Monoclonal antibodies SZ2 against GPIbα and SZ51 against P-selectin were generous gifts from Dr. Changgeng Ruan (Soochow University, Suzhou, China). Purified human VWF and botrocetin were generous gifts from Dr. Xiaoping Du (University of Illinois, Chicago, IL, USA). Ristocetin, α-thrombin, aprotinin, dimethyl sulfoxide (DMSO), anti-human gelsolin antibody, FITC-conjugated PAC-1, and dibucaine were purchased from Sigma (St. Louis, MI, USA). Non-essential amino acids, penicillin and streptomycin, L-glutamine, and L-trans-Epoxysuccinyl-leucylamido (4-guanidino) (E64) were purchased from Roche Molecular Biochemicals (Indianapolis, IN, USA). Caspase-3 inhibitor z-DEVD-fmk was purchased from Bender Medsystem (Vienna, Austria). Calpain inhibitor carbobenzoxy-valinyl-phenylalaninal (MDL28170), calpain inhibitors I and II, A23187 and GM6001 were purchased from Calbiochem (San Diego, CA, USA). Goat anti-mouse immunoglobulin (IgG) conjugated with horseradish peroxidase (GAM-HRP), goat anti-rabbit immunoglobulin (IgG) conjugated with horseradish peroxidase (GAR-HRP), FITC-conjugated goat anti-mouse IgG (FITC-GAM), and Alexa Fluor 488-conjugated goat anti-mouse IgG were purchased from Biosource (Camarillo, CA, USA). Anti-actin antibody was from Boehringer Mannheim Biochemicals (Indianapolis, IN, USA). The anti-tubulin antibody was purchased from NeoMarkers (Fremont, CA, USA). The mitochondria isolation kit was purchased from Pierce (Rockford, IL, USA). The anti-COX-1 antibody was purchased from MitoSciences (Eugene, OR, USA). Monoclonal antibodies against Bax, Bak, Bcl-2, Bcl-X_L and caspase-3 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

For studies involving human subjects, approval was obtained from the Beihang University institutional review board. Informed consent was provided according to the Declaration of Helsinki. Blood collection and preparation of washed platelets were performed as previously described [17].

For platelet aggregation studies, washed platelets were incubated with dibucaine (500 μmol/L), and then platelet aggregation was induced by the addition of ristocetin (1.25 mg/mL) plus VWF (35 μg/mL) or α-thrombin (0.1 U/mL) and measured by a turbidometric platelet aggregometer (Xinpusen, Beijing, China) at 37 °C with a stirring speed of 1000 rpm [18].

Washed platelets (3 × 10⁸ /mL) were incubated with dibucaine (125 μmol/L, 250 μmol/L, 500 μmol/L) at RT for 15 min, ΔΨm was determined using the potential sensitive dye tetramethyl-rhodamine-ethylester (TMRE). Briefly, TMRE was added into the dibucaine pre-treated platelets to a 100 nmol/L final concentration. Then samples were further incubated in the dark at 37 °C for 20 minutes (min) and analyzed by flow cytometry. TMRE signals were excited using a 488 nm krypton-argon laser line and emissions were captured using filters at 625 nm. In some experiments, platelets were pre-incubated with GM6001 (100 μmol/L) at RT for 10 min, and then incubated with dibucaine (500 μmol/L) at RT for 15 min and subjected into ΔΨm measurement [18].

Washed platelets (3 × 10⁸/mL) were incubated with dibucaine (125 μmol/L, 250 μmol/L, 500 μmol/L) at RT for 15 min. Annexin V binding buffer was mixed with pre-treated platelets and annexin V-FITC at a 50:10:1 ratio. Samples were gently mixed and incubated at RT for 15 min in the dark, then analyzed by flow cytometry. In some experiments, platelets were pre-incubated with GM6001 (100 μmol/L) at RT for 10 min, and then incubated with dibucaine (500 μmol/L) at RT for 15 min and subjected into PS exposure analysis [18].

