Topology of factor VIII bound to phosphatidylserine-containing model membranes.

Factor VIII (FVIII), a plasma glycoprotein, is an essential cofactor in the blood coagulation cascade. It is a multidomain protein, known to bind to phosphatidylserine (PS)-containing membranes. Based on X-ray and electron crystallography data, binding of FVIII to PS-containing membranes has been proposed to occur only via the C2 domain. Based on these models, the molecular topology of membrane-bound FVIII can be envisioned as one in which only a small fraction of the protein interacts with the membrane, whereas the majority of the molecule is exposed to an aqueous milieu. We have investigated the topology of the membrane-bound FVIII using biophysical and biochemical techniques. Circular dichroism (CD) and fluorescence studies indicate no significant changes in the secondary and tertiary structure of FVIII associated with the membranes. Acrylamide quenching studies show that the protein is predominantly present on the surface of the membrane, exposed to the aqueous milieu. The light scattering and electron microscopy studies indicate the absence of vesicle aggregation and fusion. Binding studies with antibodies directed against specific epitopes in the A1, A2 and C2 domains suggest that FVIII binds to the membrane primarily via C2 domain including the specific phospholipid binding epitope (2303-2332) and may involve subtle conformational changes in this epitope region.

Preparation and characterization of taxane-containing liposomes.

Drug carriers such as liposomes provide a means to alter the biodisposition of drugs and to achieve concentration-time exposure profiles in tissue or tumor that are not readily accomplished with free drug. These changes in biodisposition can improve treatment efficacy. For hydrophobic drugs, incorporation in liposome carriers can increase drug solubility markedly. The taxanes paclitaxel (taxol) and docetaxel (Taxotere) are members of one of the most important new classes of oncology drugs. However, their poor solubility presents pharmaceutical challenges, and emerging data suggest that specific tissue exposure profiles, such as low drug concentrations for extended times, can enhance beneficial antitumor mechanisms. Incorporation of the taxanes into liposomes eliminates not only the toxic effects of cosolvents required to administer these drugs clinically but also increases drug efficacy in animal tumor models, usually through a reduction in dose-limiting tissue toxicities. Although the taxanes are poorly water soluble, the preparation of physically stabile taxane-liposome formulations requires the balancing of three factors: (1) the drug:lipid ratio, (2) the liposome composition, and (3) the duration of storage in aqueous media. Biophysical evaluation of formulation characteristics, principally using circular dichroism (CD) and differential scanning calorimetry (DSC), can provide the information necessary to develop stable taxane-liposome formulations. These techniques provide information on drug-drug and drug-lipid interactions that underlie the events that lead to taxane formulation instability. Owing to the unusually low solubility of the taxanes, special consideration is necessary to devise methods for resolving drug-containing liposomes from released or precipitated drug to obtain reliable estimates of drug incorporation and retention in liposomes.

Propofol, a general anesthetic, promotes the formation of fluid phase domains in model membranes.

The molecular site of anesthetic action remains an area of intense research interest. It is not clear whether general anesthetics act through direct binding to proteins or by perturbing the membrane properties of excitable tissues. Several studies indicate that anesthetics affect the properties of either membrane lipids or proteins. However, gaps remain in our understanding of the molecular mechanism of anesthetic action. Recent developments in membrane biology have led to the concept of small-scale domain structures in lipid and lipid--protein coupled systems. The role of such domain structures in anesthetic action has not been studied in detail. In the present study, we investigated the effect of anesthetics on lipid domain structures in model membranes using the fluorescent spectral properties of Laurdan (6-dodecanoyl-2-dimethylamino naphthalene). Propofol, a general anesthetic, promoted the formation of fluid domains in model membranes of dipalmitoyl phosphatidyl choline (DPPC) or mixtures of lipids of varying acyl chains (DPPC:DMPC dimyristoyl phosphatidyl choline 1:1). The estimated size of these domains is 20--50 A. Based on these studies, we speculate that the mechanism of anesthetic action may involve effects on protein--lipid coupled systems through alterations in small-scale lipid domain structures.