Phosphatidylserine and FVa regulate FXa structure.
Human coagulation FXa (Factor Xa) plays a key role in blood coagulation by activating prothrombin to thrombin on 'stimulated' platelet membranes in the presence of its cofactor FVa (Factor Va). PS (phosphatidylserine) exposure on activated platelet membranes promotes prothrombin activation by FXa by allosterically regulating FXa. To identify the structural basis of this allosteric regulation, we used FRET to monitor changes in FXa length in response to (i) soluble short-chain PS [C6PS (dicaproylphosphatidylserine)], (ii) PS membranes, and (iii) FVa in the presence of C6PS and membranes. We incorporated a FRET pair with donor (fluorescein) at the active site and acceptor (Alexa Fluor® 555) at the FXa N-terminus near the membrane. The results demonstrated that FXa structure changes upon binding of C6PS to two sites: a regulatory site at the N-terminus [identified previously as involving the Gla (γ-carboxyglutamic acid) and EGFN (N-terminus of epidermal growth factor) domains] and a presumptive protein-recognition site in the catalytic domain. Binding of C6PS to the regulatory site increased the interprobe distance by ~3 Å (1 Å=0.1 nm), whereas saturation of both sites increased the distance by a further ~6.4 Å. FXa binding to a membrane produced a smaller increase in length (~1.4 Å), indicating that FXa has a somewhat different structure on a membrane from when bound to C6PS in solution. However, when both FVa2 (a FVa glycoform) and either C6PS- or PS-containing membranes were bound to FXa, the overall change in length was comparable (~5.6-5.8 Å), indicating that C6PS- and PS-containing membranes in conjunction with FVa2 have comparable regulatory effects on FXa. We conclude that the similar functional regulation of FXa by C6PS or membranes in conjunction with FVa2 correlates with similar structural regulation. The results demonstrate the usefulness of FRET in analysing structure-function relationships in FXa and in the FXa·FVa2 complex.
*Department of Biochemistry and Biophysics, and Program in Molecular and Cellular Biophysics, University of North Carolina, Chapel Hill, NC 27599-7260, U.S.A.
Kinshuk Raj
KR
Srivasatava
*Department of Biochemistry and Biophysics, and Program in Molecular and Cellular Biophysics, University of North Carolina, Chapel Hill, NC 27599-7260, U.S.A.
Rinku
R
Majumder
†Division of Hematology, Departments of Medicine and Pathology, Duke University Medical Center, Durham, NC 27710, U.S.A.
William H
WH
Kane
‡Department of Molecular Pathology, Duke University Health System, Durham, NC 2771, U.S.A.
Mary Ann
MA
Quinn-Allen
*Department of Biochemistry and Biophysics, and Program in Molecular and Cellular Biophysics, University of North Carolina, Chapel Hill, NC 27599-7260, U.S.A.
Barry R
BR
Lentz
D000494:Allosteric Regulation / Q000187:drug effects / Q000502:physiology; D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D015943:Factor Va / Q000502:physiology*; D015951:Factor Xa / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D010718:Phosphatidylserines / Q000378:metabolism* / Q000494:pharmacology; D011485:Protein Binding / Q000502:physiology; D021281:Quantitative Structure-Activity Relationship
D010718:Phosphatidylserines; D015943:Factor Va; D015951:Factor Xa