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Keywords:

  • ADAM10;
  • calpain;
  • CD84;
  • platelets;
  • shedding

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Addendum
  8. Acknowledgements
  9. Disclosure of Conflict of Interest
  10. References
  11. Supporting Information

Hofmann S, Vögtle T, Bender M, Rose-John S, Nieswandt B. The SLAM family member CD84 is regulated by ADAM10 and calpain in platelets. J Thromb Haemost 2012; 10: 2581–92.

Summary.  Background and objective: Ectodomain shedding is a major mechanism to modulate platelet receptor signaling and to downregulate platelet reactivity. Proteins of the a disintegrin and metalloproteinase (ADAM) family are implicated in the shedding of various platelet receptors. The signaling lymphocyte activation molecule (SLAM) family receptor CD84 is highly expressed in platelets and immune cells, but its role in platelet physiology is not well explored. Because of its ability to form homodimers, CD84 has been suggested to mediate contact-dependent signaling and contribute to thrombus stability. However, nothing is known about the cellular regulation of CD84. Methods: We studied the regulation of CD84 in murine platelets by biochemical approaches and use of three different genetically modified mouse lines. Regulation of CD84 in human platelets was studied using inhibitors and biochemical approaches. Results: We show that CD84 is cleaved from the surface of human and murine platelets in response to different shedding inducing agents and platelet receptor agonists. CD84 downregulation occurs through ectodomain-shedding and intracellular cleavage. Studies in transgenic mice identified ADAM10 as the principal sheddase responsible for CD84 cleavage, whereas ADAM17 was dispensable. Western blot analyses revealed calpain-mediated intracellular cleavage of the CD84 C-terminus, occurring simultaneously with, but independently of, ectodomain shedding. Furthermore, analysis of plasma and serum samples from transgenic mice demonstrated that CD84 is constitutively shed from the platelet surface by ADAM10 in vivo.Conclusions: These results reveal a dual regulation mechanism for platelet CD84 by simultaneous extra- and intracellular cleavage that may modulate platelet-platelet and platelet-immune cell interactions.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Addendum
  8. Acknowledgements
  9. Disclosure of Conflict of Interest
  10. References
  11. Supporting Information

Platelet aggregation at sites of vascular damage is crucial for normal hemostasis, but in diseased vessels it can lead to occlusive thrombus formation causing life threatening disease states such as myocardial infarction or ischemic stroke [1].

Activated integrin αIIbβ3 facilitates the formation of platelet-platelet bridges by binding to multivalent adhesive molecules such as fibrinogen or von Willebrand factor (VWF), which is a prerequisite for aggregate formation. Outside-in signaling through the integrin further enhances aggregation [2], a process involving tyrosine phosphorylation of the β3 subunit [3], but stable platelet aggregation requires the coordinated interaction of additional receptors on the platelet surface [4]. The close proximity between platelets in aggregates enables contact-dependent signaling involving interaction and phosphorylation of receptors such as Eph kinases with ephrins as their ligands [5], Junctional adhesion molecules (JAMs) [6], and signaling lymphocyte activation molecule (SLAM, CD150) [7]. Another cell-surface receptor that has been proposed to be involved in the regulation of platelet-platelet interactions is the SLAM family member CD84, which undergoes homophilic interactions subsequent to integrin αIIbβ3-mediated aggregation [7].

CD84 is a type I transmembrane glycoprotein that is expressed in platelets and different immune cell populations [8,9]. It belongs to the Ig superfamily of cell surface receptors, potentially binding to the cytoplasmic adaptors SAP (SH2D1A) and EAT-2 (SH2D1B) via their immunoreceptor tyrosine switch motif (ITSM) [10,11]. SLAM family receptors share a common N-terminal ectodomain structure containing a membrane-proximal Ig constant domain and a membrane-distal Ig variable domain. As on other SLAM family members, the N-terminal Ig variable domain mediates homophilic interactions of CD84 in humans and mice [12,13]. CD84 has a calculated mass of 39 kDa but shows a higher apparent molecular weight in SDS page, as it is highly glycosylated [14,15]. Human and mouse CD84 share high homology and similar domain organization (Uniprot accession no. Q9UIB8 and Q18PI6). Human CD84 is a 328 amino acid (aa) protein with the first 21 aa comprising a cleaved signal peptide. The extracellular N-terminus is followed by a 25 aa transmembrane region and an 83 aa C-terminal intracytoplasmic region [14].

In T cells, CD84 has been shown to function as a costimulatory molecule [12]. Tyrosine phosphorylation of the C-terminus of CD84 involves the Src kinase Lck and determines the recruitment of SAP and EAT-2 [16]. Mice deficient in CD84, which were recently described, show no overt phenotype, but display a specific defect in germinal center formation and T cell:B cell interaction [17]. Even though CD84 is highly expressed in platelets [15] and tyrosine phosphorylation of the cytoplasmic tail in response to aggregation has been demonstrated [7], the role of this receptor in platelet function is unclear.

