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The antiphospholipid syndrome is caused by autoantibodies directed against beta(2)-glycoprotein I (beta(2)GPI). Dimerization of beta(2)GPI results in an increased platelet deposition to collagen. We found that apolipoprotein E receptor 2' (apoER2'), a member of the low density lipoprotein receptor family, is involved in activation of platelets by dimeric beta(2)GPI. To identify which domain of dimeric beta(2)GPI interacts with apoER2', we have constructed domain deletion mutants of dimeric beta(2)GPI, lacking domain I (DeltaI), II (DeltaII), or V (DeltaV), and a mutant with a W316S substitution in the phospholipid (PL)-insertion loop of domain V. DeltaI and DeltaII prolonged the clotting time, as did full-length dimeric beta(2)GPI; DeltaV had no effect on the clotting time. Second, DeltaI and DeltaII bound to anionic PL, comparable with full-length dimeric beta(2)GPI. DeltaV and the W316S mutant bound with decreased affinity to anionic PL. Platelet adhesion to collagen increased significantly when full-length dimeric beta(2)GPI, DeltaI, or DeltaII (mean increase 150%) were added to whole blood. No increase was found with plasma beta(2)GPI, DeltaV, or the W316S mutant. Immunoprecipitation indicated that full-length dimeric beta(2)GPI, DeltaI, DeltaII, and the W316S mutant can interact with apoER2' on platelets. DeltaV did not associate with apoER2'. We conclude that domain V is involved in both binding beta(2)GPI to anionic PL and in interaction with apoER2' and subsequent activation of platelets. The binding site in beta(2)GPI for interaction with apoER2' does not overlap with the hydrophobic insertion loop in domain V.

Background: Current paradigm attributes the low incidence of cardiovascular disorders in Mediterranean countries despite a high saturated fat intake, the "French paradox," to the antioxidant capacity of red wine polyphenols. Conceivably, other antiinflammatory pathways may contribute to at least a similar extent to the atheroprotective activity of these polyphenols. We have investigated whether gallic acid (GA), an abundant red wine polyphenol, modulates the activity of P-selectin, an adhesion molecule that is critically involved in the recruitment of inflammatory cells to the vessel wall and thus in atherosclerosis., Methods and Results: GA potently inhibited the binding of a peptide antagonist (IC50, 7.2 micromol/L) and biotin-PAA-Le(a)-SO3H, an established high-affinity ligand, to P-selectin (IC50, 85 micromol/L). Under dynamic flow conditions, GA markedly and dose dependently attenuated the rolling of monocytic HL60 cells over P-selectin-transfected Chinese hamster ovary cells (EC50, 14.5 micromol/L) while increasing the velocity of P-selectin-dependent rolling of human blood leukocytes over a platelet monolayer. In vivo tests established that GA administration to normolipidemic C57/Bl6 and aged atherosclerotic apolipoprotein E-deficient mice impaired the baseline rolling of conjugates between activated platelets and circulating monocytes over femoral vein endothelium, as judged by online video microscopy (ED50, 1.7+/-0.3 and 1.5+/-0.4 mg x kg(-1) x h(-1), respectively)., Conclusions: Our findings provide a solid mechanistic foundation through which GA intervenes in major inflammatory pathobiologies by binding and antagonizing P-selectin.

Purpose of Review: This review provides a concise update of the involvement of endothelial adhesion molecules in atherogenesis, an overview of current advances in the development of adhesion molecule blocking agents, as well as an insight into the potential of these molecules in cardiovascular therapy., Recent Findings: As endothelial adhesion molecules are deemed to play an important role in the development and progression of atherosclerotic lesions, they are interesting targets for therapeutic intervention in this process. In particular, P-selectin and vascular cell adhesion molecule 1 are widely considered to hold promise in this regard. Current research efforts centre on the design of agents that directly block the interaction of the receptor with its ligand (e.g. soluble P-selectin glycoprotein ligand 1, blocking antibodies, EWVD-based peptides) or that interfere with their synthesis (e.g. antisense oligonucleotides) or their regulatory control by nuclear factor kappa B or peroxisome proliferator-activated receptor gamma. Furthermore, adhesion molecules have been exploited as a target for the specific delivery of drug carriers (e.g. biodegradable particles with entrapped dexamethasone) or therapeutic compounds (e.g. dexamethasone) to the plaque. All approaches have been shown to be effective in blocking adhesion molecule function in in-vitro studies and in-vivo models for inflammation or atherosclerosis., Summary: Although the field has achieved considerable progress in recent years, leading to the development of a number of interesting leads, final proof of their efficacy in cardiovascular therapy is eagerly awaited.

