Reconstitution of phospholipid scramblase activity from human blood platelets.

Cellular activation, accompanied by elevation of cytoplasmic Ca2+ levels, can induce a progressive loss of plasma membrane phospholipid asymmetry, resulting from increased transbilayer movement (flip-flop) of phospholipids. While this process has been demonstrated in a variety of different cells, it is most active in blood platelets. In order to test whether this lipid scrambling process is mediated by a membrane protein, platelet membranes were solubilized in cholate and fractionated by anion exchange chromatography, and fractions were reconstituted into phospholipid vesicles by detergent dialysis in the presence of small amounts of fluorescent (NBD) phospholipids. Using dithionite reduction to monitor the transbilayer location of NBD phospholipids, it was shown that addition of Ca2+ and ionomycin to vesicles reconstituted with a particular fraction results in transbilayer movement of the fluorescent phospholipid analogs from the vesicle's inner to outer leaflet. Lipid vesicles reconstituted in the absence of membrane protein, or reconstituted with another platelet membrane protein fraction, were devoid of this activity. Heating the active fraction or incubating it with pronase or the SH reagent pyridyldithioethylamine markedly diminished the ability of the vesicles to translocate fluorescent phospholipid analogs across the bilayer in response to Ca2+ and ionophore. These results argue that a membrane protein (or proteins) from blood platelets is required to catalyze Ca2+-induced transbilayer movement of phospholipids, suggesting its (or their) involvement in the loss of lipid asymmetry that can occur during cellular activation.

Contribution of different phospholipid classes to the prothrombin converting capacity of sonicated lipid vesicles.

The influence of different neutral phospholipids and cholesterol on the procoagulant properties of sonicated vesicles containing phosphatidylserine was studied, using the prothrombinase assay. When incorporated into membranes composed of phosphatidylcholine and phosphatidylserine, a stimulating effect of phosphatidylethanolamine and an inhibiting effect of sphingomyelin was observed. Cholesterol slightly increased the activities of all vesicles tested. In lipid vesicles with a composition mimicking that of the outer leaflet of the plasma membrane of the activated platelet, the inhibitory effect of sphingomyelin was overruled by an overall stimulatory effect of phosphatidylethanolamine, suggesting an accessory role for phosphatidylethanolamine in the procoagulant properties of activated platelets.

The complex of phosphatidylinositol 4,5-bisphosphate and calcium ions is not responsible for Ca2+-induced loss of phospholipid asymmetry in the human erythrocyte: a study in Scott syndrome, a disorder of calcium-induced phospholipid scrambling.

Elevation of cytoplasmic Ca2+ levels in human erythrocytes induces a progressive loss of membrane phospholipid asymmetry, a process that is impaired in erythrocytes from a patient with Scott syndrome. We show here that porcine erythrocytes are similarly incapable of Ca2+-induced redistribution of membrane phospholipids. Because a complex of phosphatidylinositol 4,5-bisphosphate (PIP2) and Ca2+ has been proposed as the mediator of enhanced transbilayer movement of lipids (J Biol Chem 269:6347,1994), these cell systems offer a unique opportunity for testing this mechanism. Analysis of both total PIP2 content and the metabolic-resistant pool of PIP2 that remains after incubation with Ca2+ ionophore showed no appreciable differences between normal and Scott erythrocytes. Moreover, porcine erythrocytes were found to have slightly higher levels of both total and metabolic-resistant PIP2 in comparison with normal human erythrocytes. Although loading of normal erythrocytes with exogenously added PIP2 gave rise to a Ca2+-induced increase in prothrombinase activity and apparent transbilayer movement of nitrobenzoxadiazolyl (NBD)-phospholipids, these PIP2-loaded cells were also found to undergo progressive Ca2+-dependent cell lysis, which seriously hampers interpretation of these data. Moreover, loading Scott cells with PIP2 did not abolish their impaired lipid scrambling, even in the presence of a Ca2+-ionophore. Finally, artificial lipid vesicles containing no PIP2 or 1 mole percent of PIP2 were indistinguishable with respect to transbilayer movement of NBD-phosphatidylcholine in the presence of Ca2+. Our findings suggest that Ca2+-induced redistribution of membrane phospholipids cannot simply be attributed to the steady-state concentration of PIP2, and imply that such lipid movement is regulated by other cellular processes.

Continuous analysis of the mechanism of activated transbilayer lipid movement in platelets.

