Medical SciencesSer-752 -> Pro mutation in the cytoplasmic domain of integrin .83 subunit and defective activation of platelet integrin aIlbP3 (glycoprotein lib-Illa) in a variant of Glanzmann thrombasthenia (molcular genetlcs/flbnnogen receptor/Arg-Gly-Asp/poyerase chan reacdon) YI-PING CHEN*, ISABELLE DJAFFAR*, DOMINIQUE PIDARDt, BEAT (8,9), as well as for aLI32 (10). At least in the case of amb43 activation, modulation of affinity for its ligands is coordinated with conformational changes of the integrin (7,11). What remains obscure is the exact link between intracellular signals and aIIbP3 conformation modulation.Glanzmann thrombasthenia is a hereditary bleeding disorder characterized by the absence of platelet aggregation due to either an absence or a functional alteration of the integrin aII03 (12). In several cases, the genetic basis for the quantitative defect was found to be associated with alterations within the genes of ajib,83 subunits-i.e., either aib (glycoprotein lIb) (13,14) or 83 (glycoprotein IIa) (14, 15). In addition, the qualitative defects in aIlbm3 found in variants of Glanzmann thrombasthenia have also become the focus of investigations, and two mutations in Glanzmann variants that disrupted ligand binding sites of (3 have been characterized (16-18). Here we report a variant of Glanzmann thrombasthenia whose biochemical and genetic analyses are consistent with the idea that the functional defect is due to replacement of serine by proline in position 752 in the cytoplasmic domain of P3 and that this mutation impairs the coupling between cellular activation and up-regulation of arnb,83 affinity for fibrinogen. To our knowledge this is the first point mutation reported affecting integrin activation. METHODSPatient's Case. Patient P.'s case of variant Glanzmann thrombasthenia has been previously reported (19). Briefly, patient P. exhibited a life-long bleeding tendency with failure of platelets to aggregate and to bind fibrinogen in response to agonists such as ADP, collagen, or thrombin. The patient's platelets could support clot retraction. The presence of alb/3 in the patient's platelets was established by immunocytochemistry.Protein Analysis and Monoclonal Antibody Binding. Blood was collected in the presence of 5 mM EDTA, and platelets were prepared by conventional methods (20). Platelet proteins were solubilized in 2% NaDodSO4 and two-dimensional nonreduced/reduced NaDodSO4/PAGE was performed as described (21). Total platelet proteins (200 ,ug) were solubilized in 2% NaDodSO4 and separated in a first dimension in a 6% polyacrylamide tube gel. Disulfide bonds were reduced by soaking the gel in 10%6 (vol/vol) 2-mercaptoethanol, and the proteins were then separated again in a perpendicular direction in a 7-12% polyacrylamide slab gel. Proteins were detected by staining with Coomassie blue-R.The monoclonal antibody AP-2, which is specific for glycoprotein Ilb-IIla (22), the a11b13 integrin (a generous gift Abbreviations: RGDS, Arg-Gly-Asp-Ser; nt, nucleotide(s). lTo whom reprint requests...

Activation of CXCR4 by the CXC chemokine stromal cell-derived factor-1 (SDF-1) requires interaction of the amino-terminal domains of both molecules. We report that proteinases released from either mononucleated blood cells or polymorphonuclear neutrophils degranulated by inflammatory stimuli generate an SDF-1 fragment that is deleted from amino-terminal residues Lys 1 -Pro 2 -Val 3 , as characterized by mass spectrometry analysis. The proteolyzed chemokine fails to induce agonistic functions and is unable to prevent the fusogenic capacity of CXCR4-tropic human immunodeficiency viruses. Furthermore, we observed that exposure of CXCR4-expressing cells to leukocyte proteinases results in the proteolysis of the extracellular aminoterminal domain of the receptor, as assessed by flow cytometry analysis and electrophoretic separation of immunoprecipitated CXCR4. Blockade of SDF-1 and CXCR4 proteolysis by the specific leukocyte elastase inhibitor, N-methoxysuccinyl-alanine-alanine-prolinevaline-chloromethyl ketone, identified elastase as the major enzyme among leukocyte-secreted proteinases that accounts for inactivation of both SDF-1 and CXCR4. Indeed, purified leukocyte elastase generated in either SDF-1 or CXCR4 a pattern of cleavage indistinguishable from that observed with leukocyte-secreted proteinases. Our findings suggest that elastase-mediated proteolysis of SDF-1/CXCR4 is part of a mechanism regulating their biological functions in both homeostatic and pathologic processes.

