Thrombolytic Effects of the Snake Venom Disintegrin Saxatilin Determined by Novel Assessment Methods: A FeCl3-Induced Thrombosis Model in Mice

Abstract

Saxatilin, a novel disintegrin purified and cloned from the venom of the Korean snake Gloydius saxatilis, strongly inhibits activation and aggregation of platelets. Glycoprotein (GP) IIb/IIIa receptor antagonists can resolve thrombus, so saxatilin might also have thrombolytic effects. We investigated the thrombolytic effects of saxatilin in mice using a ferric chloride-induced carotid arterial thrombosis model. Thrombotic occlusion and thrombus resolution were evaluated quantitatively by measuring blood flow in the carotid artery with an ultrasonic flow meter and calculating the degree of flow restoration on a minute-by-minute basis; results were confirmed by histological examination. Saxatilin dissolved thrombi in a dose-dependent manner. Saxatilin at 5 mg/kg restored blood flow to baseline levels. As saxatilin dose increased, time to recanalization decreased. A bolus injection of 10% of a complete dose with continuous infusion of the remaining dose for 60 minutes resulted in effective recanalization without reocclusion. The thrombolytic effect of saxatilin was also demonstrated in vitro using platelet aggregometry by administering saxatilin in preformed thrombi. Bleeding complications were observed in 2 of 71 mice that received saxatilin. Fibrin/fibrinogen zymography and platelet aggregometry studies indicated that saxatilin does not have fibrinolytic activity, but exerted its action on platelets. Integrin-binding assays showed that saxatilin inhibited multiple integrins, specifically α2bβ3 (GP IIb/IIIa), α5β1, αvβ3, αvβ1, and αvβ5, which act on platelet adhesion/aggregation. Saxatilin inhibited multiple integrins by acting on platelets, and was safe and effective in resolving thrombi in mice.

Time-Dependent Thrombus Resolution After Tissue-Type Plasminogen Activator in Patients With Stroke and Mice

“…In addition, the process of thrombus dissolution during thrombolytic treatment may be dynamic, with frequent thrombolysis and rethrombosis occurring during or immediately after thrombolytic treatment. 16,33 This was also observed during the real-time monitoring of blood flow in the mice in our experimental study, with frequent alternate reocclusion and reperfusion during or immediately after tPA infusion. Therefore, the thrombus volume, which was measured based on a onetime snapshot CT image, may be insufficient for assessing the entire process of thrombus dissolution after tPA treatment.…”

“…25 We have previously shown that the size of thrombus formed by FeCl 3 treatment was similar among animals in this model and that the degree of blood flow restoration, as measured by ultrasound, is a good representation of thrombus resolution. 16 Although it has been suggested that an aged thrombus would be resistant to tPA treatment, this concept remains understudied in vivo. 10 This study, using a thrombosis model in mice, clearly demonstrated that the effect of tPA is dependent on the age of the thrombus.…”

“…16 In this study, we used these thrombus-measurement tools to investigate whether the degree of thrombus resolution differed (1) according to the time from symptom onset to intravenous tPA treatment in patients with stroke and (2) according to the time from the induction of thrombus formation to intravenous tPA treatment in mice.…”

“…The restoration of flow was assessed quantitatively using Doppler-based flow measurements. This animal model and the Doppler-based assessment system have been described previously 16 and in Methods in the online-only Data Supplement.…”

“…The occlusion site was most commonly in the middle cerebral artery M1 segment (46.2%; n=79), followed by the distal internal carotid artery (26.9%; n=46), middle cerebral artery M2 segment (18.1%; n=31), basilar artery (7.0%, n=12), and posterior cerebral artery (1.8%; n=3). The median initial National Institutes of Health Stroke Scale score was 16 (IQR,[12][13][14][15][16][17][18][19]. Compared with the good volume reduction to intravenous tPA, moderate or nonvolume reduction had higher initial National Institutes of Health Stroke Scale scores and was more likely to receive sequential intra-arterial reperfusion treatment (Table II in …”