Degradation of Circulating von Willebrand Factor and Its Regulator ADAMTS13 Implicates Secreted Bacillus anthracis Metalloproteases in Anthrax Consumptive Coagulopathy

Abstract

Pathology data from the anthrax animal models show evidence of significant increases in vascular permeability coincident with hemostatic imbalances manifested by thrombocytopenia, transient leucopenia, and aggressive disseminated intravascular coagulation. In this study we hypothesized that anthrax infection modulates the activity of von Willebrand factor (VWF) and its endogenous regulator ADAMTS13, which play important roles in hemostasis and thrombosis, including interaction of endothelial cells with platelets. We previously demonstrated that purified anthrax neutral metalloproteases Npr599 and InhA are capable of cleaving a variety of host structural and regulatory proteins. Incubation of human plasma with these proteases at 37°C in the presence of urea as a mild denaturant results in proteolysis of VWF. Also in these conditions, InhA directly cleaves plasma ADAMTS13 protein. Npr599 and InhA digest synthetic VWF substrate FRETS-VWF73. Amino acid sequencing of VWF fragments produced by InhA suggests that one of the cleavage sites of VWF is located at domain A2, the target domain of ADAMTS13. Proteolysis of VWF by InhA impairs its collagen binding activity (VWF:CBA) and ristocetin-induced platelet aggregation activity. In plasma from anthrax spore-challenged DBA/2 mice, VWF antigen levels increase up to 2-fold at day 3 post-infection with toxigenic Sterne 34F 2 strain, whereas VWF:CBA levels drop in a time-dependent manner, suggesting dysfunction of VWF instead of its quantitative deficiency. This conclusion is further supported by significant reduction in the amount of VWF circulating in blood in the ultra-large forms. In addition, Western blot analysis shows proteolytic depletion of ADAMTS13 from plasma of spore-challenged mice despite its increased expression in the liver. Our results suggest a new mechanism of anthrax coagulopathy affecting the levels and functional activities of both VWF and its natural regulator ADAMTS13. This mechanism may contribute to hemorrhage and thrombosis typical in anthrax.Bacillus anthracis, the causative agent of anthrax, is a zoonotic, Gram-positive, spore-forming Bacillus. It is capable of causing a highly lethal systemic infection in people and experimental animals upon inhalation of the spores. The pathological mechanisms underlying high mortality of inhalation anthrax infection are complex and remain insufficiently understood. The major virulence factors, lethal toxin (LT) 2 and edema toxin (ET) produced by B. anthracis can cause death of experimental animals with symptoms resembling those of anthrax infection. Therefore, many studies have been focused on the cytotoxic effects of LT, including the cells of monocyte lineage (peripheral blood monocytes, macrophages, and dendritic cells) (1) and epithelial cells (2). It has also been reported that the LT-induced cell death could cause a massive release of the pro-inflammatory cytokines, such as the tumor necrosis factor ␣ (TNF␣), which may result in lethality (3), but recent data clearly indicate that the intoxication mediate...

The prognostic value of ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 repeats, member 13) deficiency in septic shock patients involves interleukin-6 and is not dependent on disseminated intravascular coagulation

“…Third, in all our patients, we found increased VWF:Ag levels and the presence of UL-VWF multimers in plasma. An overwhelming release of ULVWF multimers from activated endothelial cells may exhaust ADAMTS13 activity by a consumption mechanism [44,46,49-54]. In septic patients, the literature is controversial either supporting this consumption process [20,21,24,25] or not [23].…”

Disruption of the Endothelial Barrier by Proteases from the Bacterial Pathogen Pseudomonas aeruginosa: Implication of Matrilysis and Receptor Cleavage

“…We now further demonstrate that LasB within pseudomonal secretomes can also efficiently and rapidly (i.e., within 1 to 6 hours) degrade the subendothelial matrix-specific protein, vWf, while degradation was minimal with PAO1∆ lasB -Sec for up to 24 h. Because the massive apoptosis induced by staurosporine was not accompanied by degradation of ECM-associated Fn and vWf, one can assume that matrilysis is not the mere consequence of proteinases released by dying cells, but rather a LasB-specific process. Interestingly, cleavage of plasma vWf by bacterial metalloproteinases was previously previously reported to be associated with anti-haemostatic and pro-haemorrhagic properties of these enzymes in vivo [68], and it is thus tempting to speculate that the capacity of LasB to erase the subendothelial vWf may participate in the dysregulation of haemostasis during bloodstream infections by P. aeruginosa [25].…”

Bacillus anthracis sinLocus and Regulation of Secreted Proteases

“…InhA1 is a major component of the B. anthracis secretome that appears to degrade host substrates with relatively little specificity (13,(17)(18)(19)38). A system in which InhA1 levels are controlled transcriptionally and posttranslationally in response to the growth phase-associated activity of SinR suggests that limitation of InhA1 is beneficial in certain environments.…”

“…Several sinIR-regulated genes of B. subtilis are associated with species-specific phenotypes, including those involving biofilm formation, motility, and competency (3,35,42,45). In B. subtilis, sinIR negatively controls the biofilm-associated extrapolysaccharide genes (eps) and the biofilm structural protein gene yqxM (15 (17)(18)(19)38). Camelysin, the other sinR-regulated secreted protease of B. anthracis, was first described as a casein-lytic protein of B. cereus (33).…”