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 membranes in that changes in surface pressure (Δπ) can be monitored in response to protein adsorption to the monolayer, thus providing a unique 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 coagulation proteins to monolayers that contain negatively charged phospholipid. From combined measurements of Δπ and Γ (the surface excess protein concentration), values of dΓ/dπ have been evaluated for different proteins with varying lipid composition of the monolayers. Using mixed, liquid-expanded monolayers at equivalent initial surface pressures (πi) and which contain different amounts of phosphatidylserine, phosphatidylcholine, and phosphatidylethanolamine, the dΓ/dπ 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 dΓ/dπ values of 14, 17, and 21 nmol m-1 mN-1, respectively. However, the trend in dΓ/dπ appears to originate from characteristics of the monolayer and not from those of the protein, since a much different adsorbate (i.e., a positively charged pyrene derivative) exhibited a similar trend in dΓ/dπ with monolayer composition. On the other hand, dΓ/dπ values of bovine prothrombin, human factor IX, human protein S, bovine protein C, and human protein C, determined using liquid-expanded phosphatidylserine monolayers, were essentially equivalent. Therefore, the five vitamin K-dependent proteins that were examined were equivalent in terms of the manner in which the γ-carboxyglutamic acid (Gla) domain of each protein perturbed the surface pressure. This study shows that Ca2+-specific membrane contact sites in the Gla domain of the five proteins tested are similar despite the naturally occurring differences in the normal Gla domain sequence of these proteins.