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Blood, 1 November 2005, Vol. 106, No. 9, pp. 3028-3034. Prepublished online as a Blood First Edition Paper on July 21, 2005; DOI 10.1182/blood-2005-01-0333.
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY Platelet aggregation induces platelet aggregate stability via SLAM family receptor signalingFrom Portola Pharmaceuticals and Millennium Pharmaceuticals, San Francisco, CA; the Division of Immunology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA; and Millennium Pharmaceuticals, Cambridge, MA.
Platelet aggregation is a dynamic entity, capable of directing its own growth and stability via the activation of signaling cascades that lead to the expression and secretion of various secondary agonists. Here we show that the signaling pathways triggered during platelet aggregation include an intrinsic pro-thrombotic activity mediated by 2 homophilic adhesion molecules, CD84 and CD150 (SLAM [signaling lymphocyte activation molecule]), which are tyrosine phosphorylated in a platelet aggregation–dependent fashion. The 2 CD84/SLAM adapter proteins, SAP (SLAM-associated protein) and EAT-2 (EWS-activated transcript-2), were found in platelets; only SAP, however, was found to immunoprecipitate with tyrosine-phosphorylated SLAM. The immobilized extracellular domain of CD84 promoted microaggregate formation, while SAP-deficient platelets demonstrated defective spreading on immobilized CD84, demonstrating a functional role in platelets for SLAM family interactions. Finally, analysis of SLAM-deficient mice revealed an overall defect in platelet aggregation in vitro and a delayed arterial thrombotic process in vivo. The data indicate that signaling of the adhesion molecules in the SLAM family, activated by proximity during aggregation, further stabilize platelet-platelet interactions in thrombosis.
Platelet aggregates provide the structural framework for the occlusive events causing acute myocardial infarction and thrombotic stroke. Because of this critical role in human pathology, it has become increasingly clear that understanding the mechanisms regulating both thrombus growth and stability will provide insights not only to an important biologic question, but also to new strategies regulating platelet thrombosis. The initial adhesion of platelets to the injured vessel wall is primarily mediated by binding of platelet membrane glycoprotein (GP) Ib-IX-V to von Willebrand factor and of GPVI and integrin  2 1 to collagen. Following platelet adhesion, binding of agonists such as thrombin, adenosine diphosphate (ADP), and thromboxane A2 induce signaling events that activate the "inside-out" receptor function of platelet integrin IIb3, allowing it to bind soluble fibrinogen, resulting in platelet cross-linking. Interestingly, the stability of platelet-platelet contacts appears to be secondary to IIb3-mediated interactions. Several secondary reactions have been identified, including IIb3 "outside-in" signaling, tyrosine phosphorylation of numerous proteins, cytoskeletal rearrangements, certain secretory reactions,1 and signaling via novel platelet proteins. These include CD40L, a tumor necrosis factor (TNF) family member, Gas6, a ligand for the Axl/Sky family of receptor tyrosine kinases, which are both involved in stabilization of platelet-rich thrombi2,3; and the Eph kinases and ephrins, specifically EphA4 and ephrinB1, which on the surface of platelets enhance the binding of IIb3 to immobilized fibrinogen in the presence of physiologic agonists.4,5 The SLAM (signaling lymphocyte activation molecule) family of homophilic adhesion receptors is a subset of the CD2 cell-surface receptor Ig superfamily and is defined by binding to the cytoplasmic adapters SAP and EAT-2, which recognize at least 2 ITSM (Immunoreceptor Tyrosine-based Switch Motifs) sequence motifs: TxYxxV/I (where x is any amino acid) in their cytoplasmic domains.6 SLAM proteins have been shown to be tyrosine phosphorylated during leukocyte activation7 and recruit the SH2 domain–containing adapter proteins SAP or EAT-2 to mediate diverse functional activities. SAP has been shown to be an adapter protein involved in SLAM family signaling, which recruits a src-like kinase to the receptor.8,9 SAP/EAT-2 binding homophilic adhesion molecules of the family include SLAM (CD150, IPO-3), CD84 (Ly9B), Ly108 (NTB-A, SF2000), CRACC (CD2-like receptor-activating cytotoxic cells)/CS-1, and 2B4 (CD244, NAIL). Although CD84 was previously shown to be expressed at relatively higher levels on platelets than on T or B cells,10 a role for SLAM family members in platelet biology has not been described. Here we show, using a proteomics approach, that CD84 becomes tyrosine phosphorylated during platelet aggregation. Of the 2 cytoplasmic tyrosines that become phosphorylated, 1 exists in an ITSM motif, a putative recognition motif for the SAP/EAT-2 adapter proteins. Cross-linking of surface CD84 also induced CD84 tyrosine phosphorylation: immobilized CD84 promoted microaggregate formation in a SAP-dependent manner. We also show that SLAM phosphorylation in platelets was aggregation dependent and that SLAM-deficient platelets did not aggregate to the maximum level observed with wt platelets. In vivo, SLAM–/– mice displayed delayed thrombus formation without effecting tail bleeding times. Based on these observations, we propose the involvement of the SLAM family of receptors as secondary mediators of aggregation. The SLAM family proteins in platelets provides an unexpected mechanism that synergizes with integrins to mediate platelet thrombosis.
