Effects of hemocoagulase agkistrodon on the coagulation factors and its procoagulant activities.

OBJECTIVE: Hemocoagulase agkistrodon (HCA), a thrombin-like enzyme (TLE) from the venom of the Chinese moccasin snake (Deinagkistrodon acutus), has been used in clinical practice as a hemostatic compound. The aim of this study was to further investigate the pharmacological properties of HCA. MATERIALS AND METHODS: Sodium dodecyl sulfate or native polyacrylamide gel electrophoresis (SDS- or N-PAGE) as well as enzyme linked immunosorbent assays (ELISAs) were conducted to study the effects of HCA on the human plasma fibrinogen and prothrombin levels, as well as its in vitro interactions with some coagulation factors. In addition, the bleeding time effects of HCA in the mouse tail-bleeding model as well as its effects on the fibrinogen levels in rabbits were determined in vivo. RESULTS: In vitro results revealed that HCA exerts its procoagulant activities by hydrolyzing fibrinogen into segments that are easier to be absorbed, reducing the risk of thrombus formation. Besides, HCA could significantly inhibit the activation of prothrombin at the concentration of 0.3 µM. Unexpectedly, we also found that HCA was able to strongly bind to factor X/Xa (in a ratio of 1:1) and thus inhibit the acceleration of active factor X to tissue plasminogen activator-catalyzed plasminogen activation, demonstrating that it could be less likely to lead to thrombus formation. Finally, in vivo results indicated that HCA could significantly shorten the bleeding time in the mouse tail-bleeding model and had no effect on the fibrinogen levels in rabbits. CONCLUSION: In summary, HCA, a unique and new family member of TLEs, may become a new clinical drug for the prevention and treatment of hemorrhage due to its unique and complex interactions with the blood system. Clarification of these features will enable us to further understand the mechanism of action of HCA and then promote its further application in clinical practice as a therapeutic drug.

Antitumor effect of COOH-terminal polypeptide of human TERT is associated with the declined expression of hTERT and NF-κB p65 in HeLa cells.

Human telomerase reverse transcriptase (hTERT) plays an important role in the development of tumors and has been investigated as a potent target for anticancer therapy. In the present study, we constructed a recombinant adenovirus, Ad-EGFP-C197 which was capable of expressing COOH­terminal polypeptide of hTERT (amino acid 936-1,132, termed as C197 for the reason that it contains 197 amino acids). Infection of HeLa cells with Ad-EGFP-C197 suppressed the activity of telomerase, decreased the expression of hTERT and NF-κB p65, and induced rapid growth delay and apoptosis of HeLa cells in vitro. In nude mice xenografted with HeLa tumors, injection of Ad-EGFP-C197 into the tumor nodule significantly slowed tumor growth and promoted tumor cell apoptosis, as well as reduced the expression of NF-κB p65 in tumor tissues. In the present study, we suggest that the antitumor effect of C197 is associated with the declined expression of hTERT and NF-κB p65. Our results highlight the potential of C197 in tumor therapy.

Effect of a structurally modified human granulocyte colony stimulating factor, G-CSFa, on leukopenia in mice and monkeys.

BACKGROUND: Granulocyte colony stimulating factor (G-CSF) regulates survival, proliferation, and differentiation of neutrophilic granulocyte precursors, Recombinant G-CSF has been used for the treatment of congenital and therapy-induced neutropenia and stem cell mobilization. Due to its intrinsic instability, recombinant G-CSF needs to be excessively and/or frequently administered to patients in order to maintain a plasma concentration high enough to achieve therapeutic effects. Therefore, there is a need for the development of G-CSF derivatives that are more stable and active in vivo. METHODS: Using site-direct mutagenesis and recombinant DNA technology, a structurally modified derivative of human G-CSF termed G-CSFa was obtained. G-CSFa contains alanine 17 (instead of cysteine 17 as in wild-type G-CSF) as well as four additional amino acids including methionine, arginine, glycine, and serine at the amino-terminus. Purified recombinant G-CSFa was tested for its in vitro activity using cell-based assays and in vivo activity using both murine and primate animal models. RESULTS: In vitro studies demonstrated that G-CSFa, expressed in and purified from E. coli, induced a much higher proliferation rate than that of wild-type G-CSF at the same concentrations. In vivo studies showed that G-CSFa significantly increased the number of peripheral blood leukocytes in cesium-137 irradiated mice or monkeys with neutropenia after administration of cyclophosphamide. In addition, G-CSFa increased neutrophil counts to a higher level in monkeys with a concomitant slower declining rate than that of G-CSF, indicating a longer half-life of G-CSFa. Bone marrow smear analysis also confirmed that G-CSFa was more potent than G-CSF in the induction of granulopoiesis in bone marrows of myelo-suppressed monkeys. CONCLUSION: G-CSFa, a structurally modified form of G-CSF, is more potent in stimulating proliferation and differentiation of myeloid cells of the granulocytic lineage than the wild-type counterpart both in vitro and in vivo. G-CSFa can be explored for the development of a new generation of recombinant therapeutic drug for leukopenia.