Caspase Inhibition Reduces Hepatic Tissue Factor-Driven Coagulation In Vitro and In Vivo.

Tissue factor (TF) is the primary activator of the blood coagulation cascade. Liver parenchymal cells (ie, hepatocytes) express TF in a molecular state that lacks procoagulant activity. Hepatocyte apoptosis is an important feature of acute and chronic liver diseases, and Fas-induced apoptosis increases hepatocyte TF procoagulant activity in vitro. We determined the impact of a pan-caspase inhibitor, IDN-7314, on hepatocyte TF activity in vitro and TF-mediated coagulation in vivo. Treatment of primary mouse hepatocytes with the Fas death receptor ligand (Jo2, 0.5 µg/ml) for 8 h increased hepatocyte TF procoagulant activity and caused release of TF-positive microvesicles. Pretreatment with 100 nM IDN-7314 abolished Jo2-induced caspase-3/7 activity and significantly reduced hepatocyte TF procoagulant activity and release of TF-positive microvesicles. Treatment of wild-type C57BL/6 mice with a sublethal dose of Jo2 (0.35 mg/kg) for 4.5 h increased coagulation, measured by a significant increase in plasma thrombin-antithrombin and TF-positive microvesicles. Total plasma microvesicle-associated TF activity was reduced in mice lacking hepatocyte TF; suggesting TF-positive microvesicles are released from the apoptotic liver. Fibrin(ogen) deposition increased in livers of Jo2-treated wild-type mice and colocalized primarily with cleaved caspase-3-positive hepatocytes. Pretreatment with IDN-7314 reduced caspase-3 activation, prevented the procoagulant changes in Jo2-treated mice, and reduced hepatocellular injury. Overall, the results indicate a central role for caspase activity in TF-mediated activation of coagulation following apoptotic liver injury. Moreover, the results suggest that liver-selective caspase inhibition may be a putative strategy to limit procoagulant and prothrombotic changes in patients with chronic liver disease.

A nucleotide binding site in caspase-9 regulates apoptosome activation.

ATP or dATP is a required activator of Apaf-1 for formation of the Apoptosome and thereby activation of caspase-9 (Csp9) [Zou, H., Henzel, W. J., Liu, X., Lutschg, A., and Wang, X. (1997) Cell 90, 405-413]. Here we demonstrate that dATP or ATP may have an additional role in controlling Apaf-1-mediated Csp9 activation. In the presence of cytochrome c (CytC), dATP or ATP binds to Apaf-1 and triggers heptamerization of Apaf-1 leading to the activation of Csp9. At concentrations greater than 1 mM, dATP or ATP also functions as a negative regulator of apoptosis by binding to and inhibiting Csp9. The affinity labeling reagent, 3'-O-(5-fluoro-2,4-dinitrophenyl)-ATP (FDNP-ATP), was used to probe the binding of nucleotides to Csp9. Similar to ATP, but with a much more profound effect, FDNP-ATP binds to the full-length proCsp9 potently, with an IC(50) of approximately 5-11 nM. Neither ATP nor FDNP-ATP exhibits any effect on the prodomain-truncated enzyme DeltaproCsp9 or p18/p10. FDNP-ATP covalently labels proCsp9 with a stoichiometry of 1:1, resulting in DNP-ATP-proCsp9 that is incapable of forming a productive Apoptosome with Apaf-1. Activity assays show that ATP and dATP, but not ADP or AMP, bind to the processed Csp9 p35/p10. This nucleotide binding site might play an important and previously unrecognized role in regulating proCsp9 activation.

Conformational restrictions in the active site of unliganded human caspase-3.

Caspases are cysteine proteases that play a critical role in the initiation and regulation of apoptosis. These enzymes act in a cascade to promote cell death through proteolytic cleavage of intracellular proteins. Since activation of apoptosis is implicated in human diseases such as cancer and neurodegenerative disorders, caspases are targets for drugs designed to modulate their action. Active caspases are heterodimeric enzymes with two symmetrically arranged active sites at opposite ends of the molecule. A number of crystal structures of caspases with peptides or proteins bound at the active sites have defined the mechanism of action of these enzymes, but molecular information about the active sites before substrate engagement has been lacking. As part of a study of peptidyl inhibitors of caspase-3, we crystallized a complex where the inhibitor did not bind in the active site. Here we present the crystal structure of the unoccupied substrate-binding site of caspase-3. No large conformational differences were apparent when this site was compared with that in enzyme-inhibitor complexes. Instead, the 1.9 A structure reveals critical side chain movements in a hydrophobic pocket in the active site. Notably, the side chain of tyrosine204 is rotated by approximately 90 degrees so that the phenol group occupies the S2 subsite in the active site. Thus, binding of substrate or inhibitors is impeded unless rotation of this side chain opens the area. The positions of these side chains may have important implications for the directed design of inhibitors of caspase-3 or caspase-7.