Hybridoma Lupus Anticoagulants Distinguish between Bilayer and Nonbilayer Phase Lipid Systems<sup>a</sup>

Rauch, Joyce; Tannenbaum, Marion; Tannenbaum, H; Ramelson, H; Cullis, P. R.; Tilcock, Colin; Hope, Michael J.; Janoff, Andrew S.

doi:10.1111/j.1749-6632.1986.tb20896.x

Cited by 27 publications

(36 citation statements)

References 5 publications

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“…The observation that lupus anticoagulants and some antiphospholipid antibodies block the function of coagulation and anticoagulation complexes preferentially on PE-containing membranes (42,66) also adds to the potential interest in the effects reported here. These antibodies are associated with an increased risk of thrombosis (67)(68)(69), and there is no detailed understanding of the best in vitro assays to detect the antibodies responsible for the pathogenic response.…”

Section: Fig 12mentioning

confidence: 66%

A Chimeric Protein C Containing the Prothrombin Gla Domain Exhibits Increased Anticoagulant Activity and Altered Phospholipid Specificity

Smirnov

Safa

Regan

et al. 1998

Journal of Biological Chemistry

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To determine the structural basis of phosphatidylethanolamine (PE)-dependent activated protein C (APC) activity, we prepared a chimeric molecule in which the Gla domain and hydrophobic stack of protein C were replaced with the corresponding region of prothrombin. APC inactivation of factor Va was enhanced 10 -20-fold by PE. Protein S enhanced inactivation 2-fold and independently of PE. PE and protein S had little effect on the activity of the chimera. Factor Va inactivation by APC was approximately 5-fold less efficient than with the chimera on vesicles lacking PE and slightly more efficient on vesicles containing PE. The cleavage patterns of factor Va by APC and the chimera were similar, and PE enhanced the rate of Arg 506 and Arg 306 cleavage by APC but not the chimera. APC and the chimera bound to phosphatidylserine:phosphatidylcholine vesicles with similar affinity (K d Ϸ 500 nM), and PE increased affinity 2-3-fold. Factor Va and protein S synergistically increased the affinity of APC on vesicles without PE to 140 nM and with PE to 14 nM, but they were less effective in enhancing chimera binding to either vesicle. In a factor Xa one-stage plasma clotting assay, the chimera had ϳ5 times more anticoagulant activity than APC on PE-containing vesicles. Unlike APC, which showed a 10 fold dependence on protein S, the chimera was insensitive to protein S. To map the site of the PE and protein S dependence further, we prepared a chimera in which residues 1-22 were derived from prothrombin and the remainder were derived from protein C. This protein exhibited PE and protein S dependence. Thus, these special properties of the protein C Gla domain are resident outside of the region normally hypothesized to be critical for membrane interaction. We conclude that the protein C Gla domain possesses unique properties allowing synergistic interaction with factor Va and protein S on PE-containing membranes.

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Section: Fig 12mentioning

confidence: 66%

A Chimeric Protein C Containing the Prothrombin Gla Domain Exhibits Increased Anticoagulant Activity and Altered Phospholipid Specificity

Smirnov

Safa

Regan

et al. 1998

Journal of Biological Chemistry

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“…Therefore, the presence of PE on membranes that are involved in coagulation has been documented. The potential specificity of lupus anticoagulants for PE has been validated in at least one study in which human monoclonal antibodies were prepared from the lymphocytes of a patient, and these antibodies all exhibited a high degree of specificity toward PE (21), especially in the hexagonal phase. It remains to be seen whether these observations will aid in identifying patients whose antibody titers and specificities contribute to a high thrombotic risk.…”

Section: Resultsmentioning

confidence: 99%

“…In the presence of APC, the thrombin generation began sooner in the lupus anticoagulant plasma than in the normal control, that is, the reverse of what was seen in the absence of APC. In normal plasma, thrombin formation component of the surface of the outer phospholipid leaflet after platelet activation (27), it is a major phospholipid in brain and hence in cephalin, it is required for optimal APC function (20), and it can interact with lupus anticoagulant antibodies (21). Before comparing the response of the lupus patient plasmas, however, it was useful to compare the role of membrane composition on the dose-response relationships of clotting times of normal plasma in the presence and absence of APC (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Consistent with this possibility, we demonstrated recently that APC anticoagulant activity was dramatically enhanced by phosphatidylethanolamine (PE) or, to a lesser extent, cardiolipin (20). Both of these phospholipids have been shown by others to be major targets of the antiphospholipid antibodies (1,21), raising the possibility that PE provides a mechanism for selectively inhibiting the protein C pathway in patients with antiphospholipid antibodies, even when they exhibit low levels of lupus anticoagulant activity.…”

Section: Introductionmentioning

confidence: 99%

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On the role of phosphatidylethanolamine in the inhibition of activated protein C activity by antiphospholipid antibodies.

et al. 1995

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Phosphatidylethanolamine (PE) is an important membrane component for supporting activated protein C anticoagulant activity but has little influence on prothrombin activation. This difference constitutes a potential mechanism for selective inhibition of the protein C anticoagulant pathway by lupus anticoagulants and/or antiphospholipid antibodies. In this study, we demonstrate that the presence of PE augments lupus anticoagulant activity. In the plasma of some patients with lupus anticoagulants, activated protein C anticoagulant activity is more potently inhibited than prothrombin activation. As a result, in the presence of activated protein C and PE, these patient plasmas clot faster than normal plasma. Patients with minimal lupus anticoagulant activity are identified whose plasma potently inhibits activated protein C anticoagulant activity. This process is also PE dependent. In three patient plasmas, these phenomena are shown to be due to immunoglobulins. The PE requirement in the expression of activated protein C anticoagulant activity and the PE dependence of some antiphospholipid antibodies provide a mechanistic basis for the selective inhibition of the protein C pathway. Inhibition of activated protein C function may be a common mechanism contributing to increased thrombotic risk in certain patients with antiphospholipid antibodies. (J. Clin. Invest. 1995. 95:309-316.)

