The adsorption of prothrombin to phospholipid monolayers quantitated by ellipsometry.
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.
D000327:Adsorption; D001665:Binding Sites; D002122:Calcium Chloride / Q000494:pharmacology; D006801:Humans; D008433:Mathematics; D010713:Phosphatidylcholines / Q000378:metabolism*; D010718:Phosphatidylserines / Q000378:metabolism*; D011516:Prothrombin / Q000378:metabolism*; D012965:Sodium Chloride / Q000494:pharmacology
D010713:Phosphatidylcholines; D010718:Phosphatidylserines; D012965:Sodium Chloride; C042511:1,2-dioleoylphosphatidylserine; D011516:Prothrombin; C017251:1,2-oleoylphosphatidylcholine; D002122:Calcium Chloride