Size‐fractionated heparins have differential effects on human neutrophil function in vitro

Abstract

Background and purpose: Heparin is known to possess a range of activities, other than effects on blood coagulation, many of which are anti-inflammatory. Effects with potential anti-inflammatory applications include the inhibition of elastase release from neutrophils, as well as the adhesion of these cells to vascular endothelium. In the present study we aimed to investigate whether fractionation of heparin may yield molecules with enhanced or specific effects on human neutrophil function. Experimental approach: Fractions of defined molecular size were obtained from heparin by different methods and assessed for their effects on elastase release induced by formyl Met-Leu-Phe (fMLP), from neutrophils, in some cases following the priming of these cells with tumour necrosis factor-a (TNF-a). Effects of the fractions on neutrophil adhesion to interleukin-1b (IL-b)-stimulated human umbilical vein endothelial cells (HUVECs) were also examined. Key results: Elastase release was inhibited by very low molecular weight fractions of heparin, with an apparent minimum chain length of 10 saccharides required for full effect. In contrast, neutrophil-endothelial adhesion was unaffected by these fractionated heparins, suggesting that certain non-anticoagulant actions of heparin may be lost by such an approach. Conclusions and implications: These data suggest that an optimum chain length of heparin possibly exists for certain nonanticoagulant actions of heparin, which may prove to be useful in the design of novel drugs with specific anti-inflammatory actions.

Pharmacology of Heparin and Related Drugs

“…Heparin has been reported to inhibit the activation of a number of inflammatory cell types (Slungaard et al, 1990;Ahmed et al, 1992;Rohrer et al, 1992;Bazzoni et al, 1993;Inase et al, 1993;Piccardoni et al, 1996;Teixeira et al, 1996;Brown et al, 2003;Lever et al, 2007). These effects can at least partially be attributed to the binding and neutralization of mediators and enzymes released during the inflammatory response (Tyrrell et al, 1999) that would otherwise lead to inflammatory cell activation, although inhibition of the release of inflammatory mediators from cells may also contribute to the net effect (Rao et al, 1991;Ahmed et al, 1994;Brown et al, 2003;Ji et al, 2004;Zeng et al, 2004) (see below).…”

“…Some of these oligosaccharides possess anti-inflammatory activity (Lever et al, 2007;Shastri et al, 2013). Interestingly, one tetrasaccharide has been shown to be anti-inflammatory after oral dosing in allergic sheep (Ahmed et al, 2012(Ahmed et al, , 2013, although the same tetrasaccharide failed to show any consistent effect in patients with allergic asthma undergoing an allergen challenge (Duong et al, 2008).…”

“…Moreover, the knowledge that minimum GAG chain lengths exist for the effective binding of specific proteins, including thrombin and PF4, has already been exploited in the development and use of anticoagulant heparins with more predictable effects (Gray et al, 2008;Petitou et al, 2009). We previously described that very-LWMH preparations, of defined and homogenous chain length, have differential and size-dependent effects on neutrophil degranulation (Lever et al, 2007), and a simple approach such as molecular weight limitation could alone go some way toward removing activities that are unwanted for a specific indication…”

“…In vivo model of airway inflammation 2-and 3-O-sulfation not necessary for elastase inhibition (Griffin et al, 2014) Elastase release from neutrophils 10 monosaccharides minimum length (Lever et al, 2007) Solid phase assays, NMR, and affinity chromatography Heparin/HS binds Hlp at two distinct sites, conserved in mycobacterial homologs (Portugal et al, 2008) Viral proteins HIV-1 gp120 Inhibition of antibody binding and HIV-1 replication O-sulfation important (Rider et al, 1994) …”

“…Heparin has been shown to bind to the surface of neutrophils (Leculier et al, 1992) and to inhibit their degranulation (Brown et al, 2003;Lever et al, 2007) and homotypic aggregation (Laghi Pasini et al, 1984;Freischlag et al, 1992;Bazzoni et al, 1993;Brown et al, 2003), as well as the production of superoxide anions, the activity of lysosomal enzymes (Bazzoni et al, 1993), the release of elastase (Brown et al, 2003), and the ability of neutrophils to activate platelets (Bazzoni et al, 1993;Piccardoni et al, 1996). Furthermore, neutrophil activation in response to thrombin-stimulated platelet products is also inhibited, as is thrombin-induced platelet aggregation under certain conditions (Piccardoni et al, 1996).…”