Serpin family protein proteinase inhibitors regulate the activity of serine and cysteine proteinases by a novel conformational trapping mechanism that may itself be regulated by cofactors to provide a finely-tuned time and location-dependent control of proteinase activity. The serpin, antithrombin, together with its cofactors, heparin and heparan sulfate, perform a critical anticoagulant function by preventing the activation of blood clotting proteinases except when needed at the site of a vascular injury. Here, we review the detailed molecular understanding of this regulatory mechanism that has emerged from numerous X-ray crystal structures of antithrombin and its complexes with heparin and target proteinases together with mutagenesis and functional studies of heparin-antithrombinproteinase interactions in solution. Like other serpins, antithrombin achieves specificity for its target blood clotting proteinases by presenting recognition determinants in an exposed reactive center loop as well as in exosites outside the loop. Antithrombin reactivity is repressed in the absence of its activator because of unfavorable interactions that diminish the favorable RCL and exosite interactions with proteinases. Binding of a specific heparin or heparan sulfate pentasaccharide to antithrombin induces allosteric activating changes that mitigate the unfavorable interactions and promote template bridging of the serpin and proteinase. Antithrombin has thus evolved a sophisticated means of regulating the activity of blood clotting proteinases in a time and locationdependent manner that exploits the multiple conformational states of the serpin and their differential stabilization by glycosaminoglycan cofactors.

Protease nexin-1 (PN-1) is a serpin that inhibits plasminogen activators, plasmin, and thrombin. PN-1 is barely detectable in plasma but is expressed by platelets. Here, we studied platelet PN-1 in resting and activated conditions and its function in thrombosis. Studies on human platelets from healthy donors and from patients with a Gray platelet syndrome demonstrate that PN-1 is present both at the platelet surface and in ␣-granules.

Serine protease inhibitors, termed serpins, are key regulators in many biologic events. Protease nexin-1 (PN-1) is a serpin that is barely detectable in plasma but found in many organs and produced by most cell types, including monocytes, platelets, and vascular cells. It has a large inhibition spectrum because it is the most efficient tissue inhibitor of thrombin but also a powerful inhibitor of plasminogen activators and plasmin. It has a high affinity for glycosaminoglycans, such as heparan sulfates, which potentiate its activity toward thrombin and target it to the pericellular space. PN-1 has been previously largely described as a crucial regulator of the proteolytic activity in nerves and of central and peripheral nervous system function. In contrast, little was known about its involvement in hemostasis and vascular biology. This article reviews recent data underlining its emerging role as a key factor in the responses of vessels to injury. IntroductionSerine proteases play a predominant role in the homeostasis of the cardiovascular system, including coagulation, fibrinolysis, and tissue remodeling. In blood, they circulate as inactive proforms or zymogens, and once activated, are quickly and irreversibly inhibited by circulating inhibitors, in particular by serine protease inhibitors, termed serpins. In contrast, at the tissue level, it is still not clear how endogenous inhibitors control the activity of the different proteases. In the context of vascular biology, both the thrombus, which can be considered as a neo-tissue, and the arterial wall are good examples of the importance of the regulation of serine protease activity by serpins.Serpins form a large family of structurally related proteins present in the plasma and/or in tissues. Some of them are mainly synthesized by the liver and directly secreted into the bloodstream. Others are synthesized by peripheral cells, including circulating cells, mesenchymal stromal cells of the vascular wall, and cardiac myocytes. Although not all serpins are protease inhibitors, 1 many of the members of this large superfamily act by interacting with the active site of the target proteases via the reactive site, which is positioned on an exposed loop. The protease trapped in the complex is then irreversibly inhibited. Serpins are direct or indirect key regulators of numerous cell and tissue responses to changes in environmental conditions. 1 Via their molecular flexibility and their antiprotease activity, serpins play a key role in the modulation of cell adhesion, migration, proliferation, metabolism, and death. They are also involved in the prevention of proteolytic degradation of the extracellular matrix and of fibrin formation and lysis. 2 One of the better-known and most studied members of the serpin superfamily is serpinC1,or antithrombin (AT), which circulates in the bloodstream and is the principal inhibitor of blood coagulation proteases. Moreover, particular emphasis has been placed on serpinE1 or plasminogen activator inhibitor-1 (PAI-1), an inhibitor of tis...