在体内小鼠模型中，血管外凝血独立于活化血小板表面调节止血-动脉网

Extravascular coagulation regulates haemostasis independently of activated platelet surfaces in an in vivo mouse model

Nature 等信源发布 2025-03-08 09:56

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Abstract

摘要

While the conventional understanding of haemostatic plug formation is that coagulation proceeds efficiently on the surface of activated platelets at the vascular injury site to form a robust haemostatic plug, this understanding does not explain the clinical reality that platelet dysfunction results in a mild bleeding phenotype, whereas coagulation disorders exhibit severe bleeding phenotypes, particularly in deep tissues.

虽然对止血栓形成的传统理解是，凝血在血管损伤部位的活化血小板表面高效进行，从而形成一个坚固的止血栓，但这种理解无法解释临床上血小板功能障碍导致轻微出血表型，而凝血障碍则表现出严重出血表型（尤其是在深层组织）的现实。

Here, we introduce an in vivo imaging method to observe internal bleeding and subsequent haemostatic plug formation in mice and report that haemostatic plug formation after internal bleeding, coagulation occurs primarily outside the blood vessel rather than on platelets. Experiments in mice with impaired platelet surface coagulation, depleted platelets, haemophilia A or reduced tissue factor expression suggest that this extravascular coagulation triggers and regulates haemostatic plug formation.

在这里，我们介绍了一种活体成像方法，用于观察小鼠体内出血及随后的止血栓形成，并报告指出在内出血后的止血栓形成过程中，凝血主要发生在血管外而不是在血小板上。对血小板表面凝血功能受损、血小板耗竭、甲型血友病或组织因子表达减少的小鼠进行的实验表明，这种血管外凝血触发并调控了止血栓的形成。

Our discovery of the important role of extravascular coagulation in haemostasis may contribute to refining the treatment of haemostatic abnormalities and advancing antithrombotic therapy..

我们对外血管凝血在止血中的重要作用的发现可能有助于完善止血异常的治疗，并推动抗血栓治疗的进步。

Introduction

介绍

When bleeding occurs, a haemostatic plug forms to prevent blood loss. Traditionally, it is believed that platelet activation and coagulation occur simultaneously at the injury site, relying on phosphatidylserine (PS) on activated platelets for effective clot formation

当出血发生时，会形成止血栓以防止血液流失。传统上认为，血小板激活和凝血在损伤部位同时发生，依靠活化血小板上的磷脂酰丝氨酸（PS）来实现有效的血栓形成。

，

. However, this view does not explain why platelet dysfunction rarely results in severe bleeding, whereas coagulation disorders often do, especially in deep tissues

然而，这种观点无法解释为什么血小板功能障碍很少导致严重出血，而凝血障碍却常常如此，尤其是在深层组织中。

。

The ancestors of platelets are believed to be amebocytes found in horseshoe crab haemolymph

血小板的祖先被认为是存在于马蹄蟹血淋巴中的变形细胞。

. These cells release serine proteases, such as prochelicerase C and prochelicerase B

这些细胞释放丝氨酸蛋白酶，如前螯合酶C和前螯合酶B。

, which are thought to be the ancestors of coagulation factors. These proteases initiate a cascade of reactions for wound healing

，这些蛋白酶被认为是凝血因子的祖先，它们启动了伤口愈合的一系列反应。

. Furthermore, activated amebocytes release serine proteases, antimicrobial peptides, and lectins

此外，活化的变形细胞释放丝氨酸蛋白酶、抗菌肽和凝集素。

, but unlike human platelets, the cells themselves cannot seal wounds or initiate cascade reactions on their surface

，但与人类血小板不同，这些细胞本身无法封闭伤口或在其表面启动级联反应。

，

. Given that coagulation factors are evolutionarily more primitive than platelets are, we hypothesize that coagulation, rather than platelets, is the trigger for haemostasis.

鉴于凝血因子在进化上比血小板更为原始，我们假设凝血而非血小板是止血的触发因素。

Recent advancements in in vivo imaging techniques have been utilized to examine the process of thrombus formation

近年来，体内成像技术的进步已被用于研究血栓形成的过程。

，

. Two common in vivo imaging techniques for observing thrombus formation are as follows: (1) microscopy after oxidative endothelial injury from chemicals such as ferric chloride

两种常见的用于观察血栓形成的体内成像技术如下：（1）使用如氯化铁等化学物质引起氧化性内皮损伤后的显微镜成像

，

and (2) microscopy following laser-induced endothelial injury

（2）激光诱导内皮损伤后的显微镜检查

，

. In the oxidative injury model, vascular endothelial desquamation is typically observed after chemical stimulation, although some reports note intact vascular endothelium

在氧化损伤模型中，化学刺激后通常会观察到血管内皮脱落，尽管一些报告指出血管内皮保持完整。

. Conversely, the conventional laser injury model maintains an intact vascular endothelium, with tissue factor release at the laser site driving thrombus formation

相反，传统的激光损伤模型保持了完整的血管内皮，激光部位的组织因子释放驱动血栓形成。

，

. Both methods lack bleeding. The presence of a thrombus without bleeding is deemed pathological, leading previous in vivo imaging methods to focus on assessing pathological thromboses rather than haemostasis. In contrast, Bergmeier et al. described a method for inducing external bleeding by applying penetrating laser injury to exposed blood vessels, followed by observation of haemostasis.

两种方法均无出血。在没有出血的情况下出现血栓被认为是病理性的，因此以前的体内成像方法侧重于评估病理性血栓形成，而非止血过程。相反，Bergmeier等人描述了一种通过应用穿透性激光损伤暴露的血管来诱导外部出血的方法，随后对止血过程进行观察。

，

. However, to date, no methods have been reported for confirming haemostasis following internal bleeding, which frequently occurs in everyday life outside traumatic events. In this study, we developed an in vivo imaging method to observe the early haemostatic response after internal bleeding by disrupting the vascular endothelium via two-photon excitation.

然而，迄今为止，尚未有报道任何方法可以确认内出血后的止血情况，而这种情况在日常生活中经常发生在非创伤事件之外。在本研究中，我们开发了一种活体成像方法，通过双光子激发破坏血管内皮来观察内出血后的早期止血反应。

Our method confirmed that the haemostatic response remained unaffected in mice with impaired platelet surface coagulation. In contrast, a delayed haemostatic response was observed in mice with low tissue factor expression. These findings indicate that extravascular coagulation acts as the primary trigger for haemostasis during internal bleeding, whereas coagulation on the activated platelet surface is not essential for this process.

我们的方法证实，在血小板表面凝血功能受损的小鼠中，止血反应仍然不受影响。相反，在组织因子表达较低的小鼠中观察到延迟的止血反应。这些发现表明，血管外凝血是内出血期间止血的主要触发因素，而活化血小板表面的凝血对于此过程并非必需。

These novel findings clarify the discrepancy between the traditional understanding of haemostasis and clinical observations regarding platelet dysfunction. These findings could also contribute to improved management of haemostatic abnormalities and to advances in antithrombotic therapy..

这些新颖的发现阐明了传统凝血观念与临床观察到的血小板功能障碍之间的差异。这些发现还可能有助于更好地管理凝血异常，并推动抗血栓治疗的进步。

Results

结果

Fibrin is formed outside blood vessels

纤维蛋白在血管外形成

We developed an in vivo imaging method to observe bleeding and haemostatic plug formation in veins and arteries, with a focus on platelets and coagulation after internal bleeding. In C57BL/6J (wild-type) mice, we observed that venous and arterial haemostatic plugs contained platelet aggregates inside the vessel, fibrin in platelet-free areas outside the vessel, and coexisting platelet and fibrin clots at the boundaries (Fig. .

我们开发了一种活体成像方法，用于观察静脉和动脉中的出血及止血栓形成，重点是内出血后的血小板和凝血过程。在C57BL/6J（野生型）小鼠中，我们观察到静脉和动脉的止血栓在血管内部包含血小板聚集体，在血管外部的无血小板区域存在纤维蛋白，并且在边界处有血小板和纤维蛋白凝块共存（图.）。

1a, b

）。

Fig. 1: Fibrin, a component of haemostatic plugs, is formed outside blood vessels.

图1：纤维蛋白是止血栓的组成部分，在血管外形成。

Representative images of haemostatic plug formation after venous (

静脉后止血栓形成的代表性图像（

) and arterial (

）和动脉（

) internal bleeding. The mice were intravenously injected with dextran-rhodamine B, Alexa Fluor 488-conjugated fibrinogen and the platelet imaging antibody X649 prior to observation. After two-photon excitation injury (circle of dots), platelet aggregate formation in the lumen (magenta triangle) and extravascular plasma leakage (red triangle) were observed.

）内出血。观察前，小鼠被静脉注射了右旋糖酐-罗丹明B、Alexa Fluor 488标记的纤维蛋白原和血小板成像抗体X649。在双光子激发损伤后（点圈），观察到管腔内血小板聚集体形成（洋红色三角形）和血管外血浆渗漏（红色三角形）。

After arterial internal bleeding, red blood cell leakage was observed (white asterisk). Fibrin is formed in the extravascular area (green triangle). The haemostatic plug consisted of a platelet aggregate in the lumen, extravascular fibrin, and platelets and fibrin at the boundary of the lumen and extravascular space (white triangle).

