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Neutrophil Cathepsin G Modulates the Platelet Surface Expression of The Neutrophil Cathepsin G Modulates the Platelet Surface Expression of the Glycoprotein (GP) Ib-IX Complex by Proteolysis of the von Willebrand Factor Binding Site on GPIba and by a Cytoskeletal-Mediated Redistribution of the Remainder of the Complex By Charles A. LaRosa, Michael J. Rohrer, Stephen E. Benoit, Marc R. Barnard, and Alan D. Michelson The effects of neutrophil cathepsin G on the glycoprotein Iz,and prior fixation ofplatelets. Experiments with Serratia (GP) Ib-fX complex of washed platelets were examined. Ca- protease-treated and Bernard-Soulier platelets showed that thepsin G resulted in a concentration- and time-dependent neither platelet surface GPlb nor cathepsinG-induced prote- decrease in the platelet surface GPL-IX complex, as deter- olysis of GPlb were required for the cathepsin G-induced mined by flowcytometry, binding of exogenous von Wille- redistribution of the remnant of the GPlb-IX complex or the brand factor (vWF) in the presence of ristocetin, and risto- cathepsin G-induced increase in platelet surface P-selectin. cetin-induced platelet agglutination. Cathepsin G resulted in In summary, neutrophil cathepsin G modulates the platelet proteolysis of the vWF binding site on GPlba (defined by surface expression of the GPb-IXcomplex both by proteoly- monoclonal antibody [MoAbl 6D1).as determined by in- sis of the vWF binding site on GPlba and by a cytoskeletal- creased supernatant glycocalicin fragment (a proteolytic mediated redistribution of the remainder of the complex. product of GPlba); decreased total platelet contentof GPlb; Prior studies show that, although thrombospondin 1, anti- and lack of effect of either cytochalasin B (an inhibitor of serine proteases, and plasma are all inhibitors ofcathepsin actin polymerization), prostaglandin l2(an inhibitor of plate- G, the effects of cathepsin G on platelets, including an in- let activation), or prior fixation of the platelets. However, crease in surface GPllb-llla, occur during close contact be- cathepsin G resulted in minimal decreases in the bindingto tween neutrophils and platelets in a protective microenvi- fixed platelets of MoAbsTM60 (directed againstthe throm- ronment leg. thrombosis and localinflammation). Taken bin bindingsite onGPlba) and WM23 (directed against the together, the data suggest that, in a protective microenvi- macroglycopeptide portion of GPlba). In contrast to itspro- ronment, neutrophils play a role in transforming platelets teolytic effect on GPlba, the cathepsin G-induced decrease from a state favoring adhesion to damaged vessel walls (me- in platelet surface GPlX and theremnant ofthe GPlb-IX com- diated by vWF binding to the GPL-IX complex) to a state plex (definedby MoAbsFMC25 and AKIl was via a cytoskele- favoring platelet-to-platelet aggregation(mediated by fi- tal-mediated redistribution, as determined by lack of change brinogen bindingto theGPllb-llla complex) and platelet-to- in the total platelet contentof GPlX and the GPb-IXcom- leukocyte adhesion (mediated by P-selectinl. plex; complete inhibition by cytochalasin B, prostaglandin 0 1994 by The American Society of Hematology. LATELETS ARE ACTIVATED by cathepsin G, a ser- spondin 1,22 and antiserine pro tease^.^' However, recent stud- P ine protease released from the azurophilic granules of ies23.?4 have provided evidence that the cathepsin G-mediated stimulated neutrophils.”6 The potency of cathepsin G as a effects of neutrophils on platelets can occur during close platelet agonist is similar to that of thrombin,6 a physiologi- contact between neutrophils and platelets in a protective mi- cally important platelet a~tivator.~.’Thrombin results in in- croenvironment (eg, thrombosis and local inflammation). creased platelet surface expression of P-selectin (reflecting The thrombin-induced decrease in the platelet surface ex- a granule secretion)” and the glycoprotein (GP) IIb-IIIa pression of GPIb isnot the result of prote~lysis’~.~~or a complex (a receptor for fibrinogen, von Willebrand factor conformational change,’‘ but is the result of a cytoskeletal- [vWF], fibronectin, and vitronectin)““‘ and decreased plate- mediated translocation of the entire GPIb-IX complex to let surface expression of the GPIb-IX complex (a receptor the membranes of the open surface canalicular system.ln In for vWF).”.~’ It has recently been reported that neutrophil contrast, a number of proteases (eg, calcium-dependent pro- cathepsin G, to a similar or greater extent than thrombin, tease,2h Serratia marcescens protea~e,~~.