Human annexin V (PP4), a member of the family of calcium, membrane binding proteins, has been crystallized in the presence of calcium and analysed by crystallography by multiple isomorphic replacement at 3 A and preliminarily refined at 2.5 A resolution. The molecule has dimensions of 64 x 40 x 30 A3 and is folded into four domains of similar structure. Each domain consists of five alpha‐helices wound into a right‐handed superhelix yielding a globular structure of approximately 18 A diameter. The domains have hydrophobic cores whose amino acid sequences are conserved between the domains and within the annexin family of proteins. The four domains are folded into an almost planar array by tight (hydrophobic) pair‐wise packing of domains II and III and I and IV to generate modules (II‐III) and (I‐IV), respectively. The assembly is symmetric with three parallel approximate diads relating II to III, I to IV and the module (II‐III) to (I‐IV), respectively. The latter diad marks a channel through the centre of the molecule coated with charged amino acid residues. The protein has structural features of channel forming membrane proteins and a polar surface characteristic of soluble proteins. It is a member of the third class of amphipathic proteins different from soluble and membrane proteins.

Crystal structure analysis and refinement at 2.0 A resolution of a rhombohedral crystal form of human annexin V at high calcium concentration revealed a domain motion compared to the previously analysed hexagonal crystal form. Five calcium ions were located on the convex face of the molecule. Three strongly bound calciums are liganded at protruding interhelical loops and Asp or Glu residues in homologous positions in repeats I, II and IV. Five proteinaceous oxygens and one solvent molecule form the coordination polyhedron in each case. The unoccupied seventh site is suggested as the phospholipid headgroup binding site. Two more weakly bound sites were identified by lanthanum labelling. The structural features suggest that annexin V attaches with its convex face to membranes by specified calcium mediated interactions with at least three phospholipids. The adjacent membrane bilayer may thus become locally disordered and permeable to allow calcium inflow through the central polar channel of the molecule.

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The serpin antithrombin III (AT III), the most important natural inhibitor of thrombin activity, has been shown to exert marked anti-inflammatory properties and proven to be efficacious in experimental models of sepsis, septic shock, and disseminated intravascular coagulation. Moreover, clinical observations suggest a possible therapeutic role for AT III in septic disorders. The molecular mechanism, however, by which AT III attenuates inflammatory events is not yet entirely understood. We show here that AT III potently blocks the activation of nuclear factor B (NF-B), a transcription factor involved in immediate early gene activation during inflammation. AT III inhibited agonist-induced DNA binding of NF-B in cultured human monocytes and endothelial cells in a dose-dependent manner, suggesting that AT III interferes with signal transduction leading to NF-B activation. This idea was supported by demonstrating that AT III prevents the phosphorylation and proteolytic degradation of the inhibitor protein IB␣. In parallel to reducing NF-B activity, AT III inhibited the expression of interleukin-6, tumor necrosis factor-␣, and tissue factor, genes known to be under the control of NF-B. The observation that chemically modified AT III that lacks heparin-binding capacity had no effect on NF-B activation supports the current understanding that the inhibitory potency of AT III depends on the interaction of AT III with heparinlike cell surface glycosaminoglycans. This hypothesis was underscored by the finding that the AT III ␤-isoform, known to have higher affinity for glycosaminoglycans, is more effective in preventing NF-B transactivation than ␣-AT III. These data indicate that AT III can alter inflammatory processes via inhibition of NF-B activation. IntroductionAntithrombin is one of the most important endogenous regulators of coagulation, acting as the major inhibitor of thrombin and interfering with several plasma proteases such as kallikrein and factors IXa, Xa, XIa, and XIIa. 1 Apart from its role in hemostasis, there is accumulating evidence that antithrombin III (AT III) exerts anti-inflammatory properties and improves survival in animalsepsis models and disseminated intravascular coagulation (DIC). [2][3][4] There are a number of compelling reasons why AT III may also be an effective therapeutic agent in patients with sepsis. [5][6][7] AT III was shown to reduce leukocyte-endothelial interaction, 8 to prevent microvascular leakage, 9 and to ameliorate ischemia/reperfusion injury. 10 These effects, however, seem to result only in part from interference of AT III with thrombin activity because inhibition of thrombin generation alone did not prove similarly effective. 11,12 The beneficial effects of AT III rather appear mainly to result from direct, thrombin-independent effects on vascular cells. AT III was found to stimulate nitric oxide synthesis in vascular smooth muscle cells, 13 to inhibit migration and adhesion of neutrophils, 14 and to attenuate cytokine production in monocytes and endothelial cells (ECs) 15...