Use of surface plasmon resonance for studies of protein-protein and protein-phospholipid membrane interactions - Application to the binding of factorVIII to von Willebrand factor and to phosphatidylserine-containing membranes

The surface plasmon resonance phenomenon is used for real time measurements of protein-protein and protein-membrane interactions. In the present study two surface plasmon resonance-based binding assays permitting study of the interaction of coagulation factor WI (fVIII) with von Willebrand factor (v Wf) and phospholipid have been developed. These interactions of fVIII are r equired for maintenance of fVIII concentration in circulation and for the a ssembly of the functional factor Xase complex, respectively. With these bin ding assays, the role of the light chain (LCh) in fVIII binding to vWf and to immobilized phospholipid monolayers and intact vesicles containing 25% p hosphatidylserine (PS) and 4% PS was examined. The finding that K-d of LCh binding to vWf (3.8 nM) is 9.5 times higher than that of fVIII (0.4 nM), in dicates that the heavy chain (HCh) is required for the maximal affinity of Nm for vWf. In contrast, affinities of LCh for 25/75 PS/phosphatidylcholine (PC) monolayers and 4/76/20 PSPC-phosphatidylethanolamine (PE) vesicles ar e similar to that of fVIII, indicating that LCh is solely responsible for t hese interactions. It was also examined how removal of the acidic region af fects the binding affinity of the remaining part of LCh for vWf and phospho lipid. It was demonstrated that the loss of the LCh acidic region upon thro mbin cleavage leads to an 11 and 160-fold increase in the dissociation rate constant (k(off) value) and a 165 and 1500-fold increase in the K-d value of the binding of fVIII fragment A3-C1-C2 to vWf compared to that of LCh an d fVIII, respectively. In contrast, the binding affinity of A3-C1-C2 for PS -containing membranes was 8-11-fold higher than that of LCh. Possible confo rmational change(s) in C2 domain upon removal of the acidic region were stu died using anti-fVIII monoclonal antibody NMC-VIII/5 with an epitope within the C2 domain of LCh as a probe. The determined lower binding affinity of A3-C1-C2 for NMC-VIII/5 immobilized to a sensor chip than that of LCh, indi cates that these conformational changes do occur. (C) 1999 Elsevier Science B.V. All rights reserved.