洗涤血小板各组分促人/鼠成纤维细胞增殖初探

目的 分析人血小板及其洗涤后获得的各组分补体灭活前后对小鼠成纤维细胞(L929)和人成纤维细胞(HDP)的增殖作用.方法 将所制备的新鲜人富血小板血浆(PRP)3份及血小板浓缩液(PC)5份(50 mL/份),分别用20 mL0.9％生理盐水洗涤2次,重悬以获得贫血小板血浆(PPP)、生理盐水上清、洗涤血小板(WP)3个组分,同未经处理的PRP及PC一道,分别冻融留取对照样品后作补体灭活处理,ELASA方法测试补体灭活前后血小板源性生长因子(PDGF-AB)、转化生长因子(TGF-β1)、血管内皮生长因子(VEGF)、表皮生长因子(EGF)含量,CCK-8试剂盒测定补体灭活前后对L929/HDP细胞的增殖作用.结果 PRP及洗涤各组分补体灭活前后PDGF-AB、VEGF及EGF含量比较,P＞0.05,PC及各组分补体灭活前后PDGF-AB、VEGF、EG及TGF-β1含量比较均无明显差异(P＞0.05).PRP及WP促L929细胞增殖比较:PRP为0.68±0.12 vs2.13±0.18(P ＜0.05),WP为0.69±0.12 vs 0.66±0.13(P ＞0.05);PC及WP促L929细胞增殖比较:PC为0.42±0.05 vs 1.94±0.19(P＜0.05),WP为1.69±0.13 vs 1.93±0.19 (P ＞0.05).补体灭活前后,PRP及WP促HDP细胞增殖:PRP为1.36±0.09 vs 1.30±0.11(P＞0.05),WP为1.26±0.04 vs 1.33±0.10(P ＞0.05);PC洗涤组分促细胞增殖:PC为1.32±0.07 vs 1.28±0.09 (e ＞0.05),WP为1.39±0.13 vs 1.44±0.13(P ＞0.05).结论 补体灭活对PRP与PC及洗涤获得的各组分PGFs含量影响较小,PGFs含量与促L929/HDP细胞的增殖作用存在非线性正相关关系,PGFs促细胞增殖 可能存在种间差异.     

血小板蛋白质组学的研究进展

人类血液中的血小板在各种生命过程中扮演重要角色,并广泛参与人体的各种生物过程,如血栓、炎症、伤口愈合和血栓形成等。血小板无细胞核,因而对它的研究无法采用传统的细胞和分子生物学方法。相比之下,蛋白质组学、基因组蛋白质等研究将对血小板的生物学功能研究产生重大的影响,且血小板蛋白质组学已被应用于解析血小板蛋白质组的各种复杂生物过程。本文对血小板蛋白质组学研究的最新进展作一综述,诸如蛋白质组学及其相关研究技术,与血小板相关的蛋白质组学及血小板蛋白质组学和疾病等。     

Validation of flow cytometric analysis of platelet function in patients with a suspected platelet function defect

Essentials

• The diagnosis of mild platelet function disorders (PFDs) is challenging.
• Validation of flow cytometric testing in patients with suspected PFDs is required.
• Flow cytometry has added value to light transmission aggregometry (LTA) in diagnosis of PFDs.
• There is fair agreement in diagnosing PFDs between LTA and flow cytometry.

Summary

Background

Light transmission aggregometry (LTA) is the most commonly used test for the diagnosis of platelet function disorders (PFDs), but has moderate sensitivity for mild PFDs. Flow cytometry has been recommended for additional diagnostics of PFDs but is not yet standardized as a diagnostic test. We developed a standardized protocol for flow cytometric analysis of platelet function that measures fibrinogen binding and P‐selectin expression as platelet activation markers in response to agonist stimulation.

Objectives

To determine the additional value of flow cytometric platelet function testing to standard LTA screening in a cross‐sectional cohort of patients with a suspected PFD.

