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Blood, 15 November 2003, Vol. 102, No. 10, pp. 3637-3645. Prepublished online as a Blood First Edition Paper on July 31, 2003; DOI 10.1182/blood-2003-02-0496. This Article Abstract Full Text (PDF) All Versions of this Article: 2003-02-0496v1 102/10/3637    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Thai, L. M. Articles by Jackson, D. E. Search for Related Content PubMed PubMed Citation Articles by Thai, L. M. Articles by Jackson, D. E. Related Collections Hemostasis, Thrombosis, and Vascular Biology Cell Adhesion and Motility Signal Transduction Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY Physical proximity and functional interplay of PECAM-1 with the Fc receptor FcRIIa on the platelet plasma membrane Le M. Thai, Leonie K. Ashman, Stacey N. Harbour, P. Mark Hogarth, and Denise E. Jackson From the Austin Research Institute, Austin Hospital, Heidelberg, Victoria, Australia; University of Newcastle, Callaghan, New South Wales, Australia.    Abstract Top Abstract Introduction Materials and methods Results Discussion References   We and others have recently defined that Platelet Endothelial Cell Adhesion Molecule-1 (PECAM-1/CD31) functions as a negative regulator of platelet-collagen interactions involving the glycoprotein VI/Fc receptor gamma chain (GPVI/FcR- chain) signaling pathway.1,2 In this study, we hypothesized that PECAM-1 may be physically and functionally associated with FcRIIa on the platelet membrane. The functional relationship between PECAM-1 and FcRIIa was assessed by determining the effect of anti-PECAM-1 monoclonal antibody Fab fragments on FcRIIa-mediated platelet aggregation and heparin-induced thrombocytopenia (HITS)-mediated platelet aggregation. Preincubation of washed platelets with monoclonal antibody fragments of 2BD4 directed against PECAM-1 and IV.3 directed against FcRIIa completely blocked FcRIIa-mediated platelet aggregation and HITS-mediated platelet aggregation, whereas anti-CD151 antibody had no blocking effect. Coengagement of FcRIIa and PECAM-1 resulted in negative regulation of FcRIIa-mediated phospholipase C2 activation, calcium mobilization, and phosphoinositide 3-kinase-dependent signaling pathways. In addition, the physical proximity of FcRIIa and PECAM-1 was confirmed by using fluorescence resonance energy transfer and coimmunoprecipitation studies. These results indicate that PECAM-1 and FcRIIa are colocalized on the platelet membrane and PECAM-1 down-regulates FcRIIa-mediated platelet responses. (Blood. 2003;102:3637-3645)    Introduction Top Abstract Introduction Materials and methods Results Discussion References   Platelets are central to the physiologic process of hemostasis and pathologic process of thrombosis. The mechanisms underlying the role of platelets in these processes are highly complex, integrated, and regulated, involving the interaction of multiple surface platelet receptors, including the glycoprotein Ib-IX-V (GPIb-IX-V) complex, IIb3, and glycoprotein VI/Fc receptor gamma chain (GPVI/FcR- chain) complex with subendothelial proteins, and plasma ligands.3 The low-affinity immunoglobulin G (IgG) Fc receptor, FcRIIa, and the collagen receptor GPVI/FcR- chain complex are 2 IgG-bearing immunoreceptor tyrosine-based activation motif (ITAM)-containing receptors expressed on the human platelet surface. These ITAM-containing receptors bear tandem ITAM motifs (D/ExYxxL/I-x7-YxxL/I) either within their cytoplasmic tails or within an associated protein and are the actual signal transducing element of the receptor. On receptor clustering, activation of receptor-associated tyrosine kinases of the Src family occurs, which phosphorylate the ITAM motif. The phosphorylated ITAM then serves as a docking site for Src-homology 2 (SH2) domain-containing adapter proteins and enzymes, including Syk protein-tyrosine kinase, thereby recruiting molecules to the membrane and to the proximity of their substrates, including phospholipase C (PLC), the Ras activating enzyme, Sos and phosphoinositide 3-kinase (PI-3K).4-6 In contrast to the GPVI/FcR- chain complex, FcRIIa is unique where the ligand binding domain and the intracellular ITAM are contained in the one polypeptide, whereas the GPVI/FcR- chain complex consists of GPVI noncovalently associating with the ITAM-containing FcR- chain.7,8 FcRIIa is the sole Fc platelet receptor existing as a dimer with each molecule being a 40-kDa molecular weight (MW) type I membrane glycoprotein possessing 2 extracellular immunoglobulin domains that bind IgG (KD 10-6), a transmembrane domain, and an ITAM-containing intracellular domain. The extracellular domain contains a polymorphism at amino acid position 131 (Arg/His) and has been implicated in immune complex-related diseases such as systemic lupus erythematosus.9 In addition, in vitro and in vivo murine models have demonstrated that FcRIIa is involved in heparin-induced thrombocytopenia (HITS).10 In vitro activation of platelet FcRIIa via clustering with aggregated human IgG or activatory anti-FcRIIa antibodies causes platelet activation, secretion, shape change, and aggregation through an ITAM signaling pathway.11 Such tyrosine kinase-dependent activity pathways are regulated by phosphatases in which activity is dependent on immunoreceptor tyrosine-based inhibition motif (ITIM) sequences. Platelets also express several ITIM-containing surface receptors, including PECAM-1, signal regulatory protein (SIRP), and protein zero related (PZR).12-14 PECAM-1 is a 130-kDa MW transmembrane protein member of the immunoglobulin-ITIM superfamily with an extracellular domain containing 6 Ig-domains, a transmembrane domain, and a dual ITIM-containing intracellular domain.15,16 It is now recognized that regulation of cell signaling involves a balance between activatory and inhibitory signals as observed in many immune cells.17 In particular, the negative regulation of ITAM-containing receptors by their inhibitory counterparts, ITIM-containing receptors, determines both the threshold and magnitude of activatory responses and corresponding cellular effects. For regulation of one receptor signaling pathway by another, a close physical proximity and coengagement between these receptors is required for induction of interactions between target substrates, adaptors, and effector molecules in their individual signaling pathways. Previous studies have defined physical associations of several platelet receptors, including GPIb with FcRIIa, GPIb and the GPVI/FcR- chain complex, GPIb and protein-disulfide isomerase, and CD151 with integrin IIb3.18-20 More importantly, a functional relationship has been demonstrated between the ITAM-containing GPVI/FcR- chain complex that is negatively regulated by the ITIM-containing PECAM-1 receptor on both murine and human platelets to down-modulate platelet-collagen interactions.1,2 It is therefore conceivable that PECAM-1 may also negatively regulate FcRIIa in human platelets, particularly because platelets lack the inhibitory coreceptor, FcRIIB, that is known to negatively regulate FcRIIA in other cell types. However, the fact that ITIM-containing receptors are known to have different affinities for different ITAM-containing receptors, and, unlike the GPVI/FcR- chain complex, the ITAM is associated with the ligand binding polypeptide of FcRIIa, prompted us to investigate the possible existence of a physical and functional relationship between FcRIIa and PECAM-1. The importance of FcRIIa in both pathophysiologic conditions and healthy platelet function further necessitates these studies. In light of evidence indicating the potential existence of a physical and functional relationship between platelet FcRIIa and PECAM-1, we hypothesize that PECAM-1 negatively regulates FcRIIa-mediated platelet