ROLE OF ARGININE-132 AND LYSINE-133 IN HEPARIN-BINDING TO AND ACTIVATION OF ANTITHROMBIN

The binding of heparin to antithrombin greatly accelerates the rate of inhibition of the target proteinases thrombin and factor Xa. Accelera tion of the rate of inhibition of factor Xa involves a conformational change in antithrombin that is translated from the heparin binding sit e to the reactive center loop, A mechanism has been proposed for gener ation and propagation of the conformational change in which the bindin g of the negatively charged heparin reduces ionic repulsions be tween positively charged residues on and adjacent to the D-helix in the hepa rin binding site of antithrombin (van Boeckel, C. A. A., Grootenhuis, P. D. J., and Visser, A, (1994) Nature Struct, Biol, 1, 423-425), This charge neutralization is proposed to elongate the D-helix and initiat e the conformational change which is then translated to the reactive c enter loop. Several basic residues, including arginine 132 and lysine 133, were predicted to be important both in heparin binding and in thi s mechanism of heparin activation, To test both the helix extension me chanism and the role of these two residues in heparin binding and fact or Xa inhibition, we individually changed arginine 132 and lysine 133 to uncharged methionine by site-directed mutagenesis. The K-d values f or binding of R132M and K133M variants to the high affinity pentasacch aride were weakened only 2.3- and 4.5-fold respectively, suggesting a location for R132 and K133 peripheral to the main pentasaccharide bind ing site. However, the K-d values for long chain high affinity heparin were weakened at least 17-fold for both R132M and K133M, indicating i nvolvement of each residue in binding extended chain heparin species. These reductions in affinity were ionic strength-dependent. The rates of inhibition of factor Xa and thrombin by each variant, however, were indistinguishable from those of control antithrombin, and the acceler ations of the rate of inhibition produced by heparin were normal. We c onclude that neither arginine 132 nor lysine 133 plays an important ro le in the binding of heparin pentasaccharide or in the mechanism of he parin activation, suggesting that D-helix extension through charge neu tralization is not the mechanism for transmission of conformational ch ange from the heparin binding site to the reactive center region, Argi nine 132 and lysine 133 do, however, play a role in tight binding of l onger chain heparin species through ionic interactions.