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“…For example, specificities of individual antibodies to phosphatidylinositol phosphate were identified that either did or did not crossreact with phosphatidylinositol or phosphatidylinositol bisphosphate (28). Monoclonal antibodies to cardiolipin can react with cardiolipin or even phosphatidylethanolamine in the hexagonal HI, phase, but not with the lipids in the bilayer configuration (29). Recently it was shown that the TEPC 15 IgA myeloma antibody, which binds to phosphocholine but also recognizes phosphatidylcholine (30), interacts differently with phosphatidylcholine and phosphatidylethanolamine in a lipid monolayer system (31).…”

Section: Discussionmentioning

confidence: 99%

Antibodies to cholesterol.

Swartz

Gentry²,

Amende³

et al. 1988

Proc. Natl. Acad. Sci. U.S.A.

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Cholesterol-dependent complement activation has been proposed as a factor that might influence the pathogenesis of atherosclerosis. Although antibodies to cholesterol conjugates have been reported, cholesterol is widely regarded as a poorly immunogenic substance. Monoclonal IgM complement-fixing antibodies to cholesterol were obtained in the present study after immunizing mice with liposomes containing high amounts of cholesterol (71 mol % relative to phosphatidylcholine) and lipid A as an adjuvant.Clones were selected for the ability of secreted antibodies to react with liposomes containing 71% cholesterol but not with liposomes containing 43% cholesterol. The antibodies also reacted with crystalline cholesterol in a solid-phase enzymelinked immunosorbent assay. Binding of monoclonal antibodies to the surface of crystalline cholesterol was demonstrated by electron microscopy by utilizing a second antibody (antilgM) labeled with colloidal gold. The immunization period required to induce monoclonal antibodies was very short (3 days) and a high fraction of the hybrid cells (at least 70%) were secreting detectable antibodies to cholesterol. The results demonstrate that cholesterol can be a highly immunogenic molecule and that complement-flxing antibodies to cholesterol can be readily obtained.Cholesterol has been widely studied as a fundamental and ubiquitous constituent of cell membranes and lipoproteins (1,2). Despite occasional reports that conjugates of cholesterol can be immunogenic (3)(4)(5), because of its widespread distribution and important biological roles cholesterol has generally been assumed to be a nonimmunogenic or poorly immunogenic molecule. However, since 1977 several laboratories have reported activation of the classical and alternative pathways of complement by cholesterol (6-11). The exact mechanism (or mechanisms) of complement activation by cholesterol has not been determined. In one case human IgG antibodies to cholesterol were implicated in the activation of the classical pathway (9). On the other hand, it has been proposed that nonimmune activation of the alternative pathway of rabbit complement (7) or human complement (8, 9) also occurs. The issue of the immunogenicity of cholesterol is an important one, since antibodies to cholesterol have been reported to protect against induced atherosclerosis in rabbits (4). On the other hand, activation of the classical pathway of complement by cholesterol has been proposed as a possible mechanism in the pathogenesis of atherosclerosis (6,9,11).In previous studies we induced antibodies to the phospholipid constituents of lipid model membranes (liposomes) (reviewed in ref. 12). Production of antibodies to liposomes was achieved by utilizing lipid A as a powerful adjuvant in the liposomal lipid bilayer. In the present work we used lipid A as an adjuvant to induce antibodies to liposomes containing high concentrations of cholesterol. We found that cholesterol is an excellent immunogen and that murine monoclonal antibodies to cholesterol are eas...

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Hybridoma Lupus Anticoagulants Distinguish between Bilayer and Nonbilayer Phase Lipid Systems^a

Cited by 27 publications

References 5 publications

A Chimeric Protein C Containing the Prothrombin Gla Domain Exhibits Increased Anticoagulant Activity and Altered Phospholipid Specificity

A Chimeric Protein C Containing the Prothrombin Gla Domain Exhibits Increased Anticoagulant Activity and Altered Phospholipid Specificity

On the role of phosphatidylethanolamine in the inhibition of activated protein C activity by antiphospholipid antibodies.

Antibodies to cholesterol.

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Hybridoma Lupus Anticoagulants Distinguish between Bilayer and Nonbilayer Phase Lipid Systemsa

Cited by 27 publications

References 5 publications

A Chimeric Protein C Containing the Prothrombin Gla Domain Exhibits Increased Anticoagulant Activity and Altered Phospholipid Specificity

A Chimeric Protein C Containing the Prothrombin Gla Domain Exhibits Increased Anticoagulant Activity and Altered Phospholipid Specificity

On the role of phosphatidylethanolamine in the inhibition of activated protein C activity by antiphospholipid antibodies.

Antibodies to cholesterol.

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Product

Resources

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Hybridoma Lupus Anticoagulants Distinguish between Bilayer and Nonbilayer Phase Lipid Systems^a