Influence of γ-Secretase Inhibitor 24-Diamino-5-Phenylthiazole DAPT on Platelet Activation

DAPT (24-diamino-5-phenylthiazole) [1] is a known inhibitor of γ-secretase [2,3]. The enzyme cleaves the signaling molecule CD44 [4,5,6], a multifunctional transmembrane glycoprotein and (co)receptor [7,8] expressed in a wide variety of normal and cancer cells [7,9] and contributing to the regulation of diverse cellular functions including cell adhesion, proliferation, growth, survival, motility, migration, angiogenesis, and differentiation [7,8,10,11,12,13].

CD44 is involved in the signaling of macrophage migration inhibitory factor (MIF) [14,15], which is expressed and released from blood platelets [16]. Platelets are required for primary hemostasis following vascular injury and contribute to the pathophysiology of acute thrombotic occlusion following atherosclerotic plaque rupture [17,18]. Platelets are involved in the pathophysiology of arterial thrombosis, vascular inflammation and atherogenesis [17,19]. They are activated by an increase of cytosolic Ca²⁺ concentration ([Ca²⁺]_i) [20], resulting from Ca²⁺ release from intracellular stores [21] with subsequent activation of the Ca²⁺ release-activated channel (CRAC) Orai1 (CRACM1) in the plasma membrane [20,22,23,24]. Most recently CD44 has been disclosed as a negative regulator of platelet activation [25]. Specifically, CD44 deficiency has been shown to augment in platelets thrombin and collagen related peptide induced increase of Orai1 surface abundance, [Ca²⁺]_i, degranulation, α_IIbβ₃ integrin activation, caspase-3 activity, cell membrane scrambling, and cell shrinkage. Moreover, CD44 deficiency augmented platelet adhesion and in vitro thrombus formation under high arterial shear rates [25].

Inhibitors of γ-secretase were expected to enhance the abundance of CD44 with the respective down-regulation of platelet activation and apoptosis.

Nothing is known about any potential effects of DAPT on platelet function. The present study thus explored whether DAPT modifies the effect of collagen related peptide on platelet activation with Ca²⁺ entry and phospholipid scrambling of the cell membrane.

All animal experiments were conducted according to the German law for the welfare of animals and were approved by the authorities of the state of Baden-Württemberg. Experiments were performed with blood platelets isolated from wild type mice. The mice had free access to water and control chow (Ssniff, Soest, Germany).

Platelets were obtained from 10- to 12-week-old mice of either sex. The mice were anesthetized and 800 µl blood was drawn from the retro-orbital plexus into tubes with 200 µl acid-citrate-dextrose buffer before the mice were sacrificed [26]. Platelet rich plasma (PRP) was obtained by centrifugation at 260 g for 5 minutes. Afterwards PRP was centrifuged at 640 g for 5 minutes to pellet the platelets. Where necessary apyrase (0.02 U/ml; Sigma-Aldrich) and prostaglandin I₂ (0.5 µM; Calbiochem) were added to the PRP to prevent activation of platelets during isolation. After two washing steps the pellet of washed platelets was resuspended in modified Tyrode-HEPES buffer (pH 7.4, supplemented with 1 mM CaCl₂). Where indicated, CRP (kindly provided by R.Farndale, University of Cambridge, Cambridge, UK) was added.

Orai1 surface expression was analyzed in platelets by flow cytometry. Washed platelets were incubated with 2 µg/ml CRP without BSA, and subsequently fixed with 1% paraformaldehyde for 30 minutes on ice. After rinsing three times, platelets were incubated for 90 minutes with Orai1 rabbit anti-mouse antibody (Abcam), washed once in Tyrode buffer, and stained in 1:250 diluted CF™ 488A-labeled anti-rabbit secondary antibody (Sigma, USA) for 60 minutes. Samples were immediately analyzed on a FACS Calibur flow cytometer (BD Biosciences).

For the measurement of the cytosolic Ca²⁺ concentration the platelet preparation was washed once in Tyrode buffer (pH 7.4), stained with 3 µM Fluo-3AM (Biotinium, USA) in the same buffer and incubated at 37°C for 30 minutes. Following the indicated experimental treatment, relative fluorescence was measured utilizing a BD FACS Calibur (BD Biosciences, Heidelberg, Germany) [27].

Fluorophore-labeled antibodies were utilized for the detection of P-selectin expression (Wug.E9-FITC) and the active form of α_IIbβ₃ integrin (JON/A-PE). Washed mouse platelets (1x10⁶) were suspended in modified Tyrode buffer (pH 7.4) containing 1 mM CaCl₂ and antibodies (1:10 dilution) and subsequently subjected to the respective treatments and for the indicated time periods at room temperature (RT). The reaction was stopped by addition of PBS and the samples were immediately analyzed on a BD FACSCalibur.

The abundance of reactive oxygen species (ROS) was determined utilizing 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA). To load the platelets, DCFDA (Sigma, Schnelldorf, Germany) was added to the cell suspension at a final concentration of 10 µM. Following the indicated experimental treatments, fluorescence was measured by flow cytometry utilizing a BD FACSCalibur (BD Biosciences, Heidelberg, Germany).