Methods

Platelet function was assessed with flow cytometry and LTA in 107 patients suspected of a PFD in whom von Willebrand disease and coagulation factor deficiencies were excluded. Both tests were compared in terms of agreement and discriminative ability for diagnosing patients with PFDs.

Results

Out of 107 patients, 51 patients had an elevated bleeding score; 62.7% of the patients had abnormal platelet function measured with flow cytometry and 54.2% of the patients were abnormal based on LTA. There was fair agreement between LTA and flow cytometry (κ = 0.32). The discriminative ability of flow cytometric analysis in patients with an elevated bleeding score was good (AUC 0.82, 0.74–0.90), but moderate for LTA (AUC 0.70, 0.60–0.80). Both tests combined had a better discriminative ability (AUC 0.87, 0.80–0.94).

Conclusion

Flow cytometric analysis of platelet function has added value in diagnostics of PFDs in patients with unexplained bleeding tendency.     

Increased platelet activation and thrombosis in transgenic mice expressing constitutively active P2Y12

Summary. Background: In our previous in vitro study, we reported a constitutively active chimeric P2Y₁₂ (cP2Y₁₂) and found that AR‐C78511 is a potent inverse agonist at this receptor. The role of cP2Y₁₂ in platelet activation and thrombosis is not clear. Objectives: To investigate the physiologic implications of cP2Y₁₂ for platelet activation and thrombus formation, and to evaluate the antiplatelet activity of AR‐C78511 as an inverse agonist. Methods and Results:  We generated transgenic mice conditionally and platelet‐specifically expressing cP2Y₁₂. High‐level expression of cP2Y₁₂ in platelets increased platelet reactivity, as shown by increased platelet aggregation in response to multiple platelet agonists. Moreover, transgenic mice showed a shortened bleeding time, and more rapid and stable thrombus formation in mesenteric artery injured with FeCl₃. The constitutive activity of cP2Y₁₂ in platelets was confirmed by decreased platelet cAMP levels and constitutive Akt phosphorylation in the absence of agonists. AR‐C78511 reversed the cAMP decrease in transgenic mouse platelets, and exhibited a superior antiplatelet effect to that of AR‐C69931MX in transgenic mice. Conclusions:  These findings further emphasize the importance of P2Y₁₂ in platelet activation, hemostasis, and thrombosis, as well as the prothrombotic role of the constitutive activity of P2Y₁₂. Our data also validate the in vivo inverse agonist activity of AR‐C78511, and confirm its superior antiplatelet activity over neutral antagonists.     

Negative regulators of platelet activation and adhesion

Summary

Platelets are small anucleated cells that constantly patrol the cardiovascular system to preserve its integrity and prevent excessive blood loss where the vessel lining is breached. Their key challenge is to form a hemostatic plug under conditions of high shear forces. To do so, platelets have evolved a molecular machinery that enables them to sense trace amounts of signals at the site of damage and to rapidly shift from a non‐adhesive to a pro‐adhesive state. However, this highly efficient molecular machinery can also lead to unintended platelet activation and cause clinical complications such as thrombocytopenia and thrombosis. Thus, several checkpoints are in place to tightly control platelet activation and adhesiveness in space and time. In this review, we will discuss select negative regulators of platelet activation, which are critical to maintain patrolling platelets in a quiescent, non‐adhesive state and/or to limit platelet adhesion to sites of injury.

     

