Inhibition of platelet aggregation by vanilloid-like agents: investigation of possible mechanisms

Platelets are non-nucleated cell that play a central role in maintaining the haemostatic process. They also contribute to thrombotic events, and the initiation and progression of atherosclerosis. Antiplatelet medications such as aspirin have been shown to have a beneficial effect in the primary and secondary prevention of cardiovascular diseases. However, their use can have significant side effects, including gastrointestinal ulceration, gastritis and bleeding. It has been shown that vanilloid-like agents, including plant-derived vanilloids (capsaicin (CAP) and dihydrocapsaicin (DHC)), and endogenous vanilloids (Narachidonoyl-dopamine (NADA) and N-oleoyldopamine (OLDA)), individually inhibit in vitro aggregation in platelets obtained from healthy donors. Thus, the overall aim of this thesis was to investigate the possible mechanism(s) by which vanilloids inhibit platelet aggregation, both individually and in combination. This is important specially since some studies have shown that CAP has a gastro-protective effect against mucosal damage induced by aspirin, indomethacin and ethanol. Moreover, regular chilli consumption has been reported to decrease the incidence of peptic ulcers, control glucose and insulin levels, making chilli a potential nutraceutical. This thesis consists of six chapters. Chapter 1 is a review and critique of the literature on platelet structure and function, transient receptor potential vanilloid-1 (TRPV1) channels and cannabinoid (CB) receptors in health and disease, vanilloids and their clinical applications, the manifestations of systemic lupus erythematosus (SLE), and the role platelets in SLE. Chapters two through five present the related background, research experiments and data generated that detail the mechanistic investigations of the inhibitory effects of vanilloids on platelet function. The last Chapter 6 is a comprehensive discussion of all the investigations conducted during candidature, and includes directions for future work stemming from this research. Vanilloid-like agents mediate their actions on neurons and other cells through TRPV1 channels and/or CB receptors. The aims of the investigations described in Chapter 2 were to firstly to confirm TRPV1 expression on human platelets, and then to determine whether vanilloid inhibition of in vitro platelet aggregation was receptor mediated, i.e., through TRPV1 channels, and/or CB1 or CB2 receptors. Platelets were obtained from healthy volunteers for all experiments unless otherwise stated. Expression of TRPV1 in platelets was confirmed, although to my knowledge, this is the first time this has been demonstrated using confocal microscopy. Furthermore, the inhibitory effects of vanilloids on in vitro platelet aggregation induced by collagen, Adenosine diphosphate (ADP) and arachidonic acid (AA) were found not to be TRPV1-, CB1- or CB2- receptor mediated. However, blocking TRPV1 and CB2 receptors appear to enhance OLDA and CAP inhibitory action on platelets. After excluding a definite role for TRPV1, CB1 and CB2 receptors in the action of vanilloid on platelets, other possible mechanisms were investigated in Chapter 3. My previous Masters work showed that the endovanilloids, OLDA and NADA, as well as the high concentrations (100-25 ˜í¬¿M) of plant-derived vanilloid, CAP, significantly inhibit in vitro ADP-induced platelet aggregation. Furthermore, CAP, DHC and NADA inhibited AA-induced aggregation, whereas NADA and OLDA only inhibited aggregation induced by a low concentration of collagen (4 ˜í¬¿g/mL, but not 8 ˜í¬¿g/mL). Thus, the focus of this thesis was whether vanilloids exert their action on platelets by interfering with 1) ADP receptors by measuring vasodilatorstimulated phosphoprotein (VASP) phosphorylation level, dense- (5- hydroxytryptamine (5-HT)) release, and/or ˜í¬±-granules (platelet factor 4 (PF4) and ˜í‚â§- thromboglobulin (˜í‚â§-TG)) release, and/or 2) the AA metabolic pathway in platelets. Furthermore, the effect of vanilloids on platelet-derived microparticles (PMP) was also determined. NADA significantly increased VASP phosphorylation (17% ¬¨¬± 2.2, p<0.05) compared to control (no treatment control), indicate a potential involvement of ADP receptor. In addition, OLDA also increased VASP phosphorylation by 13.4%¬¨¬± 2.7, p=0.12 but the result was not statistically significant. However, none of the vanilloids tested produced a significant effect on PF4, ˜í‚â§-TG or 5-HT release from ADP-activated platelets. Under AA stimulation, thromboxane B2 (TXB2) formation decreased significantly in the presence of 50 ˜í¬¿M CAP (10.7%, p<0.001), whereas in the presence of 50 ˜í¬¿M DHC, the decrease in TXB2 was not significant (4.6%, p=0.8). In contrast, OLDA and NADA had no effect