Mitochondrial membrane potential was assessed by flow cytometry [28]. Briefly, platelets were incubated in 10 µM DAPT for 30 min, and then treated with 5 µg/ml collagen related peptide for 60 minutes. Pre-incubated platelets were stained with 10 µM of TMRE (Invitrogen, USA) for 30 minutes at room temperature in the dark, and the TMRE-fluorescence was measured by flow cytometry utilizing a BD FACSCalibur.

Phosphatidylserine exposure was determined in platelets with and without 10 minutes CRP treatment. To this end, the platelet preparation was centrifuged at 660 g for 5 minutes followed by washing once with Tyrode buffer (pH 7.4) with 1 mM CaCl₂, staining with 1:20 dilution of Annexin-V FITC (Mabtag, Germany) in Tyrode buffer (pH 7.4) with 2 mM CaCl₂ and incubation at 37°C for 30 minutes. Annexin-V binding reflecting surface exposure of phosphatidylserine was evaluated by flow cytometry utilizing a BD FACSCalibur. In parallel, the forward scatter (FSC) of the platelets was determined by flow cytometry as a measure of platelet size.

Aggregation was determined utilizing flow cytometry as previously described [29]. To this end platelets were labeled with CD9-APC and CD9-PE monoclonal antibodies (1:100 dilution, Abcam) for 15 minutes at room temperature. Following incubation, differently labeled samples were washed twice, mixed 1:1, and incubated in 10 µM DAPT (Sigma, Germany) for 30 min at 37°C while shaking at 600 rpm for 10 minutes. Pre-incubated platelets were activated with 2 µg/ml collagen related peptide at 37°C while shaking at 1000 rpm. At the indicated time points, samples were fixed by addition 0.5% paraformaldehyde (Carl Roth, Germany) in phosphate-buffered saline. The fixed samples were measured utilizing a BD FACSCalibur (BD Biosciences, Heidelberg, Germany). For quantification, a quadrant was set in the dot plot of respective channels on non-stimulated platelets. The appearance of double-colored events in the upper right quadrant (Q2) was quantified as percentage of total amount of labeled events (Q1+Q2+Q4) at every time point analyzed.

Data are provided as means ± SEM; n represents the number of independent experiments. All data were tested for significance using ANOVA with Tukey's test as post-test or unpaired student's t-test as appropriate. Results with p<0.05 were considered statistically significant.

The present study explored whether γ-secretase inhibitor DAPT (24-diamino-5-phenylthiazole) influences platelet activation. To this end, murine platelets were isolated from wild type mice and activated with collagen related peptide (CRP).

In a first series of experiments the abundance of Orai1 protein in the platelet cell membrane was quantified. As illustrated in Fig. 1A and D, without CRP treatment Orai1 protein expression in the platelet membrane was similarly low in presence and absence of γ-secretase inhibitor DAPT (10 µM). Activation of platelets with 2 µg/ml CRP was followed by a significant increase of Orai1 protein abundance. After short time treatment (100 seconds) Orai1 protein abundance was not significantly different in presence or absence of DAPT (Fig. 1B, D). After 15 minutes CRP treatment, the increase of Orai1 abundance in the platelet plasma membrane was, however, significantly blunted in the presence of 10 µM DAPT (Fig. 1C, D).

In order to test, whether DAPT modified cytosolic Ca²⁺ concentration ([Ca²⁺]_i) in murine platelets, [Ca²⁺]_i was determined utilizing Fluo-3 fluorescence. As illustrated in Fig. 2A and C, prior to CRP treatment [Ca²⁺]_i was similar in the absence and presence of DAPT. Treatment with CRP (2 µg/ml) was within 100 seconds followed by a significant increase of [Ca²⁺]_i in platelets, an effect significantly blunted in the presence of DAPT (Fig. 2B, C).

Platelet degranulation was estimated from the increase of P-selectin abundance on the platelet surface, which was determined utilizing specific antibodies and flow cytometry. As illustrated in Fig. 3A and C, the P-selectin abundance was negligible at the surface of resting platelets and not significantly modified by DAPT treatment. CRP (2 µg/ml) treatment was followed by a sharp increase of P-selectin abundance, an effect significantly blunted in the presence of 10 µM DAPT (Fig. 3B, C).

Similarly, the abundance of active integrin α_IIbβ₃ was negligible at the surface of resting platelets (Fig. 3D, F) and was significantly increased by treatment with CRP (2 µg/ml). Again, the effect was significantly blunted in the presence of 10 µM DAPT (Fig. 3E, F).

Further experiments were performed to test, whether DAPT modifies the abundance of reactive oxygen species (ROS). ROS generation was quantified utilizing DCFDA fluorescence. As shown in Fig. 4, following a 2 µg/ml CRP treatment, ROS generation was increased, an effect significantly blunted in the presence of DAPT after 7 minutes CRP treatment.

In a further series of experiments the effect of DAPT and CRP on mitochondrial membrane potential (Δψm) was determined with TMRE by flow cytometry. As illustrated in Fig. 5A and C, Δψm was similar in resting platelets both, with or without presence of DAPT. However, TMRE fluorescence was decreased by treatment with 5 µg/ml CRP, an effect again significantly less pronounced in the presence than in the absence of DAPT (Fig. 5B, C).

