木瓜蛋白酶对凝血功能的抑制作用及机制的体外研究

目的：观察木瓜蛋白酶体外对凝血功能的抑制作用,并探讨其可能机制。方法：将不同剂量木瓜蛋白酶分别与贫血小板血浆（PPP）和富血小板血浆（PRP）作用,分为生理盐水组、10U/L组和20U/L组,分别以血凝仪测定PPP和PRP的凝血酶原时间（PT）和活化部分凝血活酶时间（APTT）,以血气分析仪和血凝仪分别测定PPP的Ca2＋浓度和凝血因子V、VII、VIII、IX、X和XI活性（FV：C、FVII：C、FVIII：C、FIX：C、FX：C和FXI：C）;将新鲜全血与前述3种浓度木瓜蛋白酶作用,采用硅化管法测定全血凝血时间（CT）。同时测定0、20、40、60和80U/L木瓜蛋白酶PPP的PT和APTT值。结果：10 U/L和20U/L木瓜蛋白酶组PPP和PRP的PT和APTT值、全血CT值分别显著高于生理盐水组和10U/L木瓜酶组（P〈0.01）,FV：C和FVIII：C水平分别显著低于生理盐水组和10U/L木瓜酶组（P〈0.05）;三组PPP与PRP之间PT和APTT值、各组间Ca2＋浓度以及其余凝血因子活性差异均无显统计学意义（P〉0.05）。PPP的PT和APTT值均与木瓜蛋白酶剂量呈显著正相关（r=0.995和0.991,P〈0.01）。结论：木瓜蛋白酶可通过抑制凝血因子V和VIII活性,从而对凝血功能有剂量依赖性的抑制作用,具有抗凝的功效。     

Antiplatelet action of ilexoside D,a triterpenoid saponin fromIlex pubescens

The anti-platelet activity of ilexoside D isolated from the roots ofIlex pubescens Hook. et Arn. was investigated inin vitro andex vivo models of platelet aggregation induced by ADP, thrombin or collagen in rats.In vitro ilexoside D inhibited more effectively platelet aggregation induced by ADP and thrombin than by collagen as compared with aspirin.Ex vivo ilexoside D also inhibited platelet aggregation induced by ADP and collagen, but not by thrombin, and the inhibitory action of ilexoside D was more effective than that of aspirin. However,in vitro ilexoside D inhibited very poorly the generation of malonyldialdehyde, which is known to be concomitantly released with thromboxane A₂ during platelet aggregation. These results suggest that the anti-platelet activity of ilexoside D may not be responsible for prostaglandin synthesis in platelets.     

A chemically stable analogue, 9 beta-methyl carbacyclin, with similar effects to epoprostenol (prostacyclin, PGI2) in man

The effects of 9 beta-methyl carbacyclin, a chemically stable analogue of epoprostenol (prostacyclin, PGI2) were studied, in comparison with epoprostenol, both in vitro and in vivo in man. In vitro 9 beta-methyl carbacyclin and epoprostenol inhibited platelet aggregation induced by ADP, collagen, the endoperoxide analogue U46619 and arachidonic acid. The potency of 9 beta-methyl carbacyclin relative to epoprostenol was comparable in ADP and collagen-aggregated platelet rich plasma (PRP), 9 beta-methyl carbacyclin being 0.01 times as active as epoprostenol. The anti-aggregatory potencies of the two compounds were comparable in PRP and whole blood. The phosphodiesterase inhibitor isobutyl methyl xanthine enhanced the anti-aggregatory activity of both compounds in vitro. 9 beta-methyl carbacyclin and epoprostenol elevated platelet cyclic AMP, 9 beta-methyl carbacyclin being 0.04 times as active as epoprostenol. In a placebo controlled trial both drugs produces significant headache and facial flushing when compared with placebo. Nasal stuffiness, abdominal discomfort and nausea were reported on all three treatments. Both drugs caused significant and comparable increase in heart rate and decrease in pre-ejection (PEP) and PEP/left ventricular ejection time (LVET) ratio compared with placebo. Systolic and diastolic blood pressure, LVET and QS2 index were unchanged. Platelet aggregation responses to ADP were significantly inhibited by all three doses of both drugs compared with placebo. Bleeding time was significantly longer during epoprostenol infusion than either placebo or 9 beta-methyl carbacyclin infusion. Neither drug had significant effect, compared with placebo, on kaolin activated clotting time in PPP, PRP or in PRP in the presence of heparin, prothrombin time, partial thromboplastin time, thrombin clotting time, fibrinogen, fibrinogen degradation products or euglobulin clot lysis time. The pharmacodynamic effects and duration of action of 9 beta-methyl carbacyclin and of epoprostenol are similar; 9 beta-methyl carbacyclin is approximately 100 times less potent than epoprostenol in man.     

