国产蛇毒激活血浆蛋白C的研究

目的:从9种国产蛇毒中筛选具有激活血浆蛋白C作用的蛇毒。方法:运用活化部分凝血活酶时间(APTT)和发色底物实验分别观察抗凝活性和酰胺酶活性,综合抗凝活性和酰胺酶活性确定蛋白C蛇毒激活作用。结果:在9种国产蛇毒中烙铁头蛇毒及蝮蛇毒在1.5mg/L浓度下即使人血浆纯蛋白C产生酰胺酶活性,并使APTT显著延长。结论:9种国产蛇毒中烙铁头蛇毒及蝮蛇毒具有激活人体血浆蛋白C成为活化蛋白C(APC)的作用。     

Activated protein C: Antithrombotic properties and influence on fibrinolysis in an animal model

The in vivo antithrombotic and profibrinolytic properties of human plasma protein C were investigated in a metallic coil induced thrombus model in the rat. Protein C, purified by immunoadsorption, was activated by thrombin and thereafter administered to the animals. Half-life of activated protein C (APC) revealed to be 15–16 min for the antigen, but only 7–8 min for the anticoagulant activity. Thrombus formation was reduced by APC in a dose-dependent statistically significant manner. A profibrinolytic effect of APC was observed when administered in combination with human tissue plasminogen activator. APC alone caused a reduction of thrombus weight. However, both effects were statistically not significant.     

Inherited abnormalities in the protein C activation pathway

The protein C (PC) anticoagulant pathway plays a crucial role in the regulation of fibrin formation via proteolytic degradation of the procoagulant cofactors factor Va and VIIIa by activated PC (APC). PC circulates in plasma as a zymogen, which is activated, on the surface of endothelial cells by the thrombin-thrombomodulin complex. Another endothelial cell-specific protein, the endothelial cell PC/APC receptor (EPCR), binds PC on the endothelial cell surface and further enhances the rate of PC activation. Normal APC generation depends on the precise assemblage, on the surface of endothelial cells, of at least four proteins: thrombin, thrombomodulin (TM), PC and EPCR. Therefore, any change in the efficiency of this assemblage may cause reduced APC generation and an increase in the risk of thrombosis. In the last years, several reports have suggested the association between mutations in TM and EPCR genes and venous and arterial thrombosis. Surprisingly, no studies have been reported linking mutations with levels of circulating APC, the final product of the interaction between thrombin, TM, PC and EPCR. Here, we describe the previously reported mutations in the TM and EPCR genes, and present the design and evaluation of a new strategy to investigate TM, EPCR, PC and prothrombin gene mutations in arterial and venous thrombosis.     

Sequential co-immobilization of thrombomodulin and endothelial protein C receptor on polyurethane: activation of protein C

In an effort to control the surface-mediated activation of thrombin and clot formation, proteins and molecules which mimic the anticoagulant properties of the vascular endothelial lining were immobilized on material surfaces. When immobilized on biomaterial surfaces, thrombomodulin (TM), an endothelial glycoprotein that binds thrombin and activates protein C (PC), was shown to generate activated PC (APC) and delay clot formation. However, TM-mediated activation of PC on biomaterial surfaces was shown to be limited by the transport of PC to the surface, with maximum activation obtained at a surface density of ～40 fmole TM cm(-2). This work investigates surface immobilized with TM and endothelial protein C receptor (EPCR), a natural cofactor to TM which increases the rate of activation of PC on the native endothelium. A sequential and ordered immobilization of TM and EPCR on polyurethane at an enzymatically relevant distance (<10 nm) resulted in higher amounts of APC compared with surfaces with immobilized TM or with TM and EPCR immobilized randomly and at TM surface densities (1400 fmole cm(-2)) which were previously shown to be transport limited. Ordered TM and EPCR samples also showed increased time to clot formation in experiments with platelet-poor plasma, as measured by thromboelastography. Surfaces immobilized with TM and its natural cofactor EPCR at an enzymatically relevant distance are able to overcome transport limitations, increasing anticoagulant activation and time to clot formation.     