动脉内部出血后，观察到红细胞泄漏（白色星号）。血管外区域形成纤维蛋白（绿色三角形）。止血栓由腔内的血小板聚集体、血管外的纤维蛋白以及腔与血管外空间边界处的血小板和纤维蛋白组成（白色三角形）。

Scale bar = 20 μm. Green: fibrin/fibrinogen. Magenta: platelets. Red: plasma..

比例尺 = 20 μm。绿色：纤维蛋白/纤维蛋白原。洋红色：血小板。红色：血浆。

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Haemostasis after arterial internal bleeding was completed by cessation of plasma leakage following cessation of blood cell leakage

动脉内出血后的止血是通过血细胞泄漏停止后血浆泄漏的停止来完成的。

In our experiments, venous internal bleeding included plasma leakage, identified by fluorescent dextran leakage, with minimal blood cell leakage. In contrast, arterial internal bleeding resulted in both blood cell and plasma leakage, with red blood cells visible as black silhouettes (Fig.

在我们的实验中，静脉内出血包括血浆渗漏，通过荧光葡聚糖渗漏来识别，而血液细胞渗漏极少。相比之下，动脉内出血则导致血液细胞和血浆同时渗漏，红细胞可见为黑色轮廓（图。

1a, b

). To confirm how blood cell and plasma leakage ceases during haemostasis after arterial internal bleeding, we analysed images of significant vascular damage (25–40 mm) that allowed clear observation of blood cell leakage. Consequently, haemostasis was achieved first with the cessation of blood cell leakage, followed by the cessation of plasma leakage.

为了确认在动脉内出血后血细胞和血浆渗漏是如何在止血过程中停止的，我们分析了显著血管损伤（25-40毫米）的图像，这些图像可以清晰观察到血细胞的渗漏。结果显示，止血首先通过血细胞渗漏的停止实现，随后血浆渗漏也停止。

Low plasma leakage results in slight fluctuations in the dextran signal, making it difficult to determine when leakage stops. However, during blood cell leakage, clear extravascular leakage of fluorescent dextran was observed in all mice. Although blood cell leakage recurred, it ceased when platelets clog the vascular injury site (Fig. .

低血浆泄漏导致右旋糖酐信号出现轻微波动，难以确定泄漏何时停止。然而，在血细胞泄漏期间，所有小鼠均观察到荧光标记的右旋糖酐明显渗出血管外。尽管血细胞泄漏反复发生，但当血小板堵塞血管损伤部位时，泄漏停止（图。

）。

Fig. 2: Haemostasis was achieved by the cessation of plasma leakage following the cessation of blood cell leakage.

图 2：通过停止血浆渗漏继而停止血细胞渗漏来实现止血。

Representative images of haemostatic plug formation after arterial internal bleeding from a large injury. The mice were intravenously injected with dextran-rhodamine B, Alexa Fluor 488-conjugated fibrinogen, and the platelet imaging antibody X649 prior to observation. After two-photon excitation injury (circle of dots), extravascular plasma leakage (red triangle) and red blood cell leakage were observed (white asterisk).

大损伤导致动脉内出血后止血栓形成的代表性图像。小鼠在观察前被静脉注射了右旋糖酐-罗丹明B、Alexa Fluor 488标记的纤维蛋白原以及血小板成像抗体X649。在双光子激发损伤（点圈）后，观察到血管外血浆渗漏（红色三角形）和红细胞渗漏（白色星号）。

Vasoconstriction temporarily reduced blood leakage, but it resumed when vasoconstriction was released. Fibrin begins to form in slow-flowing areas of the extravascular space (green triangle). Exacerbated blood cell leakage stopped when platelets clogged the site of vessel rupture (magenta triangle).

血管收缩暂时减少了血液泄漏，但在血管收缩解除时再次出现泄漏。纤维蛋白开始在血管外间隙血流缓慢的区域形成（绿色三角形）。当血小板堵塞了血管破裂处时，加剧的血细胞泄漏停止了（洋红色三角形）。

Fibrin formation proceeded in the area of slowed flow caused by platelet blockage, resulting in the cessation of plasma leakage. Scale bar = 20 μm. Green: fibrin/fibrinogen. Magenta: platelets. Red: plasma..

由于血小板堵塞导致流速减缓的区域中，纤维蛋白形成继续进行，从而停止了血浆泄漏。比例尺=20微米。绿色：纤维蛋白/纤维蛋白原。洋红色：血小板。红色：血浆。

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To investigate platelet entrapment outside the vessel, we observed bleeding sites in wild-type mice via two-photon imaging, which is known for its ability to visualize collagen

为了研究血管外的血小板滞留，我们通过双光子成像观察了野生型小鼠的出血部位，这种成像技术以其能够可视化胶原蛋白而闻名。

. We observed that leaking platelets were attached to fibrin where collagen was disrupted. Similar findings were noted in platelet-specific anoctamin-6 (TMEM16F) knockout (Ano6 cKO) mice, a murine model of Scott’s syndrome characterized by impaired expression of PS on platelets

我们观察到泄漏的血小板附着在胶原蛋白破裂处的纤维蛋白上。在血小板特异性anoctamin-6（TMEM16F）基因敲除（Ano6 cKO）小鼠中也发现了类似的结果，这种小鼠是Scott综合征的模型，其特征是血小板上PS表达受损。

. This finding indicated that fibrin entrapped leaked platelets independently of platelet activation (Fig.

这一发现表明，纤维蛋白独立于血小板激活而捕获了渗漏的血小板（图。

）。

Fig. 3: Platelets are trapped by extravascular fibrin where collagen is damaged.

图3：血小板被血管外纤维蛋白捕获，胶原蛋白受损处。

Representative images of haemostatic plugs after large arterial injury. The mice were intravenously injected with dextran-rhodamine B, Alexa Fluor 488-conjugated fibrinogen, and the platelet imaging antibody X649 prior to observation. Vessel observation before the arterial injury and after haemostatic plug formation via two-photon imaging revealed collagen (blue triangle) and platelets attached to the fibrin network (green triangle), where collagen ruptured.

大型动脉损伤后止血栓的代表性图像。小鼠在观察前被静脉注射了葡聚糖-罗丹明B、Alexa Fluor 488标记的纤维蛋白原以及血小板成像抗体X649。通过双光子成像对动脉损伤前和止血栓形成后的血管进行观察，发现胶原（蓝色三角形）和附着在纤维蛋白网络上的血小板（绿色三角形），其中胶原破裂。

Similar findings were observed in Ano6 cKO mice with impaired platelet coagulation. Scale bar = 20 μm. In the confocal images, green indicates fibrin/fibrinogen, magenta indicates platelets, and red indicates plasma. In two-photon images, yellow-green indicates fibrin/fibrinogen, red indicates plasma, and blue indicates collagen..

在血小板凝血功能受损的Ano6条件性敲除（cKO）小鼠中观察到了类似的结果。比例尺=20微米。在共聚焦图像中，绿色表示纤维蛋白/纤维蛋白原，洋红色表示血小板，红色表示血浆。在双光子图像中，黄绿色表示纤维蛋白/纤维蛋白原，红色表示血浆，蓝色表示胶原蛋白。

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Irreversible platelet activation was not observed in the early phase of haemostatic plug formation

在止血栓形成的早期阶段，未观察到不可逆的血小板激活。

We then investigated the activation status of platelets in haemostasis by examining CD62P, a marker of irreversibly activated platelets. While CD62P was detected in platelets that clog the liver vasculature after intravenous thrombin injection (positive control, Fig.

我们接着通过检测CD62P（一种不可逆激活的血小板标志物）来研究止血过程中血小板的激活状态。在静脉注射凝血酶后，肝脏血管中堵塞的血小板检测到了CD62P（阳性对照，图。

), it was not detected in the in vivo bleeding/haemostasis model, in accordance with our hypothesis (Fig.

），在体内出血/止血模型中未检测到，这与我们的假设一致（图。

). These findings suggest that platelets exhibit limited activation during the early phase of haemostatic plug formation, with coagulation predominantly occurring outside the vessel.

）。这些发现表明，在止血栓形成的早期阶段，血小板的激活有限，凝血主要发生在血管外。

Fig. 4: Irreversible platelet activation does not occur in early haemostatic plugs.

图4：早期止血栓中不会发生不可逆的血小板激活。

Representative images of platelets that clog the liver vasculature after systemic thrombin injection (

注射全身性凝血酶后导致肝脏血管堵塞的血小板代表性图像（

) and haemostatic plugs that formed after large arterial injury (

）以及在大动脉损伤后形成的止血栓（

). For arterial haemostatic plug observation, the mice were injected with Alexa Fluor 488-conjugated fibrinogen, PE-conjugated rat anti-mouse CD62P antibody, and platelet imaging antibody X649 prior to observation. For platelet observation after thrombin injection, the mice were injected with FITC-dextran, PE-conjugated rat anti-mouse CD62P antibody, and X649 antibody prior to observation.

）。为了观察动脉止血栓，小鼠在观察前被注射了Alexa Fluor 488标记的纤维蛋白原、PE标记的大鼠抗小鼠CD62P抗体和血小板成像抗体X649。为了在凝血酶注射后观察血小板，小鼠在观察前被注射了FITC-葡聚糖、PE标记的大鼠抗小鼠CD62P抗体和X649抗体。

CD62P expression was detected on platelets that clog the liver vasculature (.

