A new method to quantify β-antithrombin glycoform in plasma reveals increased levels during the acute stroke event

Abstract

Introductionβ-antithrombin, the minor antithrombin glycoform in plasma, is probably the major thrombin inhibitor in vivo because of its high heparin affinity. The levels and variability of this glycoform in general population and its relevance in thromboembolic diseases is unknown since there is no specific method to measure this glycoform in clinical samples. MethodsPlasma and recombinant α- and β-antithrombins were purified by heparin affinity chromatography. An anti-FXa chromogenic method in presence of pentassacharide was used with two NaCl concentrations (15mM and 1.1M). This method was applied to plasma samples from 97 healthy subjects and 117 consecutive patients with ischemic cerebrovascular disease during the acute event and one year later. SERPINC1 sequencing was done in cases with antithrombin deficiency. ResultsHigh salt concentrations specifically restricted the pentassacharide-induced activation of antithrombin to the β glycoform. β-antithrombin displayed a normal distribution in the general population (89.5%-103.5%), with no significant variations according to age or sex. In patients, whole antithrombin values remained within the normal range. Only five cases had antithrombin deficiency during the thrombotic event, one carrying the L99F mutation in SERPINC1. Interestingly, both β-antithrombin and the β/whole antithrombin ratio were significantly higher in patients during the acute event but normalized after one year. ConclusionsWe have developed a rapid, simple, sensitive and specific method to quantify β-antithrombin activity using 1μL of plasma. β-antithrombin significantly increases in patients with ischemic cerebrovascular disease during the acute event, probably by its release from the vasculature.