The binding of low molecular weight heparin to hemostatic enzymes.
A low molecular weight preparation of porcine heparin (specific anticoagulation activity = 125 units/mg) was fractionated to obtain a mucopolysaccharide product of 6500 daltons (specific anticoagulant activity = 373 units/mg) that is homogeneous with respect to its interaction with antithrombin. This material was treated with fluorescamine in order to introduce a fluorescent tag into the mucopolysaccharide. Initially, we showed that the fluorescamine-heparin conjugate and the unlabeled mucopolysaccharide interacted with antithrombin in a virtually identical fashion. Subsequently, we demonstrated that labeled heparin could be utilized in conjunction with fluorescence polarization spectroscopy to monitor the binding of mucopolysaccharide to thrombin, factor IXa, factor Xa, and plasmin. The interaction of this complex carbohydrate with thrombin exhibited a stoichiometry of 2:1 with KH1T DISS = KH2T DISS = 8 x 10(-7) M. The formation of mucopolysaccharide . factor IXa complex is characterized by a stoichiometry of 1:1 with KHIXa DISS = 2.58 x 10(-7) M. The binding of heparin to factor Xa or plasmin occurred with low avidity. Therefore, the stoichiometries of these processes could not be established. However, our experimental data were compatible with a single-site binding residue with KHXa DISS = 8.73 x 10(-6) M and KHPL DISS = approximately 1 x 10(-4) M, respectively.
D000818:Animals; D000991:Antithrombins / Q000378:metabolism; D005164:Factor IX / Q000378:metabolism*; D015949:Factor IXa; D005170:Factor X / Q000378:metabolism*; D015951:Factor Xa; D005341:Fibrinolysin / Q000378:metabolism*; D005450:Fluorescamine; D006493:Heparin; D006801:Humans; D007700:Kinetics; D008970:Molecular Weight; D011485:Protein Binding; D013552:Swine; D013917:Thrombin / Q000378:metabolism*
D000991:Antithrombins; D005450:Fluorescamine; D005164:Factor IX; D005170:Factor X; D006493:Heparin; D015949:Factor IXa; D013917:Thrombin; D015951:Factor Xa; D005341:Fibrinolysin