Patients with prolonged clotting times caused by lupus anticoagulant (LAC) are at risk for thrombosis. This paradoxal association is not understood. LAC is frequently caused by anti-beta2-glycoprotein I (beta 2GPI) antibodies. Antibody-induced dimerization of beta 2GPI increases the affinity of beta 2GPI for phospholipids, explaining the observed prolonged clotting times. We constructed dimers of beta 2GPI that mimic effects of beta 2GPI-anti-beta 2GPI antibody complexes, and we studied their effects on platelet adhesion and thrombus formation in a flow system. Dimeric beta 2GPI increased platelet adhesion to collagen by 150% and increased the number of large aggregates. We also observed increased platelet adhesion to collagen when whole blood was spiked with patient-derived polyclonal anti-beta 2GPI or some, but not all, monoclonal anti-beta 2GPI antibodies with LAC activity. These effects could be abrogated by inhibition of thromboxane synthesis. A LAC-positive monoclonal anti-beta 2GPI antibody, which did not affect platelet adhesion, prevented the induced increase in platelet adhesion by beta 2GPI dimers. Furthermore, increased platelet adhesion disappeared after preincubation with receptor-associated protein, a universal inhibitor of interaction of ligands with members of the low density lipoprotein receptor family. Using co-immunoprecipitation, it was shown that dimeric beta 2GPI can interact with apolipoprotein E receptor 2 (apoER2'), a member of the low density lipoprotein receptor family present on platelets. These results demonstrate that dimeric beta 2GPI induces increased platelet adhesion and thrombus formation, which depends on activation via apoER2'.

Anti-beta(2)-glycoprotein I antibodies are thought to cause lupus anticoagulant activity by forming bivalent complexes with beta(2)-glycoprotein I (beta(2)GPI). To test this hypothesis, chimeric fusion proteins were constructed of the dimerization domain (apple 4) of factor XI and beta(2)GPI. Both a covalent (apple 4-beta(2)GPI) and a noncovalent (apple 4-C321S-beta(2)GPI) chimer were constructed. As controls, apple 2-beta(2)GPI and apple 4-C321S-beta(2)GPI-W316S, in which beta(2)GPI-W316S is not able to bind to phospholipids, were made. In a phospholipid binding assay, apple 4-beta(2)GPI and apple 4-C321S-beta(2)GPI were able to bind to phospholipids with an affinity 35 times higher than that of plasma-derived beta(2)GPI and apple 2-beta(2)GPI. Apple 4-C321S-beta(2)GPI-W316S did not bind at all. Only apple 4-beta(2)GPI and apple 4-C321S-beta(2)GPI were able to bind to adhered platelets as shown by immunofluorescence. Using the prothrombin time, which was the most responsive coagulation assay, the clotting time was approximately doubled when 200 microg/ml apple 4-beta(2)GPI or apple 4-C321S-beta(2)GPI was added. Addition of 200 microg/ml plasma-derived beta(2)GPI, apple 2-beta(2)GPI, or apple 4-C321S-beta(2)GPI-W316S did not affect clotting time. Clotting time could be corrected with the addition of extra phospholipids, which is indicative for lupus anticoagulant activity. An additional increase in clotting times for apple 4-beta(2)GPI or apple 4-C321S-beta(2)GPI was achieved by the addition of monoclonal antibodies against beta(2)GPI. In conclusion, dimerization of beta(2)GPI explains the in vitro observed effects of beta(2)GPI-anti-beta(2)GPI antibody complexes.

The presence of antiphospholipid antibodies (aPL) in plasma is an important risk factor for the development of thrombosis. A major breakthrough came in 1990 with the finding that aPL do not react with phospholipids per se, but are directed towards beta2-glycoprotein I, a plasma protein with affinity for anionic phospholipids. Afterwards other plasma proteins with affinity for phospholipids have been identified as possible antigens for aPL. Human beta2-glycoprotein I is a heavily glycosylated plasma protein that has been implicated in the binding of aPL to negatively charged phospholipids. We recently solved the crystal structure of beta2-glycoprotein I. In this review we will discuss what the 3-dimensional structure teaches us about the role of beta2-glycoprotein I in the pathogenesis of the thrombotic complications characteristic of the antiphospholipid syndrome.