检测到CD62P表达在堵塞肝脏血管的血小板上。

) but was not detected in the in vivo internal bleeding/haemostasis model (

`) 但在体内内出血/止血模型中未被检测到 (`

). Scale bar = 20 μm. Green: fibrin/fibrinogen. Magenta: platelets. Red: plasma.

）。比例尺=20微米。绿色：纤维蛋白/纤维蛋白原。洋红色：血小板。红色：血浆。

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Quantitative analysis of haemostatic plug formation in wild-type, Ano6 cKO, platelet-depleted, FVIII KO, and low-tissue-factor mice

野生型、Ano6 cKO型、血小板减少型、FVIII敲除型和低组织因子小鼠的止血栓形成定量分析

To investigate the role of platelets and coagulation in haemostatic plug formation, we analysed images from wild-type, platelet-depleted (administered antibodies for platelet depletion with platelet counts of approximately 3% of the predose level), and three genetically modified mouse strains, Ano6 cKO; coagulation factor VIII-deficient (FVIII KO), which represent severe haemophilia A; and low-tissue-factor-expressing (L-TF) mice, which are indicative of an impaired extrinsic coagulation pathway, were used.

为了研究血小板和凝血在止血栓形成中的作用，我们分析了来自野生型、血小板耗竭型（通过施用血小板耗竭抗体，血小板计数约为给药前水平的3%）以及三种基因修饰小鼠品系的图像：Ano6条件性敲除（cKO）、凝血因子VIII缺陷型（FVIII KO，代表严重的A型血友病）和低组织因子表达型（L-TF，指示外源性凝血途径受损）小鼠。

We focused on lesions within specific vascular injury ranges—25–40 μm for veins (wild-type, .

我们专注于特定血管损伤范围内的病灶——静脉为25-40微米（野生型，。

= 6 lesions of 5 animals; Ano6 cKO,

= 6个病灶，涉及5只动物；Ano6 cKO，

= 5 lesions of 5 animals; platelet-depleted,

= 5个动物的5个病灶；血小板减少，

= 5 lesions of 5 animals; FVIII KO,

= 5个病变，涉及5只动物；FVIII敲除，

= 6 lesions of 4 animals; and L-TF mice,

= 4只动物的6个病灶；以及L-TF小鼠，

= 16 lesions of 4 animals) and 10–25 μm for arteries (wild-type,

= 16个病灶来自4只动物）和10-25微米用于动脉（野生型，

= 6 lesions of 6 animals; Ano6 cKO,

= 6个病变，涉及6只动物；Ano6 cKO，

= 7 lesions of 6 animals; platelet-depleted,

= 6只动物的7处病灶；血小板减少，

= 5 lesions of 5 animals; FVIII KO,

= 5个病灶，来自5只动物；FVIII敲除，

= 5 lesions of 5 animals; and L-TF mice,

= 5个病灶来自5只动物；以及L-TF小鼠，

= 6 lesions of 6 animals)—ensuring accuracy in our evaluations. Representative images and movies are shown in Figs.

= 6个动物的6个病灶）——确保我们评估的准确性。代表性图像和电影见图。

，

, and Supplementary Movies

，以及补充电影

–

. Figures

. 图表

，

, Supplementary Figs.

，补充图。

and

和

show time-series data obtained from individual mice, while Figs.

显示从单个老鼠获得的时间序列数据，而图。

7b–f

and

和

8b–h

present extracted data related to platelet aggregates, fibrin, and bleeding.

呈现与血小板聚集体、纤维蛋白和出血相关的提取数据。

Fig. 5: Haemostatic plug formation after venous internal bleeding in wild-type, Ano6 cKO, platelet-depleted, FVIII KO, and L-TF mice.

图5：野生型、Ano6 cKO、血小板耗竭、FVIII KO和L-TF小鼠在静脉内出血后形成的止血栓。

Representative images of haemostatic plug formation after venous internal bleeding in wild-type, Ano6 cKO, platelet-depleted, FVIII KO, and L-TF mice. The mice were intravenously injected with dextran-rhodamine B, Alexa Fluor 488-conjugated fibrinogen, and the platelet imaging antibody X649 prior to observation.

野生型、Ano6 cKO型、血小板耗竭型、FVIII敲除型和L-TF小鼠在静脉内出血后止血栓形成的代表性图像。观察前，这些小鼠被静脉注射了葡聚糖-罗丹明B、Alexa Fluor 488标记的纤维蛋白原以及血小板成像抗体X649。

Scale bar = 20 μm. Green: fibrin/fibrinogen. Magenta: platelets. Red: plasma..

比例尺 = 20 μm。绿色：纤维蛋白/纤维蛋白原。洋红色：血小板。红色：血浆。

Full size image

全尺寸图像

Fig. 6: Haemostatic plug formation after arterial internal bleeding in wild-type, Ano6 cKO, platelet-depleted, FVIII KO, and L-TF mice.

图6：野生型、Ano6 cKO、血小板减少、FVIII KO和L-TF小鼠在动脉内出血后形成的止血栓。

Representative images of haemostatic plug formation after arterial internal bleeding in wild-type, Ano6 cKO, platelet-depleted, FVIII KO, and L-TF mice. The mice were intravenously injected with dextran-rhodamine B, Alexa Fluor 488-conjugated fibrinogen, and the platelet imaging antibody X649 prior to observation.

野生型、Ano6 cKO、血小板减少、FVIII KO 和 L-TF 小鼠在动脉内出血后形成止血栓的代表性图像。观察前，这些小鼠被静脉注射了葡聚糖-罗丹明 B、Alexa Fluor 488 标记的纤维蛋白原和血小板成像抗体 X649。

Scale bar = 20 μm. Green: fibrin/fibrinogen. Magenta: platelets. Red: plasma..

比例尺 = 20 μm。绿色：纤维蛋白/纤维蛋白原。洋红色：血小板。红色：血浆。

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Fig. 7: Extravascular coagulation is important for platelet aggregate formation and the cessation of plasma leakage after venous internal bleeding.

图7：血管外凝血对血小板聚集形成以及静脉内出血后血浆渗漏的停止非常重要。

Measurements of platelet aggregates, fibrin, and plasma leakage were taken from experimental mice (

从实验小鼠身上测量了血小板聚集物、纤维蛋白和血浆渗漏 (

) (wild-type,

) (野生型，

= 6 lesions from 5 animals; Ano6 cKO,

= 来自5只动物的6个病灶；Ano6 cKO，

= 5 lesions from 5 animals; platelet-depleted,

= 5个病变来自5只动物；血小板减少，

= 5 lesions from 5 animals; FVIII KO,

= 5个病变来自5只动物；FVIII敲除，

= 6 lesions from 4 animals; and L-TF mice,

= 来自4只动物的6个病灶；以及L-TF小鼠，

= 16 lesions from 4 animals). Platelet aggregates were assessed by height from the vascular endothelium. Fibrin was quantified as the ratio of the fibrin area over time, normalized to the end of the observation period. Plasma leakage was expressed relative to the maximum signal outside the vessel. Box-and-whisker plot of the peak platelet aggregate height (.

= 16个病灶来自4只动物）。血小板聚集体通过血管内皮的高度进行评估。纤维蛋白的量化是通过纤维蛋白区域随时间的比值，并归一化到观察期结束时。血浆渗漏则相对于血管外最大信号表达。峰值血小板聚集体高度的箱线图（。

). Peak platelet aggregate height at 0–120 and 120–300 s (

). 峰值血小板聚集高度在 0–120 和 120–300 秒 (

). The increase in the fibrin area accelerated, as indicated by the ratio (

`). 纤维蛋白区域的增加加速，如比率所示 (`

). Time required for the fibrin area to reach half the area at the end of observation (

观察结束时纤维蛋白区域达到一半面积所需的时间 (

). Area under the curve (AUC) of plasma leakage (

). 血浆渗漏的曲线下面积 (AUC) (

). *<0.05, **<0.01.

Wilcoxon signed-rank test;

威尔科克森符号秩检验；

，

–

Steel–Dwass test and Monte Carlo simulation.

Steel–Dwass检验和蒙特卡罗模拟。

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Fig. 8: Extravascular coagulation is important for the formation of platelet aggregates and the cessation of blood cell and plasma leakage after arterial internal bleeding.

图8：血管外凝血对于血小板聚集的形成以及动脉内出血后血细胞和血浆渗漏的停止非常重要。

Measurements of platelet aggregates, fibrin, and plasma leakage were taken from experimental mice (

对实验小鼠进行了血小板聚集、纤维蛋白和血浆渗漏的测量 (

) (wild-type,

)（野生型，

= 6 lesions of 6 animals; Ano6 cKO,

= 6个动物的6个病灶；Ano6 cKO，

= 7 lesions of 6 animals; platelet-depleted,

= 6只动物的7处病灶；血小板减少，

= 5 lesions of 5 animals; FVIII KO,

= 5个病变，涉及5只动物；FVIII基因敲除，

= 5 lesions of 5 animals; and L-TF mice,

= 5个病变，涉及5只动物；以及L-TF小鼠，

= 6 lesions of 6 animals). Platelet aggregates were assessed by height from the vascular endothelium. Fibrin was quantified as the ratio of the fibrin area over time, normalized to the end of the observation period. Plasma leakage was expressed relative to the maximum signal outside the vessel. Box-and-whisker plot of the peak platelet aggregate height (.