Purification of platelet RNA and expression of SLAM family members in platelets
Approval was obtained from Millenium Pharmaceutical's Western Institutional Review Board for these studies. Informed consent was provided according to the Declaration of Helsinki. Platelets from a healthy volunteer were collected by apheresis and included a filtration step that removed lymphocyte contaminants. These instruments typically give a platelet concentrate with less than 0.01% leukocyte contamination. We confirmed this by polymerase chain reaction (PCR) analysis of RNA prepared from these platelet concentrates and found leukocyte RNA contaminants were undetectable in our preparation. This procedure yielded approximately 250 mL platelets ( Platelet preparation for aggregation and spreading Blood was drawn from C57Bl/6 (Charles River Laboratory, Germantown, MD), C57Bl/6 SAP–/–11 (backcrossed to C57Bl/6 mice 6 times), C57Bl/6 SLAM–/–12 (backcrossed to C57Bl/6 mice 7 times), and C57Bl/6 diYF1 mice. Platelets from human and mouse blood were prepared as previously described.13 Both human and murine washed platelets were resuspended in Tyrode-HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer with 1 mM CaCl2 and MgCl2 at a cell density of 1 to 3 x 108 cells/mL. Platelet aggregation Aggregations were measured in a Chronolog lumi-aggregometer. Aggregations of washed human platelets (3 x 108 cells/mL) were initiated with 0.5 U thrombin (Haematologic Technologies, Essex Junction, VT), 4 to 10 µg/mL collagen (Chronolog, Haverton, PA), or 5 µM-human TRAP (thrombin receptor activating peptide, SFLLRN, SynPep) and inhibited in the presence of 2 µM eptifibatide (Integrilin, Millennium Pharmaceuticals). Aggregation of murine platelet-rich plasma (PRP) was initiated with 1 to 2.5 mM mTRAP (murine thrombin receptor activating peptide, AYPGKF, SynPep), 1 to 10 µg/mL collagen or 0.5 to 10 µM ADP, and inhibited in the presence of 5 or 10 µM ML00341819.14 Platelet lysate preparation Platelets were lysed in an equal volume of 2 x lysis buffer (20 mM Tris[tris(hydroxymethyl)aminomethane]-HCl, pH 8, 2% Triton X-100, 4 mM EDTA [ethylenediaminetetraacetic acid], 250 mM NaCl, 20% glycerol, 1 mM phenylmethylsulfonyl fluoride [PMSF], 2 mM Na2VO4, 2 µg/mL leupeptin, and 2 µg/mL aprotinin), and incubated on ice for 30 minutes. Samples were sonicated for 40 seconds and the insoluble fraction was removed by centrifugation at 14 000g for 20 minutes at 4°C. Phosphopeptide identification by in-gel digestion and mass spectrometry
The solubilized TRAP aggregated platelet proteins were incubated with Flow cytometry analysis
Human washed platelets (1 x 106) were left resting or activated with 5 µM TRAP for 5 minutes at 37°C without stirring. Fixed unpermeabilized platelets were stained with Immunoprecipitation and cross-linking of surface CD84
Immunoprecipitations were performed with protein G beads and 2.5 µg/mL CD84 extracellular domain Glu-fusion protein generation An expression construct containing the cDNA corresponding to the peptide epitope EYMPME (Glu-Glu tag) and the extracellular domain of CD84 (CD84-Ec) was cloned into the mammalian expression vector pSecTag2/Hygro A (Invitrogen, Carlsbad, CA) by PCR. Supernatants from 293T cells were purified over a Glu-antibody column (Covance Research Products, Denver, PA).16 Purity of the protein was assessed by SDS-PAGE and Coomassie staining. Platelet spreading For spreading experiments, glass coverslips were incubated with 50 µg/mL CD84-Ec for 2 hours at 37°C. Following washing, the coverslips were blocked with 0.5% fatty acid–free bovine serum