Isolation of bound thrombin consisting of thrombin and fibrin N-terminal fragment from clot lysate.

It has been reported that thrombin is liberated from fibrin clots by the action of fibrinolytic enzymes. It has also been reported that the liberated thrombin complexes with fibrin fragment E or (DD)E, which are denoted as bound thrombin. However, bound thrombin has not been isolated from clot lysate, and the structural characteristics of isolated bound thrombin have not been specified. In this study, we attempted to isolate the bound thrombin from clot lysate and to clarify its structural features. Rabbit fibrinogen was clotted with bovine thrombin, and clot lysate was prepared with urokinase. The bound thrombin was isolated from clot lysate by serial chromatography using a Sepharose 4B column immobilizing an anti-bovine thrombin antibody and a Sepharose 4B column immobilizing an anti-rabbit fibrinogen antibody. SDS-PAGE under unreduced conditions demonstrated that there were two different protein bands in the isolated bound thrombin. On a C4 reverse-phase HPLC, the bound thrombin from clot lysate was resolved by 4 M urea into alpha-thrombin and a fibrin fragment, the N-terminal regions of which were identified as alpha-, beta- and gamma-chains. Thus, in the bound thrombin, thrombin molecule would bind to rabbit fibrin fragment consisting of N-terminal central domain.

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.

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.

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.

Habutobin recognizes Thr(7) 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.

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

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.

Mice with early onset of death (EOD) due to lupus glomerulonephritis.

Both MRL-lpr/lpr (lpr) and BXSB mice fall victim to autoimmune disease as a function of age. To combine their properties, brother-sister mating of (female lpr x male BXSB)F1 mice was done. Mice for mating were selected according to indicators of early onset of glomerulonephritis and subsequent early death (i.e., EOD). This mating was continued for more than 16 generations. The EOD mice thus established had homozygous H-2k/k, lpr/lpr, and possible yaa/- (in the case of males). The average life span of males was 83 days while that of females was 126 days. After 12 weeks of age, the majority (> 80%) of male EOD mice were characterized by the abnormality of urine due to glomerulonephritis. We then characterized how glomerulonephritis was evoked, especially in terms of expanding lymphocyte subsets in various immune organs. Similar to the case of parental lpr mice, the major expanding cells were CD4-8-B220+ TCRint cells in the immune organs and kidney. In addition, myeloid cells were found to infiltrate the kidney. This massive infiltration of both TCRint cells and myeloid cells might be responsible for the onset of acute glomerulonephritis. Even after more than 50 generations, these EOD mice still carry both lpr and yaa genes. These results suggest that EOD mice might be a very useful tool for the study of acute lupus glomerulonephritis which is evoked by the genetic abnormalities.

Calorie restriction delays the crescentic glomerulonephritis of SCG/Kj mice.

Reduced dietary calories can delay the onset and diminish the severity of murine autoimmunities of numerous inbred and hybrid mutant strains. We sought to determine whether the precipitous, autoimmune, crescentic glomerulonephritis of recombinant inbred SCG/Kj mice could be abrogated similarly by calorie restriction. Weanling SCG/Kj mice develop hematuria and proteinuria, and 50% die as 16-week-old young adults. In this study, 113 4-week-old SCG/Kj mice were fed either ad libitum a milled chow (Group A, n = 50), or a semipurified diet (Group B, n = 29), or were fed a calorie-restricted semipurified diet (Group C, n = 34), so that mice of Group C consumed approximately 32% fewer calories, but similar amounts of essential dietary constituents as those of Group B. Calorie restriction of Group C provided modest (P = 0.05) or substantial survival advantage (P = 0.001) compared to the ad libitum feeding of Groups B or A, respectively. Progression to severe glomerular pathology was delayed among Group C mice, with more than a 5-week delay to heavy proteinuria (>100 mg/dl), a >4-week delay to hematuria, and a >5-week delay to median mortality, representing a 20% or 25% extension of median life span, compared to ad libitum-fed Group B and A mice, respectively. Mean glomerular histopathology scores were also lower in calorie-restricted mice compared to the ad libitum-fed cohorts (P = 0.001). Titers of anti-ss-DNA, ds-DNA, and ANCA autoantibodies developed in weanlings prior to the full imposition of calorie restriction and were not reduced significantly by calorie restriction.

Prevention of crescentic glomerulonephritis in SCG/Kj mice by bone marrow transplantation.

