The hemostatic derangement produced by T-2 toxin in cynomolgus monkeys

T-2 toxin, a mycotoxin produced by several strains of the genus Fusarium, has been implicated as a cause of serious illness in both animals and man. Hemorrhage is a feature of the syndromes which have been described. An LD20 dose of T-2 was administered im to adult cynomolgus monkeys. This resulted in prolongation of prothrombin and activated partial thromboplastin times and a decrease in multiple coagulation factors. These changes were detected within hours of toxin administration, were maximal at 24 hr, and returned to normal over the next 3 days. Fibrin-fibrinogen degradation products were not detected at any time point. Repeated phlebotomy produced a significantly greater increase in platelet count in control monkeys, which could be taken as evidence for an effect of toxin on platelet kinetics. In treated animals, the hematocrit level declined by about 10%, but a similar decrease occurred in control animals. The white blood cell count increased 4 to 5 times over pretreatment values. Despite the changes in multiple laboratory parameters, treated monkeys did not exhibit clinical evidence of hemorrhage. In three animals which died as a result of toxicosis, necropsy revealed mild petechial hemorrhage involving the colon and heart, as well as necrosis of lymphoid tissues.     

Cytotoxicity and effects of T2-toxin on plasma proteines involved in coagulation,fibrinolysis and kallikrein-kinin system

The activity of both the coagulation and fibrinolytic systems was markedly depressed 24 h after a sublethal dose of T-2 toxin. T-2 toxin was active as an anticoagulant at low doses, which did not affect the basal state of the animals. The kallikrein-kinin system was also affected by depletion of the prekallikrein, which indicates increased bradykinin levels in plasma. At the same time there was an increased activity of some clinically relevant enzymes in serum, indicating tissue injuries caused by T-2 toxin. All effects observed in this study reached their maximum within 24 h after administration, which corresponds to the time animals usually die when receiving a lethal dose. T-2 toxin does not, however, seem to affect the protease enzymes by reduced protein synthesis, because of early onset of the effects, nor does it act as a trigger itself. The effect of T-2 toxin on plasma protease enzymes is probably secondary to cytotoxic effects in the vascular endothelium.     

T-686, a novel inhibitor of plasminogen activator inhibitor-1, inhibits thrombosis without impairment of hemostasis in rats

The aim of this study was to evaluate the antithrombotic potential of T-686 ((3E,4E)-3-benzylidene-4-(3,4,5-trimethoxy-benzylidene)-pyrrolidine-2,5-dione), a novel inhibitor of plasminogen activator inhibitor-1 (PAI-1), in rat thrombosis models. T-686 (0.1–100 mg/kg per day, p.o.) dose dependently decreased the weight of venous thrombi induced by a combination of stasis and hypercoagulability. The antithrombotic effect was enhanced by repeated administration of T-686. Warfarin (0.1 mg/kg per day for 3 days) also prevented thrombus formation. The antithrombotic action by warfarin was accompanied by prolongation of coagulation time, while no effect on coagulation time was observed in T-686-treated rats. T-686 lowered the activity of PAI-1 in plasma. In the arterio-venous shunt model, pretreatment with T-686 (10 mg/kg per day) or ticlopidine (100 mg/kg per day) for 8 days inhibited thrombus formation by 33% and 44%, respectively. T-686 had no effect on collagen-induced platelet aggregation ex vivo, while ticlopidine inhibited platelet aggregation. T-686 did not affect bleeding time at 10–100 times the antithrombotic dose, while warfarin dose dependently prolonged bleeding time at and around the antithrombotic dose. These results suggest that T-686 prevents thrombus formation in rats without impairment of hemostasis.     

