嗜血性无脊椎动物的天然凝血因子Ⅹa抑制物

综述了血液凝固途径和Ⅹa因子在血凝过程中的重要作用,Ⅹa因子位于内外源凝血途径的交汇处,在凝血过程中起关键作用,故其可成为抗凝治疗新药开发中非常重要的靶点。凝血因子Ⅹa抑制物是近年来抗凝血因子的研究热点。自1955年发现水蛭素以来,从嗜血性无脊椎动物体内分离出了许多天然凝血因子Ⅹa抑制物,运用基因工程技术对这些物质的特性进行了广泛深入的研究,并对它们进行重组改造,使之成为生物活性更强、功能更多的抗凝血化合物,在抗血栓形成方面具有广泛应用前景,可为临床开发抗凝血药物提供新的方向。     

抗凝血酶概述

抗凝血酶亦称肝素辅助因子,是血浆凝血系统中一种重要的生理性丝氨酸蛋白酶抑制物.蚊虫在吸血过程中其唾液腺分泌一组抗凝血活性物质,这些物质不仅可干扰宿主的凝血反应和炎症反应,更是一组具有潜在药理活性的物质.论文对抗凝血酶及蚊虫抗凝血酶的分离与纯化、克隆与表达、活性测定及抗凝血酶药物的研究进展进行综述,为抗凝血酶的深入研究提...     

基于蛋白组学体外分析益母草水煎液对补体和凝血级联通路相关活性因子的作用

旨在通过蛋白组学探索益母草水煎液对人真皮微血管内皮细胞(human dermal microvascular endothelial cells, HDMECs)凝血与抗凝血相关因子表达的调节作用。MTT法筛选益母草水煎液对HDMECs的安全浓度；使用同位素标记相对和绝对定量(isobaric tags for relative and absolute quantification, iTRAQ)技术分析50和100 μg·mL^-1益母草水煎液作用24 h后HDMECs蛋白表达谱的变化，通过对比各组蛋白谱的变化筛选出差异显著的相关通路，分析该通路中筛选到的可信差异表达蛋白(differentially expressed proteins, DEPs)，并选取可信DEPs中与凝血与抗凝血相关的拮抗因子进行RT-PCR和ELISA验证。结果表明：1 mg·mL^-1以下益母草水煎液对HDMECs无毒副作用；益母草水煎液能够同时调节与凝血相关的血小板活化等过程和与抗凝血相关的肝素结合过程。对筛选出的补体和凝血级联通路中的5种可信DEPs分析显示，与空白组相比，50 μg·mL^-1益母草水煎液组凝血酶原(F2)、抗凝血酶-Ⅲ(AT-Ⅲ)、组织型纤溶酶原激活剂(t-PA)、凝血因子Ⅴ(F5)和激肽原(KNG)同时显著下调，100 μg·mL^-1益母草组F2、t-PA、AT-Ⅲ、KNG显著下调；与50 μg·mL^-1益母草水煎液组相比，100 μg·mL^-1益母草水煎液组F2、AT-Ⅲ、t-PA、F5和KNG等凝血级联相关因子均显著升高。结果提示，益母草水煎液可显著改变HDMECs蛋白表达谱，并可能通过调控补体和凝血级联通路相关因子F2、AT-Ⅲ、t-PA、F5、KNG蛋白的表达对凝血与抗凝血相关拮抗因子发挥双向调控作用。     

动物凝血和抗凝血功能的检查及临诊意义

1止血和凝血机制 在生理状态下，血液中有少量纤维蛋白生成，并覆盖在血管内膜上，保护血管内皮，以维持血管的正常通透性，同时生成的纤维蛋白又被纤维蛋白溶酶所溶解，凝血与纤溶处于动态平衡状态，保持机体不出血，也无血栓形成，血液是流体状态而循环于全身。当这种平衡失调时，便可导致出血不止或形成血栓，即止血与凝血障碍。止、凝血障碍的机制十分复杂，其涉及微血管、血小板、各凝血因子、抗凝因子及纤维蛋白溶解系统等诸多因素。在正常时止、凝血系统与抗凝血和纤维蛋白溶解系统处于动态平衡状态，遂能既无出血也无血栓形成。若止、凝血活性减弱或抗凝血及纤溶活性增强则会引起低凝状态发生出血症状。相反会引起高凝状态或导致血栓形成。前者临床上统称为出血性疾病，后者统称为血栓性疾病。     

