Isolation of an inhibitor selective for collagen-stimulated platelet aggregation from the soft tick Ornithodoros moubata.

L Waxman,T.M Connolly

doi:10.1016/s0021-9258(18)53341-x

Abstract

Soluble extracts from the soft tick Ornithodoros moubata were found to inhibit collagen-, ADP-, and thrombin-stimulated platelet aggregation. One inhibitory component was purified to homogeneity by a combination of gel filtration, ion-exchange, and reverse phase high pressure liquid chromatography. The purified activity, named moubatin, is a protein of molecular weight 17,000 and it inhibits the aggregation of washed human platelets stimulated by collagen with an IC50 of approximately 50 nM in the standard assay. At a concentration of moubatin that maximally inhibited collagen-stimulated platelet aggregation, no inhibition of aggregation initiated by other effectors, including arachidonic acid, thrombin, ristocetin, and the calcium ionophore A23187, was observed. Moubatin also inhibits collagen-dependent aggregation in plasma. At a higher concentration of moubatin (> 1 microM) it was also possible to demonstrate an inhibitory effect on the final extent of aggregation induced by a low concentration of ADP. Although moubatin selectively inhibits platelet activation by collagen, it has only a minimal effect on the adhesion of platelets to collagen. The amino acid sequences of peptides derived from proteolytic cleavage of moubatin suggest that moubatin is a unique protein, consistent with its novel functional activity.

Highlights

We recentlyidentified and purified fromOrnithodoros moubutu ticks a potentand highly specific inhibitor of blood coagulation factorXa called TAP (9)
Thrornbin was from American Diagnostica; human fibrinogen and A23187 were from Calbiochem; Endoprotease Lys-Cwas purchased from age of moubatin suggest that moubatin is a unique Boehringer
We recently reported on a 16-kDa protein isolated from the leech H. officinalis, LAPP, that blocks both platelet adhesion to collagen and collagen-induced aggregation (7, 8)

Summary

RESULTS

N o t shown), and it was not pursued further. The inhibitorof collagen-dependent aggregation eluted in a single peak with. Using chro- T h i s step resolved two peaksof inhibitory activity elutingat mogenic substrates and purified proteolytic enzymes ftrhoem -0.125 and 0.25 M NaCl (Fig. 2). Each of these two peaks was coagulation cascade, inhibitors of factor Xa and thrombin purified separately. Aggregation inhibitory essentialforclotformation and antiplateletactivities had activity eluted ina single peak for each sample with the same been reported in hard ticks (14), we tested whether the gel apparent molecular mass (-20 kDa) (data not shown). Each of the peaks was sequenced and seven unique peptides were obtained asshown inTable I Several of these sequences were confirmed by analysis of peptides derived from a cyanogen bromide digest of moubatin.

Platelet Aggregation Inhibitor from Ticks

Findings

DISCUSSION