图片显示了动脉中形成的血块。 在红细胞和白细胞之间显示出白色的尖状血小板。 活化的血小板利用其尖刺状的“手臂”相互抓住，并与血液中称为纤维蛋白的蛋白质链相连，形成团块，也将红细胞和白细胞结合在一起。 一项实验研究表明，一种在人体血液样本中检测血小板功能的新技术比目前使用的方法更快、更容易、更精确。 《自然生物医学工程》发表了这项由埃默里大学科学家领导的研究。 研究人员展示了这项技术的概念验证，首次详细观察了患者血液样本中活化血小板产生的分子力。 研究结果表明，这项技术有可能评估抗血小板药物对个体的影响，并更清楚地了解接受体外循环手术的患者的出血风险。 这项技术只需要大约一滴血就可以进行检测，而目前的检测只需要一汤匙。 这种超敏性可能使该技术成为诊断患有罕见先天性血小板疾病的婴儿的有价值的工具。 这一突破是基于十多年前在埃默里化学系和华莱士大学教授khalidsalaita实验室开发的合成dna张力探针。 埃默里大学和乔治亚理工大学的库尔特生物医学工程系。 “科学家的梦想”张力探测器可以探测到只有几皮牛顿大小的细胞力，大约是回形针重量的10亿倍。 研究人员发现了一种方法，通过利用一种名为crispr-associated 12a的酶的能量来放大探针的信号。 然后使用读板器检测机械信号，这是一种已经在临床测试中常规使用的工具。 “这个项目是出于基本的好奇心，”共同通讯作者salaita说。 “我们想知道我们是否可以测量细胞施加的最微小的力。 令人兴奋的是，我们现在正基于这种基本的好奇心开发诊断工具来帮助患者。 这是科学家的梦想。 这篇论文的第一作者是美国心脏协会salaita实验室的博士后段玉欣。 埃默里医学院麻醉学系教授、围手术期凝血领域的顶尖专家罗曼·斯尼钦斯基是这篇论文的共同通讯作者。 斯涅辛斯基说：“一般来说，血小板功能很重要，但目前我们用来测量血小板功能的工具相对来说还是比较原始的。”。 “这项新技术提供了一种更容易、更快、更便宜的测量血小板功能的方法，同时也为我们提供了以前没有的关键信息。 合著者包括：斯涅辛斯基实验室的高级助理fania szlam；胡岳松，萨拉塔实验室的研究生；李仁浩，埃默里医学院儿科、血液学/肿瘤学系教授；陈文春，埃默里医学院博士后；以及埃默里大学和乔治亚理工大学库尔特生物医学工程系副教授柯永刚。 血小板的重要性血小板是无色的盘状血细胞碎片，其作用是结合在受伤血管的部位止血。 然而，在某些情况下，血小板可能无法发挥最佳功能。 当血小板较弱或活性低于最佳水平时，血液可能无法正常凝结，导致出血失控。 但如果血小板“过度活跃”，它们可能会变得过于粘稠，导致自发血栓，从而导致心脏病发作或中风。 调节血小板功能对某些疾病的高危人群尤其重要。 抗血小板药物，如氯吡格雷、替卡格雷甚至阿司匹林，是美国最常见的处方药。 然而，对一些患者来说，这些药物可能效果不佳，调整剂量或改用其他药物可能有助于更好地预防心脏病发作。 在心脏手术中，血小板功能变得更加失调。 手术室团队必须在体外循环过程中进行平衡，使血液不凝结，然后在手术结束时使用促凝干预措施，包括输注血小板，来止血。 这可能很困难，因为使用体外循环机会使血小板紧张和减弱。 斯涅辛斯基说：“几十年来，人们一直在科学文献中写到心脏手术中血小板功能障碍的问题，但用我们一直使用的工具来测量它真的很困难。”。 由于我们无法很好地测量血小板功能，因此很难用有效的方法进行研究。 “‘图中的一小部分’血糖仪是目前用于评估血小板功能的标准工具。 它测量血液样本中血小板聚集或聚集的速度和程度。 斯涅辛斯基说：“这些数据只提供了血小板功能的一小部分，并不是最有趣的部分。”。 他解释说，当血小板被激活时，它会改变形态并长出微小的假臂。 血小板利用这些臂抓住血液中称为纤维蛋白原的蛋白质链形成凝块。 斯涅辛斯基说：“聚集测定法告诉你血小板聚集在一起。”。 “但它并不能告诉你它们的激活水平，也不能告诉你他们用来抓住其他凝固蛋白以及彼此的力量。 “放大信号”salaita实验室是使用由连接在表面的双链dna合成链制成的张力探针来可视化和测量细胞施加的机械力的领导者。 dna的双链可以被编程为与血小板细胞结合。 当细胞结合并对锚定的dna施加力时，dna分裂成两股，留下一股粘在表面。 产生的物理拖船被转换成荧光信号。 然而，解读这一信号的一个主要挑战是，这些物理牵引是微弱的、短暂的和罕见的。 他们需要显微镜来检测。 在新冠肺炎大流行期间，酶crispr12a（cas12a）作为sars-cov-2病毒的诊断工具而脱颖而出。 细菌利用cas12a防御噬菌体或攻击细菌的病毒。 cas12a酶可以装载单链“引导”rna，该rna被编程为与互补的单链dna结合。 然后，这种酶通过破坏其周围的其他单链dna来与单链dna反应。 salaita实验室决定将cas12a与其张力探针结合起来，看看这种酶是否能放大血小板施加的机械力的信号。 实验室开发了所谓的mechano-cas12a辅助张力传感器。 “它像黑帮杀手一样运作，”萨莱塔说。 他解释说：“如果cas12a看不到目标，它就会安静而不活动。”。 “但它一看到特定的单链dna，就会发疯，开始破坏它遇到的任何单链dna。 这种激活会产生大量的荧光信号输出。 单克隆抗体是精确和超灵敏的，能够测量样本中2000个血小板产生的细胞牵引力。 所得到的信号足够稳健，可以通过常规荧光计进行测量，荧光计是常规血液测试中常用的工具。 mcats还与读板器配合使用，读板器是一种设计用于同时处理数十个样本的仪器，用于进行研究所需的高通量读数。 测试其临床潜力为了测试单克隆抗体在测量血小板功能活性方面的功效，研究人员从健康的志愿者身上采集了血样。 他们首先验证了mcats的反应对血小板的机械力是敏感的。 接下来，他们在健康的血液样本中添加了不同的抗血小板药物，从非处方阿司匹林到一组不同的处方药。 mcats的结果显示，抗血小板治疗降低了血小板的机械活性，其程度与聚集度测定中观察到的降低程度相似。 研究人员还获得许可，可以在体外循环手术前后采集7名患者的血样进行调查。 结果显示，每个患者样本的血小板活性的mcats读数与他们需要输注血小板以尽量减少手术后出血的可能性相关。 研究人员现在正在招募一项前瞻性研究的参与者，以进一步探索单克隆抗体作为一种诊断工具。 被诊断为血小板紊乱的患者将在治疗前后进行血样检测，以评估治疗效果。 斯涅辛斯基说：“最重要的是，mcats是一种使用非常小的样本来测量血小板功能的全新方法。”。 “它告诉我们一些我们以前无法测量的特定信息，这可以为我们提供一种新的方法来了解血小板功能障碍的情况以及控制它的最佳方法。 salaita补充道：“血液检查可以根据血小板计数和代谢浓度等数据，基本了解你的健康状况。”。 “现在我们正在添加有关血小板力学的信息。 这就像在你的仪表板上安装了一个全新的表盘来监测你的健康状况。 这篇论文是由美国国立卫生研究所、美国国家科学基金会和美国心脏协会资助的。