A possible mechanism for the inhibition of blood platelet aggregation by pyridoxal-5'-phosphate.

Pyridoxal-5'-phosphate (PLP) the coenzyme form of vitamin Bg. has been shown to inhibit platelet aggegation [l-31 but the mechanism of its inhibitory action remains to be elucidated. Because of its negative charge and hydrophilic nature PLP does not cross the cell membrane and has been used as a probe for several membrane systems [3]. Since PLP inhibits platelet activation induced by a variety of agonists it was proposed that interaction of PLP with the platelet surface may inhibit a common step(s) crucial for normal platelet function. PLP may affect a primary event(s) involved in signal transduction pathways which would result in an immediate and effective inhibition. Ionised Ca2+ and Na+/H+ exchange play key roles in platelet function and were shown to be major processes in platelet signal transduction [4,5]. In this study we have investigated the effect of PLP on receptor-mediated platelet activation by ADP or thrombin, and on non-receptor-mediated platelet activation by the calcium ionophore Br-A23187. We studied the effect of PLP on two basic platelet functions, namely, mobilisation of Ca2+ and aggregation. We studied the effect of alkalinisation of the platelet cytosol on inhibition of aggegation by PLP. We compared the effects of PLP and dimethyl amiloride (DMA, a specific Na+/H+ exchange blocker) on platelet aggegation. Platelet rich plasma (PRP) was isolated from citrated human blood. Aggregation induced in PRP by ADP (5 pM), thrombin (0.025 unitlml) or Br-A23187 (10-40 pM) was studied in PRP on the Chronolog 430 aggrometer (37 OC). Platelet cytosolic Ca2+ concentration was measured in EDTA-washed platelets prepared as described previously (6), resuspended in Hepes buffer, pH 7.4 and loaded with the fluorescence probe flu0-3lAM. Fluorescence was measured at 37 OC in the Hitachi F-4500 fluorescence spectrophotometer, and recorded at 505 nm excitation and 530 nm emission in the presence of 1 mM external Ca2+ or in a nominally Ca2+-free buffer. Sample fluorescence was measured after the addition of the agonist in presence or absence of PLP (1-5 mM). PLP was dissolved in 0.15 N NaOH and the pH was adjusted to that of the suspending medium. Br-A23187, fluoI3AM and DMA were dissolved in DMSO, monensin was dissolved in ethanol and methylamine in water. PLP( 1-5 mM), monensin(5-80 pM) or methylamine (5-60 mM) were added 30 s before agonist addition. Platelets (PRP) were incubated with DMA for 2 min before agonist addition. There was a concentration-dependent inhibition by PLP of ADP or thrombin-induced aggregation with full inhibition at 5 mM PLP. Likewise PLP also inhibited (but not completely) the ADP or thrombin-induced Ca2+ mobilisation in the platelets. On the other hand PLP did not inhibit platelet aggregation or Ca2+ mobilisation induced by Br-A23 187; rather it effectively potentiated both processes in the presence of extracelluiar Ca2+. Cytosolic alkalinisation imposed on platelets by treatment with monensin or methylamine relieved the PLP inhibition of platelet aggregation by ADP or thrombin. DMA inhibited platelet aggregation induced by ADP or thrombin and potentiated aggregation induced by Br-A23187. The inhibition by DMA was also relieved by treatment with monensin or methylamine On the basis of these results, it may be concluded that (i) PLP had a direct effect on receptor-mediated pathways of platelet activation, and (ii) PLP may have a direct effect on Na+/H+ exchange or on the mechanisms that couple receptor activation to Na+/H+ exchange leading to cytosolic acidification which would facilitate exchange of H+ for Ca2+ mediated by the ionophore in the presence of extracellular Ca2+. This was supported by the finding that artificial alkalinisation overcame the PLP inhibition of platelet aggregation and the finding that DMA had a similar effect on platelet aggregation induced by any one of the above agonists.