The role of surface charge on the accelerating action of heparin on the antithrombin III-inhibited activity of alpha-thrombin.

C C Heuck,U Schiele,D Horn,D Fronda,E Ritz

doi:10.1016/s0021-9258(18)89113-x

Abstract

We have compared surface charge and the surface charge density on the polyanions heparin and potassium polyvinyl sulfate (KPVS), as well as on hydrolyzed heparin and KPVS, with their accelerating effect on the inhibitory action of antithrombin III on thrombin. Polyelectrolyte titration of thrombin with KPVS or heparin at pH 7.4 clearly indicates an electrostatic interaction. In contrast, at the same pH no electrostatic interaction is observed between polyanions and antithrombin III. KPVS accelerates the inhibitory action of antithrombin III to the same extent as heparin on the basis of charge equivalence. Heparin and KPVS with a mean distance between two charged centers of less than 0.75 and 0.95 nm, respectively, accelerate strongly whereas hydrolysates with lower charge densities are far less active. The following observations are indicated. Intramolecular neutralization of oppositely charged residues occurs within thrombin, antithrombin III, and partially hydrolyzed heparin. Heparin acts on the antithrombin III-thrombin reaction through cooperative electrostatic binding to thrombin and nonelectrostatic interaction with antithrombin III. This indicates a quasi-catalytic action of the polyelectrolyte. Hydrolysis of only a few N-sulfate residues within the heparin molecule decreases the linear surface charge density to such an extent that the accelerating action is drastically reduced. The loss of accelerating capacity agrees with the sudden loss of counterion condensation due to the decrease of the linear surface charge density beyond limits postulated by Manning in a theory of polyelectrolytes.

Highlights

We have compared surface charge and the surface analysis was carried out to examine the respective roles of charge density on the polyanions heparin and potas- surface charge on humanthrombin,antithrombin 111, and sium polyvinyl sulfate (KPVS), as well as on hydro- heparin in intermolecular interactions
To further extend the lyzed heparin andKPVS, with their accelerating effectconclusions drawn from such experiments we studied on the inhibitoryaction of antithrombin I11on throm- synthetic polyanions
Surface Charge of Thrombin and Antithrombin III-The charge density uersm pH curve for thrombin as determined by polyelectrolyte titration is shown in Fig. 1.The experimental curve almost coincides with the theoretical curve calculated from the content of ionized amino acids in the protein (Table6)

Summary

EXPERIMENTAL PROCEDURES'

KPVS accelerates the inhibitory action of antithrombin 111to the same extent as heparin on the basis of charge equivalence. Heparin fractions from porcine mucosa isolated by gel filtration on Sephadex G-100 (molecularweight, 8,000, less than 0.75 and 0.95 nm, respectively, accelerate 12,000,14,000,16,000and 18,000& 2,000 as estimated from ultracenstrongly whereas hydrolysates with lower charge detnri-fugation) were kindly provided by Dr Herr Antithrombin 111 was isolated from serum of healthy donors thrombin and nonelectrostatic interaction with anti- by affinity chromatography with heparin-Sepharose gel chromatogthrombin 111.This indicates a quasi-catalytic actioonf raphy and ion-exchange chromatography as described by Millerthe polyelectrolyte. The amount of heparin added in 10 p1 of buffer solution was between 15.5 X 10"' and 620.1 X 10"' charge equivalents. In other experiments the same amount (charge equivalents) of KPVS or of hydrolysates either of heparin (M,16,000) or of KPVS was added to replace heparin

RESULTS

DISCUSSION

I : metachromasy