The anti-coagulant properties of an extract from the venom sac of the oriental hornet

An extract from the venom sac of the oriental hornet inhibits the in vitro formation of thromboplastin and thrombin by human plasma and inactivates already formed tissue thromboplastin. The extract has a strong fibrinogenolytic and fibrinolytic activity when tested on purified human fibrinogen, but almost no lytic activity when tested on human plasma. The fibrinolysis is inhibited by soybean trypsin inhibitor as well as by human serum, presumably due to its anti-trypsin activity. Intravenous administration of the sac extract to dogs causes decreased clottability of the whole blood, or the recalcified plasma, and depresses prothrombin consumption and thromboplastin generation. There is a significant decline of the platelet count but a rather mild decrease of fibrinogen. All parameters tend to revert to normal after 2–3 hr. Intraperitoneal envenomation causes severe heparinemia with prolonged clotting, effects which can be reversed by the addition of protamin sulfate. The anticoagulant properties of the venom are abolished by heating at acid or alkaline pH and neutralized by anti-venom immune serum.     

Blood Compatibility of Cetyl Alcohol/Polysorbate-Based Nanoparticles

Purpose Pegylated and nonpegylated cetyl alcohol/polysorbate nanoparticles (E78 NPs) are being tested as drug carriers for specific tumor and brain targeting. Because these nanoparticle formulations are designed for systemic administration, it is important to test the compatibility of these lipid-based NPs with blood and blood cells. Methods The hemocompatibility of E78 NPs was evaluated with a particular focus on hemolytic activity, platelet function, and blood coagulation. Human red blood cell lysis was determined by measuring hemoglobin release. Activation and aggregation of human platelets were determined using flow cytometry and aggregometry, respectively. Finally, the whole blood clotting time was measured using human blood. Results E78 NPs did not cause in vitro red blood cell lysis at concentrations up to 1 mg/mL. In addition, under conditions tested, E78 and polyethylene glycol (PEG)-coated E78 NPs (PEG-E78 NPs) did not activate platelets. In fact, both NP formulations very rapidly inhibited agonist-induced platelet activation and aggregation in a dose-dependent manner. Additionally, E78 NPs significantly prolonged in vitro whole blood clotting time at a concentration of 500 μg/mL or greater. Conclusions It was concluded that PEG-coated and nonpegylated E78 NPs have potential blood compatibility at clinically relevant doses. Based on the calculated nanoparticle-to-platelet ratio, the concentration at which E78 NPs could potentially affect platelet function in vivo was approximately 1 mg/mL.     

Purification and characterization of anticoagulant protein from the Tabanus,Tabanus bivittatus

Tabanus anticoagulant protein (TAP) was isolated from the whole body of the tabanus, Tabanus bivittatus, using three purification steps (ammonium sulfate fractionation, gel filtration on Bio-Gel P-60, and ion exchange chromatography on DEAE Sephadex gel). The purified TAP, with a molecular weight of 65 kDa, was assessed to be homogeneous by SDS-polyacrylamide gel electrophoresis, and an isoelectric point of 7.9 was determined by isoelectric focusing. The internal amino acid sequence of the purified protein was composed of Ser-Leu-Asn-Asn-Gln-Phe-Ala-Ser-Phe-Ile-Asp-Lys-Val-Arg. The protein was activated by Cu2+ and Zn2+, and the optimal conditions were found to be at pH 3-6 and 40-70 degrees C. Standard coagulation screen assays were used to determine thrombin time and activated partial thromboplastin time. Chromogenic substrate assays were performed for thrombin and factor Xa activity. TAP considerably prolonged human plasma clotting time, especially activated partial thromboplastin time in a dose-dependent manner; it showed potent and specific antithrombin activity in the chromogenic substrate assay. Specific anti-factor Xa activity in TAP was not detected. Overall, this result suggested that TAP has significant anticoagulant activity on blood coagulation system.     