Platelet receptors can be downregulated from the platelet surface by internalization [18,19] or ectodomain shedding and the latter mechanism has been described for a number of major receptors, including GPIbα [20], GPVI [21,22], GPV [23,24], Semaphorin 4D [25], P-Selectin [19], JAM-A [26] or CD40-L [27]. However, Fong et al. [28] recently provided evidence that many more surface proteins, including CD84, might be proteolytically downregulated in activated platelets, but the underlying mechanisms were not addressed in detail in that study.

Members of the a disintegrin and metalloproteinase (ADAM) family have been identified to be involved in the proteolysis of some prominent platelet receptors, with ADAM17 mediating the cleavage of GPIbα [20], whereas shedding of GPV or GPVI can occur through either ADAM10 or ADAM17, depending on the shedding-inducing stimulus [29,30]. A role for both metalloproteinases was also shown for JAM-A shedding [26].

Another mechanism to regulate platelet receptor signaling is proteolytic cleavage of the receptor by intracellular proteases such as calpain [31]. Calpain is a cysteinyl protease that is in part regulated by intracellular calcium levels and cleaves a number of proteins, many of them involved in the regulation of the cytoskeleton [32]. Different studies have demonstrated that calpain cleaves the intracellular part of β3 integrin [33], FcγRIIa [34] and PECAM-1 [35]. Platelets express calpain-1, which accounts for ∼80% of calpain protease activity in these cells, and calpain-2, mediating ∼20% of calpain protease activity [36]. Deficiency of calpain-1 in platelets resulted in impaired aggregation and clot retraction, which could be attributed to enhanced PTP1B activity, but tail bleeding times of calpain-1-deficient mice were unaltered [36,37]. More recently, Kuchay et al. [38] discovered that calpain-1 regulates platelet spreading on collagen and fibrinogen through Rho GTPases. Importantly, calpain activity can be induced under the same conditions that activate metalloproteinases in platelets (e.g. in response to calmodulin inhibitors) [34,39], suggesting that intra- and extracellular cleavage events might be simultaneously operative in the downregulation of receptor signaling in platelets.

We studied the regulation of CD84 in platelets by biochemical and genetic approaches. We report that the receptor is proteolytically regulated by calpain and ADAM10, but not ADAM17, in mouse platelets in vitro and in vivo.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Addendum
  8. Acknowledgements
  9. Disclosure of Conflict of Interest
  10. References
  11. Supporting Information

Mice

Animal studies were approved by the local authorities (Bezirksregierung Unterfranken). Generation of Adam10fl/fl mice has been described previously [30]. Adam10fl/fl mice were crossed with mice carrying the Cre recombinase under the platelet factor 4 (PF4) promoter [40]. Adam10fl/fl, PF4-Cre mice are referred to as Adam10−/−. Generation of Cd84−/− mice will be described elsewhere (Sebastian Hofmann, Timo Vögtle and Bernhard Nieswandt, manuscript in preparation). Adam17ex/ex mice were previously described [41]. To restrict lack of ADAM17 to the hematopoietic system, we used bone marrow chimeric (BMc) Adam17ex/ex [30].

Reagents

3,3,5,5-tetramethylbenzidine (Becton Dickinson, Heidelberg, Germany), Nonidet P-40, Thrombin (Roche Diagnostics, Mannheim, Germany), ECL solution (GE Healthcare, Freiburg, Germany), apyrase (grade 3), Carbonyl cyanide m-chlorophenylhydrazone (Sigma, Schnelldorf, Germany), W7, N-ethylmaleimide (NEM), GM6001, Calpeptin, MDL28170, ALLN (Calbiochem, Bad Soden, Germany), horseradish peroxidase-conjugated streptavidin (Dianova, Hamburg, Germany) and Convulxin (Enzo, Lörrach, Germany) were used. Collagen-related peptide was generated as described previously [42]. Rhodocytin was isolated as described [43]. The following antibodies were purchased: Anti-murine CD84 rabbit polyclonal antibody (Santa Cruz Biotechnology, Heidelberg, Germany), biotinylated anti-murine CD84 monoclonal antibody mCD84.7 (Biolegend, Fell, Germany), biotinylated anti-human CD84 2G7 (eBioscience, Frankfurt, Germany) and anti-human CD84-FITC (Miltenyi, Bergisch Gladbach, Germany). Anti-murine CD84 monoclonal antibody JER1 and other antibodies were generated and modified in our laboratories as described [44]. The integrin αIIbβ3 blocking antibody JON/A has been described [45]. Anti-human CD84 monoclonal antibody MAX.3 was a kind gift from R. Andreesen, Regensburg [15].

Generation of bone marrow chimeric mice

Recipient C57BL/6 mice of an age between 5 and 6 weeks were lethally irradiated with 10 Gy. Femur and tibia of donor mice were prepared. Bone marrow was flushed and at least 4 million cells were intravenously injected per recipient mouse.

Determination of platelet count and surface protein expression

Platelet counts were determined in a Sysmex KX-21N cell counter (Sysmex, Norderstedt, Germany). To measure surface protein expression, platelets were stained for 15 min with saturating amounts of fluorophore-conjugated antibodies and immediately analyzed on a FACSCalibur (Becton Dickinson).

Platelet preparation

Mouse platelets  Mice were bled under isofluran anesthesia from the retro-orbital plexus. Blood was collected in TBS containing 20 U mL−1 heparin, and washed platelets were prepared as described [30].

