Platelet aggregation

There are controversial reports on effects of snake venom on platelet functions.

Venom from variety of snakes has been tested; some snakes inhibit platelet aggregation and others potentiate the process. Nathan I et al, was the first report in 1982 to demonstrate that snake venom from Echis colorata induces impairment of platelet aggregation (Nathan et al., 1982). LAAO present in the snake venom was the shown to be the causative agent. Addition of catalase, completely inhibited the impairment of platelet aggregation (Nathan et al., 1982). This is an indication that hydrogen peroxide produced by the enzyme played an important role in the impairment of platelet aggregation.

Later on Li et al, worked on LAAO from king cobra (Li et al., 1994). They purified LAAO from king cobra and addition of this protein to the platelet rich plasma system completely induced platelet aggregation. This observation was in contrast with the findings of Nathan et al in 1982 (Nathan et al., 1982). Enzyme induced aggregation was completely abolished by the addition of catalase. There are two pathways described for platelet aggregation namely ADP release and formation of thromboxane A2 and prostaglandin endoperoxides. Using of creatine phosphate, which is an ADP scavenging system, did not inhibit the platelet aggregation induced by LAAO (Li et al., 1994). This shows that LAAO induced platelet aggregation is independent of ADP scavenging system. LAAO induced platelet aggregation was completely rescued by the addition of indometacin, aspirin, arachidonic acid (Li et al., 1994). Indometacin or aspirin is a cyclooxygenase inhibitor and this clearly indicates that LAAO mediated platelet aggregation is mediated by formation of prostaglandins or thromboxane A2

(Li et al., 1994). Mepacrine, an inhibitor of the activation of endogenous phospholipase A2, completely inhibited the LAAO induced toxic effect (Li et al., 1994). Therefore, it clearly shows that activation of endogenous phospholipases is necessary for the aggregation process. EDTA, a chelator of calcium ions and verapamil, an inhibitor of calcium influx, inhibit LAAO induced platelet aggregation (Li et al., 1994). Prostoglandin E1 which is an activator of adenylate cyclase, nitroprusside activator of soluble guanylcyclase completely, inhibit LAAO induced aggregation effects by increasing the cyclic AMP and cyclic GMP concentrations (Li et al., 1994). Because cyclic AMP and cyclic GMP inhibit calcium mobilization, and calcium is required for platelet aggregation because it is required for thromboxane A2 synthesis (Li et al., 1994). On a whole, LAAO induces platelet aggregation by formation of hydrogen peroxide, which will in turn activate thromboxane A2 followed by activation of cyclooxygenases, which are prerequisite for platelet aggregation.

A report by Ali et al in 2000 demonstrates that LAAO from leaf nosed viper (Eristocophis macmahoni) induced platelet aggregation in platelet rich plasma in a concentration dependent fashion (Ali et al., 2000). Addition of catalase completely rescued platelets from aggregation. Interestingly, when they used whole venom from the snake, they could observe quite opposite effects, that is inhibition of platelet aggregation (Ali et al., 2000). Takastuka et al in 2001, worked on L-amino acid oxidase from Agkistrodon halys blomhoffii and looked at its effects on platelet aggregation (Takatsuka et al., 2001). They put forward the hypothesis that LAAO did not induce platelet aggregation but, instead, it inhibited the effect. LAAO could inhibit agonist and shear stress induced platelet aggregation effects in a concentration dependent manner. Addition of catalase, an oxidative scavenger, completely quenched the impairment of platelet aggregation (Takatsuka et al., 2001). Shear stress or agonist induced platelet aggregation effects are due to the continuous interaction of platelet integrin alphaIIb/beta3 and fibrinogen. Continuous generation of hydrogen peroxide produced by LAAO, inhibited the interaction between platelet integrin alphaIIb/beta3 and fibrinogen and hence leading to the impairment of platelet aggregation (Takatsuka et al., 2001).These results in turn suggest that bite from the above snake causes prolonged bleeding from the vessels, which, in turn, leads to fatal effects.

Sakurai et al, examined the effect of LAAO from Naja naja kaouthia venom on platelet functions (Sakurai et al., 2001). They noted that LAAO did not induce platelet aggregation at various concentrations. However, it has been shown that it could inhibit the platelet aggregation induced by ADP, collagen or ristocetin in a dose dependent manner. Aggregation induced by shear stress, has been completely inhibited by LAAO. Addition of catalase, completely quenched the event of inhibition of platelet aggregation (Sakurai et al., 2001). These results indicate that hydrogen peroxide produced by the enzyme is the main governing factor. Another report was about L-amino acid oxidase from Trimeresurus jerdonii snake venom, which has been shown to induce platelet aggregation in platelet rich plasma system. Addition of catalase rescued platelets from aggregation (Lu et al., 2002).

Stabeli et al in 2004, reported that L-amino acid oxidase from Brothropus alternus snake venom known as Balt LAAO-1 could induce platelet aggregation in platelet rich plasma and also in washed platelets in a concentration dependent manner (Stabeli et al., 2004). Recently, Toyama et al, demonstrated that LAAO from Crotalus durissus cascavella venom induced platelet aggregation in platelet rich plasma in a dose dependent manner (Toyama et al., 2006). Addition of catalase rescued platelets from undergoing aggregation. Apart from that, they observed that indometacin, which is an inhibitor of activation of endogenous phospholipase A2 could block the effect of platelet aggregation induced by LAAO (Toyama et al., 2006). Aspirin, which is a general inhibitor of cyclooxygenase pathway was able to inhibit aggregation induced by LAAO (Toyama et al., 2006). These results suggest that hydrogen peroxide generated by LAAO is the main causative agent, which in turn activates inflammatory enzymes such as thromboxane that leads to platelet aggregation.

To conclude, LAAOs from the snake venom potentiates platelet aggregation and at the same time can inhibits the aggregation process. Literature reports suggest that platelet aggregation induced by LAAO is mainly due to the production of hydrogen peroxide, as the addition of catalase, completely rescues platelets from aggregation.

There are also possibilities that produced hydrogen peroxide may activate other inflammatory enzymes that are responsible for platelet aggregation. This was evident

as the addition of inhibitors of phospholipases could inhibit the platelet aggregation induced by LAAO. Before coming to specific conclusion, it would be worthwhile to see, if these specific inhibitors could inhibit the activity of L-amino acid oxidases.

Impairment of platelet aggregation by LAAOs has been observed in some snake venoms and produced hydrogen peroxide is the main factor for the impairment.

Oxidative scavengers completely abolish the impairment. On a whole, LAAOs are unique enzymes with efficient physiological functions. A thorough understanding of the mechanisms of their action on platelet functions could lead to a better understanding of the snake venom.