The contribution of residues 192 and 193 to the specificity of snake venom serine proteinases. (1/50)

Snake venom serine proteinases, which belong to the subfamily of trypsin-like serine proteinases, exhibit a high degree of sequence identity (60-66%). Their stringent macromolecular substrate specificity contrasts with that of the less specific enzyme trypsin. One of them, the plasminogen activator from Trimeresurus stejnegeri venom (TSV-PA), which shares 63% sequence identity with batroxobin, a fibrinogen clotting enzyme from Bothrops atrox venom, specifically activates plasminogen to plasmin like tissue-type plasminogen activator (t-PA), even though it exhibits only 23% sequence identity with t-PA. This study shows that TSV-PA, t-PA, and batroxobin are quite different in their specificity toward small chromogenic substrates, TSV-PA being less selective than t-PA, and batroxobin not being efficient at all. The specificity of TSV-PA, with respect to t-PA and batroxobin, was investigated further by site-directed mutagenesis in the 189-195 segment, which forms the basement of the S(1) pocket of TSV-PA and presents a His at position 192 and a unique Phe at position 193. This study demonstrates that Phe(193) plays a more significant role than His(192) in determining substrate specificity and inhibition resistance. Interestingly, the TSV-PA variant F193G possesses a 8-9-fold increased activity for plasminogen and becomes sensitive to bovine pancreatic trypsin inhibitor.  (+info)

Prevention of rat cerebral aneurysm formation by inhibition of nitric oxide synthase. (2/50)

BACKGROUND: Cerebral saccular aneurysm is a major cause of subarachnoid hemorrhage, one of the cerebrovascular diseases with the highest mortality. The mechanisms underlying the development of aneurysms, however, still remain unclear. We have made a series of reports on an animal model of experimentally induced cerebral aneurysms that resemble human cerebral aneurysms in their location and morphology, suggesting that the arterial wall degeneration associated with aneurysm formation develops near the apex of arterial bifurcation as a result of an increase in wall shear stress. Using the animal model and human specimens, we examined the role of nitric oxide (NO) in the degenerative changes and cerebral aneurysm formation. METHODS AND RESULTS: Inducible NO synthase (iNOS) was immunohistochemically located at the orifice of human and rat aneurysms. Nitrotyrosine distribution was also seen in the human aneurysm. Although no iNOS immunostaining was found in normal arteries, iNOS immunoreactivity was observed in parallel with the development of early aneurysmal changes in rats. In contrast, during the early development of aneurysm, endothelial NOS immunostaining in the endothelium was weakened compared with that in the control arteries. An NOS inhibitor, aminoguanidine, attenuated both early aneurysmal changes and the incidence of induced aneurysms. A defibrinogenic agent, batroxobin, which may diminish shear stress by reduction of blood viscosity, prevented iNOS induction as well as early aneurysmal changes. CONCLUSIONS: The evidence suggests that NO, particularly that derived from iNOS, is a key requirement for the development of cerebral aneurysm. The iNOS induction may be caused by an increase in shear stress near the apex.  (+info)

Effect of batroxobin against dog heart ischemia/reperfusion injury. (3/50)

AIM: To study the effect of batroxobin(Bat) on dog heart ischemia/reperfusion (I/R) injury. METHODS: Dog heart I/R injury was induced by occluding the left anterior descending coronary artery for 30 min and restoring blood perfusion for 90 min. Bat was intravenously injected before heart ischemia and 15 min before reperfusion. Plasma creatine kinase (CK), lactate dehydrogenase (LDH), and myocardial malondiaedehyde (MDA) concentrations were measured. The pathologic changes of I/R myocardium were observed. RESULTS: Bat reduced the mortality rate of I/R dog (I/R group 65.0% vs Bat-I group 30.0% and Bat-II group 28.6%, P < 0.05). Myocytes of I/R heart showed intracellular edema, damaged mitochondria, and concentrated nucleus. Bat decreased these changes. In Bat-I and Bat-II group, plasma CK and LDH level were reduced, the +dp/dtmax and -dp/dtmax at 30 min after ischemia and 90 min after reperfusion were elevated, and left ventricular end dilation pressure (LVEDP) was lowered. The myocardial MDA contents were decreased by 42.3% and 38.1% (P < 0.01) in Bat-I and Bat-II group, respectively. CONCLUSION: Bat may exert an apparent role against dog heart ischemia/reperfusion injury and improve myocardial function.  (+info)

Influence of batroxobin on cerebral ischemia-reperfusion injury in gerbils. (4/50)