Human platelets  Blood (nine volumes) from healthy volunteers was collected in sodium citrate (one volume) and PRP was obtained by centrifugation at 300 × g for 20 min. PRP was centrifuged at 380 × g in the presence of prostacyclin (0.1 μg mL−1), apyrase (0.02 U mL−1) and 2 mm ethylenedi-aminetetraacetic acid (EDTA) for 20 min at RT. After two washing steps, pelleted platelets were resuspended in modified Tyrode-HEPES buffer [30] containing 2 mm CaCl2 and 0.02 U mL−1 apyrase.

Shedding of CD84 from the platelet surface

Washed platelets resuspended at a concentration of ∼3 × 108 platelets mL−1 in Tyrodes-HEPES buffer containing 2 mm CaCl2 and 0.02 U mL−1 apyrase were treated in the presence or absence of the broad range metalloproteinase inhibitor GM6001 (100 μm, 15 min, 37 °C) for 1 h with CCCP (100 μm), W7 (150 μm) or for 20 min with NEM (2 mm) at 37 °C and immediately analyzed on a FACSCalibur. Where indicated, platelets were pretreated with calpeptin (5 μg mL−1), ALLN (50 μm) or MDL28170 (50 μm) for 15 min at 37 °C. Alternatively, platelets were treated for 1 h with convulxin (1 μg mL−1), CRP (40 μg mL−1), rhodocytin (2 μg mL−1), thrombin (0.5 U mL−1) or PAR4 peptide (NH2-AYPGKF; 1–4 mm). Where indicated, aggregation of mouse platelets was inhibited with saturating concentrations of the integrin αIIbβ3 blocking antibody JON/A F(ab)2 [45].

Detection of cleaved soluble CD84 by ELISA

Soluble mouse CD84  Washed platelets were resuspended at a concentration of ∼3 × 108 platelets mL−1 in Tyrodes-HEPES buffer containing 2 mm CaCl2 and 0.02 U mL−1 apyrase. To induce CD84 shedding, platelets were treated as described in the preceding paragraph. Platelets were centrifuged and supernatants were incubated on JER1-coated (10 μg mL−1) ELISA plates for 2 h at 37 °C. After extensive washing, plates were incubated with biotinylated antibody mCD84.7 (10 μg mL−1) for 1 h at 37 °C. After extensive washing, plates were incubated with HRP-labeled streptavidin for 45 min at 37 °C and developed using 3,3,5,5-tetramethylbenzidine (TMB). The reaction was stopped by addition of 2N H2SO4 and absorbance at 450 nm was recorded on a Multiskan (Thermo Scientific, Dreieich, Germany). Alternatively, plasma or serum samples were applied to JER1-coated ELISA plates.

Soluble human CD84  Human platelet samples were treated as described above for mouse platelets. MAX.3 (10 μg mL−1) was used as coating antibody and biotinylated 2G7 (5 μg mL−1) as secondary antibody.

Western blotting

Platelets were lysed in 4x Laemmli buffer containing 1% Nonidet P-40. Proteins were separated by SDS-PAGE and blotted onto polyvinylidene difluoride membranes. After blocking with 5% fat-free milk in TBST, the membrane was incubated with either polyclonal anti-CD84 antibody M-130 overnight at 4 °C or peroxidase-conjugated monoclonal antibody JER1. As secondary antibody for polyclonal M-130, goat anti-rabbit IgG HRP (1 h at room temperature, DAKO, Hamburg, Germany) was used. Bound antibodies were visualized by ECL.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Addendum
  8. Acknowledgements
  9. Disclosure of Conflict of Interest
  10. References
  11. Supporting Information

Ectodomain shedding of CD84 by metalloproteinases in human and murine platelets

Recently, Fong et al. [28] identified soluble CD84 (sCD84) in the supernatant of activated human platelets in a mass spectrometric approach, suggesting that the receptor can be downregulated from the platelet surface by proteolytic cleavage. To study this process in more detail, we stimulated washed human platelets with thrombin or the GPVI agonist collagen related peptide (CRP) and measured surface expression of CD84 by flow cytometry (Fig. 1A). Indeed, substantial downregulation of CD84 surface levels in response to CRP and modest, non-significant, downregulation in response to thrombin were detected, and these effects were inhibited in the presence of the broad range metalloproteinase inhibitor, GM6001. These data confirmed that CD84 surface expression in human platelets is downregulated in response to agonist stimulation in a metalloproteinase-dependent manner.

image

Figure 1.  Agonist-induced shedding of CD84 from human platelets is metalloproteinase-dependent. Washed human platelets were incubated with CRP (40 μg mL−1) or thrombin (0.5 U mL−1) for 1 h at 37 °C in the presence or absence of the broad range metalloproteinase inhibitor GM6001 (100 μm). (A) Platelets were stained with the FITC-labeled CD84 antibody for 15 min and analyzed directly by flow cytometry. (B) Alternatively, supernatants were applied on a MAX.3-coated ELISA plate. CD84 was detected using 2G7-biotin as secondary antibody, followed by HRP-conjugated streptavidin. Results of all experiments are mean ± SD (n = 3 individuals, representative of two individual experiments), *P < 0.05, **P < 0.01, ***P < 0.001.

