Current models for cellular plasma membranes focus on spatial heterogeneity and how this heterogeneity relates to cell function. In particular, putative lipid raft membrane domains have been postulated to exist based in large part on the results that a significant fraction of the membrane is detergent insoluble and that molecules facilitating key membrane processes like signal transduction are often found in the detergent-resistant membrane fraction. Yet, the in vivo existence of lipid rafts remains extremely controversial because, despite being sought for more than a decade, evidence for their presence in intact cell membranes is inconclusive. In this review, a variety of experimental techniques that have been or might be used to look for lipid microdomains in intact cell membranes are described. Experimental results are highlighted and the strengths and limitations of different techniques for microdomain identification and characterization are assessed.

We propose a model that accounts for the time courses of PEG-induced fusion of membrane vesicles of varying lipid compositions and sizes. The model assumes that fusion proceeds from an initial, aggregated vesicle state ((A) membrane contact) through two sequential intermediate states (I(1) and I(2)) and then on to a fusion pore state (FP). Using this model, we interpreted data on the fusion of seven different vesicle systems. We found that the initial aggregated state involved no lipid or content mixing but did produce leakage. The final state (FP) was not leaky. Lipid mixing normally dominated the first intermediate state (I(1)), but content mixing signal was also observed in this state for most systems. The second intermediate state (I(2)) exhibited both lipid and content mixing signals and leakage, and was sometimes the only leaky state. In some systems, the first and second intermediates were indistinguishable and converted directly to the FP state. Having also tested a parallel, two-intermediate model subject to different assumptions about the nature of the intermediates, we conclude that a sequential, two-intermediate model is the simplest model sufficient to describe PEG-mediated fusion in all vesicle systems studied. We conclude as well that a fusion intermediate "state" should not be thought of as a fixed structure (e.g., "stalk" or "transmembrane contact") of uniform properties. Rather, a fusion "state" describes an ensemble of similar structures that can have different mechanical properties. Thus, a "state" can have varying probabilities of having a given functional property such as content mixing, lipid mixing, or leakage. Our data show that the content mixing signal may occur through two processes, one correlated and one not correlated with leakage. Finally, we consider the implications of our results in terms of the "modified stalk" hypothesis for the mechanism of lipid pore formation. We conclude that our results not only support this hypothesis but also provide a means of analyzing fusion time courses so as to test it and gauge the mechanism of action of fusion proteins in the context of the lipidic hypothesis of fusion.

Activation of prothrombin by factor X(a) requires proteolysis of two bonds and is commonly assumed to occur via by two parallel, sequential pathways. Hydrolysis of Arg(322)-Ile(323) produces meizothrombin (MzII(a)) as an intermediate, while hydrolysis of Arg(273)-Thr(274) produces prethrombin 2-fragment 1.2 (Pre2-F1.2). Activation by human factor X(a) of human prothrombin was examined in the absence of factor V(a) and in the absence and presence of bovine phosphatidylserine (PS)/palmitoyloleoylphosphatidylcholine (25:75) membranes. Four sets of data were collected: fluorescence of an active site probe (DAPA) was sensitive to thrombin, MzII(a), and Pre2-F1.2; a synthetic substrate (S-2238) detected thrombin or MzII(a) active site formation; and SDS-PAGE detected both intermediates and thrombin. The fluorescence data provided an internal check on the active site and SDS-PAGE measurements. Kinetic constants for conversion of intermediates to thrombin were measured directly in the absence of membranes. Both MzII(a) and Pre2-F1.2 were consumed rapidly in the presence of membranes, so kinetic constants for these reactions had to be estimated as adjustable parameters by fitting three data sets (thrombin and MzII(a) active site formation and Pre2 appearance) simultaneously to the parallel-sequential model. In the absence of membranes, this model successfully described the data and yielded a rate constant, 44 M(-1) s(-1), for the rate of MzII(a) formation. By contrast, the parallel-sequential model could not describe prothrombin activation in the presence of optimal concentrations of PS-containing membranes without assuming that a pathway existed for converting prothrombin directly to thrombin without release from the membrane-enzyme complex. The data suggest that PS membranes (1) regulate factor X(a), (2) alter the substrate specificity of factor X(a) to favor the meizothrombin intermediate, and (3) "channel" intermediate (MzII(a) or Pre2-F1.2) back to the active site of factor X(a) for rapid conversion to thrombin.