responses through attenuation of signaling via the recruitment of protein-tyrosine phosphatases (PTPs) such as SHP-2 (Src homology 2-containing protein-tyrosine phosphatase 2) subsequent to the phosphorylation of the dual PECAM-1 ITIMs, resulting in dephosphorylation of important signaling effector and adaptor molecules involved in the FcRIIa ITAM-mediated signaling pathway. To address this hypothesis using in vitro studies, we investigated the physical proximity and possible colocalization of FcRIIa and PECAM-1 on the surface of human platelets using distinct complementary biochemical techniques, flow cytometry, fluorescence resonance energy transfer (FRET), coimmunoprecipitation, and Western blot studies. We next examined the functional effect of PECAM-1 on FcRIIa-mediated platelet aggregation induced by aggregated IgG, activatory anti-FcRIIa antibody cross-linking, and heparin-induced thrombocytopenia antibody. Finally, we delineated the molecular signaling mechanisms that govern the above functional relationship by using immunoprecipitation/Western blot analysis to profile important protein-tyrosine phosphorylated signaling molecules in FcRIIa and PECAM-1 signaling pathways. These studies show for the first time that FcRIIa and PECAM-1 are physically and functionally associated on the surface of human platelets. More importantly, they provide a greater insight into the function and regulation at the signaling level of the physiologically and pathophysiologically important sole platelet Fc receptor, FcRIIa, and potential novel therapeutic targets.    Materials and methods Top Abstract Introduction Materials and methods Results Discussion References   Aggregated human IgG Human IgG (10 mg; Sigma Chemical, St Louis, MO) was aggregated as described previously.18 Monoclonal antibodies The following monoclonal antibodies (mAbs) were used in this study: PECAM-1.1 (IgG2a), PECAM-1.2 (IgG1), PECAM-1.3 (IgG1) were kindly provided by Dr Peter Newman (Blood Research Institute, Milwaukee, WI)21; anti-PECAM-1 mAbs 2BD4 (IgG1) and B2B1 (IgG1), and anti-CD151 mAbs 11B1.G4 (IgG2a) and 14A2 (IgG1)22,23; IV.3 (IgG1) was kindly provided by Prof Clark Anderson (Ohio State University, Columbus)24,25; VM16d mAb was kindly provided by Dr Michael Berndt (Monash University, Clayton, Victoria, Australia).26 Preparation and biotinylation of monoclonal antibody Fab fragments Fab fragments were prepared for the following mAbs: PECAM-1.1, PECAM-1.2, PECAM-1.3, 2BD4, B2B1, 11B1, and IV.3 with the use of a standard protocol using Ficin as previously described.27 Biotinylation of IV.3, PECAM-1.1, 2BD4, B2B1, and 11B1 Fab fragments was performed according to manufacturers' instructions. Preparation of human platelets Platelet-rich plasma (PRP) and washed platelets were prepared from freshly obtained human blood as previously described.16 Binding of aggregated human IgG to washed platelets The binding of aggregated human IgG and IV.3 to human platelets was assessed as previously described.18 Acquisition flow cytometry analysis was performed with an Epics XL-MCL (Beckman-Coulter, Fullerton, CA) using a platelet-specific fluorescein isothiocyanate (FITC) protocol with forward scatter (FSC), side scatter (SSC), and fluorescent parameters in log scale. Aggregated human IgG-mediated platelet aggregation Aggregated IgG induction of platelet aggregation mediated through FcRIIa was analyzed using a Packs-4 Platelet Aggregation Chromogenic Kinetic System (Helena Laboratories, Beaumont, TX). For aggregated IgG-mediated platelet aggregation, 225 µg/mL aggregated IgG was added to 450 µL washed platelets (3 x 108/mL) in Ringer-citrate-dextrose (RCD) pH 7.4. In blocking experiments, 10 µg/mL final concentration of nonbiotinylated anti-FcRIIa (IV.3 Fab), biotinylated anti-PECAM-1 (PECAM-1.1, 2BD4, and B2B1 Fab), and biotinylated anti-CD151 (11B1 Fab) was added to the washed platelets with stirring for 5 minutes, prior to the addition of aggregated IgG to induce platelet aggregation. IV.3-mediated platelet aggregation This assay system involves the simultaneous coengagement of FcRIIa and PECAM-1 using optimal concentrations of biotinylated anti-FcRIIa (IV.3) and biotinylated anti-PECAM-1 (2BD4 Fab) with cross-linking induced by the addition of Neutravidin (Pierce, Rockford, IL). Specificity control of Neutravidin to induce FcRIIa-mediated aggregation through cross-linking of biotinylated IV.3 mAb was applied using biotinylated IV.3 mAb without the addition of Neutravidin. Anti-CD151 (biotinylated 11B1 Fab) was used as a negative control for inhibition of platelet aggregation. Cross-linking of receptors was induced by 10 µg/mL Neutravidin, resulting in either platelet aggregation or inhibition. HITS-mediated platelet aggregometry The assay system used was essentially the same as that in FcRIIa-mediated platelet aggregation studies using aggregated human IgG. However, HITS-positive donor sera along with 1 U/mL heparin instead of aggregated IgG were used to induce platelet aggregation. A volume of 450 µL of 3 x 106/mL PRP with 20 µL HITS-positive donor sera and 1 U/mL heparin were used in these experiments. Fluorescence resonance energy transfer (FRET) IV.3, PECAM-1.1, PECAM-1.2, and VM16d mAb F(ab')2 were labeled with Alexa Fluor 546 by using an Alexa Fluor 546 Protein Labeling Kit (Molecular Probes, Eugene, OR) and fluorescein 5-isothiocyanate isomer 1 (FITC) (Sigma Chemical) according to manufacturer's instructions. Washed platelets (100 µL of 3 x 108/mL) in RCD, pH 6.5, containing 0.2% (vol/vol) bovine serum albumin (BSA) were incubated with the respective donor and acceptor fluorochrome-labeled F(ab)'2 fragments directed to the receptors at 10 µg/mL for 20 minutes. Samples were then centrifuged for 1500g for 5 minutes at room temperature, then washed with 200 µL RCD, pH 6.5, containing 0.2% (wt/vol) BSA and resuspended in 0.5 mL RCD, pH 7.4, for FRET detection by using an LS-50B luminescence spectrometer (Perkin Elmer, Bucks, England). Calcium mobilization Washed platelets (4 mL of 3 x 108 cells/mL) in RCD, pH 6.5, were loaded with 3 µM fluorescent calcium indicator, Fura-2/AM (Molecular Probes) as previously described.28 To induce calcium mobilization, 5 µg/mL biotinylated IV.3 IgG was added and allowed 5 minutes for binding prior to the addition of 10 µg/mL Neutravidin to induce clustering of FcRIIA. Biotinylated anti-PECAM-1 Fab and a negative control biotinylated anti-CD151 Fab at 5 µg/mL were used for coengagement of PECAM-1 and CD151 as required. IV.3 cross-linking with and without PECAM-1 coengagement and immunoprecipitation/Western blot studies A volume of 0.5 mL washed human platelets (3 x 108/mL) in RCD, pH 7.4, was stirred at 200g in an aggregometer cuvette at 37°C for 1 minute in a Payton dual channel aggregometer module (Paton Scientific, Victor Harbour, South Australia). Biotinylated IV.3 IgG (10 µg/mL) was then added and allowed to bind to the platelets for 5 minutes before cross-linking by the addition of 10 µg/mL Neutravidin. For PECAM-1 coengagement, 10 µg/mL biotinylated anti-PECAM-1, 2BD4, or PECAM-1.1 Fab fragments were added after the addition of biotinylated IV.3 IgG. Samples were cross-linked with 10 µg/mL Neutravidin for 0, 15 seconds, 30 seconds, 1 minute, 2 minutes, 5 minutes, and up to 15 minutes before stopping the reaction with the addition of 0.5 mL Triton lysis buffer. Specific details of immunoprecipitation procedures, Western blotting, and stripping are as previously described.16 Chemical cross-linking of cell surface antigens and digitonin lysis of platelet preparations Chemical cross-linking of platelet cell surface antigens and digitonin lysis conditions are essentially as previously described.18 Following lysis, samples were centrifuged at 11 000g for 10 minutes at 4°C, and digitonin-soluble supernatants were removed for preclearing and immunoprecipitation studies. Statistical analysis of data Acquisition flow cytometry analysis data were analyzed statistically with Excel (Microsoft, Redmond, WA) for statistical significance using Student t test (P < .05 as significant), standard deviation, and error.    