补体C3和C4与老年髋部骨折术前深静脉血栓形成的相关性及其诊断价值

摘要:目的探讨补体 C3和C4与老年髖部骨折术前深静脉血栓形成(DVT)的相关性及其诊断价值。方法连续纳入 北京积水潭医院老年髋部骨折患者并观察,行术前血管造影或彩超以筛查DVT并确诊DVT的患者共50例纳入DVT组,在未确诊DVT患者中以1:1的比例纳入与DVT组性别、年龄匹配的患者作为对照组(非DVT组)。用免疫比浊法测定血清C3和C4浓度并进行统计学分析;绘制ROC曲线以评估各指标对DVT的诊断价值。结果DVT 组、非DVT组血清C3浓度分别为( 1.162+0.223)g/L.(0.869+0.206)g/L;C4浓度分别为(0.307+0.070) g/L.(0.257+0.079)g/L, DVT组C3.C4水平均高于非DVT组(P均<0.05)。ROC曲线表明,C3的ROC曲线下面积(AUCRO)为0.839,当cut-off 值为0.893 g/L时，敏感性为70.0%(95%C1:56.3%~ 80.%) ,特异性为94.0% (95%CI:83.8% ~98.4%);C4的AUCROC为0.683,当cut-off值为0.267g/L时,敏感性为58.0%( 95%Cl:44.2%~ 70.6%) ,特异性为76.0% (95%CI:62.6% ~85.7%)。纳入C3、Fib .D-二聚体以及Caprini 评分构建回归模型计算的组合因子AUCHOC为0.943。结论补体 C3和C4与老年髋部骨折术前DVT相关联并具有诊断价值。     

川崎病患儿血小板α-颗粒膜蛋白及血小板参数变化的观察与探讨

目的探讨川崎病(Kawasaki Disease,KD)患儿急性期血小板α-颗粒膜蛋白(CD62P)、血小板参数的动态变化与本病发病机制及冠状动脉并发症之间的关系.方法应用荧光标记单克隆抗体和流式细胞仪以及自动血球记数仪分别对30例KD急性期患儿血清CD62P及50例KD急性期患儿血小板参数进行了检测,并与20例健康体检儿童进行对照.结果 KD组急性期血浆CD62P表达明显高于对照组(P<0.01),血小板计数(PLT)、血小板压积(PCT)、血小板分布宽度(PDW)水平也明显高于对照组(P<0.01).结论 KD急性期患儿血浆CD62P呈高表达,PLT、PCT、PDW水平明显增高,提示急性期KD患儿体内血小板代谢旺盛,释放因子较多,活化程度显著增高,表现为高聚集、高黏附,高凝固及低血浓度的血液流变特点;而此期也正是临床KD患儿动脉血栓及冠状动脉瘤形成的高峰期,提示活化的血小板参与或促发了冠状动脉血栓的形成;因此动态检测CD62P、血小板参数的变化,及时采取有效抗炎抗凝措施,对于减轻KD患儿血管炎症,防止血栓形成,预防冠状动脉并发症的发生具有重要的临床意义.     

血小板功能检测及其应用的研究进展

血小板在正常止血过程中十分重要,当血小板数量或功能发生变化时,机体易于发生出血或血栓。目前,检测血小板功能的方法日益增加,包括测定血小板粘附、聚集和活化的能力等。近年来随着对血小板激活机制的深入研究以及生物化学、免疫和分子生物学技术的不断发展,检测血小板的方法巳不断用于临床相关疾病的诊断和抗血小板药物治疗的研究。     

Impedance aggregometric analysis of platelet function of apheresis platelet concentrates as a function of storage time

Multiple electrode (impedance) aggregometry (MEA) allows reliable monitoring of platelet function in whole blood. The aims of the present study were to implement MEA for analyzing aggregation in platelet concentrates and to correlate results with storage time and blood gas analysis (BGA). We investigated the influence of platelet counts, calcium concentrations and agonists on platelet aggregation. Samples of apheresis concentrates up to an age of 12 days were investigated by MEA and BGA. For ASPI- and TRAPtest MEA was reproducible for a platelet count of 400 per 10^?9?L and a calcium concentration of 5?mmol L^?1. Platelets at the age of 2–4 days yielded steady aggregation. Platelet concentrates exceeding the storage time for transfusion showed steady aggregation up to 10 days, but a significant decline on day 12. Weak correlation was found regarding pCO₂ and MEA as well as regarding glucose concentration and MEA. Our results indicate that MEA is applicable for evaluation of aggregation in stored apheresis concentrates. Prolonged storage seems not to be prejudicial regarding platelet aggregation. Platelet concentrates showed acceptable BGA throughout storage time. Further studies are required to evaluate the application of MEA for quality controls in platelet concentrates.     