Effect of resistin on vascular endothelium secretion dysfunction in rats.

Resistin, a novel adipokine, was recently suggested to be involved in the development of endothelial dysfunction. However, the mechanisms of how resistin works are still unknown. This study was performed to investigate the relationship between resistin and phosphatidylinositol 3-kinase (PI3K), with the aim of gaining insight to the mechanisms by which resistin induces changes of secretion function of vascular endothelium. This study was conducted on 60 male 4-week-old Sprague-Dawley rats, which were randomly divided into four groups: resistin group (RS; n = 8), normal saline group (NS; n = 8), high-fat diet group (HF; n = 36), and control group (CO; n = 8). The resistin group was administered two injections of rat recombinant resistin. The diet-induced hyperresistinemia rats were selected from the HF group after the HF group was administered a high-fat diet for 8 weeks. The diet-induced hyperresistinemia rats were randomized into the antibody group (AB; n = 8) and hyperresistinemia group (HR; n = 8). The antibody group was given injections of resistin antibody twice per day and for 3 days. Immunohistochemistry was employed to examine the expression of PI3K p85alpha subunit and endothelial nitric oxide synthase (eNOS) in thoracic artery endothelium. In the resistin group, the levels of endothelin (ET), plasminogen activator inhibitor (PAI), and von Willebrand factor (vWF) were higher and NO was lower than those in the normal saline group. The NO level increased and ET, PAI, and vWF levels decreased in the antibody group when compared with the hyperresistinemia group. After administration of resistin antibody, the expression of PI3Kp85alpha and eNOS proteins in the antibody group was significantly increased but still differed significantly from those in the control group. PI3K grey value was correlated with resistin, PAI-1, vWF, NO, and the expression of eNOS (p < .05), after controlling for the effect of insulin. Resistin can affect the protein expression of PI3Kp85alpha, stimulate release of PAI-1, vWF, and ET, and down-regulate eNOS. The effect of resistin on PI3K signaling pathway might contribute to the development of endothelial secretion dysfunction in young rats.     

Role of activated protein C in Helicobacter pylori-associated gastritis

The protein C (PC) pathway has recently been suggested to play a role in the regulation of the inflammatory response. To further extend the anti-inflammatory effect of activated PC (APC) in vivo, particularly its biological relevance to human disease, the activity of APC in the mucosa of patients with Helicobacter pylori-associated gastritis and the effect of vacuolating cytotoxin (VacA), cytotoxin-associated antigen (CagA), and H. pylori lipopolysaccharide (LPS) on PC activation were evaluated. This study comprised 35 patients with chronic gastritis. There were 20 patients with and 15 without H. pylori infection. The levels of PC and APC-PC inhibitor (PCI) complex were measured by immunoassays. The level of PC was significantly decreased and the level of APC-PCI complex was significantly increased in biopsy specimens from gastric corpus and antrum in patients with H. pylori-associated gastritis as compared to H. pylori-negative subjects. The concentrations of VacA, CagA, and LPS were significantly correlated with those of the APC-PCI complex in biopsy mucosal specimens from the gastric corpus and antrum. H. pylori LPS, VacA, and CagA induced a dose-dependent activation of PC on the surface of monocytic cells. APC inhibited the secretion of tumor necrosis factor alpha (TNF-alpha) induced by H. pylori LPS. Overall, these results suggest that H. pylori infection is associated with increased APC generation in the gastric mucosa. The inhibitory activity of APC on TNF-alpha secretion may serve to protect H. pylori-induced gastric mucosal damage.     

