Habutobin splits the Arg16-Gly17 bond in the A alpha chain of rabbit fibrinogen.

We reported previously that habutobin, a thrombin-like enzyme from Trimeresurus flavoviridis venom, clotted only rabbit fibrinogen, whereas human, monkey, bovine, dog, rat and guinea-pig fibrinogens were unaffected. In the present study, we investigated the cleavage site of the rabbit A alpha chain by habutobin. The fibrinopeptide released by habutobin was identical to the fibrinopeptide A released by thrombin, and its amino acid sequence corresponded to A alpha 1-16 of rabbit fibrinogen. It was clarified therefore that habutobin cleaves the Arg16-Gly17 bond in the A alpha chain of rabbit fibrinogen.

Effect of Habu (Trimeresurus flavoviridis) antivenom on changes of hemostatic parameters following administration of crude venom from T. flavoviridis in rabbits.

We carried out experiments to examine the efficacy of Habu antivenom in relation to variations of hemostatic parameters induced by the administration of crude venom to rabbits. For neutralization of the crude venom by Habu antivenom, the correlation between the concentrations of Habu antivenom (Y) and crude venom (X) was expressed by the equation: Y = -0.473 + 0.539X. We examined the variations in hemostatic parameters in the state which crude venom was neutralized by Habu antivenom following the administration of crude venom (1 mg/kg) from Trimeresurus flavoviridis. Although the hemostatic parameters [level of fibrinogen, antithrombin III (AT-III) activity and alpha 2-plasmin inhibitor (alpha 2-PI) activity] underwent decreases after the administration of crude venom, they revealed fluctuations within the normal range after the Habu antivenom administration. The AT-III activity, however, decreased gradually until 90 min after the antivenom administration. These results suggested that Habu antivenom was effective in improving the abnormal coagulant activity induced by crude venom. However, the neutralization effect towards the coagulant activity of crude venom by the Habu antivenom did not continue for a long time and did not lead to recovery of the AT-III activity. Since excessive doses of antivenom can induce serious medical problems, we expect that simultaneous use of antivenom and AT-III preparation, instead of excessive and single use of Habu antivenom could provide a useful therapy for snake bites from the present study.

Inhibition of habutobin activities by habu antivenom.

This study investigated whether habu antivenom inhibits the clotting activity of habutobin, a thrombin-like enzyme from Trimeresurus flavoviridis venom. Habu antivenom, which is available as a commercial antibody against the crude venom of T. flavoviridis, has been used to treat envenoming by T. flavoviridis (the habu snake). The present study was undertaken to determine whether habu antivenom inhibits the activities of habutobin, which involve digestion of the A alpha chain and release of fibrinopeptide A (FPA) in rabbit fibrinogen. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that habu antivenom inhibited the habutobin-induced digestion of the A alpha chain in rabbit fibrinogen. The results of FPA measurements using competitive enzyme-linked immunoassay (CELIA) revealed that habu antivenom inhibited the release of FPA from rabbit fibrinogen induced by habutobin. In addition, a correlation was noted between the digestion of the A alpha chain and release of FPA from rabbit fibrinogen. Analysis of the inhibition kinetics of habu antivenom against the habutobin activity yielded a competitive double-reciprocal plot.

Inhibitory effect of habu antivenom on fibrinopeptide A release induced by Trimeresurus flavoviridis crude venom.

The correlation between the clotting activity of crude venom and concentration of fibrinopeptide A (FPA) released by the crude venom in rabbit plasma was evaluated and expressed as the coefficient of correlation (r = 0.850). The venom-induced FPA release was inhibited by habu antivenom. For such inhibition of FPA release, the correlation between the concentration of habu antivenom (Y) and that of crude venom (X) could be expressed by the equation Y = 7.115 + 0.709X. An absence of venom-induced FPA release in rabbit plasma had suggested that the clotting activity of crude venom could be neutralized by the habu antivenom. It is suggested that determinations of the FPA level in the plasma are effective in providing an indication of the reliability for serotherapy using habu antivenom.

Hemostatic disturbances observed in patients with snakebite in south China.

To investigate the hematological disorders after snakebite, we measured the maximum platelet aggregation rate (MAR), antithrombin III (AT-III) activity, alpha(2)-plasmin inhibitor (alpha(2)-PI) activity, concentration of fibrinogen (Fg) and fibrin degradation products (FDP) in 25 samples from 17 patients with snakebite in south China. The results obtained in the patients before application of antivenom and patients with Ophiophagus hannah (Oh.) bite were as follows: (1) the mean MAR values were significantly decreased in the case of the snakebites from Vipera russellii (Vr.) and Trimeresurus mucrosquamatus (Tm.); (2) the mean activities of AT-III were decreased in all patients in the present study; 3) the mean activities of alpha(2)-PI were significantly decreased in patients bitten by Deinagkistrodon acutus (Da.), Agkistrodon halys (Ah.), Vr., Trimeresurus stejnegeri (Ts.), Tm. and Naja naja atra (Nn.); (4) the mean concentrations of Fg were markedly decreased in patients bitten by Da., Ah., Vr., Ts. and Tm.; and (5) the mean levels of FDP were significantly increased in cases of Da., Vr. and Ts. bite, but not in Ah., Tm., Nn. and Oh. bite. The results of the present study indicate that disorders of platelet aggregation and the coagulation-fibrinolysis system are liable to occur in patients with snakebite from Da., Ah., Vr., Ts., Tm. and Nn. Furthermore, it appeared that disseminated intravascular coagulation (DIC) was evoked in some patients. Specific antivenom was found to be useful for improving the hemostatic disturbances after snakebite from Ah. and Nn.

