蛋白C、蛋白S、活化蛋白C抵抗与缺血性脑血管病的相关性研究

目的研究蛋白C(PC)、蛋白S(PS)、活化蛋白C抵抗(APCR)与急性缺血性脑血管病间的关系.方法对804例急性脑血管患者及168例非脑血管病患者行PC、PS、APCR检测.结果在缺血性卒中组PC、PS活性分别为(115.8±12.5)%和(112.6±13.8)%,明显低于出血性卒中组的(119.2±11.3)%和(112.1±16.5)%及对照组的(120.2±12.8)%和(122.4±15.8)%(P＜0.01).进一步比较发现,＜45岁的缺血性卒中组的PC、PS分别为(54.67±8.9)%和(40.49±9.1)%,明显低于其他各组,PS在45岁以上的缺血性卒中组为(119.2±15.6)%,明显低于对照组的(122.4±15.8)%,(P＜0.01),其余各组间无显著差异.APCR总发生率6.6%(64/972),其中缺血性卒中组为7.14%(46/644),出血性卒中组为6.25%(10/160),两组比较无明显差异(P＞0.05),45岁以下缺血性卒中组APCR的发生率为46.3%(25/54),明显高于其他各组.结论PC、PS活性降低,APCR是缺血性脑血管病发生的重要因素,特别与45岁以下的缺血性卒中的发生密切相关.     

血浆蛋白C、蛋白S与脑梗死的相关性研究

目的研究血浆蛋白C、蛋白S与脑梗死发病的关系。方法对64例脑梗死患者及15例正常对照组进行血浆蛋白C(PC)、蛋白S(PS)检测,并分析其与脑梗死严重程度、年龄及其它脑梗死危险因素之间的关系。结果实验组血浆PC浓度(4.97±1.82μg/ml)与对照组血浆PC浓度(4.74±1.95μg/ml)之间差别无统计学意义;首发组血浆PC浓度(5.28±1.85μg/ml)与复发组血浆PC浓度(4.03±1.40μg/ml)之间差别有统计学意义(P=0.016);实验组血浆PS浓度(7.47±3.87μg/ml)与对照组血浆PS浓度(12.06±3.99μg/ml)之间差别有统计学意义,中老年组血浆PS浓度(6.58±3.33μg/ml)与青年组血浆PS浓度(9.28±4.33μg/ml)之间差别有统计学意义。结论PC降低是复发性脑梗死的危险因素之一;游离PS降低是脑梗死的危险因素;PS水平的降低导致PC功能的降低,进而凝血功能障碍,导致脑梗死的发生。     

抗磷脂抗体、活化蛋白C抵抗与脑梗死的关系研究

目的探讨抗磷脂抗体（APA）、活化蛋白C抵抗（APCR）与脑梗死之间的关系及其临床意义。方法对157例脑梗死（CI）患者和82例正常对照组（NC）分别采用ELISA法检测ACA-IgG、IgM、IgA,血浆部分凝血活酶时间-狼疮抗凝物法（PTT-LA）筛选狼疮抗凝物（LA）,活化的部分凝血活酶时间±活化蛋白C（APTT±APC）检测APCR。结果CI组血清中ACAIgG、IgM、IgA以及LA的阳性率均明显高于NC组（P＜0．05）；CI组总APA阳性率（25．5％）显著高于NC组（4．9％）（P＜0．005）；CI组中APCR阳性率[5．1％（8/157）]与NC组[1．2％（1/82）]没有显著性差异（P＞0．05）；APA阳性脑梗死患者中APCR阳性率[7．5％（3/40）]与APA阴性脑梗死中APCR阳性率[4．3％（5/117）]也没有显著性差异（P＞0．05）。结论抗磷脂抗体与脑梗死密切相关,促使脑梗死患者血液呈高凝状态,是脑梗死发生的危险因素之一；APCR的发生在脑梗死患者中未见增加,APA不是导致获得性APCR的主要原因。     

