Kiyohiko Kinjoh

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.

Hematological studies on DIC-like findings observed in patients with snakebite in south China

To clarify the characteristics of the hematological disturbances evoked by snakebite, we measured the antithrombin III (AT-III) activity, alpha2-plasmin inhibitor (alpha2-PI) activity, fibrinogen concentration (Fg) and level of fibrin degradation products (FDP) in 21 patients envenomed by several snakes in south China between August 1998 and October 1999. The hematological changes observed were as follows: the mean activities of AT-III were decreased in patients bitten by Ophiophagus hannah (Oh.), Bungarus fasciatus (Bf.), Hydrophis cyanocinctus (Hc.), Rhabdophis subminiatus (Rs.), and Trimeresurus stejnegeri (Ts.), while those of alpha2-PI were decreased in all patients in the present study; Fg was not detectable in the case of Rs. bite, and the Fg concentration after Ts., Oh., Hc. and Bf. bites also decreased markedly thereby increasing the mean levels of FDP in all patients. It thus appeared that DIC-like syndrome was caused in patients envenomed by snakebite. In the present study, we found that patients who were bitten by Rs., which is still being classified as a non-venomous snake, exhibited complete defibrinogenation and severe hemorrhage without any evidence of severe multiple organ damage. We also found that patients with Ts. bite showed marked hemostatic disturbance without severe multiple organ damage. It is considered that such a discrepancy between the hematological findings and clinical symptoms could be a characteristic phenomenon of the DIC-like syndrome induced by snakebite, especially by Rs. and Ts. bites.

Urokinase-type plasminogen activator and plasminogen activator inhibitor antigen in tissue extracts of paranasal sinus mucous membranes affected by chronic sinusitis and antrochoanal polyps

Abstract We examined the effect of pH on the extraction of urokinase-type plasminogen activator (u-PA) and plasminogen activator inhibitor-1 (PAI-1) from paranasal sinus mucous membrane associated with chronic sinusitis and antrochoanal polyps. The specific activity of u-PA extracted with buffer at pH 7.4 was stronger than that extracted with buffer at pH 4.2. The antigen level of u-PA extracted with the acidic buffer was significantly higher than that extracted with the neutral buffer. In contrast, the difference in antigen levels of PAI-1 extracted with the acidic buffer and neutral buffer was not significant. Based on these results, we inferred that the u-PA-PAI-1 complex was extracted by the acidic buffer and the activity of u-PA was therefore decreased.

Pharmacokinetics of habutobin in rabbits

Following the administration of habutobin, the fibrinogen level in the circulating blood of the rabbits decreased. These results showed that the activity of habutobin was retained in vivo. The plasma level of habutobin was determined by a ELISA-double sandwich method. The pharmacokinetics of habutobin from Trimeresurus flavoviridis venom was studied in rabbits following i.v. administration of 50 micrograms kg-1 of habutobin. The time course of the plasma concentration of habutobin fitted a two-compartment open model. The half-life of the distribution phase was 4.43 +/- 1.28 min and that of the elimination phase was 50.42 +/- 7.89 min. The area under the plasma concentration-time curve (AUC) was 38.69 +/- 6.68 micrograms min ml-1. The total body clearance was 3.82 +/- 1.08 ml min-1. When the steady state was reached, the concentration ratio of habutobin in the tissue (Ct) to that in the plasma (Cc), Ct:Cc was 0.47:1. These findings suggest that relatively little habutobin tended to remain in the tissue.

Production of a monoclonal antibody against the thrombin-like enzyme, habutobin, from Trimeresurus flavoviridis venom

We succeeded in producing a monoclonal antibody to the thrombin-like enzyme, habutobin, which was purified from crude venom of the snake Trimeresurus flavoviridis. The monoclonal antibody obtained belonged to IgG1, and its light chain consisted of a kappa-chain. The monoclonal antibody reacted specifically with habutobin and crude venom from T. flavoviridis but did not react with human thrombin or bovine thrombin on Western blotting. The concentration of habutobin and crude venom of T. flavoviridis, in vitro, could be measured by means of ELISA using the monoclonal antibody. Furthermore, the ELISA-double sandwich method employing this monoclonal antibody may represent a reliable method for determining the habutobin levels in the circulating blood.

Application of a monoclonal antibody to estimate rabbit fibrinopeptide A released by habutobin

We reported previously that habutobin, one of the type A thrombin-like enzymes, releases fibrinopeptide A alone from rabbit fibrinogen. To evaluate the effective action of habutobin in experiments using rabbit for the treatment of thrombosis, we attempted to develop an immunological method for measuring the fibrinopeptide A level in the circulating blood of rabbit. The purified rabbit fibrinopeptide A was coupled to keyhole limpet hemocyanin and BALB/c mice were immunized with the resultant fibrinopeptide A-hemocyanin conjugate. The spleen cells of an immunized mouse were fused with myeloma cells (P3-X63-Ag8-U1). As a result, one hybridoma (a-F-7) was selected, which secreted an antibody against rabbit fibrinopeptide A. Using this monoclonal antibody, we developed a competitive enzyme-linked immunoassay for estimating rabbit fibrinopeptide A. It was able to measure rabbit fibrinopeptide A contained in bentonite defibrinated plasma. This competitive enzyme-linked immunoassay should be useful for determining the fibrinopeptide A level in the circulating blood of rabbits, using plasma defibrinated by bentonite.

