Purification and characterization of a thrombin inhibitor from the salivary glands of a malarial vector mosquito, Anopheles stephensi.

ABSTRACT

A coagulation inhibitor was identified and isolated from the salivary glands of a malarial vector mosquito, Anopheles stephensi. The salivary gland extract prolonged activated partial thromboplastin time (APTT) and prothrombin time (PT) in assays with human plasma. The inhibition assay of the factors in the coagulation cascade by using synthetic chromogenic substrates showed that the anticoagulant in the mosquito salivary glands is a thrombin inhibitor, but not an inhibitor of factor Xa. The anticoagulant was purified to homogeneity from the mosquito thorax which contains the salivary glands by means of a combination of thrombin affinity and anion exchange chromatography. All of the anticoagulant activity was recovered from the fraction bound to the thrombin affinity column and no activity was detected in the unbound fraction. This result indicated that the thrombin inhibitor is the sole anticoagulant in the salivary glands of A. stephensi. This also suggested a noncovalent, reversible interaction between thrombin and its inhibitor. Size exclusion chromatography and SDS-PAGE estimated the molecular weight of the inhibitor as 45 kDa.