Isolation and properties of two forms of thrombin inhibitor from the nymphs of the camel tick Hyalomma dromedarii (Acari: Ixodidae)

Two forms of the nymphal thrombin inhibitors (NTI) 3.2 kDaand 14.9 kDa were purified by chromatography on CM-cellulose,Sephacryl S-300 and Sephadex G-50 columns and designated NTI-1 and NTI-2respectively. The NTI-2 turned out to be homogenous monomeric protein in bothnative-PAGE and denatured SDS-PAGE with M(r) value of 14.9 kDaapproximately and its pI value ranged from 7.2 to 7.5. The NTI-1 and NTI-2displayed anticoagulant activity since they prolonged both the activatedpartialthromboplastin time (APTT) and the prothrombin time (PT) of the camel plasma ina concentration-dependent manner. The potency of NTI-1 toward thrombin was5-fold higher than that toward FXa, while NTI-2 was 3-fold active toward FXathan thrombin. However, both of them did not inhibit any of the other examinedproteases. The types of inhibition of thrombin by NTI-1 and NTI-2 were non-competitive and competitive with inhibition constants (Ki) values of 11.7μM and 211 nM respectively. One binding site wasdeduced on thrombin for each inhibitor.