Topology of the binding site of blood-clotting factors in model membranes. A fluorescence study.
Factors II, X and IX are blood-clotting proteins which bind to phospholipid interfaces in the presence of Ca2+ to activate coagulation. The topology of their binding site on the membrane was investigated in two ways. First, the transition temperature changes of equimolar mixtures of dipalmitoylglycerophosphocholine/phosphatidylserine and dimyristoylglycerophosphocholine/dipalmitoylglycerophosphoserin e were examined by the fluorescence polarization of 1,6-diphenylhexatriene. Results show that Ca2+ triggers a shift of about 3-4 degrees C and that blood-clotting factors further increase this shift by about 1.5 degree C. This suggests that in the gel phase, Ca2+ induces some aggregation of the phosphatidylserine molecules which is reinforced by blood proteins. Second, isothermal energy transfer experiments were performed with natural lipids in their fluid phase. The tryptophan residues of the factors were the energy donors, and pyrene covalently bound to a fatty acid chain of either phosphatidylcholine or phosphatidic acid was the energy acceptor. These pyrene-phospholipids probe either the neutral or the acidic component of phospholipid mixtures. It is concluded that the binding sites of the factors are constituted by both types of lipids and that their composition depends on the membrane. Factor II exhibits some specificity for acidic phospholipids and seems to be surrounded by non-interacting zwitterionic lipids. Factor IX appears to be surrounded by statistically the same amount of charged and zwitterionic lipids. We also demonstrate that binding can also occur without Ca2+. This Ca2+-independent binding probably involves electrostatic and hydrophobic forces but its physiological significance remains to be elucidated.
D001665:Binding Sites; D001779:Blood Coagulation Factors / Q000378:metabolism*; D002118:Calcium / Q000502:physiology; D004161:Diphenylhexatriene; D004735:Energy Transfer; D005164:Factor IX / Q000378:metabolism; D005170:Factor X / Q000378:metabolism; D005454:Fluorescence Polarization; D006801:Humans; D008560:Membrane Fluidity; D008563:Membrane Lipids / Q000097:blood*; D010743:Phospholipids / Q000097:blood*; D011516:Prothrombin / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013696:Temperature
D001779:Blood Coagulation Factors; D008563:Membrane Lipids; D010743:Phospholipids; D004161:Diphenylhexatriene; D011516:Prothrombin; D005164:Factor IX; D005170:Factor X; D002118:Calcium