Pseudomonas aeruginosa, a major lung pathogen in cystic fibrosis (CF) patients, secretes an elastolytic metalloproteinase (EPa) contributing to bacterial pathogenicity. Proteinase-activated receptor 2 (PAR2), implicated in the pulmonary innate defense, is activated by the cleavage of its extracellular N-terminal domain, unmasking a new N-terminal sequence starting with SLIGKV, which binds intramolecularly and activates PAR2. We show that EPa cleaves the N-terminal domain of PAR2 from the cell surface without triggering receptor endocytosis as trypsin does. As evaluated by measurements of cytosolic calcium as well as prostaglandin E(2) and interleukin-8 production, this cleavage does not activate PAR2, but rather disarms the receptor for subsequent activation by trypsin, but not by the synthetic receptor-activating peptide, SLIGKV-NH(2). Proteolysis by EPa of synthetic peptides representing the N-terminal cleavage/activation sequences of either human or rat PAR2 indicates that cleavages resulting from EPa activity would not produce receptor-activating tethered ligands, but would disarm PAR2 in regard to any further activating proteolysis by activating proteinases. Our data indicate that a pathogen-derived proteinase like EPa can potentially silence the function of PAR2 in the respiratory tract, thereby altering the host innate defense mechanisms and respiratory functions, and thus contributing to pathogenesis in the setting of a disease like CF.

Proteinase-activated receptor (PAR)-2 is cleaved within its aminoterminal extracellular domain by serine proteinases such as trypsin, unmasking a new aminoterminus starting with the sequence SLIGKV, which binds intramolecularly and activates the receptor. PAR-2 has been reported to be involved in inflammation within the lungs. We show that PAR-2 is expressed not only by human alveolar (A549), but also by bronchial (16HBE) epithelial cell lines, using RT-PCR and flow cytometry with a PAR-2 antibody whose epitope maps over the trypsin cleavage site. PAR-2 activation by trypsin and by the activating peptide SLIGKV-NH(2) leads to intracellular calcium mobilization in both lung epithelial cells. During lung inflammation, airspaces are burdened by neutrophils that release elastase and cathepsin G, two serine proteinases. We demonstrate that these proteinases do not activate PAR-2, but rather disarm the receptor, preventing activation by trypsin but not by SLIGKV-NH(2). Preincubation of a PAR-2-transfected cell line, as well as 16HBE and A549 cells, with either proteinase led to the disappearance of the cleavage/activation epitope recognized by the PAR-2 antibody. We hypothesize that elastase and cathepsin G disarm PAR-2 by proteolysis of the extracellular domain downstream from the trypsin cleavage/activation site, while leaving unmodified the SLIGKV-NH(2)-binding site. These findings suggest that the neutrophil serine proteinases may play a role in PAR-2-mediated lung inflammation.

The urokinase receptor (CD87) participates to the pericellular proteolytic potential of migrating cells and to the recruitment of leukocytes during inflammation. It consists of three structurally homologous domains, with the C-terminal domain D3 attached to cell membranes through a GPI anchor. CD87 is susceptible to an endoproteolytic processing removing the N-terminal domain D1 and generating truncated D2D3 membrane species, thus modulating CD87-associated functions. Full-length or truncated CD87 can be also released from cells via juxtamembrane cleavage by phospholipases and/or by yet unidentified proteinases. Using a recombinant CD87 and the CD87-positive monocytic U937 cell line and isolated blood monocytes, we show by protein immunoblotting and flow immunocytometry that the human neutrophil serine-proteinases elastase and cathepsin G cleave CD87 within the D1-D2 linker sequence, while in addition cathepsin G is highly efficient in cleaving the C terminus of D3. The combination of cathepsin G and elastase provided by degranulated neutrophils results in enzymatic cooperation leading to the release from monocytic cells of a truncated D2D3 species resembling that previously described in pathological body fluids. Using mass spectrometry analysis, the proteolytic fragmentation of synthetic peptides mapping the D1-D2 linker and D3 C-terminal domains identifies potential cleavage sites for each enzyme and suggests the existence of a mechanism regulating the CD87(D1-D2)-associated chemotactic activity. Finally, isolated or combined elastase and cathepsin G drastically reduce the capacity of cells to bind urokinase. Secretable leukocyte serine-proteinases are thus endowed with high potential for the regulation of CD87 expression and function on inflammatory cells.

