The binding of affinity-purified anticardiolipin antibodies (ACA) to liposomes that contained cardiolipin or phosphatidylserine was investigated. ACA bound to these liposomes only in the presence of plasma or serum, which indicated a requirement for a plasma component. This component--referred to as aca-cofactor--was purified; its activity to support ACA binding to liposomes that contained cardiolipin was not destroyed by heat (10 min at 90 degrees C), but was greatly diminished on incubation with trypsin. aca-cofactor bound liposomes that contained negatively charged phospholipid but had no affinity for liposomes that contained neutral phospholipid (eg, phosphatidylcholine); this binding was independent of calcium ions. aca-cofactor was essential for ACA to bind to liposomes that contained cardiolipin or phosphatidylserine and, when coated on a microtitre plate in the absence of any phospholipid, aca-cofactor was an apparent antigen for ACA in an enzyme-linked immunosorbent assay. aca-cofactor is a single chain polypeptide with an apparent molecular weight of 50 kD (non-reduced), which increases to 70 kD upon reduction, and its properties closely resemble those of beta 2-glycoprotein I (apolipoprotein H).

The asymmetric phospholipid distribution in plasma membranes is normally maintained by energy-dependent lipid transporters that translocate different phospholipids from one monolayer to the other against their respective concentration gradients. When cells are activated, or enter apoptosis, lipid asymmetry can be perturbed by other lipid transporters (scramblases) that shuttle phospholipids non-specifically between the two monolayers. This exposes phosphatidylserine (PS) at the cells' outer surface. Since PS promotes blood coagulation, defective scramblase activity upon platelet stimulation causes a bleeding disorder (Scott syndrome). PS exposure also plays a pivotal role in the recognition and removal of apoptotic cells via a PS-recognizing receptor on phagocytic cells. Furthermore, expression of PS at the cell surface can occur in a wide variety of disorders. This review aims at highlighting how PS expression in different cells may complicate a variety of pathological conditions, including those that promote thromboembolic complications or produce aberrations in apoptotic cell removal.

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SummaryPlasmas from 16 patients that were found to be positive both for anticardiolipin antibodies (ACA) and lupus anticoagulants (LA) were incubated with liposomes that contained anionic phospholipids. In 11 of these plasmas, ACA could be cosedimented with the liposomes in a dose-dependent manner, whereas LA activity of the remaining supernatant was unaffected. LA activity of purified total IgG from 6 patients was measured in three different coagulation tests, using normal plasmas from different species. Prolongation of the aPTT, KCT and dRW clotting times was observed only with normal plasma from human origin, not with bovine, rat or sheep plasma.Highly purified coagulation factors Xa, Va and prothrombin, both of human and bovine origin, were used to establish for two patient IgG's the effect of LA on the rate of thrombin formation in the presence and absence of lipid vesicles composed of 20 mole% phosphatidylserine and 80 mole% phosphatidylcholine. A strong and dose dependent inhibition by LA was observed only when human prothrombin was used as substrate in the prothrombinase complex in the presence of lipids. No inhibition was found when bovine prothrombin was used as substrate. The inhibitory effect observed in the presence of human prothrombin was independent of the source of factors Xa and Va, and was not found in the absence of lipid. Preliminary binding studies suggest that LA only associate with a lipid surface, provided that human prothrombin and calcium ions are present. These data indicate that LA are not directed to phospholipids alone, but presumably recognize an epitope which becomes exposed upon Ca2+-mediated binding of human prothrombin to phospholipids.

Phosphatidylserine (PS) is a major component of membrane bilayers whose change in distribution between inner and outer leaflets is an important physiological signal. Normally, members of the type IV P-type ATPases spend metabolic energy to create an asymmetric distribution of phospholipids between the two leaflets, with PS confined to the cytoplasmic membrane leaflet. On occasion, membrane enzymes, known as scramblases, are activated to facilitate transbilayer migration of lipids, including PS. Recently, two proteins required for such randomization have been identified: TMEM16F, a scramblase regulated by elevated intracellular Ca(2+), and XKR8, a caspase-sensitive protein required for PS exposure in apoptotic cells. Once exposed at the cell surface, PS regulates biochemical reactions involved in blood coagulation, and bone mineralization, and also regulates a variety of cell-cell interactions. Exposed on the surface of apoptotic cells, PS controls their recognition and engulfment by other cells. This process is exploited by parasites to invade their host, and in specialized form is used to maintain photoreceptors in the eye and modify synaptic connections in the brain. This review discusses what is known about the mechanism of PS exposure at the surface of the plasma membrane of cells, how actors in the extracellular milieu sense surface exposed PS, and how this recognition is translated to downstream consequences of PS exposure.

In the present paper we compare prothrombin-converting activities of platelets non-activated, or activated by collagen, thrombin or collagen plus thrombin in the absence and presence of added factor V,. In all experiments described, the rate of thrombin formation for platelets activated by the combined action of collagen and thrombin is greater than that of platelets stimulated by collagen or thrombin alone. The presence of added factor V, enhanced the rate of thrombin formation in all cases, but the higher activity observed with platelets stimulated by collagen plus thrombin remains. When platelets are activated by collagen plus thrombin in the presence of factor X, and prothrombin, a lag period of approximately 10 min is observed before the rate of thrombin formation reaches a steady state. Addition of an excess of factor V, in this experiment reduces the lag time to 3 min. This lag period is interpreted as the time required to generate extra binding sites for the prothrombinase complex at the platelet surface. These extra sites explain the difference in thrombin formation rate between these platelets and platelets activated by either collagen or thrombin only.The exposure of phospholipids at the platelet outer surface was studied with phospholipases after various activations of the platelets. It is demonstrated that activation by collagen plus thrombin is accompanied by increased susceptibility of platelet phospholipids towards phospholipase Az. Among these degradable phospholipids are 25 % of the phosphatidylserine and 30 % of the phosphatidylethanolamine. On the other hand, little or no phosphatidylserine is exposed at the membrane exterior of thrombin-treated or control platelets. We propose that the exposure of phosphatidylserine at the outer surface of platelets activated with thrombin plus collagen is essential for the rate enhancement of thrombin formation observed under these conditions. The possibility of a transbilayer movement of phospholipids in the platelet membrane as a result of the activation process will be discussed.One of the major functions of platelets in the process of hemostasis is to provide a catalytic surface for the formation of the intrinsic factor-X-activating complex and the prothrombinase complex (for a review, see [I]). Nesheim et al. [2] have shown that there is no appreciable difference in catalytic efficiency of the prothrombinase complex when phospholipid vesicles plus factor Va are substituted for activated platelets. Rosing et al. [3] showed that the role of phospholipids in the prothrombinase complex is to decrease the K,,, for prothrombin below its plasma concentration, whereas factor V, strongly increases the V,,, of the reaction [2,3]. Recently, similar results were found for the role of phospholipids in the intrinsic factor-X-activating complex [4].It has been demonstrated that the cytoplasmic surfaces of both erythrocytes and platelets, as well as liposomes prepared from phospholipids present in the inner leaflet of the plasma membrane of these cells, possess a str...

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