Protein C activation in endothelial cells is inhibited under the presence of anticardiolipin antibody in ischemic stroke patients

抗心磷脂抗体（anti - cardiolipin antibody, aCL）被认为是脑梗死发生的重要因素之一。凝血系统中存在许多与aCL相互作用可导致血栓形成的部位。然而，aCL的确切作用机制尚不清楚。人活化蛋白C通过抑制凝血辅助因子Va和viia的活性，在人血浆中发挥有效的抗凝作用。本研究旨在探讨缺血性脑卒中患者血清IgG对培养的人脐带静脉内皮细胞蛋白C活化率的影响。对象和方法：在以下组中检测蛋白C的活化率。我们有10例缺血性脑卒中患者IgG aCL阳性。其中包括7例脑梗死相关SLE患者（第一组）和3例无免疫学背景的SLE患者（第二组）。我们还检查了6例非SLE相关的无aCL的缺血性卒中患者（第3组），14例有aCL但未发生缺血性卒中的SLE患者（第4组），6例无卒中和aCL的SLE患者（第5组）和6例正常对照（第6组）。融合内皮细胞与缓冲液或患者IgG （7 mg/ml）在37℃下孵育1小时。加入纯化的人蛋白C （1 nM/孔）和凝血酶（0.04 nM/孔）。37℃孵育2小时后，以P-Glu-Pro-Arg-MNA为底物，采用酶解法测定蛋白C的活化率。蛋白C的活化程度表示为IgG部分存在和不存在时所测吸光度变化的百分比。负百分比变化表明蛋白C活化受到抑制。结果：在aCL阳性的SLE相关缺血性卒中（1组）中，7例患者中有5例显示为阴性百分比变化，这表明添加IgG部分抑制了蛋白C的激活率。这7例患者吸光度的平均变化百分比为-7.2%（范围从-21.4%到+4.5%）。3例aCL阳性的非sle卒中患者（2组）吸光度变化百分比分别为-2.0%、-9.8%和-6.6%。另一方面，IgG分数对3组（吸光度的平均变化为+0.1%）、4组（-0.5%）和5组（+0.3%）患者的蛋白C活化没有明显影响。在正常对照中，测得的吸光度的平均百分比变化为+0.6%。结论：我们的研究结果表明，IgG片段中的aCL可以干扰内皮细胞表面凝血酶、蛋白C和凝血调节蛋白之间的相互作用。蛋白C活化率的降低可能与缺血性脑卒中的发生有关。少

INTRODUCTION : Anticardiolipin antibody (aCL) is considered to be one of the contributory factors in the development of cerebral infarction. A number of sites exist in the coagulation system where interaction with aCL may lead to thrombosis. However, the precise mechanism of action of aCL remains unknown. Human activated protein C functions as a potent anticoagulant in human plasma by inhibiting the activity of coagulation cofactors Va and VIIIa. The purpose of the present study was to investigate the effect of serum IgG fraction obtained from ischemic stroke patients on the rate of protein C activation in cultured human endothelial cells from the umbilical cord veins.SUBJECTS AND METHODS : The rate of protein C activation was examined in the following groups. We had 10 ischemic stroke patients with positive IgG aCL.These comprised 7 patients with cerebral infarction related SLE (Group 1) and 3 patients without an immunological background including SLE (Group 2). We also examined in 6 … More non-SLE related ischemic stroke patients without aCL (Group 3), 14 SLE patients with aCL who did not develop ischemic stroke (Group 4), 6 SLE patients without stroke and aCL (Group 5) and 6 normal controls (Group 6). Confluent endothelial cells were incubated with buffer or IgG obtained from the patients (7 mg/ml) for 1 hour at 37ﾟC.Purified human protein C (1 nM/well) and thrombin (0.04 nM/well) were added. After 2 hours incubation at 37ﾟC,the rate of protein C activation was determined by amidolytic assay employing P-Glu-Pro-Arg-MNA as a substrate. The degree of activation of protein C was expressed as the percent change in the measured absorbance between the presence and absence of the IgG fraction. A negative percent change indicated inhibition of protein C activation.RESULTS : In SLE related ischemic stroke with positive aCL (Group 1), 5 of the 7 patients revealed a negative percent change which indicated inhibition of the rate of protein C activation by addition of the IgG fraction. The mean percent change of absorbance in these 7 patients was -7.2% (range, from -21.4% to+4.5%). In the 3 non-SLE stroke patients with positive aCL (Group 2), the percent changes in absorbance were -2.0%, -9.8% and -6.6%, respectively. On the other hand, IgG fraction did not appreciably influence the activation of protein C in the patients of Group 3 (the mean parcent change of absorbance, +0.1%), Group 4 (-0.5%) and Group 5 (+0.3%). In the normal controls, the mean percent change in the measured absorbance was +0.6%.CONCLUSION : Our results appear to indicate that aCL in IgG fractions can interfere with interactions among thrombin, protein C and thrombomodulin on the surface of endothelial cells. Impairment of the rate of activation of protein C could play a role in the development of ischemic stroke. Less