蚕蛹壳聚糖甘氨酸衍生物的制备及其抗凝血作用研究

壳聚糖是生产抗凝血物质的理想原料。采用化学方法对蚕蛹壳聚糖进行分子修饰,即在蚕蛹壳聚糖分子上引入甘氨酸和环氧氯丙烷反应的中间体,制备壳聚糖甘氨酸衍生物。通过红外光谱及核磁共振分析其分子结构,结果显示甘氨酸已接枝到蚕蛹壳聚糖分子上,为在C6-OH发生醚化反应的壳聚糖甘氨酸衍生物。抗凝血试验结果表明,蚕蛹壳聚糖甘氨酸衍生物具有抗凝血活性,当其质量浓度达到2mg/mL时抗凝血效果最佳,可使血液完全不凝固。从凝血时间、凝血酶原时间、活化部分凝血活酶时间、凝血酶时间的检测结果可知,蚕蛹壳聚糖甘氨酸衍生物影响到外源性、内源性凝血系统和凝血活性酶的形成。依据抗凝血试验结果分析认为,蚕蛹壳聚糖甘氨酸衍生物中C6引入的羧基与壳聚糖C2上的氨基共同作用,产生了抗凝血效果。研究结果提示:蚕蛹壳聚糖甘氨酸衍生物可进一步开发为医用抗凝血材料。     

蚊虫抗凝血活性物质研究进展

蚊虫在吸血过程中其唾液腺分泌一组抗凝血活性物质,这些物质不仅可干扰宿主的凝血反应和炎症反应,更是一组具有潜在药理活性的物质。本文对蚊虫抗凝血活性物质进行分类并从其特点、抗凝血作用原理、研究现状等方面进行综述,为该类物质的深入研究提供借鉴。     

一例泰迪犬溴敌隆中毒的治疗

溴敌隆是高毒抗凝血性杀鼠剂,属茚满二酮类抗凝血剂,半衰期60小时,中毒潜伏期长,中毒症状至少要12~24小时后出现,需3~5天中毒达高峰期。溴敌隆结构中含4-2羟基香豆素与维生素K相似,干扰肝脏对维生素K的作用,抑制凝血酶原和凝血因子Ⅴ、Ⅶ、Ⅸ、Ⅹ的生物合成,导致凝血障碍,其体内     

动物外用止血剂的制备

为提供外用止血剂用于兽医临床，本试验利用凝血酶原、凝血因子Xa、凝血因子V，采用正交试验设计确定了凝血酶原酶复合物各种成分的最佳浓度,从猪血中制备凝血酶，并根据兽医临床上的需要制成适合动物外用的止血剂型。该止血剂可以应用于兽医临床，是国内外兽医临床的新型外用止血剂。     

氯化钙溶液对家兔凝血系统的影响

采用凝血酶原时间（ＰＴ－期法）测定、白陶土非特异性粘附试验及加入活化因子等指标，观察了氯化钙抗凝血剂对家兔内、外源凝血系统的影响。结果表明，用０．７２ｍｏｌ／ＬＣａＣｌ２作为抗凝剂，血液凝血酶原时间延长，即外源性凝血系统受抑制。白陶土不能激活内源性凝血系统而诱发凝血，即内源性凝血系统亦受到抑制。ａ因子也不能使之启动     

桑叶中抗凝血活性成分的初步分离与纯化

采用乙醇分级沉淀和Sephadex G100凝胶层析的方法,结合凝血指标检测,对桑叶中的抗凝血活性成分进行了分离纯化。结果表明:桑叶的抗凝血活性主要集中在桑叶的水提取液中,能够显著延长人体血浆的活化部分凝血活酶时间(APTT)值。桑叶水提取液经30%乙醇沉淀、凝胶层析和醋酸纤维素薄膜电泳表明其抗凝血活性成分是一种多糖组分。试验还表明,高浓度乙醇处理对桑叶多糖的抗凝血活性没有影响。     

Congenital factor XI deficiency in a domestic shorthair cat

A 6-month-old, female, domestic shorthair cat was examined after onychectomy and ovariohysterectomy because of bleeding from the paws. Prolonged activated partial thromboplastin time was discovered, Coagulation factor analyses revealed deficiency of factor XI coagulant activity. Plasma mixing studies indicated factor deficiency or dysfunction rather than factor inhibition. Feline factor XI deficiency in one adult cat has been previously reported but was attributed to factor XI inhibitors. The signalment, lack of primary disease, and the finding of persistent factor XI deficiency in the absence of coagulation inhibitors were considered compatible with congenital factor XI deficiency in the cat of this report.     

