ADSORPTION OF VITAMIN-K-DEPENDENT BLOOD-COAGULATION PROTEINS TO SPREAD PHOSPHOLIPID MONOLAYERS AS DETERMINED FROM COMBINED MEASUREMENTS OF THE SURFACE PRESSURE AND SURFACE PROTEIN-CONCENTRATION

Spread phospholipid monolayers are particularly useful as model membra nes in that changes in surface pressure (Delta pi) can be monitored in response to protein adsorption to the monolayer, thus providing a uni que manner of assessing protein-membrane contact. In the present study , spread monolayers below their collapse pressures have been utilized to evaluate Ca2+-specific adsorption of several vitamin K-dependent co agulation proteins to monolayers that contain negatively charged phosp holipid. From combined measurements of Delta pi and Gamma (the surface excess protein concentration), values of d Gamma/d pi have been evalu ated for different proteins with varying lipid composition of the mono layers. Using mixed, liquid-expanded monolayers at equivalent initial surface pressures (pi(i)) and which contain different amounts of phosp hatidylserine, phosphatidylcholine, and phosphatidylethanolamine, the d Gamma/d pi of bovine prothrombin was shown to decrease monotonically with increasing protein affinity for the monolayer. For example, K-D values of 7, 20, and 60 nM produced d Gamma/d pi values of 14, 17, and 21 nmol m(-1) mN(-1), respectively. However, the trend in d Gamma/d p i appears to originate from characteristics of the monolayer and not f rom those of the protein, since a much different adsorbate (i.e., a po sitively charged pyrene derivative) exhibited a similar trend in d Gam ma/d pi with monolayer composition. On the other hand, d Gamma/d pi va lues of bovine prothrombin, human factor IX, human protein S, bovine p rotein C, and human protein C, determined using liquid-expanded phosph atidylserine monolayers, were essentially equivalent. Therefore, the f ive vitamin K-dependent proteins that were examined were equivalent in terms of the manner in which the gamma-carboxyglutamic acid (Gla) dom ain of each protein perturbed the surface pressure. This study shows t hat Ca2+-specific membrane contact sites in the Gla domain of the five proteins tested are similar despite the naturally occurring differenc es in the normal Gla domain sequence of these proteins.