graphic image shows a blood clot forming in an artery. the white, spikey platelets are shown amid red and white blood cells. activated platelets use their spikes like “arms” to grip onto one another and to stringy chains of proteins in the blood called fibrin, forming clumps that also bind up red and white blood cells.a novel technique to test platelet function within a person’s blood sample is faster, easier and more precise than methods currently in use, an experimental study shows.nature biomedical engineering published the research, led by scientists at emory university.the researchers demonstrated the proof-of-concept for the technique, which provides the first detailed look at the molecular forces generated by activated platelets in patient blood samples.the study results show that the technology holds the potential to assess the effects of antiplatelet drugs on individuals and to gain a clearer picture of bleeding risks for patients undergoing cardiopulmonary bypass surgery.the technique requires only about a drop of blood to run tests, compared to the tablespoon needed for current assays. this ultrasensitivity may make the technology a valuable tool for the diagnosis of babies suffering from rare, congenital platelet disorders.the breakthrough is based on synthetic-dna tension probes developed more than a decade ago in the laboratory of khalid salaita, professor in emory’s department of chemistry and in the wallace h. coulter department of biomedical engineering at emory and georgia tech.‘a scientist’s dream’the tension probes can detect cellular forces on the magnitude of just a few piconewtons, or about a billion times less than the weight of a paper clip. the researchers found a way to amplify the signal of the probes by tapping the power of an enzyme known as crispr-associated 12a. the mechanical signal is then detected using a plate-reader, a tool already routinely used in clinical testing.“this project started out of basic curiosity,” says salaita, co-corresponding author. “we wanted to know whether we could measure the tiniest forces exerted by cells. it’s exciting that we are now building on this basic curiosity to develop diagnostic tools to help patients. it’s a scientist’s dream.”first author of the paper is yuxin duan, an american heart association postdoctoral fellow in the salaita lab.roman sniecinski, a professor in emory school of medicine’s department of anesthesiology and a leading expert in the field of perioperative coagulation, is co-corresponding author of the paper.“platelet function in general is important and yet the current tools that we have to measure it are relatively primitive,” sniecinski says. “this new technique offers an easier, faster and cheaper way to measure platelet function, while also providing us with key information that we didn’t have before.”co-authors include: fania szlam, a senior associate in the sniecinski lab; yuesong hu, a graduate student in the salaita lab; renhao li, a professor in emory school of medicine’s department of pediatrics, hematology/oncology; wenchun chen, a postdoctoral fellow in emory school of medicine; and yonggang ke, an associate professor in the coulter department of biomedical engineering at emory and georgia tech.the importance of plateletsplatelets are colorless, disc-shaped blood-cell fragments whose job is to bind at the site of an injured blood vessel to stop the bleeding. in some