albumin (BSA) (Sigma, St Louis, MO)/phosphate-buffered saline (PBS) for 1 hour at room temperature. Washed human (0.5-1 x 108 cells/mL) platelets were adhered to the coated coverslips for 30 minutes at 37°C. Similarly, washed murine platelets (0.5-1 x 108 cells/mL) were adhered in the absence or presence of 1 µM ADP added immediately prior to plating. When required, platelets were treated with the P2Y1 antagonist (100 µM MRS2179) for 10 minutes prior to plating. Nonadhered platelets were removed by washing, and the adhered platelets were fixed, permeabilized, and stained with tetramethyl-rhodamine-5(and 6)-isothiocyanate (TRITC)–conjugated phalloidin. Platelets were viewed on a Nikon Eclipse E1000 microscope with 40 x/0.95 NA or 100 x/1.4 NA objectives (Nikon, Tokyo, Japan). Images were collected with a Hamamatsu camera (Hamamatsu City, Japan), and imaging was performed using Simple PCI (Technical Instruments, Cranberry Township, PA). Photo images displayed are representative of 3 different images from at least 3 experiments. Arterial thrombosis Ferric chloride injury model was performed as previously described13 (Figure 6). Briefly, anaesthetized mice were injected with rhodamine 6G to fluorescently label platelets in vivo. Mesenteric arteries were injured with 10% ferric chloride, and the thrombotic process was recorded in real time for 40 minutes or until vessel occlusion occurred. Statistical analyses All comparisons were done using unpaired Student t test. P values below .05 were considered significant.
Identification of platelet CD84
To identify novel membrane receptors that become tyrosine phosphorylated during platelet aggregation, we used a proteomics approach. In preliminary experiments, we confirmed that many platelet proteins become tyrosine phosphorylated during aggregation. Washed platelets were induced to aggregate by TRAP-induced stimulation, and the platelet aggregates were detergent solubilized and the tyrosine-phosphorylated proteins were captured on an
Aggregation- and immuneclustering-induced tyrosine phosphorylation of CD84
CD84 contains 2 extracellular IgG motifs and 4 cytoplasmic tyrosines: 273Y, 275Y, 290Y, and 310Y. Two of these are embedded within ITSM (TxYxxV/I where x is any amino acid) motifs, 271TI273YTYI276 and 308TV310YSEV313. The LC/MS/MS data presented in Table 1 indicated that 290Y and 310Y were phosphorylated in platelet aggregates, demonstrating that one of the CD84 ITSM motifs becomes tyrosine phosphorylated as a consequence of platelet aggregation. Confirmation that platelet aggregation induced CD84 tyrosine phosphorylation was obtained by Western blotting of CD84-immunocomplexes isolated from resting or aggregated platelets with an
CD84 signaling in intact platelets was further investigated by measuring CD84 tyrosine phosphorylation following receptor ligation with an -CD84-IgG or nonspecific IgG. Cross-linking of bound -CD84 IgG, but not nonspecific IgG, together with either whole -mouse IgG or its Fab'2 fragment, resulted in CD84 tyrosine phosphorylation (Figure 1C, upper panel). As -CD84 IgG by itself was unable to induce phosphorylation, antibody-induced CD84 tyrosine phosphorylation was likely to have occurred specifically through ligation and clustering of surface CD84 and not the Fc portion of the activating IgGs. CD84 extracellular domain induces platelet spreading through homophilic interactions
It has previously been established that CD84 is an adhesion receptor and functions by homophilic interactions and by clustering.19 These data include the demonstration that a recombinant protein expressing the extracellular domain of CD84 binds to platelets, and this binding is blocked by an Identification of additional SLAM family members and adapter proteins in human platelets
