全血电阻法检测血小板聚集程度对抗血小板治疗效果的评价

目的研究全血电阻法检测血小板聚集程度对抗血小板治疗效果的评价。方法比较全血电阻法和经典的血浆光学法测定血小板聚集程度的重复性。采用全血电阻法，分别以二磷酸腺苷（ADP）、花生四烯酸和胶原为诱导剂，检测30名健康志愿者和60例服用血小板聚集抑制剂的患者的血小板聚集程度。采用Kappa分析比较全血电阻法和经典的血浆光学法检测血小板聚集程度的一致性。结果全血电阻法的平均CV值为4．8％，优于血浆光学法（CV值为7．7％）。采用全血电阻法，分别以ADP、花生四烯酸和胶原为诱导剂，健康志愿者的平均聚集程度分别为（9．6±3．4）Ω、（10．4±2．8）Ω和（13．5±2．2）Ω。服用阿司匹林（100mg／d）的患者，以花生四烯酸为诱导剂，95％的患者血小板聚集程度为0Ω。服用血小板聚集抑制剂患者的血小板聚集程度显著低于健康志愿者（ADP为诱导剂，t=2．391，P〈0．05；花生四烯酸为诱导剂，t=4．057，P〈0．01；胶原为诱导剂t=2．047，P〈0．05）。同时服用阿司匹林和氯吡格雷两种抑制剂的患者的血小板聚集程度显著低于单独服用阿司匹林的患者（t=3．646，P〈0．01）。另外，以花生四烯酸为诱导剂，阿司匹林抵抗的患者的血小板聚集开始时间，有比健康志愿者延长的特点。经Kappa分析，表明全血电阻法和经典的血浆光学法两种方法检测患者对阿司匹林（Kappa=1）和氯吡格雷（Kappa=0．67）两种血小板聚集抑制剂的效果的一致性良好。结论采用全血电阻法和多种诱导剂测定血小板聚集能力对帮助临床判断抗血小板治疗的效果，辅助个体化用药是很有意义的。     

血小板激活时其胞质内钙离子变化的测定

目的建立测定血小板激活时细胞内钙离子变化的方法。方法摸索荧光染料Ｆｌｕｏ－３染色血小板内钙离子的适宜条件，应用流式细胞仪测定标记了Ｆｌｕｏ－３／ＡＭ的血小板激活后细胞内钙离子的变化。结果Ｆｌｕｏ－３标记血小板时，随着染料浓度增加和孵育时间延长，基线荧光强度和钙离子载体Ａ２３１８７刺激后的最大荧光强度均提高，但比值基本一致，不受染料条件的影响。无论二磷酸腺苷（ＡＤＰ）还是凝血酶作诱导剂时，染色和未染色血小板之间的最大聚集率均无差异。ＡＤＰ可引起血小板内钙离子增高，并随着ＡＤＰ浓度增加，钙离子荧光强度增强。结论建立了流式细胞仪测定血小板激活时细胞内钙离子变化的方法。     

反复脑梗死患者服阿司匹林后血小板聚集功能的变化

目的比较反复脑梗死患者同一次脑梗死患者服用阿司匹林后血小板聚集功能的差异，来探讨阿司匹林预防脑血管方面的作用。方法40名至少二次脑梗死的患者和28名一次脑梗死的患者服用阿司匹林（100mg／d）至少7天以上，用二磷酸腺苷和花生四烯酸作诱导剂，测定血小板聚集功能。结果花生四烯酸作诱导剂时，反复脑梗死患者同一次脑梗死患者的血小板聚集功能有显著差异（P〈0．01）。结论服用阿司匹林需检测血小板聚集功能，反映阿司匹林能否有效预防脑梗死复发。     

