Distinct Roles of the Extrinsic and the Intrinsic Coagulation Pathways In Different Murine Thrombosis Models,
Authors: Zampolli Antonella , Marchese Patrizia , Gruber Andras , Ruf Wolfram , Ruggeri Zaverio M. 
Published: October 2019

Abstract
Abstract 3278
In vivo vascular lesion models are used to elucidate the pathophysiological mechanisms of thrombosis. We found that the intrinsic and extrinsic coagulation pathways exhibit a distinct relative contribution to arterial occlusion initiated by a FeCl3- or nitrogen dye pulse laser (NDPL)-induced injury evaluated comparatively in the carotid artery (CA), the femoral artery (FA) or the femoral vein (FV). Methods. C57Bl/6J (BL6), IL4Rα/GPIbα-tg (IL4) and VWF−/− mice between 8 and 12 weeks of age were anesthetized with 2% isofluorane, and the right jugular vein was cannulated for IV delivery of fluids. Injury in the exposed left CA was induced by applying a 0.7 ml drop of 7 or 8 % FeCl3 for 3 minutes; variations of blood flow in the injured artery were then monitored with a miniaturized ultrasound flow probe for an additional 30 min. In all experiments, FeCl3 concentrations refer to the hexahydrate form of the salt. The time to first occlusion (flow rate <0.1 ml/min) and the normalized area under the flow rate curve, defined as the Flow Index (FI), were used as endpoints for statistical analysis. In the FA and FV, respectively, vascular injury by FeCl3 was induced by applying a 9% drop for 2.5 minutes or 4% drop for 1 minute; then, platelet and fibrin deposition in forming thrombi were visualized by epifluorescence microscopy of injected green calcein-labeled platelets and AlexaFluor 546-labelled anti-fibrin antibody. The NDPL injury was induced in both FV and FA with two series of 30 s laser light pulses, the second upstream of and 30 s after the first. Images selected at regular time intervals from a 20 min real-time video recording were used for measuring the integrated density of formed thrombi with the ImageJ software. Results. In the CA, 8% FeCl3 reliably caused a stable occlusion that was completely abolished by lepirudin, a thrombin inhibitor, at a calculated 42 μM concentration in blood. Also, IL4 mice, lacking a functional extracellular GPIbα domain, and VWF−/− mice failed to develop a normal thrombus. In C57Bl/6J mice, injection of the anti-mouse factor XI (mFXI) monoclonal antibody (mAB) 14E11 inhibited thrombus formation in a dose dependent manner, and completely when injected at a dose of 0.5 μg/g. In contrast, the anti-mouse tissue factor (mTF) mAB 21E10 had no effect when injected at 25 μg/g; the latter amount of anti-mTF mAB was variably inhibitory only when a presumably milder lesion was induced by a reduced 7% FeCl3 concentration. Of note, however, even at the lower dosage of 9 μg/g the anti-mTF mAB completely prevented CA occlusion when co-injected with an individually non-effective dose (0.065 μg/g) of anti-mFXI mAB. Essentially the same results, albeit obtained with a different methodology, were observed by evaluating thrombus formation in the FA and FV after FeCl3-induced injury. Findings were remarkably different in the NDPL model of FA and FV thrombosis. In this case, platelet aggregate formation was transient and almost completely reversed by 20 minute post-injury, while fibrin deposition at the site of injury increased progressively in time. Fibrin deposition was essentially absent in the IL4 mouse but, contrary to what we observed in the same vascular beds after FeCl3 injury, it was only partially inhibited by injecting the anti-mFXI mAB at dosage as high as 1 μg/g. Moreover, and again in contrast with the results after FeCl3 injury, the anti-mTF mAB injected at 25 μg/g by itself significantly inhibited fibrin deposition. Conclusions. Our findings indicate that distinct thrombogenic vascular lesions elicit a varied response by the extrinsic and intrinsic coagulation pathways, as reflected by the different effects of inhibiting initiating events mediated by TF or FXI activation by FXIIa (the reaction blocked by mAB 14E11), respectively. Moreover, and again in a lesion-specific manner, the two coagulation pathways exhibit a variably relevant synergistic function in leading to thrombin generation as evidenced by fibrin deposition. These results, and the underlying mechanisms presently under investigation, suggest that the thrombogenic potential of different pathological lesions in human vascular disease is also likely varied and may be influenced by lesion-specific characteristics. Consequently, more direct mechanistic evidence must be generated to establish how a given animal thrombosis model may reflect the pathogenesis of acute vascular occlusion in human disease.

Disclosures:
Gruber: Aronora, LLC: Consultancy, Equity Ownership.