Molecular characterization of anti-platelet aggregating proteins in salivary gland extracts of Hyalomma anatolicum ticks

Tick saliva is a cocktail of potent anti-haemostatic, anti-inflammatory and immunomodulatory molecules that help the ticks to obtain a blood meal from their vertebrate hosts. Thrombin is considered as an activator for stimulation of blood platelets at different receptor sites and thrombin induced platelet aggregation depends on the interaction among thrombin exosites and the receptors on platelet membrane. Thus, the present study was planned to isolate and characterize the salivary antiplatelet aggregating proteins from Hyalomma anatolicum ticks. A hundred pairs of salivary glands were dissected out and extract was prepared by fractionation of proteins by gel filtration chromatography. The fractions so obtained were tested for thrombin induced platelet aggregation and platelet-collagen adhesion inhibition activities. Proteins in fraction nos. 28, 29, 30, 31, 32, 42, 58, 111 and 112 were found to inhibit platelet aggregation. These proteins also showed significant inhibitory effects on platelet adhesion to collagen when compared to agonist thrombin. This indirectly indicates inhibition of glycoprotein 1b (GP1b), glycoprotein VI (GPVI) and integrin alpha 2 beta 1 receptors of platelets as these proteins are involved in aggregation of platelets for formation of platelet plug. On electrophoretic separation of fractions by SDS-PAGE, the proteins/peptides present in initial fractions were having approximate molecular weight in the range of 6.5 to 113.4kDa. However, in the fraction no. 112 only single prominent protein band of approximate 65.4kDa was present and is expected to inhibit platelet aggregation possibly through inhibition of platelet receptors such as proteins platelet activating receptors-1 and 4 playing direct role in platelet aggregation. In conclusion, there are possibilities of exploring these anti-platelet aggregating proteins/peptides for therapeutics against cardio-vascular disorders as well as for raising anti-tick vaccine.