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Total surface levels of CD84, as measured by flow cytometry, may also be influenced by receptor internalization or exposure of additional CD84 proteins originating from intracellular pools. To circumvent this limitation, we established an ELISA system, using two monoclonal antibodies directed against distinct epitopes on the extracellular domain of the receptor to directly measure soluble human CD84 in the platelet supernatant. As shown in Fig. 1(B), marked release of soluble human CD84 from thrombin- and CRP-stimulated human platelets was confirmed by this approach. Similar results were obtained with the snake venom convulxin, which activates GPVI signaling, and also with another snake venom, rhodocytin, which activates the hemITAM receptor CLEC-2 (data not shown).

Next, we analyzed CD84 regulation in murine platelets. In a first set of experiments, we stimulated washed mouse platelets with thrombin, CRP, convulxin (CVX) or the CLEC-2 activating snake venom protein rhodocytin (RC), and determined release of sCD84 with a newly established ELISA system, designed to detect the extracellular domain of mouse CD84 (sCD84). High levels of sCD84 were measured in the supernatant of wild-type platelets in response to stimulation with each of these agonists, compared with the untreated control (Fig. 2A). In contrast, virtually no sCD84 was detected when the experiments were performed in the presence of GM6001, strongly suggesting that CD84 cleavage was mediated by metalloproteinases. The ELISA yielded only background signals when the same experiments were performed with platelets from Cd84−/− mice, confirming the specificity of the system. When platelet aggregation was blocked by inhibition of integrin αIIbβ3 with F(ab)2 fragments of the JON/A antibody [45], less sCD84 was detected after CRP and thrombin stimulation. This indicated that cleavage of CD84 was, at least in part, dependent on platelet aggregation (Fig. 2B). Of note, addition of JON/A F(ab)2 after the agonist incubation period did not reduce the sCD84 signal in ELISA, excluding that the F(ab)2 interfered with the signal (data not shown).

image

Figure 2.  Metalloproteinase-dependent shedding of CD84 from mouse platelets. (A) Washed wild-type (control) mouse platelets were incubated with CVX (1 μg mL−1), CRP (40 μg mL−1), rhodocytin (2 μg mL−1) or thrombin (0.5 U mL−1) for 1 h at 37 °C in the presence or absence of the broad range metalloproteinase inhibitor GM6001 (100 μm). Supernatants were applied on a JER1-coated ELISA plate. CD84 was detected using mCD84.7-biotin as secondary antibody, followed by HRP-conjugated streptavidin. Platelet supernatants from Cd84−/− mice yielded either low background signals, or were not detectable (n.d.) (B) Washed mouse platelets were incubated with CRP (40 μg mL−1) or thrombin (0.1 U mL−1) in the presence or absence of 25 μg mL−1 JON/A F(ab)2 and ELISA was performed as described above. (C) Washed mouse platelets were incubated with CCCP (100 μm) or W7 (150 μm) for 1 h at 37 °C or NEM (2 mm) for 20 min at 37 °C in the presence or absence of the broad range metalloproteinase inhibitor GM6001. ELISA was performed as described above. DMSO treatment served as control. Results of all experiments are mean ± SD (n = 4 mice per group, representative of three individual experiments).

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To further assess the mechanisms underlying CD84 ectodomain shedding, we treated washed mouse platelets with different agents that are known to induce shedding of multiple platelet membrane receptors by distinct mechanisms [39] and measured CD84 cleavage from the platelet surface by sCD84 ELISA. The calmodulin inhibitor W7 induces the dissociation of calmodulin from receptors, thereby facilitating ectodomain shedding (e.g. of GPIbα [29,30] and GPV [23] by ADAM17 and GPVI by ADAM10 [29,30]). N-ethylmaleimide (NEM) is a thiol-modifying reagent that induces shedding by directly activating ADAM10 and ADAM17 independently of platelet activation [39]. Both reagents induced marked ectodomain shedding of CD84 as revealed by detection of high levels of sCD84 in the platelet supernatant compared with the untreated control and this effect was virtually abolished in the presence of GM6001 (Fig. 2C). Carbonyl cyanide m-chlorophenylhydrazone (CCCP) induces mitochondrial injury by uncoupling oxidative phosphorylation and triggers receptor shedding mainly in an ADAM17-dependent manner [20,30]. Compared with W7 and NEM, CCCP induced only a mild GM6001-sensitive increase in sCD84 in the platelet supernatant. Similar results were obtained with human platelets (Fig. S1).

