Blood Coagulation

The process of tissue factor initiated blood coagulation is discussed. Reactions of the blood coagulation cascade are propagated by complex enzymes containing a vitamin K-dependent serine protease and an accessory cofactor protein that are assembled on a membrane surface in a calcium-dependent manner.These complexes are 10⁵ 10⁹-fold more efficient in proteolyses of their natural substrates than enzymes alone. Based upon data acquired using several in vitro models of blood coagulation, tissue factor initiated thrombin generation can be divided into two phases: an initiation phase and a propagation phase. The initiation phase is characterized by the generation of nanomolar amounts of thrombin, femto- to picomolar amounts of factors VIIa, IXa, Xa, and XIa, partial activation of platelets, and almost quantitative activation of procofactors, factors V and VIII. The duration of this phase is primarily influenced by concentrations of tissue factor and TFPI. The characteristic features of the propagation phase are: almost quantitative prothrombin activation at a high rate, completion of platelet activation, and solid clot formation. This phase is primarily regulated by antithrombin III and the protein C system. Thrombin generation during the propagation phase is remarkably suppressed in the absence of factor VIII and IX (hemophilia A and B, respectively) and at platelet counts <5% of mean plasma concentration. The majority of data accumulated in in vitro models and discussed in this review are in good agreement with the results of in vivo observations.     

重症肺炎影响极低出生体重儿血小板及凝血功能的研究

目的:通过观察我院收治的极低出生体重新生儿肺炎的临床治疗,研究重症肺炎对该类疾病患儿的血小板及凝血功能的影响,探讨其临床研究价值.方法:将47例一般肺炎的极低出生体重新生儿设为对照组,45例重症肺炎的极低出生体重新生儿设为实验组,对比分析两组患儿的血小板参数的检测指标并行患儿凝血功能检查.结果:在血小板参数方面,实验组在MPV和PDW的数据较对照组高,而在PLT明显较低;在凝血功能方面,实验组在PT、APTT和TT等方面的数据较对照组高,而在FIB方面明显较低.结论:临床上对动态监测重症肺炎患儿血小板的变化情况,对判断该疾病患儿的预后具有积极的参考与预警意义,而对重症肺炎患儿出现凝血系统功能紊乱及时把握具体发病机制,对症进行凝血功能恢复治疗,值得临床进一步研究与探讨.     

Receptors of the PAR Family as a Link between Blood Coagulation and Inflammation

Blood coagulation plays a key role among numerous mediating systems that are activated in inflammation. Receptors of the PAR family serve as sensors of serine proteinases of the blood clotting system in the target cells involved in inflammation.Activation of PAR-1 by thrombin and of PAR-2 by factor Xa leads to a rapid expression and exposure on the membrane of endothelial cells of both adhesive proteins that mediate an acute inflammatory reaction and of the tissue factor that initiates the blood coagulation cascade. Certain other receptors (EPR-1, thrombomodulin, etc.), which can modulate responses of the cells activated by proteinases through PAR receptors, are also involved in the association of coagulation and inflammation together with the receptors of the PAR family. The presence of PAR receptors on mast cells is responsible for their reactivity to thrombin and factor Xa and defines their contribution to the association of inflammation and blood clotting processes.     

Circadian Variations in Blood Coagulation Parameters, Alpha-Antitrypsin Antigen and Platelet Aggregation and Retention in Clinically Healthy Subjects

Ten clinically healthy subjects (5 men and 5 women), 31 11 yrs of age, were studied at six timepoints (0800, 1200, 1600, 2000, 0000, 0400) distributed over a 1-week span. Circadian rhythms in platelet aggregation in response to adenosine diphosphate (ADP) and adrenalin (A), platelet adhesiveness measured as retention in a glass bead column, prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT), fibrinogen, Factor VIII activity and alpha-1-antitrypsin antigen showed circadian rhythms. The plasma concentrations of plasminogen, alpha-2-macroglobulin, and antithrombin III (AT III) antigen, Factor V and fibrinogen degradation products showed no circadian rhythm by ANOVA or cosinor analysis. The phase relations of the rhythms of different coagulation parameters are of interest in the physiology and pathobiology of the coagulation-fibrinolytic system. The extent of the circadian rhythm (range of change) described is not of a magnitude to lead to diagnostic problems in the clinical laboratory. The timing of these rhythms, however, may determine transient risk states for thromboembolic phenomena, including myocardial infarction and stroke. Several but not all coagulation parameters suggest a transient state of hypercoagulability during the morning hours. The recognition of these rhythmic, and thus in the time of the occurrence predictable temporary risk states for thromboembolic phenomena, may lead to timed treatment and/or effective prevention.     