Dithionite reduction of fluorescent (NBD) phospholipids was used as the basis of a continuous assay of transbilayer lipid movement to the cell surface during platelet activation. This assay reveals that virtually all previously internalized phosphatidylserine passes through the external leaflet of the membrane within 90 s after activation with Ca2+ and ionophore or with thrombin and thapsigargin. We demonstrate that this lipid scrambling is reversible, bidirectional, and insensitive to the lipid headgroup. Prolonged activation gradually results in inactivation of the scramblase. The assay also reveals that activation of the scrambling activity is sensitive to the sulfhydryl reagent pyridyldithioethylamine, suggesting the involvement of a protein in the process of activated transbilayer lipid scrambling.

Calcium-induced transbilayer scrambling of fluorescent phospholipid analogs in platelets and erythrocytes.

The non-random distribution of phospholipids in the plasma membrane of human platelets and erythrocytes is at least partially maintained by the ATP-dependent aminophospholipid translocase, but can be disturbed by a calcium-induced scrambling of lipids. Using fluorescent NBD-phospholipid analogs, we demonstrate that in both cells the aminophospholipid translocase has a slightly higher preference for the naturally occurring L-isomer of the polar headgroup of phosphatidylserine as compared to the D-isomer. Calcium-induced outward movement of internalized phosphatidylserine probe, however, is not affected by the stereochemical configuration of the serine headgroup and is virtually identical to both the inward and outward movement of the phosphatidylcholine probe. The data also indicate that both in platelets and red blood cells the calcium-induced transbilayer movement is bidirectional and involves all major phospholipid classes, with reorientation rates of sphingomyelin being appreciably lower than that of the other phospholipid classes. While our results largely support earlier observations on red cells, they clearly differ from a recent study on platelets which suggested that calcium-induced scrambling is restricted to aminophospholipids and would not involve cholinephospholipids. The present results indicate that the same mechanism is responsible for calcium-induced lipid scrambling in red blood cells and platelets.

Assembly of the prothrombinase complex on lipid vesicles depends on the stereochemical configuration of the polar headgroup of phosphatidylserine.

The conversion of prothrombin into thrombin is an imperative step in the sequence of reactions leading to the formation of a hemostatic plug. This reaction is catalyzed by the prothrombinase complex, composed of factors Xa and Va, which is assembled on a phospholipid surface through Ca-mediated interactions with the lipid polar headgroups. In this paper we describe experiments indicative for a major role of the stereochemical configuration of phosphatidylserine in the binding of the prothrombinase complex to a phospholipid surface. Using two stereoisomers of phosphatidylserine, i.e., L-alpha-glycerophosphoryl-L-serine (PLS) and L-alpha-glycerophosphoryl-D-serine (PDS), we demonstrate that membranes containing PLS are appreciably more favorable than membranes containing PDS in promoting assembly of the prothrombinase complex and catalysis of prothrombin conversion. Ellipsometric analysis of the binding of factor Va and factor Xa to a surface composed of phosphatidylcholine and 10 mol % of either PLS or PDS reveals that the apparent Kd for factor Va increases about 25-fold when substituting PDS for PLS. For factor Xa a 5-fold increase in Kd was observed on replacing PDS for PLS. When PLS is replaced by phosphatidyl-beta-lactate (PLac), a phospholipid resembling PS but lacking the amino group, a similar decrease in prothrombinase activity is found as observed with PDS, implicating the importance of both the amino group and the stereoconfiguration of the serine moiety for the assembly of the prothrombinase complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Thapsigargin amplifies the platelet procoagulant response caused by thrombin.

The platelet procoagulant response involves an increase in surface-exposed phosphatidylserine, which allows binding and assembly of enzyme complexes of the coagulation pathway resulting in acceleration of the clotting process. This response essentially requires the presence of extracellular Ca2+, and varies in extent with the type of agonist used. In the present paper we demonstrate that the moderate procoagulant response of human platelets caused by thrombin is strongly amplified by the presence of thapsigargin, an inhibitor of the microsomal Ca(2+)-ATPase. Thapsigargin, like thrombin, has only a weak effect on procoagulant activity. The large increase in procoagulant activity observed with the combined action of these two agonists is associated with increased shedding of microvesicles from the platelet plasma membrane as well as with inhibition of transport of a fluorescent-labeled analog of phosphatidylserine from the outer to the inner leaflet of the plasma membrane by the aminophospholipid translocase. The latter two observations support current concepts regarding the mechanism of development of procoagulant activity. Although the synergistic effect of thapsigargin on thrombin-induced procoagulant activity is at least in part due to the high levels of intracellular [Ca2+] evoked by these agonists, the data clearly indicate that a rise of the intracellular [Ca2+] is insufficient to completely explain this response. The present findings suggest that additional factors control expression of procoagulant activity upon stimulation of platelets by thrombin.

Phosphorylation of surface E-selectin and the effect of soluble ligand (sialyl Lewisx) on the half-life of E-selectin.

