The Relationship between Counterion Activity Coefficients and the Anticoagulant Activity of Heparin

Abstract

A series of sodium heparinate preparations have been characterised in terms of their ionogenic groups, utilizing a fluorescence titration technique. Single ion activity coefficients of the counterion were determined in pure polyelectrolyte solutions and polyelectrolyte/NaCl solutions. The results are discussed in relation to the anticoagulant activity of each heparin preparation. There appears to be a correlation between the linear charge parameter of the polyanion, and anticoagulant activity.Heparin is a naturally occurring mucopolysaccharide of molecular weight 5000-50 000. It is initially synthesized as an alternating copolymer of N-acetylglucosamine and glucuronic acid' but in subsequent pathways glucosamine residues are N-deacetylated and N-sulfated; glucuronic acid residues epimerise to iduronic acid which together with glucosamine are ester sulfated.2 T h e anticoagulant activity of heparin has been investigated by several researchers i n order t o correlate the biological role with polydispersity i n molecular size,3 variation in t h e ratio of glucuronic t o iduronic acid,4 alterations i n t h e a m o u n t of ester and N -~u l f a t i o n ,~ and differing degree of N-acetylation.6 Although these chemical and physical differences can affect t h e anticoagulant activity, at present none of these criteria have been shown t o be paramount, a n d for instance molecular weight dependence of anticoagulant activity has given rise to a number of conflicting report^.^,^,^ T h i s implies that the anticoagulant activity of heparin might be related t o a variety of chemical and physical factor^.^ In this present investigation, preparations of sodium salts of heparin were examined in an attempt to relate the cationic binding strength both in the pure polyelectrolyte system and also with a d d e d sodium chloride t o t h e anticoagulant activity of heparin. Stivala e t a l l o have already proposed that a correlation exists between Cu(I1) binding and t h e anticoagulant activity of heparin. Also it has been noted that a relationship exists between the counterion salt form of a heparin preparation a n d its efficacy as a prophylactic drug for d e e p venous thrombosis a n d local haematoma treatment."-13 There is, therefore, a necessity t o provide a better understanding of t h e parameters governing anticoagulant activity.It is generally known14 that polyelectrolytes characterised by t h e dimensionless linear charge parameter, 5,where e is t h e protonic charge, t is t h e bulk dielectric constant of the solvent, k is Boltzmann's constant, T i s the absolute temperature, and b is t h e average distance between charged groups on t h e polyelectrolyte chain; tcriticd is given by (zizP(-l, where zi is t h e charge carried by t h e counterion and zp is t h e charge carried by t h e charged site of the polyelectrolyte.T h e Manning model for the infinite line charge gives rise to limiting laws for thermodynamic parameters, and these laws received extensive experimental testing, a n d have been found t o give good agreeme...

Kinetic Analysis of the Clotting System in the Presence of Heparin and Depolymerized Heparin

“…This observation seems to question previous statements relat ing differences of observations to the molec ular weight of heparins [ 15,16]. Other stud ies revealed an increase in anticoagulant ac tivity with anionic charge density of heparin [17,18], However, some partially depolymerized heparins may have a concomitantly reduced surface charge density. In fact, the negative surface charge density of the depolymerized heparin used in this study (0.79 nm/charge equivalence, as can be calculated from the number of 1.27 charged residues per monosaccharide unit) falls into a range, in which unfractionated heparin is loosing its accelerating effect of inhibition on throm bin independent of its molecular weight [26].…”

Thermodynamics of Charged Oligopeptide-Heparin Interactions

“…The number of saccharides occluded per bound peptide (n) was estimated to be 2, based on comparison of the structures of heparin and the oligopeptide, KWK-CO2. The length of a fully extended tetrasaccharide was determined to be 20.4 Á (Diakun et al, 1978), and we estimate that the fully extended length of KWK-CO2 is ~10-13 Á. However, at the low peptide binding densities used in our experiments (<6% saturation of the heparin by oligopeptides), accurate determination of the equilibrium binding constant, K0bS, does not require precise knowledge of the occluded site size, since the statistical effects of overlap are minor (<±2% difference in Kobs for 1.3 < n < 2.6) (data not shown).…”

Binding of magnesium and lanthanum ions to heparin in the presence of sodium ions. A relationship between 13C chemical shift displacements and counterion condensation theory

“…This non-adjustable parameter is determined by the structure of the polyelectrolyte, namely, the axial separation, b , between charged groups. For heparin, the latter quantity was determined from molecular models of the disaccharide repeat :equence in its most extended form, from which a value of 2.9 A was obtained (15). This compares with a value of 3.0A from X-ray scattering measurements (20).…”

“…The determination of the quantity, b , and hence, 5, assumes an idealized repeating structure of heparin, which may not be true, since heparin is a highly heterogeneous polymer (21), and its composition may vary substantially with the source. This means that the SO3-/C02-ratio may vary from sample to sample, and so b (or 5) also can vary (15). Moreover, the linear chargedensity parameter chosen is for a polyelectrolyte in its most extended form.…”