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Monoclonal Antibody IAC-1 Is Specific for Activated 2 1 and Binds to Amino

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Monoclonal Antibody IAC-1 Is Specific for Activated 2 1 and Binds to Amino HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY Monoclonal antibody IAC-1 is specific for activated ␣2␤1 and binds to amino acids 199 to 201 of the integrin ␣2 I-domain Anne Schoolmeester, Karen Vanhoorelbeke, Shinya Katsutani, Hilde Depraetere, Hendrik B. Feys, Johan M. W. Heemskerk, Marc F. Hoylaerts, and Hans Deckmyn In this study we describe the first monoclo- (3) the epitope for IAC-1 is T199Y200K201, does not interfere with platelet-collagen bind- nal antibody, integrin activated conforma- which is located at the opposite site of the ing, it defines a new class of antibodies that tion-1 (IAC-1), to recognize the active form metal ion-dependent adhesion site in a re- is distinct from those belonging to the “cat- of the platelet-collagen receptor, the integrin gion not involved in the I-domain “shifts” ion- and ligand-induced binding sites” ␣2␤1. IAC-1 has the following properties: (1) that occur upon ligand binding; (4) IAC-1 (CLIBSs) and the “ligand mimetic” group. IAC-1 fails to bind to resting platelets but strongly binds to recombinant ␣2 I-domain, These characteristics make IAC-1 a very readily interacts with platelets stimulated by therefore suggesting that the neo-epitope powerful tool to study ␣2␤1 activation under Downloaded from http://ashpublications.org/blood/article-pdf/104/2/390/1699771/zh801404000390.pdf by guest on 23 September 2021 the glycoprotein VI–specific agonist, con- appears to be exposed by an “unmasking” dynamic and physiologically relevant condi- vulxin, and by other agonists; (2) similar of I-domain–covering regions upon activa- tions. (Blood. 2004;104:390-396) concentration response relationships for tion; (5) IAC-1 binds to platelets during adhe- binding of IAC-1 and soluble collagen were sion to collagen under shear conditions, observed in convulxin-stimulated platelets; demonstrating activation of ␣2␤1; (6) as IAC-1 © 2004 by The American Society of Hematology Introduction Integrins are a large family of receptor molecules that mediate apparent affinity for soluble collagen was lower in platelets stimulated attachment of cells to each other and to the surrounding extracellu- by adenosine diphosphate (ADP) compared with platelets stimulated by lar matrix (ECM). Integrin engagement activates signaling path- thrombin or a glycoprotein VI (GPVI)–specific collagen-related peptide, ways involved in adhesion-dependent processes including cell suggesting 2 different activation states of ␣2␤1. migration, proliferation, survival, and differentiation. Conse- In the present study, we describe for the first time the quently, they participate in diverse (patho)physiologic processes as identification of an anti-␣2 I-domain monoclonal antibody (mAb) diverse as hemostasis, immunity, and inflammatory disorders such that binds to activated but not to resting platelets. Thus, these data as atherosclerosis. A unique aspect of integrin function consists of provide direct evidence of a conformational switch for ␣2␤1 during affinity regulation of their extracellular domain for biologic ligands platelet activation. The mAb is termed integrin activated conforma- by signals from within the cell (inside-out signaling).1 tion-1 (IAC-1). We further show that IAC-1 recognizes both the 2 ␣ ␤ The ␣2␤1 integrin is expressed on a variety of cell types, mainly intermediate and high-affinity states of 2 1, as its binding was ␣ serving as a collagen receptor.3 The I-domain, inserted into the induced by ADP and by strong agonists. Using chimeric 2 head region of the ␣2 subunit, plays a central role in ligand binding I-domains, we mapped the IAC-1 epitope to amino acids and recapitulates many of the ligand-binding properties of the T199Y200K201 in the loop between ␤-strand C and ␣-helix 3 at 4-6 the bottom of the I-domain, distant from the metal ion-dependent intact integrin. The crystal structure of the ␣2 I-domain, deter- 7 adhesion site (MIDAS). This region is not involved in the I-domain mined both in the absence and presence of an ␣2␤1-specific collagen-related peptide,8 has revealed that ligand binding to the shifts observed upon ligand binding. Taken together, our findings I-domain results in a change in metal coordination leading to indicate that a neo-epitope is generated upon platelet activation by ␣ reorganization of the upper surface and propagation of conforma- “unmasking” areas of the 2 I-domain covered by other regions of ␣ ␤ tional changes to the opposite pole of the domain.8 These structural 2 1 rather than by a shift in the I-domain itself. differences are thought to reflect different conformations of ␣2␤1. However, no information is available on how such changes in the I-domain might be induced through inside-out activation. Materials and methods 9 Kainoh et al were the first to demonstrate ␣2␤1-mediated, activation- dependent platelet adhesion to collagen. This was later extended by Jung Materials 10-13 14 and Moroi and Wang et al, who demonstrated platelet activation- Gi9, an inhibitory anti ␣2 I-domain mAb, was purchased from Immunotech dependent binding of soluble collagen to ␣2␤1. Interestingly, the (Marseille, France). Fluorescein isothiocyanate (FITC)–conjugated CD49b From the Laboratory for Thrombosis Research, Interdisciplinary Research Onderzoek in de Industrie grant IWT 21394 (H.B.F.). Center (IRC), K.U. Leuven Campus Kortrijk, Belgium; the Department of An Inside Blood analysis of this article appears in the front of this issue. Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), University of Maastricht, The Netherlands; and the Center for Molecular and Reprints: Hans Deckmyn, Laboratory for Thrombosis Research, IRC, K.U. Vascular Biology, K.U. Leuven, Belgium. Leuven Campus Kortrijk, E. Sabbelaan 53, B-8500 Kortrijk, Belgium; e-mail: [email protected]. Submitted December 12, 2003; accepted March 4, 2004. Prepublished online as Blood First Edition Paper, March 23, 2004; DOI 10.1182/blood-2003-12-4224. The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby Supported by grant Geconcerteerde Onderzoeksaltie GOA/03/009, a postdoctoral marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734. fellowship of the Fonds voor Wetenschappelijk Onderzoek Flanders (K.V.), and Vlaams Instituut voor de Bevordering Van Het Wetenschappelyk-Technologisch © 2004 by The American Society of Hematology 390 BLOOD, 15 JULY 2004 ⅐ VOLUME 104, NUMBER 2 BLOOD, 15 JULY 2004 ⅐ VOLUME 104, NUMBER 2 IAC-1 BINDS TO ACTIVATED INTEGRIN ␣2␤1 391 mAb (AK-7, anti-␣2 I), phycoerythrin (PE)–conjugated CD62P (anti–P- Platelet preparation selectin), and FITC-conjugated PAC-1 (anti–activated ␣ ␤ ) mAbs were IIb 3 Blood samples from healthy volunteers, free of acetylsalicylic acid for the purchased from BD Bioscience (San Diego, CA). An unrelated mouse last 10 days, were collected on acid citrate dextrose (ACD). Platelet-rich monoclonal immunoglobulin G 1 (IgG1), MOPC-21–FITC, and horse- plasma (PRP) was obtained by centrifugation. Washed platelets were radish peroxidase (HRP)–conjugated goat antimouse IgG (GAM-HRP) prepared essentially as described.16 were purchased from Sigma (St Louis, MO); FITC-conjugated rabbit antimouse (RAM-FITC) was purchased from Dako (Glostrup, Denmark); and streptavidin-HRP was purchased from Roche (Mannheim, Ger- Static adhesion and aggregation of platelets many). Platelet agonists were Horm collagen (Nycomed, Munich, Ger- Static adhesion of washed human platelets (3 ϫ 105/␮L) to human collagen many), acid soluble human collagen type I (Sigma), convulxin (Kordia, type I (25 ␮g/mL in PBS) coated on a microtiter plate was tested in the Leiden, The Netherlands), ADP and U46619 (Sigma), collagen-related presence or absence of various concentrations of anti-␣ I-domain mAbs. peptide (CRP; a kind gift from Dr Farndale, Cambridge, United Kingdom), 2 Platelet binding was determined by measuring endogenous phosphatase and thrombin (Kordia). activity after lysis of the platelets with Triton X-100 using P-nitrophenyl Collagen type I and anti-␣ I-domain mAbs were labeled with FITC or 2 phosphate (Merck, Darmstadt, Germany) as substrate.6 NHS-SS–biotin (sulfosuccinimidyl-2-(biotinamido)ethyl-1,3-dithiopropi- Platelet aggregation was monitored using an ELVI-400 platelet aggre- Downloaded from http://ashpublications.org/blood/article-pdf/104/2/390/1699771/zh801404000390.pdf by guest on 23 September 2021 onate) according to the instructions of the supplier (Pierce, Rockford, IL). gometer (Elvi Logos, Milano, Italy) at 37°C under stirring conditions. Recombinant ␣ 2 I-domain was expressed in Escherichia coli either as a Aggregation of washed platelets was induced by human collagen type I maltose binding protein (MBP) or as a His-tag fusion protein and purified as (threshold collagen concentration to induce full aggregation). In inhibition 6,15 described. assays, platelets were preincubated with the mAbs (10 ␮g/mL final concentration) for 5 minutes before agonist addition. Production of anti-␣2 I-domain mAbs 15 Flow cytometry Anti-␣2 I-domain mAbs were produced essentially as described. Briefly, BALB/c mice were immunized by injection with purified ␣2 I-MBP. Two Reaction mixtures of PRP or washed platelets (5 ␮L in a total volume of 50 days after the final intraperitoneal antigen boost, the spleen cells were fused ␮L), agonist, CD62P-PE, and FITC-labeled mAb or FITC-labeled collagen with Sp2/0 mouse myeloma cells and cultured in HAT (hypoxanthine in buffer A (10 mM HEPES [N-2-hydroxyethylpiperazine-NЈ-2-ethanesul- aminopterin thymidine) selection medium (Gibco, Paisley, United King- fonic acid], 150 mM NaCl, 1 mM MgCl2, 5 mM KCl, pH 7.4) were dom). Cloning was performed by a limiting dilution method using 96-well incubated for 15 minutes. The reaction was stopped by adding 10 volumes microtiter plates with mouse peritoneal macrophage feeder layers. Hybrid- of 0.2% formyl saline, and a 1:10 dilution was analyzed on a FACS Calibur omas producing anti-␣2 I-MBP mAbs were identified in an enzyme-linked flow cytometer (Becton Dickinson).
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