Activation of Factor VIII by Thrombin Increases Its Affinity for Binding to Synthetic Phospholipid Membranes and Activated Platelets

Abstract

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.

The factor VIII C1 domain contributes to platelet binding

“…Alternatively, conformational changes within the C2 domain(s) could increase the affinity of FVa and FVIIIa for membranes. 14 In the current study, the FVIII C1C2 protein blocked FVIII clotting activity at a 4-fold lower concentration than C2. In this 1-stage assay, 50% inhibition was achieved at 10 5 molar excess of C2 over FVIII, essentially the same ratio that produced 50% inhibition of FVIII in an intrinsic X-ase assay.…”

“…The FVIIIa light chain binds to phospholipid vesicles 14,15 and activated platelets 10,14 with a higher affinity than C2, suggesting the C1 domain may contribute to membrane binding. A molecular model of the FVIII C1 domain, 21 constructed from its homology with C2, indicated that C1 lacks the loops that expose adjacent hydrophobic residues (M2199-F2200 and L2251-L2252) in the analogous region of C2.…”

“…9 A hydrophobic surface on the FVIII(a) C2 domain 11 becomes buried in the phospholipid membrane upon binding, 12,13 and basic C2 residues make favorable charge-charge interactions with negatively charged PS head groups. Although FVIIIa and the light chain bind to PS-containing vesicles and activated platelets with similar affinities, the affinity of the recombinant C2 domain is 5-to 100-fold lower, 10,14,15 suggesting possible roles for the C1 and/or A3 domains.…”

“…9 A hydrophobic surface on the FVIII(a) C2 domain 11 becomes buried in the phospholipid membrane upon binding, 12,13 and basic C2 residues make favorable charge-charge interactions with negatively charged PS head groups. Although FVIIIa and the light chain bind to PS-containing vesicles and activated platelets with similar affinities, the affinity of the recombinant C2 domain is 5-to 100-fold lower, 10,14,15 suggesting possible roles for the C1 and/or A3 domains.To address the potential role of the C1 domain in FVIII(a) attachment to platelets, a recombinant human FVIII C1C2 protein (residues 2020-2332) was produced in Escherichia coli, refolded, and characterized. The binding of C1C2 to platelet surfaces both before and after platelet activation was compared with C2 11,15 binding using flow cytometry.…”

Isolation of a Drosophila Gene Coding for a Protein Containing a Novel Phosphatidylserine-Binding Motif

“…6) and synaptotagmin I (7) in the regulation of signaling pathways and membrane fusion, respectively. Previous studies have revealed the presence of primary sequences responsible for the binding to PS, such as the C2 domain in protein kinase C (8-10), synaptotagmin I (7), and factor VIII (11), and the γ-carboxyglutamic acid domain in factor Xa (12) and Gas6 (13), the latter of which is presumably involved in phagocyte recognition of apoptotic cells (14). Many PS-binding proteins require Ca 2+ , and this ion has been shown to bridge PS and protein kinase C (8,10).…”