= 6个动物的6个病灶）。通过血管内皮的高度评估血小板聚集体。纤维蛋白的量化是通过纤维蛋白区域随时间的比值，并归一化到观察期结束时。血浆渗漏表示为相对于血管外最大信号的相对值。血小板聚集体峰值高度的箱线图（。

). Peak platelet aggregate height at 0–120 and 120–300 s (

). 峰值血小板聚集高度在 0–120 和 120–300 秒 (

). The increase in the fibrin area accelerated, as indicated by the ratio (

). 纤维蛋白区域的增加加速，如比率所示 (

). Time required for the fibrin area to reach half the area at the end of observation (

观察结束时，纤维蛋白区域达到一半面积所需的时间 (

). Area under the curve (AUC) of plasma leakage (

). 血浆渗漏的曲线下面积 (AUC) (

). The time from the start of bleeding to the cessation of blood cell leakage (

). 从出血开始到血细胞泄漏停止的时间 (

). The extent of platelet aggregate extrusion from the vessel was evaluated by measuring the distance from the vessel wall to the platelets, as shown in (

）。通过测量从血管壁到血小板的距离来评估血小板聚集体从血管中挤出的程度，如图（

). †<0.1, *<0.05, **<0.01.

Wilcoxon signed-rank test;

威尔科克森符号秩检验；

，

–

Steel–Dwass test and Monte Carlo simulation.

钢铁-德瓦斯检验和蒙特卡洛模拟。

Full size image

全尺寸图像

Extravascular coagulation is important for stable platelet aggregate formation

血管外凝血对于稳定的血小板聚集形成非常重要。

Statistical analysis of platelet aggregates formed after venous internal bleeding revealed a significantly lower peak height in L-TF mice than in wild-type (

静脉内出血后形成的血小板聚集体的统计分析显示，L-TF小鼠的峰值高度显著低于野生型小鼠（

= 0.0024), Ano6 cKO (

= 0.0005), and FVIII KO mice (

= 0.0005)，以及FVIII敲除小鼠（

= 0.0012) (Fig.

= 0.0012)（图。

). A comparison of aggregates formed between 0–120 s (early-stage) and between 120–300 s (late-stage) revealed a lower peak height in the late-stage for both FVIII KO (

). 对在0-120秒（早期）和120-300秒（晚期）之间形成的聚集体进行比较显示，FVIII KO的晚期峰值高度较低（

= 0.031) and L-TF mice (

= 0.031) 和 L-TF 小鼠 (

< 0.0001) (Fig.

<0.0001)（图。

). After arterial bleeding, peak heights did not differ among mouse types (Fig.

动脉出血后，小鼠类型之间的峰值高度没有差异（图。

), except in platelet-depleted mice. When comparing early- and late-stage platelet aggregates, the peak height tended to be lower in the late-stage in FVIII KO and L-TF mice (

)，但在血小板减少的小鼠中除外。比较早期和晚期的血小板聚集体时，FVIII敲除小鼠和L-TF小鼠在晚期时峰值高度往往较低（

= 0.063) (Fig.

= 0.063)（图。

). These findings suggest the importance of extravascular coagulation in venous and arterial platelet aggregates.

）。这些发现提示了血管外凝血在静脉和动脉血小板聚集体中的重要性。

Extravascular coagulation proceeds independently of the activated platelet surface

血管外凝血过程独立于活化血小板表面进行。

As bleeding progresses, the formation of extravascular fibrin increases, except in L-TF mice. Thus, to assess coagulation efficiency, we quantified fibrin formation acceleration rather than the total fibrin area. After venous internal bleeding, FVIII KO mice exhibited significantly lower acceleration compared to wild-type (.

随着出血的进展，血管外纤维蛋白的形成增加，L-TF小鼠除外。因此，为了评估凝血效率，我们量化了纤维蛋白形成加速情况，而不是总的纤维蛋白面积。静脉内出血后，FVIII敲除小鼠表现出比野生型显著更低的加速。

= 0.0025), Ano6 cKO (

= 0.044), and platelet-depleted mice (

= 0.044），以及血小板耗竭的小鼠（

= 0.020) (Fig.

= 0.020)（图。

7天

). No significant difference was observed between wild-type and Ano6 cKO mice (

）。在野生型和Ano6 cKO小鼠之间未观察到显著差异（

= 0.30). Following arterial internal bleeding, while the acceleration in platelet-depleted mice tended to be greater than that in FVIII KO mice (

= 0.30）。动脉内部出血后，血小板减少的小鼠的加速趋势往往大于FVIII敲除小鼠（

= 0.098), no significant differences were found among the groups (Fig.

= 0.098)，组间未发现显著差异（图。

8天

). These findings suggest that extravascular fibrin formation occurs independently of activated platelet surfaces in both veins and arteries.

）。这些发现表明，在静脉和动脉中，血管外纤维蛋白的形成都不依赖于活化的血小板表面。

Extravascular fibrin formation is important for the cessation of both blood cell and plasma leakage

血管外纤维蛋白的形成对于停止血细胞和血浆渗漏都非常重要。

Under our experimental conditions, minimal blood cell leakage occurred after venous injury. In terms of arterial internal bleeding, the time from bleeding onset to cessation of blood cell leakage was significantly longer in platelet-depleted and L-TF mice than in wild-type and Ano6 cKO mice (wild-type vs.

在我们的实验条件下，静脉损伤后出现极少量的血细胞泄漏。对于动脉内出血，血小板耗竭和L-TF小鼠的血细胞泄漏从开始出血到停止的时间显著长于野生型和Ano6 cKO小鼠（野生型 vs.

platelet-depleted, .

血小板减少，。

= 0.014; wild-type vs. L-TF,

= 0.014；野生型 vs. L-TF，

= 0.0045; Ano6 cKO vs. platelet-depleted,

= 0.0045；Ano6 cKO 对比血小板耗竭，

= 0.0064; and Ano6 cKO vs. L-TF,

= 0.0064；以及 Ano6 cKO 对比 L-TF，

= 0.0018) (Fig.

= 0.0018)（图。

8克

). Platelet extravasation revealed a significantly greater distance from the vascular endothelium to extravasated platelets in L-TF mice than in wild-type (

）。血小板外渗显示，在L-TF小鼠中，从血管内皮到外渗血小板的距离显著大于野生型小鼠（

= 0.0054) and Ano6 cKO mice (

= 0.0054) 和 Ano6 cKO 小鼠 (

= 0.033) (Fig.

= 0.033)（图。

8小时

). Additionally, plasma leakage from internal venous bleeding was significantly greater in L-TF mice than in wild-type (

此外，L-TF小鼠的内静脉出血导致的血浆渗漏明显多于野生型小鼠（

< 0.0001), FVIII KO (p = 0.0023), and Ano6 cKO mice (

<0.0001)、FVIII KO（p=0.0023）和Ano6 cKO小鼠（

= 0.0005) (Fig.

= 0.0005)（图。

), whereas arterial internal bleeding was significantly greater in L-TF mice than in wild-type mice (

)，而L-TF小鼠的动脉内出血明显多于野生型小鼠(

= 0.027), with a trend towards platelet-depleted mice (

= 0.027)，血小板减少的小鼠呈现出一种趋势（

= 0.064) (Fig.

= 0.064)（图。

). These findings highlight the importance of extravascular fibrin formation in achieving complete haemostasis.

这些研究结果强调了血管外纤维蛋白形成在实现完全止血中的重要性。

Discussion

讨论

Haemostatic plug formation is traditionally thought to involve efficient coagulation on the surface of activated platelets

传统上认为，止血栓的形成涉及在活化血小板表面的有效凝血。

，

, suggesting that platelet dysfunction should lead to severe bleeding. However, in the clinic, platelet dysfunction disorders, such as thrombasthenia or Scott syndrome, cause minimal bleeding, whereas coagulation disorders, such as haemophilia, lead to severe joint or muscle bleeding

，这表明血小板功能障碍应导致严重出血。然而，在临床上，血小板功能障碍疾病（如血小板无力症或Scott综合征）仅引起轻微出血，而凝血障碍疾病（如血友病）则导致严重的关节或肌肉出血。

. The aim of this study was to elucidate the sites and roles of coagulation in haemostatic plug formation through in vivo imaging of internal bleeding. These findings suggest that extravascular coagulation, rather than coagulation on activated platelets, is crucial in initiating and regulating haemostasis following internal bleeding..

本研究的目的在于通过体内成像技术观察内出血情况，以阐明凝血在止血栓形成中的位置和作用。这些发现表明，在内出血后的止血启动和调控中，血管外凝血而非活化血小板上的凝血起着关键作用。

In vascular injury induced by conventional one-photon excitation, damage occurs not only in the targeted area but also in the superficial and deep surrounding regions. In contrast, our method of inflicting vascular injury with two-photon excitation can induce localized damage at the targeted depth without injuring the areas traversed by the laser.