人血小板冻干前负载海藻糖技术的研究

为研究人血小板冻干前负载海藻糖技术与方法，筛选出最佳负载海藻糖实验条件并进一步研究血小板在温度37℃条件下、氪载海藻糖4小时后的平均体积、体外激活程度和聚集反应性的变化，绘制血小板胞内海藻糖负载效率及浓度随温度、时间、胞外海藻糖浓度变化曲线，筛选合适的负载条件，分别以凝血酶、ADP、胶原、瑞斯托霉素4种物质作为血小板激活诱导剂，用血小板聚集仪分别检测血小板负载海藻糖前后的聚集反应性，用流式细胞仪检测分析血小板负载海藻糖前后其膜表面糖蛋白分子CD62p、PAC-1的表达率，加可逆性激活抑制剂PGE-1、腺苷后血小板激活被抑制的程度。结果表明：海藻糖负载效率与孵育时间（2小时后）、温度（30-40℃）呈良好线性关系，在37℃条件下经4小时孵育后负载效率可达60％，载入到胞内海藻糖浓度随胞外海藻糖浓度（〈50mmol／L）的升高而递增；同负载前相比较，血小板平均体积（MPV）、血小板对4种激活诱导剂的最大聚集率均无显著性差别（P〉0.01），经4小时负载海藻糖后血小板膜表面CD62p表达率升高，但联合添加可逆性激活抑制剂PGE-1、腺苷后CD62p表达率显著下降。结论：37℃、4小时、胞外海藻糖浓度小于50mmol／L为合适负载条件，添加可逆性血小板激活抑制剂后．冻干前负载海藻糖过程对血小板的体外激活和聚集活性没有显著影响．     

血小板激活时其胞质内钙离子变化的测定

建立测定血小板激活时细胞内钙离子变化的方法。摸索荧光染料Ｆｌｕｏ－３染色血小板内钙离子的适宜条件，应用流式细胞仪测定标记了Ｆｌｕ－３／ＡＭ的血小板激活后细胞内钙离子的变化。结果Ｆｌｕｏ－３标记血小板时，随着染料浓度增加孵育时间延长，基线荧光强度和钙离子载体Ａ２３１８７刺激后的最大荧光强度均提高，但比值基本一致，不受染料条件的影响。     

比浊法血小板聚集试验的影响因素研究

目的 在不同的实验条件下,观察影响比浊法血小板聚集试验的因素.方法 选择30例健康对照组和204例病例组,观察不同的血小板计数及诱导剂浓度对糖尿病患者餐前与餐后、采血后的检测时间、诱导剂的种类互补等不同试验条件下的血小板聚集率.结果 随着血小板计数的减少或增加,血小板聚集率相应的减少或增加;二磷酸腺苷(ADP)、胶原(COL)、花生四烯酸(AA)浓度增加,血小板最大聚集率增大,相应的阿司匹林抵抗(AR)或氯吡格雷抵抗(CR)的检出率增高;服用不同的抗血小板药物,糖尿病患者对以ADP、COL、AA为诱导剂测定的血小板聚集率,其影响不同;未空腹及检测时间超过3 h重复性较差;服药2周后,COL、AA诱导的血小板聚集率下降不明的患者,随着时间的延长,6个月后易重新出现CR.结论 血小板计数、诱导剂浓度、糖尿病疾病、空腹状态、检测时间与比浊法检测血小板聚集率相关,以ADP、COL、AA作诱导剂检测血小板聚集率较单一的ADP更全面、准确.     

不同浓度血小板激活剂激活血小板微粒膜表达PAC-1、CD62p的比较

目的　比较不同浓度的激活剂激活血小板形成的血小板微粒 (PMP)膜表达PAC 1、CD6 2p的差异。方法　抽取 10例健康人静脉血 ,以枸橼酸钠抗凝 ,离心得富血小板血浆 ,分别以不同浓度的ADP(5、10、2 0 μmol/L)、凝血酶 (0 .1、0 .5、1.0U/ml)、胶原 (5、10、2 0 μg/ml)作诱导剂 ,激活同一标本中的血小板 ,比较其PMP表达PAC 1和CD6 2p的情况。 结果　随着激活剂浓度的增加 ,CD6 2p PMP、PAC 1 PMP的百分率都逐渐增加 ,同一诱导剂各浓度间有显著性差异 (P <0 .0 1)。结论　不同刺激强度的血小板诱导剂ADP、凝血酶、胶原 ,随浓度的增加 ,其诱导血小板所形成的CD6 2p PMP、PAC 1 PMP的百分率逐渐增加。     

