The adsorption of prothrombin to phospholipid monolayers quantitated by ellipsometry

Area of Science:

• Biochemistry
• Surface Science
• Biophysics

Background:

• Prothrombin is a key protein in blood coagulation.
• Phospholipid interactions with proteins are crucial for biological processes.
• Understanding protein-lipid interactions is vital for developing hemostatic agents.

Purpose of the Study:

• To investigate the calcium-dependent binding of prothrombin to dioleoylphosphatidylserine (DOPS) and dioleoylphosphatidylcholine (DOPC) monolayers.
• To quantify the binding affinity and capacity of prothrombin on different phospholipid compositions.
• To assess the impact of phospholipid composition on prothrombin's procoagulant activity.

Main Methods:

• Automated ellipsometry was used to measure protein binding to phospholipid monolayers on chromium slides.
• Direct measurements of bound and free protein concentrations were obtained.
• Thrombin generation assays were performed to evaluate procoagulant activity.

Main Results:

• Pure DOPS monolayers exhibited high-affinity prothrombin binding (Kd = 6 x 10(-10) M).
• Incorporation of DOPC significantly reduced binding affinity, showing biphasic behavior.
• Monolayers with 20% DOPS and 80% DOPC showed reduced affinity (Kd = 1.6 x 10(-7) M).
• Thrombin generation capacity was comparable between monolayers and vesicles but diffusion-limited in monolayers.
• Calcium-dependent multilayer formation was influenced by the DOPS/DOPC ratio.

Conclusions:

• High-affinity prothrombin-phospholipid and phospholipid-phospholipid interactions occur in pure DOPS monolayers.
• These interactions are significantly disrupted when the monolayer contains more than 20-30% DOPC.
• Phospholipid composition critically affects prothrombin binding and procoagulant activity.