The hemostatic derangement produced by T-2 toxin in guinea pigs

T-2 toxin produced significant coagulation abnormalities when administered parenterally to Hartley strain guinea pigs. The animals developed depressed activity of all coagulation factors except fibrinogen. Platelet aggregation in whole blood was depressed in response to ADP and collagen. The animals also exhibited an initial rise followed by a fall in hematocrit level, leukocytosis, and a decrease in platelet count. These changes were detectable within hours of toxin administration, reached a maximum at 24 hr, and returned to normal over the next 2 days. Pretreatment of animals with vitamin K1 had no effect on the activity of coagulation factors. The activated partial thromboplastin time of dilutions of plasma from animals given T-2 toxin with plasma from control animals revealed a pattern which pointed to a deficiency of coagulation factors as the principal cause of prolonged clotting times in treated animals. The presence of a weak circulating anticoagulant could not be ruled out. The addition of T-2 to plasma and blood of normal animals in a concentration of 1 microgram/ml had no effect on clotting times or platelet aggregation.     

The effects of the purified thrombin-like and anticoagulant principles of Agkistrodon acutus venom on blood coagulation in vivo.

The effects of the purified thrombin-like and anticoagulant principles of the snake venom of Agkistrodon acutus on blood coagulation of rabbits in vivo were studied. The thrombin-like principle caused a marked prolongations of whole blood coagulation time and one-stage plasma prothrombin time and a marked decrease of the fibrinogen concentration, while no significant change in the two-stage plasma prothrombin level was detected. It is concluded that the retardation of blood clotting by the thrombin-like principle was chiefly due to the decrease of plasma fibrinogen level. The anticoagulant principle 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 effects of the purified thrombin-like enzyme and anticoagulant principle of Trimeresurus gramineus venom on blood coagulation in vivo

Chaoho Ouyang and Fun-Yun Yang. The effects of the purified thrombin-like enzyme and anticoagulant principle of Trimeresurus gramineus venom on blood coagulation in vivo. Toxicon14, 197–201, 1976.—The effects of the purified thrombin-like enzyme and anticoagulant principle of Trimeresurus gramineus venom on in vivo blood coagulation of rabbits were studied. The thrombin-like enzyme caused a marked prolongation of whole blood coagulation time and one-stage plasma prothrombin time and a marked decrease of the fibrinogen concentration, while no significant change in the two-stage plasma prothrombin level was detected. It is concluded that the retardation of blood clotting by the thrombin-like enzyme was chiefly due to the decrease of plasma fibrinogen level. The anticoagulant principle 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.     

Effect of whole blood clotting time in rats with ionized hypocalcemia induced by rapid intravenous citrate infusion

Although the toxic effects of citrate including hemodynamic and cardiovascular changes result from a decrease in ionized calcium levels in serum due to chelating action, these effects of citrate on blood coagulation have not yet been fully clarified. The present study examines whether serum citrate and ionized calcium levels affect whole blood clotting time in rats using the test tube method in which citrate is administered by rapid intravenous infusion. Citrate was infused via the tail vein into 10 rats at 3, 4 or 5 mmol/kg/hr for 1 hr, and then whole blood clotting time, serum citrate and ionized calcium levels were determined. Whole blood clotting time did not significantly change at citrate infusion rates of 3 and 4 mmol/kg/hr. However, at 5 mmol/kg/hr, whole blood clotting time was significantly prolonged by a factor of 2.1 relative to the untreated group, when the serum citrate level was 10.03 +/- 1.39 mmol/l (59.0-fold higher than that in the untreated group) and the serum-ionized calcium level was 0.29 +/- 0.02 mmol/l (0.2-fold lower than that in the untreated group). These results suggest that whole blood clotting time is significantly prolonged in rats with severe ionized hypocalcemia.     

