence for a direct role of the C2 domain in the association between FVIII and FXa.
Highlights
Factor VIII (FVIII) is activated by proteolytic cleavages with thrombin and factor Xa (FXa) in the intrinsic blood coagulation pathway
In order to examine the influence of anti-FVIII monoclonal antibodies, FXa-dependent FVIII activation in the presence of each antibody was performed in the presence of PL
NMC-VIII/10, C5, and JR8 did not inhibit FVIII cleavage in the absence of PL (Fig. 2B, lanes 5–7, respectively). All of these findings indicated that the inhibitory effect of ESH8 on FXa-induced activation of FVIII could be attributed to a PL-independent mechanism, in particular inhibition of FVIII light chain (LCh) proteolysis at Arg1689 and Arg1721
Summary
FVIII was affinity-purified using monoclonal antibody NMC-VIII/10, recognizing the FVIII A3 domain. Elution from the monoclonal antibody column was performed with 1 M KI and 40% ethylene glycol as described previously [25]. The specific activity of the purified FVIII was 2700 units/mg. Enzyme-linked immunosorbent assay (ELISA) demonstrated that the purified FVIII was free of vWF antigen [26]. LCh and HCh fragments of FVIII, together with A1, A2, and thrombin-cleaved 72-kDa LCh fragments, were prepared from plasma FVIII as described previ-. NMC-VIII/10 and C5 bind to the acidic region of the A3 and A1 domains, respectively
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
