Effects of methanol and formic acid on human platelet aggregation

Alcohol drinking is known to have diverse effects on the risk of cardiovascular disease that depend on the amount of ethanol consumed; the risk is reduced by moderate drinking but is increased by excessive drinking [1, 2]. The mechanism of the former beneficial effect is explained primarily by ethanol-induced increases in blood HDL cholesterol levels [3]. Attenuation of blood coagulability by drinking also lowers cardiovascular risk. Inhibition of platelet aggregation by ethanol [4, 5] is known to underlie the decreased blood coagulability in drinkers compared with nondrinkers, as is the decrease in blood levels of coagulation factors, including fibrinogen, in drinkers [6].

Methanol, another variant of alcohol, is used often in industry and is known to cause optic nerve injury and metabolic acidosis through the production of formic acid in vivo [7]. However, to the best of our knowledge, there have been no reports on effects of methanol and its metabolite, formic acid, on platelet function. Brain hemorrhage has been observed in 13.5% of patients with methanol intoxication [8]. Thus, there is a possibility of blood coagulation disorder caused by methanol intoxication.

Transplasmalemmal Ca²⁺ entry is a crucial step in the platelet activation process [9]. Our previous study demonstrated the diverse effects of ethanol on transplasmalemmal Ca²⁺ entry into the platelets through various pathways [10]. The purpose of the present study was to determine whether and how methanol and formic acid affect Ca²⁺ entry into and subsequent aggregation of platelets following different stimuli. For this purpose, we used three known platelet stimulants: thrombin, thapsigargin, and 1-oleoyl-2-acetyl-sn-glycerol (OAG).

Although environmental and toxic concentrations of methanol did not affect platelet aggregation and Ca²⁺ entry into platelets, methanol at pharmacological doses showed diverse effects on platelet aggregation that depended on the aggregating stimulant. Thrombin-induced aggregation was increased by methanol (0.5–2%), while thapsigargin-induced aggregation was inhibited by a higher concentration (2%) of methanol but was not significantly changed by lower concentrations. Platelet aggregation is known to depend strongly on Ca²⁺ entry [9], and thus we suspected that Ca²⁺ entry pathways would be affected by methanol. In this study, we used three platelet activating stimulants that act through different mechanisms. Thrombin activates its receptors, resulting in hydrolysis of phosphoinositides and subsequent production of two signal transduction messengers, inositol trisphosphate and diacylglycerol. The former induces mobilization of Ca²⁺ from its intracellular stores, which triggers transmembranous Ca²⁺ entry, so-called capacitative Ca²⁺ entry, through TRPC (transient receptor potential canonical) 1/4/5 channels [17]. The latter facilitates TRPC3/6/7 channel-mediated Ca²⁺ entry [18, 19]. Thapsigargin and OAG were used as mimics of inositol trisphosphate and diacylglycerol, respectively. However, methanol did not affect Ca²⁺ entry induced by any of the stimulants used in this study. Therefore, the effects of methanol on platelet aggregation are thought to be independent of Ca²⁺ entry, and methanol is suggested to affect signaling pathways in platelets that are downstream of the increase in intracellular Ca²⁺ concentrations. Further studies are needed to clarify the mechanisms for the diverse effects of methanol on platelet aggregation induced by thrombin and thapsigargin.

Blood concentrations of methanol in its acute intoxication have been reported to be about 0.2% [13, 14]. Thus, the concentrations of methanol that exerted significant actions on platelet aggregation in the present study were higher than those attained under an environmental condition and those observed in patients with acute methanol intoxication. Therefore, it is unlikely that methanol influences blood coagulability in vivo through its direct action on platelet aggregation even under a toxic condition.