急性脑梗死患者蛋白S基因多态性和血浆蛋白S水平与其家族史关系的研究

目的探讨急性脑梗死(ACI)患者蛋白S基因(PROS1)多态性和血浆总蛋白S(tPS)和游离蛋白S(fPS)水平与其家族史的关系。方法对93例ACI患者,其中散发(NFCI)组83例,有家族史(FCI)组10例,采用聚合酶链式反应-限制性片段长度多态性分析法(PCR-RLFP)检测PROS1A2148G多态性;采用酶联免疫吸附双抗体夹心法(ELISA)测定血浆tPS和fPS水平;并与正常对照组进行比较。结果ACI组PROS1A2148G的AA、AG和GG型频率分别为18.3%、48.4%和33.3%,与正常对照组(22.0%、48.0%和30.0%)差异无统计学意义(均P>0.05)。FCI组A2148GGG基因型频率(50%)高于正常对照组(30%),但差异无统计学意义(P>0.05)。ACI组GG基因型患者血浆tPS水平和fPS水平[(13.67±1.49)、(6.07±0.61)μg/ml]显著低于AA型和AG型[(15.73±1.24)、(6.72±0.55)和(15.15±1.48)、(6.63±0.66)μg/ml](均P<0.01),且FCI组血浆fPS水平[(6.12±0.69)μg/ml]显著低于NFCI组[(6.48±0.75)μg/ml](P<0.05)。结论PROS1A2148GGG基因型导致FCI患者血浆fPS水平降低,其基因型可能与ACI家族史有关。     

糖化血红蛋白及高敏C反应蛋白与脑梗死急性期患者颈动脉粥样硬化的关系

目的 探讨脑梗死急性期患者糖化血红蛋白(HbA1c)、高敏C反应蛋白(hs-CRP)与颈动脉粥样硬化斑块的关系. 方法 连续收集广州医学院第二附属医院神经内科自2010年1月1日至12月31日收治的110例脑梗死急性期患者(急性脑梗死组)和同期98例健康体检者(正常对照组),通过颈动脉彩超测量颈总动脉内中膜厚度(IMT)并根据其值将脑梗死组分为3个亚组(正常组、轻度粥样硬化组、重度粥样硬化组),同时观察有无颈动脉斑块形成;全自动生化仪器检测HbA1c、hs-CRP水平. 结果 与正常对照组相比,急性脑梗死组HbA1c、hs-CRP水平[(4.8±0.3)％vs(5.9±0.6)％;(0.78±0.11) mg/Lvs(3.25±0.72) mg/L]明显升高,差异有统计学意义(P＜0.05).轻度粥样硬化组HbA1c水平[(5.1±0.5)％]明显高于正常组[(4.3±0.3)％],重度粥样硬化组HbA1c、hs-CRP水平[(7.1±0.9)％;(1.37±1.5)mg/L]高于正常组、轻度粥样硬化组[(1.11±1.1) mg/L;(1.02±0.3) mg/L].脑梗死急性期患者颈动脉IMT值与HbA1c、hs-CRP水平及二者乘积均呈明显正相关关系(r=0.437,P=0.000;r=0.215,P=0.000; r=0.729,P=0.000). 结论 糖耐量异常和炎症的联合作用与脑梗死急性期患者颈动脉粥样硬化有一定的相关性.     

血清C反应蛋白水平与抑郁症患者病情严重程度的关系

目的:探讨血清C反应蛋白(CRP)水平与抑郁症患者病情的关系。方法:检测159例抑郁症患者(患者组)及159名健康对照者(对照组)血清CRP水平,采用汉密尔顿抑郁评定量表(HAMD)评定患者病情。采用多元线性回归分析血清CRP水平与HAMD评分的关系。结果:患者组血清CRP水平[(3.4±3.2) mg/L]明显高于对照组[(1.2±0.3) mg/L](P0.05);多元线性回归分析显示,随着血清CRP水平升高,患者HAMD评分明显增加(P 0.05);这种线形相关在女性患者中更加显著(P0.05);高血清CRP水平女性患者其HAMD的焦虑/躯体化、认知障碍、阻滞和绝望感评分更高(P均0.05)。结论:血清CRP水平可以反映抑郁症患者病情,并且在女性患者中更为显著。     

