Vitamin E and platelet function
Platelets play a critical role in the pathophysiology of reperfusion (Gawaz, 2004). Platelet function is not static during ischemia-reperfusion. Instead, during ischemia regional platelet aggregability is increased. Systemic and regional platelet aggregability also increases during myocardial reperfusion. The mechanism of these responses is unknown but may be related to regional endothelial dysfunction created by ischemia. The response observed could also be explained by the release of proaggregatory mediators in the coronary and/or systemic circulation during ischemia-reperfusion (Gurbel et al., 1995).
Reperfusion induces an important inflammatory response, characterized by a massive production of free radicals and by the activation of the complement and leucocyte neutrophils (Gourdin et al., 2009). Platelets and neutrophils act synergistically in provoking postreperfusion cardiac dysfunction (Lefer et al., 1998). Activated platelets play an important role in the process of myocardial ischemia-reperfusion injury, and platelet-derived P-selectin is a critical mediator (Xu et al., 2006), whereas platelet P-selectin promotes platelet interactions with leukocytes. Because platelets release potent proinflammatory chemokines and modulate leukocyte function, platelet accumulation in the postischemic microvasculature might significantly contribute to the manifestation of I/R injury (Massberg et al., 1998).
Reperfusion of the tissue, subsequent to ischemia, results in burst of oxygen consumption with consequent generation of oxygen derived free radicals; the oxidant-anti oxidant status of the tissue is thrown out of balance and multi dimensional free radical mediated damage ensues. Since vitamin E is a potent natural anti-oxidant, its administration is expected to restore the imbalance.
Effect of administration of 600 mg vitamin E each day, for six days, was observed on activity of some of the anti-oxidant enzymes and levels of malondialdehyde (as an index of free radical mediated damage) in the platelets of patients reperfused after myocardial infarction. It has been found that vitamin E administration significantly lowers the level of malondialdehyde in the patients. Vitamin E administration increases the activities of anti oxidant enzymes (superoxide dismutase, glutathione reductase and catalase) tested both in the patients and healthy controls. However, lowering of lipid peroxidation upon administration of vitamin E is specific for patients. These findings exhibit beneficial role of vitamin E administration in the management of the patients reperfused after myocardial infarction (Dwivedi et al., 2005).
The results of Chen et al, (2002) suggested that the reduction of myocardial I/R injury with vitamin E supplementation may be the result of the inhibition of polymorphonuclear neutrophil (PMN) CD11b expression.
Vitamin E supplementation in healthy subjects or patients with hypercholesterolemia was shown to diminish platelet function as assessed by ex vivo platelet aggregation of 11-dehydrothromboxane B2, a marker of in vivo platelet activation (Calzada et al., 1997; Davi et al., 1997). Celestini et al., (2002) demonstrated that vitamin E can potentiate the antiplatelet activity of aspirin by inhibiting the early events of platelet activation pathway induced by collagen.
A study from our laboratory was to examine the possibility that vitamin E administration could exert an effect on blood elements and platelet aggregation.
Materials and Methods
Albino rats of both sexes weighing 180- 220 gm fed on a standard rat diet and fasted for 1824 hours before sacrifice were used in this study.
In vivo study: A total of 30 rats were used in this study. They were divided in two groups. Group I: Saline control group; rats in this group were injected with saline instead of vitamin E, daily for 5 consecutive days. Group II: vitamin E treated group; rats were injected with vitamin E (300 mg/kg b.w.) intraperitoneally for five consecutive days. After 5 days all injections were stopped for 2 days. By the seventh day rats were anesthetized with pentobarbitone in a dose of 40 mg/kg b.w.
Collection of blood samples: blood samples were obtained by arterial puncture from the abdominal aorta. One ml samples were collected into tubes containing EDTA for examination of RBCs and platelet counts, hemoglobin content and hematocrite values.