On the other hand, formic acid, the final metabolite of methanol in vivo, at toxic concentrations showed inhibitory effects on platelet aggregation and corresponding Ca²⁺ entry in platelets in response to thrombin and thapsigargin. Therefore, formic acid inhibits platelet aggregation through attenuating capacitative Ca²⁺ entry. Since thrombin is a physiological agonist for platelet aggregation, there is a possibility that bleeding tendency due to attenuation of platelet aggregation occurs in patients with methanol poisoning, and further clinical studies are needed to test this hypothesis. Platelet aggregation induced by OAG was also attenuated in the presence of formic acid, while corresponding Ca²⁺ entry in platelets induced by OAG was not affected by formic acid at lower concentrations (0.005 and 0.01%) and was rather augmented by formic acid at a higher concentration (0.02%). Thus, in addition to the inhibitory action on capacitative Ca²⁺ entry, there may be another inhibitory mechanism, besides action on Ca²⁺ entry, for formic acid-induced inhibition of platelet aggregation. This is the first study showing the inhibitory action of formic acid on Ca²⁺ entry into platelets and subsequent aggregation of them. Further studies are also needed to clarify the Ca²⁺ entry-independent pathway for platelet aggregation that is inhibited by formic acid.

The pH of the solution suspending platelets was lowered by the addition of formic acid (0.005–0.02%) in a concentration-dependent manner. We previously reported that thapsigargin-induced Ca²⁺ entry into and subsequent aggregation of platelets were decreased under the condition of extracellular acidosis (pH 6.9) and were increased under the condition of extracellular alkalosis (pH 7.9) [20]. Thus, platelet aggregation induced by thapsigargin depends on extracellular pH in the range of pH from 6.9 to 7.9. Interestingly, the degrees of inhibition of Ca²⁺ entry and subsequent aggregation by extracellular acidosis (pH 6.9) [20] were comparable to the degrees of inhibition by formic acid at 0.01% (Figs. 4 and 5), which caused a similar (slightly lower) degree of intracellular acidosis (pH 7.017 ± 0.013) in the present study. Therefore, we concluded that formic acid-induced inhibition of Ca²⁺ entry and subsequent aggregation resulted mainly from extracellular acidosis induced by formic acid. This explanation is plausible because metabolic acidosis occurs in patients with severe methanol poisoning [21].

Table 1 compares the effects of methanol and ethanol on Ca²⁺ entry and subsequent platelet aggregation. In our previous study, ethanol at its toxic concentrations (0.5–2%) inhibited platelet aggregation in a concentration-dependent manner irrespective of platelet stimuli [10]. The effects of ethanol on Ca²⁺ entry were complicated. Ethanol decreased Ca²⁺ entry induced by thapsigargin and increased that induced by OAG and did not significantly affect that induced by thrombin [10]. Thus, ethanol has diverse actions on Ca²⁺ entry mediated by TRPC1/4/5 and TRPC3/6/7 channels, and these actions were thought to be canceled when platelets were stimulated with thrombin, which activates both TRPC1/4/5 and TRPC3/6/7 channels. In addition to these actions on Ca²⁺ entry, ethanol has an inhibitory effect on platelet aggregation independent of Ca²⁺ entry. Thus, the effects of ethanol on Ca²⁺ entry into and aggregation of platelets are quite different from the effects of methanol reported in the present study. Figure 6 shows representative recordings of thrombin-induced platelet aggregation in the presence of the same concentration (2%) of methanol and ethanol, which increased and decreased the aggregation, respectively. Since both methanol and ethanol are membrane-permeable, they are speculated to affect not only different types of Ca²⁺ channels in the plasmalemma but also various intracellular organelles. Further studies are needed to determine the reasons for the differing actions of methanol and ethanol on Ca²⁺ entry into and aggregation of platelets, and to clarify how these actions are related to the differences in the structures of these alcohol variants.

Methanol, at pharmacological doses (0.5% or higher) but not at toxic doses, had diverse actions on platelet aggregation—levels of aggregation induced by thrombin and thapsigargin were increased and decreased, respectively, by methanol. On the other hand, methanol showed no significant effects on Ca²⁺ entry in platelets. Thus, methanol has diverse effects on platelet aggregation, depending on the aggregation stimuli, without affecting Ca²⁺ entry into platelets. Formic acid at toxic doses (0.01% or higher) showed significant inhibitory actions on platelet aggregation and corresponding Ca²⁺ entry in platelets, which may be caused by extracellular acidosis. Therefore, it is suggested that, in methanol intoxication, platelet aggregation is not affected by methanol itself but is inhibited by its metabolite, formic acid, resulting in bleeding tendency, which may be involved in etiology of complicated brain hemorrhage [8].