Antithrombin, a plasma serpin, is relatively inactive as an inhibitor of the coagulation proteases until it binds to the heparan side chains that line the microvasculature. The binding specifically occurs to a core pentasaccharide present both in the heparans and in their therapeutic derivative heparin. The accompanying conformational change of antithrombin is revealed in a 2.9-Å structure of a dimer of latent and active antithrombins, each in complex with the high-affinity pentasaccharide. Inhibitory activation results from a shift in the main sheet of the molecule from a partially six-stranded to a five-stranded form, with extrusion of the reactive center loop to give a more exposed orientation. There is a tilting and elongation of helix D with the formation of a 2-turn helix P between the C and D helices. Concomitant conformational changes at the heparin binding site explain both the initial tight binding of antithrombin to the heparans and the subsequent release of the antithrombin-protease complex into the circulation. The pentasaccharide binds by hydrogen bonding of its sulfates and carboxylates to Arg-129 and Lys-125 in the D-helix, to Arg-46 and Arg-47 in the A-helix, to Lys-114 and Glu-113 in the P-helix, and to Lys-11 and Arg-13 in a cleft formed by the amino terminus. This clear definition of the binding site will provide a structural basis for developing heparin analogues that are more specific toward their intended target antithrombin and therefore less likely to exhibit side effects.Heparin, a sulfated polysaccharide, is second only to insulin as a natural therapeutic agent and is the initial-choice anticoagulant in the treatment and prevention of thromboembolic disease. It functions in life as a component of the heparans that line the inner walls of the microvascular system (1), but heparin as a drug is a heterogeneous animal extract administered by injection to circulate in the bloodstream. Both heparin and the natural heparans contain a specific pentasaccharide fragment (2, 3) that binds and activates the plasma proteinase inhibitor antithrombin.In nature, this binding to heparans substantially localizes the function of antithrombin to inhibition of the serine proteases of the coagulation cascade within the bloodstream, allowing their coagulant activity in damaged tissue outside the vascular system. The heparans and the longer-chain heparins (4) activate the inhibition of thrombin by antithrombin by bringing them into close apposition, but there is also a direct activation of inhibition due to an overall conformational change (5) induced by the binding to the core pentasaccharide present in both heparin and heparans. This pentasaccharide-induced change alters the conformation of the reactive site loop of antithrombin (6, 7) and gives a 300-fold increase in inhibitory activity against the key coagulation protease factor Xa. Linked to this is a change in affinity at the heparin binding site (see Fig. 1), and as antithrombin contacts the pentasaccharide, it moves from an initial low-affinit...

We present a comprehensive alignment and phylogenetic analysis of the serpins, a superfamily of proteins with known members in higher animals, nematodes, insects, plants, and viruses. We analyze, compare, and classify 219 proteins representative of eight major and eight minor subfamilies, using a novel technique of consensus analysis. Patterns of sequence conservation characterize the family as a whole, with a clear relationship to the mechanism of function. Variations of these patterns within phylogenetically distinct groups can be correlated with the divergence of structure and function. The goals of this work are to provide a carefully curated alignment of serpin sequences, to describe patterns of conservation and divergence, and to derive a phylogenetic tree expressing the relationships among the members of this family. We extend earlier studies by Huber and Carrell as well as by Marshall, after whose publication the serpin family has grown functionally, taxonomically, and structurally. We used gene and protein sequence data, crystal structures, and chromosomal location where available. The results illuminate structure-function relationships in serpins, suggesting roles for conserved residues in the mechanism of conformational change. The phylogeny provides a rational evolutionary framework to classify serpins and enables identification of conserved amino acids. Patterns of conservation also provide an initial point of comparison for genes identified by the various genome projects. New homologs emerging from sequencing projects can either take their place within the current classification or, if necessary, extend it.