Regulation of glycosaminoglycan function by osmotic potentials. Measurement of water transfer during antithrombin activation by heparin.
The sulfated glycosaminoglycan heparin is an important anticoagulant, widely used to treat and to prevent arterial thrombosis. Heparin triggers conformational changes in, and the functional activation of, the serine proteinase inhibitor antithrombin. We investigated water-transfer reactions during the activation process to explore the possibility that functional interaction between antithrombin and sulfated glycosaminoglycans can be regulated by osmotic potentials. Volume of water transferred upon heparin binding was measured from differences in free energy change, Delta(Delta G), with osmotic stress, pi. Osmotic stress was induced with chemically inert probes that are geometrically excluded from the water-permeable spaces of antithrombin and from intermolecular spaces formed during the association reaction. The free energy change, Delta G, for the antithrombin/heparin interaction was calculated from the dissociation constant, determined by functional titrations of heparin with antithrombin at fixed concentrations of the coagulation protease factor Xa. The effect of osmotic stress was independent of the chemical nature of osmotic probes but correlated with their radius up to radius >17 A. In mixtures including a large and a small probe, the effect of the large probe was not modified by the small probe added at a large molar excess. With an osmotic probe of 4-A radius, the Delta(Delta G)/pi slope corresponds to a transfer of 119 +/- 25 water molecules to bulk solution on formation of the complex. Analytical characterization of water-permeable volumes in x-ray-derived bound and free antithrombin structures revealed complex surfaces with smaller hydration volumes in the bound relative to the free conformation. The residue distribution in, and atomic composition of, the pockets containing atoms from residues implicated in heparin binding were distinct in the bound versus free conformer. The results demonstrate that the heparin/antithrombin interaction is linked to net water transfer and, therefore, can be regulated in biological gels by osmotic potentials.
McGee
M P
MP
Internal Medicine Department, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157, USA. mmcgee@wfubmc.edu
Liang
J
J
D000991:Antithrombins / Q000737:chemistry / Q000378:metabolism*; D001692:Biological Transport; D006025:Glycosaminoglycans / Q000378:metabolism*; D006493:Heparin / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D009997:Osmotic Pressure; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D013816:Thermodynamics; D014867:Water / Q000378:metabolism*
D000991:Antithrombins; D006025:Glycosaminoglycans; D014867:Water; D006493:Heparin