Structural basis for the anticoagulant activity of heparin. 2. Relationship of anticoagulant activity to the thermodynamics and fluorescence fading kinetics of acridine orange-heparin complexes

Abstract

Complexing heparin or dermatan sulfate with the fluorescent probe acridine orange provides a means of studying electrostatic as well as static and dynamic conformational aspects of these glycosaminoglycans via the thermodynamic and photochemical (fluorescence fading) properties of these complexes. The cooperative binding constants (Kq), fluorescence fading rate parameters (r''), and anticoagulant activities of heparins fractionated according to anionic density all showed qualitatively the same dependence upon anionic density. When Kq and r'' were plotted against anticoagulant activity, empirical relationships were observed. Interestingly, the corresponding values for unfractionated dermatan sulfate fell on the lines defined by the heparin fractions. Temperature-dependence, studies demonstrated that differences in fading rate observed for heparins of different anionic densities are entropic in origin and reflect differences in the ability to assume a special configuration. Differences in activation entropy for fluorescence fading can be empirically correlated with anticoagulant activity. The latter correlation suggests a physical similarity in the roles played by anionic density in both fluorescence fading and anticoagulant activity.

Structure-activity relationships of heparin. Independence of heparin charge density and antithrombin-binding domains in thrombin inhibition by antithrombin and heparin cofactor II.

“…Except the VLA heparins, which are inactive with AT, the activities of the heparins with both inhibitors show the same correlation with charge density. That such a charge-density effect is seen at all with the HA heparins is strong, direct evidence that, as predicted earlier from indirect evidence (10), the correlation with Z2 arises from a domain that is independent of the AT-binding domain on the heparin. Moreover, the slope of the lines, which is a measure of the change in activity with Z2, is insensitive to the nature of the inhibitor and is not changed by AT-affinity fractionation.…”

“…Hog mucosal heparin (176 U. S. Pharmacopeia, units per milligram; Diosynth BV, Holland) was fractionated by two-phase partition (10,12) to obtain heparin fractions, previously characterized in detail (9)(10)(11)(12), that vary systematically in linear charge density, Z. The highly purified and more homogeneous fractions have similar molecular-weight distributions, with an average molecular weight of 15,000, and contain no detectable dermatan sulfate or other contaminating glycosaminoglycans.…”

“…A short sequence of defined structure (2) is necessary for AT-heparin binding and, hence, activity with AT (3,4). Because additional properties such as molecular weight (5,6), thrombin affinity (6)(7)(8), and charge density (9)(10)(11)(12), can also markedly affect the activity of a heparin, the molecule must have multiple functional domains (6,7).…”

Peptide–glycosaminoglycan cluster formation involving cell penetrating peptides

“…Clustering models have been proposed previously to explain cationic dye quenching phenomena which predominately involve dye-dye contacts with each dye on a separate heparin chain. [43][44][45][46] While the structural features of the CPPs responsible for formation of stable heparin clusters have not been investigated in detail from this study, it appears to stem from the ability of the peptide to form nonionic, likely hydrophobic interactions with other heparinbound peptides. The presence of non-ionic contributions to the peptide-heparin clusters was confirmed by the sensitivity of heparin affinity chromatography to the nature of the eluting salt solution with changes in elution profile and a differing order of elution observed with NaCl and GdnHCl (see Figure 1).…”

Structure, function, and pathology of proteoglycans and glycosaminoglycans in the urinary tract

“…Glycosaminoglycans are highly charged, and the very high density of charge at this surface will lead to a strong ordering of water molecules at the surface of the bladder such as is seen in physicochemical studies of the binding properties of glycosaminoglycan chains [18,31,32]. This physical phenomenon of water ordering, which is thermodynamically equivalent to exclusion of solutes, provides a plausible mechanism for bladder defenses.…”