人血浆高密度脂蛋白对大鼠内毒素血症的治疗及防护作用研究

目的:观察人血浆高密度脂蛋白(HDL)对大鼠内毒素血症的治疗和防护效果。方法:采用鲎试剂法和放射免疫分析法于3个时点动态测定对照组、治疗组和防护组大鼠血浆内毒素(ET)水平和肿瘤坏死因子(TNF)浓度并观察血压及存活时间。结果:①输注ET后对照组大鼠血压进行性下降(P<0.01);治疗组大鼠输注HDL后其血压虽也降低但下降程度明显弱于对照组(P<0.01);防护组大鼠在输注ET后血压无明显下降(P>0.05)。治疗组及防护组大鼠存活时间均明显长于对照组(P<0.01)。②3组大鼠血浆ET水平在各时点均无明显变化(P>0.05)。③治疗组及防护组大鼠血浆TNFα水平于第3时点均明显降低(P>0.05)。结论:人血浆HDL能减轻或抑制内毒素血症大鼠血压下降并明显延长其存活时间,对内毒素血症具有良好的治疗和防护作用,此作用可能与其抑制TNF释放有关。     

一个纤维蛋白原γ链Arg275His突变导致的遗传性异常纤维蛋白原血症家系

目的　对一个遗传性异常纤维蛋白原血症家系进行表型和基因型分析。方法　采集家系3代5人外周血,吸取上层血浆用血凝仪检测活化部分凝血酶原时间、凝血酶原时间、凝血酶时间、蛋白C活性、蛋白S活性和抗凝血酶活性,纤维蛋白原活性和抗原分别用Clauss法和免疫比浊法进行检测。以常规酚-氯仿法抽提家系所有成员外周血基因组DNA,PCR扩增纤维蛋白原基因FGA、FGB和FGG所有外显子及其侧翼序列,PCR产物纯化后直接测序以检测基因突变。结果　先证者活化部分凝血酶原时间、凝血酶原时间正常,凝血酶时间超出正常上限值2倍以上,纤维蛋白原活性明显下降,抗原也低于正常范围,且活性显著低于抗原;其母表型检测结果与之相似。基因分析显示先证者呈纤维蛋白原FGG基因第8外显子g.5 6 78G>A杂合碱基置换,导致Arg2 75 His错义突变,该突变来源于母系。结论　纤维蛋白原γ链Arg2 75 His杂合错义突变是引起该家系异常纤维蛋白原血症的原因。     

The fibrinolytic system of the vascular wall

The vascular endothelium produces both PAs and a PAI. The activities of these components in the circulation must be regulated precisely to ensure that normal vascular homeostasis is not compromised. The blood contains a number of molecules that may function in this way by either promoting or inhibiting the synthesis, release and/or activity of the PAs and PAI. It is clear that the regulation of this system is considerably more complex than previously thought. For example, the initiation of fibrin dissolution is influenced by a number of additional factors including fibrin itself, pro-activators, PAI, platelet components (including the PAI), and possibly by APC generated at the endothelial cell surface. Despite the many recent advances discussed above, little is known about the temporal control of the events leading to plasminogen activation during thrombus formation and dissolution. Obviously, such information must be obtained before more effective treatments of abnormal vascular fibrinolytic activity can be developed. In this chapter, we have described a number of reagents and assays that should aid in the quantification of the PAs and the PAI in plasma. Eventual utilization of these assays in a clinical setting may be valuable for the diagnosis and subsequent treatment of abnormalities of the vascular fibrinolytic system.     

大鼠实验性心肌梗塞时血浆蛋白C,纤溶活性变化及人参皂甙作用的研究

通过观察实验性大鼠急性心肌梗塞（ＡＭＩ）时血浆蛋白Ｃ（ＰＣ）活性和组织型纤溶酶原激活剂（ｔ－ＰＡ）及纤溶酶原激活剂的特异性抑制因子（ＰＡＩ）活性的变化，初步探讨了心肌缺血时ＰＣ活性变化与纤溶系统的关系以及人参皂甙对其变化的影响。结果表明，１．ＡＭＩ时血浆ＰＣ活性显著低于正常对照组，且与缺血程度呈负相关。２．ＡＭＩ时ｔ－ＰＡ活性降低，ＰＡＩ活性增高。３．人参皂甙可提高ＡＭＩ时血浆ＰＣ活性，降低ＰＡＩ     

