Inhibition by Hydrogen Sulfide of Rabbit Platelet Aggregation and Calcium Mobilization

Abstract

Hydrogen sulfide (H₂S), a gasotransmitter, plays a variety of roles in the mammalian body including the cardiovascular system. Given evidence that H₂S donors including NaHS inhibit human platelet aggregation, we examined and characterized the effects of NaHS on rabbit platelet aggregation and cytosolic Ca²⁺ mobilization. Rabbit platelet aggregation was determined in platelet-rich plasma (PRP) and washed platelets. Intracellular Ca²⁺ levels were monitored in Fura2-loaded washed platelets. NaHS prevented rabbit platelet aggregation induced by collagen or ADP, and the effective concentration range of NaHS was 0.1–0.3 mM in PRP and 1–3 mM in washed platelets. In washed platelets, NaHS attenuated cytosolic Ca²⁺ mobilization induced by collagen or ADP and also reduced platelet aggregation induced by ionomycin, a Ca²⁺ ionophore. The anti-platelet effect of NaHS was blocked by an adenylyl cyclase inhibitor and enhanced by a phosphodiesterase inhibitor. H₂S thus suppresses rabbit platelet aggregation by interfering with both upstream and downstream signals of cytosolic Ca²⁺ mobilization in a cAMP-dependent manner.

Hydrogen sulfide (H₂S), a toxic gas smelling like rotten eggs, is now considered the third gasotransmitter after nitric oxide (NO) and carbon monoxide. H₂S is formed endogenously from L-cysteine by distinct enzymes including cystathionine-γ-lyase and cystathionine-β-synthase in the mammalian body, and plays a variety of roles in the nervous, gastrointestinal, circulatory and respiratory systems.^1–10) In the cardiovascular system, H₂S, like NO, causes vasorelaxation through activation of ATP-sensitive potassium channels,^1,4) although it also acts as a vasoconstrictor under certain conditions.^10,11) Interestingly, there is a paper indicating that H₂S inhibits aggregation of human platelets,¹²⁾ as NO does. The effect of H₂S in human platelets appears to be independent of cAMP or cyclic GMP generation,¹²⁾ although H₂S is known to activate adenylyl cyclase in neurons and vascular smooth muscle cells^3,13) or inhibit phosphodiesterase.¹⁴⁾ A recent paper has also indicated that H₂S inhibits human platelet adhesion through its antioxidative activity.¹⁵⁾ In the present study, we examined and characterized the effect of NaHS, an H₂S donor, on the platelet aggregation or cytosolic Ca²⁺ mobilization in rabbit platelet-rich plasma and/or washed platelet suspension.

MATERIALS AND METHODS

Chemicals

NaHS was purchased from Kishida Chemical Co., Ltd. (Osaka, Japan), and collagen (Collagenreagent Horm®) was from Hormon-Chemie (Munich, Germany). ADP was obtained from Wako Pure Chemicals Industries, Ltd. (Osaka, Japan), and ionomycin, a Ca²⁺ ionophore, and dipyridamole, a phosphodiesterase inhibitor, were from Sigma-Aldrich (St. Louis, MO, U.S.A.). SQ22,536, an adenylyl cyclase inhibitor, was purchased from Calbiochem (Darmastadt, Germany), and Fura2-AM was from Dojindo Laboratories (Kumamoto, Japan). Collagen was diluted with the SKF buffer (Hormon-Chemie). NaHS was dissolved in saline, and ionomycin, Fura2-AM and dipyridamole were dissolved in 100% dimethyl sulfoxide (DMSO) and then diluted with distilled water. All other chemicals were dissolved in distilled water.

Animals

Male JW/CSK rabbits weighing 3–4 kg were purchased from Japan SLC Inc. (Shizuoka, Japan). They were housed in a temperature-controlled room under a 12-h day per night cycle at about 24°C and had free access to food and water. All experimental protocols were approved by the Committee for the Care and Use of Laboratory Animals of Kinki University and were in accordance with the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (NIH Publication No. 85-23, revised 1996).

