Platelet aggregation inhibitors from hematophagous animals

Abstract

Salivary glands from blood-sucking animals (e.g., mosquitoes, bugs, sand flies, fleas, ticks, leeches, hookworms, bats) are a rich source of bioactive molecules that counteract hemostasis in a redundant and synergistic manner. This review discusses recent progress in the identification of salivary inhibitors of platelet aggregation, their molecular characterization, and detailed mechanism of action. Diversity of inhibitors is remarkable, with distinct families of proteins characterized as apyrases that enzymatically degrade ADP or as collagen-binding proteins that prevent its interaction with vWF, or platelet integrin α2β1 or GPVI. Molecules that bind ADP, TXA(2), epinephrine, or serotonin with high affinity have also been cloned, expressed, and their structure determined. In addition, a repertoire of antithrombins and an increasingly number of RGD and non-RGD disintegrins targeting platelet αIIbβ3 have been reported. Moreover, metalloproteases with fibrinogen(olytic) activity and PAF phosphorylcholine hydrolase are enzymes that have been recruited to the salivary gland to block platelet aggregation. Platelet inhibitory prostaglandins, lysophosphatydilcholine, adenosine, and nitric oxide (NO)-carrying proteins are other notable examples of molecules from hematophagous salivary secretions (herein named sialogenins) with antihemostatic properties. Sialogenins have been employed as tools in biochemistry and cell biology and also display potential therapeutic applications.

Fig. 1

Platelet activation. Pro-aggregatory molecules ADP, thrombin, and collagen activate platelets through specific receptors, leading to PLA2 and PLC activation, granule release, production of TXA2 and activation of integrin αIIbβ3. Inhibitory signals include endothelial apyrase (not shown), prostacyclin, and NO (for details see text).

Fig. 2

Platelet inhibition by sialogenins. (A) ADP-binding proteins, e.g., RPAI-1. Low concentrations of ADP (< 0.5 μM)-induced platelet aggregation including shape change is dose-dependently inhibited by RPAI-1 which has only marginal effects at higher concentrations of ADP (> 1 μM) (Francischetti, et al., 2000). (B) Collagen (1 μg/ml)-induced platelet aggregation is attenuated by RPAI-1 without abolishing shape change (Francischetti, et al., 2002). (C) TXA2-binding protein, e.g., moubatin. Collagen-induced platelet aggregation is attenuated by moubatin without abolishing shape change (Mans and Ribeiro, 2008). (D) Serotonin-binding protein, e.g., ABP. Serotonin (0.5 μM)-induced potentiation of aggregation by low doses of collagen (0.75 μg/ml) is blocked by ABP (0.8 μM) (Andersen, et al., 2003). (E) Collagen-binding protein, e.g., aegyptin. Aggregation induced by collagen (2 μg/ml) is accompanied by delay in onset time for shape change at 60 nM aegyptin; no shape change or aggregation is observable at higher concentrations of the inhibitor (Calvo, et al., 2007). (F) NO-delivery, e.g., nitrophorins. ADP (3 μM)-induced platelet aggregation and shape change is attenuated by NP2 (0.4 μM) when added before stimulus. NP2 also promotes disaggregation when added (arrow) after addition of ADP (Andersen, et al., 2004). (G) Disintegrin targeting αIIbβ3; e.g., monogrins. ADP (20 μM)-induced platelet aggregation is prevented by monogrins (0.3 μM), while initiation of shape change is not affected (Mans, et al., 2008). Concentrations of inhibitors are given next to each tracing of platelet aggregation. ΔT, transmittance.

Fig. 3

Disintegrin from blood-sucking arthropods. (A) Clustal alignment for variabilin (Wang, et al., 1996) and ixodegrin (gi 51011476) (Francischetti, et al., 2005, Francischetti, et al., 2009) (B) Clustal alignment and putative disulfide pattern for savignygrin (gi 21435983) (Mans, et al., 2002), disagregin (gi 545738) (Karczewski, et al., 1994), and monogrin (gi 114152966) (Mans, et al., 2008). (C) The predicted secondary folding for savignygrin is shown. The RGD sequence is indicated in gray.

Fig. 4

Hematophagy and inhibition of platelet aggregation. Platelets adhere to collagen at sites of vascular injury through vWF, GPIb and specific receptors collagen receptors GPVI and integrin α2β1. This event is accompanied by release of secondary mediators (e.g., ADP, epinephrine and serotonin) and generation of TXA2. During this process, thrombin may be produced through exposure of tissue factor. These agonists activate the signaling pathway, culminating with activation of integrin αIIbβ3, which binds fibrinogen (Gross and Weitz, 2009, Watson, et al., 2005). The target for each family of inhibitor is indicated by a number. The target for PAF phosphorylcholine hydrolase is not shown. The name of each inhibitor is shown beside each picture exemplifying a different hematophagous animal found in different genus and/or species. Epi, epinephrine; 5HT, serotonin.