Glycoprotein (GP) Ib was purified from lysates of human platelets prepared in the presence or absence of inhibitors of the endogenous calcium-activated neutral protease (CANP) by immunoaffinity chromatography, employing the GPIb-specific murine monoclonal antibody, AP1, coupled to Sepharose CL4B. When derived from lysates prepared in the presence of EDTA or leupeptin, the eluate from the AP1-affinity column contained a 240,000-260,000-mol-wt protein in addition to GPIb. In SDS PAGE, this protein was stained by Coomassie Blue R, but not by the periodic acid-Schiff reagent, and it was not labeled with 125I in intact platelets by the lactoperoxidase-catalyzed method. When derived from lysates prepared in the absence of CANP inhibitors, the eluate contained only GPIb and its proteolytic derivative, glycocalicin. A change in the electrophoretic mobility of GPIb consistent with its association with the 240,000-260,000-mol-wt protein was confirmed by crossed immunoelectrophoresis. By an immunoblot technique involving transfer of proteins eluted from the AP1-affinity column and separated by SDS PAGE onto a nitrocellulose membrane, the 240,000-260,000-mol-wt protein bound polyclonal goat antibody raised against rabbit macrophage actin-binding protein (ABP). On the basis of these results, we conclude the GPIb is tightly associated with ABP under conditions in which the endogenous CANP is inhibited, and that this apparent transmembrane complex of GPIb-ABP can be isolated in lysates of nonactivated human platelets.

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 ␣ IIb ␤ 3 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 ligandoccupied conformers of ␣ IIb ␤ 3 . 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 Ϸ5.5-fold over basal values measured on nontreated platelets, whereas this binding raised only by Ϸ3- Thrombosis and inflammation are processes which result from complex relationships between various vascular cell types, i.e. endothelial cells, leukocytes, and platelets (1, 2). As part of such a cell cooperation network, polymorphonuclear neutrophils contribute to vessel injury not only by their own, but also through interactions with platelets. Thus, neutrophils are found admixed with platelets in the core of vascular occlusions in several experimental models (1, 3), and more importantly, a neutrophil-dependent platelet deposition has been described in arterial injuries (4 -6). Neutrophil-mediated platelet activation can be demonstrated in vitro by adding specific neutrophil agonists such as the formyl-Met-Leu-Phe (fMLP) peptide, tumor necrosis factor-␣, or interleukin-8 to autologous neutrophil-platelet mixed suspensions (7-10). Cathepsin G, a serine proteinase stored in the azurophilic granules of neutrophils and released upon their stimulation, has been established as the major mediator of this cell-to-cell interaction (11-13). Acting similarly to ␣-thrombin, another serine proteinase agonist of platelets, cathepsin G-induced platelet activation results in massive exocytosis and aggregation reactions. The

SummaryDisruption of cell/ECM interactions resulting from uncontrolled pericellular proteolysis leads to detachment-induced cell apoptosis (anoikis), contributing to the morbid evolution of inflammatory vascular diseases. During cardiovascular infections, bacterial proteinases might induce vascular cells to enter a similar pathway. We focused on LasB, the predominant metalloproteinase secreted by the haematotropic pathogen Pseudomonas aeruginosa. While the exosecretome of the LasBdeficient pseudomonal strain PAO1lasBD had limited impact on human vascular cell adherence and viability, secretomes from the LasBexpressing reference strain, PAO1, or clinical isolates from patients with cardiac infection all induced anoikis, as did purified LasB. Immunofluorescence and/or immunoblotting analysis of heart valve myofibroblast cultures or whole tissue revealed an extensive, LasB-dependent degradation of ECM-associated fibronectin and vitronectin, that preceded cell de-adherence, whereas type I collagen showed limited degradation. Moreover, LasB produced a rapid endoproteolysis of the cell-associated urokinase receptor/uPAR, leaving a truncated receptor that is unable to support cell adherence and survival via interactions with vitronectin and integrins. Conversely, major myofibroblast integrins showed no or only minor alterations. Thus, among P. aeruginosa-secreted metalloproteinases, LasB can induce vascular cell anoikis through simultaneous proteolysis of ECM components and cell receptors, suggesting the uPAR-vitronectin axis as a major target in this process.