Preparation of Platelet-Rich Plasma and Washed Platelets

Rabbit blood (10–15 mL) was collected from the auricular artery into a plastic syringe containing 3.8% sodium– citrate (9 : 1, v/v). The collected blood was centrifuged at 200×g for 10 min at room temperature, and the supernatant was used as platelet-rich plasma (PRP). The pellet was further centrifuged at 1500×g for 10 min at room temperature, and the supernatant was used as platelet-poor plasma (PPP). The number of platelets was adjusted to 4×10⁵ cells/µL by diluting PRP with PPP. Washed platelets were prepared as previously reported.¹⁶⁾ Briefly, platelets was washed three times with a N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES)-Tyrode buffer for washing (NaCl 137 mM, KCl 2.68 mM, NaH₂PO₄ 3.75 mM, MgCl₂ 0.98 mM, HEPES 3.78 mM, glucose 5.56 mM, bovine serum albumin (BSA) 3.5 g/L, apyrase 3 U/mL, pH 6.7), and finally resuspended in a HEPES-Tyrode resuspension buffer that contains 0.35 g/L BSA and no apyrase (pH 7.35). The number of platelets was adjusted to 3×10⁵ cells/µL. CaCl₂ at 1.5 mM and fibrinogen at 0.4 mg/mL (only for ADP stimulation) were added immediately before the experiments.

Platelet Aggregation Assay

Platelet aggregation was measured turbidimetrically with constant stirring (1000 rpm) at 37°C, using Mebanix Aggregometer PAM8C (Mebanix Co., Ltd., Tokyo, Japan). PRP (4×10⁵ cells/µL) or washed platelet preparations (3×10⁵ cells/µL) were preincubated for 1 min at 37°C, and NaHS was added for 3-min incubation. Platelets were then stimulated with collagen (10 µg/mL), ADP (10 µM) or ionomycin (0.3 µM). SQ22,356 (100 µM) and dipyridamole (30 µM) were added 30 and 10 min before collagen stimulation, respectively. Platelet aggregation was evaluated by measuring the peak of the aggregation curves. Data are expressed as the percentage of the maximal aggregation.

Lactate Dehydrogenase (LDH) Release Assay

To check the cell toxicity of NaHS, the released LDH was determined after the treatment of washed platelets with NaHS at 3 mM, using an LDH cytotoxicity detection kit (TaKaRa Bio, Otsu, Japan).

Determination of Cytosolic Ca²⁺ Mobilization

For determination of cytosolic Ca²⁺ concentration ([Ca²⁺]_i), washed platelets (3×10⁵ cells/µL) in a volume of 1 mL were incubated with 6 µM Fura2-AM for 60 min at room temperature, and, after washing, resuspended in the HEPES-Tyrode resuspension buffer (3×10⁵ cells/µL), described above. Washed platelet preparations in a volume of 669 µL, after 1-min preincubation at 37°C, was treated with 21 µL of NaHS for 3 min. Immediately after addition of CaCl₂ at 1.5 mM and fibrinogen at 0.4 mg/mL (only for ADP stimulation), as shown above, the platelets were stimulated with 10 µL of collagen at 3 µg/mL, or 10 µL of ADP at 10 µM. Fluorescence with alternative excitation wavelengths, 340 and 380 nm, and an emission wavelength, 500 nm, and aggregation were monitored simultaneously in the platelet suspension (700 µL) at 37°C, using a intracellular ion measurement apparatus (CAF-110, Nihon-Bunkoh, Tokyo, Japan). The ratio of the emitted signals at 500 nm with excitation at 340 and 380 nm was calculated as a parameter of [Ca²⁺]_i. Data are expressed as the proportion to the maximal value caused by ionomycin at 100 µM (for calibration).

