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Methods to investigate the nonanticoagulant functions of heparin and related polysaccharides

Student thesis: Doctoral ThesisDoctor of Philosophy

Background: There is increased interest in the development of heparin and related polysaccharides for clinical indications beyond their current anticoagulant remit. These indications range from anti-inflammatory and anti-complement agents, anti-metastatic therapeutics, prevention of protracted labour and easing pulmonary conditions such as chronic pulmonary obstructive disease and asthma. Anticoagulant activity may not be necessary and may cause adverse side effects when heparin is used for these conditions. Removal of anticoagulant activity such as selective de-sulfation have been carried out by a few pharmaceutical manufacturers of heparin and some of these products are now in clinical trials. Currently there is limited information on the structure/function relationships between non-anticoagulant heparins and these indications. The aim of this project was to study the structure and function relationship between heparins for some of these new indications. Methods and results: Heparin materials with different structures were prepared and characterised using a range of physicochemical and biological assays. Three different non-anticoagulant functions of these heparin were investigated: 1/ Potential pro-inflammatory properties in the kallikrein-kinin system were investigated for sulfate modified heparin and chondroitin sulfates. Of the samples investigated only over-sulfated chondroitin sulfates significantly increased generation of kallikrein and bradykinin, indicating a high degree of sulfation is required to stimulate the kallikrein-kinin system. 2/ A low molecular weight heparin, dalteparin has been shown to shorten protracted labour. There is some evidence that heparin may act by influencing myometrium contraction. However, at present there is no establish potency assays for this indication. A prototype calcium release method using uterine smooth muscle cells was developed as a surrogate system to study contraction. Further refinement of this assay method is required to confirm suitability for assessment of the ability of heparin to influence oxytocin induced contraction of smooth muscle cells. 3/ Histones, a basic protein, are released from stimulated neutrophils as part of neutrophil extracellular traps (NETs). The ability of modified heparins to influence the inflammatory responses to histones was studied in an in vitro whole blood model. Histones were found to be pro-inflammatory, increasing complement activation and cytokine release. Heparin and modified heparins, with reduced or no anticoagulant activity, were able to attenuate the responses induced by histones. A molecular weight and sulfate dependency of heparin were demonstrated. Conclusions: A panel of well characterised heparin and related polysaccharides have been prepared for study of structure and function relationship in inflammatory responses. Preliminary methods have been developed to study calcium release as a marker for contractility of uterine smooth muscle cells. Generation of kallikrein and bradykinin were sulfate but not molecular weight dependent and both kallikrein and bradykinin assays would be useful as adverse effect screening tests for suspected adulterated heparins and novel modified polysaccharides. The anti-histone effect of heparin was found to be both sulfate and molecular weight dependent and measurement of C3a production in whole blood would be a sensitive method to investigate the role of heparin and related polysaccharides in NETosis.
Original languageEnglish
Awarding Institution
Award date1 May 2019

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