E-selectin (ELAM-1) is an adhesion molecule for leukocytes that is transiently expressed on endothelial cells. Following cell surface expression of E-selectin on human umbilical vein endothelial cells (HUVEC) stimulated with tumor necrosis factor, the induced E-selectin molecules are rapidly degraded. The kinetics of turnover of surface disposed E-selectin were investigated. The rapid disappearance of surface E-selectin is temperature dependent and sensitive to the lysosomotropic agent chloroquine. The half-life of E-selectin is not affected by inclusion of soluble sialyl Lewis x (sLex) ligands in the medium. Surface E-selectin is phosphorylated on one or more serine residues, but this modification is not obviously related to internalization.

E-selectin and intercellular adhesion molecule-1 are released by activated human endothelial cells in vitro.

Endothelial cells respond to several cytokines by a rapid increase in expression of the adhesion molecules E-selectin and intercellular adhesion molecule-1 (ICAM-1), followed by a gradual decline. The fate of these molecules, which was so far unknown, was studied. Specific sandwich ELISA for the detection of soluble (s)E-selectin and sICAM-1 were developed. In supernatant, centrifuged 3 hr at 100,000 g to remove microparticles, from human umbilical vein endothelial cells (HUVEC) activated with tumour necrosis factor (TNF), interleukin-1 (IL-1) or lipopolysaccharide (LPS), E-selectin and ICAM-1 molecules could be detected. Biochemical analysis revealed that sE-selectin migrated as a band of approximately 94,000 MW. The amount of soluble adhesion molecules released was directly correlated with cell surface expression. Maximal release of E-selectin was observed 6-12 hr after activation of HUVEC and decreased to below detection limit 24 hr after activation. After activation, release of ICAM-1 gradually increased with ICAM-1 cell surface expression, and reached a plateau after 24 hr, which was constant for 3 days. Since E-selectin and ICAM-1 are highly expressed at inflammatory sites, the resulting high concentrations of released E-selectin and ICAM-1 may affect interactions of leucocytes with endothelial cells. The physiological role, however, of the release of E-selectin and ICAM-1 remains to be elucidated.

Evidence for endocytosis of E-selectin in human endothelial cells.

E-selectin is an inducible adhesion molecule on endothelial cells. The internalization of this glycoprotein was investigated on tumor necrosis factor (TNF)-activated cultured human umbilical vein endothelial cells (HUVEC). Kinetics of intercellular adhesion molecule-1 (ICAM-1) were studied in parallel experiments. Internalization studies were performed with radioiodinated antibodies in an acid elution endocytosis assay, and by immunohistology; both approaches gave equivalent results. [125I]ENA1, a monoclonal antibody (mAb) specific for E-selectin, was internalized at a rate of approximately 1.7% of the membrane-bound [125I]mAb per minute. In contrast, less than 0.1% of membrane-bound [125I]RR1/1, an mAb specific for ICAM-1, was internalized per minute. TNF-activated HUVEC were immunostained and examined by light microscopy (LM) and electron microscopy (EM). LM revealed the presence of ENA1, but not RR1/1, after 30 minutes of incubation with these mAb in cytoplasmic vesicles, which were characterized as multivesicular bodies by EM. Without previous mAb exposure of the endothelial cells, both high amounts of E-selectin and bovine serum albumin complexed to colloidal gold, used as a marker for fluid-phase internalization, were detected in the same organelles, thus arguing against mAb interaction-induced E-selectin internalization. Furthermore, the amount of E-selectin surface expression was not influenced by ongoing mAb presence, also arguing against mAb interference with normal E-selectin kinetics. Taken together, these results indicate that TNF-activated HUVEC constitutively internalize E-selectin. Physiological significance of E-selectin internalization in the regulation of E-selectin membrane expression, and in clearing E-selectin ligands from the circulation, needs further investigation.

A comparison of substrates for human umbilical vein endothelial cell culture.

We have studied culture conditions which facilitate the growth of stable, non-proliferating, human umbilical vein endothelial cell (HUVEC) monolayers. Gelatin and fibronectin coatings, with or without glutaraldehyde cross-linking, on both plastic and glass were investigated for initial attachment of HUVEC and growth characteristics. The presence during culture of intercellular (IC) junctions demonstrated by silver staining, expression of platelet endothelial cell adhesion molecule-1 (PECAM-1) and maintenance of a cobblestone appearance of HUVEC monolayers were assessed over time. Glutaraldehyde cross-linked fibronectin and gelatin coatings on glass and glutaraldehyde cross-linked gelatin or untreated fibronectin coatings on plastic served as good substrates for short term culture. Long term (20 days) cultures of HUVEC which maintained silver and PECAM-1 staining of IC junctions and a cobblestone appearance could be achieved if glutaraldehyde cross-linked gelatin coatings on glass were used as substrates.