Antithrombotic effect of a novel protein from Fusarium sp. CPCC 480097 in a rat model of artery-vein bypass thrombosis

Context: The incidence and mortality of thrombotic disorders are rapidly increasing throughout the world. Therefore, attempts have been made to develop new anticoagulant and antithrombotic drugs. Our previous studies showed that a novel protein, named Fu-P, had fibrinogenolytic activity and much higher fibrinolytic activity on the fibrin plate than urokinase in vitro.

Objective: The antithrombotic activities of Fu-P in vivo are investigated here for the first time.

Materials and methods: Antithrombotic activity of Fu-P was studied in a rat model of artery-vein bypass thrombosis. The anticoagulant activity of Fu-P was measured by clotting assay of activated partial thrombinplastin time and prothrombin time (PT). The effects of Fu-P on the factor Xa and thrombin were assayed using the chromogenic substrate S-2765 and S-2238.

Results: Intravenous injection of Fu-P produced a 58.4% inhibition ratio of thrombus formation at 0.1?mg/kg body weight, while heparin produced 42.5% inhibition ratio of thrombus formation at 0.6?mg/kg body weight. Fu-P significantly prolonged fibrinogen clotting time, activated partial thrombinplastin time and thrombin time, which also prolonged PT. The inhibition assay of the coagulant factors using chromogenic substrates S-2238 and S-2765 showed that Fu-P was not the inhibitor of the thrombin and Xa.

Discussion and conclusion: These findings demonstrated that the novel fibrinolytic enzyme (Fu-P) might also be used as a natural agent for thrombolytic therapy or thrombosis prevention.     

原矛头蝮蛇毒的抗凝作用

目的 研究原矛头蝮蛇毒(PMV)对血液系统的作用。方法 将PMV 0.2 mg·kg^-1一次性尾静脉注射给予SD大鼠。注射后0.5, 1, 3, 6和24 h分别取下腔静脉血,制备抗凝全血、抗凝血浆和富血小板血浆(PRP)。利用血细胞计数板对抗凝全血和PRP进行血小板计数;将抗凝血浆用生理盐水稀释5倍,在412 nm测定血红蛋白含量;采用凝血酶时间(TT)、凝血酶原时间(PT)、活化部分凝血活酶时间(APTT)和纤维蛋白原(FIB)含量测定试剂盒分别测定抗凝血浆的TT, APTT, PT和FIB含量;用发色底物法测定抗凝血浆酶切发色底物S-2251的活性。给昆明小鼠一次性尾静脉注射PMV 0.28 mg·kg^-1,在0.5, 1, 3, 6和24 h测定尾部出血时间。结果 给予PMV 0.2 mg·kg^-1 30 min大鼠抗凝全血血小板计数减少至正常对照组的1/3(P<0.01),PRP中血小板计数减少至正常对照组的1/20(P<0.01),抗凝血浆血红蛋白含量增加约6倍(P<0.01);给予PMV 6 h APTT明显延长(P<0.05),3和6 h TT明显延长(P<0.01),1和3 h PT明显缩短(P<0.01);FIB含量和抗凝血浆酶切发色底物S-2251的活性无明显变化。给予小鼠PMV 0.28 mg·kg^-1 30 min小鼠尾部出血时间达(2341±742)s,较正常对照组小鼠(81±11)s明显延长(P<0.01),1 h逐渐缩短(P<0.01),24 h仍未恢复至正常水平(P<0.05)。结论 PMV具有明显的抗凝作用。     

Effects of synthetic oligonucleotides on human complement and coagulation1

Oligodeoxynucleotide phosphorothioates (PS-oligos) are being studied as novel therapeutic agents based on their ability to inhibit gene expression. Preclinical studies produced unanticipated complement and coagulation effects in monkeys receiving high-dose PS-oligo. In the present in vitro studies, PS-oligo inhibited normal human blood clotting as well as subsequent assays for prothrombin fragment PF₁₊₂ and hemolytic complement. PS-oligo treatment of normal donor plasma produced concentration-dependent prolongations of clotting times, with the activated partial thromboplastin time more sensitive than prothrombin time or thrombin clotting time. PS-oligo treatment of normal donor serum similarly reduced hemolytic complement activity in a concentration-dependent manner. Reduced hemolysis correlated with increased levels of complement fragment C4d. The anti-heparin drug protamine sulfate inhibited in vitro effects of PS-oligo in both complement and coagulation assays, suggesting that charged residues in intemucleotide linkages of PS-oligo mediated the observed activities. Therefore, oligonucleotides with varying intemucleotide linkages, nucleotide sequence, or secondary structure were compared. Both complement and coagulation effects appeared to be independent of nucleotide sequence but were strongly related to the nature of intemucleotide linkages. Several of these modified oligonucleotides have been shown previously to retain potent antisense activity and thus may represent viable alternatives for antisense therapeutics.     

