Interaction of bovine blood clotting factor Va and its subunits with phospholipid vesicles.
Thrombin-activated factor Va and factor Va subunit binding to large-volume vesicles was investigated by a technique based on the separation by centrifugation of phospholipid-bound protein from the bulk solution. This technique allows the direct measurement of free-protein concentration. It is concluded that the phospholipid binding site on factor Va is located on a basic factor Va subunit with Mr 80 000 (factor Va-LC). The effects of phospholipid vesicle composition, calcium concentration, pH, and ionic strength on the equilibrium constants of factor Va- and factor Va-LC-phospholipid interaction were studied. Factor Va and factor Va-LC binding to phospholipid requires the presence of negatively charged phospholipids. It is further demonstrated that the following occur: (a) Calcium ions compete with factor Va and factor Va-LC for phospholipid-binding sites. (b) The dissociation constant of protein-phospholipid interaction increases with the ionic strength, whereas the maximum protein-binding capacity of the phospholipid vesicle was not affected by ionic strength. (c) The dissociation constant for factor Va-phospholipid interaction depends on pH when the vesicle consists of phosphatidic acid. It is concluded that factor Va-phospholipid interaction is primarily electrostatic in nature, where positively charged groups on the protein directly interact with the phosphate group of net negatively charged phospholipids. The results suggest that factor Va, like factor Xa and prothrombin, has the characteristics of an extrinsic membrane protein.
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002417:Cattle; D005165:Factor V / Q000378:metabolism*; D015943:Factor Va; D005170:Factor X / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008081:Liposomes; D046911:Macromolecular Substances; D009994:Osmolar Concentration; D010713:Phosphatidylcholines; D011485:Protein Binding; D011516:Prothrombin / Q000378:metabolism; D013917:Thrombin / Q000378:metabolism
D008081:Liposomes; D046911:Macromolecular Substances; D010713:Phosphatidylcholines; D015943:Factor Va; D005165:Factor V; D011516:Prothrombin; D005170:Factor X; D013917:Thrombin; C017251:1,2-oleoylphosphatidylcholine; D002118:Calcium