Statistical Analysis

Data are expressed as the means±S.E.M. Statistical analyzed significance was analyzed by Bartlett’s test followed by Dunnett’s test or Tukey’s test (Excel Statictics ver. 5, Social Survey Research Information Co., Ltd., Tokyo, Japan), and set at a p<0.05 level.

RESULTS

NaHS Inhibits Rabbit Platelet Aggregation Caused by Collagen or ADP in PRP and Washed Platelet Suspension

In rabbit PRP, the H₂S donor NaHS at 0.3–1 mM and at 0.1–0.3 mM reduced the platelet aggregation induced by collagen at 10 µg/mL and by ADP 10 µM, respectively, showing the “bell-shaped” concentration–response relationship (Fig. 1A). In washed rabbit platelets, NaHS at 0.3–3 mM and 1–3 mM significantly inhibited the aggregation induced by collagen at 3 µg/mL and ADP at 10 µM, respectively, and the concentration–response relationship in washed platelets was not bell-shaped (Fig. 1B). NaHS was thus more potent as an anti-platelet agent in PRP than in washed platelets, whereas it showed greater maximal inhibition in washed platelets than in PRP. Since NaHS even at the highest concentration, 3 mM, only slightly caused a leak of LDH from washed platelets (data not shown), the anti-platelet effect of NaHS at high concentrations was not attributable to the toxic effect.

Fig. 1. Effect of NaHS on Rabbit Platelet Aggregation Induced by Collagen or ADP

Platelet aggregation was induced by collagen 10 µg/mL or ADP at 10 µM in platelet-rich plasma (PRP) (A), and by collagen 3 µg/mL or ADP at 10 µM in washed platelets (B). Data show the mean with S.E.M. of 5–9 experiments. ** p<0.01 vs. vehicle (Dunnett’s test).

Effects of NaHS on the Cytosolic Ca²⁺ Mobilization Induced by Collagen or ADP in Washed Rabbit Platelets

To test if H₂S modulates cytosolic Ca²⁺ mobilization in platelets, [Ca²⁺]_i and platelet aggregation in washed rabbit platelets were simultaneously monitored. NaHS at 0.3–3 mM caused a concentration-dependent inhibition of the increase in [Ca²⁺]_i and platelet aggregation caused by collagen at 3 µg/mL or ADP at 10 µM in parallel (Figs. 2A, B).

Fig. 2. Effect of NaHS on Cytosolic Ca²⁺ Mobilization in Washed Rabbit Platelets

Washed platelets were stimulated with collagen 3 µg/mL (A) or ADP at 10 µM (B), and [Ca²⁺]_i levels and aggregation in platelets were monitored simultaneously. Data show the mean with S.E.M. of 8–10 experiments. * p<0.05, ** p<0.01 vs. the control in the absence of NaHS (0 mM) (Dunnett’s test).

Effects of NaHS on the Ionomycin-Induced Aggregation of Rabbit Platelets

To ask whether NaHS inhibits the downstream signals of cytosolic Ca²⁺ mobilization in rabbit platelets, we examined the effect of NaHS on ionomycin-induced platelet aggregation in washed platelets. In this study, platelets were stimulated with ionomycin at 0.3 µM that was around the EC₅₀ value in our preliminary experiments. NaHS suppressed the ionomycin-induced platelet aggregation in washed platelets (Fig. 3). The effective concentration range of NaHS was equivalent for inhibition of aggregation induced by ionomycin and by collagen or ADP in washed platelets (Figs. 1B, 3).

Fig. 3. Effect of NaHS on Rabbit Platelet Aggregation Induced by Ionomycin

Platelet aggregation was induced by ionomycin at 0.3 µM in washed platelets. Data show the mean with S.E.M. of 6–7 experiments. ** p<0.01 vs. vehicle (Dunnett’s test).