Endotoxin-induced liver injury in aged and subacutely hypervitaminotic A rats

The plasma disappearance of endotoxin and endotoxin-induced hepatic injury were studied in two rat models: the aging rat and the subacutely hypervitaminotic A rat. The choice of these models was based on their respective association with a decreased or increased Kupffer cell endocytic activity. The half-life of endotoxin (E. coli O26: B6, phenol extracted) in plasma was significantly prolonged in aged rats as measured by both the Limulus assay (t1/2 = 2.1 +/- 0.1 h in 3-6-month-old, and 3.3 +/- 0.3 h in 24-36-month-old rats) and 51Cr-labeled endotoxin radioactivity assay (t1/2 = 5.3 +/- 0.3 h in 3-6-month old and 7.7 +/- 0.6 h in 24 36-month-old rats). In subacute hypervitaminosis A, the half-life of endotoxin was significantly decreased in the Limulus assay (t1/2 = 2.1 +/- 0.1 h in 3-6-month old and 1.4 +/- 0.2 h in subacutely hypervitaminotic A rats), but not in the radioactivity assay (t1/2 = 5.3 +/- 0.3 h in 3-6-month-old and 5.0 +/- 0.4 h in subacutely hypervitaminotic A rats). Hundred percent mortality was observed at a dose of 2 mg endotoxin/100 g body wt. in old rats, but not in young rats. Only 1 of 7 young subacutely hypervitaminotic A rats died following injection of this dose of endotoxin. The dose of endotoxin which caused only minimal parenchymal liver cell injury in young rats induced substantial parenchymal cell injury in old rats and subacutely hypervitaminotic A rats as determined by both histological and biochemical parameters. It is concluded that some basic characteristics of experimental animals, such as age and nutritional status, can dramatically influence the sensitivity to endotoxin and this is not necessarily correlated with the rate of endotoxin clearance.     

Some species differences in the false prolongation of prothrombin times and activated partial thromboplastin times in toxicology

Changes in plasma activated partial thromboplastin times (APTT) and prothrombin times (PT) in mice, rats, rabbits, dogs, monkeys and human were examined for up to 96 h at storage temperatures of 4 and 25°C. Prolongation of APTT in rats was rapid and marked, with times doubling within 24 h post-sampling. Plasma APTT of human and monkey were also affected, but to a lesser extent. No effect was observed in mice, rabbits and dogs. On the other hand, the magnitude of PT changes was much smaller than that observed with APTT in all species. No significant differences were noted between the results from samples stored at 4°C or 25°C for either test.The false prolongation of APTT is clearly undesirable in a toxicity study, especially in rats. It is important therefore to minimise these changes by performing this test under strict time-controlled conditions.     

Interaction of blood components with heparin-immobilized polyurethanes prepared by plasma glow discharge

The blood compatibility of poly(ethylene oxide) (PEO)-grafted and heparin (Hep) immobilized polyurethanes was investigated using in vitro plasma recalcification time (PRT), activated partial thromboplastin time (APTT), platelet adhesion and activation, and peripheral blood mononuclear cell (PBMC) adhesion and activation. In the experiment with plasma proteins, the PRT of the polyurethane (PU) surface was prolonged by PEO grafting and further prolonged by heparin immobilization. The APTT was prolonged on PU-Hep, suggesting the binding of immobilized heparin to antithrombin III. The percentage of platelet adhesion on PU was not much different from that on acrylic acid- and PEO-grafted PUs (PU-C, PU-6, PU-33), yet was substantially decreased by heparin immobilization (PU-6-Hep, PU-33-Hep). The release of serotonin from adhering platelets was slightly suppressed on PEO-grafted PUs yet significantly suppressed on heparin-immobilized PUs. In the PBMC experiments, the adhesion and activation of the cells were significantly suppressed on heparin-immobilized PUs, and the amount of interleukin-6 (IL-6) released from PBMCs stimulated with surface-modified PUs decreased with a decrease in PBMC adhesion.     

