Evidence for a NO synthase in porcine platelets which is stimulated during activation/aggregation

Abstract: We tried to characterize the porcine platelet nitric oxide (NO) synthase and its L-arginine (L-arg)/NO metabolism. Using RT-PCR we could show a constitutive endothelial NOS (ecNOS) and an inducible NOS (iNOS) similar mRNA in platelets. The NOS protein could be evidenced by an ecNOS specific antibody which also bound in platelets. This finding could be confirmed by Western blot showing an ecNOS in the membrane but not the cytosolic fraction; iNOS protein could not be detected. Using NADPH-diaphorase staining we could show NO synthase in preactivated platelets but not in resting platelets, indicating that the platelet NOS may be activated during platelet activation/aggregation. Porcine L-arg plasma levels (9.31 × 10^–5 mol/l ± 10%) could be shown to be in the same range as human plasma levels. Moreover, we could show that the NO precursor L-arg and hydroxy-L-arginine (OHarg) concentration dependently inhibited collagen induced platelet aggregation. Summarizing these results confirm the existence of and further characterize porcine platelet NO synthases.     

Effects of 5-Hydroxytraptamine on Aggregation,Release Reaction and Mobilization of Cytosolic Free Calcium Concentration in Rabbit Normal and Washed Single Platelets

In rabbit platelet rich plasma (PRP), 5-hydroxytraptamine (5-HT,0.03～3μmol/L) induced decrease in light transmission (DLT) dose-de pendently with centralization, as revealed by electron microscopy. However, 5-HT did not induced platelet aggregation and release reaction. 5-HT-induced DLT was inhibited by methysergide (0.3～30μmol/L), indomethacin (0.3～10μmol/L) and PGE_1 (0.01～0.3μmol/L) in a dose-dependent manner, but not EGTA(0.3～3mmol/L). Collagen(Coll), arachidonic acid (AA), adenosine diphosphate (ADP) and a stable thromboxane A2 analoge(STA_2) also induced DLT before aggregation by themselves, which was also inhibited by PGE_1, but not inhibited by EGTA except for Coll However,Coll-, AA-, STA_2-and ADP-induced DLT were reduced by pretreatment of PRP with 5-HT dose-depen-dently. The duration of DLT by Coll and AA were decreased from 151.4±5.93 sec and 15(?)38±0.60sec to 45.44±4.04 sec and 9.00±1.25 sec respectively ((?)±s(?) P<0.01), but not by ADP and STA_2, 3μmol/L of ADP-and 0.3μmol/L of STA_2-induced aggregation which was not accompanied with release reaction were enhanced by 5-HT pretreatment, but in those (Coil 5μg/ml, AA 100～200μmol/L and STA_2 1～3 μmol/L) with release reaction, the amount of adenosine triphosphate(ATP)were suppressed significantly (P<0.001) by 5-HT pretreatment without the effect on the magnitude of aggregation, The mobilization of cytosolic free calcium concentration ([Ca~(2+)]i) was observed after 5-HT treatment in single washed platelet, the results indicated that the basic level of [Ca~(2+)] i was 64.78±3.24nmol/L and this level was increased dose-dependently and significantly at 30 sec after administration of 5-HT and the time of peak value of [Ca~(2+)] i was at 90～100 sec.The similar time courses of suppression of ATP released during aggregation, in cases of Coll(5μg/ml), AA (200μmol/L) and STA_2(3μmol/L), by 5-HT were also found in the present experiment.     

大鼠肝细胞Ⅰ，Ⅲ型前胶原基因表达及PDGF的影响

目的观察大鼠肝细胞Ⅰ，Ⅲ型前胶原基因的表达及ＰＤＧＦ对其表达的影响．方法应用原位杂交技术检测分离培养的ＳＤ大鼠肝细胞（ｎ＝３０）内Ⅰ，Ⅲ型前胶原基因的表达．同时观察１０μｇ／Ｌ（ｎ＝３０）和３０μｇ／Ｌ（ｎ＝３０）ＰＤＧＦ促进前胶原基因表达的作用．测定基因表达颗粒总面积占细胞总面积的百分比，并作比较分析．结果无论正常肝细胞或是在两种浓度的ＰＤＧＦ存在时，肝细胞内均可见到Ⅰ，Ⅲ型前胶原基因的表达．正常肝细胞Ⅰ，Ⅲ型前胶原基因表达面积的百分比（％）为７７±１９和７５±２１；加１０μｇ／ＬＰＤＧＦ后为１１５±１９和１１２±１０，而加３０μｇ／Ｌ后为１５２±３４及１８１±２８，且在后者中表达明显增强（Ｐ＜００５及Ｐ＜００１）．结论ＰＤＧＦ在转录水平上促进肝细胞胶原的合成．     

