title: Efficient thrombin generation requires molecular phosphatidylserine, not a membrane surface.
abstract: Activation of prothrombin to thrombin is catalyzed by a "prothrombinase" complex, traditionally viewed as factor X(a) (FX(a)) in complex with factor V(a) (FV(a)) on a phosphatidylserine (PS)-containing membrane surface, which is widely regarded as required for efficient activation. Activation involves cleavage of two peptide bonds and proceeds via one of two released intermediates or through "channeling" (activation without the release of an intermediate). We ask here whether the PS molecule itself and not the membrane surface is sufficient to produce the fully active human "prothrombinase" complex in solution. Both FX(a) and FV(a) bind soluble dicaproyl-phosphatidylserine (C6PS). In the presence of sufficient C6PS to saturate both FX(a) and FV(a2) (light isoform of FV(a)), these proteins form a tight (Kd = 0.6 +/- 0.09 nM at 37 degrees C) soluble complex. Complex assembly occurs well below the critical micelle concentration of C6PS, as established in the presence of the proteins by quasi-elastic light scattering and pyrene fluorescence. Ferguson analysis of native gels shows that the complex migrates with an apparent molecular mass only slightly larger than that expected for one FX(a) and one FV(a2), further ruling out complex assembly on C6PS micelles. Human prothrombin activation by this complex occurs at nearly the same overall rate (2.2 x 10(8) M(-1) s(-1)) and via the same reaction pathway (50-60% channeling, with the rest via the meizothrombin intermediate) as the activation catalyzed by a complex assembled on PS-containing membranes (4.4 x 10(8) M(-1) s(-1)). These results question the accepted role of PS membranes as providing "dimensionality reduction" and favor a regulatory role for platelet-membrane-exposed PS.
authors.affiliation: Program in Molecular and Cellular Biophysics, Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7260, USA.
authors.forename: Rinku
authors.initials: R
authors.lastname: Majumder
authors.forename: Gabriel
authors.initials: G
authors.lastname: Weinreb
authors.forename: Barry R
authors.initials: BR
authors.lastname: Lentz
mesh_terms: D001120:Arginine / Q000031:analogs & derivatives / Q000737:chemistry; D001792:Blood Platelets / Q000502:physiology; D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000502:physiology*; D003619:Dansyl Compounds / Q000737:chemistry; D004789:Enzyme Activation; D005165:Factor V / Q000737:chemistry / Q000378:metabolism; D015943:Factor Va / Q000378:metabolism; D015951:Factor Xa / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008823:Micelles; D008970:Molecular Weight; D010718:Phosphatidylserines / Q000378:metabolism / Q000502:physiology*; D011516:Prothrombin / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D013917:Thrombin / Q000378:metabolism*
chemical_list: C402786:1,2-dicaproylphosphatidylserine; D003619:Dansyl Compounds; D008823:Micelles; D010718:Phosphatidylserines; C022475:prothrombinase complex; C019162:dansylarginine N-(3-ethyl-1,5-pentanediyl)amide; D015943:Factor Va; D005165:Factor V; D011516:Prothrombin; D001120:Arginine; D013917:Thrombin; D015951:Factor Xa; D002118:Calcium