Platelet hyperreactivity and a prothrombotic phenotype in mice with a gain‐of‐function mutation in phospholipase Cγ2

Summary. Background: Agonist‐induced platelet activation involves different signaling pathways leading to the activation of phospholipase C (PLC) β or PLCγ2. Activated PLC produces inositol 1,4,5‐trisphosphate and diacylglycerol, which trigger Ca²⁺ mobilization and the activation of protein kinase C, respectively. PLCβ is activated downstream of Gq‐coupled receptors for soluble agonists with only short interaction times in flowing blood. In contrast, PLCγ2 becomes activated downstream of receptors that interact with immobilized ligands such as the collagen receptor glycoprotein (GP) VI or activated integrins. Objective and methods: We speculated that PLCγ2 activity might be optimized for sustained but submaximal signaling to control relatively slow platelet responses. To test this hypothesis, we analyzed platelets from mice heterozygous for a gain‐of‐function mutation in the Plcg2 gene (Plcg2^Ali5/+). Results: Plcg2^Ali5/+ platelets showed enhanced Ca²⁺ mobilization, integrin activation, granule secretion and phosphatidylserine exposure upon GPVI or C‐type lectin‐like receptor‐2 stimulation. Furthermore, integrin α_IIbβ₃ outside‐in signaling was markedly enhanced in the mutant platelets, as shown by accelerated spreading on different matrices and faster clot retraction. These defects translated into virtually unlimited thrombus formation on collagen under flow in vitro and a prothrombotic phenotype in vivo. Conclusions: These results demonstrate that the enzymatic activity of PLCγ2 is tightly regulated to ensure efficient but limited platelet activation at sites of vascular injury.     

Endogenous glutathione and platelet function in platelet concentrates stored in plasma or platelet additive solution

Platelet function was studied in platelet concentrates by assay of the thrombin-induced release of endogenous serotonin and presence of the swirling phenomenon in relation to endogenous glutathione (GSH) and cysteine. In platelets stored in plasma, addition of cysteamine resulted in only a moderate fall in GSH after 5 days of storage, from an average of 14.91 to 11.46 nmol per 109 platelets. Exogenously added GSH had no effect, and addition of buthionine sulfoximine (BSO) resulted in almost complete depletion of GSH, to an average of 0.65 nmol per 109 platelets. Addition of cysteamine or GSH resulted in increased endogenous cysteine whereas BSO had no effect. In platelets stored in a platelet additive solution (T-sol), complete depletion of GSH was found in the presence of cysteamine, GSH and BSO. Endogenous serotonin was unchanged during storage both in plasma and in additive solution (2.8 nmol per 109 platelets). Despite almost total depletion of endogenous GSH, the thrombin-induced release of serotonin after 5 days' storage was significantly affected only in the presence of BSO in platelets stored in additive solution (mean values 72.3% vs. 63.3% of endogeneous serotonin, P < 0.05). Similarly, addition of cysteamine or GSH had no significant effect on swirling but BSO reduced the swirling score after 5 days' storage in platelet additive solution compared with plasma. After 10 days' storage, there was a significant reduction in swirling in the concentrates where BSO was added (P < 0.05).     