Matching blood donations to type-specific product needs: A recruitment technique

The conversion of multiple whole blood donors to apheresis donors is a challenge since a rapidly expanding apheresis donor base could erode homologous collections. We addressed this concern with a plan to enhance apheresis recruitment as well as donations among homologous donors with types O and B blood. Focusing the donor's attention on blood type as it relates to type-specific product needs was the basis of our approach. A matrix was used to recruit the desired types for the desired procedures (whole blood, platelet/plasma apheresis). The matrix instructed donors of blood types O, A-, and B- to primarily give whole blood and to give apheresis as a secondary donation. Donors AB, A+, and B+ were primarily directed to apheresis donations, whole blood donation being secondary. A+ and O- donors only gave their secondary donation if they were at maximum donations with the primary donation. The collections by blood type in percentages for 12 months of 1992/93 for whole blood were O+ 38.9, 0- 7.3, A+ 29.5, A- 5.7, B+ 11.9, B- 2.1, AB+ 3.7, AB+ 0.7. For apheresis it was 0+ 36.2, 0- 6.7, A+ 33.0, A- 6.6, B+ 10.4, B- 1.2, AB+ 4.9, AB+ 1.0. In 1992/93, A+ and B+ apheresis collections as compared to total apheresis collections increased by 4.9% and 13.7%, respectively. For O group apheresis donations, a decrease of 2.5% was shown and A+ whole blood donations decreased by 5.35%. During the same period of time, total apheresis collections increased by 3,058 units. We demonstrated that integration of apheresis recruitment with type-specific whole blood recruitment yielded significant increases of type-specific products.     

Flow cytometry analysis of platelet antibodies

We have devised assays to detect both circulating alloantibodies to platelets (indirect assay) and platelet-association IgG and IgM (direct assay) using a flow cytometric technique. A variety of patients with immune thrombocytopenia were studied. Employment of a confocal lens in the flow cytometer increased the discrimination power of the instrument. Patients with autoimmune thrombocytopenia (idiopathic thrombocytic purpura [ITP], systemic lupus erythematosus (SLE), lymphoma, leukemia, and drug-induced thrombocytopenia showed a significant increase in platelet-associated antibody. Circulating antibodies to platelets (alloantibodies) were demonstrated in cases of platelet refractoriness and neonatal isoimmune purpura. Day-today precision of the assays ranged from 3% to 6% (coefficient of variation). No interference was shown in the presence of hemoglobin (5 g/L), triglycerides (10 g/L), or polyclonal and monoclonal immunoglobulinemia (50 g/L: IgG, IgA, IgM). The sensitivity of the direct assay was 500 attograms of IgG or IgM platelet.     

喉癌患者血小板聚集功能的测定

目的 探讨喉癌与血小板聚集功能的关系。方法 采用比浊法，对50例喉癌患进行血小板聚集功能的检测并以良性肿瘤病人、喉慢性炎性疾病病人及正常人作为对照，应用SAS计算机系统进行分析。结果 与其他组病人相比，喉癌组血小板聚集功能增强并且随着临床分期的增高有增强趋势。结论 喉癌病人的血小板聚集功能有一定的增强；测定血小板聚集功能对恶性肿瘤的疗效判定、预后评估及预测转移有一定的意义；血小板抑制剂有望成为治疗肿瘤的新方法。     