Washed platelets (3 × 10⁸/mL) were incubated with dibucaine (500 μmol/L) at RT for 15 min. To analyze P-selectin surface expression, platelets pre-treated with dibucaine were firstly incubated with SZ51 at RT for 30 min, and then further incubated with FITC-GAM in the dark at RT for 30 min. The treated platelets were analyzed by flow cytometry. For PAC-1 binding, platelets pre-treated with dibucaine were incubated with FITC-labeled soluble PAC-1 at RT for 20 min in the dark. Then platelets were fixed with 1% cold paraformaldehyde, further incubated at 4 °C in the dark for 30 min, and analyzed by flow cytometry. A23187 was set as a positive control. As negative controls, platelets were incubated with mouse IgG and then incubated with FITC-GAM [18].

Washed platelets (3 × 10⁸/mL) were treated with dibucaine (125 μmol/L, 250 μmol/L, 500 μmol/L) at RT for 15 min, and lysed with an equal volume of lysis buffer containing 0.1 mmol/L E64, 1 mmol/L phenylmethylsulfonyl fluoride (PMSF) and 1/100 aprotinin on ice for 30 min, and the whole lysate was resolved by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotted with anti-gelsolin antibody (1:2500) or anti-caspase-3 antibody (1:1000), respectively. To evaluate Bcl-2 family protein expression, lysate was resolved by SDS-PAGE, and immunoblotted with anti-Bak (1:500), anti-Bax (1:500), anti-Bcl-2 (1:500), or anti-Bcl-X_L (1:500), respectively. Anti-actin antibody was used to confirm that protein inputs in each lane were similar. In some experiments, platelets were pre-incubated with GM6001 (100 μmol/L) at RT for 10 min, or calpain inhibitor MDL28170 (100 μmol/L) at RT for 10 min, or caspase-3 inhibitor z-DEVD-fmk (50 μmol/L), or control DMSO at RT for 15 min, and then were further incubated with dibucaine (500 μmol/L) at RT for 15 min and subjected into western blot analysis [18].

In the isolation and analysis of the platelet mitochondrial fraction assay [25], platelets were treated with dibucaine (500 μmol/L) or DMSO at RT for 15 min, and then were suspended in mitochondrion isolation buffer A for 2 min, buffer B for 5 min, and buffer C for 5 min followed by centrifugation. The supernatants were centrifuged at 12,000 g for 20 min. The pellets containing the mitochondria were washed once with buffer C, then were incubated in lysis buffer for 30 min, and centrifuged at 12,000 g for 5 min. The samples were subjected to Western blot analysis.

Platelets (3 × 10⁸/mL) were pre-incubated with dibucaine (500 μmol/L) or DMSO at RT for 15 min. The Lab-Tek cover slips (Nunc) were pre-coated with 20 μg/mL of VWF and blocked with 5% BSA. Platelets pre-treated with dibucaine were incubated with botrocetin (5 μg/mL) and allowed to adhere to the prepared cover slips as described previously [18]. Platelets were fixed with 4% paraformaldehyde, permeabilized and blocked with BSA. Samples were incubated with SZ2 (10 μg/mL) firstly, and then with Alexa Fluor 488-conjugated goat anti-mouse IgG. Samples were scanned under a confocal microscope (63 × objective lens) (Lab-Tek MicroImaging, Inc. LSM510).

Caspase-3 activity was determined using the caspase-3 activity kit (Beyotime Institute of Biotechnology, Haimen, China) as described previously [26]. Briefly, platelets (3 × 10⁸/mL) were pre-treated with caspase-3 inhibitor z-DEVD-fmk (50 μmol/L) or DMSO at RT for 15 min, or calpain inhibitor MDL28170 (100 μmol/L) at RT for 10 min, and then were further incubated with dibucaine (500 μmol/L) at RT for 15 min. Caspase-3 activity assays were performed on 96-well microtitre plates by incubating 10 μL platelet lysate per sample in 80 μL reaction buffer and 10 μL caspase-3 substrate (Ac-DEVD-pNA) (2 mmol/L). Samples were incubated at 37 °C for 4 h and were measured with an enzyme-linked immunosorbent assay (ELISA) reader at an absorbance of 405 nm. The specific caspase-3 activity, normalized for total proteins of platelet lysates, was then expressed as fold of the baseline caspase-3 activity of the control DMSO-treated sample.