缺血缺氧性脑病新生儿的神经行为评分与血浆神经肽Y水平的相关性

目的探讨缺血缺氧性脑病(HIE)新生儿的神经行为评分与血浆神经肽Y水平的相关性。方法选取62例HIE新生儿作为观察组,另选取60例相同出生日龄的健康新生儿作为对照组。采用新生儿神经行为量表(NBNA)测定神经行为评分,利用放射免疫分析法检测血浆神经肽Y水平。结果观察组NBNA评分[(34.01±0.64)分]显著低于对照组[(38.92±0.71)分;P0.05],血浆神经肽Y水平[(192.74±13.62)ng/L]显著高于对照组[(65.75±10.01)ng/L;P0.05)。治疗后,HIE新生儿NBNA评分[(37.43±0.82)分]显著高于治疗前[(34.01±0.64)分;P0.05],血浆神经肽Y水平[(74.45±9.82)ng/L]显著低于治疗前[(192.74±13.62)ng/L;P0.05)。HIE新生儿NBNA评分与血浆神经肽Y表达水平呈明显负相关(r=-0.527,P0.05)。结论与正常新生儿相比,HIE新生儿的神经行为评分与血浆神经肽Y水平存在明显异常,且二者具有明显负相关。     

脑梗死患者血浆蛋白C、蛋白S动态观察

目的　动态观察脑梗死患者血浆蛋白C抗原 (PC :Ag)和蛋白S(PS)水平。方法　测定 94例脑梗死患者血浆PC :Ag、总蛋白S(TPS)和游离蛋白S(FPS)并与 5 2名健康者相对照 ,动态观察 41例脑梗死患者PC :Ag和PS浓度。结果　脑梗死组PC :Ag、TPS、FPS降低发生率明显高于对照组 (P <0 0 5 )。PC :Ag浓度动态观察提示脑梗死急性期PC :Ag浓度呈非线性改变 ,在发病第二周出现一“反跳”性增高。结论　部分脑梗死患者存在PC和PS缺乏现象 ,多数为继发性缺陷 ,发现这些患者并区别其为原发性或继发性对明确脑梗死的病因、治疗和预防有重要意义。     

急性脑梗死患者血清S100β蛋白和C反应蛋白含量变化的研究

目的 探讨急性脑梗死(ACI)患者血清S100β蛋白和C反应蛋白(CRP)含量变化的意义及其机制.方法 测定54例ACI患者、21名正常对照者血清S100β蛋白和CRP的含量,观察比较ACI患者在不同病情、不同梗死面积时的血清S100β蛋白和CRP含量变化,采用Speannan法对二者进行相关性分析.结果 ACI患者血清S100β蛋白[(0.442±0.207) μg/L]和CRP[(16.35±7.51) mg/L]浓度明显高于正常对照组[(0.058±0.022) μg/L,(5.06±2.03) mg/L](均P＜0.05).并且随着神经功能缺损程度评分的增高,血清S100β和CRP含量亦明显升高;S100β蛋白水平和CRP水平呈正相关(r=0.887,P＜0.01).结论 S100β蛋白与CRP一样可作为预测ACI患者病情严重程度及预后的生化指标,二者相结合进行评价意义更大.     

