Activation of factor VIII by thrombin increases its affinity for binding to synthetic phospholipid membranes and activated platelets.
Membrane-bound thrombin-activated factor VIII (fVIIIa) functions as a cofactor for factor IXa in the factor Xase complex. We found that binding of heterotrimeric fVIIIa (A1.A2.A3-C1-C2) to synthetic vesicles with a physiologic content of 4% phosphatidylserine (PS), 76% phosphatidylcholine, and 20% phosphatidylethanolamine occurs with a 10-fold higher affinity than that of factor VIII (fVIII). The increased affinity of fVIIIa for PS-containing membranes resulted from the reduced rate of fVIIIa dissociation from the vesicles compared with that of fVIII. Similar affinities of A3-C1-C2, A1.A2. A3-C1-C2, and A3-C1-C2.heavy chain for interaction with PS-containing membranes demonstrate that removal of the light chain (LCh) acidic region by thrombin is responsible for these increased affinities of fVIIIa and its derivatives. Similar kinetic parameters of fVIII and its LCh and C2 domain for binding to PS-containing membranes and to activated platelets indicated that the C2 domain is entirely responsible for the interaction of fVIII with membranes. We conclude that the increased fVIIIa affinity for PS-containing membranes is a result of conformational change(s) within the C2 domain upon removal of the acidic region of the LCh. This conclusion is based on the finding that binding of the monoclonal antibody ESH8 to the C2 domain, which is known to prevent this conformational transition, resulted in fVIIIa binding to PS/phosphatidylcholine/phosphatidylethanolamine vesicles (4/76/20) with a lower affinity similar to that of fVIII. In addition, stabilization of the low affinity binding conformation of the C2 domain of fVIIIa by this antibody led to an inhibition of the fVIIIa activity in the factor X activation complex.
Holland Laboratory, American Red Cross, Rockville, Maryland 20855, USA.
E L
EL
Saenko
D
D
Scandella
A V
AV
Yakhyaev
N J
NJ
Greco
D001665:Binding Sites; D001667:Binding, Competitive; D001792:Blood Platelets / Q000378:metabolism*; D015949:Factor IXa / Q000378:metabolism; D005169:Factor VIII / Q000378:metabolism*; D015944:Factor VIIIa / Q000378:metabolism*; D007700:Kinetics; D008567:Membranes, Artificial; D010743:Phospholipids / Q000378:metabolism*; D015539:Platelet Activation; D011485:Protein Binding; D013917:Thrombin / Q000494:pharmacology*
D008567:Membranes, Artificial; D010743:Phospholipids; D015944:Factor VIIIa; D005169:Factor VIII; D015949:Factor IXa; D013917:Thrombin