Creation of modified heparins to probe platelet-endothelial glycocalyx interactions necessary for inflammation.

Platelets play a critical role in innate immunity (Page and Pitchford 2013); being recruited independently of other cell types (Cleary et al., 2019) and are also essential for efficient leukocyte recruitment to inflamed or infected lungs (Pitchford et al., 2005; Amison et al., 2018). Mechanisms by which platelets adhere and 'prepare' the surface of the respiratory endothelium for efficient leukocyte recruitment require greater understanding. The luminal side of the endothelium is coated by a glycocalyx structure that normally acts as a barrier to circulating cells. Significantly, platelet-dependent leukocyte adhesion to endothelium (as observed by intravital microscopy) can be inhibited by low molecular weight (LMW) heparin (Riffo-Vasquez et al., 2016). LMW heparin can inhibit the binding of pro-inflammatory mediators to the glycocalyx (Lipowsky & Lescanic 2017). Uniquely as a circulating cell, platelets contain the CXC chemokine PF4 (CXCL4). PF4 binds with high affinity to heparin, a highly sulfated glycosaminoglycan (GAG), and to negatively charged cell surface GAGs (e.g. heparan sulfate) of the glycocalyx (Loscalzo et al., 1985; Petersen et al., 1995; Sachais et al., 2004). It has been postulated that the activity of PF4 appears to involve binding to surface GAGs (Kowalska et al., 2010). However, the role of PF4 in platelet preparation of the glycocalyx for leukocyte recruitment, and an understanding of the surface chemistry by which heparin can interfere has not been examined, even though the modification of pulmonary endothelial glycocalyx is necessary during lung injury arising from infection (Schmidt et al., 2012).
The National Institute of Biological Standards and Controls (NIBSC), have a unique panel of non-anticoagulant modified heparins and related polysaccharides that have anti-inflammatory properties (Hogwood et al., 2019). Expertise from NIBSC will be necessary to allow the PhD student to probe the structure-activity relationship of platelet released PF4 binding to GAGs within the endothelial glycocalyx to promulgate cell adhesion and the use of these heparins is necessarily interconnected to later project objectives at KCL.
This project requires an interdisciplinary approach, where the student will learn techniques in pharmacology/physiology (in vitro and in vivo functional assays), synthetic chemistry (creation of heparin analogues), electron microscopy (EM), stochastic optical resolution microscopy (STORM), and analytical structural chemistry approaches (eg. Fourier transform infrared spectroscopy, FTIR).