下调硫氧还蛋白相互作用蛋白表达对急性脑梗死大鼠脑保护作用

目的探讨下调硫氧还蛋白相互作用蛋白(TXNIP)表达对急性脑梗死大鼠脑保护作用。方法 48只SD大鼠随机分为假手术组、模型组、阴性对照组和TXNIP干扰组。构建大鼠缺血再灌注损伤模型。建模3 d时,行神经功能评分,分别检测大鼠脑梗死面积和神经细胞凋亡水平,以及脑组织中TXNIP基因和TXNIP、ASK1、p-ASK1和caspase-1蛋白表达。结果 TXNIP干扰组大鼠在建模3 d时,神经功能评分[(1. 65±0. 10)分]低于模型组[(2. 22±0. 55)分]和阴性对照组[(2. 49±0. 97)分](F=42. 046,P=0. 000)。TXNIP干扰组大鼠脑梗死面积[(16. 40±1. 32)%]和神经细胞凋亡率[(22. 61±2. 33)%]低于模型组[(24. 74±2. 38)%和(71. 53±6. 25)%]和阴性对照组[(23. 48±2. 72)%和(68. 23±3. 05)%](F=192. 936,F=473. 627; P 0. 05]。TXNIP干扰组大鼠脑组织中TXNIP mRNA和蛋白、p-ASK1和caspase-1蛋白相对表达量低于模型组和阴性对照组,而ASK1蛋白相对表达量高于模型组和阴性对照组(P 0. 05)。结论下调TXNIP基因表达可减少脑梗死面积和神经细胞凋亡,其机制可能与抑制促进凋亡相关蛋白ASK1磷酸化介导的细胞凋亡有关。     

Activated protein C stimulates osteoblast proliferation via endothelial protein C receptor

Introduction

Bone is continually remodeled by the action of osteoblasts, osteocytes, and osteoclasts. Resting osteoblasts are able to proliferate and differentiate into mature osteoblasts when physiologically required, as after tissue injury. Activated protein C (APC) is a serine protease that functions in anticoagulation, anti-inflammation, anti-apoptosis, cell proliferation, and wound repair. In this study, we examined the effect of APC on osteoblast proliferation and differentiation.

Materials and Methods

We examined the presence of protein C in human fracture hematoma by immunohistochemical staining. We then evaluated the effect of APC, diisopropyl fluorophosphate-inactivated APC (DIP-APC) or protein C zymogen on normal human osteoblast (NHOst) proliferation using tetrazolium salt assay in the presence or absence of aprotinin, hirudin, protein C, antibody against protein C, endothelial protein C receptor (EPCR) or protease-activated receptor (PAR)-1. Finally, activation of p44/42 MAP kinase was evaluated by Western blot analysis.

Results

Both APC and DIP-APC increased osteoblast proliferation in a dose-dependent manner, while protein C did not. The APC-induced increased proliferation of osteoblast was not affected by aprotinin, hirudin, and anti-protein C antibody which inhibits the protease activity of APC. Treatment with protein C or anti-EPCR antibody which inhibits APC binding to EPCR inhibited APC-mediated osteoblast proliferation, while treatment with anti-PAR-1 antibody did not. APC promoted the phosphorylation of p44/42 MAP kinase within osteoblasts; this effect was inhibited by the anti-EPCR antibody.

Conclusions

APC stimulates osteoblast proliferation by activating p44/42 MAP kinase through a mechanism that requires EPCR but not PAR-1 or the proteolytic activity of APC. APC generated at fracture sites may contribute to fracture healing by promoting osteoblast proliferation.     

Recombinant human protein C: comparative functional studies with human plasma protein C

Protein C (PC) is the central protein in a major antithrombotic regulatory mechanism. Hereditary deficiencies of PC are associated with thrombosis. Therapeutic PC replacement may be an important treatment if pure functional human protein C is available in sufficient quantity. Human PC has been produced on a commercial scale using recombinant techniques. To study the functional properties of recombinant protein C (r-PC), we undertook a comparative investigation of the basic properties of r-PC and plasma protein C (n-PC). Both were isolated by immunopurification methods. Protac C activation proceeded at the same rate and kinetics for both forms. With thrombin-thrombomodulin (T-TM) activation, r-PC is significantly better than the activation of n-PC (for r-PC: Kcat/Km = 378 vs. n-PC: Kcat/Km = 35). No difference in the anticoagulant (aPTT prolongation) or profibrinolytic activities (inactivation of PAI-1 and PAI-3) were observed between activated r-PC and n-PC. Based on these functional studies, recombinant protein C has similar properties to the plasma form of protein C. However, T-TM activation of r-PC occurs faster than the n-PC. The mechanism is unknown, but may be due to the presence of larger amounts of single chain protein C which exists in a conformation more rapidly activated by the T-TM complex.     