In vivo and in vitro hemostatic activity of Chromolaena odorata leaf extract

Context: Chromolaena odorata (L.) R.M.King & H.Rob. (Asteraceae) or Siam weed has long been used to stop bleeding in Thailand and many countries. Only the aqueous leaf extract was investigated in in vivo and there have been conflicting results of in vitro hemostatic mechanisms of this plant. Objective: The most appropriate C. odorata leaf extract that promoted the highest hemostatic activity and the hemostatic mechanisms of these plant extracts will be investigated. Materials and methods: The lyophilized aqueous leaf extract and alcoholic (50, 70, and 95% ethanol) extracts from the fresh and dried leaves were investigated both in vivo and in vitro. The bleeding time in male Wistar rats was measured to investigate the hemostatic effect. The hemostatic mechanisms were tested using in vitro platelet aggregation and blood coagulation tests in sheep plasma. Results: All extracts displayed significantly reducing bleeding time (<2.5?min) in rats but did not induce platelet aggregation or blood clotting in the in vitro study. The in vitro blood clotting times of all extracts were > 0.6?min. Ethanol extract (70%) from the dried leaves proved to be the extract producing the highest hemostatic activity in vivo with the bleeding time of 1.85?min. Discussion and conclusion: The in vivo study with rats confirmed the significant ability of this plant extract to stop bleeding. However, the sufficient amount of calcium and active compounds which are aggregating and clotting agents to enhance blood coagulation and platelet aggregation in in vitro tests should be further studied.     

Therapeutic Effect of Dexamethasone in T-2 Toxicosis

T-2 Toxin is a mycotoxin that induces toxemia characterized by numerous hematological and biochemical changes. We have previously shown that prostaglandin (PG) production in brain tissue is increased following T-2 toxin. The present study was designed in order to test the effect of dexamethasone on brain prostaglandins and survival of rats subjected to T-2 toxin. Furthermore, the effect of BW 755c, a dual inhibitor of the cyclooxygenase and lipoxygenase pathways of arachidonate metabolism, on the survival of rats exposed to T-2 toxin was also examined. The present study demonstrated that dexamethasone increases the survival of rats exposed to a highly lethal T-2 toxicosis. This effect was demonstrated at low as well as high doses and at different times after T-2 administration. Dexamethasone depressed PGE2 levels in the brain cortex 6 hr after T-2 toxin but abolished the reduction of PGE2 in brain cortex seen 24 hr after T-2. BW 755c had no consistent effect on the survival of rats in T-2 toxicosis. It is suggested that dexamethasone might be a useful therapeutic agent in T-2 toxicosis in animals and humans, but its mechanism of action remains obscure.     

In vivo effects of the purified thrombin-like and anticoagulant principles of Agkistrodon acutus (Hundred pace snake) venom

The thrombin-like enzyme of Agkistrodon acutus venom caused a marked prolongation of whole blood coagulation time and one-stage plasma prothrombin time and a marked decrease of fibrinogen level, while no significant change in the two-stage plasma prothrombin level was detected. The retardation of blood clotting by the thrombin-like enzyme was chiefly due to the decrease of plasma fibrinogen level. The thrombin-like enzyme did not cause significant change in blood pressure, heart rate, EKG or respiration at a defibrinating dose (0·1 mg/kg, i.v.). It also did not induce platelet aggregation. Fibrin formed by the thrombin-like enzyme was not cross-linked. The thrombin-like enzyme could further digest the -chain of fibrin and the fibrin formed by this enzyme was more susceptible to plasmin degradation than fibrin formed by thrombin.

The anticoagulant principle of Agkistrodon acutus venom caused a marked, but transient prolongation of whole blood coagulation time and one-stage plasma prothrombin time with no significant change in the two-stage plasma prothrombin level or plasma fibrinogen level. Combining these results with our previous in vitro findings, it is concluded that the retardation of blood clotting by the anticoagulant principle might be due to the interference in the interaction between prothrombin and its activation factors. The anticoagulant principle did not affect platelet aggregation induced by ADP. It also did not cause significant change in blood pressure, heart rate, EKG or respiration at an i.v. dose of 1–10 mg/kg.     