ADP诱导的血小板内钙离子浓度变化的研究

ADP被认为是动脉血栓形成的一个重要因素。在体外，ADP可诱发血小板内Ca2 浓度增高，为了探讨其机制及在临床诊断中的价值，我们应用流式细胞仪[1]和激光扫描共聚焦显微镜[2]观察负载ca2 荧光深外Fluo-3／AM的血小板在ADP作用下细胞内Ca2 变化。材料和方法1材料1．1试剂1．1．IADP（上海生物化学试剂厂产品），聚乙烯亚胺（日本TsurogaWomen’sJunior学院HiroakiNishio博士惠赠），A23187（美国Sigma公司），EGTA（美国Ser－va公司产品）。这些试剂均用不含Ca’”、Mg’”的PBS缓冲液配制。1．1．ZnU。－3／＊M（美国分子探…     

尼莫地平和阿司匹林对急性脑梗死患者GMP-140和TXB_2的影响

目的探讨急件脑梗死（ＡＩ）与血小板活化和花生四烯酸代谢的关系，以及尼莫地平和阿司匹林干预的影响。方法２８例ＡＩ患者随机分为尼莫地平组１０例，阿司匹林组８例和对照组１０例。所有患者于发病当大、第２、３和７天采血，测定血小板表面ａ－颗粒胰蛋白（ＧＭＰ—１４０）分子数、血浆内ＧＭＰ—１４０含量和血栓烷Ｂ２（ＴＸＢ２）浓度。结果ＡＩ患者发病当大血小板表面ＧＭＰ—１４０分子数和血浆ＧＭＰ－１４０浓度均无显著升高。而在发病后第２、３人对照组明显升高，第７大趋于正常。尼莫地平组第２、３天升高程度明显低于对照组，而阿司匹林组与对照组比较无显著差异。对照组血浆ＴＸＢ２浓度于发病当天升高，然后逐步下降，阿司匹林组下降显著，而尼莫地个组与对照组比较无差异性。结论　钙拮抗剂可以干预 ＡＩ 后血小板活化程度，对花生四烯酸代谢无显著影响，阿司匹林可影响花生四烯酸代谢，但对血小板活化无显著影响。     

不同血小板激活剂在薄片法聚集实验中的应用比较

本研究的目的是筛选和评价可用于薄片法血小板聚集试验的血小板激活剂。应用下列方法进行实验 :①测定常用血小板激活剂 ,包括ADP、胶原、肾上腺素、花生四烯酸和瑞斯托霉素及阳离子没食子酸丙酯溶液 (c PG)在塑料薄片上诱导 15名健康献血者血小板聚集强度和时间 ;②测定添加血小板抑制剂PGI2 、cAMP或EDTA前后常用血小板激活剂及c PG在诱导 15名健康献血者血小板聚集时间 ;③测定不同浓度肝素对c PG诱导的 15名健康献血者小板聚集时间的影响 ;④检测c PG诱导健康献血者不同浓度血小板的聚集强度和时间 ;⑤检测c PG诱导服用阿司匹林患者血小板聚集时间。结果表明 :①在塑料薄片上c PG诱导血小板的聚集强度最强 ,肉眼清晰可见 ,需要时间较短 ;②瑞斯托霉素、花生四烯酸和c PG均可检测出抑制剂PGI2 和cAMP对血小板的抑制 ,其中c PG检测能力相对最强 ;③ 0 .5 - 3U ml肝素对c PG诱导的血小板聚集时间无明显影响 ;④健康献血者血小板稀释至 30× 10 9 L仍可被c PG诱导出明显的肉眼可见的聚集 ;⑤c PG诱导服用阿司匹林患者血小板聚集时间明显延长。结论 :与常用血小板激活剂比较 ,c PG应用于薄片法血小板聚集试验中具有明显优势。     

Effect of common agonists on cytoplasmic ionized calcium concentration in platelets. Measurement with 2-methyl-6-methoxy 8-nitroquinoline (quin2) and aequorin

