非抗凝肝素衍生物对脓毒症小鼠高凝状态改善作用机制研究

Mechanism of non-anticoagulant heparin derivatives on hypercoagulable state in septic mice

目的研究非抗凝肝素衍生物(ROH)对脓毒症小鼠高凝状态的影响和可能的机制。方法体外实验采用生色底物法检测ROH通过抗凝血酶Ⅲ(ATⅢ)及肝素辅因子Ⅱ(HCⅡ)对凝血酶(Ⅱa)活性的抑制作用。体内实验,将小鼠随机分为4组:正常组、模型组、肝素组、ROH组,每组5只。除正常组外,其余3组小鼠均腹腔注射脂多糖(LPS)溶液(15 mg·kg^(-1))复制脓毒症模型。肝素组和ROH组分别于造模前15 min尾静脉注射400 U·kg^(-1)肝素溶液和30 mg·kg^(-1)ROH溶液。正常组和模型组则尾静脉注射等体积的生理盐水。建模5 h后,采用剪尾法测定出血时间;采用酶联免疫吸附法(ELISA)检测血浆中D-二聚体、组织因子途径抑制物(TFPI)、组织因子(TF)、可溶性血栓调节蛋白(sTM)的含量。结果体外检测发现ROH不依赖ATⅢ抑制Ⅱa活性,但能通过激活HCⅡ发挥拮抗Ⅱa的活性。正常组、模型组、肝素组、ROH组小鼠尾出血时间分别为(6.56±2.72),(1.68±0.62),(13.93±2.39)和(13.08±2.65)min;这4组小鼠血浆中D-二聚体的含量分别为(1.16±0.19),(1.46±0.18),(1.26±0.13)和(1.23±0.19)ng·mL^(-1)。造模11 h后,这4组的TFPI含量分别为(38.97±1.49),(39.25±3.73),(42.51±2.66)和(42.83±4.75)μg·L^(-1);这4组的TF含量分别为(153.07±15.49),(172.84±3.31),(147.26±22.45)和(161.60±16.07)ng·L^(-1);这4组的sTM含量分别为(5.87±0.75),(6.58±0.39),(6.39±0.64)和(5.66±0.63)ng·mL^(-1)。与正常组比较,模型组尾部出血时间显著缩短(P<0.05),肝素组和ROH组相较于模型组小鼠的尾出血时间均显著延长,差异均有统计学意义(均P<0.05)。造模后,各组TFPI、TF及sTM较正常组均有显著升高或呈升高趋势,在11 h,ROH组sTM含量明显低于模型组(P<0.05)。结论体外非抗凝的ROH可以通过抑制凝血激活和改善生理抗凝作用,发挥改善脓毒症导致小鼠高凝状态的作用。

Objective To investigate the effect of non-anticoagulant heparin derivatives(ROH),a non-acnticoagulant heparin derivative,on the hypercoagulable state of septic mice and the possible mechanisms.Methods In vitro experiments used chromogenic substrate method to detect the inhibition effect of ROH on thrombin(Ⅱa)activity through antithrombinⅢ(ATⅢ)and heparin cofactorⅡ(HCⅡ).During the in vivo experiment,the mice were randomly divided into 4 groups:normal group,model group,heparin group and ROH group.Except the normal group,the other three groups of mice were injected intraperitoneally with LPS solution(15 mg·kg^(-1))to replicate the sepsis model.Heparin group and ROH group were injected with heparin solution(400 U·kg^(-1))and ROH solution(30 mg·kg^(-1))through caudal vein 15 min before modeling.Normal group and model group were injected with equal volume of normal saline through caudal vein.After 5 h of modeling,the bleeding time was measured by tail cutting method.The contents of D-dimer,tissue factor pathway inhibitor(TFPI),tissue factor(TF)and soluble thrombomodulin(sTM)in the plasma of mice in each group were measured by enzyme-linked immunosorbent assay(ELISA).Results In vitro,it was found that ROH did not inhibitⅡA activity dependent on ATⅢ,but could antagonizeⅡA activity by activating HCⅡ.The tail bleeding time of normal group,model group,heparin group and ROH group were(6.56±2.72),(1.68±0.62),(13.93±2.39)and(13.08±2.65)min,respectively;the contents of D-dimer in the plasma of the four groups were(1.16±0.19),(1.46±0.18),(1.26±0.13)and(1.23±0.19)ng·mL^(-1),respectively.After 11 hours after modeling,the TFPI contents of the four groups were(38.97±1.49),(39.25±3.73),(42.51±2.66)and(42.83±4.75)μg·L^(-1);the TF contents of the four groups were(153.07±15.49),(172.84±3.31),(147.26±22.45)and(161.60±16.07)ng·L^(-1),respectively;the contents of s TM in these four groups were(5.87±0.75),(6.58±0.39),(6.39±0.64)and(5.66±0.63)ng·mL^(-1),respectively.Compared with the normal group,the tail bleeding time in the model group was significantly shorter(P<0.05),and the tail bleeding time in the heparin group and ROH group was significantly longer than that in the model group(P<0.05).After modeling,TFPI,TF and s TM in each group were significantly higher than those in the normal group.After 11 hours,the content of s TM in ROH group was significantly lower than that in model group(P<0.05).Conclusion Non-anticoagulant ROH in vitro can improve the hypercoagulability of septic mice by inhibiting coagulation activation and improving physiological anticoagulation.