人胎盘抗凝蛋白变体抗凝与抗血栓形成的效应与作用时间及剂量的关系

背景肝素作为最常用的抗凝药物已广泛用于临床,但其存在明显的出血副作用,与纤维蛋白结合的凝血酶活性作用有限,并发血小板减少症等不良反应.将水蛭素C端结构域与人胎盘抗凝蛋白连接在一起构建抗凝蛋白人胎盘抗凝蛋白变体,比较其抗凝抗栓作用与肝素的差异. 目的观察抗凝蛋白质人胎盘抗凝蛋白变体抗凝和抑制动脉血栓形成的作用的量效与时效关系,并了解该药的安全性. 设计完全随机分组设计,对照实验. 单位一所市级医院心脏科. 材料实验于2000-07/2001-04在江苏省人民医院动物实验室完成.选用纯种雄性新西兰白兔32只,随机将白兔分成4组人胎盘抗凝蛋白变体大剂量组、人胎盘抗凝蛋白变体小剂量组、普通肝素组和生理盐水组,每组8只.方法肝素和人胎盘抗凝蛋白变体都用生理盐水稀释.①人胎盘抗凝蛋白变体大剂量组人胎盘抗凝蛋白变体0.7 mg/kg静脉推注,继以0.35 mg/(kg·h)维持静脉滴注2 h;人胎盘抗凝蛋白变体小剂量组人胎盘抗凝蛋白变体0.3 mg/kg静脉推注,继以0.15 mg/(kg·h)维持静脉滴注2 h;普通肝素组肝素75 IU/kg静脉推注后,以37.5 IU/(kg·h)维持静脉滴注2 h;生理盐水组采用与药物组相同体积的生理盐水和给药方法.②分别在用药前,用药后15和30及60min及停药后2 h从股静脉采血测血常规、活化部分凝血活酶时间、凝血酶时间.③用药后15 min用球囊剥脱股动脉内皮并监测股动脉远端的血压,记录脉压为0即血栓完全闭塞血管的时间.最后剪取球囊损伤的股动脉,剥离血管测量所形成血栓的长度、血栓的湿质量和干质量.④人胎盘抗凝蛋白变体毒副作用观察实验过程中监测动物的血压、心率、呼吸等生命体征,并将部分动物实验后,行腹部解剖,观察内脏出血的情况,检测血白细胞数目. 主要观察指标①人胎盘抗凝蛋白变体对凝血系统和血栓形成的影响.②人胎盘抗凝蛋白变体毒副作用. 结果白兔32只均进入结果分析.①抗凝效应活化部分凝血活酶时间值人胎盘抗凝蛋白变体大小剂量组在用药后15 min时最长(136.86±39.46),(122.90±4.19)s],明显长于生理盐水组,短于普通肝素组(95.14±24.64),(180.00±0.00)s,P＜0.05,0.01].用药后30 min,人胎盘抗凝蛋白变体大剂量组仍保持其抗凝疗效,明显长于生理盐水组(124.61±40.19),(85.57±27.67)s],人胎盘抗凝蛋白变体小剂量组明显短于普通肝素组(112.94±43.17),(179.39±1.83)s,P＜0.05].用药后60 min,人胎盘抗凝蛋白变体大小剂量组明显短于普通肝素组(P＜0.05).停药后2 h虽长于生理盐水组,但差异不明显(P＞0.05).凝血酶时间用药后15和30及60 min普通肝素组明显长于人胎盘抗凝蛋白变体大小剂量组(P＜0.05).②对动脉血栓形成的影响血栓湿重人胎盘抗凝蛋白变体组明显小于普通肝素组(P＜0.05).血栓干质量人胎盘抗凝蛋白变体大小剂量组明显小于普通肝素组,人胎盘抗凝蛋白变体大剂量组明显小于人胎盘抗凝蛋白变体小剂量组(P＜0.05).血栓长度人胎盘抗凝蛋白变体小剂量组明显小于生理盐水组(P＜0.05),人胎盘抗凝蛋白变体大剂量组明显小于普通肝素组(P＜0.05).完全闭塞时间人胎盘抗凝蛋白变体大小剂量组明显高于生理盐水组(P＜0.05).③人胎盘抗凝蛋白变体的毒副作用人胎盘抗凝蛋白变体大小剂量组白兔表现近似于其他两组,血液动力学的无明显改变,无内脏出血的情况. 结论人胎盘抗凝蛋白变体是一种安全有效的抗凝和抗栓制剂,人胎盘抗凝蛋白变体有明显的抗凝血作用,在用药后15 min抗凝作用最强,但与肝素相比,抗凝作用较弱,用药60min后抗凝作用变得很弱;人胎盘抗凝蛋白变体抗血栓形成作用强于肝素,所形成血栓的大小明显小于肝素作用后,而且呈剂量依赖性关系,大剂量人胎盘抗凝蛋白变体的抗栓作用明显强于小剂量组.