Paired comparison of apheresis platelet function after storage in two containers

Platelet quality after storage strongly depends on the pre-storage quality as well as on the storage conditions determined by the storage container. In this paired study, we evaluated two different containers (MedSep CLX and Delmed DPL-110). The Fresenius AS104 cell separator was used to prepare 17 platelet concentrates that were split and distributed into the containers to be compared. Cell counts, blood gas analysis, morphological scores, glucose and lactate levels, platelet activation, and platelet aggregation were measured before splitting at the day of preparation and after storage at day 3 and day 5. At day 3, there was no significant difference between the two bags apart from increased lactate and decreased pCO(2) concentrations in the CLX bags. At day 5 there were significantly higher lactate concentrations, pO(2) levels, and aggregation after stimulation in the CLX group, while the glucose and pCO(2) concentrations were significantly lower in these platelet concentrates as compared to the DPL-110 group. However, these parameters did not influence the functional parameters tested. While the platelet quality decreased during storage in all bags, the functional changes were nearly identical in both bags tested. We conclude that both bags are equivalent for 5-day storage of platelet concentrates.     

Dynamics of platelet thrombus formation

Summary.  Platelet aggregation and thrombus formation at sites of atherosclerotic plaque rupture is a dynamic process that can lead to intermittent or permanent obstruction to blood flow, resulting in ischemic tissue injury and organ dysfunction. There is a growing body of evidence suggesting that the dynamics of platelet aggregation and initial thrombus development are regulated by two distinct, complementary processes, involving: (i) rheological (biomechanical) and (ii) soluble-agonist -dependent mechanisms. Rheological-dependent platelet aggregation occurs between discoid platelets and requires the biomechanical adhesive and signaling function (mechanotransduction) of the major platelet adhesion receptors, GPIb and integrin α_IIbβ₃. Soluble agonists further potentiate platelet activation, stimulating global platelet shape change and degranulation, and play a major role in stabilizing formed aggregates. Unraveling the dynamics of platelet aggregation and thrombus formation in vivo requires consideration of the cooperative interplay between rheological- and soluble agonist-dependent platelet aggregation mechanisms.     

溃疡性结肠炎患者体内血小板功能状态研究

目的 探讨溃疡性结肠炎患者血小板功能状态。方法 对28例活动期溃疡性结肠炎患者、ll例缓解期溃疡性结肠炎患者、30例正常对照者的血小板黏附功能(PAdT)、血小板的聚集功能(PAgT)、血栓素B2(TXB2)进行检测。结果 28例活动期溃疡性结肠炎患者血小板的黏附、聚集功能、血栓素B2均显著高于缓解期溃疡性结肠炎患者和正常对照组(P＜0．05)；缓解期溃疡性结肠炎患者与正常对照组比较差异无统计学意义(P＞0．05)。结论 活动期溃疡性结肠炎患者体内血小板的活性增强，提示血小板的活化直接参与了结肠黏膜的急性炎症。     

丹七胶囊对血小板聚集功能影响的临床研究

目的通过研究丹七胶囊对血小板聚集功能的影响,探讨丹七胶囊影响血小板聚集功能的生理机制。方法32名健康志愿者随机均分为空白对照组和低剂量、高剂量和超高剂量组,3个剂量组连续口服丹七胶囊(空白对照组服用安慰剂)1周,每日3次,于服药1周后采集空腹静脉血,检测血常规、凝血常规、生化常规和血小板最大聚集率(MA),诱导剂分别为二磷酸腺苷(ADP)、肾上腺素(EPI)、花生四烯酸(AA)、瑞斯托霉素(RIS)。结果服药1周后,3个剂量组的血常规、凝血常规、生化常规指标比较差异均无统计学意义(P>0.05)。高剂量组和超高剂量组中ADP和EPI诱导的MA较空白对照组下降,差异有统计学意义(P<0.05)。结论丹七胶囊可降低ADP和EPI诱导的血小板聚集功能,其可能通过抑制血小板腺苷酸环化酶活性发挥其抗血小板作用。     