在传统单光子激发诱导的血管损伤中，不仅目标区域会受损，浅层和深层周围区域也会受到影响。相比之下，我们使用双光子激发造成血管损伤的方法可以在目标深度引发局部损伤，而不会伤及激光经过的区域。

In this model, blood leaks from the vessel into the surrounding undamaged tissue. In the external bleeding model of Bergmeier et al., haemostatic plugs are observed exclusively within the intravascular space and at the boundary between intravascular and extravascular regions.

在此模型中，血液从血管渗漏到周围未受损的组织。在Bergmeier等人的外部出血模型中，止血栓仅在血管内空间以及血管内和血管外区域的边界处被观察到。

，

. In contrast, in our internal bleeding model, haemostatic plugs are more widely distributed, extending from the intravascular area into the extravascular space. To determine whether the haemostatic plug observed in our model, which is composed of extravascular fibrin, intravascular platelet aggregate, and a clot consisting of both platelets and fibrin at the vascular boundary (Fig. .

相比之下，在我们的内出血模型中，止血栓分布更广，从血管内区域延伸到血管外空间。为了确定在我们的模型中观察到的止血栓是否由血管外纤维蛋白、血管内血小板聚集体以及血管边界处由血小板和纤维蛋白组成的凝块构成（图。

1a, b

), is specific to our model, we punctured the mouse medial saphenous vein or artery with a 30 G needle and applied compression to halt bleeding. The resulting plug, containing extravascular fibrin and intravascular platelet aggregates, was similar to our model (Supplementary Fig.

我们用30号针头刺穿小鼠的内侧隐静脉或动脉，并施加压迫以止血。由此形成的栓塞包含血管外纤维蛋白和血管内血小板聚集体，与我们的模型相似（补充图）。

). Thus, we conclude that the plugs observed in our model resemble those resulting from vascular injury in mammals.

）。因此，我们得出结论，我们模型中观察到的栓塞类似于哺乳动物血管损伤后产生的栓塞。

A clot of platelets and fibrin formed at the intra/extravascular boundary following the formation of extravascular fibrin, which was similarly observed in Ano6 cKO mice (Fig.

在血管内外边界处形成的血小板和纤维蛋白凝块，这是在细胞外纤维蛋白形成后产生的，同样在Ano6 cKO小鼠中也观察到了这一现象（图。

). Extravascular fibrin formed even in the platelet-depleted and Ano6 cKO mice (Figs.

`). 即使在血小板耗尽和Ano6 cKO小鼠中也形成了血管外纤维蛋白（图。`

and

和

). These findings indicate that coagulation on the surface of platelets plays a minor role in fibrin formation in our internal bleeding/haemostasis model. Notably, platelets involved in early haemostasis did not express CD62P, a marker of irreversibly activated platelets that serve as a site of coagulation (Fig. .

这些发现表明，在我们的内出血/止血模型中，血小板表面的凝血在纤维蛋白形成中起次要作用。值得注意的是，参与早期止血的血小板未表达CD62P，这是不可逆激活血小板的标志物，也是凝血发生的位点（图。

), which aligns with prior findings of no PS exposure on platelets even up to 30 min after bleeding

），这与之前的研究结果一致，即即使在出血后30分钟内，血小板上也没有PS暴露。

. In contrast, Bergmeier et al. reported that fibrin formed primarily on platelets with PS exposure in an external bleeding model

相比之下，Bergmeier 等人报告称，在外部出血模型中，纤维蛋白主要形成于暴露磷脂酰丝氨酸 (PS) 的血小板上。

. This difference may stem from the fact that, in our internal bleeding model, blood leakage into an enclosed space rich in tissue factor allows coagulation to proceed even in the absence of activated platelets. Sufficient thrombin generation occurs not only on activated platelets but also on tissue factor-bearing cells.

这种差异可能源于这样一个事实：在我们的内出血模型中，血液泄漏到富含组织因子的封闭空间后，即使在没有活化血小板的情况下，凝血过程仍可进行。足够的凝血酶生成不仅发生在活化的血小板上，也发生在携带组织因子的细胞上。

, and extravascular cells support the formation of a dense, fibrinolysis-resistant fibrin network

，细胞外基质细胞支持形成密集的、抗纤溶的纤维蛋白网络

. Our findings and previous reports suggest that early after internal bleeding, platelet activation is limited, preventing coagulation within blood vessels while allowing rapid fibrin formation outside vessels. This mechanism may reduce the risk of pathological thrombosis and prevent exsanguination..

我们的研究结果和之前的报告表明，在内出血后的早期阶段，血小板的激活是有限的，这防止了血管内的凝血，同时允许在血管外快速形成纤维蛋白。这种机制可能降低病理性血栓的风险，并防止大出血。

In our study, venous internal bleeding primarily caused plasma leakage with minimal blood cell leakage, whereas arterial internal bleeding involved leakage of both. During arterial internal bleeding, haemostasis occurred first with cessation of blood cell leakage, followed by cessation of plasma leakage (Figs. .

在我们的研究中，静脉内出血主要导致血浆渗漏，伴有极少量的血细胞渗漏，而动脉内出血则涉及两者的渗漏。在动脉内出血过程中，首先发生止血，血细胞渗漏停止，随后血浆渗漏停止（图。

b，

，

8a, g

8a，g

). L-TF and platelet-depleted mice had a significantly longer time to cease blood cell leakage (Fig.

). L-TF 和血小板耗竭的小鼠停止血细胞渗漏的时间显著延长（图。

8克

). Although plasma leakage did not differ between wild-type and Ano6 cKO mice, it increased in L-TF mice with venous bleeding (Fig.

尽管野生型和Ano6 cKO小鼠之间的血浆渗漏没有差异，但在静脉出血的L-TF小鼠中有所增加（图。

) and in L-TF and platelet-depleted mice with arterial bleeding (Fig.

) 在L-TF和血小板减少的小鼠中，动脉出血情况相同 (图。

). These findings highlight the critical role of extravascular coagulation in haemostasis in both veins and arteries, with platelets potentially contributing additional roles in arterial haemostasis other than PS exposure. While fibrin formation rates were not significantly different among the wild-type, Ano6 cKO, and platelet-depleted mice (Figs. .

这些研究结果强调了血管外凝血在静脉和动脉止血中的关键作用，血小板除了PS暴露外，可能在动脉止血中还发挥了其他作用。而纤维蛋白形成速率在野生型、Ano6 cKO和血小板耗竭的小鼠中没有显著差异（图。

7天

and

和

8天

), the platelet-depleted mice spent more time reaching half the final fibrin amount during arterial internal bleeding (Fig.

)，在动脉内出血期间，血小板耗尽的小鼠达到最终纤维蛋白量一半的时间更长（图。

), indicating that platelets help regulate blood flow at injury sites, promoting effective coagulation.

），这表明血小板有助于调节受伤部位的血流，促进有效的凝血。

To elucidate the role of extravascular coagulation in platelet aggregate formation (Fig.

为了阐明血管外凝血在血小板聚集形成中的作用（图。

1a, b

), we compared the heights of platelet aggregates among various mouse groups. In veins, L-TF mice presented significantly lower maximum platelet aggregate heights (Fig.

），我们比较了各组小鼠血小板聚集体的高度。在静脉中，L-TF小鼠的最大血小板聚集体高度显著较低（图。

), emphasizing the role of extravascular coagulation through the TF-triggered extrinsic pathway. In the arteries, the maximum platelet aggregate height did not significantly differ among the wild-type, Ano6 cKO, FVIII KO, and L-TF mice (Fig.

），强调了通过TF触发的外源性途径的血管外凝血作用。在动脉中，野生型、Ano6 cKO、FVIII KO 和 L-TF 小鼠的最大血小板聚集高度没有显著差异（图。

). However, when comparing early-stage (0–120 s) and late-stage (120–300 s) platelet aggregates in both veins and arteries, FVIII KO and L-TF mice exhibited lower late-stage platelet aggregates (Figs.

然而，在比较静脉和动脉中早期（0-120秒）和晚期（120-300秒）的血小板聚集时，FVIII敲除小鼠和L-TF小鼠表现出较低的晚期血小板聚集（图。

and

和

). These findings suggest that in veins, platelet aggregate formation involves TF-triggered coagulation from the early-stage, whereas in arteries, coagulation becomes significant in the late-stage, with early activation driven by shear stress

这些发现表明，在静脉中，血小板聚集体的形成从早期就涉及组织因子（TF）触发的凝血过程，而在动脉中，凝血在后期才变得显著，早期激活则由剪切应力驱动。

，

. Platelets are regulated by both shear stress and extravascular coagulation. Extravascular thrombin activates platelets via PAR1 receptors

血小板受剪切应力和血管外凝血的共同调节。血管外凝血酶通过PAR1受体激活血小板。

(and PAR3 and PAR4 receptors in mice

（以及小鼠中的PAR3和PAR4受体

), whereas thrombin and factor Xa activate the vascular endothelium via PAR1 and PAR2

），而凝血酶和因子Xa通过PAR1和PAR2激活血管内皮。

. This dual regulation may explain the reduced platelet aggregate height in mice with impaired extravascular coagulation. The mechanism by which extravascular coagulation influences haemostatic plug formation, including intravascular platelet aggregate formation, seems reasonable, as it helps prevent excessive thrombus formation once haemostasis is achieved..

