Inactivation of human coagulation factor X by a protease of the pathogen Capnocytophaga canimorsus
Inactivation of human coagulation factor X by a protease of the pathogen Capnocytophaga canimorsus
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DOI：
10.1111/jth.13605
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发表时间：
2017-03-01
影响因子：
10.4
通讯作者：
Cornelis, G. R.
中科院分区：
文献类型：
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作者：
Hack, K.;Renzi, F.;Cornelis, G. R.
研究背景犬二氧化碳噬纤维菌是一种革兰氏阴性细菌，存在于犬的口腔植物群中，并在被咬、舔或抓伤的人中引起暴发性脓毒症。在患者中，出血异常，如瘀点、暴发性紫癜或弥散性血管内凝血（DIC）是常见的。方法采用自动血栓图和凝血时间测定法测定犬C.犬型流感病毒5（Cc 5），一种从致命的人类感染中分离出来的菌株。用凝血因子缺乏血浆测定凝血因子活性。用放射性标记的酶原和蛋白质印迹法监测因子X切割。进行编码推定的丝氨酸蛋白酶的Cc 5基因的诱变以鉴定切割FX的蛋白酶。用亲和层析进行蛋白纯化。Edman降解允许检测FX的N-末端切割。结果Cc 5通过对FX的裂解，抑制凝血酶的生成，增加凝血酶原时间和活化部分凝血活酶时间。不能合成S46丝氨酸蛋白酶家族的7型二肽基肽酶（DPP 7）的突变体不能蛋白水解FX。纯化的蛋白酶（CcDPP 7）切割FX重链和轻链的N-末端，并在体内静脉注射后是活跃的。结论这是，据我们所知，第一项研究表明FX灭活细菌蛋白酶的详细机制，它是第一个功能研究关联DPP 7蛋白酶与潜在的致病性结果。
BackgroundCapnocytophaga canimorsus is a Gram-negative bacterium that is present in 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 To investigate 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 cleavage was monitored with the radiolabeled zymogen and western blotting. Mutagenesis of Cc5 genes encoding putative serine proteases was performed to identify the protease that cleaves FX. Protein purification was performed with affinity chromatography. Edman degradation allowed the 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 and the activated partial thromboplastin time of human plasma via FX cleavage. A mutant that was 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 intravenous 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.