Albumin reduces thrombogenic potential of single-walled carbon nanotubes

Reduction of thrombogenicity of carbon nanotubes is an important prerequisite for their biomedical use. We assessed the thrombogenic activity of carboxylated single-walled carbon nanotubes (c-SWCNTs) and covalently PEGylated c-SWNCTs (PEG-SWCNTs) by testing the clotting time of platelet poor plasma and platelet aggregation in whole blood samples, and evaluated the impact of human serum albumin on thrombogenicity of carbon nanotubes. Both types of SWCNTs exhibited considerable thrombogenic activity. SWCNTs accelerated plasma clotting, with a lesser effect seen for PEG-SWCNTs. Treatment of SWCNTs with albumin did not affect the SWCNT-induced shortening of clotting time. In whole blood, no discernible differences in the effect of c-SWCNTs and PEG-SWCNTs on platelets were observed. Upon addition of SWCNTs to blood, dose- and time-dependent formation of agglomerates of nanotubes and platelets was demonstrated. Pretreatment of SWCNTs with albumin reduced the platelet aggregation: the number of single platelets increased, and the size of platelet-SWCNT agglomerates decreased dramatically. Hence, addition of albumin may serve to attenuate the adverse, thrombogenic effect of CNTs.     

CGS 12970: a novel, long acting thromboxane synthetase inhibitor.

CGS 12970 is a potent selective inhibitor of human platelet thromboxane synthetase in vitro (IC50 = 12 nM). It is four orders of magnitude less potent as an inhibitor of sheep seminal vesicle cyclooxygenase, bovine aorta prostacyclin synthetase and human leucocyte 15-lipoxygenase. The compound inhibited collagen-induced thromboxane B2 production by human platelets in vitro without an effect on the accompanying platelet aggregation induced by collagen, ADP, platelet activating factor, thrombin, arachidonic acid or the prostaglandin mimetic, U 46619. Administration of CGS 12970 to rats inhibited collagen-induced thromboxane B2 production but had no effect on platelet aggregation ex vivo. It also had no effect on platelet aggregation induced by thrombin or on plasma clotting times. A single oral dose of 1 or 3 mg kg-1 to rabbits inhibited thromboxane B2 production in clotting blood ex vivo for 12 or 24 h respectively. CGS 12970 inhibited thromboxane B2 production in vivo induced by intravenous administration of collagen to rats or calcium ionophore to guinea-pigs. In both cases there was a concomitant elevation of immunoreactive 6-keto-prostaglandin F1 alpha but no effect on the induced thrombocytopenia. As with other thromboxane synthetase inhibitors, CGS 12970 prolonged tail bleeding time in the rat. However, CGS 12970 was not as potent as other thromboxane synthetase inhibitors in this test. In addition to these usual effects of thromboxane synthetase inhibitors, CGS 12970 inhibited the thrombocytopenia induced by the Forssman reaction or ADP administration. In these tests the effect of the compound was of short duration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Assessment of potential therapies for acute T-2 toxicosis in the rat

The efficacy of a variety of approaches for the treatment of animals with acute T-2 toxicosis was assessed utilizing young female rats. A single large dose of the water soluble salt of methylprednisolone significantly prolonged survival times in T-2 toxin treated animals. The use of diltiazem hydrochloride, dazemgrel, N-acetylcysteine, dimethyl sulfoxide, adenosine triphosphate (ATP), ATP combined with magnesium chloride, ascorbic acid, and aprotinin did not prolong survival times at the dosages administered. Trichodermin, a trichothecene similar in structure and biochemical activity to T-2 toxin but much less acutely toxic, had a detrimental effect on survival times whether given 1 hr prior to or after T-2 toxin.     

真空采血管内壁处理剂对血清TT3和TT4及TSH检测的影响

目的：评价一种真空采血管内壁处理剂对甲功三项检测结果的影响。方法采用内壁处理剂处理的玻璃管与未经过内壁处理剂处理的玻璃管生产的无抗凝真空采血管,对20例健康人进行采血测试,考察两种无抗凝管的血清分离效果及在三个不同的检测平台上总三碘甲状原氨酸（TT3）、总甲状腺素（TT4）、促甲状腺激素（TSH）检测值的差异。结果经过内壁处理剂处理的玻璃无抗凝管与未经过内壁处理的玻璃无抗凝管采血后分离血清,经过内壁处理的玻璃无抗凝管没有纤维蛋白挂壁情况;TT3、TT4、TSH在贝克曼DXI800、西门子Centaur、雅培i2000检测平台上检测,经过内壁处理的玻璃无抗凝管的TT3均值分别为3.10、2.38、2.07 nmol/L,标准差分别为0.42、0.38、0.31;TT4均值分别为102.44、102.34、103.15 nmol/L,标准差分别为15.49、16.25、17.56;TSH均值分别为2.25、2.21、2.29 mIU/L,标准差分别为1.29、1.27、1.38;未经内壁处理的玻璃无抗凝管的TT3均值分别为2.06、1.99、2.08 nmol/L,标准差分别为0.28、0.48、0.30;TT4均值分别为102.82、103.46、101.80 nmol/L,标准差分别为16.48、16.66、16.17;TSH均值分别2.26、2.25、2.23 mIU/L,标准差分别为1.33、1.28、1.28。TT3在贝克曼DXI800检测平台差异具有统计学意义（P＜0.01）。结论在某些检测平台上,真空采血管内壁处理剂L-720会对血清TT3检测造成干扰。     

