Inactivation of human coagulation factor X by a protease of the pathogen Capnocytophaga canimorsus

BACKGROUND: Capnocytophaga canimorsus is a Gram-negative bacterium, which belongs to the oral flora of dogs and causes fulminant sepsis in humans who have been bitten, licked or scratched. In patients, bleeding abnormalities, such as petechiae, purpura fulminans or disseminated intravascular coagulation (DIC) occur frequently.

OBJECTIVE: We investigated whether C. canimorsus could actively contribute to these bleeding abnormalities.

METHODS: Calibrated automated thrombogram and clotting time assays were performed to assess the anticoagulant activity of C. canimorsus 5 (Cc5), a strain isolated from a fatal human infection. Clotting factor activities were measured with factor deficient plasma. Factor X (FX) cleavage was monitored using the radiolabeled zymogen and western blot. Mutagenesis of Cc5 genes encoding putative serine proteases was performed to identify the protease cleaving FX. Protein purification was done by affinity chromatography. Edman-degradation allowed detection of N-terminal cleavage of FX. Tail bleeding times were measured in mice.

RESULTS: We found that Cc5 inhibited thrombin generation and increased the prothrombin time (PT) and the activated partial thromboplastin time (aPTT) of human plasma via FX cleavage. A mutant, unable to synthesize a type 7 dipeptidyl peptidase (DPP7) of the S46 serine protease family, failed to proteolyse FX. The purified protease (CcDPP7) cleaved FX heavy and light chains from the N-terminus and was active in vivo after i.v. injection.

CONCLUSIONS: This is to our knowledge the first study demonstrating a detailed mechanism for FX inactivation by a bacterial protease and it is the first functional study associating DPP7 proteases with a potentially pathogenic outcome. This article is protected by copyright. All rights reserved.