Calcium-mediated activation of calpain induces apoptosis in several types of nucleated cells [9,10]. In particular, it has been reported that ONO-3403 (ethyl N-allyl-N-[(E)-2-methyl-3-[4-(4-amidinophenoxycarbonyl)phenyl]propenoyl]amino acetate methansulfonate), a specific stimulator of calpain, induces apoptosis cascades in NIH3T3 mouse fibroblasts [8], suggesting that the calpain activator dibucaine may induce platelet apoptosis. To test this hypothesis, we first determined the effectiveness of dibucaine, an established calpain activator [27]. Platelets were incubated with dibucaine and subjected to Western blot analysis. The results showed that talin and filamin A, both of which are recognized substrates of calpain, were dose-dependently cleaved in platelets pre-treated with dibucaine (data not shown). Furthermore, cleavage of filamin A was inhibited by calpain inhibitors (Supplementary Figure I), indicating that dibucaine is a calpain-specific activator. In addition, dibucaine was able to induce cleavage of filamin A in the absence of extracellular calcium (Supplementary Figure II), consistent with previous findings [27]. Therefore, dibucaine was selected in the following studies.

There are two signal transduction pathways leading to apoptosis in nucleated cells: the receptor-mediated apoptotic pathway and the mitochondria-linked apoptotic pathway. However, platelet apoptosis commonly arises from the mitochondria-mediated signaling pathway characterized by depolarization of ΔΨm [17]. So effects of dibucaine on depolarization of ΔΨm in platelets were investigated by flow cytometry with the cell-permeable lipophilic cationic dye TMRE. Figure 1a and b demonstrated clearly that dibucaine, but not the vehicle control DMSO, dose-dependently elicited depolarization of ΔΨm in platelets, as monitored by decreased fluorescence in TMRE-stained platelets, and an increase of depolarized platelets.

Depolarization of ΔΨm occurs in both platelet apoptosis and platelet activation induced by strong agonists [16–21]. Whether depolarization of ΔΨm resulted from platelet apoptosis was further investigated. Although platelets are anucleated cells, the Bcl-2 family proteins, interacting with the mitochondrial outer membrane, are well-characterized regulators of platelet apoptosis and act as a checkpoint upstream of caspases and a mitochondria gateway where a variety of apoptotic signals converge [17,18]. Therefore, to further investigate whether dibucaine can elicit platelet apoptosis through the mitochondria-mediated pathway, the expression profiles of the Bcl-2 family proteins including Bax, Bak, Bcl-2, and Bcl-X_L were analyzed. As shown in Figure 1c and d, the expression levels of pro-apoptotic proteins Bax and Bak increased significantly in platelets following dibucaine induction. Conversely, decreased expression levels of anti-apoptotic proteins Bcl-2 and Bcl-X_L were detected simultaneously, consistent with previous studies [28]. In addition, there was an obvious increase in mitochondrial membrane-bound Bax.

It is well known that Bcl-2 family proteins regulate the mitochondrial inner transmembrane potential as well as the release of cytochrome c, which results in the activation of caspases, especially caspase-3, the executioner of apoptosis [17,18]. Furthermore, both calpains and caspase-3 are activated and involved in platelet apoptosis induced by A23187 or α-thrombin [11,22,23]. However, it is still unknown whether calpains function upstream of caspases or not in platelet apoptosis induced by dibucaine. To answer this question, gelsolin, a cytoskeletal regulatory protein and also the confirmed substrate of caspase-3, was employed to analyze the activation state of caspase-3 in Western blot assays. As demonstrated in Figure 2a, dibucaine dose-dependently elicited the presence of the 48 kDa cleaved fragment of gelsolin, indicating that caspase-3 was activated.