cases, however, platelets may not function optimally. when platelets are weak, or less active than optimal, the blood may not clot properly, leading to uncontrolled bleeding. but if platelets are “hyperactive” they may become too sticky and cause spontaneous blood clots that can lead to heart attack or stroke.regulating platelet function is especially critical to people at higher risk for some conditions. antiplatelet drugs, such as clopidogrel, ticagrelor and even aspirin, are among the most commonly prescribed medications in the united states. in some patients, however, these drugs may not work well and adjustments in doses or changing to another drug might better help prevent heart attacks.during cardiac surgery, platelet function becomes even more dysregulated. the operating-room team must perform a balancing act of making blood not clot during cardiopulmonary bypass, then using procoagulant interventions, including transfusions of platelets, to stop the bleeding when the surgical procedure is finished. this can be difficult because the use of the cardiopulmonary bypass machine can stress and weaken blood platelets.“for decades, people have written in the scientific literature about this problem of platelet dysfunction during cardiac surgery,” sniecinski says, “but it’s really difficult to measure it with the tools that we’ve been using. and since we haven’t been able to measure platelet function well, that’s made it difficult to study it in effective ways.” ‘a small part of the picture’aggregometry is a standard tool currently used to assess platelet function. it measures the speed and degree at which platelets in a blood sample clump together, or aggregate.“this data provides only a small part of the picture of platelet function and it’s not the most interesting part,” sniecinski says.when a platelet gets activated, he explains, it changes its morphology and grows tiny pseudo “arms.” platelets use these arms to grip onto chains of proteins in the blood called fibrinogen to form clots.“aggregometry tells you that platelets are clumping together,” sniecinski says. “but it doesn’t tell you about their level of activation the amount of force they’re using to hold on to other coagulation proteins, as well as each other.” amplifying the signalthe salaita lab is a leader in visualizing and measuring the mechanical forces applied by cells using tension probes made from synthetic strands of double-stranded dna tethered to a surface.the double-strands of dna can be programmed to bind to platelet cells. when the cells bind and apply force to the anchored dna, the dna splits into two strands, leaving one strand stuck to the surface. the resulting physical tug is converted into a fluorescent signal.a major challenge to reading this signal, however, is that these physical tugs are faint, fleeting and infrequent. they require a microscope to detect them.during the covid-19 pandemic , the enzyme crispr 12a, or cas12a, came to the fore as a diagnostic tool for sars-cov-2 virus. bacteria use cas12a to defend against phages, or viruses that attack bacteria. the cas12a enzyme can be loaded with single-stranded “guide” rna that is programmed to bind to a complementary single-stranded dna. the enzyme then reacts to the single-stranded dna by destroying other single-stranded dna surrounding it.the salaita lab decided to combine cas12a with its tension probes to see if the enzyme would amplify the signal for the mechanical forces exerted by blood platelets. the lab developed what it calls the mechano-cas12a assisted tension sensor, or mcats.