Results Top Abstract Introduction Materials and methods Results Discussion References   Effect of mAbs on aggregated human IgG and IV.3 binding to platelets To establish a possible physical proximity relationship between FcRIIa and PECAM-1, aggregated IgG binding to FcRIIa studies were conducted in the presence and absence of anti-PECAM-1 Fab fragments. These studies have revealed significant reduction (P < .05) of binding of aggregated IgG (75 µg/mL) to FcRIIa with treatment of platelets with 4 different anti-PECAM-1 Fab fragments (PECAM-1.1 and PECAM-1.3, B2B1 and 2BD4) (Figure 1A). Specifically, a 2.5-fold reduction in aggregated IgG binding was observed following pretreatment with anti-PECAM-1 Fab fragments, whereas a 6-fold decrease in aggregated IgG binding to FcRIIa was observed with FcRIIa-specific blocking mAb, IV.3 Fab (10 µg/mL). Conversely, pretreatment with an antibody recognizing a platelet tetraspanin family member, CD151, had no observable effect in terms of inhibition of aggregated IgG binding. CD151 has a similar copy number to PECAM-1 on platelets on the basis of 11B1 binding.23 View larger version (19K): [in this window] [in a new window]   Figure 1.. Inhibition of aggregated IgG binding to human platelets by anti-PECAM-1 antibodies. (A) Binding of aggregated human IgG to washed platelets was measured by flow cytometry. Platelets were (1) untreated or (2) incubated with aggregated human IgG. Platelets were also incubated with aggregated human IgG after pretreatment with 10 µg/mL Fab fragments of (3) IV.3 mAb; (4) PECAM-1.1 mAb; (5) PECAM-1.3 mAb; (6) B2B1 mAb; (7) 2BD4 mAb; or negative control (8) CD151 mAb. * denotes statistical significance (P < .05) (n = 3). (B) Binding of biotinylated IV.3 to washed platelets in the presence of anti-PECAM-1 Fab fragments was measured by flow cytometry. Platelets were (1) untreated or (2) incubated with biotinylated IV.3 Fab (10 µg/mL). Platelets were also incubated with biotinylated IV.3 Fab (10 µg/mL) after pretreatment with increasing doses of anti-PECAM-1 2BD4 Fab fragments (3) 5 µg/mL, (4) 10 µg/mL, (5) 20 µg/mL, (6) 40 µg/mL, and IV.3 binding detected with streptavidin-PE and measured with flow cytometry. Results are representative of 3 experiments. Error bars represent mean ± SEM.   To exclude the possibility that anti-PECAM-1 Fab fragments interfere with FcRIIa ligand binding, we performed a dose-response of nonbiotinylated anti-PECAM-1 Fab fragments (0-40 µg/mL) in the presence of biotinylated IV.3 Fab fragments and detected IV.3 binding to platelets with streptavidin-phycoerythrin (PE) and flow cytometry. As shown in Figure 1B, the anti-PECAM-1 Fab fragments had no effect on the alteration of the IV.3 binding to FcRIIa on the platelet membrane. Overall, these results suggest the existence of either a close physical or functional proximity between FcRIIa and PECAM-1, whereas no such relationship can be observed between FcRIIa and CD151. Analysis of receptor colocalization by FRET On the basis of the ability of anti-PECAM-1 mAbs to inhibit the binding of aggregated human IgG to washed human platelets, we hypothesized that PECAM-1 and FcRIIa were located in close physical proximity on the platelet membrane. To test the proximity we used FRET between antibodies bound to FcRIIa and PECAM-1 and coimmunoprecipitation studies. By using FITC-conjugated anti-FcRIIa IV.3 F(ab')2 and Alexa Fluor 546-conjugated anti-PECAM-1.1 as the donor and acceptor fluorochromes, respectively, significant (100%) FRET was observed relative to background controls (Figure 2). FITC-conjugated anti-FcRIIA IV.3 F(ab')2 and Alexa Fluor 546-conjugated anti-GPIb VM16d was also positive (100%) for FRET as shown previously (Figure 2).18 Relatively lower FRET (6%-20%) was observed with donor and acceptor fluorochromes labeling of the same receptor for all 3 receptors (Figure 2), indicating the proximity of FcRIIa to PECAM-1 and to GPIb is much greater than that between identical receptors. View larger version (23K): [in this window] [in a new window]   Figure 2.. Fluorescence resonance energy transfer showing close physical proximity of FcRIIa with platelet glycoprotein receptors, PECAM-1, and GPIb Fluorescence resonance energy transfer efficiency values between platelet membrane epitopes using monoclonal antibody F(ab')2 fragments to glycoprotein Ib (VM16d), PECAM-1 (PEC-1.1, PEC-1.2), and FcRIIa (IV.3). Energy transfer was measured between FITC-labeled (donor excitation) and Alexa-546-labeled (acceptor emission) epitopes using a luminescence spectrometer. Results are representative of 4 experiments.   Physical association between FcRIIa and PECAM-1 Immunoprecipitation with anti-PECAM-1.1 and anti-FcRIIa IV.3 IgG mAbs from chemically cross-linked and non-cross-linked digitonin-lysed platelets and blotting for PECAM-1 reveals the presence of PECAM-1 bands (Figure 3A). In contrast, platelet lysates immunoprecipitated with healthy mouse IgG1 and anti-CD151, 11B1.G4 IgG mAb, displayed no PECAM-1 bands. Conversely, when similarly treated platelet lysates were blotted for CD151 with 11B1.G4 IgG mAb, only anti-CD151, 11B1.G4 immunoprecipitates displayed the presence of CD151 (Figure 3B). In addition, cross-linking decreased the yield of monomeric PECAM-1 but induced the formation of dimeric and oligomeric forms of PECAM-1 (results not shown) in both digitonin-lysed platelets immunoprecipitated with anti-PECAM-1 and anti-FcRIIa mAb, as previously described.29 Taken as a whole, these results indicate that FcRIIa is constitutively associated with PECAM-1, whereas CD151 is not physically associated with either receptor, on the platelet membrane. View larger version (48K): [in this window] [in a new window]   Figure 3.. Coimmunoprecipitation of PECAM-1 and FcRIIa from digitonin-lysed platelets. (A) Washed platelets were treated with and without chemical cross-linking prior to digitonin lysis and immunoprecipitation using antibodies against the polypeptides: normal mouse IgG1, FcRIIa (IV.3), PECAM-1 (PEC-1.1), and CD151 (11B1). Immunoblot shown reveals presence of PECAM-1 antigen. (B) Same as panel A. The only exception is that the immunoblot is probed for the presence of CD151 under nonreduced conditions. NM indicates normal mouse IgG; NXL, nonchemically crosslinked; and XL, chemically crosslinked.   Effect of mAbs on aggregated IgG-mediated platelet aggregation Whereas aggregated human IgG induced rapid and robust platelet aggregation, there was complete inhibition of aggregated IgG-induced platelet aggregation following pretreatment of washed platelets with anti-PECAM-1 IgG mAb, PECAM-1.1 (Figure 4). Similar results were obtained with anti-PECAM-1 mAbs, 2BD4 and B2B1 (results not shown). Similarly, the anti-FcRIIa, IV.3 IgG mAb (10 µg/mL) displayed the same inhibitory outcome (Figure 4A). Conversely, as predicted from initial inhibition of aggregated IgG binding studies, anti-CD151, 11B1.G4 mAb IgG, does not inhibit platelet aggregation (Figure 4A). View larger version (48K): [in this window] [in a new window]   Figure 4.. Inhibition of aggregated IgG FcRIIA-mediated platelet aggregation by anti-PECAM-1 intact and Fab fragments. (A) Addition of aggregated IgG to washed platelets results in a lag phase of 2 minutes followed by platelet aggregation. Pretreatment of platelets with anti-CD151 mAb 11B1 (10 µg/mL) did not block aggregated IgG-mediated platelet aggregation. Pretreatment of platelets with either anti-FcRIIA mAb IV.3 (10 µg/mL) or anti-PECAM-1.1 mAb (10 µg/mL) completely blocked aggregated IgG-mediated platelet aggregation. (B) Platelet aggregation of washed platelets induced by subthreshold concentration of aggregated IgG. Pretreatment of washed platelets with biotinylated anti-CD151 mAb 11B1 Fab (10 µg/mL) for 5 minutes prior to Neutravidin cross-linking did not block aggregated IgG-mediated platelet aggregation. Pretreatment of washed platelets with either FcRIIA mAb IV.3 Fab (10 µg/mL) or biotinylated anti-PECAM-1 mAb 2BD4 Fab (10 µg/mL) for 5 minutes prior to 10 µg/mL Neutravidin cross-linking completely blocked aggregated IgG-mediated platelet aggregation.   