An investigation, in vitro, of the actions of three Western Australian snakes on the blood coagulation of the dog, cat, horse and wallaby

Venoms of the tiger snake and brown snake were procoagulant, in vitro, when tested with cat, dog, horse and wallaby plasma. In the absence of calcium and phospholipid the coagulant activity of tiger snake venom was minimal. In contrast, brown snake venom alone had marked procoagulant activity. This activity, however, was enhanced by the presence of calcium and phospholipid. Death adder venom exerted an anticoagulant effect. Apparent species' differences in susceptibility to the coagulant venoms were noted. However, the probable explanation of these differences was attributed to variation in the control values of the special studies rather than to a difference in the postulated actions of the venoms on prothrombin. A possible role for clotting studies in suspected snake bite in veterinary practice is suggested.     

The effects of desmopressin on hemostatic parameters in the normal dog.

Seven normal unanesthetized dogs were infused with desmopressin in saline (doses 0.2, 0.4 and 0.6 micrograms/kg i.v.) or saline alone, on separate occasions in order to determine the effects of desmopressin on circulating factor VIII coagulant and factor VIII-related antigen activities, on partial thromboplastin times and prothrombin times, on blood platelet count and packed cell volume, and on serum osmolarity. Desmopressin caused a rapid dose-dependent elevation in both factor VIII activities peaking at 30-60 minutes postinfusion. At a dosage of 0.6 microgram/kg desmopressin induced rises in factor VIII coagulant activity and factor VIII-related antigen of 150% and 198% of the preinfusion levels respectively, while the half disappearance times were approximately five and six hours respectively. The other parameters were not significantly affected by even the highest dose of desmopressin used. These results indicate that normal dogs respond to desmopressin in a manner similar to man. The major differences are in the dosage required to produce comparable effects (higher in dogs), and in the fact that the factor VIII-related antigen activity consistently responds to a greater degree than the coagulant activity. It is concluded that desmopressin may have particular value in stimulating elevations in plasma factor VIII-related antigen in dogs deficient in this plasma protein. Six dogs were pretreated with the endorphin inhibitor, naloxone, (0.04 mg/kg I.V.) or sterile saline, three minutes prior to infusion with desmopressin at a dose of 0.6 microgram/kg i.v.(ABSTRACT TRUNCATED AT 250 WORDS)     

The relationship between factor XI coagulant and factor XI antigenic activity in cattle.

Factor XI protein, isolated from normal bovine plasma, was used to raise antiserum in rabbits. The antisera was partially purified and used in a neutralization-inhibition assay to investigate the relationship between factor XI coagulant activity and antigenic material in the plasma of normal cattle and cattle homozygous and heterozygous for factor XI deficiency. Factor XI antigen was reduced in both the homozygous and heterozygous animals to levels comparable to the factor XI coagulant activity. The reduction of immunologically cross-reactive material to normal factor XI suggests that the factor XI coagulation defect is associated with the absence of a normal protein.     

Endogenous Anticoagulants

Blood coagulation is a complex and highly coordinated process that is constantly altered and impacted by procoagulant and anticoagulant “players.” It is vital that these components work in concert to maintain a balance to keep coagulation in check. Several important endogenous anticoagulants will be discussed in this review including tissue factor pathway inhibitor, antithrombin, protein C, and protein S in origin, structure, mechanism of action, effects of deficiency, and current knowledge in veterinary medicine.     

Cyclooxygenase (COX) inhibitors and the intestine

Nonsteroidal anti-inflammatory drugs (NSAIDs) have long been used for the treatment of pain and inflammation because of their inhibitory effects on cyclooxygenase (COX). For almost as long as NSAIDs have been in use, multiple adverse effects have been noted. Assessment of many of these adverse effects have been complicated because of the discovery of multiple splice variants of the cox gene, and a greater array of COX inhibitors, especially the COX-2 selective inhibitors have become available. Some of these adverse effects cannot be readily explained by the effect of these drugs on COX. This has sparked a new field of investigation into the COX-independent effects of the COX inhibitors. The major noncyclooxygenase targets of the COX inhibitors of particular relevance to inflammation and the gastrointestinal tract are phosphatidylinositol 3'-kinase Akt signaling, uncoupling of oxidative phosphorylation, PPARgamma, nuclear factor KB, mitogen activated protein kinases, and heat shock proteins.     