Effect of T-2 toxin on regional blood flow and vascular resistance in the conscious rat

The acute effect of T-2 toxemia on local blood flow and vascular resistance in hindquarter, mesenteric, and renal vascular beds was continuously measured by the directional pulsed Doppler technique in conscious, male Sprague-Dawley rats. Intravenous injection of T-2 toxin (1 mg/kg) in the conscious rat reduced blood flow and increased vascular resistance in all blood vessels studied but had no significant effect on mean arterial pressure or heart rate. The blood flow in hindquarters gradually decreased to a minimum of -77 +/- 9% (mean +/- SE) 6 hr after the toxin injection. The hindquarter vascular resistance concomitantly increased to a maximum value of +323 +/- 69% above the resistance before toxin administration. Mesenteric and renal blood flow initially increased (slightly) and then gradually decreased. The maximum drop of blood flow, -90 +/- 13% and -76 +/- 13% for the mesenteric and renal vascular beds, respectively, was achieved 4 hr after T-2 toxin injection and the blood flow values remained low for up to 6 hr. Simultaneously with the impairment of blood flow the mesenteric and renal vascular resistance increased to reach the maximal values of +404 +/- 99% and +556 +/- 15%, respectively. In addition, plasma renin activity was markedly elevated (+653 +/- 160%) at the time of reduced renal blood flow. Intravenous injection of the same value of vehicle (10% ethanol in saline) had no significant effect on any of the cardiovascular variables studied. Two of five rats in the T-2 toxin-treated group died within 5 hr after the T-2 toxin injection and only one animal survived 24 hr while all the control animals survived over 24 hr. The results suggest that strong vasoconstriction in skeletal muscle, mesenteric, and renal vascular beds leads to impairment of local blood flow. The ischemia in vital organs together with the earlier reported decrease in cardiac output by T-2 toxin might then be the cause of rapid death in acute T-2 toxemia.     

Enhancement of Platelet Aggregation by Ursolic Acid and Oleanolic Acid

The pentacyclic triterpenoid ursolic acid (UA) and its isomer oleanolic acid (OA) are ubiquitous in food and plant medicine, and thus are easily exposed to the population through natural contact or intentional use. Although they have diverse health benefits, reported cardiovascular protective activity is contentious. In this study, the effect of UA and OA on platelet aggregation was examined on the basis that alteration of platelet activity is a potential process contributing to cardiovascular events. Treatment of UA enhanced platelet aggregation induced by thrombin or ADP, which was concentration-dependent in a range of 5–50 μM. Quite comparable results were obtained with OA, in which OA-treated platelets also exhibited an exaggerated response to either thrombin or ADP. UA treatment potentiated aggregation of whole blood, while OA failed to increase aggregation by thrombin. UA and OA did not affect plasma coagulation assessed by measuring prothrombin time and activated partial thromboplastin time. These results indicate that both UA and OA are capable of making platelets susceptible to aggregatory stimuli, and platelets rather than clotting factors are the primary target of them in proaggregatory activity. These compounds need to be used with caution, especially in the population with a predisposition to cardiovascular events.     