Effects of Inhibition of Adenylyl Cyclase or Phosphodiesterase on the Anti-platelet Effect of NaHS in Rabbit PRP

We next tested possible involvement of the cAMP pathway in the anti-platelet effects of NaHS in PRP. SQ22,356, an inhibitor of adenylyl cyclase, at 100 µM abolished the inhibitory effect of NaHS on platelet aggregation by collagen at 10 µg/mL (Fig. 4A). Dipyridamole, a phosphodiesterase inhibitor, at 30 µM accelerated the anti-platelet effect of NaHS at 0.1 mM, a subeffective concentration, although it alone had no effect on the collagen-induced platelet aggregation (Fig. 4B).

Fig. 4. Effects of SQ22,356, an Adenylyl Cyclase Inhibitor, and Dipyridamole, a Phosphodiesterase Inhibitor on Platelet Aggregation Induced by Collagen in Rabbit Platelet-Rich Plasma (PRP)

Platelets were stimulated with collagen at 10 µg/mL in the presence of SQ22,356 at 100 µM (A) or dipyridamole at 30 µM (B). Data show the mean with S.E.M. of 6–7 experiments. Statistical analyzed significance was analyzed by Tukey’s test.

DISCUSSION

In the present study, we demonstrated that, as it did in human platelets,¹²⁾ NaHS prevented rabbit platelet aggregation induced by collagen or ADP, and that the potency of NaHS as an anti-platelet agent in PRP was much higher than that in the washed platelets, implying that the plasma milieu might affect the effect of NaHS. Our findings that NaHS suppressed cytosolic Ca²⁺ mobilization in response to collagen or ADP, and inhibited the ionomycin-induced platelet aggregation, suggest that NaHS interferes with both upstream and downstream signals of cytosolic Ca²⁺ mobilization. Finally, the results that the anti-platelet effect of NaHS was blocked by the adenylyl cyclase inhibitor and enhanced by the phosphodiesterase inhibitor, point to the critical role of cAMP in NaHS inhibition of rabbit platelet activation. Thus, H₂S donors including NaHS appear to exhibit anti-platelet activity through multiple mechanisms.

The effective concentrations of NaHS in PRP, 0.1–0.3 mM (100–300 µM), shown in the present study are close to the reported H₂S concentrations, 30–300 µM, in the mammalian blood or tissues over the past decade.¹⁷⁾ However, very recent studies using H₂S gas-sensing electrodes or HPLC analysis of head-space gas have indicated that sulfide does not circulate at micromolar levels and is rapidly consumed by blood or tissues.¹⁷⁾ Therefore, the physiological roles of endogenous H₂S in regulation of platelet activity have yet to be clarified. Nonetheless, the therapeutic significance of H₂S donors including ACS14 as anti-platelet or anti-thrombotic agents has been reported.¹⁸⁾ It is particularly of interest that combined application of NaHS and dipyridamole, a commercially available anti-platelet drug, exerted synergistic effects in the present study (see Fig. 4B). It is noteworthy that NaHS was less potent as an anti-platelet in washed platelets than in PRP (see Fig. 1). There are two possibilities to be considered; 1) one is the influence of various substances including fibrinogen present in the plasma and on the platelet surface; 2) the other is the difference in extracellular calcium concentrations, which are very low in PRP that contains approximately 0.38% sodium citrate, but 1.5 mM in washed platelet suspension. Therefore, we have to consider that platelet aggregation in PRP is mainly dependent on the intracellular calcium mobilization from the calcium store, while washed platelet aggregation occurs through intracellular and extracellular calcium mobilization. Thus, it is likely that NaHS at low and high concentrations might inhibit the calcium release from the intracellular store and the extracellular calcium influx, respectively. The reason why NaHS showed a bell-shaped concentration–response curve in PRP, but not washed platelets, is still open to question. In general, concentrations of H₂S greater than 1 mM are toxic for animals including humans, and therefore it may be difficult to apply mM order of NaHS for the therapeutic purposes. In this context, it might be better to consider combined administration of H₂S donors and another anti-platelet agent with different pharmacological properties, such as phosphodiesterase inhibitors including dipyridamole, NO donors or COX inhibitors, although the effective concentration range of NaHS in PRP was 0.1–0.3 mM (Fig. 1A).