这种双重调节可能解释了血管外凝血功能受损小鼠的血小板聚集高度减少的原因。血管外凝血影响止血栓形成（包括血管内血小板聚集形成）的机制似乎是合理的，因为它有助于在实现止血后防止过度血栓形成。

Limitations

限制条件

This study has several limitations. First, the vascular injury was observed under restricted conditions, limiting our understanding of how it affects haemostatic plug and thrombus formation in different vessels or different extents of damage. Second, during arterial internal bleeding, vasoconstriction causes discrepancies in the timing of bleeding exacerbation among mice, highlighting the need to understand its role in haemostatic plug formation.

本研究有几个局限性。首先，血管损伤是在受限条件下观察到的，这限制了我们对其在不同血管或不同程度损伤中如何影响止血栓和血栓形成的理解。其次，在动脉内出血期间，血管收缩会导致小鼠之间出血加重的时间不一致，突显了需要了解其在止血栓形成中的作用。

Third, we injected human fibrinogen conjugated with a fluorescent dye for in vivo observation, which means that we may have underestimated fibrin thrombi since we did not observe mouse fibrin clots. Finally, the anaesthetic urethane may influence the results by inducing sympathetic nervous system excitement, and its use poses challenges for replicating study conditions, as it is no longer recommended in animal experiments.

第三，我们注射了结合荧光染料的人纤维蛋白原用于体内观察，这意味着我们可能低估了纤维蛋白血栓，因为我们没有观察到小鼠的纤维蛋白凝块。最后，麻醉剂氨基甲酸乙酯可能通过诱导交感神经系统兴奋影响结果，并且由于在动物实验中已不再推荐使用，其应用为复制研究条件带来了挑战。

The results of the evaluation of the effects of anaesthetic urethane compared with alternative anaesthetics (a combination of medetomidine, butorphanol and midazolam) are given in the .

评估麻醉剂乌拉坦与替代麻醉剂（美托咪啶、布托啡诺和咪达唑仑的组合）效果的比较结果见。

Supplementary Information

补充信息

。

Conclusion

结论

Our understanding of the impact of extravascular coagulation on haemostasis after internal bleeding is shown in Fig.

我们对血管外凝血对内出血后止血的影响的理解如图所示。

. Our findings, together with those of previous reports

我们的发现，连同之前报告的那些

，

, suggest that the platelet surface is the site of coagulation in external bleeding and that the extravascular tissue is the site of coagulation in internal bleeding, which may explain the phenotype of platelet dysfunction, with frequent nasal and gum bleeding

，表明血小板表面是外出血凝血的部位，而血管外组织是内出血凝血的部位，这可能解释了血小板功能障碍的表型，伴随频繁的鼻出血和牙龈出血。

(external bleeding) and coagulation disorders with frequent deep tissue bleeding

外部出血和凝血障碍，常伴有深层组织出血

(internal bleeding). These insights could inform clinical treatment strategies, such as administering coagulation factors with plasma transfusion in severe trauma, aligning with findings from the PAMPer

（内部出血）。这些见解可以为临床治疗策略提供信息，例如在严重创伤中配合血浆输注给予凝血因子，这与PAMPer的研究结果一致。

and COMBAT

和战斗

clinical trials that highlight benefits in emergency settings. While many clotting factor replacements and mimetics are designed for prolonged retention in the bloodstream, optimizing their concentration outside the vasculature could enable drug development that enhances haemostasis while reducing thrombosis risk.

在急诊环境中突出益处的临床试验。尽管许多凝血因子替代品和拟态物被设计为在血液中长时间保留，优化它们在血管外的浓度可能有助于药物开发，在增强止血的同时降低血栓形成的风险。

Additionally, if fibrinolytic drugs used for thrombosis treatment could be engineered to remain inactive outside the vascular system, it may become possible to manage thrombosis more safely..

此外，如果用于治疗血栓的纤溶药物能够被设计为在血管系统外保持非活性，那么可能就可以更安全地控制血栓。

Fig. 9: Extravascular coagulation triggers and regulates haemostasis.

图 9：血管外凝血触发并调节止血。

When vascular continuity is lost, blood cells and plasma leak out of the vessels (

当血管连续性丧失时，血细胞和血浆会从血管中泄漏出来 (

). Extravascular fibrin and platelets captured by fibrin inhibit blood cell and plasma leakage (

). 被纤维蛋白捕获的血管外纤维蛋白和血小板抑制血细胞和血浆泄漏 (

). Coagulation proceeds extravascularly, and the generated thrombin accelerates extravascular fibrin formation and activates platelets in the lumen (

`). 凝血过程在血管外进行，生成的凝血酶加速血管外纤维蛋白的形成并激活管腔中的血小板 (`

). As extravascular fibrin grows, plasma leakage ceases, resulting in complete haemostasis (

). 随着血管外纤维蛋白的增加，血浆渗漏停止，从而达到完全止血 (

）。

Full size image

全尺寸图像

In conclusion, we demonstrated the critical role of extravascular coagulation in the formation of haemostatic plugs following internal bleeding via in vivo imaging. These findings have the potential to enhance our understanding of bleeding symptoms in clinical settings and contribute to the development of more effective and safer therapeutic drugs.

总之，我们通过体内成像展示了血管外凝血在内出血后形成止血栓中的关键作用。这些发现有可能增强我们对临床出血症状的理解，并有助于开发更有效、更安全的治疗药物。

Furthermore, this research method proved to be a valuable tool for evaluating such drugs..

此外，这种研究方法被证明是评估此类药物的宝贵工具。

Methods

方法

Mice

老鼠

Male C57BL/6J (wild-type) mice, FVIII-deficient (FVIII KO) mice (B6;129S-F8tm1Kaz/J)

雄性C57BL/6J（野生型）小鼠，FVIII缺陷型（FVIII KO）小鼠（B6;129S-F8tm1Kaz/J）

and low-TF-expressing (L-TF) mice

低TF表达（L-TF）小鼠

, all with a matching genetic background, were used. These mice were aged 8–13 weeks and weighed 22–25 g. Platelet-specific anoctamin-6 (TMEM16F) knockout (Ano6 cKO) mice were generated by crossing anoctamin-6 flox/flox (B6.Cg-Ano6tm1.1Naga)

，所有小鼠具有相同的遗传背景，被使用。这些小鼠年龄为8-13周，体重22-25克。血小板特异性anoctamin-6（TMEM16F）敲除（Ano6 cKO）小鼠通过交叉anoctamin-6 flox/flox（B6.Cg-Ano6tm1.1Naga）生成。

and Pf4-iCre transgenic mice

和 Pf4-iCre 转基因小鼠

. Wild-type mice received an intravenous injection of 2 mg/kg R300 antibody (Emfret Analytics GmbH & Co., KG, Würzburg, Germany)

野生型小鼠接受了一次2 mg/kg R300抗体（Emfret Analytics GmbH & Co., KG, 德国维尔茨堡）的静脉注射。

四十三

to obtain platelet-depleted mice. Wild-type mice were purchased from Japan SLC, Inc. (Shizuoka, Japan). FVIII KO and Pf4-iCre mice were obtained from The Jackson Laboratory (Maine, USA). Anoctamin-6 flox/flox mice were provided by Riken BRC (Ibaraki, Japan). L-TF mice were provided by Prof. Nigel Mackman (University of North Carolina at Chapel Hill).

获取无血小板的小鼠。野生型小鼠购自日本SLC公司（静冈，日本）。FVIII敲除小鼠和Pf4-iCre小鼠购自杰克逊实验室（美国缅因州）。Anoctamin-6 flox/flox小鼠由日本理化学研究所生物资源中心（茨城县，日本）提供。L-TF小鼠由北卡罗来纳大学教堂山分校的Nigel Mackman教授提供。

Mice obtained from other facilities were housed in the Institute of Experimental Animal Science at Nara Medical University for at least one week to allow for acclimatisation. The mice were kept under controlled conditions (20–26 °C, 40–60% humidity) with a 12-h light/dark cycle and had ad libitum access to food and water.

从其他设施获得的小鼠被安置在奈良医科大学实验动物科学研究所至少一周，以适应环境。小鼠在受控条件下饲养（20-26°C，40-60%湿度），具有12小时明暗周期，并可自由获取食物和水。

Anaesthesia was induced via intraperitoneal injection of urethane (1.5 mg/kg). After observation, the mice were euthanized by cervical dislocation. The depth of anaesthesia was confirmed by the hindlimb retraction reflex and the abdominal skin pain reflex. All animal experiments were approved by the Institutional Animal Care and Use Committee and strictly followed the guidelines for animal experiments of Nara Medical University (Permit Nos.

通过腹腔注射乌拉坦（1.5 mg/kg）诱导麻醉。观察后，通过颈椎脱位处死小鼠。麻醉深度通过后肢屈曲反射和腹部皮肤疼痛反射确认。所有动物实验均经机构动物护理和使用委员会批准，并严格遵循奈良医科大学的动物实验指南（许可编号）。

13320, 13323, and 13387)..

13320、13323 和 13387）。

In vivo imaging of internal bleeding and haemostatic plug formation after two-photon excitation injury

体内成像：双光子激发损伤后的内出血和止血栓形成

An A1R-MP microscope (Nikon, Japan) was used for visual analysis of bleeding and haemostatic plug formation in the testicular artery and medial saphenous vein. After sufficient anaesthesia was achieved, two small incisions were made in the median abdomen and medial thigh to observe blood vessels on the surface of the testis and under the skin of the thigh.

