Results of the present study clearly demonstrate the inhibitory effect of tocotrienols on in vivo platelet thrombus formation and ex vivo platelet aggregation using the Folt's stenosed canine coronary artery model. Significant and marked inhibition of collagen- or ADP-induced platelet aggregation was observed with α-tocotrienol and TRF (α-, + γ-, + δ-tocotrienols) treatments. This inhibition exceeded that achieved with α-tocopherol under similar conditions (Figs. 2, 3). This is the first report in which the effects of tocotrienols on platelet aggregation in vivo and ex vivo have been evaluated using the Folts' Model . Although α-tocopherol, α-tocotrienol and TRF treatments were all effective in inhibiting collagen- and ADP-induced platelet aggregation in platelet rich plasma, the inhibition achieved with TRF was significantly greater than that observed with either α-tocopherol or α-tocotrienol. As mentioned earlier, the TRF used in this study contained α-, γ- and δ-tocotrienols (15%, 25%, and 60%, respectively), and was free of α-tocopherol.
Treatment with all of the tocols produced significant and pronounced increases in tocopherol concentrations in plasma and platelets. The effect of tocol treatments on tocotrienol levels was much more modest. It is important to recognize that α-tocopherol is the major form of vitamin E in plasma and platelets, even after treatment with pure α-tocotrienol or TRF25, due to bioconversion of tocotrienols to α-tocopherol [33–39, 41]. The conversion of tocotrienols to α-tocopherol in plasma or platelets following IV injection in the current study suggests that uptake of tocotrienols by plasma or platelets is relatively rapid in the Folts model. To our knowledge, the time dependency of tocotrienol uptake in plasma or platelets has not been reported, and the results reported herein suggest that the majority of tocotrienols are ultimately converted to tocopherols as reported earlier [35, 37, 41], although the site of this conversion cannot be determined from the present data. Moreover, it is also unclear if the biological effects described above (i.e. decreased platelet aggregation, abrogation of CRF's) are secondary to tocotrienols, tocopherols, or a combination of both. The magnitude of the increase in plasma or platelet tocopherol concentrations in the present study is greater than that achieved in most dietary supplementation studies using α-tocopherol or tocotrienols, and it could be speculated that tocotrienols are utilized more efficiently than tocopherols by plasma or platelets. The increase in plasma tocopherols was also greater than that seen in most dietary supplementation studies using various formulations of α-tocopherol in TRF25 or Palmvitee capsules [35, 37, 41]. Whether the greater plasma and platelets levels of tocopherols in the present study are the result of a unique effect of tocotrienols, the intravenous route of administration, the formulation used (TRF25, Palmvitee), or an effect of improved recovery during assay of these vitamin E compounds, cannot be determined from these data.
Although, the present study does not delineate the mechanism of action of tocopherols or tocotrienols on platelet function, previous reports have suggested that α-tocopherol inhibits platelet thromboxane A2 production, increases vascular PGI2 production, inhibits the platelet release reaction, inhibits platelet calcium mobilization, alters platelet membrane fluidity, and inhibits platelet phospholipase A2[46, 47]. It has been also demonstrated that tocotrienols are highly effective at reducing expression of adhesion molecules on endothelial cells and inhibiting monocyte adhesion to endothelial cells .
Recently, it was reported that α-tocopherol inhibits platelet-mononuclear cell interaction, platelet aggregation and platelet protein kinase activity induced with either phorbol 12-myristate 13-actate or thrombin in humans . Dietary supplementation of α-Tocopherol significantly inhibited the superoxide production, lipid oxidation, IL-1β secretion monocyte-endothelial cell due to inhibition of protein kinase activity . In humans, α-Tocopherol also partially inhibits platelet protein kinase C (PKC), and this action of α-tocopherol on platelet function provides new insights into the anti-thrombotic and atherogenic properties of α-tocopherol .
Moreover, the mixture of α-, γ-, δ-tocopherols were more effective in preventing platelet aggregation as compared to α-tocopherol alone observed in humans . This inhibition of platelet aggregation was associated with increased release of nitric oxide due to activation of endothelial constitutive nitric-oxide synthase and protein kinase C . Similarly, the effects of α-tocopherol and γ-tocopherol differ with respect to low-density-lipoprotein oxidation, superoxide activity, platelet aggregation and arterial thrombogenesis in human studies . γ-Tocopherol is more potent than α-tocopherol in these effects also . However, most studies only show an effect in cultured cells or under ex vivo conditions. Importantly, cell culture studies are often conducted under conditions of vitamin E deficiency. This might partially explain the inconsistency observed between cell culture studies and studies performed in animals or humans .
Combined treatment of diabetic rats with α-tocopherol and acetylsalicyclic acid (aspirin) had a greater inhibitory effect on platelet aggregation, and reduced nitric oxide production, than either treatment alone [47, 54]. Combination therapy also improved balance of thromboxane and prostacyclin compared to untreated diabetic rats [47, 54]. Consequently, this combination therapy appears to induce beneficial physiologic changes that may protect tissues from detrimental thrombotic and ischemic phenomena . It has previously been demonstrated that tocopherol levels in platelets are depressed in diabetic subjects and that these low levels may contribute to the increased incidence of atherosclerosis and thrombotic events in diabetic patients [55–57]. Supporting this concept is the demonstration that dietary α-tocopherol has been demonstrated to reverse abnormalities of platelet function in diabetic rats and patients [49–52]. The above mentioned properties and other positive biological effects of tocopherols have been reviewed comprehensive by Reiter et al. .
Recently, tocotrienols were found to be potent neuroprotective agents against stroke [31, 59]. Specifically, incorporation of tocotrienols into the diet of hypertensive rats protected them against stroke-induced injury [31, 59]. This protective property of tocotrienols was due its inhibition of pp66 (c-Src gene) kinase activation and 12-Lipoxygenase, which protect against glutamase- and stroke-induced neurodegeneration . This protective effect of tocotrienols (in nanomolar concentrations) is independent of their antioxidant activity because tocopherols were effective only at higher (micromolar) concentrations . Recently, it was reported that γ-tocotrienol was the most cardioprotective of all the isomers, followed by α- and δ-tocotrienols . It was also suggested that, although these isomers possess comparable antioxidant properties, their abilities to potentiate signal transduction could be different . On the other hand, our previous and recent findings showed that tocotrienols exhibit varying degrees of biological activity with δ-tocotrienol showing the most potency, followed by γ-tocotrienol, and then by α-tocotrienol [23, 29]. The present results show that a mixture of tocotrienols containing mainly γ-, and δ-tocotrienol, is more potent than α-tocotrienol with respect to inhibition of collagen- or ADP-induced platelet aggregation. Further studies are required to clarify the potency of γ-tocotrienol vs δ-tocotrienol.