According to the previously reported potent dual l-peptide PMI of p53-MDM2/MDMX interactions, a series of d-amino acid mutational PMI analogues, PMI-1-4, with enhanced proteolytic resistence and in vitro tumor cell inhibitory activities were reported, of which Liposome-PMI-1 showed a stronger inhibitory activity against the U87 cell lines than Nutlin-3. This d-amino acid mutation strategy may give a hand for enhancing the potential of peptide drugs.

01 Apr 1997·Journal of Biological ChemistryQ2 · BIOLOGY

Human Neutrophil Elastase Proteolytically Activates the Platelet Integrin αIIbβ3 through Cleavage of the Carboxyl Terminus of the αIIb Subunit Heavy Chain

Q2 · BIOLOGY

Article

Author: Si-Tahar, Mustapha ; Balloy, Viviane ; Moniatte, Marc ; Van Dorsselaer, Alain ; Chignard, Michel ; Pidard, Dominique ; Kieffer, Nelly

Neutrophil elastase (NE) and cathepsin G are two serine proteinases released concomitantly by stimulated polymorphonuclear neutrophils. We previously demonstrated that while NE by itself does not activate human platelets, it strongly enhances the weak aggregation induced by a threshold concentration of cathepsin G (threshold of cathepsin G) (Renesto, P., and Chignard, M. (1993) Blood 82, 139-144). The aim of this study was to delineate the molecular mechanisms involved in this potentiation process. Two main pieces of data prompted us to focus on the activation of the platelet fibrinogen receptor, the alphaIIbbeta3 integrin. First, previous studies have shown this integrin to be particularly prone to proteolytic regulation of its function. Second, we found that the potentiating activity of NE on the threshold of cathepsin G-induced platelet aggregation was strictly dependent on the presence of exogenous fibrinogen. Using flow cytometry analysis, NE was shown to trigger a time-dependent binding of PAC-1 and AP-5, two monoclonal antibodies specific for the activated and ligand-occupied conformers of alphaIIbbeta3. Furthermore, the potentiated aggregation was shown to result from an increased capacity of platelets to bind fibrinogen. Indeed, the combination of NE and threshold of cathepsin G increased the binding of PAC-1 approximately 5.5-fold over basal values measured on nontreated platelets, whereas this binding raised only by approximately 3-fold in threshold of cathepsin G-stimulated platelets (p < 0.05). By contrast, phosphatidic acid accumulation, pleckstrin phosphorylation, and calcium mobilization produced by the combination of NE and threshold of cathepsin G were not significantly different from those measured with threshold of cathepsin G alone (p > 0.05), indicating that the phospholipase C/protein kinase C pathway is not involved in the potentiation of aggregation. The foregoing data, as well as the requirement of catalytically active NE to trigger alphaIIbbeta3 activation and potentiate threshold of cathepsin G-initiated platelet aggregation, led us to examine whether the structure of this integrin was affected by NE. Immunoblot and flow cytometry analysis revealed a limited proteolysis of the carboxyl terminus of the alphaIIb subunit heavy chain (alphaIIbH), as judged by the disappearance of the epitope for the monoclonal antibody PMI-1. Mass spectrometry studies performed on a synthetic peptide mapping over the cleavage domain of alphaIIbH predicted the site of proteolysis as located between Val837 and Asp838. Treatment by NE of ATP-depleted platelets or Chinese hamster ovary cells expressing human recombinant alphaIIbbeta3 clearly established that activation of the integrin was independent of signal transduction events and was concomitant with the proteolysis of alphaIIbH. In support of this latter observation, a close correlation was observed between the kinetics of proteolysis of alphaIIbH on platelets and that of expression of the ligand binding activity of alphaIIbbeta3 (r2 = 0.902, p

01 Mar 1996·Biokhimiia (Moscow, Russia)

[Conformational changes of the platelet membrane glycoprotein IIb-IIIa complex stimulated by a monoclonal antibody to the N-terminal segment of glycoprotein IIIa].

Article

Author: Vyzova, T V ; Khaspekova, S G ; Lukin, V V ; Tikhomirov, O Iu ; Mazurov, A V ; Kouns, W ; Berndt, M

During platelet activation, the glycoprotein (GP) complex IIb-IIIa (alpha 11b beta 3-integrin) changes its conformation, resulting in binding of adhesive proteins of RGD containing an amino acid sequence as well as in expression of new ligand-induced binding sites (LIBS) on the GPIIb-IIIa molecule. Like its F(ab)-fragments, the monoclonal antibody CRC54, whose epitope is located in the N-terminal part of the GPIIIa molecule, binds to no more than 10% of GPIIb-IIIa on the resting platelet surface. However, the binding of CRC54 increases considerably during activation of platelets by thrombin, platelet adhesion on plastic, GPIIb-IIIa interaction with RGDS-peptide as well as during dissociation of the complex in the presence of EDTA. These finding suggest that CRC54 is specifically directed against the LIBS epitope on the GPIIIa molecule. This epitope differs from those of other known conformation-dependent antibodies against GPIIb-IIIa (LIBS1, LIBS6, PMI-1, pl55 and p180), since those antibodies did not block the CRC54 binding to GPIIb-IIIa on the surface of adhering platelets. Unlike whole platelets, the binding of GPIIb-IIIa from lysates of platelets treated with Triton X-100 with immobilized CRC54 did not depend on the presence of the RGDS peptide. Under these conditions another anti-LIBS-antibody, p180 specifically directed against GPIIb, preserved its ability to discriminate the RGDS-occupied and resting conformations of GPIIb-IIIA. CRC54 and its F(ab) fragments induced platelet aggregation in both platelet-enriched plasma and in suspensions of washed platelets. CRC54 also stimulated the binding to platelets of GPIIb-IIIa ligand fibrinogen, labelled with 125I as well as adhesion of 51Cr-labelled platelets to immobilized ligands-fibrinogen and fibronectin. The CRC54-dependent aggregation was fully blocked by RGDS-peptide and antibody CRC64 inhibiting the GPIIb-IIIa binding to the ligands. However, the platelet activation inhibitor, prostaglandin EI, and the mixture of metabolic inhibitors, deoxyglucose-sodium azide, only party inhibited the CRC54-dependent aggregation. Incubation of platelets with CRC54 induced the binding to platelets of the anti-GPIIb LIBS antibody p180 and of the anti-GPIIb-IIIa activation-dependent antibody p155. The binding of GPIIb-IIIa from lysates of CRC54-treated platelets with immobilized p180 and p155 was also several times as high as that of GPIIb-IIIa from control platelet lysates. The data obtained indicate that the GPIIb-IIIa transition to the active state and its interaction with ligands induces conformational changes in the N-terminal part of GPIIIa and that the CRC54 binding to the N-terminal part of GPIIIa stimulates conformational changes in GPIIb-IIIa, complex interaction with ligands and platelet aggregation.

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Neuritis	Phase 1	-	Precision Molecular Inc.	-
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