To determine if other SLAM family members and the SLAM adapter protein SAP were expressed in platelets, we performed high throughput molecular profiling of RNA isolated from double-phoresed human platelets using custom arrays, representing 25 000 genes. The sequences for the genes represented on the custom arrays were derived both from sequencing of platelet and human umbilical vein endothelial cell (HUVEC) cDNA libraries as well as all the nonoverlapping human genes present in the public gene database. Analysis of the data obtained from these custom assays revealed that RNA from platelets hybridized to probes for the following members of the SLAM family: CD84, SLAM, CD48 and 2B4, with no signal detected for 2 members of the CD2 family of cell-surface receptors that are expressed in leukocytes: CD58 or BLAME (B-lymphocyte activator macrophage expressed). Comparing the relative expression of these SLAM family members in platelets to peripheral blood leukocytes (PBLs) showed that CD84 was expressed at a relative level of 1.16, SLAM at 0.25, CD48 at 0.78, and 2B4 at 0.11. A strong signal for the SLAM adapter protein SAP also was detected (relative expression to PBLs was 0.38). Since in platelets SLAM message was as abundant as CD84 and since
Expression of SLAM, SAP, and EAT-2 and aggregation-induced tyrosine phosphorylation of SLAM in murine platelets
In an effort to study SLAM family function and SLAM family signaling in platelets, we turned to a model where both immunoreagents and mouse models were available. The expression of SLAM, SAP, and EAT-2 in mouse platelets was confirmed by Western blotting (Figure 4A-B). To determine if SLAM was also tyrosine phosphorylated upon aggregation, SLAM immunoprecipitates isolated from resting and activated wt (wild-type) murine platelets were probed with an Contribution of SLAM and SAP to stabilization of platelet aggregates in vitro and thrombus formation in vivo SLAM–/– and SAP–/– platelets were analyzed by agonist-induced aggregation reactions (Figure 5). Compared to wt platelets, the aggregation response of SLAM–/– platelets was blunted when stimulated with low (1 mM, data not shown) or high (2.5 mM) doses of mTRAP (Figure 5).22 A similar defect was observed when platelets were stimulated with collagen; however, response to ADP was normal. SAP–/– platelet aggregations were not impaired at low (data not shown) or high concentrations of mTRAP, collagen, or ADP (Figure 5iv-5vi). The roles for SLAM and SAP were further examined in vivo using a thrombosis model (Figure 6A) and by tail-bleeding measurements (Figure 6B). In vivo platelet function was assessed by real-time continuous analysis of the thrombotic profile post ferric-chloride injury of the mesenteric arteries of 3-week-old mice (Figure 6A). Female SLAM–/– mice revealed a delay in the time for appearance of first thrombus and occlusion of the vessel (time to occlusion (± SEM, n = 7) was 11.78 ± 0.48 minutes (female wt) and 21.73 ± 2.4 (female SLAM–/–, P vs wt < .01). Compared to wt mice, there also was an increase in embolization of small thrombi during the thrombotic process. Unlike the in vitro aggregation phenotype, the thrombosis defect was sex specific and was apparent only in females. Factors within the vasculature of male mice most likely differ from that of female mice to compensate for this difference. Both SLAM–/– and SAP–/– mice demonstrated normal tail-bleeding times (Figure 6B).
