Background: Ticks puncture human and animals´ skin and secrete saliva into the bloodstream of the host, containing anticoagulant proteins. We studied disagregin, a potent platelet-inhibiting protein derived from the salivary glands of Ornithodoros moubata, an African soft tick. Conventional αIIbβ3 antagonists contain an RGD-sequence for integrin binding. In contrast, disagregin contains an RED-sequence, hypothesizing a differential mode of inhibitory action.

Aims: We aimed to compare the inhibitory effects of native disagregin and its RGD-mutant on platelet activation and to unravel the molecular basis of disagregin-αIIbβ3 integrin interactions.

Methods: Disagregin (MW: 6952 Da) was synthesized by solid-phase peptide synthesis using tert-butyloxycarbonyl(Boc)-chemistry and native chemical ligation. Effects of both disagregins on platelet aggregation were assessed by light transmission aggregometry in human platelet-rich plasma. Whole-blood thrombus formation was investigated by perfusing blood over collagen type I with and without tissue factor (500 pM) at high wall-shear rate (1000s^-1) in the presence of disagregin, disagregin-RGD or eptifibatide, a clinically approved αIIbβ3 integrin inhibitor. Thrombi formed on microspots were assessed by 9 parameters, reflecting platelet adhesion, thrombus formation and fibrin formation. Parameters were analysed by Friedman tests.

Results: Disagregin showed dose-dependent inhibition of collagen- and adenosinediphosphate (ADP)-induced classical platelet aggregation with IC₅₀ values of 65 and 100 nM, respectively. Multiparameter assessment of thrombus formation showed suppressed platelet adhesion and aggregate formation in presence of disagregin or disagregin-RGD, while eptifibatide treatment allowed platelets to form small aggregates.
Time to fibrin formation was delayed when adding disagregin or disagregin-RGD (p< 0.01) or eptifibatide (p< 0.05). However, surface covered by fibrin was lower in presence of disagregin and disagregin-RGD (p< 0.01), while addition of eptifibatide did not affect fibrin surface area coverage.

Conclusions: Both αIIbβ3-blocking disagregins have potent inhibitory effects on platelet adhesion, thrombus formation and fibrin formation in a dose-dependent manner. Eptifibatide was less effective, at concentrations fully blocking classical platelet aggregation.

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ISTH Congress Abstracts - https://abstracts.isth.org/abstract/inhibition-of-platelet-adhesion-thrombus-formation-and-fibrin-formation-by-a-potent-%ce%b1iib%ce%b23-integrin-inhibitor/