凝血靶向通用效应因子tTF/SA融合蛋白的表达及活性鉴定

制备凝血靶向通用效应因子tTF/SA融合蛋白,并鉴定其生物学活性。利用PCR技术构建tTF与链霉亲和素SA的融合基因,克隆至表达载体pET22b( ),在E.coliBL21(DE_3)中表达,镍亲和层析柱纯化tTF/SA融合蛋白。凝血实验和FⅩ活化实验鉴定融合蛋白中tTF的活性,ELISA鉴定融合蛋白中SA与生物素Biotin结合的活性。获得序列正确的tTF/SA/pET22b( )重组子,融合基因在E.coliBL21(DE_3)中高效表达。纯化后的融合蛋白具有活化FⅩ、引起血液凝固的能力,且能与生物素结合。融合基因已成功在E.coliBL21(DE_3)中表达,tTF/SA融合蛋白具有TF和SA活性。融合蛋白tTF/SA可作为通用效应因子,与生物素化的肿瘤组织血管特异性载体联用,实现选择性诱发肿瘤组织血管栓塞的多点治疗。     

玉蜀黍轴及苞叶对凝血作用的影响

采用体外血浆复钙时间法,以维生素k1作为促凝血和灯盏花素作为抗凝血作用阳性对照,对玉蜀黍轴及苞叶石油醚、乙酸乙酯和正丁醇提取物及玉蜀黍苞叶甲醇总浸膏对体外血浆复钙时间的影响进行测定。结果显示玉蜀黍轴正丁醇及苞叶乙酸乙酯提取物,均可显著缩短体外血浆复钙时间(P<0.001);玉蜀黍苞叶石油醚提取物可显著延长体外血浆复钙时间(P<0.001)。提示玉蜀黍轴正丁醇及苞叶乙酸乙酯提取物具有较好的促凝血活性,玉蜀黍苞叶石油醚提取物具有较好的抗凝作用。     

羟考酮在腹腔镜胆囊切除术后镇痛中的应用及对凝血功能的影响

目的:分析羟考酮在腹腔镜胆囊切除术后镇痛中的应用及对凝血功能的影响。方法:选择2015年5月~2016年5月于我院行腹腔镜胆囊切除术患者106例,依据抽签法分成对照组与实验组,各有53例,对照组予以芬太尼镇痛,实验组予以羟考酮镇痛,比较两组痛觉指标[5-羟色胺(5-HT)、P物质]、疼痛数字评分(NRS)、凝血功能[凝血酶原时间(PT)、纤维蛋白原(Fg)、活化部分凝血酶原时间(APTT)、血小板计数(PLT)]、血流动力学、炎症因子、应激指标、麻醉效果和安全性。结果:干预后,实验组5-HT、P物质水平、NRS评分、PT、Fg、APTT、PLT以及血流动力学、炎症因子、应激指标均低于对照组,差异具有统计学意义(P0.05)。两组麻醉效果比较差异无统计学意义(P0.05)。实验组不良反应率显著低于对照组(P0.05)。结论:腹腔镜胆囊切除术应用羟考酮的镇痛作用与芬太尼相当,但能够更有效减轻术后血液的高凝状态。     