Comparison of the inhibitory effect of T-2 toxin on bovine platelet function with that of other known platelet inhibitors

The inhibitory effects of T-2 toxin on bovine platelet function and thromboxane A₂ production were compared with those of the known inhibitors of human platelet function, acetylsalicylic acid, dipyridamole and verapamil. T-2 toxin (1 × 10⁻³M) effectively inhibited bovine platelet aggregation (33.2–64.3%), whereas neither acetylsalicylic acid nor dipyridamole did so. T-2 toxin appeared to be a less effective inhibitor of platelet aggregation than the calcium channel blocker, verapamil. T-2 toxin (1 × 10⁻³M) added to platelet suspensions together with verapamil, produced an additive inhibitory response. T-2 toxin (2.5 × 10⁻⁴M) effectively inhibited the release of thromboxane A₂ from ADP-stimulated bovine platelets as did acetylsalicylic acid and verapamil but not dipyridamole. T-2 toxin appears to inhibit bovine platelets by a biochemical mechanism distinct from that of the other inhibitors.     

葛根素对凝血功能的影响

目的:通过研究不同剂量葛根素对凝出血时间、血小板聚集以及血流变学的作用,探讨其对凝血功能方面的影响及作用机制。方法:分别将3种不同剂量的葛根素药液灌胃给予正常小鼠,测凝血时间和出血时间;剂量转换后再将3种不同剂量的葛根素药液灌胃给予正常大鼠测定血小板聚集率和血液流变学指标。结果:不同剂量葛根素组可显著延长出、凝血时间;明显抑制血小板聚集率;能显著降低高、中、低切变率下的全血黏度。结论:葛根素有较强的抗凝血作用,其抗凝作用与其抑制血小板聚集作用和改善血流变有关。     

Lonomia obliqua caterpillar envenomation causes platelet hypoaggregation and blood incoagulability in rats

Envenomation caused by Lonomia obliqua is a public health hazard in Southern Brazil. Envenomed victims present severe hemorrhagic syndrome that can progress to intracranial hemorrhage and death. To understand the mechanisms that lead to hemorrhage, we investigated the platelet dysfunction and blood coagulation disturbances following experimental envenomation in rats. L. obliqua bristle extract was injected (s.c.) and blood collected at different times post-venom administration for determination of platelet response and analysis of blood coagulation. Rats presented hypofibrinogenemia and platelet hypoaggregation in platelet rich plasma (PRP). After addition of exogenous fibrinogen to PRP, platelet hypoaggregation was not corrected. Interestingly, normoaggregation was observed when platelets were separated from plasma. In addition, incubation of plasma from envenomed rats inhibits aggregation response of normal washed platelets. These results indicate that an aggregation inhibitor is generated in plasma during envenomation. Moreover, rats presented an increase in nitric oxide plasmatic levels which coincided with maximum inhibition in platelet aggregation. Animals also showed blood incoagulability and a significant increase in thrombin, plasmin and urokinase plasmatic activities. Despite this intravascular thrombin generation, only a slight decrease in platelet numbers was detected. Certainly, the platelet hypoaggregation and blood incoagulability described herein contribute to systemic bleeding observed in patients.     

荫风轮、风轮菜总皂甙对血小板功能影响及机理的研究

荫风轮总皂甙(TSCP)、风轮菜总皂甙(TSCC)均明显增强ADP诱导的血小板聚集、提高血小板粘附率。TSCP作用比TSCC稍强。效射免疫法测定血浆、血小板中cAMP、TXB_2含量,结果:TSCP可使TXB_2显著升高,此作用可能是该药促进血小板聚集的主要机理。     

