New Functional Tools for Antithrombogenic Activity Assessment of Live Surface Glycocalyx

Abstract

Objective It is widely accepted that the presence of a glycosaminoglycan-rich glycocalyx is essential for endothelialized vasculature health; in fact, a damaged or impaired glycocalyx has been demonstrated in a number of vascular diseases. Currently, there are no methods that characterize glycocalyx functionality, thus limiting investigators’ ability to assess the role of the glycocalyx in vascular health. Approach and Results We have developed novel easy to use in vitro assays that directly quantify live endothelialized surface’s functional heparin weights and their anti-coagulant capacity to inactivate Factor Xa and thrombin. Using our assays, we characterized two commonly used vascular models: native rat aorta and cultured human umbilical vein endothelial cell (HUVEC) monolayer. We determined heparin contents to be about 10,000 ng/cm2 on the native aorta, and about ten-fold lower on cultured HUVECs. Interestingly HUVECs demonstrated a five-fold lower anti-coagulation capacity in inactivating both Factor Xa and thrombin relative to native aortas. We verified the validity and accuracy of the novel assays developed in this work using LC-MS analysis. Conclusions Our assays are of high relevance in the vascular community as they can be utilized to establish the anti-thrombogenic capacity of many different types of surfaces such as vascular grafts and transplants. This work will also advance the capacity for glycocalyx-targeting therapeutics development to treat damaged vasculatures.

Endothelial Functions

“…76 Endothelial glycocalyx plays important roles in preserving healthy endothelial functions, including anticoagulation, mechanotransduction, and shear stress-mediated NO production. Dimitrievska et al 77 developed for the first time simple assays that can evaluate glycocalyx function by measuring its antithrombogenic capacity in vitro. This assay will pave the way for the development of a new diagnostic tool of endothelial function and a novel therapeutic approach targeting glycocalyx.…”

Thrombotic Regulation From the Endothelial Cell Perspectives

“…How to determine the functions of heparin on live ECs remains challenging. Dimitrievska et al 69 developed a method that can quantify functional heparin weight on live endothelialized surface and their anticoagulant capacity to inactivate FXa and thrombin. Using this novel approach, they reported a striking difference (≈10-folds) in heparin weight on native aorta and cultured human umbilical vein ECs, which is consistent with the lower anticoagulation capacity of human umbilical vein ECs in inactivating both FXa and thrombin relative to native aortas.…”

“…Using this novel approach, they reported a striking difference (≈10-folds) in heparin weight on native aorta and cultured human umbilical vein ECs, which is consistent with the lower anticoagulation capacity of human umbilical vein ECs in inactivating both FXa and thrombin relative to native aortas. 69 This method can be valuable for future study of endothelial anticoagulation activity and its molecular regulation in vitro.…”

Lung regeneration

“…Assays that evaluate the ability of the engineered vasculature to inactivate factor Xa and thrombin [42] or report of a minimum time to thrombosis for heparinized or un-heparinized whole blood could be useful in determining how the construct would fare in vivo .…”