As illustrated in Fig. 6A and C, the percentage of annexin-V positive platelets was again negligible in untreated platelets, irrespective of the presence of DAPT. CRP (5 µg/ml) within 10 min significantly enhanced the percentage of annexin-V binding platelets, an effect again significantly blunted in the presence of DAPT (10 µM) (Fig. 6B, C).

Platelet volume was estimated from forward scatter, which was determined by flow cytometry. As illustrated in Fig. 6D and F, prior to stimulation with CRP, forward scatter was again similar in the absence and presence of DAPT. CRP (5 µg/ml) treatment within 10 min was followed by a significant decrease of forward scatter, an effect significantly less pronounced in the presence than in the absence of DAPT (Fig. 6E, F).

To elucidate the effect of DAPT and CRP on platelet aggregation, platelets were labeled with two distinct dyes and the coincidence of the two dyes estimated by flow cytometry. As illustrated in Fig. 7, aggregation of resting platelets was similarly low in DAPT treated and untreated platelets. CRP (2 µg/ml) treatment within a few min significantly increased the platelet aggregation, an effect significantly blunted by prior DAPT treatment.

The present observations disclose a novel effect of the γ-secretase inhibitor DAPT (24-diamino-5-phenylthiazole), i.e. the attenuation of platelet activation following treatment with collagen related peptide (CRP). In the absence of CRP, treatment of platelets with DAPT had little effect on the tested platelet properties. In contrast, DAPT significantly blunted the effect of CRP on degranulation, integrin activation, cell membrane scrambling and cell shrinkage. Phosphatidylserine translocation to the platelet surface following cell membrane scrambling is expected to foster the procoagulant function of platelets and is thus decisive in hemostasis [30].

The effects of DAPT could be explained by inhibition of γ-secretase which would be expected to enhance the CD44 protein abundance and thus augment the inhibitory effect of CD44 on platelet activation and apoptosis [25].

Activation of platelets with thrombus formation and apoptosis is in large part secondary to increase of cytosolic Ca²⁺ activity ([Ca²⁺]_i) [23,31] and the negative effects of DAPT on activation and cell membrane scrambling is paralleled by a significantly blunted increase of [Ca²⁺]_i. The increase of [Ca²⁺]_i following CRP treatment could be contributing to oxidative stress. Along those lines, DAPT blunted the increase of reactive oxygen species (ROS) following CRP treatment. The oxidative stress is paralleled by mitochondrial depolarization. CD44 counteracts oxidative stress [32] and could thus contribute to or even account for the attenuation of ROS formation by DAPT.

An increase of [Ca²⁺]_i leads to platelet activation, which fosters the development of arterial thrombosis [20]. An increase of [Ca²⁺]_i further triggers phospholipid scrambling of the cell membrane with translocation of phosphatidylserine to the platelet surface [19,33,34,35]. The inhibition of CRP-induced phosphatidylserine exposure may thus result from a decrease of Ca²⁺ entry. Phosphatidylserine exposed at the platelet surface stimulates thrombin formation, which in turn augments the activation of platelets [33,34,35,36]. Phosphatidylserine exposing platelets are further bound to and phagocytosed by macrophages [37]. Both, platelet activation and apoptosis contribute to the stimulation of platelet aggregation, which is again slightly, but significantly blunted by DAPT.

Upon stimulation, platelets release the CD44 activating ligand macrophage migration inhibitory factor (MIF) [16], which inhibits, as part of an autocrine negative feedback loop, platelet cell membrane scrambling. MIF limits activation-induced apoptosis via interaction with CXCR7 [28]. CD44 is considered a target in the treatment of inflammatory disease [15]. To the extent that γ-secretase degrades CD44 [4,5,6], pharmacological inhibition γ-secretase could strengthen the negative feedback loop and thus down-regulate platelet activation and phospholipid scrambling of the platelet plasma membrane. However, even though CD44 participates in the signaling of MIF [14,15], and lack of CD44 augments the activation of platelets by several agonists such as CRP [25], the observed blunting effect of DAPT on platelet activation and cell membrane scrambling is not necessarily due to increased CD44 abundance and activity.

In conclusion, the γ-secretase inhibitor DAPT attenuates the CRP-induced increase of Orai1 abundance, [Ca²⁺]_i, oxidative stress, degranulation, integrin activation, translocation of phosphatidylserine to the platelet cell membrane surface and platelet shrinkage, and thus attenuates platelet activation and apoptosis.

We thank Efi Faber for providing technical assistance as well as Tanja Loch for meticulous preparation of the manuscript. This study was supported by the Deutsche Forschungsgemeinschaft - Klinische Forschergruppe [DFG-KFO 274] ‘Platelets—Molecular Mechanisms and Translational Implications', as well as the Tuebingen Platelet Investigative Consortium (TuePIC), and the Open Access Publishing Fund of Tuebingen University.

The authors of this manuscript state that they have no conflicts of interest to declare.

G. Liu and G. Liu contributed equally and thus share first authorship.