Evaluation of a citrate-based anticoagulant with platelet inhibitory activity for feline blood cell Counts

Abstract: Aggregation of feline platelets in vitro results in difficulty assessing platelet number. A citrate-based anticoagulant containing the platelet inhibitors theophylline, adenosine, and dipyridamole (CTAD; Diatube-H, Becton Dickinson, Oxford, UK) has been developed for use in human platelet studies and heparin assays. To evaluate the efficacy of CTAD in reducing platelet aggregation in feline blood samples, aliquots of blood from 51 cats were anticoagulated with EDTA, CTAD, and for 12 samples, citrate solution. Samples preserved in CTAD had significantly higher (P ≤ .001) platelet counts, as determined by an impedance counter, hemacy-tometer, and smear estimation, than samples preserved in EDTA. In addition, subjective assessment of blood smears showed significantly fewer platelet aggregates (P<.001) in CTAD-treated samples compared with EDTA samples. Although values were similar, automated platelet counts and smear estimates of platelet number were significantly higher (P < .05) and platelet aggregation was significantly less (P < .05) in CTAD samples than in citrate samples. These results suggest that the platelet inhibitory activity of CTAD reduced feline platelet aggregation. Automated total WBC counts in CTAD samples were significantly lower (P<.001) than automated counts in EDTA samples but were similar to manual WBC counts in EDTA samples. Differences in both platelet and WBC counts between CTAD and EDTA or citrate samples were clinically relevant. Mean platelet volume and MCV were significantly lower (P< .05) in CTAD samples than in EDTA samples. No effect was seen on cell morphology or staining characteristics. The anticoagulant CTAD offers an advantage over both EDTA and citrate for feline hematologic analysis, by decreasing pseudothrombocytopenia and pseudoleukocytosis.     

野菊花抗血小板聚集有效成分的筛选

以野菊花为材料,顺次用石油醚、氯仿、乙酸乙酯、正丁醇、水抽提,抽提物终浓度相当于1g草药/ml,对5μmol/L ADP诱导的血小板聚集分别抑制30.8±20.2％、49.3±29.4％、51.9±22.8％、43.5±18.0％、13.7±1.6％。野菊花乙酸乙酯抽提物抑制5μmol/L ADP诱导的血小板聚集IC_(50)=1mg/ml。乙酸乙酯抽提物用硅胶柱制备分离得C1、C2、C3、C4、C5、C6六种结晶,经TLC检识分别为一个斑点。定性分析知为基黄酮化合物。在0.5mg/ml浓度下,对2μmol/L ADP诱导的血小板聚集后三种物质分别抑制78.9％、85.3％、83.5％。     

P2Y12 receptor gene mutation associated with postoperative hemorrhage in a Greater Swiss Mountain dog

A novel hereditary disorder of platelets was identified across 5 generations of a family of Greater Swiss Mountain dogs. The first dog identified with the mutation bled excessively following routine ovariohysterectomy and required multiple transfusions. Coagulation screening assays, platelet counts, and von Willebrand factor antigen activity were within reference intervals. Flow cytometric studies indicated that platelets from the affected dog expressed normal levels of glycoproteins IIb and IIIa and responded to 2 platelet-activating agents, convulxin and platelet-activating factor, but not to ADP. Based on DNA studies, a 3 base-pair deletion predicted to result in elimination of a serine from the extracellular domain was identified in the gene encoding P2Y12, an ADP receptor protein located on platelet membranes. Flow cytometric analysis of platelets and studies of DNA performed concurrently on 2 unrelated Greater Swiss Mountain dogs were unremarkable. The mutation was subsequently identified in the sire, the maternal grand-dam, a maternal great grandparent, a paternal great grandparent, and a great-great grandparent. The sire was homozygous, but had not yet been identified as having a hemostatic disorder; the other 4 dogs were carriers. This is the first report of a mutation in the gene encoding the ADP receptor P2Y12 in a domestic animal. P2Y12 is the same receptor targeted by ticlopidine and clopidogrel, platelet inhibitors used in lieu of aspirin in people at risk for cardiovascular disease; thus, spontaneous bleeding is not expected unless there are other contributing factors. This disorder is particularly troublesome because spontaneous hemorrhage is absent to mild in affected dogs; however, following routine surgical procedures or trauma, excessive bleeding could occur and have possible fatal consequences.     