To exclude any Fc-mediated effects, Fab fragments were used in subsequent platelet experiments. Pretreatment with biotinylated anti-PECAM-1 Fab (2BD4) (10 µg/mL) and cross-linking of PECAM-1 with Neutravidin (10 µg/mL) results in inhibition of aggregated IgG-induced platelet aggregation (Figure 4B). Nonbiotinylated anti-FcRIIA F(ab) (IV.3) exhibits potent inhibition of platelet aggregation, whereas no inhibition of platelet aggregation is observed with biotinylated anti-CD151 Fab (11B1.G4) (10 µg/mL) (Figure 4B). This inhibitory effect mediated by anti-PECAM Fab fragments on FcRIIa-mediated platelet aggregation required cross-linking of PECAM-1. Effect of mAbs on FcRIIa-mediated platelet aggregation FcRIIa-mediated platelet aggregation was performed using biotinylated IV.3 mAb (10 µg/mL) and cross-linking with Neutravidin (10 µg/mL). The addition of biotinylated anti-PECAM-1 Fab (2BD4) prior to cross-linking with Neutravidin allows the simultaneous coengagement of PECAM-1. Coengagement of PECAM-1 induced by addition of biotinylated anti-PECAM-1 Fab (2BD4) with biotinylated IV.3 mAb followed by cross-linking with Neutravidin resulted in a dose-dependent inhibition of platelet aggregation with biotinylated 2BD4 anti-PECAM-1 Fab fragments (Figure 5A). Conversely, when biotinylated anti-CD151 Fab (11B1.G4) was used to simultaneously cross-link CD151 and FcRIIa, no inhibition of platelet aggregation was observed (Figure 5B). Similarly, in the absence of Neutravidin, no platelet aggregation response was observed, signifying the specificity of biotinylated anti-PECAM-1 Fab to inhibit FcRIIa-mediated platelet aggregation (Figure 5B). View larger version (47K): [in this window] [in a new window]   Figure 5.. Inhibition of FcRIIa-mediated platelet aggregation by anti-PECAM-1 Fab fragments. (A) Washed platelets (3 x 108 platelets/mL) were pretreated with varying concentrations of biotinylated Fab fragments of anti-PECAM-1 (2BD4) (0-10 µg/mL as indicated) and anti-FcRIIa IV.3 mAb (10 µg/mL) for 5 minutes. Following pretreatment, where indicated cross-linking (XL) was initiated by addition of 10 µg/mL Neutravidin to induce FcRIIa-mediated platelet aggregation. (B) Washed platelets (3 x 108 platelets/mL) were pretreated with 10 µg/mL biotinylated Fab antibody fragments of mAbs directed to CD151 (11B1), PECAM-1 2BD4, or FcRIIa (IV.3) for 5 minutes. Following pretreatment, cross-linking was initiated in appropriate cuvettes by addition of 10 µg/mL Neutravidin to induce FcRIIa-mediated platelet aggregation.   Effect of mAbs on HITS-mediated platelet aggregation In humans, the sole Fc receptor for IgG, FcRIIa, in platelets plays an important role in the pathophysiology of drug-induced thrombocytopenia, including HITS. On the basis of these features, we hypothesized that PECAM-1 may negatively regulate HITS-mediated platelet aggregation via its down-regulation of FcRIIa-mediated signaling events. To test this possibility, we examined the effect of anti-PECAM-1 Fab fragments in HITS-mediated platelet aggregation. As shown in Figure 6, 3 x 108 washed platelets/mL pretreated with 10 µg/mL biotinylated anti-PECAM-1 Fab fragments (2BD4) and cross-linking with 10 µg/mL Neutravidin resulted in 5-fold inhibition of platelet aggregation induced by 20 µL HITS-positive donor sera. Similar results were observed with anti-PECAM-1 Fab fragments, PECAM-1.1 and B2B1 (results not shown). In addition, pretreatment with nonbiotinylated anti-FcRIIa Fab fragments (IV.3) (10 µg/mL) also inhibited HITS-mediated platelet aggregation (Figure 6). In contrast, no inhibitory effect was observed with biotinylated anti-CD151 Fab fragments (11B1.G4) (10 µg/mL). Taken together, these studies strongly suggest the negative inhibitory effect of PECAM-1 activation on FcRIIa-mediated platelet aggregation induced by HITS antibodies. View larger version (51K): [in this window] [in a new window]   Figure 6.. Inhibition of FcRIIa-mediated HITS platelet aggregation by cross-linked anti-PECAM-1 Fab fragments. Platelet-rich plasma (3 x 106 platelets/mL) was pretreated with various biotinylated Fab antibody fragments directed to CD151 (11B1) (10 µg/mL); nonbiotinylated anti-FcRIIa (IV.3) (10 µg/mL), and anti-PECAM-1 (2BD4) (10 µg/mL) for 5 minutes prior to cross-linking with 10 µg/mL Neutravidin and stimulation with HITS-positive donor sera and 1 U/mL heparin to induce HITS-mediated platelet aggregation.   Coengagement of FcRIIa and PECAM-1 leads to negative regulation of phospholipase C2 and PI 3-kinase-dependent signaling pathways On clustering, FcRIIa is tyrosine phosphorylated and recruits Syk protein-tyrosine kinase (PTK) via its intrinsic ITAMs The recruitment of Syk PTK allows tyrosine phosphorylation of PLC2 and recruitment of phosphatidylinositol 3 (PI 3) kinase, whose activation results in downstream rapid accumulation of phosphatidylinositol-3, -4, -5 phosphate [PtdIns(3,4,5)P3] and Akt phosphorylation. Activation of the PLC2 signaling pathway results in calcium mobilization to promote platelet secretion and engagement of integrin IIb3 to initiate platelet aggregation. On the basis of our studies with PECAM-1, we hypothesize that coengagement of PECAM-1 and its recruitment and activation of protein-tyrosine phosphatases such as SHP-2 delivers a negative feedback signal to down-modulate FcRIIa-mediated signaling events. To test this hypothesis, we examined a number of phosphorylation profiles of key signaling molecules (antiphosphotyrosine content, tyrosine phosphorylation of PLC2, phosphoSer/Thr Akt) and calcium mobilization associated with FcRIIa signaling by using immunoprecipitation, Western blot, and calcium mobilization studies. The antiphosphotyrosine blot of total tyrosine phosphorylated platelet proteins following IV.3 cross-linking of FcRIIA alone or with PECAM-1 coengagement revealed induction of several tyrosine phosphorylated platelet proteins following clustering of FcRIIA alone (Figure 7A, lanes 1-6). In contrast, coengagement of PECAM-1 with FcRIIA cross-linking induced a significant reduction in tyrosine phosphorylation over time of at least 4 identifiable tyrosine phosphorylated platelet proteins as indicated by arrows with approximate molecular weights of p64, p55, p45, and p40 (Figure 7A, lanes 8-13). After 1 minute of coengagement with PECAM-1, a significant decrease in tyrosine phosphorylation of these 4 platelet proteins was seen, as observed in lanes 12 and 13. Three of the 4 platelet proteins displayed inducible protein-tyrosine phosphorylation subsequent to clustering of FcRIIa after 15 seconds with IV.3 cross-linking as indicated in lanes 2 to 6 (Figure 7A). Overall, these results indicate a reduction in tyrosine phosphorylation of platelet proteins on PECAM-1 coengagement consistent with its inhibitory activity. View larger version (44K): [in this window] [in a new window]   Figure 7.. Coengagement of PECAM-1 with IV.3 cross-linking leads to a reduction in tyrosine phosphorylation of platelet proteins, induction of tyrosine phosphorylation of PECAM-1, and recruitment of SHP-2 protein-tyrosine phosphatase. (A) Washed platelets were stimulated by FcRIIa cross-linking with and without coengagement of PECAM-1 over 5 minutes at 37°C with stirring. Immunoblotting of 100 µg platelet whole proteins with horseradish peroxidase (HRP)-conjugated antiphosphotyrosine antibody, RC20, and enhanced chemiluminescence (ECL) development. Arrows indicate proteins with reduced antiphosphotyrosine content. (B, top panel) Washed platelets were stimulated by FcRIIa cross-linking with and without coengagement of PECAM-1 over a time course of 0 to 5 minutes at 37°C with stirring. Immunoprecipitation of PECAM-1 with monoclonal anti-PECAM-1 antibody (PECAM-1.3) followed by immunoblotting with HRP-conjugated antiphosphotyrosine antibody (RC20) and ECL development. (B, middle panel) Blot was stripped and reprobed for SHP-2 antigen with polyclonal anti-SHP-2 antibody. (B, bottom panel) Blot was stripped and reprobed for PECAM-1 antigen content with polyclonal anti-PECAM-1 (SEW16) antibody.   