J. Biol. Chem., Vol. 259, Issue 22, 13993-13998, Nov, 1984
The adsorption of prothrombin to phospholipid monolayers quantitated by ellipsometry
JM Kop, PA Cuypers, T Lindhout, HC Hemker and WT Hermens
We investigated by means of an automated ellipsometer the calcium-
dependent binding of prothrombin from a buffer solution to monolayers of
dioleoylphosphatidylserine (DOPS) and dioleoylphosphatidylcholine (DOPC)
deposited on chromium slides. This technique allows direct measurements of
bound and free protein concentrations and is not hampered by
calcium-induced aggregation of vesicles. For pure DOPS a dominant class of
binding sites exists with a dissociation constant, Kd = (6 +/- 2) X 10(-10)
M (mean +/- S.D.) and maximal binding of prothrombin, gamma max = 0.26 +/-
0.03 micrograms/cm2. Incorporation of a small fraction of DOPC in the
monolayer causes a large decrease in the binding affinity with a pronounced
biphasic behavior of the binding curve. For monolayers consisting of 20%
DOPS and 80% DOPC the binding curve becomes monophasic with Kd = (1.6 +/-
0.6) X 10(-7) M and gamma max = 0.22 +/- 0.03 micrograms/cm2. The
procoagulant activity of the monolayers was tested by measuring the
generation of thrombin after addition of prothrombin and activated
coagulation factors X and V. The thrombin-generating capacity of monolayers
and single-bilayer vesicles is comparable but is apparently diffusion
limited in the monolayer system. The calcium-dependent formation of stacked
multilayers according to the Blodgett technique appeared to be strongly
influenced by the DOPS/DOPC ratio in the phospholipid monolayer. From these
results it is concluded that for pure DOPS monolayers high-affinity
prothrombin-phospholipid and phospholipid-phospholipid interactions exist
which are radically disturbed when the monolayer contains more than 20-30%
of DOPC.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
