Antithrombin Deficiency

Antithrombin is part of a family of serine protease inhibitors known as serpins. Serpins generally consist of a highly conserved structure of amino acid chains organized into three beta-sheets, nine alpha sheets, and a reactive center loop (RCL) designated as the sequence of amino acids which serve as the reactive site for protease interaction[rx]. The RCL loop in antithrombin exists along the amino acid chain sequence at the 393 arginine residue and 394 serine residue near the carboxyl-terminal of the amino acid sequence. It is the region within the antithrombin that creates the antithrombin-protease complex for inhibition.[rx]

Synthesis of antithrombin occurs primarily in the liver initially as an immature protein chain made up of 464 amino acid residues. The amino acid chain is then cleaved at the N-terminal by 32 amino acids, thereby creating a mature 432 amino acid sequence protein. The mature protein contains three disulfide bonds that intermolecularly link six cysteine residues together. This configuration allows for four potential glycosylation sites to exist within the molecule.[rx] Depending on the number of occupied glycosylation sites, anti-thrombin further subcategorized into two isoforms: alpha antithrombin and beta antithrombin.[rx]

Alpha antithrombin refers to antithrombin in which oligosaccharides bind all four glycosylation sites. It is the predominant configuration of antithrombin, presenting as around 90% of the antithrombin in the plasma.[rx] Beta antithrombin, however, refers to antithrombin with three of the four sites occupied, with the oligosaccharide chain at Asn135 missing.[rx] This change in configuration increases the affinity of beta antithrombin binding to heparin at a designated heparin-binding domain.[rx] The binding of heparin dramatically increases the affinity for antithrombin to bind to serine protease, enhancing the functional efficiency of antithrombin to inhibit clot formation.

Mechanism

Antithrombin has two specific binding sites- the reactive site consisting of the reactive center loop (RCL) which binds proteases such as thrombin, factor Xa, IXa, and the heparin-binding domain which, as the name suggests, binds heparin.[rx]

The reactive center loop of antithrombin located at the arginine residue at 393 and the serine residue at 394 near the carboxyl-terminal functions to bind to the active site of proteases through a complex mechanism involving a conformational change of the antithrombin reactive site.[8] Strands from beta-sheet A of the antithrombin site separate halfway along their length and the RCL subsequently rearranges at the point of entry into sheet A along with various other conformational changes, which serve to increase the mobility of the RCL. The increased mobility provides a docking site for the protease, which in turn creates an irreversible complex.[rx]

The inhibitor-protease complex is then rapidly removed from the circulation no more than 5 minutes after formation, which removes thrombin from the circulation, disrupting the clotting effect of the coagulation cascade. While the exact mechanism is still uncertain, evidence may suggest receptors on hepatocytes to be involved in the removal of the complex from the plasma.[rx]

The formation of the antithrombin-protease complex, while irreversible, is a naturally slow and inefficient reaction. The process can be rapidly increased up to 1000-fold with the presence of sulfated polysaccharides in the form of heparin and heparan sulfate.[8] Heparin contains a unique pentasaccharide sequence within its glycosaminoglycan chain consisting of negatively charged sulfate groups, which is responsible for its high affinity towards the antithrombin heparin-binding domain.

The heparin-binding domain located on the surface of antithrombin, on the other hand, contains positively charged arginine and lysine which bind to the negatively charged domains of the heparin pentasaccharide sequence through partially an allosteric mechanism.[rx]

The successful binding of heparin activates a conformational change within the antithrombin, which increases its affinity towards protease, promotes the formation of the antithrombin-protease complex, and ultimately inhibits blood coagulation.[rx]

Allosteric activation induced by heparin to the antithrombin serpin structure has been the object of extensive study, and while the kinetics of the reaction is quite complex, generally the allosteric activation of antithrombin induces structural changes within the RCL, the heparin-binding site, and the hydrophobic core that constitutes the antithrombin. The exact interactions involved in conformational change are still a topic for research and revision.[rx]