''Biochemical studies on blood anticoagulant during the developmental stages of camel tick Hyalomma dromedarii"

Abstract

Six anticoagulants were isolated from the camel tick embryos by ion-exchange chromatography on DEAE-cellulose column and designated Pl, P2, P3, P4, PS and P6. They were purified by gel filtration on Sephadex G-50 with molecular weights ranged from 390 to 790 dalton. All of them reacted positively with the ninhydrin and displayed a maximum ultraviolet absorption between 205 and 215 nm. The embryonic peptide anticoagulant (P6) was the most potent anticoagulant since it inhibited the activity of both thrombin and FXa. The types of inhibition of thrombin and FXa by P6 anticoagulant were competitive and uncompetitive with inhibition constants (Ki) values of 66.08 and 65.16 pM respectively.

Two forms of thrombin inhibitors (NTI-1 and NTI-2) were purified from nymphs of the camel tick H. dromedarii by chromatography on CM-cellulose, Sephacryl S-300 and Sephadex G-50 columns, with molecular weights 3286 ± 56 Da and 15484 ± 416 Da respectively. The NTI-2 was a homogenous preparation and a single polypeptide chain with a molecular mass of 13620 ± 247 Da as detected from native and SDS-PAGE, its pi value ranged from 7.2 to 7.5. Both thrombin inhibitors NTI-1 and NTI-2 prolonged both the activated partial thromboplastin time (APIT) and the prothrombin time (PT) of the camel plasma in a concentration-dependent manner. The potency of NTI-1 toward thrombin was 5-fold higher than that toward FXa, while NTI-2 was more potent toward FXa than toward thrombin (3-fold). However, both NTI-1 and NTI-2 did not inhibit any of the proteases. The types of inhibition of thrombin by NTI-1 and NTI-2 were non competitive and competitive with inhibition constants (Ki) values of 26.04 and 14.05 JlM respectively.

One form of FXa inhibitor was purified from nymphs of the camel tick H. dromedarii by chromatography on DEAE-cellulose and Sephacryl S-300 columns with molecular weight of 14108 ± 379 Da. The homogenous preparation was a single polypeptide chain with a molecular weight of 13820 ± 130 Da and pi value ranged from 7.6 to 7.9. It prolonged both the activated partial thromboplastin time (APIT) and the prothrombin time (Pf) of the camel plasma in a concentration-dependent manner. Its activity was 3 fold lower toward thrombin than FXa, but it did not inhibit any of the proteases. The type of inhibition of FXa by the nymphal FXa inhibitor were uncompetitive with inhibition constant (Ki) value of 1.137 JtM.

The results of this study will contribute in two main trends; (a) vaccination of the host against the tick by the purified anticoagulants, (b) therapeutic purposes of thrombosis.