An inhibitor selective for collagen-stimulated platelet aggregation from the salivary glands of hard tick Haemaphysalis longicornis and its mechanism of action

Soluble materials of salivary glands from Haemaphysalis longicornis were found to inhibit collagen, ADP, and thrombin-stimulated platelet aggregation. One inhibitory component was purified to salivary gland homogeneity by a combination of gel filtration, ion-exchange, and C-g reverse phase HPLC. The purified activity, named longicornin, is a protein of molecular weight 16 000 on SDS-PAGE under both reduced and nonreduced conditions. Collagen-mediated aggregation of platelets in plasma and of washed platelets (IC50 was approximately 60 nmol/L) was inhibited with the same efficacy. No inhibition of aggregation stimulated by other effecters, including ADP, arachidonic acid, thrombin, ristocetin, calcium ionophore A23187, thromboxane A2 mimetic U46619 and 12-O-phorbol- 13-myristate acetate, was observed. Longiconin had no effect on platelet adhension to collagen. Not only platelet aggregation but also release reaction, and increase of intracellar Ca2+ level of platelets in response to collagen were completely eliminated by longicornin. Increasing amounts of collagen are able to overcome the inhibition of aggregation by longicornin, indicating that longicornin probably shares with collagen a common receptor. In addition, collagen fibers did not emit fluorescence after incubation with isothocyanate-conjugated longicornin, indicating that longicornin did not bind directly to collagen fibers. The identification and isolation of longicornin demonstrates the existence of a new type of platelet inhibitor that should be useful to better understand the mechanism of collagen stimulation of platelets.