Calpain and metalloproteinases cleave CD84

To further analyze the mechanisms underlying CD84 regulation in platelets, we assessed CD84 processing in response to shedding-inducing agents by Western blotting using two different antibodies: JER1 (anti-CD84N-term) and M-130, which was raised against the intracellular C-terminal part of CD84 (anti-CD84C-term). The band of the full-length CD84 protein appeared between 55 and 72 kDa under non-reducing conditions as previously reported by others [7,15]. While in unstimulated platelets, M-130 detected only the full-length protein, an additional band at a size of approximately 15 kDa appeared in NEM-treated platelets (Fig. 3A, lower left). As simultaneously the band intensity of the full-length protein decreased, we hypothesized that this 15 kDa band represents the C-terminal remnant of CD84 that is generated by shedding of the receptor ectodomain. This assumption was confirmed by the finding that GM6001 abrogated the appearance of this additional band in the lysate of NEM-treated platelets (Fig. 3A, lower left).

image

Figure 3.  Dual regulation of CD84 by intra- and extracellular cleavage. (A) Washed platelets from wild-type mice were pre-incubated at 37 °C for 15 min in the presence or absence of the broad range metalloproteinase inhibitor GM6001 (100 μm) and/or calpeptin (5 μg mL−1), an inhibitor of calpain. Afterwards, shedding was induced with CCCP (100 μm, 1 h), W7 (150 μm, 1 h) and NEM (2 mm, 20 min). DMSO or buffer served as control. CD84 was detected by Western blotting with anti-CD84 C-term antibody M-130 and anti-CD84 N-term antibody JER-1. GPIIIa was used as a loading control. (B) Washed platelets from wild-type mice were pre-incubated at 37 °C for 15 min in the presence or absence of calpeptin (5 μg mL−1). Shedding was induced with W7 (150 μm, 1 h) or NEM (2 mm, 20 min). Soluble CD84 was detected by ELISA. Results are mean ± SD (n = 4 mice per group, representative of three individual experiments).

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In Fig. 2(B) we have shown that W7 and, to a lower extent, also CCCP can trigger GM6001-sensitive release of sCD84 into the supernatant. Thus, it was surprising that the C-terminal remnant could not be detected in W7 and CCCP-treated platelets, albeit the band of the full-length protein was clearly reduced in intensity compared with the untreated control, or even absent after W7 treatment (Fig. 3A, lower left). One possible explanation was that CD84, in addition to ectodomain shedding, may be cleaved in the C-terminus and that this cleavage interferes with binding of the anti-CD84C-term antibody, M-130. This assumption was also supported by the fact that a shift in molecular weight of CD84 was detectable with the anti-CD84N-term antibody in the lysates of platelets treated with W7 or CCCP. Moreover, this shift, as well as the lack of binding of the anti-CD84C-term antibody, was not influenced by GM6001.

One candidate enzyme for mediating this intracellular cleavage is calpain, because shedding by metalloproteinases often occurs concomitantly with activation of calpains [39] and calmodulin-binding proteins are frequently substrates for calpains [46]. This hypothesis was further supported by the analysis of the CD84 C-terminus with an online prediction tool (http://calpain.org) [47], which identified a potential cleavage site for calpain in murine CD84, between amino acids 268 and 272 (sequence: V-S-R-N-A), as well as in human CD84.

To test whether calpain indeed mediated the cleavage of the CD84 C-terminus, we used the calpain-inhibitor calpeptin. Strikingly, pre-incubation of platelets with calpeptin abolished the shift in the molecular weight of CD84 seen in W7 and CCCP-treated platelets with the anti-CD84N-term antibody, JER1, (Fig. 3A, upper right) and also allowed the detection of the C-terminal remnant with the anti-CD84C-term antibody, M-130 (Fig. 3A, lower right). The band intensity for this remnant was strong after W7 and NEM treatment, and weak for CCCP-treated platelets. Importantly, all bands detected by M-130 and JER1 were specific as no signal was obtained when the experiments were performed with Cd84−/− platelets (Fig. S2). These results were in agreement with the results obtained with our sCD84 ELISA system. When platelets were pre-incubated with calpeptin and GM6001, the appearance of the 15 kDa remnant (anti-CD84C-term) as well as the shift in molecular weight and the decrease in band intensity detected by anti-CD84N-term were abolished, showing additive effects of the two inhibitors (Fig. 3A, right). To corroborate our findings using calpeptin and to exclude that its effects on the shift of the CD84 full-length band in Western blotting with the anti-CD84N-term antibody were caused by inhibition of other enzymes than calpain [48], we also tested two other membrane-permeable calpain inhibitors, MDL28170 and ALLN, on CD84 cleavage after platelet stimulation. Calpeptin, MDL28170 and ALLN exerted the same effects on CD84 cleavage (Fig. S3), confirming the role of calpain in this process. To estimate whether CD84 ectodomain shedding was affected by calpain inhibition, we also performed ELISA measurements (Fig. 3B) and found that sCD84 levels were unaltered in the presence of calpeptin.

These results demonstrated that CD84 is proteolytically regulated by two independent mechanisms: ectodomain shedding by metalloproteinase(s) and intracellular cleavage by calpain. Apparently, shedding was functional under calpain-inhibiting conditions and vice versa. A summary of these results is shown schematically in Fig. 7.