Rho GTPases in platelet function

Summary. The Rho family of GTP binding proteins, also commonly referred to as the Rho GTPases, are master regulators of the platelet cytoskeleton and platelet function. These low‐molecular‐weight or ‘small’ GTPases act as signaling switches in the spatial and temporal transduction, and amplification of signals from platelet cell surface receptors to the intracellular signaling pathways that drive platelet function. The Rho GTPase family members RhoA, Cdc42 and Rac1 have emerged as key regulators in the dynamics of the actin cytoskeleton in platelets and play key roles in platelet aggregation, secretion, spreading and thrombus formation. Rho GTPase regulators, including GEFs and GAPs and downstream effectors, such as the WASPs, formins and PAKs, may also regulate platelet activation and function. In this review, we provide an overview of Rho GTPase signaling in platelet physiology. Previous studies of Rho GTPases and platelets have had a shared history, as platelets have served as an ideal, non‐transformed cellular model to characterize Rho function. Likewise, recent studies of the cell biology of Rho GTPase family members have helped to build an understanding of the molecular regulation of platelet function and will continue to do so through the further characterization of Rho GTPases as well as Rho GAPs, GEFs, RhoGDIs and Rho effectors in actin reorganization and other Rho‐driven cellular processes.     

Role of mitochondria in the maintenance of platelet function during in vitro storage

Aims/Objectives: Platelets undergo structural and biochemical alterations during in vitro storage and these are collectively called platelet storage lesions (PSL). The mitochondrion is an important cell organelle involved not only in energy production but also in the regulation of cellular functions and viability. This implies that some platelet functions may be regulated by mitochondria; hence, preservation of mitochondrial functions may be important for the maintenance of platelet quality in stored platelet concentrates (PCs). This work describes the effects of various compounds on mitochondrial functions important for the maintenance of platelet quality in in vitro stored PCs. Methods: PCs were stored at 22 °C with gentle agitation in the presence or absence of 2,4‐dinitrophenol, antimycin A, acetyl‐l ‐carnitine and ascorbic acid. The effects of these products on platelet quality were assessed by analysing glucose and lactate concentrations, pH of the storage medium, shape of the platelets, mitochondrial membrane potential and depolarisation, surface expression of CD62P and collagen‐induced platelet aggregation. Results: 2,4‐Dinitrophenol and antimycin A increased PSL levels, whereas acetyl‐l ‐carnitine reduced the level of changes in pH and mitochondrial depolarisation. Ascorbic acid in the storage medium resulted in improved levels of collagen‐induced platelet aggregation. However, none of the examined reagents suppressed CD62P expression in platelets. Conclusions: These results suggest that preservation of mitochondrial function is fundamental, but not fully sufficient, for the maintenance of platelet in vitro quality during storage. Further research is necessary to develop methods for preserving both mitochondrial and platelet functions in in vitro stored PCs.     

Vitronectin stabilizes thrombi and vessel occlusion but plays a dual role in platelet aggregation.

The role of vitronectin (Vn) in thrombosis is currently controversial; both inhibitory and supportive roles have been reported. To monitor directly the function of Vn in thrombotic events at the site of vascular injury, we studied Vn-deficient (Vn-/-) and wild-type (WT) control mice with two real-time intravital microscopy thrombosis models. In the mesenteric arteriole model, vessel injury was induced by ferric chloride. We observed unstable thrombi and a significantly greater number of emboli in Vn-/- mice. Vessel occlusion was also delayed and frequent vessel re-opening occurred. In the cremaster muscle arteriole model, vessel injury was induced by a nitrogen dye laser. We observed significantly fewer platelets, lower fibrin content, and unstable fibrin within the thrombi of Vn-/- mice. To define further the role of Vn in thrombus growth, we studied platelet aggregation in vitro. Consistent with our in vivo data, the second wave of thrombin-induced aggregation of gel-filtered platelets was abolished at a low concentration of thrombin in Vn-/- platelets. Interestingly, adenosine diphosphate (ADP)-induced platelet aggregation was significantly increased in Vn-/- platelet-rich plasma (PRP) and this effect was attenuated by adding purified plasma Vn. We also observed increased platelet aggregation induced by shear stress in Vn-/- whole blood. These data demonstrate that Vn is a thrombus stabilizer. However, in contrast to released platelet granule Vn which enhances platelet aggregation, plasma Vn inhibits platelet aggregation.