on TXB2 formation compared to AA alone. In ADP- and AA- timulated platelets, vanilloids had no effect on PMP release. Taken together, these results suggest that NADA and possibly OLDA inhibits in vitro platelet aggregation through interference with the ADP receptor, P2Y12, as VASP phosphorylation increased significantly in its presence. Moreover, CAP and perhaps DHC, inhibit the AA pathway, as TXB2 formation was significantly decreased. Finally, no changes in circulating PMP in the presence of CAP, DHC, OLDA and NADA were observed, suggesting that they do not affect the pathway that leads to PMP formation. Although the precise mechanism(s) that produce PMP is (are) not well understood, alterations in phospholipid symmetry and cytoskeleton rearrangement appear to be essential. Pepper fruits (chilli) are the main source of vanilloids, CAP and DHC, which are usually present in 60:40 ratio. In Chapter 4, the effects of CAP and DHC both individually, and in combination (CAP:DHC, 60:40), on AA-, ADP-, and collageninduced in vitro platelet aggregation were investigated and compared. Additionally, their effects on platelet count and TXB2 formation were determined to assess the combination toxicity toward platelets and their mechanism of action, respectively. Under AA stimulation, 12.5 ˜í¬¿M CAP and DHC inhibited aggregation by 23.2% and 25.3%, respectively compared to control (both p<0.01). Interestingly, combination of CAP and DHC (7.5:5 ˜í¬¿M) produced further inhibition 57.5%, p<0.001, compared to control (no treatment control). However, CAP and DHC individually, and in combination, had no effect on ADP- or collagen-induced platelet aggregation. Incubation of platelets with vanilloids, individually or in combination, did not significantly affect the platelet count. The 60:40 CAP:DHC (7.5:5 ˜í¬¿M) combination significantly inhibited (p<0.001) TXB2 formation compared to the individual vanilloids. These results indicate that CAP and DHC in combination act synergistically to inhibit the AA metabolic pathway. Therefore, the combination of chilli pepper-derived vanilloids exhibits a stronger antiplatelet effect than the individual vanilloids, which opens up a new opportunity for research. Finally, a pilot study was conducted that investigated the effect of vanilloid-like agents, CAP, DHC, OLDA and NADA (0-50 ˜í¬¿M) on ADP-(5 ˜í¬¿M) and collagen-(4 ˜í¬¿g/mL) induced aggregation of platelets obtained from SLE patients (Chapter 5). As all patients were on non-steroidal anti-inflammatory drugs, AA-induced aggregation has been excluded. These patients have a higher risk of thrombotic events and the development of atherosclerosis compared to the general population that may be associated with enhanced platelet activation. CAP, DHC, OLDA and NADA, in contrast to their inhibitory effects on platelets from healthy individuals, had no effect on ADP-induced aggregation of platelets from SLE patients. Similarly, CAP, DHC and OLDA did not influence aggregation induced by collagen. However, NADA inhibited collagen-induced aggregation in a concentration-dependent manner (0 vs 50 ˜í¬¿M; %AUC, 44.8¬¨¬±6.5 vs 34.7¬¨¬±7.8, p<0.001) and (%MAX, 60.8¬¨¬±7.7% vs 37.8¬¨¬±9.7%, p<0.001 n = 5). These results suggest that the pathway(s) through which CAP, DHC and OLDA inhibit platelet aggregation in healthy platelets may be impaired in SLE, and/or affected by the medications used to treat the manifestations of SLE. In summary, through these studies I have generated new data and knowledge on the mechanism of action of vanilloid-like agents on platelets and platelet aggregation. The major outcomes are as follows. First, TRPV1 expression on platelets has been confirmed using confocal microscopy. Second, inhibition of in vitro platelet aggregation by vanilloid-like agents appears to be independent of a direct interaction with TRPV1 channels or CB receptors. Third, CAP and DHC appear to inhibit in vitro platelet aggregation by interfering with the AA-pathway, whereas NADA and OLDA do so by interfering with and/or blocking the ADP receptor, P1Y12. Fourth, CAP, DHC, NADA and OLDA have no effect on the release of PMP from ADP- and AA- stimulated platelets. Fifth, low concentrations of a CAP and DHC combination (60:40) have a greater inhibitory effect on in vitro platelet aggregation through the AA-pathway, compared to a higher concentration of the individual vanilloid. Finally, unlike findings in platelets from healthy individuals, only NADA appears to have an inhibitory effect on platelets from patients with SLE. The data presented in this thesis can be used as a basis for the design of future studies that may investigate the effect of vanilloid-like agents using in vivo models as well as on other patients with high risk of thrombosis and atherosclerosis to test the viability of chilli pepper as nutraceutical.