Factor XA binding to phosphatidylserine-containing membranes produces an inactive membrane-bound dimer.
Factor Xa (FXa) has a prominent role in amplifying both inflammation and the coagulation cascade. In the coagulation cascade, its main role is catalyzing the proteolytic activation of prothrombin to thrombin. Efficient proteolysis is well known to require phosphatidylserine (PS)-containing membranes that are provided by platelets in vivo. However, soluble, short-chain PS also triggers efficient proteolytic activity and formation of an inactive FXa dimer in solution. In this work, we ask whether PS-containing membranes also trigger formation of an inactive FXa dimer. We determined the proteolytic activity of human FXa toward human Pre2 as a substrate both at fixed membrane concentration (increasing FXa concentration) and at fixed FXa concentration (increasing membrane concentration). Neither of these experiments showed the expected behavior of an increase in activity as FXa bound to membranes, but instead suggested the existence of a membrane-bound inactive form of FXa. We found also that the fluorescence of fluorescein attached to FXa's active site serine was depolarized in a FXa concentration-dependent fashion in the presence of membranes. The fluorescence lifetime of FXa labeled in its active sites with a dansyl fluorophore showed a similar concentration dependence. We explained all these observations in terms of a quantitative model that takes into account dimerization of FXa after binding to a membrane, which yielded estimates of the FXa dimerization constant on a membrane as well as the kinetic constants of the dimer, showing that the dimer is effectively inactive.
Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.
Tilen
T
Koklic
Rinku
R
Majumder
Gabriel E
GE
Weinreb
Barry R
BR
Lentz
D019281:Dimerization; D015951:Factor Xa / Q000737:chemistry*; D019793:Fluorescein / Q000737:chemistry; D005453:Fluorescence; D005454:Fluorescence Polarization; D006801:Humans; D007700:Kinetics; D008954:Models, Biological; D009842:Oligopeptides; D010713:Phosphatidylcholines / Q000737:chemistry; D010718:Phosphatidylserines / Q000737:chemistry*; D055503:Protein Multimerization; D011516:Prothrombin / Q000737:chemistry; D013917:Thrombin / Q000737:chemistry; D053835:Unilamellar Liposomes / Q000737:chemistry*
D009842:Oligopeptides; D010713:Phosphatidylcholines; D010718:Phosphatidylserines; D053835:Unilamellar Liposomes; C048812:kinetensin; D011516:Prothrombin; C012007:prethrombins; D013917:Thrombin; D015951:Factor Xa; C017251:1,2-oleoylphosphatidylcholine; D019793:Fluorescein