The relationship between microvessel density, the expression of vascular endothelial growth factor (VEGF), and the extension of nasopharyngeal carcinoma.

OBJECTIVE: The present study was aimed at clarifying whether the microvessel density (MVD) and the expression of vascular endothelial growth factor (VEGF) were related to the degree of local invasion and metastasis in nasopharyngeal carcinoma (NPC). STUDY DESIGN: We measured the MVD and examined whether VEGF was expressed in NPC tissue using histological study combined with immunohistochemistry. METHODS: MVD and VEGF expression was measured in 73 specimens of NPC, 15 benign tumors of nasopharyngeal region, and 20 nasopharyngeal tissue without tumor. MVD and VEGF expression in NPC was compared between a metastasis group (49 specimens) and a non-metastasis group (24 specimens). RESULTS: Both MVD and VEGF expression were markedly increased in NPC tissue as compared with those in benign tumors of nasopharyngeal region. Both MVD and VEGF expression in NPC tissue with metastasis were statistically significantly increased as compared with those in NPC without metastasis. Therefore, the invasion and metastasis of NPC cells were closely related to MVD and the expression of VEGF in NPC tissue. CONCLUSION: The metastatic potency of NPC tissue and the prognosis of the patients with NPC can be estimated by measuring MVD and the expression of VEGF in NPC tissue. Drugs that have inhibitory actions on angiogenesis could be useful to prevent metastasis of NPC cells in the patients.

Analysis of IgE turnover in non-sensitized and sensitized rats.

BACKGROUND: Although the levels of immunoglobulin E (IgE) in the circulating blood are often elevated in patients with allergic diseases, such levels cannot always be considered as pathognomonic signs of allergy. The induction of allergic reactions in the tissue was inferred to be related to the amount of IgE passing through the vascular wall. AIMS: We attempted to clarify which compartment, the intravascular or extravascular, plays an important role in the regulation of the turnover of rat IgE. METHODS: The level of DNP-specific rat IgE in the serum was estimated by IgE-capture enzyme-linked immunosorbent assay, and the turnover of IgE was analyzed from its pharmacokinetic parameters. RESULTS: The transfer rate constants from the central to tissue compartment (Kct) were larger than those from the tissue to central compartment (Ktc) irrespective of the sensitized state. The value of the distribution volume of the tissue compartment (Vt) was larger than that of the distribution volume of the central compartment (Vc) irrespective of the sensitized state. CONCLUSIONS: These Findings suggest that the short half-life of rat IgE in the circulation could be attributable to the distribution of IgE from the intravascular to the extravascular compartment.

The nucleotide sequence of dinitrophenyl-specific IgE and Fc(epsilon)RI alpha-subunit obtained from FE-3 hybridoma cells.

FE-3 cells were established by Hanashiro et al. by hybridizing mouse myeloma cells (Sp2/0-Ag14/SF) with rat spleen cells that were freshly isolated from Brown-Norway rats sensitized with DNP-As. FE-3 cells can constitutively secrete IgE without stimulation by cytokines. Our preliminary experiments demonstrated that the IgE secretion was decreased at 3 days after start of culture and the addition of exogenous IgE into culture media depressed the secretion of IgE. Thus, we hypothesized that the IgE production in FE-3 cells may be regulated by a signal transduction through the binding of IgE to its high affinity receptor (Fc(epsilon)RI) or to an IgE binding protein on the cell surface. In this study, we aimed to identify the nucleotide sequence of IgE FE-3 and compared with those of mouse IgE and IgE IR162 to find a structural heterogeneity in the Fc region of IgE FE-3. We also tested if the mRNA of Fc(epsilon)RI was expressed in FE-3 cells using the reverse transcriptase-polymerase chain reaction (RT-PCR) method with the combination of sequencing analysis. Consequently, the cDNA sequence of IgE FE-3 was identical to that of the CH3 and CH4 domains in the epsilon-chain of rat IgE IR162, whereas the cDNA of Fc(epsilon)RI was identical to that of mouse, suggesting that the genes of IgE FE-3 and Fc(epsilon)RI was derived from that of rat spleen cells and mouse myeloma cells, respectively.