Protein-tyrosine phosphorylation is an early event in FcRIIa-mediated signal transduction. To test if PECAM-1 cross-linking attenuated FcRIIa-mediated signal transduction, we performed a time course (0-5 minutes) of IV.3 ± PECAM-1 cross-linking, lysed the platelet suspensions, immunoprecipitated PECAM-1, and detected tyrosine phosphorylated PECAM-1 by immunoblotting with an antiphosphotyrosine antibody, RC20. As shown in Figure 7B (top panel), weak inducible tyrosine phosphorylation of PECAM-1 was observed following IV.3 cross-linking over time (lanes 1-6) and strongly inducible after 15 seconds following IV.3 ± PECAM-1 (lanes 8-13). Cross-linking of PECAM-1 alone with mAbs results in weak inducible tyrosine phosphorylation of PECAM-1 as previously described.28 In addition, as shown in Figure 7B (middle panel), inducible recruitment of SHP-2 protein-tyrosine phosphatase parallels the induction of tyrosine phosphorylation of PECAM-1 observed over time. The anti-PECAM-1 blot confirmed the proper PECAM-1 antigen loading in PECAM-1 immunoprecipitates (Figure 7B, bottom panel). These studies verify that induction of the tyrosine phosphorylation of PECAM-1 ITIMs leads to the recruitment of SHP-2 following clustering of FcRIIa and PECAM-1 in platelets. As recruitment of Syk by FcRIIa leads to recruitment of PLC2 and subsequent downstream activation of signaling events, we tested the tyrosine phosphorylation status of PLC2 following clustering of FcRIIA and coengagement of FcRIIa and PECAM-1. As shown in Figure 8A (top panel), following clustering of FcRIIa, there is an inducible tyrosine phosphorylation of PLC2 evident at 30 seconds and peaking at 1 minute. Following coengagement of FcRIIa and PECAM-1, there was almost complete inhibition of the tyrosine phosphorylation of PLC2 (lanes 9-13). In contrast, coengagement of FcRIIa and CD151 showed no difference in the level of tyrosine phosphorylation of PLC2 (Figure 8B, top panel). PLC2 antigen loading is consistent in all lanes (Figure 8A-B, bottom panels). To exclude the possibility that competition between biotinylated anti-IV.3 and biotinylated anti-PECAM-1 Fab fragments for a limiting amount of Neutravidin accounts for the lower level of tyrosine phosphorylated PLC-2 over time, we have performed a dose response of Neutravidin (0-100 µg/mL) at a 1 minute timepoint. As shown in Figure 8C, increasing doses of Neutravidin resulted in equivalent levels of tyrosine phosphorylation of PLC-2 following IV.3 cross-linking alone. Following cross-linking of IV.3 and PECAM-1, the reduced tyrosine phosphorylation of PLC-2 is not affected by super-saturating concentrations of Neutravidin. These studies have shown that coengagement of PECAM-1 with FcRIIa leads to dephosphorylation of tyrosine phosphorylated PLC2, an inhibitory signaling event characteristic of PECAM-1-mediated signaling involving SHP-2 recruitment and activation. View larger version (56K): [in this window] [in a new window]   Figure 8.. Coengagement of PECAM-1 with FcRIIa cross-linking with IV.3 results in dephosphorylation of PLC2. (A, top panel) Washed platelets were stimulated by FcRIIa cross-linking with and without coengagement of PECAM-1 over 5 minutes at 37°C with stirring. Immunoprecipitation of PLC2 with polyclonal anti-PLC2 antibody followed by immunoblotting with HRP-conjugated antiphosphotyrosine antibody (RC20) and ECL development. (A, bottom panel) Blot was stripped and reprobed for PLC2 antigen content with PLC2 antibody. (B, top panel) Same experimental conditions as in panel A, with the exception that coengagement of CD151 was performed. (B, bottom panel) Blot was stripped and reprobed for PLC2 antigen content with PLC2 antibody. (C, top panel) Similar experimental conditions as in panel A, with the exception that a single 1-minute timepoint and a dose response with Neutravidin (0-100 µg/mL) were performed. (C, bottom panel) Blot was stripped and reprobed for PLC2 antigen content with PLC2 antibody.   In addition, as recruitment of Syk by FcRIIa also leads to recruitment of PI 3-kinase and subsequent downstream activation of signaling events, we tested the Ser/Thr phosphorylation status of Akt, a known downstream effector of PI 3-kinase-dependent signaling events following coengagement of FcRIIa and PECAM-1. Following clustering of FcRIIa, there is an inducible Ser/Thr phosphorylation of Akt evident by 30 seconds of stimulation (Figure 9A, lanes 3-6). In contrast, there was complete inhibition of Ser/Thr phosphorylation of Akt following coengagement of PECAM-1 with FcRIIa and probing with antiphosphoSer/Thr Akt (lanes 9-13). Equal protein loading is observed in an Akt Western blot (Figure 9B). These results most likely suggest that recruited SHP-2 dephosphorylates Syk, an essential upstream effector of PI 3-kinase recruitment and activation by FcRIIa, thereby initiating a block in PI 3-kinase-dependent signaling events. View larger version (32K): [in this window] [in a new window]   Figure 9.. Coengagement of PECAM-1 with FcRIIa cross-linking with IV.3 results in dephosphorylation of serine/threonine kinase, Akt. (A) Washed platelets were stimulated by FcRIIa cross-linking with and without coengagement of PECAM-1 over a time course of 0 to 5 minutes at 37°C with stirring. Platelet lysate (200 µg) was electrophoresed on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel, followed by immunoblotting with a mixture of antiphosphoSer473 Akt and antiphosphoThr308 Akt (1/2000) followed by HRP-conjugated antirabbit (1/10 000) and ECL development. (B) Blot was stripped and reprobed for Akt antigen content with goat anti-Akt antibody (0.3 µg/mL) and HRP-conjugated antigoat (1/4000) and ECL development.   As recruitment of Syk by FcRIIa leads to tyrosine phosphorylation of PLC2, generation of secondary messengers inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) and subsequent calcium mobilization, we tested the effect on calcium mobilization following coengagement of FcRIIa and PECAM-1. Following IV.3 cross-linking, there was release of calcium from intracellular stores. In contrast, coengagement of PECAM-1 with FcRIIa resulted in inhibition of intracellular calcium release, whereas coengagement of CD151 with FcRIIa cross-linking displayed no inhibitory effect (Figure 10). Inactivation of PLC2 leading to inhibition of Ca2+ mobilization is likely to be mediated on PECAM-1 inhibitory signaling involving down-regulation of phosphorylation of PLC2 by Syk, decrease in PIP3 (PI 3-kinase, phosphatidylinositol 3,4,5-trisphosphate) production as a result of down-regulation of PI 3-kinase activity. View larger version (19K): [in this window] [in a new window]   Figure 10.. Inhibition of FcRIIa-mediated calcium mobilization mediated by anti-PECAM-1 Fab fragments. Calcium mobilization was measured after FcRIIa cross-linking of Fura-2 labeled washed platelets in the presence and absence of coengagement of CD151 and PECAM-1. The Fura-2 ratio was determined using a luminescence spectrometer and recorded for 500 seconds.      