Physiological significance of antithrombin D-helix interaction with vascular GAGs

• 1. Interaction of D-helix of antithrombin with therapeutic heparins promotes the protease-inhibitory function of the serpin

• 2. Interaction of D-helix of antithrombin with vascular GAGs unveils the anti-inflammatory signaling function of the serpin

Antithrombin (AT) is an anticoagulant serpin involved in the regulation of proteolytic activities of coagulation proteases. AT also possesses a direct anti-inflammatory function. The anticoagulant function of AT is mediated through its reactive-center loop (RCL)-dependent inhibition of coagulation proteases, but anti-inflammatory function of AT is mediated via its D-helix-dependent interaction with vascular glycosaminoglycans (GAGs). In vitro assays have established that therapeutic heparins promote the anticoagulant function of AT by binding D-helix and activating the serpin, however, the contribution of vascular GAGs to D-helix-dependent anticoagulant function of AT has remained poorly understood in vivo. Here, we explored this question by utilizing two AT mutants, (AT-4Mut) which exhibits neither affinity for heparin nor D-helix-dependent anti-inflammatory signaling but possesses normal protease-inhibitory function and an inactive signaling-selective AT mutant in which its P1-Arg425 is deleted (AT-R425del). In vivo properties of mutants were compared to wildtype AT (AT-WT) in an siRNA-mediated AT-deficient mouse model. The siRNA knockdown efficiently reduced expression of AT and induced robust procoagulant and pro-inflammatory phenotypes in mice. Infusion of both AT-WT and AT-4Mut rescued the procoagulant phenotype of AT-deficient mice as evidenced by restoration of the plasma clotting time and inhibition of fibrin deposition. AT-WT also attenuated inflammation as evidenced by reduced VCAM-1 expression and leukocyte infiltration in the liver and lungs; however, AT-4Mut failed to attenuate inflammation. Interestingly, AT-R425del also effectively attenuated inflammation in AT-depleted mice. These results suggest interaction of AT D-helix with vascular GAGs may primarily be responsible for anti-inflammatory signaling rather than protease-inhibitory function of the serpin.