Warfarin: a review with emphasis on its use in the horse

Warfarin or dicoumarol prevents the production of functional clotting factors II, VII, IX and X. Navicular disease and thrombophlebitis are examples of equine thrombotic diseases in which warfarin has been used therapeutically. The initiation of anticoagulant therapy is relatively simple but attending veterinarians must be aware of the potential risks in order to minimize them. These risks include epistaxis, bleeding into the gastrointestinal tract and at the venipuncture site, and increased susceptibility to hematoma formation following local trauma. Vitamin K, especially vitamin K₁ is a swift and specific antidote for warfarin toxicity.     

Factor X deficiency in a Jack Russell terrier

Deficiency in factor X (Stuart-Prower factor) was identified in a 7-month-old spayed female Jack Russell Terrier following recurrent bleeding episodes. Various relatives were screened for factor X deficiency and low and subnormal levels were identified in the father and paternal grandmother, respectively. Factor X deficiency has been previously documented in a family of American Cocker Spaniels, in which the inheritance pattern appeared to be an autosomal dominant trait with variable expression. This is the first report describing this coagulopathy in the Jack Russell Terrier.     

Failure of sodium pentobarbital anesthesia to alter 1-desamino-8-D-arginine vasopressin-induced elevations of plasma factor VIII/ von Willebrand factor in normal dogs.

The vasopressin analog 1-desamino-8-D-arginine stimulates elevations in plasma Factor VIII/ von Willebrand factor in normal dogs. In order to study the effects of general anesthesia on this response, six dogs were anesthetized with sodium pentobarbital or given an equivalent amount of saline then challenged with an intravenous dose of 1-desamino-8-D-arginine (0.6 micrograms/kg body weight). Factor VIII coagulant activity, von Willebrand factor antigen, and ristocetin cofactor activity were quantitated before anesthesia (or saline infusion), 20 min after induction (pre-1-desamino-8-D-arginine), and at 30 and 60 min post-1-desamino-8-D-arginine. Anesthesia did not significantly affect the elevations in plasma Factor VIII/ von Willebrand factor induced by 1-desamino-8-D-arginine. Sodium pentobarbital appeared however to prevent the rise in Factor VIII coagulant activity seen following saline treatment. The results of this study suggest that when 1-desamino-8-D-arginine is to be used in normal dogs to boost basal plasma von Willebrand factor levels, it is not necessary to administer it prior to induction of general anesthesia with sodium pentobarbital.     

Comparative stability of canine and feline hemostatic proteins in freeze‐thaw‐cycled fresh frozen plasma

Objective – To evaluate the stability of canine and feline hemostatic proteins in freeze‐thaw‐cycled (FTC) fresh frozen plasma (FFP). Design – Prospective study. Setting – Veterinary Teaching Hospital. Animals – Nine blood donor dogs and 10 blood donor cats. Interventions – Whole blood was collected and separated into packed RBC and plasma units according to standard methods. Each unit of plasma was divided into 2 equal aliquots and frozen (?41°C). One aliquot from each donor (FTC) was then thawed and then refrozen (?41°C) until time of analysis. The second aliquot (nonfreeze‐thaw‐cycled; NFTC) remained frozen until time of analysis. The hemostatic proteins assessed included coagulation factors, anticoagulant factors (antithrombin and Protein C), and adhesive proteins (fibrinogen and von Willebrand Factor). The coagulant activities of factors II, VII, VIII, IX, X, XI, and XII were measured in modified one‐stage activated partial thromboplastin time or prothrombin time assays. Antithrombin and Protein C activities were measured in chromogenic substrate assays. Clottable fibrinogen was measured via the Clauss method, and von Willebrand Factor concentration (vWF:Ag) was measured in an ELISA. A paired t‐test was utilized to identify differences in factor activity or concentration between FTC FFP and NFTC FFP. Measurements and Main results – No clinically or statistically significant differences (all P>0.05) were identified between FTC FFP and NFTC FFP. Conclusions – Refreezing FFP within 1 hour of initial thawing appeared to have no deleterious effects on the hemostatic protein activity or content of that unit. Transfusion of FTC FFP is expected to provide the recipient with comparable replacement of hemostatic proteins as FFP that has remained frozen.