Discussion Top Abstract Introduction Materials and methods Results Discussion References   The concept that ITIM-bearing molecules have the capacity to negatively regulate cell activation induced by coaggregation with receptors bearing ITAMs has been demonstrated in a variety of immune cells. In human platelets, 2 different ITAM-associated receptor complexes are known. These receptors are the collagen GPVI receptor noncovalently coupled to a signal receptor ITAM-containing molecule, FcR- chain, and FcRIIa containing an intrinsic ITAM in its intracellular domain. On the basis of this study, we have demonstrated that PECAM-1 and FcRIIa are located in close physical proximity and are colocalized on the platelet membrane. This novel observation is supported by functional studies with PECAM-1-mediated signaling events inducing negative regulation of FcRIIa-mediated signaling events and FcRIIa-mediated platelet aggregation. From a clinical perspective, we found that PECAM-1 is an important negative regulator of HITS-mediated platelet aggregation via its potent inhibition of FcRIIa-mediated signaling events. There are several examples of physical associations of platelet receptors on the platelet membrane that have been described, including GPIb association with FcRIIa18 and GPIb association with protein-disulfide isomerase.19 On the basis of this study, we have shown the existence of a close physical relationship between FcRIIa and PECAM-1 with potent inhibition of aggregated IgG binding with all 4 anti-PECAM-1 mAb Fab fragments directed to various Ig-domains. In contrast, no physical relationship between FcRIIa and CD151 was established with anti-CD151 mAb Fab, 11B1.G4. Other physical studies using FRET and coimmunoprecipitation further supported the concept of a physical relationship and colocalization of these 2 receptors on the platelet plasma membrane. The FRET-based analysis indicated that FcRIIa and PECAM-1 are located in close physical proximity as required for energy transfer to occur. The coimmunoprecipitation studies with and without chemical cross-linking using membrane impermeable, DTSSP (3,3'-dithiobis(sulfosuccinimidyl propionate)), revealed that FcRIIa and PECAM-1 are constitutively associated on the platelet membrane under resting conditions. This interaction on an activated platelet may lead to FcRIIa influencing conformational changes in PECAM-1, or vice versa, to modulate ligand binding properties. Again, no such relationship appears to exist between FcRIIa and CD151. The sensitivity of FRET in a system in which both donor and acceptor fluorochromes are used is known to be dependent on a number of factors. These factors include relative receptor density of the 2 receptors under analysis, brightness of donor and acceptor fluorochromes with preferential overlapping of the emission, and absorption peaks of the donor and acceptor fluorochromes, respectively, and relative distribution of receptors on the platelet membrane. The Forster upper limit of critical distance for energy transfer (8-10 nm) is dependent upon the donor and acceptor pair and on the distance between fluorochromes attached to respective antigens. On the basis of estimates of copy number, the density difference between PECAM-1 and FcRIIa is approximately 5 to 1. Taking these factors into consideration, we used the lower density FcRIIa as the donor and the higher density PECAM-1 as the acceptor. One could also argue that the significant FRET observed with PECAM-1 and FcRIIa labeled acceptor and donor fluorochromes may be due to an artifact of high receptor density, whereby FRET is due to chance association rather than true physical proximity. This association can be ruled out, as it can be calculated that one molecule of PECAM-1 would be found in an area of 1800 nm2 from the statistics of 10 000 copies of PECAM-1 on 22 µm2 area of a platelet surface. On the assumption of even receptor distribution in a lattice-like arrangement, receptors are, therefore, estimated to be 60 nm apart, much greater than the required Forster upper limit of 8 to 10 nm for FRET. The chance of random FRET with respect to FcRIIa is even lower given the 5-fold lower density. In addition, our results of FRET between the same receptor were significantly lower than that between FcRIIa and PECAM-1, and FcRIIa and GPIb, indicating that homoassociation of receptors leading to FRET was not significant. Functional studies using 3 different approaches involving aggregated IgG, biotinylated IV.3 mAb IgG cross-linking of FcRIIa, and HITS antibody-induced platelet aggregation indicated the potent inhibitory functional effects of anti-PECAM-1 Fab fragments in negatively regulating FcRIIa-mediated platelet aggregation. In contrast, the tetraspanin family member, CD151, was observed to have no functional relationship with FcRIIa. These 3 different approaches have different mechanisms of activation of FcRIIa to induce platelet aggregation. The use of a more specific and potent method involving the cross-linking of FcRIIa with or without PECAM-1 engagement using biotinylated anti-FcRIIa Fab and biotinylated anti-PECAM-1 Fab and Neutravidin have shown consistent inhibitory activity of PECAM-1 on FcRIIa-mediated platelet aggregation. For this reason, this method was used in subsequent Ca2+ mobilization and signaling studies. Numerous in vitro studies have concluded that antibodies to the heparin:PF4 complex activate human platelets via FcRIIa.30-33 In this study, we provide evidence that platelet aggregation induced by HITS antibodies is mediated by FcRIIa in vitro as revealed by the inhibition of platelet aggregation by intact and Fab anti-FcRIIa mAb, IV.3, in both washed platelets and plasma-rich plasma. More significantly, activation of PECAM-1 using anti-PECAM-1 intact mAb and Neutravidin biotinylated anti-PECAM-1 Fab inhibited platelet aggregation, whereas anti-CD151 Fab fragments had no effect. Because PECAM-1 can negatively modulate collagen GPVI-dependent signaling events involving the FcR- chain1,2 and FcRIIa as revealed in this study, it is possible that the same mechanism of inhibition involving activation of PECAM-1 and its ITIM coinhibitory signaling pathways is involved in inhibition of HITS antibody-induced ITAM-containing FcRIIa-mediated platelet aggregation. To define the molecular mechanisms of PECAM-1-mediated negative regulation of FcRIIa-mediated platelet responses, we analyzed the profiles of signaling molecules associated in FcRIIa-mediated signaling pathways. We hypothesized that PECAM-1 may negatively regulate the FcRIIa-mediated signaling pathway on its activation, phosphorylation of its dual ITIMs, recruitment and activation of SHP-2 protein-tyrosine phosphatase, and subsequent dephosphorylation of multiple key proteins involved in FcRIIa-mediated signaling pathway. Our studies show that PECAM-1 coengagement in FcRIIa-mediated signaling results in inducible tyrosine phosphorylation of the dual PECAM-1 ITIMs, recruitment of SHP-2,34 and dephosphorylation of protein-tyrosine signaling species known to be involved in this signaling pathway, including PLC2,35 Akt, and inhibition of Ca2+ mobilization. These signaling events are a prerequisite for integrin IIb3 activation and platelet aggregation. Taken together, these results provide further evidence of the inhibitory role of PECAM-1 in FcRIIa-mediated platelet aggregation. However, there is current controversy in relation to recent findings about the actual role of SHP-2. Unlike SHP-1 protein-tyrosine phosphatase, which primarily acts as a negative regulator, SHP-2 can act as a positive or negative regulator in cell signaling, depending on the cell type and signaling complexes formed.36,37 These 2 closely related SH2 domain-containing