Heparin is a widely-used and important drug, particularly for the aging US population. It is a carbohydrate-based anticoagulant that used by elderly patients for many applications, including thrombotic disorders, in surgery, and during kidney dialysis. The long-term goal of this project is to create synthetic heparin drugs that are safer, more effective, and better tailored to different applications than the currently-available drugs. Heparin has a highly-variable structure due to the abundance of sulfate groups along its backbone, and its chemical structure determines its biological effect. Heparin is difficult to dose for elderly patients, and has well- documented bleeding side effects and contamination issues. In addition, different heparin applications require different rates of drug clearance;for example, the anticoagulant effect during surgery should be eliminated quickly after administration stops, while in deep vein thrombosis treatment the effect should be prolonged. Although there are several heparin drugs on the market, there is a significant need for heparins with homologous structures and controlled rates of clearance. We propose to determine the effects of different structural motifs on the rate of heparin clearance with the aim of developing structurally-defined heparin analogs with varied cellular internalization and clearance rates. To achieve this goal, we will use a unique chemoenzymatic method to synthesize radioactively-labeled heparin constructs having defined sulfation patterns, sulfation densities and lengths. The internalization rates of these constructs will be tested in an experimental cell line (Flp-In 293 cells), and their binding affinities to the heparin clearance receptor, HARE, will be determined using biochemical assays. The constructs will also be tested in human endothelial cells, which are known to internalize and degrade heparin in vivo. Lastly, we will prepare stable isotopically-labeled constructs having different internalization rates and test their clearance and anticoagulant activity in a mouse model. From these studies, we hope to elucidate the specific carbohydrate structures that control heparin clearance, which will be a powerful tool in creating anticoagulant drugs that are tailored to specific applications and patients.
Heparin is a common anticoagulant used during surgery, kidney dialysis and the treatment of thrombosis. This project aims to elucidate how different heparin structures control the rates at which heparin is cleared from the body, which would allow the creation of heparins that are safer and specifically tailored to different treatments.
|Chappell, Elizabeth P; Liu, Jian (2013) Use of biosynthetic enzymes in heparin and heparan sulfate synthesis. Bioorg Med Chem 21:4786-92|
|Pempe, Elizabeth H; Xu, Yongmei; Gopalakrishnan, Sandhya et al. (2012) Probing structural selectivity of synthetic heparin binding to Stabilin protein receptors. J Biol Chem 287:20774-83|