The Dual Regulatory Role of Amino Acids Leu480 and Gln481 of Prothrombin

Prothrombin (FII) is activated to α-thrombin (IIa) by prothrombinase. Prothrombinase is composed of a catalytic subunit, factor Xa (fXa), and a regulatory subunit, factor Va (fVa), assembled on a membrane surface in the presence of divalent metal ions. We constructed, expressed, and purified several mutated recombinant FII (rFII) molecules within the previously determined fVa-dependent binding site for fXa (amino acid region 473–487 of FII). rFII molecules bearing overlapping deletions within this significant region first established the minimal stretch of amino acids required for the fVa-dependent recognition exosite for fXa in prothrombinase within the amino acid sequence Ser⁴⁷⁸–Val⁴⁷⁹–Leu⁴⁸⁰–Gln⁴⁸¹–Val⁴⁸². Single, double, and triple point mutations within this stretch of rFII allowed for the identification of Leu⁴⁸⁰ and Gln⁴⁸¹ as the two essential amino acids responsible for the enhanced activation of FII by prothrombinase. Unanticipated results demonstrated that although recombinant wild type α-thrombin and rIIa^S478A were able to induce clotting and activate factor V and factor VIII with rates similar to the plasma-derived molecule, rIIa^SLQ→AAA with mutations S478A/L480A/Q481A was deficient in clotting activity and unable to efficiently activate the pro-cofactors. This molecule was also impaired in protein C activation. Similar results were obtained with rIIa^ΔSLQ (where rIIa^ΔSLQ is recombinant human α-thrombin with amino acids Ser⁴⁷⁸/Leu⁴⁸⁰/Gln⁴⁸¹ deleted). These data provide new evidence demonstrating that amino acid sequence Leu⁴⁸⁰–Gln⁴⁸¹: 1) is crucial for proper recognition of the fVa-dependent site(s) for fXa within prothrombinase on FII, required for efficient initial cleavage of FII at Arg³²⁰; and 2) is compulsory for appropriate tethering of fV, fVIII, and protein C required for their timely activation by IIa.     

The Human Platelet as a Model for the Glutamatergic Neuron: Platelet Uptake of L-Glutamate

Abstract: L-Glutamate uptake into human platelets revealed two components: a high-affinity system ( K_mH = 3.1 μM), which was sodium-dependent, and a low-affinity site ( K_mL = 88 μM) displaying temperature rather than sodium dependency. These kinetic properties were similar to those found in crude synaptosomal preparations and brain slices. However, V_max values were far higher in brain(V_maxH= 325 ± 96, V_maxH= 3759 ± 1116 pmol/mg wet weight per min) than in platelets (V_maxH, = 14 ± 6, V_maxL= 313 ± 63 pmol/mg platelet protein per 10 min), indicating a denser population in brain than in platelets of qualitatively similar sites. Pharmacological analysis substantiated the resemblance of nerve endings and platelets: the specific uptake inhibitors threo-3-hydroxy-DL-aspartate and DL-aspartate-β-hydroxamate as well as D-and L-glutamate and L-aspartate showed similar—though not identical—IC₅₀ values in both preparations; a spectrum of compounds devoid of inhibitory effects in synaptosomes also did not interfere with glutamate uptake in platelets. Uptake parameters were studied in a population of human volunteers to determine the variability of platelet glutamate uptake. Whole blood could be stored up to 6 h after venipuncture without any appreciable change in experimental values. Percentage of variation between 0.09 and 0.28 for three repetitive (weekly) assays in single subjects indicated that glutamate uptake measurements in human platelets are sufficiently suited for future clinical studies.     

The influence of Annexin32, a new Ca2+-dependent phospholipid-binding protein, on coagulation time and thrombosis

The pharmacodynamics of Annexin32, a new Ca2+-dependent phospholipid-binding protein, was studied by measuring coagulation time in rabbits and venous thrombosis in rabbits and rats. Rabbits and rats were given Annexin32 by intravenous administration. Then Kaolin partial thromboplastin time (KPTT), thrombosis in vitro and in vivo were assayed. The results showed that KPTT of rabbits was prolonged (p < 0.01), and the length and weight of thrombus in vitro were reduced (p < 0.01) after administration of Annexin32 at 1 mg/kg. It also inhibited thrombosis in vivo and reduced the weight of venous thrombus significantly in rats (p < 0.01). All these results suggested that Annexin32 possesses the characteristic of antithrombotic effect and fewer side effects on coagulation time.     