An early study using human platelets showed that the inhibitory effect of NaHS at 3 mM on human platelet aggregation in PRP caused by ADP at 2 µM was not inhibited by SQ22,536, an inhibitor of adenylyl cyclase, at 100 µM,¹²⁾ being inconsistent with our present study in which the effect of NaHS at 0.3 mM on rabbit platelet aggregation in PRP caused by collagen at 10 µg/mL (see Fig. 4). Apart from species differences, the discrepancy might be attributable to differences in the concentrations of NaHS (3 mM vs. 0.3 µM) and in platelet stimulating reagents (ADP vs. collagen). Interestingly, the latest study using the H₂S-releasing aspirin, ACS14, has suggested that H₂S actually increases intracellular cAMP levels in human platelets, being consistent with the previous evidence that H₂S activates adenylyl cyclase in neurons and vascular smooth muscle cells^3,13) or inhibits phosphodiesterase.¹⁴⁾ Our study using rabbit platelets strongly indicates the involvement of cAMP in the anti-platelet effect of NaHS. Activation of protein kinase A (PKA) following increased cAMP levels blocks the release of Ca²⁺ from intracellular stores possibly via direct phosphorylation of inositol 1,4,5-trisphosphate (IP₃) receptors.¹⁹⁾ PKA also phosphorylates transient receptor potential channel 6 (TRPC6) that plays a role in store-operated Ca²⁺ entry.¹⁹⁾ These reports are consistent with the present findings that NaHS inhibited the [Ca²⁺]_i increase caused by collagen or ADP in rabbit washed platelets (see Fig. 2). On the other hand, PKA phosphorylates and inhibits the small G-protein Rap1B, a potent regulator of integrin activity, and actin-binding proteins such as vasodilator-stimulated phosphoprotein (VASP) that is involved in fibrinogen binding and platelet aggregation, but not cytosolic Ca²⁺ mobilization.¹⁹⁾ It is noteworthy that ACS14, but not aspirin, causes phosphorylation of VASP in human platelets.¹⁸⁾ These reports are consistent with the present finding that NaHS also blocked ionomycin-induced aggregation in rabbit platelets, suggesting that NaHS interferes with not only the upstream but also downstream signals of cytosolic Ca²⁺ mobilization probably through the cAMP/PKA pathway. Nonetheless, in our preliminary experiments, we could not find appropriate conditions in which elevation of cAMP levels in rabbit PRP following addition of NaHS was detectable (data not shown), whereas our ongoing study still focuses on effects of NaHS on cAMP levels in PRP and washed platelets from rabbits and other species. These experiments would also be useful to clarify the reason for the difference in the potency of NaHS in inhibiting aggregation of PRP and washed platelets.

Most recently, Morel et al. reported that H₂S inhibits the adhesion of human platelets,¹⁵⁾ most probably by changing the adhesive properties of fibrinogen and collagen.²⁰⁾ An independent group has also shown that ACS14, an H₂S-releasing aspirin, but not aspirin, decreases activation of αIIbβ3 integrin, the fibrinogen receptor, and fibrinogen binding.¹⁸⁾ These mechanisms might also contribute to the anti-platelet effect of NaHS in rabbit platelets.

In conclusion, H₂S suppresses rabbit platelet aggregation by interfering with both upstream and downstream signals of cytosolic Ca²⁺ mobilization in a cAMP-dependent manner. Our study thus supports a notion that H₂S-releasing compounds including NaHS and ACS14 may be available for anti-platelet or anti-thrombotic therapy.

Acknowledgments

This research was supported in part by Grant-in-Aid for Scientific Research from Japan Society for the Promotion of Science and by “Antiaging Center Project” for Private Universities from Ministry of Education, Culture, Sports, Science and Technology of Japan (2008–2012).

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