title: Soluble phosphatidylserine binds to two sites on human factor IXa in a Ca2+ dependent fashion to specifically regulate structure and activity.
abstract: Clinical studies have demonstrated a correlation between elevated levels of FIX and the risk of coronary heart disease, while reduced plasma FIX causes hemophilia B. FIXa interacts with FVIIIa in the presence of Ca2+ and phosphatidylserine (PS)-containing membranes to form a factor X-activating complex (Xase) that is key to propagation of the initiated blood coagulation process in human. We test the hypothesis that PS in these membranes up-regulates the catalytic activity of this essential enzyme. We used a soluble form of phosphatidylserine, 1, 2-dicaproyl-sn-glycero-3-phospho-L-serine (C6PS), as a tool to do so. C6PS and PS in membranes are reported to regulate the homologous FXa nearly identically. FIXa binds a molecule of C6PS at each of with two sites with such different affinities (∼100-fold) that these appear to be independent. A high affinity C6PS binding site (Kd∼1.4 µM) regulates structure, whereas a low-affinity binding site (Kd∼140 µM) regulates activity. Equilibrium dialysis experiments were analyzed globally with four other data sets (proteolytic and amidolytic activities, intrinsic fluorescence, ellipticity) to unequivocally demonstrate stoichiometries of one for both sites. Michaelis-Menten parameters for FIXa proteolytic activity were the same in the presence of C6PS or PS/PC membranes. We conclude that the PS molecule and not a membrane surface is the key regulator of both factors Xa and IXa. Despite some minor differences in the details of regulation of factors Xa and IXa, the similarities we found suggest that lipid regulation of these two proteases may be similar, a hypothesis that we continue to test.
authors: Department of Biochemistry & Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America.
authors: Rinku
authors: R
authors: Majumder
authors: Department of Biochemistry & Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America.
authors: Tilen
authors: T
authors: Koklic
authors: Department of Biochemistry & Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America.
authors: Tanusree
authors: T
authors: Sengupta
authors: Department of Biochemistry & Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America.
authors: Daud
authors: D
authors: Cole
authors: Department of Biochemistry & Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America.
authors: Rima
authors: R
authors: Chattopadhyay
authors: Department of Biochemistry & Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America.
authors: Subir
authors: S
authors: Biswas
authors: Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America.
authors: Dougald
authors: D
authors: Monroe
authors: Department of Biochemistry & Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America.
authors: Barry R
authors: BR
authors: Lentz
mesh_terms: D001665:Binding Sites; D001777:Blood Coagulation; D002118:Calcium / Q000737:chemistry* / Q000378:metabolism; D002863:Chromogenic Compounds / Q000737:chemistry; D002942:Circular Dichroism; D015949:Factor IXa / Q000737:chemistry* / Q000378:metabolism; D005170:Factor X / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D010718:Phosphatidylserines / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D059748:Proteolysis; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012996:Solutions; D013050:Spectrometry, Fluorescence
chemical_list: D002863:Chromogenic Compounds; D010718:Phosphatidylserines; D011994:Recombinant Proteins; D012996:Solutions; D005170:Factor X; D015949:Factor IXa; D002118:Calcium