The influence of Annexin32, a new Ca2+-dependent phospholipid-binding protein, on coagulation time and thrombosis

The pharmacodynamics of Annexin32, a new Ca2+-dependent phospholipid-binding protein, was studied by measuring coagulation time in rabbits and venous thrombosis in rabbits and rats. Rabbits and rats were given Annexin32 by intravenous administration. Then Kaolin partial thromboplastin time (KPTT), thrombosis in vitro and in vivo were assayed. The results showed that KPTT of rabbits was prolonged (p < 0.01), and the length and weight of thrombus in vitro were reduced (p < 0.01) after administration of Annexin32 at 1 mg/kg. It also inhibited thrombosis in vivo and reduced the weight of venous thrombus significantly in rats (p < 0.01). All these results suggested that Annexin32 possesses the characteristic of antithrombotic effect and fewer side effects on coagulation time.     

小鼠骨髓造血干细胞、外周血组成随年龄的变化趋势及其相关性分析

造血干细胞是具有自我更新能力并能分化为血液中各种血细胞组分的多能干细胞。近来研究显示,不同造血干细胞表面标志物标记的造血干细胞具有分化为不同血细胞的趋势,但是这种分化的内在关系仍不清楚。对小鼠CD34~-/Sca-1~+骨髓造血干细胞、外周血组成随小鼠年龄增长的变化情况进行了分析,结果显示:随着年龄的增长,骨髓中的CD34~-/Sca-1~+骨髓造血干细胞比率显著增加;而外周血各组分则随年龄变化呈现不同的趋势。对不同年龄段小鼠的骨髓造血干细胞及其他组分与外周血组分的同步分析发现,外周血中血小板密度变化趋势与CD34~-/Sca-1~+骨髓造血干细胞变化情况相关系数为0.804 8;外周血中淋巴细胞密度变化趋势与CD34~+/Sca-1~-骨髓细胞的变化情况相关系数为0.947 97;外周血中白细胞密度变化趋势与CD34~+/Sca-1~+骨髓细胞变化情况相关系数为0.763 1(大于0.9为极度相关,0.7到0.9为高度相关)。     

Activating Blood Circulation,Anti-Inflammatory and Diuretic Effects of Leonurus japonicus Extract on a Rat Model of Trauma Blood Stasis and Its Phytochemical Profiling

Leonurus japonicus Houtt. has been traditionally used to treat many ailments. This study evaluated the activating blood circulation, anti-inflammatory, and diuretic effects of L. japonicus extract (LJ) and identified its phytochemicals. In this work, the phytochemicals in LJ were identified using liquid chromatography mass spectrometry. Rats were randomly assigned to three groups (n=8): Control group was treated with saline, while the Model group (saline) and LJ group (426 mg/kg) had induced traumatic injury. All rats were treated with once by daily oral gavage for one week. The biochemical indices and protein expression were measured. Herein, 79 constituents were identified in LJ, which were effective in elevating body weight, food consumption, water intake, and urinary excretion volume, as well as in ameliorating traumatic muscle tissues in model rats. In addition, LJ prominently decreased the contents of plasma viscosity, platelet aggregation rate, thrombin time, prothrombin time, activated partial thromboplastin time, fibrinogen, thromboxane B2 (TXB2), TXB2/6-keto-prostaglandin F1α (6-keto-PGF1α), urokinase-type plasminogen activator (u-PA), plasminogen activator inhibitor 1 (PAI-1), PAI-1/tissue-type PA (t-PA), and PAI-1/u-PA, while significantly increasing antithrombin III, 6-keto-PGF1α, and t-PA contents. Furthermore, LJ notably inhibited tumor necrosis factor alpha, interleukin 6 (IL-6), IL-8, angiotensin II, antidiuretic hormone, aldosterone, aquaporin 1 (AQP1), AQP2, and AQP3 levels, and markedly elevating IL-10 and natriuretic peptide levels. Finally, LJ markedly reduced the protein expression of AQP1, AQP2, and AQP3 compared to the model group. Collectively, LJ possessed prominent activating blood circulation, anti-inflammatory, and diuretic effects, thus supporting the clinical application of L. japonicus.     

