Abstract
Background
We have isolated, variegin, a unique direct thrombin inhibitor (DTI) from tropical bont tick Amblyomma variegatum. Variegin inhibits thrombin active site and exosite-1 with an inhibitory constant of 0.3 nM (9-fold better than bivalirudin). It is also >5 orders of magnitude more selective for thrombin than other blood coagulation serine proteases. Variegin has a plasma half-life of 50 minutes (compared with bivalirudin 25 minutes and heparin ∼ hours).
Purpose
We aimed to develop variegin into a parenteral anticoagulant for percutaneous coronary intervention (PCI) and tested variegin in several pre-clinical models.
Methods
In rats, variegin was tested for efficacy (anticoagulation intensity) in a FeCl3-induced carotid artery thrombosis model while safety (bleeding risk) was tested in a tail incision model that recapitulated the time-frame of PCI (∼1 hour) in humans (time-response model). In pigs, an extracorporeal circuit with modified Badimon chambers containing coronary stents was used to assess efficacy, while bleeding risk was evaluated through needle-induced injury on a superficial ear vein, with or without concurrent administration of aspirin and ticagrelor (DAPT). Unfractionated heparin (UFH) and bivalirudin at dosages recommended for PCI were used as references. Ex vivo clotting analyses including thrombin generation test, rotational thromboelastometry, activated partial thromboplastin time and clot waveform analysis were performed in human blood spiked with DAPT and the three anticoagulants.
Results
In the rat time-response model, a single variegin bolus conferred better antithrombotic effect than a continuous infusion of bivalirudin and more rapid recovery of haemostasis than a single bolus of heparin. In the porcine ex vivo model, without DAPT, UFH, bivalirudin and 1 mg/kg variegin reduced stent thrombus by 35% (P<0.001), 60% (P<0.0001), and 80% (P<0.0001), compared with saline, respectively. In the presence of DAPT, UFH, bivalirudin and only 0.1 mg/kg of variegin (10-fold lowered dose) reduced stent thrombus by 65% (P<0.01), 75% (P<0.001), and 87% (P<0.0001), respectively (Fig. 1A). However, in the presence of DAPT, standard-dose UFH and bivalirudin prolonged bleeding times far longer than low-dose variegin (Fig. 1B). In human platelet rich plasma treated with DAPT, UFH showed a much more precipitous decline in thrombin generation potential than variegin (Fig. 1C). Dose response curves for inhibition of thrombin generation are also steeper in UFH and bivalirudin than in variegin, suggesting larger safety dose margin for variegin (Fig. 1D). These observations potentially account for the better preservation of haemostasis with low-dose variegin in combination with DAPT.
Figure 1
Conclusion
In the presence of aspirin and ticagrelor, a low dose of variegin, a novel direct thrombin inhibitor, achieved superior antithrombotic effect with significantly lower bleeding risk than heparin or bivalirudin in pre-clinical PCI models.
Tailoring the effect of antithrombin-targeting therapy in haemophilia A using in silico thrombin generation