Effects of human annexin Ⅴ derivative on coagulation and thrombosis: Effective time and dosage

BACKGROUND: As a common anticoagulant, heparin is widely used in clinic, but it has remarkable side effects such as severe bleeding and heparin-induced thrombocytopenia, and it cannot inactivate fibrin-bound thrombin. Annexin Ⅴ derivative (AND) is inosculated with C-terminal of hirudin and annexin Ⅴ, and its anticoagulation and anti-thrombosis effects are compared with those of heparin. OBJECTIVE: To investigate the relationship between quantitative effectiveness and time effectiveness of AND on coagulation and thrombosis, and study its reliability. DESIGN: Completely randomized grouping design and controlled study. SETTING: Cardiac Department of amunicipal hospital. MATERIALS: The experiment was conducted at the Animal Laboratory of Jiangsu Provincial People's Hospital from July 2000 to April 2001. Totally 32 male New Zealand white rabbits were randomly divided into 4groups, namely, high dosage AND group, low dosage AND group, common heparin group and saline group with 8 in each group. METHODS: Heparin and AND were diluted with saline.①High dosage AND group: 0.7 mg/kg AND was injected intravenously and followed by intravenous dripping of 0.35mg/(kg ·h) for 2 hours. Low dosage AND group: 0.3 mg/kg AND was injected intravenously and followed by intravenous dripping of 0.15 mg/(kg·h) for 2 hours. Heparin group: 75 IU/kg heparin was injected intravenously and followed by intravenous dripping of 37.5 IU/(kg·h) for 2 hours. Saline group: The same volume of saline and medication were used as those in drug groups.② Blood sample was collected from the femoral vein before administration so as to test blood routine, activated partial thromboplastin time(APTT)and prothrombin time (PT) after 15-, 30- and 60-minute administration and 2-hour withdrawal.③Saccule was separated from endothelium of femoral artery to measure blood pressure of distal femoral artery at 15 minutes after administration.Time of pulse pressure equal to 0 mmHg was recorded when the vessel was occluded completely by a thrombus. Finally the injured femoral arteries whose vessel was stripped were collected to measure its length, wet weight and dry weight. ④Observation of AND toxicity and sideeffects:During the experiment,vital signs of the animals were measured,such as blood pressure,heart rate and breath;in addition,bowelhemorrhage was observed and the number of leucocytes was counted after dissection of some of the animals. MAIN OUTCOME MEASURES:①Effect of AND on blood coagulation system and arterial thrombosis.②AND toxicity and side effects. RESULTS: All the 32 white rabbits entered the final analysis. ① Anticoagulant effect: APTT: Fifteen minutes after administration, APTT in AND group was the longest,which was(136.86±39.46)s in high dosage AND group and (122.90±34.19) s in low dosage ANDgroup.Moreover, APTT was longer than that in saline group (95.14±24.64) s], but shorter than that in common heparin group (180.00±0.00) s, P ＜ 0.05, 0.01]. At 30 minutes after administration, AND in high dosage group still had coagulation,and APTT was (124.61±40.19) s in high dosage group, which was longer than that in saline group (85.57±27.67) s], but APTT was (112.94±43.17) sin low dosage group, which was shorter than that in common heparin group (85.57±27.67) s, P ＜ 0.05]. APTT was shorter in high and low dosage groups than in common heparin group at 60 minutes after administration (P ＜ 0.05), and longer than that in saline group 2 hours after drug withdrawal, but there was not significant difference (P ＞ 0.05). PT: PT in common heparin group was longer than that in high and low dosage groups at 15,30 and 60 minutes after administration (P ＜ 0.05).② Effect on arterial thrombosis: Wet weight of thrombus: It was lighter in AND group than in common heparin group (P ＜ 0.05). Dry weight of thrombus:Thrombus was lighter in high and low dosage groups than in common heparin group, and was lighter in high dosage group than in low dosage group (P ＜ 0.05). Thrombus length: It was shorter in low dosage group than in saline group (P ＜ 0.05), and shorter in high dosage groupthan in common heparin group (P ＜ 0.05). Time of complete occlusion: It was longer in high and low dosage groups than in saline group (P ＜ 0.05). ③ AND toxicity and side effects: The behavior of rabbits in high and low dosage groups was similar to that in other two groups. Obvious hemodynamic changes were not found, and bowel hemorrhage was not observed, either. CONCLUSION: AND is an effective anticoagulant and anti-thrombosis agent; the highest anticoagulation effect occurs at 15 minutes afteradminis tration. However, the anticoagulant effect is poor as compared to heparin.The effect is poorer after 60-minute administration. Effect of AND on thrombus is stronger than that of heparin, but the size of thrombus is smaller than that of heparin, and the dosage-dependence manner was found. In addition, the anti-thrombus effect of AND is stronger in high dosage group than in low dosage group.