Human beta 2-glycoprotein I is a heavily glycosylated plasma protein which has been implicated in the binding of antiphospholipid antibodies to negatively charged phospholipids; a process considered as an important risk factor for the development of thrombosis. We have solved the crystal structure of beta 2-glycoprotein I. In this review we will discuss what the three-dimensional structure teaches us about the role of beta 2-glycoprotein I in the pathogenesis of the antiphospholipid syndrome., (Copyright 2000 Academic Press.)

Alphaherpesvirus glycoproteins E and I (gE and gI, respectively) assemble into a hetero-oligomeric complex which promotes cell-to-cell transmission, a determining factor of virulence. Focusing on gI of feline herpesvirus (FHV), we examined the role of disulfide bonds during its biosynthesis, its interaction with gE, and gE-gI-mediated spread of the infection in vitro. The protein's disulfide linkage pattern was determined by single and pairwise substitutions for the four conserved cysteine residues in the ectodomain. The resulting mutants were coexpressed with gE in the vaccinia virus-based vTF7-3 system, and the formation and endoplasmic reticulum (ER)-to-Golgi transport of the hetero-oligomeric complex were monitored. The results were corroborated biochemically by performing an endoproteinase Lys-C digestion of a [35S]Cys-labeled secretory recombinant form of gI followed by tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the peptides under reducing and nonreducing conditions. We found that (i) gI derivatives lacking Cys79 (C1) and/or Cys223 (C4) still assemble with gE into transport-competent complexes, (ii) mutant proteins lacking Cys91 (C2) and/or Cys102 (C3) bind to gE but are retained in the ER, (iii) radiolabeled endoproteinase Lys-C-generated peptide species containing C1 and C4 are linked through disulfide bonds, and (iv) peptides containing both C2 and C3 are not disulfide linked to any other peptide. From these findings emerges a model in which C1 and C4 as well as C2 and C3 form intramolecular disulfide bridges. Since the cysteines in the ectodomain have been conserved during alphaherpesvirus divergence, we postulate that the model applies for all gI proteins. Analysis of an FHV recombinant with a C1-->S substitution confirmed that the C1-C4 disulfide bond is not essential for the formation of a transport-competent gE-gI complex. The mutation affected the posttranslational modification of gI and caused a slight cold-sensitivity defect in the assembly or the intracellular transport of the gE-gI complex but did not affect plaque size. Thus, C1 and the C1-C4 bond are not essential for gE-gI-mediated cell-to-cell spread, at least not in vitro.

Alphaherpesvirus glycoproteins gE and gI form a noncovalently associated hetero-oligomeric complex, which is involved in cell-to-cell spread. In the absence of gI, feline herpesvirus (FHV) gE is transport incompetent and fully retained in the endoplasmic reticulum. Here, we assess the effect of progressive C-terminal truncations of FHV gI on the biosynthesis, intracellular transport, and function of the gE-gI complex. The truncated gI proteins were coexpressed with gE in the vaccinia virus-based vTF7-3 expression system. The results were corroborated and extended by studying FHV recombinants expressing truncated gI derivatives. The following conclusions can be drawn. (i) Deletion of the cytoplasmic tail, the transmembrane region plus the C-terminal half of the ectodomain of gI, does not affect intracellular transport of gE. Apparently, the N-terminal 166 residues of gI constitute a domain involved in gE-gI interaction. (ii) A region mediating stable association with gE is located within the N-terminal 93 residues of gI. (iii) The cytoplasmic domain of gI is not essential for gE-gI-mediated cell-to-cell transmission of FHV, as judged from plaque morphology. Deletion of the cytoplasmic tail of gI reduced plaque size by only 35%. (iv) Recombinants expressing the N-terminal 166 residues of gI display a small-plaque phenotype but produce larger plaques than recombinants with a disrupted gI gene. Thus, a complex consisting of gE and the N-terminal half of the gI ectodomain may retain residual biological activity. The implications of these findings for gE-gI interaction and function are discussed.