小剂量阿司匹林对缺血性脑血管病患者血小板聚集功能的影响

目的 :观察小剂量阿司匹林 (ASA)对ICVD患者血小板聚集功能的影响。方法 :用花生四烯酸 (AA ,5 0 0 μmol·L-1)、二磷酸腺苷 (ADP ,5 μmol·L-1)、肾上腺素 (EPN ,5 μmol·L-1)和胶原 (COL ,2 μg·mL-1)做诱导剂检测ICVD患者服用不同剂量ASA组 ( 2 5、5 0和10 0mg·d-1)的血小板聚集率。结果 :各ASA组对AA、COL诱导聚集的变异系数较大。对AA诱导聚集无显著抑制者的比例分别为2 5mg·d-1组 44 4%、5 0mg·d-1组 2 9 6%、10 0mg·d-1组 2 9% ,其中 5 0mg·d-1组中合并糖尿病的例数较显著抑制者中多。结论 :ICVD患者中小剂量ASA作用个体差异较大 ,部分与自身危险因素有关。提示临床中ASA的效果需要实验室检测并应个体化     

A multiparametric index of platelet in vitro aggregation in cerebrovascular disease

148 patients with various forms of cerebrovascular disease (CVD) were studied by means of a multiparametric analysis ofin vitro platelet aggregation, based on the following six parameters: ADP and epinephrine primary and secondary aggregation thresholds and percent maximum aggregation induced by optimal concentrations of ADP and epinephrine. These patients were assigned to four study groups, according to clinical diagnosis supported by CT scan, of transient ischemic attack and reversible neurological deficit (TIA-RIND), or completed stroke, in the presence or absence respectively of antiplatelet medical treatment at the time of the study. A statistically significant increase of thein vitro platelet aggregation was found in 44.4% of the untreated TIA-RIND patients and in 33.9% of the untreated stroke patients. However this last group showed a higher percentage of very marked hyperaggregation. Differences between the two treated study groups and controls were not signicant. No difference was found in collagen-and ristocetin-induced aggregation between the patient groups and the controls.

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Sommario 148 pazienti con varie forme di malattia cerebrovascolare, sono stati studiati con analisi multiparametriche dell'aggregazione piastrinica in vitro sulla base dei seguenti sei parametri: le soglie di aggregazione primaria e secondaria e l'aggregazione massima percentuale indotta da ADP ed Epinefrina. Questi pazienti sono stati suddivisi in 4 gruppi di studio in accordo con la diagnosi clinica confortata dai dati della TAC e cioè: TIA, RIND, o rammollimento in presenza o in assenza rispettivamente di un trattamento antiaggregante nel momento dello studio. è stato trovato un aumento statisticamente significativo dell'aggregazione in vitro delle piastrine nel 44.4% dei casi TIA, RIND non trattati e nel 33,9% dei casi di rammollimento non trattati. Quest'ultimo gruppo, però, ha dimostrato una più alta percentuale di iperaggregazione molto marcata. Le differenze tra i 2 gruppi di studio trattati con antiaggreganti e i controlli non erano significative. Inoltre nessuna differenza è stata riscontrata tra i gruppi e i controlli nell'aggregazione indotta da collageno e ristocetina.

     

高脂血症患者血小板体积及有关参数的研究

本文报道242例高脂血症患者血小板(PLT)、平均血小板数(MPLT)及平均血小板体积(MPV)等参数的测定结果,其中高胆固醇并高甘油三酯患者,PLT、MPLT及巨大血小板比例(Macro-PLT)高于正常,MPV正常;而单纯高胆固醇或高甘油三酯患者,上述指标与正常人无显著性差异。提示高胆固醇并高甘油三酯患者,存在血小板生成动力学异常,其血小板的破坏增加,生成率加速、但处于一种高水平的动态平衡之中。这可能是高脂血症出现高凝状态的原因之一。同时表明此类患者使用抗血小板疗法具有一定的合理性和必要性。     

特异性血小板抗体检测的实验研究

目的　建立一种简易、特异性强的血小板抗体检测方法。方法　利用磷酸氯喹溶液对血小板进行处理 ,去掉血小板相关抗原 (HLA抗原 ) ,用只含特异性抗原的血小板包被聚乙烯板来捕获血小板抗体。结果　与常用的SEPSA和淋巴细胞毒实验 (LCT)比较 ,该方法只表现血小板特异性抗体反应。结论　本方法能够鉴别血小板相关抗原的抗体和血小板特异性抗原的抗体 ,为临床血小板减少疾病的鉴别诊断、非溶血性发热反应和血小板输注无效的原因提供有用的参考依据