Because of controversy regarding the relationship of cytoplasmic ionized calcium concentration ([Cai2+]) to platelet activation, we studied the correlation of platelet aggregation and ATP secretion with [Cai2+] as determined by 2-methyl-6-methoxy 8-nitroquinoline (quin2) and aequorin in response to ADP, epinephrine, collagen, the Ca2+ ionophore A23187, and thrombin. Both indicators showed a concentration-dependent increase in [Cai2+] in response to all agonists except epinephrine when gel-filtered platelets were suspended in media containing 1 mM Ca2+. With epinephrine, a rise in [Cai2+] was indicated by aequorin, but not by quin2; [Cai2+] signals, aggregation, and secretion were suppressed by EGTA. ADP [0.5 microM] produced a rise in [Cai2+] that was registered by both aequorin and quin2 in platelets in Ca2+-containing media; addition of EGTA to the medium raised the threshold concentration of ADP to 5.0 microM for both indicators. Collagen produced progressive concentration-related increases in [Cai2+] and aggregation in aspirin-treated aequorin-loaded platelets. Quin2 failed to indicate a rise in [Cai2+]at lower collagen concentrations with EGTA or aspirin. [Cai2+] response to A23187 and thrombin was reduced by addition of EGTA to platelets loaded with either aequorin or quin2. With all five agonists in all conditions tested, aequorin [Cai2+] signals occurred at the same agonist concentration as that or lower than that which produced platelet shape change, aggregation, or secretion. Platelet activation was better correlated with changes in [Cai2+] indicated by aequorin than with the response of quin2, possibly because aequorin is more sensitive to local zones of [Cai2+] elevation.     

Hydrogen peroxide triggers activation of human platelets selectively exposed to nonaggregating concentrations of arachidonic acid and collagen.

The effects of H2O2 on platelet function were investigated in vitro and ex vivo. H2O2 (0.5 to 5 mumol/L) alone did not influence platelet function, but when it was combined with subthreshold concentrations of arachidonic acid or collagen, it induced platelet aggregation and serotonin release in a dose-dependent fashion. The increase in platelet aggregation was associated with thromboxane A2 production and was prevented by 100 mumol/L aspirin. The amplification of platelet response by H2O2 was also inhibited 2 hours after 300 mg aspirin was given to healthy subjects. H2O2 alone did not affect intraplatelet Ca++ influx or mobilization but, combined with subthreshold concentrations of arachidonic acid, it increased Ca++ mobilization. In platelets prelabeled with tritiated arachidonic acid, H2O2 induced tritium release in a dose-dependent fashion; this effect was prevented by mepacrine, an inhibitor of the phospholipase A2 enzyme. Platelet function was not affected by using H2O2 in combination with other agonists such as thrombin, calcium ionophore, or adenosine diphosphate. This study suggests that H2O2 triggers activation of platelets preexposed to agonists at subthreshold levels by stimulating arachidonic acid metabolism, likely by stimulating the phospholipase A2 enzyme. The stimulation of platelets by concentrations of H2O2 similar to those released by activated leukocytes may give new insights into the functional cooperation between leukocytes and platelets.     

Pathways responsible for platelet hypersensitivity in rats with diabetes. I. Streptozocin-induced diabetes

Several pathways are activated when platelets aggregate and undergo the release reaction. We have examined the relative importance of these pathways in the responses to adenosine diphosphate (ADP), thrombin, or collagen of washed platelets from rats with diabetes induced by streptozocin. ADP-induced aggregation was enhanced without the release reaction with platelets from diabetic rats. Collagen-induced aggregation and release, and the adherence of platelets to collagen-coated glass were similar with platelets from diabetic and control rats. Thrombin (1 U/ml) induced more extensive loss of tritium from 3H-arachidonic acid-labeled platelets from diabetic rats than from control rats. Platelet aggregation and the release of 14C-serotonin from prelabeled platelets was greater in response to low concentrations of thrombin (0.04 U/ml). Creatine phosphate-creatine phosphokinase (CP/CPK) and aspirin completely blocked aggregation and partially blocked the release of granule contents from platelets from control and diabetic rats exposed to this low concentration of thrombin. Thus, the enhanced platelet aggregation in response to low concentrations of thrombin was likely mediated in part by released ADP and products formed from arachidonate. In contrast, with a higher concentration of thrombin (0.0625 U/ml), CP/CPK and aspirin did not inhibit the increased sensitivity of diabetic platelets to thrombin-induced aggregation and release; the concentrations of CP/CPK completely blocked aggregation induced by ADP (10 mumol/L), and the aspirin inhibited thromboxane B2 production in response to thrombin (1 U/ml) by 99%. Thus, a thrombin-induced pathway(s) of aggregation and release independent of released ADP and the products of arachidonate metabolism is enhanced in platelets from diabetic rats.     