A Novel In vitro Model for Studying the Interactions Between Human Whole Blood and Endothelium

The majority of all known diseases are accompanied by disorders of the cardiovascular system. Studies into the complexity of the interacting pathways activated during cardiovascular pathologies are, however, limited by the lack of robust and physiologically relevant methods. In order to model pathological vascular events we have developed an in vitro assay for studying the interaction between endothelium and whole blood. The assay consists of primary human endothelial cells, which are placed in contact with human whole blood. The method utilizes native blood with no or very little anticoagulant, enabling study of delicate interactions between molecular and cellular components present in a blood vessel.We investigated functionality of the assay by comparing activation of coagulation by different blood volumes incubated with or without human umbilical vein endothelial cells (HUVEC). Whereas a larger blood volume contributed to an increase in the formation of thrombin antithrombin (TAT) complexes, presence of HUVEC resulted in reduced activation of coagulation. Furthermore, we applied image analysis of leukocyte attachment to HUVEC stimulated with tumor necrosis factor (TNFα) and found the presence of CD16⁺ cells to be significantly higher on TNFα stimulated cells as compared to unstimulated cells after blood contact. In conclusion, the assay may be applied to study vascular pathologies, where interactions between the endothelium and the blood compartment are perturbed.     

孟鲁司特钠联合肝素钠对小儿过敏性紫癜的效果及对T细胞亚群、凝血功能的影响

摘要 目的：分析孟鲁司特钠联合肝素钠对小儿过敏性紫癜的效果及对T细胞亚群、凝血功能的影响。方法：选择我院自2019年4月至2022年4月接诊的136例过敏性紫癜患儿作为研究对象,随机分为对照组和观察组,各68例。两组均在常规治疗的基础上,对照组予以肝素钠治疗,观察组予以孟鲁司特钠联合肝素钠治疗。比较两组各项临床症状的消退时间,治疗前后的外周血T细胞亚群（CD3⁺、CD4⁺、CD8⁺、CD4⁺/CD8⁺）、凝血功能指标[纤维蛋白原（FIB）、凝血酶原时间（PT）、凝血活酶时间（APTT）];根据临床症状、体征及实验室指标的改善情况,综合评价疗效,计算两组总有效率。结果：观察组关节疼痛、便血缓解、腹痛缓解和紫癜消退的时间均短于对照组（P＜0.05）;观察组治疗后外周血CD3⁺、CD4⁺、CD4⁺/CD8⁺水平较对照组高,CD8⁺水平较对照组低（P＜0.05）;观察组治疗后FIB水平较对照组低,PT、APTT均长于对照组（P＜0.05）;观察组总有效率为95.59 %,高于对照组的79.41 %（P＜0.05）。结论：孟鲁司特钠联合肝素钠能协同提高小儿过敏性紫癜的效果,调节T细胞亚群,增强免疫功能,改善凝血功能,值得临床予以重视应用。     

脐血干细胞移植治疗缺血性心脏病的临床疗效及安全性分析

目的:探讨脐血干细胞移植治疗缺血性心脏病(IHD)的临床效果和安全性。方法:选择2011年1月至2013年1月在我院接受治疗的IHD患者130例。随机分为观察组和对照组,各65例。其中对照组应用常规药物治疗,观察组应用脐血干细胞移植方法进行治疗。观察对比两组患者治疗前以及治疗后3个月的左室舒张末容量(LVEDV)、右室舒张末容量(RVEDV)、左室射血分数(LVEF)及治疗后临床疗效及生活质量改善状况。结果:观察组治疗后显效率和总有效率为49.23%,90.77%,显著高于对照组的30.77%,70.77%,差异均有统计学意义(均P0.05)。两组在治疗前LVEDV、RVEDV以及LVEF比较无差异。在治疗后观察组这三个指标得到明显改善,其中LVEDV和RVEDV减小、LVEF提高均较对照组显著,差异均有统计学意义(均P0.05)。观察组在治疗后身体功能、运动限制和总体健康评分均高于对照组,差异均有统计学意义(均P0.05)。结论:应用脐血干细胞移植治疗IHD效果好,能显著改善心功能,提高生活质量,安全性高,值得临床使用。     

Structural Basis for Activity and Specificity of an Anticoagulant Anti-FXIa Monoclonal Antibody and a Reversal Agent

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预存式自体输血与异体输血对原发性肝癌围术期患者凝血功能、炎性因子和T细胞亚群的影响

