Preparation and Structure of a New Coagulation Factor XI Catalytic Domain for Drug Discovery

Human blood coagulation factor XI （FXI） is a key enzyme in the amplification phase of blood coagulation cascade, and is recognized as an important target for anti-coagulant development in recent years. We designed a new mutant form of FXIa catalytic domain rhFXI370-607 （N73Q-N113Q-C123S）, and report here the facile preparation, protein crystallization, and crystal structure of this protein. We highlight a few unique structural features of FXIa after comparison with the trypsin family serine proteases at sequence and structural levels. This work provides a foundation to develop new small molecular FXIa inhibitors with increased potency and specificity.

Extrahepatic synthesis of coagulation factor Ⅶ by colorectal cancer cells promotes tumor invasion and metastasis

Background Blood coagulation factor Ⅶ （FⅦ） is physiologically synthesized in the liver and released into the blood. Binding of FⅦ to tissue factor （TF） is related to the metastatic potential of tumor cells, also a significant risk factor in the development of hepatic metastasis in patients with colorectal cancer （CRC）. It has been found that some cancer cells can produce FⅦ extrahepatically. However, litte is known about FⅦ and CRC. We therefore hypothesized that CRC cells may synthese FⅦ, leading to tumor invasion and metastasis.Methods We detected the expression of FⅦ protein in 55 CRC specimens by immunohistochemical staining. The FⅦ mRNA in 45 of 55 CRC cases, 6 colon cancer cell lines and one hepatoma cell line was measured by real-time reverse transcription-PCR （RT-PCR）. Transwell invasion assays were performed to evaluate the changes of cell migration and invasion of LoVo cancer cells in vitro. We further observed the likely effectors regulated by the TF/FⅦa complex Western blotting assay.Results Extrahepatic synthesis of FⅦ was detected in the cytoplasm of 32 （58.2%） CRC specimens byimmunohistochemistry, but not in normal mucosa. Liver metastasis （P=0.003） and TNM staging （P=0.005） were significantly correlated with FⅦ antigen expression. The positive ratios in stages Ⅰ, Ⅱ, Ⅲ and Ⅳ were 33.3%, 40.0%,52.4% and 87.5%, respectively. The expression of FⅦ mRNA in CRC with hepatic metastasis was significantly higher than CRC without hepatic metastasis （5.33±2.88 vs. 1.47±0.51, P=0.03）. Ectopic FⅦa induced a slight increase （1.34-fold） in the number of migrating cells, which was inhibited by the specific TF antibody. The formation of TF/FⅦacomplex resulted in a marked increase in the expression of matrix metalloproteinases （MMP）-2 （3.5-fold） and MMP-9（4.7-fold） in a time-dependent and dose-dependent manner.Conclusions Extrahepatic synthesis of FⅦ by CRC cells may promote tumor invasion and metastasis. MMPs, as downstream effectors of TF/FⅦa signaling, facilitate the development of metastasis in colon cancer.

Dynamically monitoring tissue factor and tissue factor pathway inhibitor following secondary brain injury

Objective: To study the altering rule of coagulation function at molecular level in patients with secondary brain injury (SBI). Methods: Tissue factor (TF) and tissue factor pathway inhibitor (TFPI) were studied in 32 patients 1, 2, 3 and 7 days after craniocerebral injury. Repeated cranial CT scans and platelet counts were made simultaneously. Same measurements were done in 30 normal adults except CT scan. Results: No obvious difference was found in age, sex and platelet count between the injured and the normal groups. TFPI/TF decreased markedly in the first week after injury in patients with SBI, but only decreased on the 7th day in the patients without obvious SBI. For the patients who developed delayed intracranial hematoma (DIH) or hematoma enlargement, TF rose only 1 and 2 days after injury, but TFPI had a tendency to rise again after a fall on the 3rd day. For those patients who developed no DIH, TF rose all the time within the 1st week. Conclusions: Decrease of TFPI/TF for a long time, especially within 3 days after injury, may be one of the most important reasons for SBI. High expression of TF for a relative short time and increase of TFPI after a fall within 3 days may be one of the important reasons for DIH or hematoma enlargement.