Fast functional protein C assay using Protac, a novel protein C activator

A simple and rapid clotting method for the quantitative determination of protein C (PC) in plasma consists of the conversion of PC into activated PC (APC) by means of Protac, an activator protein isolated from Agkistrodon contortrix contortrix venom, of the subsequent degradation of factors V and VIII in PC immuno-depleted plasma by the generated APC and of the measurement of the prolongation of the activated partial thromboplastin time (APTT) which is proportional to the amount of PC in the sample. In 33 normal individuals a mean PC level of 97.1% of a normal pooled plasma was found. Comparison with an enzyme-immunoassay for PC in 33 patients with liver disease revealed a good correlation (r = 0.986). Patients under warfarin therapy (n = 34) had a mean PC level of 19.8%; a comparison with the immunological assay (mean value = 55.3%) in the same population suggested that the assay did not co-estimate acarboxy forms of PC. The assay proved to be insensitive to heparin concentration lower than 1 U/ml. Due to its simplicity, it should be suitable for diagnostic routine and monitoring of patients with abnormal PC level, even if under anticoagulation.     

Functional assays for protein C activity and protein C inhibitor activity in plasma

An assay system for protein C (PC) activity and PC-inhibitor in plasma was developed. The assay was based on: (1) binding of PC to wells of a microtiter plate coated with a murine monoclonal anti-PC antibody (C3) that did not interfere with the activity or activation of PC; (2) activation of immobilized PC with Protac C; (3) incubation with or without a source of activated PC inhibitor; and (4) measurement of amidolytic activity using the substrate S-2366. The activity assay was specific for PC and sensitive to less than 1 microliter of plasma or 4 ng PC. Inhibition of activated PC by plasma followed pseudo first order kinetics. Heparin caused a dose dependent increase in the inhibition rate with half maximal stimulation at approximately 3 U/ml and maximal stimulation at heparin concentrations greater than or equal to 10 U/ml. This assay is suitable not only for determination of functional plasma levels of PC and PC inhibitor activities but also for kinetic studies of inhibition of activated PC in complex systems, such as plasma. Studies showed that urokinase interfered with the inhibition of APC by plasma inhibitor(s).     

Determination of plasma protein S--the protein cofactor of activated protein C

Protein S, an important cofactor of activated protein C, and C4b-binding protein were purified from human plasma. Specific antibodies against the purified proteins were raised in rabbits and used for the development of immunologic assays for these proteins in plasma: an immunoradiometric assay for protein S (which measures both free protein S and protein S complexed with C4b-binding protein) and an electroimmunoassay for C4b-binding protein. Ranges for the concentrations of these proteins were established in healthy volunteers and patients using oral anticoagulant therapy. A slight decrease in protein S antigen was observed in patients with liver disease (0.78 +/- 0.25 U/ml); no significant decrease in protein S was observed in patients with DIC (0.95 +/- 0.25 U/ml). Criteria were developed for the laboratory diagnosis of an isolated protein S deficiency.     

The functional significance of the autolysis loop in protein C and activated protein C

The autolysis loop of activated protein C (APC) is five residues longer than the autolysis loop of other vitamin K-dependent coagulation proteases. To investigate the role of this loop in the zymogenic and anticoagulant properties of the molecule, a protein C mutant was constructed in which the autolysis loop of the protein was replaced with the corresponding loop of factor X. The protein C mutant was activated by thrombin with approximately 5-fold higher rate in the presence of Ca2+. Both kinetics and direct binding studies revealed that the Ca2+ affinity of the mutant has been impaired approximately 3-fold. The result of a factor Va degradation assay revealed that the anticoagulant function of the mutant has been improved 4-5-fold in the absence but not in the presence of protein S. The improvement was due to a better recognition of both the P1-Arg506 and P1-Arg306 cleavage sites by the mutant protease. However, the plasma half-life of the mutant was markedly shortened due to faster inactivation by plasma serpins. These results suggest that the autolysis loop of protein C is critical for the Ca(2+)-dependence of activation by thrombin. Moreover, a longer autolysis loop in APC is not optimal for interaction with factor Va in the absence of protein S, but it contributes to the lack of serpin reactivity and longer half-life of the protease in plasma.