Degradation of circulating von Willebrand factor and its regulator ADAMTS13 implicates secreted Bacillus anthracis metalloproteases in anthrax consumptive coagulopathy.
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 degrees 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.
Chung
Myung-Chul
MC
National Center for Biodefense and Infectious Diseases, College of Sciences, George Mason University, 10900 University Boulevard, Manassas, VA 20110, USA.
Popova
Taissia G
TG
Jorgensen
Shelley C
SC
Dong
Li
L
Chandhoke
Vikas
V
Bailey
Charles L
CL
Popov
Serguei G
SG
D051722:ADAM Proteins / Q000378:metabolism*; D000071120:ADAMTS13 Protein; D000818:Animals; D000881:Anthrax / Q000201:enzymology* / Q000473:pathology; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000378:metabolism*; D001792:Blood Platelets / Q000378:metabolism / Q000382:microbiology / Q000473:pathology; D002450:Cell Communication / Q000187:drug effects; D003094:Collagen / Q000378:metabolism; D004195:Disease Models, Animal; D004211:Disseminated Intravascular Coagulation / Q000201:enzymology* / Q000382:microbiology / Q000473:pathology; D042783:Endothelial Cells / Q000378:metabolism / Q000382:microbiology / Q000473:pathology; D006487:Hemostasis / Q000187:drug effects; D006801:Humans; D007970:Leukopenia / Q000201:enzymology / Q000382:microbiology / Q000473:pathology; D008666:Metalloendopeptidases / Q000378:metabolism; D045726:Metalloproteases / Q000378:metabolism*; D051379:Mice; D010949:Plasma / Q000201:enzymology / Q000382:microbiology; D010974:Platelet Aggregation / Q000187:drug effects; D011485:Protein Binding / Q000187:drug effects; D017434:Protein Structure, Tertiary; D012310:Ristocetin / Q000494:pharmacology; D013171:Spores, Bacterial / Q000201:enzymology; D013379:Substrate Specificity / Q000187:drug effects; D013921:Thrombocytopenia / Q000201:enzymology / Q000382:microbiology / Q000473:pathology; D013927:Thrombosis / Q000201:enzymology / Q000382:microbiology / Q000473:pathology; D013997:Time Factors; D014508:Urea / Q000494:pharmacology; D014841:von Willebrand Factor / Q000378:metabolism*
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D014841:von Willebrand Factor; D012310:Ristocetin; D014508:Urea; D003094:Collagen; D045726:Metalloproteases; D051722:ADAM Proteins; C502939:ADAMTS13 protein, mouse; D008666:Metalloendopeptidases; D000071120:ADAMTS13 Protein; C099604:ADAMTS13 protein, human