Another blood samples were collected in chilled plastic tubes containing sodium citrate 3.8gm/100 ml (9 volumes of blood to 1 volume of sodium citrate) and gently shaken. These blood samples were used for study of platelet aggregation. The citrated blood was centrifuged at 1500 r.p.m. for 5 min. The supernatant platelet rich plasma (PRP) was pipetted into clean plastic tubes. The remaining blood sample was centrifuged at 10,000 r.p.m. for 10 min. to prepare platelet poor plasma (PPP). Standard PRP: the number of platelets in PRP was counted using coulter T-660 counter. The platelet number was adjusted to a standardized number of 3 x 105 platelet per ^l by dilution with autologus platelet poor plasma.
Aggregation study: platelet aggregation was performed using Chrono-Log automatic aggregometer (model 540-VC, Chrono-Log Corp, Harvertown, USA) coupled with computer and printer. ADP as an aggregating agent was used at a final concentration of 10 uM. The maximum aggregation was recorded after 3 min.
In vitro of vitamin E on platelet aggregation
Collection of blood samples: blood was collected from normal rats, anaesthetized by pentobarbitone, by arterial puncture from abdominal aorta into chilled tubes containing sodium citrate 3.8 gm%. Preparation of standard PRP was carried out as described in the in vivo experiments.
The in vitro effect of vitamin E on ADP- induced platelet aggregation was studied by exposing PRP to rising concentrations of the vitamin 1,2,3,4 and 5 mg/ ml. Equal volumes of saline were added to control samples.
Aggregation study: was carried as described above in vivo experiments.
Statistical analysis of the data was done using Student’s "t" test for unpaired data according to Fisher and Yates (1957) P < 0.05 was considered significant.
Regression study: linear regression analysis was used to relate different parameters to a certain outcome (platelet aggregation) to find out the highest beta coefficient and the most important factor affecting this outcome. This analysis was performed on SPSS windows version eight.
Results
Table 1 portrays the results of in vivo effects of vitamin E on hematological parameters. RBCs count, Hb content and PCV showed slight and insignificant changes in vitamin E treated rats compared to their saline controls. The number of platelets was insignificantly decreased in vitamin E treated animals. However platelet aggregation induced by ADP showed a significant decrease (P < 0.05) in this group (table 2 and figure 9).
Regression analysis: as seen in fig. 10, multiple regression analysis of platelet aggregation against other parameters. Only a significant negative correlation between platelet number and platelet aggregation was seen in vitamin E treated group (P < 0.03).
In vitro effect of vitamin E on platelet aggregation: the platelet aggregation effect of ADP in presence of rising concentration of vitamin E showed significant inhibition (P <0.01) only when vitamin E was added at a final concentration of 5 mg/ ml. Addition of vitamin E in smaller concentrations of 2- 4 mg/ml final concentration produced insignificant inhibition of platelet aggregation. Almost no effect was seen when vitamin E was added at a final concentration of 1 mg/ml (table 3 and figure 11).
Data are mean± SEM In parenthesis is the number of observations NS: non significant
Table 2. Red blood cell count (RBCs), hemoglobin level (Hb), packed cell volume (PCV), platelet count and platelet aggregation in saline control and vitamin E treated rats.
|
Additions to normal PRP |
|||||
|
|
Final concentration of vitamin E |
||||
|
Saline control |
1mg/ml |
2mg/ ml |
3mg/ ml |
4mg/ ml |
5mg/ ml |
|
(12) |
(8) |
(9) |
(11) |
(11) |
(13) |
|
50.08+2.6 |
50.75+2 |
47.1+2.32 |
44.36+2.85 |
44.7+3.4 |
37.23+3 |
|
P |
NS |
NS |
NS |
NS |
< 0.01 |
Data are mean± SEM In parenthesis is the number of observations NS: non significant
Table 3. In vitro effect of vitamin E on platelet aggregation of normal rat PRP in presence of different concentrations of vitamin E compared to saline control
ADP-induced platelet aggregation%
Fig. 9. Tracing of ADP- induced platelet aggregation of Vitamin E-treated rats (B) compared to saline treated rats (A).