~* pla~min,~~-~’and also results inan increased platelet surface expression of neutrophil ela~tase”~’~)decrease the platelet surface expres- P-selectid and the GPIIb-IIIa complex6,2‘and a decreased sion of GPIb by proteolysis of an a chain fragment that platelet surface expression of the GPIb-IX complex.6,2’The contains the vWF binding site. effects of cathepsin G are inhibited by plasma: thrombo- In this study, we examined the mechanism of the cathepsin G-induced decrease in the platelet surface expression of the GPIb-IX complex. We determined that cathepsin G modu- From the Division of Vascular Surgely and the Departments of lates the platelet surface expression of the GPIb-IX complex Surgery and Pediatrics, Universiry of Massachusetts Medical both by proteolysis of the vWF binding site on GPIba and School, Worcester. by a cytoskeletal-mediated redistribution of the remainder Submitted September 16, 1993; accepted March S, 1994. of the complex. Address reprint requests to Alan D. Michelson, MD, Department of Pediatrics, University of Massachusetts Medical School, 55 Lake MATERIALS AND METHODS Ave N, Worcester, MA 01655. Murine Monoclonal Antibodies (MoAbs) The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked GPZb-IX-specific MoAbs. MoAbs 6D1 (provided by Dr BW “advertisement” in accordance with 18 U.S.C. section l734 solely to S. Coller, SUNY, Stony Brook, NY) and AK2 (provided by Dr indicate this fact. Michael C. Bemdt, Baker Medical Research Institute, Melbourne, 0 1994 by The American Society of Hematology. Australia) are directed against the vWF binding site on the amino 0006-4971/94/8401-0007$3.00/0 terminal domain of platelet membrane GPIba.34-3hTM60 (provided 158 Blood, VOI 84. NO 1 (July l), 1994: pp 158-168 CATHEPSIN G MODULATESPLATELET GPlb-IX 159 by Dr Naomasa Yamamoto, Tokyo Metropolitan Institute of Medical Tyrode's buffer, pH 7.4, and stored at 4°C until flow cytometric Science, Tokyo, Japan) is directed against the thrombin binding analysis was performed (within 24 hours). site on the amino terminal domain of GpIb~r.~~.'~WM23 and AK3 Samples were analyzed in an EPICS Profile I1 flow cytometer (provided by Dr Berndt) are directed against the macroglycopeptide (Coulter Cytometry, Hialeah, FL) equipped with a 500 mW argon portion of GPn~ct.'~,~FMC25 (provided by Dr Bemdt) is directed laser (Cyonics, San Jose, CA) operated at 15 mW and a wavelength against platelet membrane GPIX.41AK1 (provided by Dr Berndt) is of 488 nm. The fluorescence of FITC and PE were detected using 525 directed against the GPIb-IX AKI only binds to the intact nm and 575 nm band pass filters, respectively. After identification of GPIb-IX complex, not to uncomplexed GPIb or GPIX." platelets by gating on both FITC positivity and their characteristic P-selectin-spec$c MoAbs. MoAb S12 (provided by Dr Rodger light scatter, binding of the biotinylated MoAb was determined by P. McEver, University of Oklahoma, Oklahoma City) is directed analyzing 5,000 individual platelets for PE fluorescence. Background against P-sele~tin."~,~P-selectin, also referred toas GMP-140,43 binding, obtained from parallel samples run with FITC-YU51 and PADGEM protein," and CD62,"6 is a component of the (x granule biotinylated mouse IgG (Boehringer Mannheim, Indianapolis, IN), membrane of resting platelets that is only expressed on the platelet was subtracted from each test sample. surface membrane after platelet degranulation and se~retion.'~.~' In some experiments, before the addition of cathepsin G, the Other MoAbs. 6F1 (provided by Dr Coller) is directed against platelets were incubated at 22°C with or without (1) 10 pmoW PG& the platelet membrane GPIa-IIa complex.4' Y2/51 (Dako Corp. Car- (Sigma, St Louis, MO) (an inhibitor of platelet activation) for 30 pinteria, CA) is directed against platelet membrane GPIIIa.4' 7E3 minutes, (2) 6 pmom cytochalasin B (Sigma) (an inhibitor of actin (provided by Dr Coller) is directed against the platelet membrane p~lymerization~~)for 15 minutes, or (3) 2.5 pg/mL Serratia mrces- GPIIb-IIIA c~mplex.''~OKM5 (provided by Dr Patricia Rao, Ortho cens metalloprotease (provided by Dr G.A. Jamieson, American Red Diagnostic Systems, Raritan, NJ) and F13 (provided by Dr Irwin D. Cross, Rockville, MD) for 30 minutes. The degree of proteolysis of Bemstein, Fred Hutchinson Cancer Research Center, Seattle, WA) platelet surface GPIb by Serratia protease was assessed byflow are directed against platelet membrane GPIV?0.5' cytometry with MoAb 6D1, as previously described?' Antibodies were biotinylated or conjugated with fluorescein iso- In other experiments, washed platelets from normal donors were thiocyanate (FlTC), as previously des~ribed.'~.~~In some experi- compared with washed platelets from a patient with Bernard-Soulier syndrome (see below). ments, 7E3 was directly conjugated with phycoerythrin (PE) by In some experiments, washed platelets were fixed with I % formal- Molecular Probes (Eugene, OR). dehyde
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