Inhibition of plasmin attenuates murine acute graft-versus-host disease mortality by suppressing the matrix metalloproteinase-9-dependent inflammatory cytokine storm and effector cell trafficking.

The systemic inflammatory response observed during acute graft-versus-host disease (aGVHD) is driven by proinflammatory cytokines, a 'cytokine storm'. The function of plasmin in regulating the inflammatory response is not fully understood, and its role in the development of aGVHD remains unresolved. Here we show that plasmin is activated during the early phase of aGVHD in mice, and its activation correlated with aGVHD severity in humans. Pharmacological plasmin inhibition protected against aGVHD-associated lethality in mice. Mechanistically, plasmin inhibition impaired the infiltration of inflammatory cells, the release of membrane-associated proinflammatory cytokines including tumor necrosis factor-α (TNF-α) and Fas-ligand directly, or indirectly via matrix metalloproteinases (MMPs) and alters monocyte chemoattractant protein-1 (MCP-1) signaling. We propose that plasmin and potentially MMP-9 inhibition offers a novel therapeutic strategy to control the deadly cytokine storm in patients with aGVHD, thereby preventing tissue destruction.

Persistent activation of Gsalpha through limited proteolysis by calpain.

Treatment of rat pituitary GH(4)C(1) cell membranes with calpain, a calcium-activated cysteine protease, increased adenylate cyclase activity, and this activity was inhibited by a calpain inhibitor, leupeptin. Calpain treatment potentiated the activity of guanosine 5'-[gamma-thio]triphosphate (GTP[S]), but did not attenuate MnCl(2) action on adenylate cyclase, suggesting that calpain acted at the G-protein level, rather than directly on adenylate cyclase. This calpain stimulation of adenylate cyclase was inhibited by an antibody raised against the C-terminal portion of G(s)alpha, but not by anti-G(i)2alpha or anti-Gbeta antibodies. Furthermore, it was shown that G(s)alpha is more susceptible to calpain-mediated proteolysis than G(i)2alpha or Gbeta. Therefore the stimulatory effect of calpain on adenylate cyclase is due to the cleavage of G(s)alpha in GH(4)C(1) cell membranes. Proteolysis of G(s)alpha by micro-calpain involved sequential cleavages at two sites, resulting in the generation of a 39 kDa fragment first, and then a 20 kDa fragment, from the C-terminus. Treatment of GH(4)C(1) cell membranes with cholera toxin increased the rate of cleavage. Cholera toxin treatment of intact GH(4)C(1) cells induced the translocation of calpain from the cytosol to the membranes, a hallmark of calpain activation. In addition, treatment of intact GH(4)C(1) cells with a calpain-specific inhibitor, benzyloxycarbonyl-Leu-leucinal, blocked the increased cAMP production and the down-regulation of G(s)alpha, which were produced by cholera toxin or pituitary adenylate cyclase-activating polypeptide. These results suggest that calpain sustains adenylate cyclase in an active form through the cleavage of G(s)alpha to an active G(s)alpha fragment. This is a novel calpain-dependent activation mechanism of G(s)alpha and, thus, of adenylate cyclase in rat pituitary cells.