We previously reported that during death receptor-mediated apoptosis, cardiolipin (CL) relocates to the cell surface, where it reacts with autoantibodies from antiphospholipid syndrome sera. Here, we analysed the intracellular distribution of CL and its metabolites during the early phase of cell death signalling triggered by Fas stimulation in U937 cells and mouse liver. We found a redistribution of mitochondrial CL to the cell surface by using confocal microscopy and flow cytometry. Mass spectrometry revealed that CL and its metabolites relocated from mitochondria to other intracellular organelles during apoptosis, with a conversion into non-mitochondrial lipids. Concomitantly, cytosolic Bid relocated to the light membranes comprised in fraction P100, including the plasma membrane and associated vesicular systems. A direct Bid-CL interaction was demonstrated by the observation that CL and monolysoCL coimmunoprecipitated with Bid especially after Fas stimulation, suggesting a dynamic interaction of the protein with CL and its metabolites.

In this study we analyzed the interaction of prion protein PrP C with components of glycosphingolipid-enriched microdomains in lymphoblastoid T cells. PrP C was distributed in small clusters on the plasma membrane, as revealed by immunoelectron microscopy. PrP C is present in microdomains, since it coimmunoprecipitates with GM3 and the raft marker GM1. A strict association between PrP C and Fyn was revealed by scanning confocal microscopy and coimmunoprecipitation experiments. The phosphorylation protein ZAP-70 was immunoprecipitated by anti-PrP after T cell activation. These results demonstrate that PrP C interacts with ZAP-70, suggesting that PrP C is a component of the multimolecular signaling complex within microdomains involved in T cell activation.

SUMMARYThis study provides evidence that cardiolipin (CL) molecules are expressed on the surface of apoptotic cells and are recognized by antiphospholipid antibodies, purified from patients with the antiphospholipid antibody syndrome (APS). CL expression on cell surface was demonstrated by high performance thin layer chromatography analysis of phospholipids from plasma membrane purified fractions and by the positive staining with the CL-specific dye nonyl-acridine orange. This finding was complemented with the observation that aCL IgG purified from patients with APS bind to the surface of apoptotic cells. This staining shows a clustered distribution mostly localized on surface blebs. Interestingly, CL exposure on the cell surface preceded the DNA fragmentation, as shown by cytofluorimetric analysis. These findings demonstrate that exposure of CL molecules on the cell plasma membrane is an early event of the apoptotic cellular program that may represent an in vivo trigger for the generation of aCL.

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SummaryThis study was undertaken to analyze antibodies to protein S (PS) in patients with an acquired PS deficiency. Plasma from symptomatic patients with acquired (n = 14) or congenital (n = 10) PS deficiency and 10 healthy donors was screened for PS antibodies by immunoblotting and for anti-phospholipid antibodies. PS antibodies (IgG) were detected in five of the patients with acquired PS deficiency. These antibodies belonged to the G1 and G4 immunoglobulin subclasses. IgG fractions from the same 5 patients were shown to inhibit PS activity. The inhibition of PS activity by the 5 IgG fractions was shown to be time-and dose-dependent and was abolished following incubation with purified PS, while no effect was found after absorption with cardiolipin micelles. In addition, anticardiolipin monoclonal or human purified antibodies, failed to exert significant PS inhibition. These findings demonstrate that anti-PS antibodies are able to inhibit PS activity and that this is independent of anti-phospholipid antibodies. Given the clinical features of the patients, these antibodies should be regarded as an expression of the broad autoimmune syndrome involving the phospholipid-binding plasma proteins.

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SUMMARYIt is well known that monocytes may play an active role in thrombogenesis, since they may express on their surface tissue factor, the major initiator of the clotting cascade. The results of this investigation demonstrate beta-2-glycoprotein I ( b 2 -GPI) mRNA expression by human peripheral blood monocytes, indicating that these cells synthesize b 2 -GPI. In addition, we show b 2 -GPI expression on cell surface of these cells by flow cytometric analysis, and the presence of this protein in cell lysate by Western blot. Interestingly, b 2 -GPI expression on monocytes is significantly increased in patients with antiphospholipid syndrome (APS) or systemic lupus erythematosus (SLE) as against healthy blood donors and correlates with tissue factor expression on monocytes. These findings support the view that monocytes are able to synthesize b 2 -GPI and suggest that patients with APS may have increased b 2 -GPI exposure on cell surface, which leads to persistently high monocyte tissue factor expression and consequently to a prothrombotic diathesis.

SummaryThis study has been undertaken to assess whether anticardiolipin and anti-(32-GPI are two distinct populations of (auto)antibodies, and to clarify whether the β2-GPI region critical for phospholipid binding is also crucial for anti-β2-GPI reactivity.Fourteen of the 62 anticardiolipin (aCL) ELISA positive sera (22.6%) were positive for anti-β2-GPI by immunoblotting, 42 (67.7%) for aCL using TLC immunostaining.IgG fractions from 5 sera gave the same anticardiolipin reactivity detected by TLC immunostaining in the corresponding sera.All anti-β2-GPI-positive sera were reactive with the phenylthio-carbamyl derivative of the protein, indicating that binding of phe-nylisothiocyanate with lysine residues does not modify the molecule antigenicity. In addition, incubation of IgG fractions with the phospholipid binding site did not modify reactivity with β2-GPI.These findings demonstrate that: a) “true” antiphospholipid antibodies are detectable in patients sera; b) aCL and anti-β2-GPI have a different immunological profile; c) the β2-GPI phospholipid-binding site is not the region recognized by the antibodies.