AIM: To study the effects of batroxobin (Bat) on neurons survival, neurobehavioral test, ATP levels and hydroxyl radical outputs in hippocampus during forebrain ischemia-reperfusion in gerbils. METHODS: The forebrain ischemia was induced by occluding the bilateral common carotid arteries for 10 min in gerbils, and ATP levels and 2, 3-dihydroxybenzoic acid (DHBA) outputs were assayed by HPLC. The neurons survival were assessed by histology, and behavioral tests of gerbils were assessed by open field test. RESULTS: The number of neurons survival in Ir at d 7 postischemic insult were (7 +/- 4)% of sham-operated gerbils, much less than that in Bat (45 +/- 16)%. The levels of explore activities of ischemic gerbils was 175% and 159% of sham-operated gerbils at d 3 and d 6 postischemic insult, much more than that in Bat (120% d 3 and 140% d 6). Hippocampal ATP levels in Ir were 64% of sham-operated gerbils at reperfusion 60 min, much less than that in Bat I and II (82% and 89% respectively). The hippocampal 2,3-DHBA outputs in Ir increased by 4.5 folds of sham-operated gerbils at reperfusion 60 min, but the 2,3-DHBA outputs in Bat I and Bat II were only 2.6 and 2.4 folds respectively. CONCLUSION: Bat possesses the inhibitory effects on DND and OH. production following cerebral ischemia-reperfusion in gerbils.  (+info)

GAP-43 expression and pathological changes of temporal infarction in rats and effects of batroxobin. (5/50)

To study the changes of the expression of growth-associated protein-43 (GAP-43) and pathology in temporal infarction of rats photochemically induced and the effects of batroxobin. METHODS: Immunohistochemical technique and hematoxylin-eosin stain was used to show the changes of the expression of GAP-43 and pathology. RESULTS: In infarction group, GAP-43 expression was markedly increased on the infarction and surrounding tissues at 24 h cerebral infarction. The expression reached peak level at 72 h after cerebral infarction and was decreased at 7 d after cerebral infarction. However, in batroxobin-treated group, GAP-43 expression was increased and the pathological changes were much slight as compared with infarction group. CONCLUSION: The expression of GAP-43 increases in infarction of temporal neocortex and batroxobin promotes the expression of GAP-43 and ameliorates the pathological changes in infarction of temporal neocortex.  (+info)

Recombinant BbetaArg14His fibrinogen implies participation of N-terminus of Bbeta chain in desA fibrin polymerization. (6/50)

We synthesized BbetaArg14His fibrinogen with histidine substituted for arginine at the Bbeta thrombin-cleavage site. This substitution led to a 300-fold decrease in the rate of thrombin-catalyzed fibrinopeptide B (FpB, Bbeta 1-14) release, whereas the rate of FpA release was normal with either thrombin or the FpA-specific enzyme, batroxobin. Both thrombin- and batroxobincatalyzed polymerization of BbetaArg14His fibrinogen were significantly impaired, with a longer lag time, slower rate of lateral aggregation, and decreased final turbidity. Moreover, desA monomer polymerization was similarly impaired, demonstrating that the histidine substitution itself, and not the lack of FpB cleavage, caused the abnormal polymerization of BbetaArg14His fibrin. Scanning electron microscopy showed BbetaArg14His fibrin fibers were thinner than normal (BbetaArg14His, approximately 70 nm; normal, approximately 100 nm; P <.0001), as expected from the decreased final turbidity. We conclude that the N-terminus of the Bbeta chain is involved in the lateral aggregation of normal desAprotofibrils and that the Arg-->His substitution disrupts these interactions in BbetaArg14His fibrinogen.  (+info)

Thrombolytic actions of reptilase. (7/50)

In thrombolytic model in vitro, reptilase (Rep, defibrase) did not show appreciable thrombolytic actions on red and white thrombi. After daily iv infusion of Rep 0.25 IU for 10 d, the time of 50% lysis of euglobulin (ELT1/2) was shortened from 9.3 +/- 0.8 to 6.7 +/- 1.0 h (P < 0.01), alteplase activity was increased from 1.9 +/- 0.7 to 3.7 +/- 0.9 IU.ml-1, and plasminogen inactivator (PI) activity reduced from 4.3 +/- 0.6 to 1.8 +/- 0.9 AU.ml-1 (all P < 0.01). The findings indicate that the thrombolytic action of Rep shown in vivo may not be from the direct action on thrombi but from the influence on alteplase and PI activity.  (+info)

Detection of soluble intermediates of the fibrinogen-fibrin conversion using erythrocytes coated with fibrin monomers. (8/50)

The presence of minimal amounts of fibrinogen-fibrin intermediates in human plasma was visualized by an agglutination reaction of glutaraldehyde-treated human erythrocytes coated with purified fibrin monomers. A degree of monomer coating was established which produced erythrocytes not agglutinated by normal plasma but by plasma containing minimal amounts of soluble complexes of fibrinogen with fibrin monomers. Under standardized conditions of coating, erythrocyte concentration, temperature, pH, and incubation time, the agglutination time varied with the ratio of soluble fibrin to fibrinogen in plasma. The test was sensitive down to a soluble fibrin concentration of 0.675% of the plasma fibrinogen concentration. Early fibrinogen and fibrin degradation products (FDP) in the plasma led to a prolongation of the agglutination time at a concentration of more than 16 mg/100 ml. Late FDP in a concentration of 100 mg/100 ml did not convert a positive test to negative. The test was not affected by heparin and protamine at concentrations of up to 12.5 and 50 NIH units/ml, respectively.  (+info)

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