ADAM10 is the principal sheddase for CD84 in murine platelets

ADAM10 and ADAM17 are both expressed in platelets [39] and therefore represent possible candidates to mediate CD84 shedding. To test this directly, we first studied platelets from Adam17ex/ex bone marrow chimeric mice, which exhibit a virtually complete loss of ADAM17 protein in hematopoietic cells, including platelets [30,41]. While shedding of GPIb in response to CCCP, W7 and NEM was abolished in Adam17ex/ex platelets (data not shown), levels of released sCD84, as determined by ELISA, were indistinguishable between wild-type and Adam17ex/ex platelets, excluding a major role of ADAM17 in CD84 ectodomain shedding under these experimental conditions (Fig. 4A). To investigate the role of ADAM10 in this process, we analyzed platelets from mice with a megakaryocyte and platelet-specific deficiency of ADAM10 (Adam10fl/fl, PF4-Cre mice, referred to as Adam10−/−) [30]. In sharp contrast to wild-type and Adam17ex/ex platelets stimulated with either agent, sCD84 was virtually undetectable in the supernatant of stimulated Adam10−/− platelets (Fig. 4B). These findings were confirmed by results of Western blot analyses, where no C-terminal remnant was detected in lysates from Adam10−/− platelets treated with NEM, W7 and CCCP in the presence or absence of calpeptin (Fig. 4C). Again, the results from Adam17ex/ex platelets did not differ from those obtained with wild-type platelets (Fig. 4D).

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Figure 4.  ADAM10 is the principal sheddase for CD84 in murine platelets. Washed platelets from wild-type (Adam17+/+) and Adam17ex/ex bone marrow chimeric mice (A) or Adam10+/+ and Adam10−/− mice (B) were treated with CCCP (100 μm, 1 h), W7 (150 μm, 1 h), NEM (2 mm, 20 min) or DMSO as a vehicle control. sCD84 in the supernatants was detected by ELISA as described. Results are mean ± SD (n = 4 mice per group, representative of two individual experiments). (C,D) Western blot detection of CD84 in the lysates of platelets from mice with the indicated genotype (A10, Adam10−/−; A17, Adam17ex/ex; WT are the respective wild-type controls). Platelets were incubated with calpeptin (5 μg mL−1, 37 °C, 15 min) or vehicle control and shedding was induced as described above (two mice pooled per group, representative of two to three individual experiments).

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Taken together, these data established ADAM10 as the principal sheddase that cleaves CD84 in murine platelets, while ADAM17 is not significantly involved in this process.

ADAM10 and calpain regulate surface expression of CD84 in response to agonist receptor stimulation

To investigate whether ADAM10 is also the principal sheddase for CD84 cleavage in response to agonist receptor stimulation, we activated wild-type and Adam10−/− platelets with CVX, CRP, thrombin or RC. Remarkably, none of these agonists induced significant ectodomain shedding of CD84 in the mutant platelets (Fig. 5A). To exclude that thrombin directly cleaved CD84 in wild-type platelets by its protease activity, platelets were also stimulated by PAR4 activating peptide. This led to generation of soluble CD84 similar to thrombin, confirming that thrombin receptor signaling induced loss of CD84 (Fig. 5B). Similar results were obtained with TRAP-6 in human platelets (data not shown). To confirm our results, and to gather information on shedding kinetics, we tested lysates from wild-type and Adam10−/− platelets that had been stimulated for different time durations by Western blotting. These experiments were performed in the presence and absence of calpeptin to also detect intracellular cleavage of CD84. Shedding, visualized by detection of the 15 kDa remnant with the anti-CD84C-term antibody, occurred within 5 min in response to all agonists and the band intensity increased over time (Fig. 5C). RC also induced strong activation of calpain, as indicated by the observation that the remnant was only detectable in the presence of calpeptin as well as by the shortened CD84 protein detected by the anti-CD84N-term antibody, in the absence but not in the presence of calpeptin. In contrast to RC, the other agonists only moderately activated calpain. No C-terminal remnant was detected in lysates of platelets deficient in ADAM10, while calpain activity was unaffected by the absence of the metalloproteinase.

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Figure 5.  ADAM10 cleaves CD84 in response to platelet receptor stimulation. (A) Washed mouse platelets from wild-type (Adam10+/+) and Adam10−/− mice were incubated with CVX (1 μg mL−1), CRP (40 μg mL−1), rhodocytin (2 μg mL−1) or thrombin (0.5 U mL−1) for 1 h at 37 °C. Soluble CD84 was detected by ELISA as described in Material and Methods. Results are mean ± SD (n = 4 mice per group, representative of three individual experiments). (B) Washed mouse platelets from Adam10+/+ and Adam10−/− mice were incubated with thrombin (0.5 U mL−1) or PAR4 activating peptide (PAR4p, 4 mm) for 1 h at 37 °C. Soluble CD84 was detected by ELISA. Results are mean ± SD (n = 4 mice per group). (C) Washed platelets from Adam10+/+ and Adam10−/− mice were pre-incubated for 15 min at 37 °C with calpeptin (5 μg mL−1) or vehicle control, prior to stimulation for 5 min, 15 min or 1 h with agonists described in (A). Platelet lysates were subjected to Western blotting (four mice pooled per group, representative of three individual experiments).