protein-tyrosine phosphatases have been demonstrated to display differential substrate specificity, including the Ig-ITIM family member, SIRP.38 Recently, SHP-2 has been shown to negatively regulate the kinetics and amplitude of PI 3-kinase activation in a receptor-specific manner.39 Several studies have highlighted differences in the functional regulation mediated by SHP-2. In fibroblast cell lines, SHP-2 acts as a positive regulator of PI 3-kinase activation by insulin growth factor 1 (IGF-1) or insulin. In contrast, in similar cell lines, SHP-2 can act as a negative regulator of epidermal growth factor (EGF)-induced PI 3-kinase activation. In platelet studies, SHP-2 appears to play a negative regulatory role in collagen GPVI activation. PECAM-1-deficient platelets showed hyperresponsive features of collagen GPVI/FcR- chain-mediated responses and increased platelet thrombus formation on type I collagen and preferential recruitment of SHP-2 by phosphorylated PECAM-1 ITIMs in platelet signaling.1,2 Overall, on the basis of our findings, there is strong evidence that a physical and functional relationship between FcRIIa and PECAM-1 exists on the surface of human platelets. The implications of these findings are of relevance in scientific and clinical terms. First and foremost, FcRIIa, with its ITAM associated with the ligand-binding polypeptide, has not been demonstrated to be regulated by PECAM-1 on platelets. In addition, the novel findings of the colocalization of FcRIIa and PECAM-1 on human platelets, albeit probably only existing in approximately 10% of the PECAM-1 receptor population, on human platelets is quite unexpected. FcRIIa has been demonstrated in our and numerous in vitro studies to be a very potent mediator of platelet activation, secretion, and aggregation. From this perspective, PECAM-1 may have a role in functional regulation of FcRIIa-mediated events in platelets in vivo. In particular, FcRIIa has been demonstrated to be involved in immune complex-mediated phagocytosis of platelets by macrophages and in high IgG content sites such as in inflammation.40,41 From a clinical point of view, FcRIIa has been implicated in immune complex-mediated diseases such as systemic lupus erythematosus (SLE) and heparin-induced thrombocytopenia and/or thrombosis (HITTS).42,43 Specifically, our findings show that PECAM-1 can abrogate FcRIIa-mediated HITS antibody-induced platelet aggregation. These in vitro studies provide a foundation to explore the consequences of PECAM-1-negative regulation of FcRIIa-mediated signaling events within the context of in vivo disease models, including HITS.    Footnotes   Submitted February 20, 2003; accepted July 21, 2003. Prepublished online as Blood First Edition Paper, July 31, 2003; DOI 10.1182/blood-2003-02-0496. Supported by grant no. 250398 (D.E.J.) from the National Health and Medical Research Council of Australia. 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 marked advertisement in accordance with 18 U.S.C. section 1734. Reprints: Denise E. Jackson, Kronheimer Bldg, Austin Research Institute, Austin Hospital, Studley Road, Heidelberg, Victoria, Australia 3084; e-mail: d.jackson{at}ari.unimelb.edu.au.    References Top Abstract Introduction Materials and methods Results Discussion References   Jones KL, Hughan SC, Dopheide SM, Farndale RW, Jackson SP, Jackson DE. Platelet endothelial cell adhesion molecule-1 is a negative regulator of platelet-collagen interactions. Blood. 2001;98: 1456-1463.[Abstract/Free Full Text] Patil S, Newman DK, Newman PJ. Platelet endothelial cell adhesion molecule-1 serves as a inhibitory receptor that modulates platelet responses to collagen. Blood. 2001;97: 1727-1732.[Abstract/Free Full Text] Andrews RK, Shen Y, Gardiner EE, Berndt MC. Platelet adhesion receptors and (patho)physiological thrombus formation. Histol Histopathol. 2001;16: 1-12.[Medline] [Order article via Infotrieve] Vely F, Vivier E. Conservation of structural features reveals the existence of a large family of inhibitory cell surface receptors and noninhibitory/activatory counterparts. J Immunol. 1997:159: 2075-2077.[Abstract/Free Full Text] Oda A, Ikeda Y, Ochs HD, et al. Rapid tyrosine phosphorylation and activation of Bruton's tyrosine/Tec kinases in platelets induced by collagen binding or CD32 cross-linking. Blood. 2000;95: 1663-1670.[Abstract/Free Full Text] Chacko GW, Duchemin AM, Coggeshall KM, Osborne JM, Brandt J, Anderson CL. Clustering of the platelet Fc gamma receptor induces noncovalent association with the tyrosine kinase p72Syk. J Biol Chem. 1994;269: 32435-32440.[Abstract/Free Full Text] Watson SP, Gibbins J. Collagen receptor signalling in platelets: extending the role of the ITAM. Immunol Today. 1998;19: 260-264.[CrossRef][Medline] [Order article via Infotrieve] Anderson CL, Chacko GW, Osborne JM, Brandt JT. The Fc receptor for immunoglobulin G (Fc gamma RII) on human platelets. Semin Thromb Haemost. 1995;21: 1-9.[Medline] [Order article via Infotrieve] Duits AJ, Bootsma H, Derksen RH, et al. Skewed distribution of IgG Fc receptor IIa (CD32) polymorphism is associated with renal disease in systemic lupus erythematosus patients. Arthritis Rheum. 1995;39: 1832-1836. Bachelot-Loza C, Saffroy R, Lasne D, Chatellier G, Aiach M, Rendu F. Importance of the FcRIIA-Arg/His-131 polymorphism in heparin-induced thrombocytopenia diagnosis. Thromb Haemost. 1998;79: 523-528.[Medline] [Order article via Infotrieve] Gratacap M-P, Herault JP, Viala C, et al. FcRIIA requires a Gi-dependent pathway for an efficient stimulation of phosphoinositide 3-kinase, calcium mobilization, and platelet aggregation. Blood. 2000;96: 3439-3446.[Abstract/Free Full Text] Newman PJ, Berndt MC, Gorski J, et al. PECAM-1 (CD31) cloning and relation to adhesion molecules of the immunoglobulin gene superfamily. Science. 1990;247: 1219-1222.[Abstract/Free Full Text] Yamao T, Noguchi T, Takeuchi O, et al. Negative regulation of platelet clearance and of the macrophage phagocytic response by the transmembrane glycoprotein SHPS-1. J Biol Chem. 2002;42: 39833-39839. Zannettino ACW, Roubelakis M, Welldon KJ, et al. Novel mesenchymal and haematopoietic cell isoforms of the SHP-2 docking receptor, PZR: identification, molecular cloning and effects on cell migration. Biochem J. 2003;370: 537-549.[CrossRef][Medline] [Order article via Infotrieve] Newman PJ. Switched at birth: a new family for PECAM-1. J Clin Invest. 1999;103: 5-9.[Medline] [Order article via Infotrieve] Jackson DE, Ward CM, Wang R, Newman PJ. The protein-tyrosine phosphatase SHP-2 binds platelet/endothelial cell adhesion molecule-1 (PECAM-1) and forms a distinct signalling complex during platelet aggregation: evidence for a mechanistic link between PECAM-1 and integrin-mediated cellular signalling. J Biol Chem. 1997;272: 6986-6993.[Abstract/Free Full Text] Ravetch JV, Lanier LL. Immune inhibitory receptors. Science. 2000;290: 84-89.[Abstract/Free Full Text] Sullam PH, Hyun WC, Szollosi J, Dong J, Foss WM, Lopez JA. Physical proximity and functional interplay of the glycoprotein Ib-IX-V complex and the Fc receptor FcRIIA on the platelet plasma membrane. J Biol Chem. 1998;273: 5331-5336.[Abstract/Free Full Text] Burgess JK, Hotchkiss KA, Suter C, et al. Physical proximity and functional association of glycoprotein Ib and protein-disulphide isomerase on the platelet plasma membrane. J Biol Chem. 2000;275: 9758-9766.[Abstract/Free Full Text] Fitter S, Sincock PM, Jolliffe CN, Ashman LK. Transmembrane 4 superfamily protein CD151 (PETA-3) associates with 1 and IIb3 integrins in haematopoietic cell lines and modulates cell-cell adhesion. Biochem J. 1999;338: 61-70. Sun QH, DeLisser HM, Zukowski MM, Paddock C, Albelda SM, Newman PJ. Individually distinct Ig homology domains in PECAM-1 regulate homophilic binding and modulate receptor affinity. J Biol Chem. 1996;271: 11090-11098.