PubMed · 7045862
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N Ia Lagutina, G A Fedulova. 1982. [Antithrombin III].. https://pubmed.ncbi.nlm.nih.gov/7045862/
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Variant forms of human antithrombin III with glutamine or threonine substitutions at Lys114, Lys125, Lys133, Lys136, and Lys139 were expressed in insect cells to evaluate their roles in heparin binding and activation. Recombinant native ATIII and all of the variants had very similar second order rate constants for thrombin inhibition in the absence of heparin, ranging from 1.13 x 10(5) M-1min-1 to 1.66 x 10(5) M-1min-1. Direct binding studies using 125I-flouresceinamine-heparin yielded a Kd of 6 nM for the recombinant native ATIII and K136T, whereas K114Q and K139Q bound heparin so poorly that a Kd could not be determined. K125Q had a moderately reduced affinity. Heparin binding affinity correlated directly with heparin cofactor activity. Recombinant native ATIII was nearly identical to plasma-purified ATIII, whereas K114Q and K139Q were severely impaired in heparin cofactor activity. K125Q and K136T were only slightly impaired. Based on these data, Lys114 and Lys139, which are outside of the putative pentasaccharide binding site, play pivotal roles in the high affinity binding of heparin to ATIII and the activation of thrombin inhibitory activity.
Autoproteolysis of blood coagulation factor Xa (FXa) results in the excision of a 4-kDa fragment (beta-peptide) from the intact subform, factor Xaalpha (FXaalpha), to yield factor Xabeta (FXabeta). In the preceding paper, we showed that generation of FXabeta leads to expression of a plasminogen binding site. FXabeta may consequently participate in fibrinolysis; therefore, the timing of subform conversion compared with thrombin production is important. In the current study we evaluated the kinetics of FXabeta generation, which showed that autoproteolysis of FXaalpha followed a second order mechanism where FXaalpha and FXabeta behaved as identical enzymes. Rate constants of 9 and 172 M-1 s-1 were derived, respectively, in the absence and presence of FXaalpha binding to procoagulant phospholipid. Under identical conditions the latter is estimated to be 6 orders of magnitude slower than thrombin generation by prothrombinase. Since heparin binding and prothrombin recognition have been previously attributed to a region of FXaalpha proximal to the beta-peptide, functional comparisons were conducted using homogeneous and stabilized preparations of FXaalpha and FXabeta. Comparisons included 1) the recognition of small substrates; 2) the rate of interaction with antithrombin/heparin; 3) the assembly of prothrombinase; and 4) the activation of prothrombin by prothrombinase. Although the beta-peptide neighbors a probable functional region in FXaalpha, conversion to FXabeta was not observed to influence these functions. The data support a model where FXaalpha is predominantly responsible for thrombin generation and where slow conversion to FXabeta coordinates coagulation and the initiation of fibrinolysis at sites of prothrombinase assembly.
A heparin-induced conformational change is required to convert antithrombin from a slow to a fast inhibitor of factor Xa. It has been proposed [van Boeckel et al. (1994) Nat. Struct. Biol. 1, 423-425] that the reactive center residue P14 is inserted into beta-sheet A in native antithrombin and is displaced from the beta-sheet by heparin binding, thereby altering the conformation of the reactive center and making it a better target for factor Xa binding. To test this hypothesis, we have characterized a P14 serine --> tryptophan antithrombin variant. From changes in tryptophan fluorescence upon heparin binding, increased affinity for heparin, and partial activation of the variant against factor Xa, we conclude that the proposed mechanism of heparin activation is correct with respect to loop expulsion and that it may consequently be possible to create more highly activated antithrombin variants through suitable hinge region substitutions.