Gierula, MagdalenaMagdalenaGierulaSalles‐Crawley, Isabelle IIsabelle ISalles‐CrawleySantamaria, SalvatoreSalvatoreSantamariaTeraz‐Orosz, AdriennAdriennTeraz‐OroszCrawley, James TBJames TBCrawleyLane, David ADavid ALaneAhnström, JosefinJosefinAhnström2019-08-022020-07-302019-07-30Journal of Thrombosis and Haemostasis, 20191538-7933http://hdl.handle.net/10044/1/72223Background: Activated protein C (APC ) -mediated inactivation of factor ( F )Va is greatly enhanced by protein S. For inactivation to occur, a trimolecular complex between FVa, APC and protein S must form on the phospholipid membrane. However, direct demonstration of complex formation has proven elusive. Objectives: To elucidate the nature of the phospholipid -dependent interaction s between APC, protein S and FVa . Methods: We evaluated binding of active site blocked APC to phospholipid -coated magnetic beads in the presence and absence of protein S and/or FVa. The importance of protein S and FV residues were evaluated functionally. Results: APC alone bound weakly to phospholipids. Protein S mildly enhanced APC binding to phospholipid surfaces, whereas FVa did not. However, FVa together with protein S enhanced APC binding ( >14 -fold), demonstrating formation of an APC/protein S/FVa complex. C4b binding protein -bound protein S failed to enhance APC binding, agreeing with its reduced APC cofactor function. Protein S variants (E36A and D95A) with reduced APC cofactor function exhibited essentially normal augmentation of APC binding to phospholipids, but diminished APC/protein S/FVa complex formation, suggesting involvement in interactions dependent upon FVa. Similarly, FVaNara (W1920R), an APC resistant FV variant, also did not efficiently incorporate into the trimolecular complex as efficiently as wild -type FVa. FVa inactivation assays suggested that the mutation impairs its affinity for phospholipid membranes and with protein S within the complex. Conclusions: FVa plays a central role in the formation of its inactivation complex. Furthermore, membrane proximal interactions between FVa, APC and protein S are essential for its cofactor function.© 2019 Owner. This is the accepted version of the following article: Gierula, M. , Salles‐Crawley, I. I., Santamaria, S. , Teraz‐Orosz, A. , Crawley, J. T., Lane, D. A. and Ahnström, J. (2019), The roles of factor Va and protein S in formation of the activated protein C/protein S/factor Va inactivation complex. J Thromb Haemost. Accepted Author Manuscript. doi:10.1111/jth.14594, which has been published in final form at https://doi.org/10.1111/jth.14594.1102 Cardiorespiratory Medicine and Haematology1103 Clinical SciencesCardiovascular System & HematologyJournal Articlehttps://www.dx.doi.org/10.1111/jth.14594PG/14/90/31219FS/10/004/28165FS/10/004/281651538-7836