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Key Points• ATG induces monocyte TF procoagulant activity dependent on complement activation but independent of de novo protein synthesis.• TF decryption requires oxidation of cell surface PDI following C5 activation and phosphatidylserine membrane exposure following C7 insertion.Lymphocyte depletion with antithymocyte globulin (ATG) can be complicated by systemic coagulation activation. We found that ATG activated tissue factor procoagulant activity (TF PCA) on monocytic cells more potently than other stimuli that decrypt TF, including cell disruption, TF pathway inhibitor inhibition, and calcium ionophore treatment. Induction of TF PCA by ATG was dependent on lipid raft integrity and complement activation. We showed that ATG-mediated TF activation required complement activation until assembly of the C5b-7 membrane insertion complex, but not lytic pore formation by the membrane attack complex C5b-9. Consistently, induction of TF PCA by ATG did not require maximal phosphatidylserine membrane exposure and was not correlated with the magnitude of complement-induced lytic cell injury. Blockade of free thiols, an inhibitory monoclonal antibody to protein disulfide isomerase (PDI), and the small-molecule PDI antagonist quercetin-3-rutinoside prevented ATG-mediated TF activation, and C5 complement activation resulted in oxidation of cell surface PDI. This rapid and potent mechanism of cellular TF activation represents a novel connection between the complement system and cell surface PDI-mediated thiol-disulfide exchange. Delineation of this clinically relevant mechanism of activation of the extrinsic coagulation pathway during immunosuppressive therapy with ATG may have broader implications for vascular thrombosis associated with inflammatory disorders. (Blood. 2013;121(12):2324-2335

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Cyclic nucleotide-gated channels (CNGCs) form non-selective cation entry pathways regulated by calmodulin (CaM), a universal Ca2+ sensor in eukaryotes. Although CaM binding has been shown to be important for Ca2+-dependent feedback regulation of CNGC activity, the CaM-binding properties of these channels have been investigated in a few cases only. We show that CNGC20 from Arabidopsis thaliana binds CaM in a Ca2+-dependent manner and interacts with all AtCaM isoforms but not with the CaM-like proteins CML8 and CML9. CaM interaction with the full-length channel was demonstrated in planta, using bimolecular fluorescence complementation. This interaction occurred at the plasma membrane, in accordance with our localization data of green fluorescent protein (GFP)-fused CNGC20 proteins. The CaM-binding site was mapped to an isoleucine glutamine (IQ) motif, which has not been characterized in plant CNGCs so far. Our results show that compared with the overlapping binding sites for cyclic nucleotides and CaM in CNGCs studied so far, they are sequentially organized in CNGC20. The presence of two alternative CaM-binding modes indicates that ligand regulation of plant CNGCs is more complex than previously expected. Since the IQ domain is conserved among plant CNGCs, this domain adds to the variability of Ca2+-dependent channel control mechanisms underlining the functional diversity within this multigene family.

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