Characterization of antihemostatic components from the salivary gland of the bed bug Cimex lectularius.

Abstract

Four anti-hemostatic components, an anti-platelet, an anti-clotting, an anti-thrombin and a vasodilator, have been characterized from the salivary glands of Cimex lectularius. Apyrase activity (E.C. 3.6.1.5.) was detected in the salivary gland of Cimex lectularius. Kinetic, chromatographic and thermal sensitivity assays indicated that the apyrase activity is the result of a true apyrase and not the result of two or more different enzyme activities. The apparent molecular mass of apyrase activity was 79,600 and 21,000. Additionally, the saliva of Cimex lectularius inhibited platelet aggregation induced by ADP. Cimex lectularius has an anti-clotting with an apparent molecular mass of 17,000. The inhibition was observed in both pathways, the extrinsic, as determined by inhibition of the prothrombin time, and in the intrinsic, as determined by the inhibition of factor X activation in the presence of factor VIII, IXa, X, phospholipids and calcium. The data obtained here exclude the inhibition by salivary homogenate of either factor VIII, IX or active factor X, and suggest the inhibition of factor X activation. Additionally, an anti-thrombin was detected which minimally affected the recalcification time of citrated plasma, suggesting that Cimex anti-thrombin may be inhibiting other functions of thrombin during hemostasis. Cimex has a nitrosyl-hemoprotein (nitrophorin) that releases nitric oxide in a pH dependent manner. The fraction containing the nitrophorin when separated by HPLC caused vasodilation of the preconstricted rabbit aortic strip. The presence of a nitrosyl-hemoprotein in Cimex salivary gland was verified by EPR. Cimex nitrophorin was purified to homogeneity, and partial amino-acid sequence data was obtained. The sequence was used to generate a PCR product to screen a salivary cDNA library which yielded a 1 kb clone whose sequence indicates homology to the human inositol polyphosphate 5-phosphatase. It is suggested that Cimex co-opted this enzyme, found in the salivary glands of insects, to the function of nitric oxide carrier, where this protein now is a major secretory protein (salivary glands). This scenario indicates the possibility that vertebrate inositol polyphosphate 5-phosphatase may be a hemoprotein regulated by nitric oxide.