General pharmacology of T-2588, a new oral cephem antibiotic

A new oral cephem antibiotic, T-2588, the pivaloyloxymethyl ester of (+)-(6R, 7R)-7-[(Z)-2-(2-amino-4-thiazolyl)-2-methoxyiminoacetamido]-3- [(5-methyl-2H-tetrazol-2-yl)methyl]-8-oxo-5-thia-1-azabicyclo[4.2. 0]oct-2-ene-2-carboxylic acid (T-2525), is mainly absorbed from the intestinal tract and biotransformed to T-2525 thereafter. General pharmacological activities of T-2588 were studied and following results were obtained. On the central nervous system, T-2588 did not show any effects at oral doses of 500-2,000 mg/kg and T-2525 produced only a slight elevation of body temperature in rabbits without any other effects at an intravenous dose of 500 mg/kg. On the respiratory and cardiovascular systems, T-2525 caused a slight hypotension and increased both respiratory rate and femoral blood flow, but no changes were observed in heart rate and electrocardiogram in dogs at an intravenous dose of 500 mg/kg. The T-2525 exerted no significant influence on blood pressure response to isoproterenol, acetylcholine or histamine, but showed a slight tendency to decrease pressor response to adrenaline in dogs at intravenous doses of 100-500 mg/kg. For the renal function in rats, T-2588 had no effects on urine volume, electrolytes and PSP excretion at oral doses of 500-2,000 mg/kg. Intravenous administration of T-2525 caused an increase of sodium excretion at 500 mg/kg and dose-dependent increases of PSP excretion at 20-500 mg/kg. Hematological studies revealed that both T-2588 at oral doses of 500-2,000 mg/kg and T-2525 at intravenous doses of 100-500 mg/kg had no effects on bleeding time in mice, blood coagulation and platelet aggregation in rats. The T-2588 exerted no effect on the gastrointestinal system in rats or mice and had no antiinflammatory activity in rats at oral doses of 500-2,000 mg/kg. The T-2525 scarcely affected the motilities of isolated smooth muscle preparations in experimental animals including stomach, ileum, colon, uterus, vas deferens and trachea at a concentration as high as 10(-3) g/ml. The T-2525 increased bile secretion in rats at intravenous doses of 100-500 mg/kg. The T-2525 slightly decreased the twitch tension of musculus gastrocnemius induced by electrical stimulation in rats at an intravenous dose of 500 mg/kg. These results indicate that T-2588 is a pharmacologically inactive antibiotic.     

Absorption and distribution of cadmium in mice fed diets containing either inorganic or oyster-incorporated cadmium

Human platelets were incubated with T-2 toxin, the most toxic component of Fusarium fungi, at doses of 5 to 500 micrograms/10(9) platelets. A dose-related inhibition of platelet aggregation and release of dense bodies (these consist mainly of serotonin-containing granules) were observed. A change in membrane permeability in the absence of shape changes was also demonstrated by a heavy metal impregnation technique. There was no correlated inhibition of thromboxane synthesis or significant alterations in platelet calcium content. The microtubular system was also unaffected. Suppressed platelet aggregation may contribute to the lethal hemorrhagic phenomenon associated with intoxication by fusarial toxins in man and animals.     

Characteristics of rat platelets and relative contributions of platelets and blood coagulation to haemostasis.

In order to understand some of the haemostatic mechanisms in rats for the interpretation of toxicological data, basic haemostatic parameters with a special emphasis on platelet functions were first measured in vitro. The results of reactions of rat platelets to many aggregating agents suggest that only ADP may be a consistently significant aggregator. The search for physiologic aggregators revealed ADP to be available from erythrocytes. Adhesion reaction also required ADP. Collagen was not considered to be essential for either reaction. Aggregation and adhesion were probably both reversible in flowing blood, while irreversible thrombi were formed in blood at rest ex vivo. Blood coagulation parameters determined revealed that the intrinsic pathway may be more important than the extrinsic one. The rate of intrinsic coagulation reaction was rapid, and plasma coagulation appeared to be of primary importance while the influence of platelet aggregation was minor. A simple model of rat haemostatic mechanism is proposed based on these results. Additionally, to define the relative contribution of platelets versus other cellular and plasma coagulation in vivo, rats were administered antiplatelet drugs (ticlopidine, suprofen and clopidogrel) and an anticoagulant (warfarin) intraperitoneally. Bleeding times (BTs) were significantly increased in all treated groups. ADP-induced platelet aggregations were significantly depressed by the administration of the three antiplatelet drugs, while kaolon-activated partial thromboplastin time and prothrombin time were greatly increased in the warfarin-treated rats. The increase in BT may be due to the inhibition of platelet activity or blood coagulation defect in rats given antiplatelet drugs or warfarin, respectively. These results suggest that platelets play a key role in haemostasis in the rat. Two possible explanations of the disparity between in vitro and in vivo results may be that functional tests used here are not adequate to cover the properties of rat platelets or that mechanisms leading to the formation of platelet thrombi in rats are ADP-dependent adhesion and ADP-induced aggregation.