Induction of t-PA Synthesis with intravenous bolus injection of vitamin A palmitate in vitamin a deficient rats

Retinoic acid and vitamin A palmitate induce tissue-type plasminogen activator (t-PA) synthesis in cultured human umbilical vein endothelial cells (HUVEC) in vitro without alteration of plasminogen activator inhibitor-] (PAI-1) synthesis. Therefore the effect of intravenous bolus injection of water-miscible vitamin A palmitate on the blood fibrinolytic system was investigated in normal and vitamin A deficient rats. In 5 normal rats, injection of 200000 IU/kg of vitamin A palmitate did not induce a significant increase in euglobulin fibrinolytic activity, t-PA antigen and PAI activity in plasma nor of t-PA and PAI-1 mRNA in the heart within 240 min after injection. In groups of 3–6 vitamin A deficient rats, injection of 200000IU/kg of vitamin A palmitate induced a significant increase in t-PA antigen in plasma (1.9-fold after 60 min and 2.9-fold after 120 min), but no significant increase in plasma euglobulin fibrinolytic activity. A 2-fold increase in PAI activity was observed 90 min after injection of both vitamin A palmitate as well as of its solvent, suggesting that this induction was non-specific. The increase in t-PA antigen coincided with a significant increase in t-PA mRNA in heart tissue: 2.3-fold after 60 min and 4.3-fold after 120 min. PAI-1 mRNA in heart tissue increased 3.6-fold 90 min after injection with vitamin A palmitate but, surprisingly, not after injection of solvent.It is concluded that intravenous bolus injection of vitamin A palmitate induces a specific increase of t-PA mRNA in the heart and of t-PA antigen secretion in plasma of vitamin A deficient rats.     

Quantitative analysis of thrombomodulin-mediated conversion of protein C to APC: Translation from in vitro to in vivo

Thrombomodulin-bound thrombin cleaves protein C (PC) zymogen in blood plasma producing activated protein C (APC), which exerts anti-coagulant, anti-inflammatory, anti-apoptotic and CNS-protective effects. Recombinant APC and thrombomodulin (TM) are both in clinical studies for management of acute conditions including sepsis. Methods that permit accurate measurement of APC in plasma are needed for clinical monitoring and mechanistic studies in animal models. However, the two existing methods require either long incubation periods with substrate, resulting in high background or they also recognize protein C inhibitor (PCI) complexed with APC (APC:PCI), which convolutes analysis of the amount of APC generated. Here we describe a robust quantitative in vivo assay that measures APC generation at both low levels of human protein C seen in chronic inflammatory disease and at physiological levels that shows a >99% fit with in vitro data.     

内皮素在NG-硝基-L-精氨酸甲酯引起的高血压的作用

在口服Ｎ＾Ｇ－硝基－Ｌ－精氨酸甲酯（Ｎ＾Ｇ－ｎｉｔｒｏ－Ｌ－ａｒｇｉｎｉｎｅ－ｍｅｔｈｌｅｓｔｅｒ,Ｌ－ＮＡＭＥ）造成的大鼠高血压模型上,观察血浆内皮素和亚硝酸盐含量,血管内皮素水平和ＮＯ合酶活性和ＥＴ受体的变化,以及应用ＥＴ受体阻断剂ＪＫＣ－３０１对高血压的影响。     

Interaction of blood components with heparin-immobilized polyurethanes prepared by plasma glow discharge

