Deletion of P1 arginine in a novel antithrombin variant (antithrombin London) abolishes inhibitory activity but enhances heparin affinity and is associated with early onset thrombosis.
A novel variant of antithrombin, the major serpin inhibitor of coagulation proteases, has been identified in a patient with early onset thrombosis and abnormal plasma antithrombin activity. Sequencing of the antithrombin genes of the patient revealed that one of the two alleles was abnormal due to an in-frame deletion of the codon for the P1 arginine residue. The abnormal antithrombin was separated from the normal inhibitor by complexing the latter with thrombin followed by heparin-agarose affinity chromatography. The purified variant, antithrombin London, was completely inactive as a thrombin or factor Xa inhibitor even after heparin activation. Surprisingly, the variant bound heparin with a K(D) reflecting an approximately 10-fold greater affinity than the normal inhibitor. Stopped-flow kinetic analysis showed that this was almost entirely due to a more favorable conformational activation of the variant than the normal inhibitor, as reflected by a decreased rate constant for reversal of the activation. Consistent with its higher than normal heparin affinity, the inactive antithrombin variant was a potent competitive antagonist of the heparin-catalyzed reaction of normal antithrombin with thrombin but did not affect the uncatalyzed reaction. These results suggest that deletion of the antithrombin P1 residue partially activates the serpin by inducing strain in the reactive center loop, which destabilizes the native loop-buried state and favors the activated loop-exposed state with high heparin affinity. The unusually severe thrombosis associated with the heterozygous mutation may be explained by the ability of antithrombin London to bind endogenous heparan sulfate or heparin molecules with high affinity and to thereby block activation of the normal inhibitor.
Raja
Srikumar M
SM
Center for Molecular Biology of Oral Diseases, College of Dentistry, University of Illinois at Chicago, 60612, USA.
Chhablani
Neetu
N
Swanson
Richard
R
Thompson
Elizabeth
E
Laffan
Mike
M
Lane
David A
DA
Olson
Steven T
ST
D017668:Age of Onset; D058833:Antithrombin Proteins; D000991:Antithrombins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D001120:Arginine / Q000737:chemistry; D001667:Binding, Competitive; D001777:Blood Coagulation; D002846:Chromatography, Affinity; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D065427:Factor Xa Inhibitors; D005192:Family Health; D005260:Female; D017353:Gene Deletion; D006493:Heparin / Q000378:metabolism; D006497:Heparan Sulfate / Q000737:chemistry; D006579:Heterozygote; D006801:Humans; D007700:Kinetics; D008297:Male; D008954:Models, Biological; D010375:Pedigree; D011485:Protein Binding; D011506:Proteins / Q000235:genetics* / Q000378:metabolism*; D013917:Thrombin / Q000037:antagonists & inhibitors / Q000378:metabolism; D013927:Thrombosis / Q000209:etiology* / Q000235:genetics*; D013997:Time Factors
D058833:Antithrombin Proteins; D000991:Antithrombins; D001120:Arginine; D065427:Factor Xa Inhibitors; D006493:Heparin; D006497:Heparan Sulfate; D011506:Proteins; D013917:Thrombin; C474022:antithrombin London