Salutary Effects of Two Verapamil Analogs in Traumatic Shock

We studied the effects of two verapamil analogs, anipamil and ronipamil, in traumatic shock. Noble-Collip drum trauma produced a shock state characterized by an eight-fold increase in plasma cathepsin D activity, a 13-fold increase in the rate of plasma myocardial depressant factor (MDF) accumulation, and a survival time of 1.9 ± 0.1 hours. Neither verapamil analog had any significant effect on attenuating the shock-induced rise in plasma cathepsin D activity. However, both anipamil and ronipamil (p < 0.01) significantly blunted the rate of MDF accumulation in the plasma. In addition, these agents significantly inhibited proteolysis in vitro. Both analogs significantly prolonged survival time to 3.1 ± 0.6 h at 0.25 mg/kg (p < 0.05) and to 4.4 ± 0.3 h at 1.0 mg/kg (p < 0.001). Anipamil appears to provide a more potent protection in this shock model; however, both verapamil derivatives possess promising anti-shock potential.     

Effects of adrenaline and alprenolol (Aptin®) on blood coagulation and fibrinolysis in man

Summary The effects of intravenous infusions of adrenaline alone, and after alprenolol (Aptin^®) pre-treatment, on whole blood clotting time, factor VIII, fibrinolysis, partial thromboplastin time (PTT), platelet count and fibrinogen have been studied in four healthy, fasting male subjects by double-blind technique using saline infusions as control. The adrenaline infusion caused increases in factor VIII levels, fibrinolysis and platelet counts and a decrease in whole blood clotting time. Alprenolol inhibited the effects of adrenaline on factor VIII and fibrinolysis in all subjects, and the effects on whole blood clotting time in all but one subject. The adrenaline-induced rise in platelet count was not significantly influenced by alprenolol pre-treatment. No effects were seen of the alprenolol infusion itself.     

Characterization of the Rabbit as an In Vitro and In Vivo Model to Assess the Effects of Fibrinogenolytic Activity of Snake Venom on Coagulation

Several in vitro investigations have demonstrated that anticoagulant effects of fibrinogenolytic snake venom metalloproteinases have been abrogated in human plasma by modifying fibrinogen with iron (Fe) and carbon monoxide (CO) to prevent catalysis or by directly inhibiting these enzymes with CO. To translate these findings, we chose to assess the rabbit as a model of envenomation with Crotalus atrox venom. It was determined with thrombelastography that 15 times the concentration of venom noted to compromise coagulation in plasma in vitro was required to cause coagulopathy in vivo, likely secondary to venom binding to blood cells and being cleared from the circulation rapidly. Unlike human plasma, rabbit plasma pre‐treated with Fe/CO was not protected from fibrinogenolysis by venom. Consequently, the administration of purified human fibrinogen (with or without Fe/CO) would be required before venom administration to rabbits. Of greater interest, venom exposed to CO had complete loss of fibrinogenolytic effect in rabbit plasma and partial loss of activity in whole blood, indicative of unbinding of CO from venom and binding to haemoglobin. Thus, venom exposed to CO could remain partially or completely inhibited in whole blood long enough for clearance from the circulation, allowing rabbits to be a useful model to test the efficacy of regional CO administration to the bite site. Future investigations are planned to test these novel approaches to attenuate venom‐mediated coagulopathy in the rabbit.     