Habutobin recognizes Thr7 in the sequence of fibrinopeptide A of rabbit fibrinogen.

Habutobin, a thrombin-like enzyme from Trimeresurus flavoviridis venom, cleaves only the Arg(16)-Gly(17) bond in the rabbit Aalpha chain and releases fibrinopeptide A (FPA). To investigate the role of amino acid residues in the rabbit FPA sequence upon habutobin action, we examined the inhibitory effects of FPA and peptides containing partial sequences of FPA on the habutobin action. Fibrinopeptides from rabbit, human, bovine and dog were isolated and rabbit FPA was fragmented using dilute HCl. Rabbit FPA inhibited the action of habutobin although FPA from human, bovine and dog did not. Among the fragments of rabbit FPA, a heptapeptide Aalpha 3-9, the N-terminal region of rabbit FPA, competitively inhibited the release of FPA by habutobin, whereas the C-terminal hexapeptide of FPA (Aalpha 11-16) exerted no effect on the habutobin action. Synthetic tripeptides Ser-Thr-Phe corresponding to Aalpha 6-8 and Ala-Thr-Phe also inhibited the habutobin action, but Ser-Asp-Phe and Ala-Thr-Gly did not. It is concluded that habutobin would recognize the region around Thr(7)-Phe(8) in the sequence of rabbit FPA (Aalpha 1-16) prior to the cleavage of the Arg(16)-Gly(17) bond.

Platelet Aggregation in Head and Neck Tumors in China

We measured the maximum aggregation rate (MAR) of platelets in 770 patients with malignant head and neck tumors, 55 patients with benign tumors of the head and neck, and 164 healthy people as a control group. The results were as follows:1. the mean MAR value of patients with malignant tumors was significantly higher than the control group mean value; 2. prior to treatment, the mean MAR value increased with advancing tumor stage;3. both MAR values of relapsed or metastasized patients and of nonsurvivors in stage III and IV increased significantly compared with survivors or patients recovering from malignant tumors. The results of the present study suggest that MAR values of patients with malignant tumors of the head and neck may serve as indicators in evaluating therapeutic procedures and prognosis.

A Monoclonal Antibody Against Bovine Thrombin Reacting to the C-Terminal Side of Thrombin

We succeeded in producing a monoclonal antibody (MAb) against bovine thrombin. The MAb belonged to mouse IgG(1), and its light chain consisted of kappa-chain. The MAb reacted with bovine and human thrombins, which were coated by coupling to poly-lysine-coated wells with glutaraldehyde, but did not react with the thrombin-like enzyme, habutobin. Furthermore, the MAb did not react with thrombin which was coated to plates without poly-lysine and glutaraldehyde. The concentration of thrombin in ovalbumin solution (10 mg/mL) could be measured by means of the enzyme-linked immunosorbent assay (ELISA) double sandwich method using the MAb and polyclonal antibody. Thrombin added to defibrinated plasma could not be detected by means of the ELISA double sandwich method using the present MAb, and this may be due to the AT-III activity in the defibrinated plasma. Postclotting thrombin could be detected by means of the ELISA-double sandwich method using the MAb. It is suggested, from the results of our experiments, that the MAb obtained reacted in a limited fashion to the C-terminal of bovine thrombin.

α2-Macroglobulin of rabbits inhibits the habutobin activity

We have investigated whether alpha 2-macroglobulin (alpha 2M) of rabbits inhibits the activity of habutobin, a thrombin-like enzyme from Trimeresurus flavoviridis venom. Rabbit alpha 2M was purified with ultracentrifugation, gel filtration on Sepharose 6B and ion exchange chromatography on DEAE-Sephacel. Inhibitory effects of rabbit alpha 2M on habutobin was determined by the fibrin forming activity, digestion of A alpha chain of fibrinogen, and the release of fibrinopeptide A from fibrinogen. As a results, purified alpha 2M showed a single band with high molecular weight, around 800,000 mol. wt by means of polyacrylamide gel electrophoresis using PhastSystem. Besides inhibiting amidolytic and caseinolytic activity of porcine trypsin, it has inhibited the activity of habutobin: that is, in the presence of rabbit alpha 2M, fibrin forming activity of habutobin was decreased and habutobin-induced digestion of A alpha chain was inhibited. In addition, rabbit alpha 2M reduced habutobin-induced release of fibrinopeptide A from rabbit fibrinogen.