Fig. 10. Correlation between platelet aggregation and platelet count among vitamin E-treated rats and saline treated rats (by multiple regression analysis)
Fig. 11. Tracing of ADP- induced platelet aggregation of normal rats PRP in presence of vitamin E in concentrations of 1, 2, 3, 4 and 5 mg/ml respectively (B- F) compared to saline control (A)
The data reported here demonstrated that administration of megadose of vitamin E (300 mg/kg for one week) to rats, produced slight and nonsignificant changes in red blood cell counts, hemoglobin content, packed cell volume. Platelet counts showed an insignificant decrease. However, the platelet aggregation responses to ADP of PRP from treated rats were significantly inhibited. This finding shows the safety of vitamin E in this supra-physiological dose of 300 mg/kg on blood parameters tested.
On addition of vitamin E to normal PRP in vitro, the platelet aggregating effect of ADP showed significant inhibition only when vitamin E was added at a final concentration of 5 mg/ ml. Addition of vitamin E in smaller concentrations of 2- 4 mg/ ml produced insignificant inhibition of platelet aggregation. Almost no effect was seen when vitamin E was added at a final concentration of 1 mg/ml.
From these data, it can be concluded that this supra physiological dose of vitamin E is safe concerning the blood parameters tested.
The observation in the present study that vitamin E when added in vitro to normal rat PRP caused significant inhibition of platelet aggregation in response to ADP; illustrate that vitamin E by itself exerts a direct antiplatelet effect. Higashi & Kikuchi (1974) were the first to demonstrate that vitamin E inhibits the aggregation of platelets using hydrogen peroxide as the aggregating stimulus. Subsequent studies by Steiner & Anastasi, (1976) demonstrated that vitamin E also inhibited platelet aggregation response to epinephrine, collagen and ATP. Moreover, Freedman and Keaney, (2001) found that platelet incorporation of vitamin E both in vitro and in vivo leads to dose-dependent inhibition of platelet aggregation in response to agonists such as arachidonic acid and phorbol ester.
Although it is best known for its antioxidant activity the exact mechanism of the antiplatelet effect of vitamin E is not exactly known and one of the following mechanisms may operate. First, it can be attributed to altered metabolism of prostaglandins. The vascular generation of prostacyclin (PGI2) is higher and the platelet thromboxane A2 generation is lower than normal. This view is supported by the findings of Steiner and Anastasi (1976) and others (Karpen et al., 1981; Pritchard et al., 1982; Pignatelli et al., 1999) that vitamin E inhibits platelet thromboxane A2 synthesis. On the other hand, PGI2 synthesis is stimulated possibly by reduction of cellular peroxide level (Gilbert et al., 1983). Second, the inhibition of platelet aggregation can be explained by the ability of vitamin E to inhibit intracellular mobilization of sequestrated calcium from the dense tubular system of the cytoplasm (Srivastava, 1986). Third, by its membrane stabilizing action, vitamin E would impair platelet release reaction. This view is supported observations of Feki et al., (2001) that vitamin E by its antioxidant effect, protects molecules and tissue against the deleterious effect of free radicals and also contributes to the stabilization of biological membranes. Fourth, unrelated to its antioxidant action, vitamin E was shown to inhibit protein kinase C (PKC) in various cell types with consequent inhibition of platelet aggregation (Azzi et al., 2002; Freedman et al., 1996; Freedman and Keaney, 2001). Further, vitamin E includes inhibitory effects are the result of specific interactions with component of the cell e.g. proteins, enzymes and membranes (Ricciarelli et al., 2002).
Vitamin E attenuated P-selectin expression on activated human platelets and thus inhibited the P-selectin-dependent function, platelet-mononuclear cell (MNC) interaction. The mechanism probably was related to the inhibition of PKC activity in platelets. Since P-selectin is an important atherothrombogenic adhesion molecule, this finding will provide us new insights into the mechanism by which dietary vitamin E inhibits thrombosis and atherogenesis and thereby reduces the risk of coronary artery diseases (Murohara et al, 2004).
Although these studies from our laboratory have shown that vitamin E administered in megadose, could provide a protective effect against the cardiac responses to the injury of post I/R. Further studies should be conducted to test the possibility of using vitamin E in cardiac surgery.