Transplantation of MHC-compatible, T-cell-depleted, bone marrow cells has successfully treated autoimmunities, immunodeficiencies, malignancies, and developmental deficiencies of the hematopoietic system. Recombinant inbred SCG/Kj mice develop spontaneous crescentic glomerulonephritis, systemic vasculitis, and a lymphoproliferative disorder early in life. To determine whether the precipitous autoimmune disease of SCG/Kj mice could be treated by bone marrow transplantation, 30 SCG/Kj mice were engrafted with T-cell-depleted, bone marrow (TCDM) from allogeneic, MHC-compatible, autoimmune-resistant C3H/He donors, and 30 SCG/Kj mice served as controls and received TCDM from syngeneic, SCG/Kj donors. A significant survival advantage was evident from SCG/Kj mice engrafted with C3H/He TCDM (p < 0.005), and an 89% extension of median survival compared to recipients of SCG/Kj TCDM. Within 28 weeks post-transplantation, 62% of mice engrafted with SCG/Kj TCDM had died with clinical signs of fatal crescentic glomerulonephritis. This result compared with only 10% of mice engrafted with C3H/He TCDM. Mice engrafted with SCG/Kj TCDM developed significantly greater titers of autoantibodies to ss-DNA, ds-DNA, and myeloperoxidase (ANCA) (p < 0.001), had shorter latencies to the development of, and a greater incidence of proteinuria, hematuria, and peripheral lymphadenopathy, and a greater mean grade of glomerular lesion (p < 0.001), than mice engrafted with C3H/He TCDM. These findings indicate that the genetic defect of the SCG/Kj strain of mice resides within the hematopoietic stem cells and provokes the speculation that bone marrow transplantation might be a useful means of treating progressive crescentic glomerulonephritis in humans.

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.

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.

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.

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.

Inhibitory effect of azelastine hydrochloride and suplatast tosilate on airway responses in sensitized rats following exposure to antigen.

In the present study, we examined the relationship between the increase in respiratory resistance following exposure to antigen and the IgE level in the identical rate sensitized with DNP-As. Additionally, we investigated the effects of the antiallergic drugs, suplatast tosilate and azelastine hydrochloride, which have been reported to suppress the production of IgE, on the increase in respiratory resistance in rats following exposure to antigen. The IgE antibody level rose to its highest value on day 10 during the course of sensitization with DNP-As, and decreased sharply on day 20. The changes of IgE antibody level in the azelastine hydrochloride-administered group were similar to those in the distilled water-administered group (control). In contrast, in the suplatast tosilate-administered group, the IgE antibody levels were lower than those in the control group at days 10 and 15. The ratio of increase in the respiratory resistance induced by the early and late phase responses in the control group reached its highest value on day 15, and then decreased gradually. In contrast, in both the azelastine hydrochloride and suplatast tosilate-administered groups, the ratio of increase in the respiratory resistance induced by the early and late phase responses remained almost unchanged, and was lower than that in the control group at day 15 or 20. In the present study, an increased peak of respiratory resistance was observed at 5 days after the appearance of an increased peak in the IgE level.

Alpha 2-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.

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.

Gastrointestinal vasculitis in autoimmune-prone (NZW X BXSB)F1 mice: association with anticardiolipin autoantibodies.

Gastrointestinal vasculitis is a fatal aspect of systemic lupus erythematosus. Whether lupus-prone strains of mice develop gastrointestinal vasculitis, and whether it is accompanied by elevated titres of anticardiolipin autoantibody is not known. Lupus-prone (NZW X BXSB)F1 (W/BF1) mice, and other strains were examined immunohistologically. Levels of anticardiolipin autoantibody and circulating immune complexes were determined by microELISA. Reciprocal haploidentical marrow transplantations between W/BF1 and autoimmune-resistent B6C3F1 mice were made. Young adult W/BF1 mice had the highest incidence of gastrointestinal vasculitis (61%), and the highest mean titre of anticardiolipin autoantibody. Lesions consisted of subintimal fibrinoid degeneration in arterioles of the duodenum, jejunum and ileum, and were prevented, or alternatively induced by reciprocal marrow transplantations between W/BF1 and B6C3F1 mice. Mice engrafted with W/BF1 marrow which developed vasculitis had higher titres of anticardiolipin autoantibodies than engrafted mice free of vasculitis (P < 0.005). This represents the first report of gastrointestinal vasculitis as an aspect of systemic autoimmunity in lupus-prone mice. The concurrent elevation of anticardiolipin autoantibody and development of vasculitis suggests that anticardiolipin autoantibodies, and their proposed thrombogenic and vascular injury consequences, contribute to development of microvasculitis in lupus-prone mice.

Effect of argatroban on the formation of artificial thrombus on dogs.

In the present study, we have investigated the effects of a synthetic antithrombin, Argatroban, and an antiplatelet agent. Ticlopidine hydrochloride, on the weight of artificial thrombus. These drugs at various concentrations were added to canine bloods, which were adjusted to 20%, 40% and 60% of haematocrit, and an artificial thrombus was formed using a modification of Chandler's method. Argatroban inhibited the formation of artificial thrombus, and this marked inhibition was observed especially in the experiment using blood with a high value of Ht. On the other hand, Ticlopidine hydrochloride did not inhibit the formation of artificial thrombus. From these results, it becomes clear that the mechanism of inhibitory action of Argatroban on artificial thrombus formation is based on the inhibition of thrombin activity and not on the inhibition of platelet aggregation. In addition, it is suggested that Argatroban inhibits the aggregation of red blood cells in the manner of direct or indirect action.

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.