使用A1R-MP显微镜（尼康，日本）对睾丸动脉和股内侧静脉的出血及止血栓形成进行视觉分析。在充分麻醉后，在腹部中线和大腿内侧切开两个小口，以观察睾丸表面和大腿皮肤下的血管。

Mice were injected via the jugular vein with dextran-rhodamine B (MW 70 kDa) (Merck KGaA, Darmstadt, Germany) (40 μg/g body weight), Alexa Fluor 488-conjugated fibrinogen (Thermo Fisher, Massachusetts, USA) (30 μg/g body weight) and X649 antibody (Emfret) (0.1 μg/g body weight), which labels platelets in mice without affecting their function.

通过颈静脉向小鼠注射了罗丹明B-葡聚糖（分子量70 kDa）（默克公司，达姆施塔特，德国）（40 μg/g体重）、Alexa Fluor 488标记的纤维蛋白原（赛默飞世尔科技，马萨诸塞州，美国）（30 μg/g体重）以及X649抗体（Emfret）（0.1 μg/g体重），该抗体可标记小鼠血小板而不影响其功能。

The mice were placed on a heated stage (Tokaihit, Shizuoka, Japan), and transverse sections of the testicular arteries and medial saphenous veins were visualized via confocal imaging (lens: Nikon Apo LWD ×40/1.15 WI λS, scanner zoom: 1.5×). The endothelium was cauterized by two-photon excitation with a pulsed laser at 800 nm (laser power: ~2064 mW) (laser source: COHERENT Verdi 18 W) (Coherent, Pennsylvania, U.S.A.), with an injury time of 0.8–1.6 s.

小鼠被放置在加热台上（东海日立，静冈，日本），通过共聚焦成像观察睾丸动脉和股静脉内侧的横截面（镜头：尼康Apo LWD ×40/1.15 WI λS，扫描变焦：1.5×）。内皮细胞通过800纳米脉冲激光（激光功率：约2064毫瓦）的双光子激发进行烧灼（激光源：COHERENT Verdi 18瓦）（相干公司，宾夕法尼亚州，美国），损伤时间为0.8至1.6秒。

Laser irradiation was stopped immediately after bleeding was obtained, and the irradiation area was 40 μm in diameter. After vascular injury, extravascular collagen was observed via two-photon microscopy at 930 nm in the same area. The total observation time was 5 min..

出血后立即停止激光照射，照射区域直径为40微米。血管损伤后，通过双光子显微镜在相同区域以930纳米观察到血管外胶原蛋白。总观察时间为5分钟。

In vivo imaging of platelet activation

体内血小板活化成像

After the appropriate depth of anaesthesia was achieved, small incisions were made to observe the testis, and Alexa Fluor 488-conjugated fibrinogen, 25 μL of PE-conjugated rat anti-mouse CD62P antibody (Emfret), and X649 antibody were injected intravenously. Platelets involved in haemostasis were then observed in the testicular artery of wild-type mice via microscopy following two-photon excitation injury.

在达到适当的麻醉深度后，做小切口以观察睾丸，并静脉注射Alexa Fluor 488标记的纤维蛋白原、25微升PE标记的大鼠抗小鼠CD62P抗体（Emfret）和X649抗体。随后通过双光子激发损伤后，利用显微镜观察野生型小鼠睾丸动脉中参与止血的血小板。

In the positive control, small incisions were made in the median upper abdomen to observe the liver after it reached the appropriate depth of anaesthesia. FITC-dextran (MW 70 kDa) (Merck KGaA) (8 μg per g body weight), PE-conjugated rat anti-mouse CD62P antibody, X649 antibody, and 1 U per body thrombin (Fuji Pharma Co., Ltd., Tokyo, Japan) were injected intravenously, and platelets were observed in the liver vasculature via microscopy..

在阳性对照中，于上腹部正中线作小切口，在达到适当麻醉深度后观察肝脏。静脉注射FITC-葡聚糖（分子量70 kDa）（默克公司）（8 μg每克体重）、PE标记的大鼠抗小鼠CD62P抗体、X649抗体和每体重单位1 U的凝血酶（富士制药株式会社，东京，日本），并通过显微镜观察肝脏血管中的血小板。

Image analysis

图像分析

For quantitative analysis of bleeding and fibrin/platelet aggregate formation, the acquired sequential images were first denoised with NIS-Elements Advanced Research (AR) version 5.21.00 (Nikon). Images with vascular damage areas of 25–40 μm for venous bleeding and 10–25 μm for arterial bleeding were then extracted.

为了对出血和纤维蛋白/血小板聚集体形成进行定量分析，首先使用NIS-Elements高级研究（AR）版本5.21.00（尼康）对获取的连续图像进行去噪处理。然后提取静脉出血血管损伤区域为25-40微米、动脉出血为10-25微米的图像。

Platelet, fibrin, and plasma signals were quantified via NIS-Elements AR. The height of adhered platelet aggregates from the endothelial end to the luminal end, defined as the platelet height, was measured (Figs. .

通过NIS-Elements AR量化血小板、纤维蛋白和血浆信号。测量了从内皮端到管腔端的粘附血小板聚集体的高度，该高度定义为血小板高度（图。

and

和

). The protrusion of platelet aggregates outside the vessel was assessed by their length beyond the vascular endothelium (Fig.

）。通过血小板聚集体超出血管内皮的长度来评估其在血管外的突出程度（图。

). Fibrin was quantified by the area of fibrin formed (Figs.

`). 纤维蛋白通过形成的纤维蛋白面积进行量化（图。`

and

和

), which was determined by the stronger signals compared with circulated blood, and bleeding was measured by the leaked blood cells and the signal of leaked plasma. The data were analysed via Microsoft® Excel for Mac version 16.79.1. The maximum platelet height was defined as the peak height.

），这一高度是通过与循环血液相比更强的信号来确定的，出血则通过泄漏的血细胞和泄漏血浆的信号来衡量。数据通过 Microsoft® Excel for Mac 版本 16.79.1 进行分析。最大血小板高度定义为峰值高度。

For bleeding analysis, the maximum intensity of the leaked plasma signal was normalized to 1. In veins, the area under the curve (AUC) of the leaked plasma signal was calculated from 1 to 181 s; in arteries, it was calculated from the time of a 0.4 bleeding signal to 246 s. The fibrin signals in the veins were normalized to a minimum of 0 and a convergence value of 1.

对于出血分析，将泄漏血浆信号的最大强度归一化为1。在静脉中，泄漏血浆信号的曲线下面积（AUC）从1秒计算到181秒；在动脉中，则从0.4出血信号的时间计算到246秒。静脉中的纤维蛋白信号被归一化为最小值0和收敛值1。

For FVIII KO mice, a quadratic fitting curve was generated from values above 0. Other strains’ signals from 0 to 120 s were used to create quadratic fitting curves. The slope of the tangent line for a signal of 0.5 was calculated for these curves. In arteries, a quadratic fitting curve was created from fibrin signals ranging from 0.2 to 0.8, and the slope for 0.5 was similarly calculated.

对于FVIII敲除小鼠，从大于0的值生成了二次拟合曲线。其他品系的信号从0到120秒被用来创建二次拟合曲线。针对这些曲线，计算了信号为0.5时切线的斜率。在动脉中，从0.2到0.8的纤维蛋白信号生成了二次拟合曲线，并同样计算了0.5时的斜率。

The time for blood cells to stop leaking was confirmed via video..

通过视频确认了血细胞停止泄漏的时间。

Statistics and Reproducibility

统计与可重复性

Statistical analyses were conducted via RStudio version 2023.12.0 + 369, with R version 4.3.2 (R Foundation for Statistical Computing, Vienna, Austria). The Wilcoxon signed-rank test was used to compare matched pairs via the ‘exactRankTests’ package (version 0.8-35), whereas the Steel–Dwass test with the Monte Carlo method for three or more independent groups was performed via the ‘NSM3’ package (version 1.18).

统计分析通过RStudio版本2023.12.0+369进行，使用R版本4.3.2（R统计计算基金会，奥地利维也纳）。使用‘exactRankTests’包（版本0.8-35）的Wilcoxon符号秩检验用于比较配对样本，而采用蒙特卡罗方法的Steel–Dwass检验用于三个或更多独立组，通过‘NSM3’包（版本1.18）完成。

。

values < 0.05 indicated statistical significance.

值 < 0.05 表示具有统计学意义。

Data availability

数据可用性

Source data underlying the graphs presented in the main figures are available in Supplementary Data

主图中展示的图表的源数据可在补充数据中获得。

and

和

. Other datasets including R scripts used in this study, are available from the corresponding author upon request.

其他数据集，包括本研究中使用的R脚本，可应要求从通讯作者处获取。

Code availability

代码可用性

In this study, the researchers did not generate any new custom code.

本研究中，研究人员没有生成任何新的自定义代码。

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Acknowledgements

致谢

The Ano6 cKO mice used in this study were generated with permission obtained from S. Nagata (Osaka University, Japan). We would like to thank T. Ohmori (Jichi Medical University, Japan), M. Ishii (Osaka University, Japan) and S. Kawabata (Kyushu University, Japan) for useful discussions. We thank American Journal Experts (AJE) for English language editing and T.