Although gelsolin is generally regarded as a specific substrate of the caspase-3, there is a report that calpain also cleaves gelsolin [24]. Therefore, the activation of caspase-3 was further analyzed. As expected, compared with the control samples, the 17 kDa fragment representing caspase-3 activation dose-dependently presented in platelets in response to the treatment of dibucaine (Figure 2b). In addition, activation of caspase-3 was observed in platelets treated with dibucaine, which was significantly inhibited by the caspase-3 inhibitor z-DEVD-fmk or calpain inhibitor MDL28170 (Figure 2d). Although Wolf et al. [24] reported that caspase-3 was not activated in platelets treated with thrombin, other groups demonstrated that activation of caspase-3 was induced by thrombin [11,22], which is not in conflict with our findings.

Depolarization of ΔΨm and activation of caspase-3 are the common characteristics of apoptosis in platelets induced by various stimuli [17,18]. In contrast, PS exposure is the characterized morphology of apoptosis in platelets stimulated by strong agonists [19]. Therefore, in order to investigate whether dibucaine is potent to elicit PS exposure during platelet apoptosis, PS exposure was measured in platelets pre-incubated with dibucaine using flow cytometric analysis. A dose-dependent PS exposure was observed in platelets treated with dibucaine (Figure 2c and d), suggesting that dibucaine is a strong apoptosis stimulator. In addition, PS exposure was significantly inhibited by the caspase-3 inhibitor z-DEVD-fmk, further supporting that this process is mediated by caspase-3 (Supplementary Figure III). Together, these data indicate that dibucaine triggers signaling cascades leading to platelet apoptosis.

We recently reported that calpains play regulatory roles in ADAM17-mediated GPIbα shedding [5]. To determine whether dibucaine-induced platelet apoptosis results from GPIbα shedding, platelets were pre-incubated with GM6001, a recognized ADAM17 inhibitor [5], then further incubated with dibucaine, and markers of the apoptotic cascades were analyzed. GM6001 effectively blocked GPIbα shedding (data not shown), however, it exerted no obvious effect on dicucaine-induced apoptosis events such as depolarization of ΔΨm, expression levels of Bcl-2 family and PS exposure (Figure 3), indicating that dibucaine-induced platelet apoptosis is independent of GPIbα shedding.

Our findings demonstrated that dibucaine induces PS exposure in platelets. However, there is also a possibility that PS exposure results from platelet activation [17]. In order to exclude the interference of platelet activation and further confirm that the mechanisms leading to platelet activation and platelet apoptosis are distinct from each other, the effects of dibucaine on platelet activation events were investigated. Platelets were incubated with dibucaine, using DMSO and A23187 as negative and positive controls, respectively, then the surface expression of P-selectin, an established marker of α-granule release and platelet activation, was analyzed. Flow cytometry results demonstrated that dibucaine does not elicit obvious surface expression of P-selectin (Figure 4a and b). To further study whether dibucaine can induce platelet activation, the activation of integrin α_IIbβ₃ was examined by flow cytometry with FITC-conjugated PAC-1, a confirmed antibody that specifically reacts with the activated integrin α_IIbβ₃. Figure 4c and d showed that no obvious PAC-1 binding was observed in platelets pre-treated with dibucaine, in contrast, PAC-1 positive platelets presented in A23187 treated samples, excluding the interference of experimental conditions. Taken together, these data suggest that dibucaine does not induce platelet activation.

Platelets are critical players in both hemostasis and thrombosis. It has also been proposed that altered calpain activity is involved in TTP [6,7]. Our findings indicated that dibucaine induces platelet apoptosis but not platelet activation. However, it is still unknown whether dibucaine affects platelet function. To answer this question, platelet aggregation and confocal assays were employed to analyze the roles of dibucaine in platelet aggregation, adhesion and spreading on VWF surface. As shown in Figure 4e, both ristocetin/VWF- and α-thrombin-induced platelet aggregations were markedly diminished in dibucaine-incubated platelets. Furthermore, both the number of adherent platelets and the area of platelet membrane spreading on VWF were obviously decreased in platelets in response to dibucaine treatment (Figure 4f and g). Collectively, these data indicate that dibucaine induces platelet dysfunction.