Factor X a (FX a ) binding to factor V a (FV a ) on plateletderived membranes containing surface-exposed phosphatidylserine (PS) forms the "prothrombinase complex" that is essential for efficient thrombin generation during blood coagulation. There are two naturally occurring isoforms of FV a , FV a1 and FV a2 . These two isoforms differ by a 3-kDa polysaccharide chain ( ). The ability of soluble PS to trigger formation of a soluble prothrombinase complex suggests that exposure of PS molecules during platelet activation is likely the key event responsible for the assembly of an active membrane-bound complex.The final step in the blood coagulation cascade involves the activation of prothrombin to thrombin, which is the central enzyme of the coagulation system. This activation requires assembly of an enzyme complex, called prothrombinase (1), which consists of blood coagulation factors X a (a serine protease) and V a (a cofactor), Ca 2ϩ , and membranous vesicles derived from stimulated platelets (2). Several studies (3-5) have suggested that phosphatidylserine (PS) 1 might play a specific role in prothrombin activation. PS is asymmetrically distributed to the cytoplasmic surface of resting platelet membranes (6) but is exposed when human platelets are activated (7). It has become clear only very recently that PS regulates the structure and function of factors X a and V a (8, 10). 2 Here we explore further the extent of this regulation.Factor V exists in plasma as an inactive, single chain glycoprotein with a molecular mass of 330 kDa. The active form of factor V, FV a , has a central domain removed to yield a heterodimer composed of two chains, a heavy chain (M r ϭ 94,000 in the bovine species; 105,000 in human) and a heterogeneous light chain (M r ϭ 74,000 in FV a1 or 71,000 in FV a2 ). The heavy and light chains form a tight complex in the presence of a calcium ion (11). The heterogeneity in the light chain is seen in both the bovine and human molecules. In the human form, it appears to arise from glycosylation of Asn 2181 at the C-terminal end of the light chain (12). Prothrombinase complexes assembled from the two molecular species derived from human plasma are observed to have somewhat different cofactor activities (13,14). This has been attributed to substantially different affinities for binding to membranes (13). However, our lab has reported that the two forms of both bovine and human FV a bind to membranes with only ϳ3-fold different affinities (12,14). This suggests that differences in the ability to support prothrombinase activity must reflect either different binding between factors FX a and FV a1 versus FV a2 or different intrinsic activities of the FX a ⅐FV a1 and FX a ⅐FV a2 complexes. Although our results have favored the former possibility (14), it has been difficult to prove this unambiguously because it is difficult to measure precisely the interaction between FX a and FV a on a membrane surface (15).The presence of FV a in a reaction mixture is critical to obtaining a maximal and physiologicall...

Activation of prothrombin to thrombin is catalyzed by a "prothrombinase" complex, traditionally viewed as factor X(a) (FX(a)) in complex with factor V(a) (FV(a)) on a phosphatidylserine (PS)-containing membrane surface, which is widely regarded as required for efficient activation. Activation involves cleavage of two peptide bonds and proceeds via one of two released intermediates or through "channeling" (activation without the release of an intermediate). We ask here whether the PS molecule itself and not the membrane surface is sufficient to produce the fully active human "prothrombinase" complex in solution. Both FX(a) and FV(a) bind soluble dicaproyl-phosphatidylserine (C6PS). In the presence of sufficient C6PS to saturate both FX(a) and FV(a2) (light isoform of FV(a)), these proteins form a tight (Kd = 0.6 +/- 0.09 nM at 37 degrees C) soluble complex. Complex assembly occurs well below the critical micelle concentration of C6PS, as established in the presence of the proteins by quasi-elastic light scattering and pyrene fluorescence. Ferguson analysis of native gels shows that the complex migrates with an apparent molecular mass only slightly larger than that expected for one FX(a) and one FV(a2), further ruling out complex assembly on C6PS micelles. Human prothrombin activation by this complex occurs at nearly the same overall rate (2.2 x 10(8) M(-1) s(-1)) and via the same reaction pathway (50-60% channeling, with the rest via the meizothrombin intermediate) as the activation catalyzed by a complex assembled on PS-containing membranes (4.4 x 10(8) M(-1) s(-1)). These results question the accepted role of PS membranes as providing "dimensionality reduction" and favor a regulatory role for platelet-membrane-exposed PS.