The blood compatibility of poly(ethylene oxide) (PEO)-grafted and heparin (Hep) immobilized polyurethanes was investigated using in vitro plasma recalcification time (PRT), activated partial thromboplastin time (APTT), platelet adhesion and activation, and peripheral blood mononuclear cell (PBMC) adhesion and activation. In the experiment with plasma proteins, the PRT of the polyurethane (PU) surface was prolonged by PEO grafting and further prolonged by heparin immobilization. The APTT was prolonged on PU-Hep, suggesting the binding of immobilized heparin to antithrombin III. The percentage of platelet adhesion on PU was not much different from that on acrylic acid- and PEO-grafted PUs (PU-C, PU-6, PU-33), yet was substantially decreased by heparin immobilization (PU-6-Hep, PU-33-Hep). The release of serotonin from adhering platelets was slightly suppressed on PEO-grafted PUs yet significantly suppressed on heparin-immobilized PUs. In the PBMC experiments, the adhesion and activation of the cells were significantly suppressed on heparin-immobilized PUs, and the amount of interleukin-6 (IL-6) released from PBMCs stimulated with surface-modified PUs decreased with a decrease in PBMC adhesion.     

Neutralization of plasminogen activator inhibitor-1 (PAI-1) by activated protein C is species-dependent

The interaction of bovine and human activated protein C (APC) with type-1 plasminogen activator inhibitor (PAI-1) was studied in a cell-free system. Human plasma and a preparation of PAI-1 obtained from human endothelial cell cultures were used as sources of PAI-1. Bovine APC was able to neutralize PAI-1 inhibitory activity present in both sources in a dose-dependent manner; the concentration of bovine APC required to produce 50% (C₅₀) neutralization of endothelial PAI-1 (0.5nM) was 4 μg/ml (64nM). Moreover, when complexes between tissue plasminogen activator (t-PA, 60ng/ml, 0.84nM) and PAI-1(18.8ng/ml, 0.35nM) were incubated with bovine APC, the amount of such complexes decreased as a function of the concentration of added APC (C₅₀ = 2 μ/ml, 32 nM), and a concomitant increase in the amount of residual t-PA activity was observed. This effect was due to the formation of APC⊎PAI-1 complexes as detected by immunoblotting with monoclonal antibodies directed against PAI-1. By this mechanism bovine APC prevented the initial reaction between PAI-1 and t-PA and interfered with the stability of complexes between t-PA and PAI-1. The latter observation suggests that complexes between t-PA and PAI-1 may dissociate in the presence of bovine APC. In contrast with these findings, when the experiments were performed in an entirely human homologous cell-free system, human APC did not form a complex with PAI-1 and no effect either on PAI-1 or on the stability of preformed t-PA ⊎ PAI-1 complexes was observed. The results indicate that the neutralization of PAI-1 by APC is a phenomenon induced by interspecies molecular interactions.     

卡托普利对自发性高血压大鼠微血管稀少和内皮功能障碍的影响

为探讨自发性高血压大鼠（SHR）的微血管改变和血管内皮细胞（VEC）功能障碍的关系及卡托普利（CPT）的干预效果，对9只SHR给予CPT（CPT组），并以10只WKY大鼠作为正常对照组（WKY组），以10只SHR作为实验纠（SHR组），观察检测人鼠的血压（BP）、回肠壁各级微动脉分支总数、血浆一氧化氮（NO）和血浆内皮素（ET）。结果显示，SHR组的微动脉分支总数、NO及K值[log(NO／ET)]均较WKY组下降（P均<0.01），而ET则增高（P＜0．01）；CPT组给药后第7口BP较给药前下降（P<0．01），其微动脉分支总数、NO及K值均较SHR组增加（P均<0．01）；SHR的BP与K值呈负相关（r=－0．5863），BP与各级微动脉分支总数呈负相关(r＝－0．7866～－0．6380），而K值与各级微动脉分支总数呈正相关（r＝0.5951～0.7529）。认为SHR的微血管改变与其内皮功能状态关系密切，CPT时改善高血压的VEC功能和微血管稀少现象，从而发挥降压作用。