Effects of Heloderma suspectum venom on blood coagulation

The effect on blood coagulation of venom from the Gila monster Heloderma suspectum was studied in vivo and in vitro. Lethal and sublethal doses of venom failed to alter the clotting and prothrombin times of rabbit and cat blood in vivo. Venom mixed with freshly drawn blood from rabbits and cats had no effect on the clotting time. When venom was incubated with human, rabbit, and cat plasma for periods longer than 5 min, prothrombin time was prolonged. Placing venom in a boiling water bath for 15 min did not alter this.     

N-(2-羟丙基)甲基丙烯酰胺聚合物-5-氟尿嘧啶接合物的体外释药规律、体内分布及抗肿瘤活性研究

考察本实验室合成的N-(2-羟丙基)甲基丙烯酰胺［N-(2-hydroxypropyl) methacrylamide，HPMA］聚合物-5-氟尿嘧啶(5-flurouracil，5-FU)接合物(P-FU)的体外释药、体内分布及抗肿瘤活性。以小鼠血浆为介质，考察P-FU中5-FU的释放规律；以小鼠H22肝癌实体瘤模型(皮下型)为肿瘤模型，考察接合物在荷瘤小鼠体内的分布情况、药代动力学规律及抑瘤活性。结果表明，37 ℃时P-FU在小鼠血浆中具有一定的稳定性，半衰期(t_1/2)为32.4 h。与5-FU相比，P-FU在荷瘤小鼠体内的循环时间明显延长(血浆中t_1/2为原药的166倍)，在肿瘤中的沉积量(AUC为5-FU的3.3倍)及滞留时间(t_1/2为5-FU的2.3倍)均有明显增加。体内药效学研究表明，P-FU组对荷瘤小鼠的肿瘤生长抑制率(69.09%)显著高于5-FU组(56.49%，P<0.05)，瘤块组织病理学观察结果也显示P-FU组小鼠肿瘤组织中细胞凋亡程度大于5-FU组。HPMA聚合物可被用于为5-FU构建一种新型实体瘤高分子给药系统。     

Crossover dose escalation study to assess safety, pharmacokinetics, and pharmacodynamics of single doses of R1663, an oral factor Xa inhibitor, in healthy male volunteers

This study investigated the safety, pharmacokinetics, and pharmacodynamics of single oral doses of R1663, a factor Xa inhibitor, in healthy volunteers. It was a single-blind, randomized, crossover, placebo-controlled, dose escalation study in 33 healthy male volunteers aged 18 to 45 years. Each volunteer was dosed on 3 occasions with R1663 or placebo. Single oral doses of R1663 were safe and well tolerated. R1663 did not affect bleeding time. Pharmacodynamic effects (prothrombin time [PT], activated partial thromboplastin time [aPTT]), parameters of thrombogram, and anti-factor Xa activity) and plasma concentrations of R1663 were dose-dependent and showed low to moderate (<40%) intersubject and intrasubject variability. Maximum factor Xa inhibition was achieved 3 hours post dose (time to maximum concentration of R1663): clotting times were prolonged up to 2.5-fold, whereas endogenous thrombin potential (ETP) and peak height were decreased by 48% and 85% from their baseline values, respectively. Pharmacodynamic parameters were strongly correlated to R1663 plasma concentrations, with IC50 values of 182 and 2680 ng/mL for peak height and ETP, respectively. Oral doses of R1663 up to 480 mg were well tolerated, with predictable pharmacodynamics and pharmacokinetics. R1663 prolonged clotting times (PT, aPTT) and inhibited thrombin generation without increasing bleeding time.     

Comparative bioavailability: A new type ofin vitro-in vivo correlation exemplified by prednisone

The average time to reach half-maximal plasma concentration of prednisolone and the average plasma concentrations of prednisolone at 0.5 and 1 hr obtained from three crossover bioavailability studies, involving testing of commercially available 5-mg prednisone tablets, were highly correlated (r0.88) with parameters derived from in vitrotablet dissolution rates performed in the spin filter apparatus of Shah. The in vitroparameters were the times to dissolve 16% or 50% of the labeled amount of prednisone or the percent of the labeled amount of prednisone dissolved in 20 min in water at 37C. Such correlations may be useful in the setting of in vitrodissolution rate specifications for commencal prednisone tablets.Supported by Contract FDA 69-22, Food and Drug Administration, Washington, D.C., and in part by Public Health Service Grant 5-P11-GM15559.