血常规在宠物临床上的意义

以研究血常规检查在宠物临床中的意义为目的。采用血常规检查,包括定量检测结果（白细胞总数计数、白细胞分类计数、红细胞计数及指数、血小板计数及指数）、定性检测结果（血涂片形态学检查）,从而使血常规检查能为宠物临床诊断、疗效的观察及疾病的预后等提供参考。     

蛋白激酶C抑制剂Staurosporine对血小板聚集、蛋白磷酸化和肌动蛋白聚合的影响

目的：进一步研究蛋白激酶C抑制剂Staurosporine在血小板聚集、蛋白磷酸化、肌动蛋白聚合中的作用。方法：以32P－Na2HPO4标记血小板；以血小板聚集仪测定血小板聚集；以SDS－PAGE分离蛋白质，进行放射自显影；以TritonX－100抽提法沉淀骨架蛋白。结果：（1）1μmol／LStaurosporine完全抑制0．5U／ml凝血酶或10μmol／LPMA诱导的血小板聚集，大部分抑制20μmol／LA23187诱导的血小板聚集。（2）1μmol／LStaurosporine几乎完全抑制0．5U／ml凝血酶、或10μmol／LPMA、或20μmol／LA23187诱导的血小板40kD和20kD蛋白磷酸化。（3）1μmol／LStaurosporine完全抑制0．5U／ml凝血酶或10μmol／LPMA诱导的血小板肌动蛋白聚合。结论：Staurosporine抑制蛋白激酶C的活性．从而抑制血小板的聚集和肌动蛋白聚合；蛋白激酶C在血小板聚集和肌动蛋白聚合中起着重要调控作用。     

Blood coagulation times in the European brown hare (Lepus europaeus)

Background:  Many causes of mortality in the European brown hare, such as bacterial and viral infections, anticoagulant poisoning, and trauma, may result in hemorrhage. There are, however, no reference values concerning blood clotting in this species.  Objectives:  The aim of this study was to determine reference values for blood coagulation times and related parameters in healthy European brown hares.  Methods:  Blood samples from 30 clinically healthy adult hares (15 males and 15 females) were obtained. Hares were physically restrained for blood collection from the cephalic vein into tubes containing citrate and EDTA.  Results:  Mean ± SD were obtained for thrombin time (TT) (13.97 ± 1.37 seconds), prothrombin time (PT) (13.32 ± 2.15 seconds), activated partial thromboplastin time (APTT) (16.73 ± 1.86 seconds), fibrinogen concentration (2.98 ± 1.06 g/L), and platelet count (355.28 ± 128.73 × 10⁹/L). Conclusions: Reference values for blood coagulation times and other parameters associated with blood clotting will be useful in the laboratory evaluation of hemorrhage in the European brown hare.     

磷脂酶C对血小板粘附性及表面膜GpIb的影响

采用旋转玻球法测定了磷脂酶C(PLC)经十二指肠给药后对家兔血小板粘附性的影响,同时利用竞争性酶联免疫法测定了PLC对人血小板表面膜糖蛋白Ib(GpIb)分子数的影响。结果表明,PLC冻干辅料及100和200U/kg的PLC对家兔血小板粘附率无明显影响,而400～1000U/kgPLC对血小板粘附率具有明显的影响。PLC冻干辅料对人血小板表面膜GpIb分子数没有明显影响,而每mL血浆(PRP)中加入0.5,1.0和2.0U的PLC对人血小板表面GpIb分子数可产生明显影响。