CD84-induced spreading is dependent on the adapter molecule SAP
Previous studies in leukocytes have shown that CD84 signaling partly depends on the binding of SAP and EAT-2 to the tyrosinephosphorylated ITSM motif. Since this motif becomes tyrosine phosphorylated by aggregation, we next asked whether CD84 signaling depended on those proteins. Because the adapter molecule SAP is expressed in murine platelets (Figure 4), we determined whether a SAP-dependent signaling pathway was involved in CD84-induced spreading using platelets from SAP–/– mice. In the setting where platelet adhesion was stimulated by CD84-Ec, SAP–/– platelets displayed reduced spreading compared to wt in the absence or presence of ADP. On the other hand, SAP–/– platelets showed normal spreading on immobilized fibrinogen (Figure 7). To confirm that
It is well established that the platelet integrin IIb3 is responsible for the primary interaction of platelets during thrombosis and hemostasis and that soluble stimuli (eg, CD40L, thromboxane A2, ADP, Gas6) are released to support this process. Published data, including those from our laboratory, have been based on the assumption that aggregate-induced signaling is induced by "outside-in" signaling through IIb3. The signaling reactions though additional receptors induced by platelet-platelet contact, particularly under conditions induced by arterial shear, have not been previously described. The data presented in this paper have led us to the identification of additional aggregation-induced signaling receptors that are induced by platelet-platelet contact. We show that the 2 members of the SLAM family of homophilic adhesion receptors, which operate at the interface of antigen-presenting and lymphoid cells in immune responses, are not only expressed on the platelet surface but also tyrosine phosphorylated in an aggregation-dependent manner, contributing to cohesion of platelet-platelet contacts, and that ligation of these receptors will induce platelet activation.
Previous studies have shown that SLAM on the surface of T cells interacts with SLAM on the surface of antigen-presenting cells, thereby inducing interleukin-2 (IL-2) and IFN-
In platelets, both SLAM and CD84 are tyrosine phosphorylated in an aggregation-dependent manner; however, there seem to be differences in SLAM/CD84 signaling in platelets compared to lymphocytes. First, SLAM tyrosine phosphorylation in murine platelets does not require SAP, a conclusion based on the similar SLAM tyrosine phosphorylation levels observed upon aggregation of wt and SAP–/– platelets (Figure 4B). Secondly, EAT-2, which also binds to the same SLAM tyrosine-phosphorylated residues as SAP in lymphocytes, is present in murine platelets and does not bind to platelet phosphorylated SLAM (Figure 4B). An additional difference is that tyrosine at position 290, which is outside the ITSM consensus sites on CD84 (Table 1), is tyrosine phosphorylated in platelets but has not been detected in lymphocytes. While we cannot rule out the dependency of platelet SLAM and CD84 signaling on SAP and EAT-2, clearly there are differences in signaling between lymphocytes and platelets.
Recently it has been reported24 that binding of the soluble ligand Gas6 to its receptors can activate downstream signaling pathways, leading to tyrosine phosphorylation of the
We thank Dr Pablo Engle for helpful discussions in the preparation of the manuscript.
Submitted January 25, 2005; accepted June 10, 2005.
Prepublished online as Blood First Edition Paper, July 21, 2005; DOI 10.1182/blood-2005-01-0333.
An Inside Blood analysis of this article appears in the front of this issue.
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: David R. Phillips, Portola Pharmaceuticals, 270 E Grand Ave, Suite 22, San Francisco, CA 94080; e-mail: dphillips{at}portola.com.
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Abstract
Introduction
1 x 109 platelets/mL). Platelets were diluted 1:4 with CGS (13 mM sodium citrate, 30 mM glucose, 0.12 M sodium chloride) containing 0.1 µg/mL PGI2. Platelets were pelleted by spinning for 20 minutes at 2000g in a Beckman Coulter Allegra 6 centrifuge at room temperature. The supernatant was removed, and the platelet pellet was resuspended in RNA-STAT-60 (Tel-Test) using 1 mL RNA-STAT-60 per 2.9 x 108 platelets, and immediately vortexed to facilitate platelet lysis. The platelet lysate was further processed according to RNA-STAT-60 protocol specifications. Total RNA was then treated with RNA Easy (Qiagen, Valencia, CA) for further purification. Five micrograms of DNAse-treated platelet RNA was converted to cRNA, fragmented, and hybridized to Millennium Pharmaceuticals (South San Francisco, CA) cardiovascular custom array per the manufacturers' suggested protocol (Affymetrix, Santa Clara, CA). Probe sets for the following genes were designed based on their published sequences: CD84 (NM_003874
secretion