Clinical and preclinical pharmacology of KC-764, a novel antiplatelet agent]

Preclinical pharmacological studies showed that KC-764 was more potent and more selective in inhibiting platelet aggregation than aspirin. The concentration of KC-764 for inhibiting PGI2 production in the aorta was 70 times higher than that for inhibiting TXA2 in platelets. Antiplatelet action of KC-764 was augmented by plasma components. This augmentation by plasma may lead to selective antiplatelet activity. KC-764 has been investigated for platelet function in patients with chronic cerebral infarction. KC-764 at 10, 20 and 40 mg b i d, inhibited platelet aggregation induced by arachidonic acid, collagen, and ADP, and its potency was almost equal to aspirin at 100-330 mg daily. Plasma TXB2 levels were markedly depressed by KC-764 but plasma 6-keto-PGF1 alpha levels were not influenced. On the contrary, aspirin depressed both plasma prostanoids. These findings suggest that KC-764 can overcome the 'aspirin dilemma'.     

The regulatory mechanism of free Ca2+ concentration in activated platelets]

The change in intracellular Ca2+ concentration ([Ca2+]i) following platelet stimulation results from mobilization, influx and restoration of Ca2+. To determine whether inositol 1,4,5 trisphosphate (IP3) is involved in Ca2+ influx, the relationship between IP3 formation (IP3) and Ca2+ influx ( delta [Ca2+]i) was investigated in platelets stimulated wtih various agonists (thrombin, ADP, PAF, STA2, etc). The ratio of IP3 to delta [Ca2+]i varied among the agonists, although delta [Ca2+]i was increased, depending on the amount of agonist. Furthermore, in spite of the similar delta [Ca2+]i, IP3 was smaller at 20 degrees C compared with that at 37 degrees C in thrombin-stimulated platelets. These results indicate that Ca2+ influx in platelets might be regulated by receptor-operated Ca2+ channel rather than by an IP3 mediated mechanism. As for Ca2+ restoration, calpain was demonstrated to play a role through Ca(2+)-ATPase activation by limited proteolysis.     

Differences in thromboxane A2 synthesis by megakaryocytes and platelets

The capacity for thromboxane A2 synthesis in response to exogenous arachidonic acid, calcium ionophore A23187, thrombin, and collagen was studied during megakaryocyte maturation. Studies were performed in (1) isolated megakaryocytes not separated, (2) isolated megakaryocytes separated into subgroups at different stages of maturation, and (3) washed platelets. When comparisons were based on equal amounts of cell protein (10(5) megakaryocytes vs 10(8) platelets), isolated megakaryocytes, not separated into subgroups, responded to exogenous arachidonic acid with synthesis of thromboxane A2 equal to that of platelets from the same animals at their respective times of maximum synthesis (30 minutes vs 10 minutes). In similar fashion, megakaryocytes and platelets synthesized thromboxane A2 from endogenous arachidonic acid at the same minimum concentration of A23187, 0.1 mumol/L, and showed equal maximum synthesis at 1 mumol/L (167 +/- 9 pmol and 150 +/- 18 pmol, respectively). In contrast, maximum thromboxane A2 synthesis in response to thrombin (10 U/ml) was three times higher in platelets than in megakaryocytes (230 +/- 15 pmol and 74 +/- 5 pmol, respectively), and synthesis in response to collagen (20 micrograms/ml) was 20 times higher in platelets (130 +/- 20 pmol vs 7 +/- 1.2 pmol). When synthesis was studied in isolated megakaryocytes at different stages of maturation, the capacity for thromboxane A2 synthesis was established in immature megakaryocytes but was not fully developed in the most immature megakaryocytes. Synthesis in response to thrombin was not significantly enhanced by megakaryocyte maturation. Thus the ability to metabolize arachidonic acid occurs early during megakaryocyte maturation, but the ability to respond to thrombin and collagen is only fully established in platelets.     

