These findings were in contrast to previous studies, which have shown that an important characteristic of clostridial myonecrosis is an absence of vascular permeability due to the formation of platelet-leucocyte and platelet-platelet aggregates within the vasculature and the up-regulation of adhesion molecules on endothelial cells, leading to an occlusion of blood flow into infected regions.. Studies using an in vivo microvascular perfusion model also have shown that treatment of mouse cremaster muscle tissue with culture supernatants from wild-type C. perfringens strains led to a decreased functional capillary density, which was reversed when cells were pretreated with anti-Gr-1, a granulocyte-specific antibody, and antiplatelet serum. These data confirmed the importance of neutrophil-platelet aggregates in blocking vascular perfusion during C. perfringens infection. Using a mouse myonecrosis infection model, we have now shown that Regorafenib a-clostripain is not essential for C. perfringens-mediated disease. Mice infected with an a-clostripain mutant did not show any alteration in disease progression or in the development of vascular leukostasis, a hallmark of C. perfringens infection. Mutation of the ccp gene also had no affect on bacterial growth or on the extracellular level of the two major toxins, a-toxin and perfringolysin O, which are implicated in clostridial myonecrosis. In agreement with previous studies our results provide good evidence that a-clostripain is the major protease produced by C. perfringens strain 13. Other bacterial cysteine proteases, which unlike a-clostripain have been shown to be essential for virulence, also are involved in influencing the levels of other virulence factors, many of which have nonredundant functions. For example, mutation of both P. gingivalis gingipains not only reduces total cysteine protease activity, but also affects the growth and expression of cell surface structures such as fimbriae and vesicles, both of which are thought to assist in colonization and evasion of the immune response. Like a-clostripain other potential C. perfringens-encoded spreading factors, such as collagenase and two different sialidases, are not to be essential for disease in the mouse myonecrosis model. These enzymes may still have a role during infection. The breakdown of host tissues is important for the release of nutrients such as amino acids, sugars and essential minerals such as sequestered iron, which are required for the growth of C. perfringens cells in the lesion.