[Abstract/Free Full Text] Ashman LK, Aylett GW, Cambareri AC, Cole SR. Different epitopes of the CD31 antigen identified by monoclonal antibodies: cell type-specific patterns of expression. Tissue Antigens. 1991;38: 199-207.[Medline] [Order article via Infotrieve] Geary SM, Cambareri AC, Sincock PM, Fitter S, Ashman LK. Differential tissue expression of epitopes of the tetraspanin C recognised by monoclonal antibodies. Tissue Antigens. 2001;58: 141-153.[CrossRef][Medline] [Order article via Infotrieve] Looney RJ, Abraham GN, Anderson CL. Human monocytes and U937 cells bear two distinct Fc receptors for IgG. J Immunol. 1986;136: 1641-1647.[Abstract] Rosenfeld SI, Ryan DH, Looney RJ, Anderson CL, Abraham GN, Leddy JP. Human Fc gamma receptors: stable inter-donor variation in quantitative expression on platelets correlates with functional responses. J Immunol. 1987;138: 2869-2873.[Abstract] Mazurov AV, Vinogradov DV, Vlasik TN, Repin VS, Booth WJ, Berndt MC. Characterization of an antiglycoprotein Ib monoclonal antibody that specifically inhibits platelet-thrombin interaction. Thromb Res. 1991;62: 673-684.[CrossRef][Medline] [Order article via Infotrieve] Mariani M, Camagna M, Tarditi L, Seccamani E. A new enzymatic method to obtain high-yield F(ab')2 suitable for clinical use from mouse IgG1. Mol Immunol. 1991;28: 69-77.[CrossRef][Medline] [Order article via Infotrieve] Henshall TL, Jones KL, Wilkinson R, Jackson DE. Src homology 2 domain-containing protein-tyrosine phosphatases, SHP-1 and SHP-2, are required for platelet endothelial cell adhesion molecule-1/CD31-mediated inhibitory signalling. J Immunol. 2001;166: 3098-3106.[Abstract/Free Full Text] Zhao T, Newman PJ. Integrin activation by regulated dimerization and oligomerization of platelet endothelial cell adhesion molecule (PECAM-1) from within the cell. J Cell Biol. 2001;152: 65-73.[Abstract/Free Full Text] Cines DB, Kaywin P, Bina M, Tomaski A, Schreiber AD. Heparin-associated thrombocytopenia. N Engl J Med. 1980;303: 788-795.[Abstract] Kelton JG, Sheridan D, Santos S, et al. Heparin-induced thrombocytopenia: laboratory studies. Blood. 1998;72: 925-930. Warkentin TE, Hayward CP, Boshkov LK, et al. Sera from patients with heparin-induced thrombocytopenia generate platelet-derived microparticles with procoagulant activity: an explanation for the thrombotic implications of heparin-induced thrombocytopenia. Blood. 1994;84: 3691-3699.[Abstract/Free Full Text] Suh JS, Malik MI, Aster RH, Visentin GP. Characterization of the humoral immune response in heparin-induced thrombocytopenia. Am J Hematol. 1997;54: 196-201.[CrossRef][Medline] [Order article via Infotrieve] Jackson DE, Kupcho KR, Newman PJ. Characterization of phosphotyrosine binding motifs in the cytoplasmic domain of platelet/endothelial cell adhesion molecule-1 (PECAM-1) that are required for the cellular association and activation of the protein-tyrosine phosphatase, SHP-2. J Biol Chem. 1997;272: 24868-24875.[Abstract/Free Full Text] Gratacap MP, Payrastre B, Viala C, Mauco G, Plantavid M, Chap H. Phosphatidylinositol 3,4,5-trisphosphate-dependent stimulation of phospholipase C-gamma2 is an early event in FcgammaRIIA-mediated activation of human platelets. J Biol Chem. 1998;273: 24314-24321.[Abstract/Free Full Text] Qu C-K, Nguyen S, Chen J, Feng G-S. Requirement of SHP-2 tyrosine phosphatase in lymphoid and hematopoietic cell development. Blood. 2001;97: 911-914.[Abstract/Free Full Text] Timms JF, Swanson KD, Marie-Cardine A, et al. SHPS-1 is a scaffold for assembling distinct adhesion-regulated multi-protein complexes in macrophages. Curr Biol. 1999;9: 927-930.[CrossRef][Medline] [Order article via Infotrieve] Mishra AK, Zhang A, Niu T, et al. Substrate specificity of protein-tyrosine phosphatase: different behaviour of SHP-1 and SHP-2 towards signal regulation protein SIRPalpha1. J Cell Biochem. 2002;84: 840-846.[CrossRef][Medline] [Order article via Infotrieve] Zhang SQ, Tsiaras WG, Araki T, et al. Receptor-specific regulation of phosphatidylinositol 3'-kinase activation by the protein tyrosine phosphatase Shp2. Mol Cell Biol. 2002;22: 4062-4072.[Abstract/Free Full Text] McKenzie SE, Taylor SM, Malladi P, et al. The role of the human Fc receptor FcRIIA in the immune clearance of platelets: a transgenic mouse model. J Immunol. 1999;162: 4311-4318.[Abstract/Free Full Text] Hulett MD, Witort E, Brinkworth RI, McKenzie IF, Hogarth PM. Multiple regions of human FcRII (CD32) contribute to the binding of IgG. J Biol Chem. 1995;270: 21188-21194.[Abstract/Free Full Text] Blasini AM, Steckman IL, Leon-Ponte M, Caldera D, Rodriguez MA. Increased proportion of responders to a murine anti-CD3 monoclonal antibody of the IgG1 class in patients with systemic lupus erythematosus (SLE). Clin Exp Immunol. 1993;94: 423-428.[Medline] [Order article via Infotrieve] Reilly MP, McKenzie SE. Insights from mouse models of heparin-induced thrombocytopenia and thrombosis. Curr Opin Haematol. 2002;9: 395-400.[CrossRef][Medline] [Order article via Infotrieve] CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this? This article has been cited by other articles: C. H. Serezani, D. M. Aronoff, R. G. Sitrin, and M. Peters-Golden Fc{gamma}RI ligation leads to a complex with BLT1 in lipid rafts that enhances rat lung macrophage antimicrobial functions Blood, October 8, 2009; 114(15): 3316 - 3324. [Abstract] [Full Text] [PDF] J. Rivera, M. L. Lozano, L. Navarro-Nunez, and V. Vicente Platelet receptors and signaling in the dynamics of thrombus formation Haematologica, May 1, 2009; 94(5): 700 - 711. [Abstract] [Full Text] [PDF] R. K. Andrews, D. Karunakaran, E. E. Gardiner, and M. C. Berndt Platelet Receptor Proteolysis: A Mechanism for Downregulating Platelet Reactivity Arterioscler Thromb Vasc Biol, July 1, 2007; 27(7): 1511 - 1520. [Abstract] [Full Text] [PDF] F. A. Lee, M. van Lier, I. A. M. Relou, L. Foley, J.-W. N. Akkerman, H. F. G. Heijnen, and R. W. Farndale Lipid Rafts Facilitate the Interaction of PECAM-1 with the Glycoprotein VI-FcR {gamma}-Chain Complex in Human Platelets J. Biol. Chem., December 22, 2006; 281(51): 39330 - 39338. [Abstract] [Full Text] [PDF] M. W. Goschnick, L.-M. Lau, J. L. Wee, Y. S. Liu, P. M. Hogarth, L. M. Robb, M. J. Hickey, M. D. Wright, and D. E. Jackson Impaired "outside-in" integrin {alpha}IIbbeta3 signaling and thrombus stability in TSSC6-deficient mice Blood, September 15, 2006; 108(6): 1911 - 1918. [Abstract] [Full Text] [PDF] S. Falati, S. Patil, P. L. Gross, M. Stapleton, G. Merrill-Skoloff, N. E. Barrett, K. L. Pixton, H. Weiler, B. Cooley, D. K. Newman, et al. Platelet PECAM-1 inhibits thrombus formation in vivo Blood, January 15, 2006; 107(2): 535 - 541. [Abstract] [Full Text] [PDF] J. L. Wee and D. E. Jackson The Ig-ITIM superfamily member PECAM-1 regulates the "outside-in" signaling properties of integrin {alpha}IIb{beta}3 in platelets Blood, December 1, 2005; 106(12): 3816 - 3823. [Abstract] [Full Text] [PDF] L.-M. Lau, J. L. Wee, M. D. Wright, G. W. Moseley, P. M. Hogarth, L. K. Ashman, and D. E. Jackson The tetraspanin superfamily member CD151 regulates outside-in integrin {alpha}IIb{beta}3 signaling and platelet function Blood, October 15, 2004; 104(8): 2368 - 2375. [Abstract] [Full Text] [PDF] S. M. Albelda, K. C. Lau, P. Chien, Z.-Y. Huang, E. Arguiris, A. Bohen, J. Sun, J. A. Billet, M. Christofidou-Solomidou, Z. K. Indik, et al. Role for Platelet-Endothelial Cell Adhesion Molecule-1 in Macrophage Fc{gamma} Receptor Function Am. J. Respir. Cell Mol. Biol., August 1, 2004; 31(2): 246 - 255. [Abstract] [Full Text] [PDF] J. M. Gibbins Platelet adhesion signalling and the regulation of thrombus formation J. Cell Sci., July 15, 2004; 117(16): 3415 - 3425. 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