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High concentrations of sCD84 in plasma

To test whether CD84 shedding occurs in vivo, we measured sCD84 levels in mouse plasma. Indeed, significant levels of sCD84 could be detected in the plasma of wild-type mice, while the ELISA yielded only background signals with plasma of Cd84−/− mice (Fig. 6A). Remarkably, sCD84 plasma levels in Adam10−/− mice were reduced by >50% compared with wild-type mice, demonstrating that shedding by platelet ADAM10 occurs in vivo and accounts for approximately half of the total sCD84 protein found in the plasma of normal healthy mice. To investigate whether ADAM17 plays a role in CD84 shedding in platelets in vivo and thus may be responsible for the sCD84 levels observed in Adam10−/− mice, we measured sCD84 levels in the plasma of bone marrow chimeras with platelets double-deficient in ADAM10 and ADAM17 (Adam10−/−/Adam17ex/ex) [30]. As depicted in Fig. 6(B), levels of sCD84 were not further reduced in plasma of double-deficient bone marrow chimeras compared with ADAM10 single-deficient mice, thus excluding a role for ADAM17 in regulating plasma levels of sCD84 in vivo. In consistence, plasma levels of wild-type and Adam17ex/ex bone marrow chimeras were indistinguishable (data not shown).

image

Figure 6.  CD84 levels in mouse plasma and serum. (A) sCD84 levels in the plasma and serum of wild-type control, Adam10−/− and Cd84−/− mice were measured by ELISA. Serum and plasma samples were obtained from the same animals and analyzed within a single experiment. (B) sCD84 levels in the plasma of Adam10−/− and Adam10−/−/Adam17ex/ex mice and their respective controls as determined by ELISA are depicted. Results of all experiments are mean ± SD (n = 4 mice per group, representative of three individual experiments).

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To test whether CD84 shedding occurs during normal blood clotting, non-anticoagulated whole blood was allowed to clot in vitro and sCD84 levels were measured in the obtained serum. In wild-type mice levels of sCD84 increased approximately 2-fold in serum compared with plasma (Fig. 6A). In sharp contrast, sCD84 concentrations in the serum of Adam10−/− mice did not differ from those found in plasma, demonstrating that ADAM10 is the only proteinase that sheds CD84 during blood clotting.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Addendum
  8. Acknowledgements
  9. Disclosure of Conflict of Interest
  10. References
  11. Supporting Information

In this study we have shown that CD84 is regulated by two distinct proteolytic mechanisms in platelets: metalloproteinase-dependent ectodomain shedding and calpain-mediated cleavage of the intracellular C-terminal domain. Studies in mice lacking ADAM10 identified this protease as the principal CD84 sheddase and revealed that this process occurs in vivo in healthy individuals.

The function of CD84 in platelet physiology is not well explored, but it has been reported that the receptor becomes tyrosine phosphorylated in response to platelet aggregation [7]. The close proximity between platelets in aggregates enables contact-dependent signaling [4]. It has been suggested that homophilic interactions of CD84 at the ‘platelet synapse’ could contribute to thrombus stability, making CD84 a potential target for antithrombotic drug discovery [49]. CD84 was recently shown to act as a co-receptor in lymphocytes that facilitates prolonged B-cell:T-cell interaction required for optimal germinal center formation [17]. However, a signaling function of CD84 in platelet activation or aggregation has not been shown to date. On the other hand, as besides platelets also many immune cell types abundantly express CD84 and the receptor undergoes homotypic interactions, it appears possible that the receptor is of functional importance in platelet-immune cell rather than in platelet-platelet interactions. Shedding of CD84 on platelets might therefore represent a novel mechanism to regulate such interactions. Future studies on platelet and immune cell function in Cd84−/− mice will be required to better understand the role of this receptor in thrombotic, inflammatory and/or immunologic processes.

Our studies clearly establish ADAM10 as the principal sheddase to mediate CD84 cleavage under all tested conditions, whereas ADAM17 plays no or only a very minor role in this process. This was surprising as previous studies have shown that other prominent platelet receptors are either cleaved only by ADAM17 (GPIbα, semaphorin 4D) [20,25] or by ADAM10 and ADAM17, depending on the shedding-inducing stimulus (GPV, GPVI) [29,30]. Importantly, ADAM10 also appears to be the only protease to mediate CD84 ectodomain shedding in clotting blood, suggesting that even under conditions of maximal agonist receptor stimulation no other proteinase can cleave the receptor, at least in mouse platelets. In contrast, Fong et al. [28] observed a significant reduction of CD84 shedding in human platelets in the presence of a selective ADAM17 inhibitor, but it was not analyzed in detail whether ADAM10 activity was also affected by this inhibitor. Thus, it cannot be entirely excluded that minor differences in the substrate selectivity of ADAM family sheddases exist between mouse and human platelets. Additional studies are necessary to address this question.

In our in vitro studies we consistently detected low amounts of sCD84 in the supernatant of unstimulated wild-type, but not of Adam10−/− or GM6001-treated platelets. Together with the elevated CD84 plasma levels of wild-type compared with Adam10−/− mice, this strongly suggests that CD84 is continuously shed from the platelet surface by ADAM10, similar to the described constitutive shedding of GPIbα by ADAM17 [20]. Residual sCD84 levels in plasma of Adam10−/− mice might be due to shedding from other cell types or trans-shedding of platelet CD84 by non-platelet ADAM10. The high basal sCD84 levels in plasma of healthy wild-type mice indicate that its use as a marker of thrombotic/inflammatory activity might be limited. Additional sCD84, which might be locally generated upon platelet activation in vivo (e.g. during thrombotic events), would not lead to a significant elevation in the systemic plasma concentration above the basal level. Glycocalicin, the shed extracellular fragment of GPIbα, is another platelet receptor fragment that has likewise been detected in considerable amounts in plasma of normal healthy mice [50], and is also not a sensitive marker of platelet activity.

