PURIFICATION AND CHARACTERIZATION OF A THROMBIN INHIBITOR FROM THE SALIVARY-GLANDS OF A MALARIAL VECTOR MOSQUITO, ANOPHELES-STEPHENSI

A coagulation inhibitor was identified and isolated from the salivary glands of a malarial vector mosquito, Anopheles stephensi. The salivar y 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 ch romogenic substrates showed that the anticoagulant in the mosquito sal ivary glands is a thrombin inhibitor, but not an inhibitor of factor X a. The anticoagulant was purified to homogeneity from the mosquito tho rax which contains the salivary glands by means of a combination of th rombin affinity and anion exchange chromatography. All of the anticoag ulant activity was recovered from the fraction bound to the thrombin a ffinity column and no activity was detected in the unbound fraction. T his result indicated that the thrombin inhibitor is the sole anticoagu lant in the salivary glands of A. stephensi. This also suggested a non covalent, reversible interaction between thrombin and its inhibitor. S ize exclusion chromatography and SDS-PAGE estimated the molecular weig ht of the inhibitor as 45 kDa. (C) 1998 Elsevier Science B.V. All righ ts reserved.