“it worked like gangbusters,” salaita says.“cas12a is quiet and inactive if it doesn’t see its target,” he explains. “but as soon as it sees a specific single-strand dna, it goes bananas and starts destroying any single-stranded dna it comes across. this activation generates a massive fluorescence signal output.”mcats is precise and ultrasensitive, able to measure cellular traction forces generated by as few as 2,000 platelets within a sample. and the resulting signal is robust enough to measure via a conventional fluorometer a tool commonly used in routine blood tests.mcats also works with a plate reader, an instrument designed to handle dozens of samples simultaneously, for the kind of high-throughput readout needed to conduct research.testing its clinical potentialto test the efficacy of mcats at measuring the activity of platelet function, the researchers drew blood samples from healthy volunteer donors. they first validated that the mcats response was sensitive to the mechanical forces of platelets.they next added to the healthy blood samples different antiplatelet drugs, ranging from over-the-counter aspirin to a panel of different prescription medications. the mcats results showed that the antiplatelet therapies reduced the mechanical activity of platelets by an amount similar to the reduction observed in aggregometry.the researchers also received permission to take blood samples for investigation from seven patients pre- and post-cardiopulmonary bypass surgery. the results showed that the mcats readings for the platelet activity of each individual patient’s sample correlated to their likelihood to need platelet transfusions to minimize bleeding after surgery.the researchers are now enrolling participants in a prospective study to further explore mcats as a diagnostic tool. people diagnosed with a platelet disorder will have their blood samples tested pre- and post-treatment to assess how well a therapy is working. “the bottom line is that mcats is a whole new way to measure platelet function using a really tiny sample,” sniecinski says. “it’s telling us something specific that we haven’t been able to measure before and that can give us a new way to understand what’s going on with platelet dysfunction and the best methods for controlling it.”“blood work up gives you a basic readout of your health based on data like platelet count and metabolic concentrations,” salaita adds. “now we’re adding information about the mechanics of platelets. that’s like getting a whole new dial on your dashboard for monitoring your health.”work on the current paper was funded by the national institutes of health, the national science foundation and the american heart association.本文来源： 分析血小板的新工具具有重大的医学潜力
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