Treatment of platelets with the calmodulin inhibitor W7 induced strong shedding and calpain-dependent degradation of the C-terminal part of CD84, indicating that CD84 is a calmodulin-binding protein. The platelet receptors GPVI [51], GPIbβ and GPV [52] bind calmodulin by their positively charged, membrane-proximal sequences within cytoplasmic domains. We also found positively charged, membrane-proximal sequences by analyzing sequence data of the murine as well as the human CD84 C-terminus, further supporting the hypothesis that CD84 is a calmodulin-binding protein. Calpain-mediated cleavage of the C-terminus may attenuate or completely terminate signaling. Calpain-mediated intracellular receptor downregulation in platelets has been described for PECAM-1 [35], FcγRIIa [34] and the β3 integrin subunit [33].

Our data clearly show a dual regulation of CD84 by ADAM10 and calpain and both processes can occur independently of each other (Fig. 7). Calpain was able to cleave both the full-length protein and the C-terminal remnant that is generated by ADAM10 activity. Although it is recognized that stimuli inducing extracellular shedding also have the potential to activate intracellular calpain cleavage [39,53], this is, to our knowledge, the first study showing that a single platelet receptor is simultaneously targeted by calpain and a metalloproteinase in response to a single stimulus (W7 or RC). It has been shown in cell culture experiments (e.g. for IL6R [54]) that γ-secretase-mediated cleavage of the C-terminal protein remnant can occur subsequently to ADAM-mediated ectodomain shedding, leading to degradation of the remnant. Our data indicate, however, that this mechanism does not play a role in the degradation of the C-terminal remnant of platelet CD84, because calpeptin, an inhibitor of calpain, was sufficient to inhibit the degradation.

image

Figure 7.  Schematic representation of the regulation of CD84 by intra- and extracellular cleavage. In the absence of sheddase and calpain activity, both antibodies detect the full-length protein (A). If intracellular cleavage occurs, the anti-CD84C-term antibody M-130 is unable to bind CD84 (B and C). In addition to intracellular cleavage, ectodomain shedding by a metalloproteinase may occur (B). In the absence of shedding (e.g. W7+ GM6001 treatment), calpain-mediated cleavage can be visualized by detection of a shortened CD84 protein with the anti-CD84N-term antibody JER1 in Western blotting (C). If shedding occurs in the absence of calpain activity (e.g. NEM treatment) a C-terminal remnant can be detected with the anti-CD84C-term antibody (D). Please note: the exact binding site of the anti-CD84C-term antibody is unknown, as are the cleavage sites; further degradation of the C-terminus may also occur.

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Receptor shedding has been proposed as a mechanism to regulate principal platelet functions, for example by modulation of adhesive properties or modification of receptor signaling, thereby regulating thrombus growth and stability [53]. Ectodomain cleavage could limit the response to agonists, and concomitantly release soluble receptor fragments into the plasma, which may serve as potential regulators of distinct biological functions [39]. Shedding of CD84 could be of specific relevance in this context, because of its broad expression on platelets and immune cells and its ability to undergo homophilic interaction. As recently shown, CD84 stabilizes B cell:T cell interaction [17]. Therefore, it is tempting to speculate that sCD84 of platelet origin might have the potential to modulate immune cell interactions, but this needs further investigation.

Taken together, we have demonstrated that the surface expression of the SLAM family receptor CD84 is tightly regulated by two proteolytic mechanisms involving ADAM10 and calpain and that ectodomain shedding of the receptor through ADAM10 constitutively occurs in vivo. These results may serve as a basis for a better understanding of the regulation of platelet-platelet and platelet-immune cell interactions.

Addendum

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Addendum
  8. Acknowledgements
  9. Disclosure of Conflict of Interest
  10. References
  11. Supporting Information

S. Hofmann, T. Vögtle, and M. Bender performed experiments, analyzed data and contributed to the writing of the manuscript. S. Rose-John analyzed data and contributed to the writing of the manuscript. B. Nieswandt planned the project, analyzed data and contributed to the writing of the manuscript.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Addendum
  8. Acknowledgements
  9. Disclosure of Conflict of Interest
  10. References
  11. Supporting Information

We thank B. Midloch and S. Hartmann for excellent technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft, Bonn (SFB 688, project A1 to BN) and the Rudolf Virchow Center. The work of SR-J. was supported by the Deutsche Forschungsgemeinschaft, Bonn (SFB877, project A1) and by the Cluster of Excellence ‘Inflammation at Interfaces’.

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  4. Materials and methods
  5. Results
  6. Discussion
  7. Addendum
  8. Acknowledgements
  9. Disclosure of Conflict of Interest
  10. References
  11. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Addendum
  8. Acknowledgements
  9. Disclosure of Conflict of Interest
  10. References
  11. Supporting Information

Figure S1. Metalloproteinase dependent shedding of CD84 in human platelets.

Figure S2. High specifity of the anti-CD84C-term antibody M-130 and the anti-CD84N-term antibody JER1.

Figure S3. Inhibition of calpain cleavage of CD84 by three different calpain inhibitors.

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