摘要 目的：对比异体输血和预存式自体输血对原发性肝癌围术期患者凝血功能、炎性因子和T细胞亚群的影响。方法：回顾性选取2018年8月~2020年12月期间在西部战区总医院行肝部分切除术的91例原发性肝癌患者的临床资料,根据输血方式的不同分为异体组和自体组,例数分别为44例、47例,观察两组患者凝血功能、炎性因子和T细胞亚群的变化情况,记录两组输血不良反应发生率。结果：自体组、异体组术后1 d活化部分凝血活酶时间（APTT）、凝血酶时间（TT）、D-二聚体（D-D）、凝血酶原时间（PT）较术前升高（P<0.05）,术后5 d的APTT、TT、PT、D-D均较术后1 d降低（P<0.05）,且两组术后1 d、术后5 d上述指标对比无明显差异（P>0.05）。自体组术后1 d、术后5 d的白介素（IL）-6、IL-8、肿瘤坏死因子-α（TNF-α）低于异体组（P<0.05）。自体组术后1 d、术后5 d 的CD3⁺、CD4⁺、CD4⁺/CD8⁺高于异体组,CD8⁺低于异体组（P<0.05）。两组患者输血不良反应发生率比较差异无统计学意义（P>0.05）。结论：原发性肝癌围术期患者采用预存式自体输血,对机体凝血功能的影响小,同时还可减轻免疫抑制,抑制机体炎性反应,具有一定临床应用价值。     

Sleep-Time Blood Pressure: Prognostic Value and Relevance as a Therapeutic Target for Cardiovascular Risk Reduction

Correlation between blood pressure (BP) level and target organ damage, cardiovascular disease (CVD) risk, and long-term prognosis is greater for ambulatory BP monitoring (ABPM) than clinical BP measurements. Nevertheless, the latter continue to be the “gold standard” to diagnose hypertension, assess CVD risk, and evaluate hypertension treatment. Independent ABPM studies have found that elevated sleep-time BP is a better predictor of CVD risk than either the awake or 24-h BP mean. A major limitation of all previous ABPM-based prognostic studies is the reliance only upon a single baseline profile from each participant at the time of inclusion, without accounting for potential changes in the level and pattern of ambulatory BP thereafter during follow-up. Accordingly, impact of the alteration over time, i.e., during long-term follow-up, of specific features of the 24-h BP variation on CVD risk has never been properly investigated. We evaluated the comparative prognostic value of (i) clinic and ambulatory BP; (ii) different ABPM-derived characteristics, e.g., asleep or awake BP mean; and (iii) specific changes in ABPM characteristic during follow-up, mainly whether reduced CVD risk is more related to the progressive decrease of asleep or awake BP. We prospectively studied 3344 subjects (1718 men/1626 women), 52.6?±?14.5 (mean?±?SD) yrs of age, during a median follow-up of 5.6 yrs. Those with hypertension at baseline were randomized to ingest all their prescribed hypertension medications upon awakening or ≥1 of them at bedtime. At baseline, BP was measured at 20-min intervals from 07:00 to 23:00?h and at 30-min intervals at night for 48-h, and physical activity was simultaneously monitored every min by wrist actigraphy to accurately derive awake and asleep BP means. Identical assessment was scheduled annually and more frequently (quarterly) if treatment adjustment was required. Data collected either at baseline or the last ABPM evaluation per participant showed that the asleep systolic BP mean was the most significant predictor of both total CVD events and major CVD events (a composite of CVD death, myocardial infarction, and stroke). Moreover, when the asleep BP mean was adjusted by the awake mean, only the former was a significant independent predictor of outcome in a Cox proportional-hazard model adjusted for sex, age, diabetes, anemia, and chronic kidney disease. Analyses of changes in ambulatory BP during follow-up revealed 17% reduction in CVD risk for each 5?mm Hg decrease in the asleep systolic BP mean (p?<?.001), independent of changes in any other clinic or ambulatory BP parameter. The increased event-free survival associated with the progressive reduction in the asleep systolic BP mean during follow-up was significant for subjects with either normal or elevated BP at baseline. The ABPM-derived asleep BP mean was the most significant prognostic marker of CVD morbidity and mortality. Most important, the progressive decrease in asleep BP mean, a novel therapeutic target that requires proper patient evaluation by ABPM and best achieved by ingestion of at least one hypertension medication at bedtime, was the most significant predictor of event-free survival. (Author correspondence: rhermida@uvigo.es)