Peptide Antagonists for P-selectin Discriminate between Sulfatide-Dependent Platelet Aggregation and PSGL-1-Mediated Cell Adhesion.

BACKGROUND: Membrane-exposed sulfatides are proposed to contribute to P-selectin-dependent platelet aggregation. Here, we demonstrated that P-selectin-mediated platelet aggregation on a collagen-coated surface under flow indeed depended on sulfatides and that this interaction differed considerably from the interaction of P-selectin with P-selectin Glycoprotein Ligand-1 (PSGL-1), which underlies leukocyte-endothelium adhesion. METHODS AND RESULTS: Upon platelet activation, sulfatides were translocated to the platelet surface to form focal hot-spots. Interestingly, P-selectin was observed to exclusively interact with liposomes with a sulfatide density higher than 21% (w/w), indicating that the binding profile of P-selectin for sulfatide-rich liposomes was dependent on sulfatide density. Sulfatide-liposome binding to P-selectin and sulfatide/P-selectin-dependent platelet aggregation was blunted by peptide antagonists, carrying the EWVDV motif within N-terminal extensions, such as CDVEWVDVSC (half maximal inhibitory concentration IC50 = 0.2 µM), but not by the EWVDV core motif itself (IC50 > 1000 µM), albeit both being equally potent inhibitors of PSGL-1/P-selectin interaction (IC50= 7-12 µM). CONCLUSIONS: Our data suggest that the sulfatide/P-selectin interaction implicates multiple binding pockets, which only partly overlap with that of PSGL-1. These observations open ways to selectively interfere with sulfatide/P-selectin-dependent platelet aggregation without affecting PSGL-1-dependent cell adhesion.

The binding site in {beta}2-glycoprotein I for ApoER2' on platelets is located in domain V.

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.

Gallic acid antagonizes P-selectin-mediated platelet-leukocyte interactions: implications for the French paradox.

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.

Blocking endothelial adhesion molecules: a potential therapeutic strategy to combat atherogenesis.

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.

Dimers of beta 2-glycoprotein I increase platelet deposition to collagen via interaction with phospholipids and the apolipoprotein E receptor 2'.

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'.