Pathways responsible for platelet hypersensitivity in rats with diabetes. II. Spontaneous diabetes in BB Wistar rats

The discovery of a group of spontaneously diabetic rats has made it possible to examine changes in diabetic animals in the absence of possible confounding toxic effects of diabetogenic agents. The responses of washed platelets to adenosine diphosphate (ADP), thrombin, or collagen have been compared with platelets from spontaneously diabetic rats (these rats were hyperglycemic), their nondiabetic littermates (normoglycemic), and control rats from the same colony. Platelets from the diabetic rats aggregated more extensively in response to ADP than did platelets from the nondiabetic littermates or control animals. In contrast, platelet aggregation and release of granule contents in response to a low thrombin concentration (0.05 U/ml) were greater with platelets from diabetic rats and nondiabetic littermates than with platelets from control rats. A similar effect of collagen on the release of platelet serotonin was observed. Except at low concentrations of thrombin, the enhanced sensitivity to thrombin-induced aggregation and release of granule contents from platelets from diabetic rats or their nondiabetic littermates could not be inhibited by creatine phosphate-creatine phosphokinase (CP/CPK) and aspirin (CP/CPK used at concentrations that inhibited aggregation induced by ADP [10 mumol/L] and aspirin at concentrations that inhibited thromboxane B2 production induced by thrombin [1 U/ml] by 99%). Loss of radioactivity from platelets labeled with 3H-arachidonic acid and the amount of thromboxane B2 formed in response to high concentrations of thrombin (1 U/ml) was greater from platelets from the diabetic rats or their nondiabetic littermates than from control animals. Thus the effect of diabetes on this aspect of arachidonate metabolism is not primarily determined by blood glucose levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synergism of platelet-aggregating agents. Role of elevation of cytoplasmic calcium.

When a pair of platelet agonists, each in subthreshold concentration, is added together or in sequence to a platelet suspension, the platelet response is enhanced. Addition of two agonists to platelets loaded with aequorin also enhanced the observed rise in cytoplasmic ionized calcium ([Ca2+]i) in response to the second agonist if the agonists were added within 20 s of each other. Enhancement of aggregation and secretion required that an increase in [Ca2+]i (as indicated by aequorin but not necessarily indo-1) followed the first agonist, but not that the [Ca2+]i remain elevated until addition of the second agonist. Enhancement was not prevented by aspirin, ADP scavengers, or chelators of extracellular Ca2+. We conclude that a rise in [Ca2+]i induced by a first agonist "primes" platelets for an augmented functional response to a second agonist, which is not, however, determined by the [Ca2+]i at the time of addition of the second agonist.     

Platelet dysfunction in uremia. II. Correction by arachidonic acid of the impaired exposure of fibrinogen receptors by adenosine diphosphate or collagen

Previous studies showed that platelets from patients with uremia have a marked decrease in their aggregation response to adenosine diphosphate (ADP) and collagen as single agents or as a pair. It is known that small amounts of arachidonic acid can enhance the sensitivity of platelets to concentrations of ADP or collagen that do not cause aggregation when used singly. Stimulation of platelets by certain agonists induces the formation of fibrinogen receptors on the platelet surface. The binding of fibrinogen that follows is essential for platelet aggregation. The platelet membrane glycoprotein IIb-IIIa complex appears to be the site of the fibrinogen receptor. Therefore, we investigated the binding of iodine 125-labeled fibrinogen to uremic platelets exposed to ADP, collagen, or arachidonic acid as single agents and as pairs. When aggregation and binding were studied in response to ADP, collagen, or the combination of ADP with collagen, uremic platelets had reduced aggregation and bound abnormally low amounts of fibrinogen. In contrast, platelets from patients with uremia bound as much 125I-fibrinogen and aggregated as well as controls when ADP or collagen were used in combination with low concentrations of arachidonic acid. Studies with a monoclonal antibody (B 79.7) suggested that the number of glycoprotein IIb-IIa molecules is the same in uremic and normal platelets. We conclude that uremia impairs the exposure of fibrinogen receptors on platelets in response to ADP or collagen without affecting the glycoprotein IIb-IIa complex quantitatively. Correction by arachidonic acid of the impaired aggregation and exposure of fibrinogen receptors by ADP or collagen suggests that abnormal release of endogenous arachidonic acid plays a role in the dysfunction of platelets in uremia.