本研究中使用的Ano6 cKO小鼠是经日本大阪大学的名古屋信夫（S. Nagata）授权生成的。我们要感谢日本慈惠医科大学的大森俊彦（T. Ohmori）、日本大阪大学的石井茂（M. Ishii）以及日本九州大学的川端重幸（S. Kawabata）提供的有益讨论。我们感谢美国期刊专家（American Journal Experts, AJE）进行英语语言编辑，以及T.

Kameoka, from Nikon Solutions Co., Ltd. for their technical assistance with microscopy..

来自尼康解决方案有限公司的龟冈市，感谢他们在显微镜技术方面的协助。

Author information

作者信息

Authors and Affiliations

作者与所属机构

Medicinal Biology of Thrombosis and Haemostasis, Nara Medical University, 840 Shijo-Cho, Kashihara, Nara, 634-8521, Japan

日本奈良县橿原市 Kashihara，Shijo-Cho 840，奈良医科大学，血栓与止血医学生物学，邮编634-8521

Asuka Sakata, Kohei Tatsumi, Naoki Matsumoto, Suguru Harada, Ryohei Kawasaki, Yukiko Okuyama-Nishida, Tetsuhiro Soeda, Keiji Nogami & Midori Shima

坂田明日香、辰巳浩平、松本直树、原田卓郎、川崎良平、奥山西田由纪子、添田哲弘、野上圭司、岛美代里

Advanced Medical Science of Thrombosis and Haemostasis, Nara Medical University, 840 Shijo-Cho, Kashihara, Nara, 634-8521, Japan

日本奈良县橿原市 Kashihara，Shijo-Cho 840，奈良医科大学血栓与止血高级医学科学系，邮编634-8521

Kohei Tatsumi

龙士康平

Product Research Department, Medical Affairs Division, Chugai Pharmaceutical Co., Ltd., 216 Totsukacho, Totsuka-ku, Yokohama, Kanagawa, 244-8602, Japan

日本中外制药株式会社医学事务部产品研究部，邮编244-8602，神奈川县横滨市都筑区都筑町216号

Naoki Matsumoto, Suguru Harada, Ryohei Kawasaki & Tetsuhiro Soeda

松本直树、原田卓鲁、川崎亮平、副岛哲弘

Department of Medicine, Division of Hematology and Oncology, University of North Carolina at Chapel Hill, 116 Manning Drive, Chapel Hill, NC, 27599, USA

美国北卡罗来纳大学教堂山分校医学院，血液学与肿瘤学部，地址：116 Manning Drive, Chapel Hill, NC, 27599, USA

Nigel Mackman

奈杰尔·麦克曼

Specialty Lifecycle Management Department, Project & Lifecycle Management Unit, Chugai Pharmaceutical Co., Ltd., 1-1 Nihonbashi-Muromachi 2-chome, Nihonbashi Mitsui Tower, Chuo-ku, Tokyo, 103-8324, Japan

日本中外制药株式会社，特殊生命周期管理部门，项目与生命周期管理单元，地址：日本东京都中央区日本桥室町2丁目1-1，日本桥三井塔楼，邮编：103-8324

Yukiko Okuyama-Nishida

奥田山子-西田

Department of Paediatrics, Nara Medical University, 840 Shijo-Cho, Kashihara, Nara, 634-8521, Japan

日本奈良县橿原市 Kashihara，四条町840号，奈良医科大学儿科部，邮编634-8521

Keiji Nogami

野上敬二

Authors

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Asuka Sakata

坂田明日香

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Kohei Tatsumi

辰巳孝平

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Naoki Matsumoto

松本直树

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Nigel Mackman

奈杰尔·麦克曼

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Suguru Harada

原田卓

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Ryohei Kawasaki

川崎良平

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Yukiko Okuyama-Nishida

奥山雪子-西田

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Tetsuhiro Soeda

副田哲弘

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Keiji Nogami

野上敬二

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Midori Shima

绿岛

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Contributions

贡献

Conceptualization: A.S.; Data curation: A.S., Naoki M., S.H., R.K., Y.N., and T.S.; Data interpretation: A.S., K.T., Naoki M., Nigel M., S.H., R.K., K.N., and M.S.; Formal analysis: A.S. and Naoki M.; Investigation: A.S.; Methodology: A.S.; Project administration: K.T., T.S., and M.S.; Resources: K.T., Nigel M., Y.N., and T.S.; Supervision: M.S.; Visualization: A.S.; Writing— original draft preparation: A.S.

概念化：A.S.；数据管理：A.S.、Naoki M.、S.H.、R.K.、Y.N. 和 T.S.；数据解释：A.S.、K.T.、Naoki M.、Nigel M.、S.H.、R.K.、K.N. 和 M.S.；正式分析：A.S. 和 Naoki M.；调查：A.S.；方法论：A.S.；项目管理：K.T.、T.S. 和 M.S.；资源：K.T.、Nigel M.、Y.N. 和 T.S.；监督：M.S.；可视化：A.S.；写作—原始草稿准备：A.S.

and Naoki M.; Writing—review and editing: A.S., Naoki M., K.T., Nigel M., S.H., R.K., Y.N., T.S., K.N., and M.S..

Naoki M.；撰写—审核与编辑：A.S.、Naoki M.、K.T.、Nigel M.、S.H.、R.K.、Y.N.、T.S.、K.N. 和 M.S.。

Corresponding author

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Correspondence to

致信给

Asuka Sakata

坂田明日香

。

Ethics declarations

伦理声明

Competing interests

利益冲突

This study was funded by Chugai Pharmaceutical. The authors declare the following competing interests: A.S., K.T., Naoki M., S.H., R.K., Y.N., T.S., K.N., and M.S.: Members of the Medicinal Biology of Thrombosis and Haemostasis established by Nara Medical University and Chugai Pharmaceutical Co., Ltd.

本研究由中外制药公司资助。作者声明以下竞争利益：A.S.、K.T.、Naoki M.、S.H.、R.K.、Y.N.、T.S.、K.N. 和 M.S.：均为奈良医科大学与中外制药有限公司设立的血栓与止血医学生物学成员。

Naoki M., S.H., R.K., Y.N., and T.S.: Employees of Chugai Pharmaceutical Co., Ltd. Naoki M., S.H. and R.K.: Ownership of stock by Chugai Pharmaceutical Co., Ltd. M.S.: Patents for inventions related to products of Chugai Pharmaceutical Co., Ltd. K.T.: Grants or research support from the Japan Blood Products Organization, the Mother and Child Health Foundation and Novo Nordisk Pharma.

Naoki M.、S.H.、R.K.、Y.N. 和 T.S.：中外制药株式会社员工。Naoki M.、S.H. 和 R.K.：持有中外制药株式会社股票。M.S.：拥有与中外制药株式会社产品相关的发明专利。K.T.：接受来自日本血液制品组织、母婴健康基金会和诺和诺德制药的资助或研究支持。

M.S.: Takeda Pharmaceutical Co., Ltd., and CSL Behring; honoraria or consultation fees from Chugai Pharmaceutical Co., Ltd.; speaker bureau from Chugai Pharmaceutical Co., Ltd.; CSL Behring, Sanofi, Bayer, Novo Nordisk Pharma, Takeda Pharmaceutical Co., Ltd., Pfizer, and Fujimoto Seiyaku Corp. K.N.: Representative of Medicinal Biology of Thrombosis and Haemostasis collaborative research laboratory; research support from Chugai Pharmaceutical Co., Ltd.; grants or research support from Chugai Pharmaceutical Co., Ltd.; Takeda Pharmaceutical Co., Ltd.; KM Biologics Co., Ltd.; Sanofi Co., Ltd.; Novo Nordisk Pharma Co., Ltd.; Bayer Co., Ltd.; AbbVie GK LLC; Janssen Pharmaceutical K.K.

M.S.：武田药品工业株式会社和CSL Behring；接受中外制药株式会社的酬金或咨询费；担任中外制药株式会社、CSL Behring、赛诺菲、拜耳、诺和诺德制药、武田药品工业株式会社、辉瑞及藤本制药公司的讲者。K.N.：血栓与止血药物生物学合作研究实验室代表；接受中外制药株式会社的研究支持；获得中外制药株式会社、武田药品工业株式会社、KM生物制品株式会社、赛诺菲株式会社、诺和诺德制药株式会社、拜耳株式会社、艾伯维有限公司、杨森制药株式会社的资助或研究支持。

Co., Ltd.; honouraria or consultation fees from Chugai Pharmaceutical Co., Ltd.; Sanofi Co., Ltd.; and CSL Behring..

有限公司；接受过来自中外制药株式会社、赛诺菲有限公司和CSL贝林的酬金或咨询费。

Peer review

同行评审

Peer review information

同行评审信息

Communications Biology

通讯生物学

thanks Eduardo Fuentes and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: Christina Karlsson Rosenthal. A peer review file is available.

感谢爱德华多·富恩特斯和其他匿名评审员对这项工作的同行评审所做出的贡献。主要处理编辑：克里斯蒂娜·卡尔松·罗森塔尔。同行评审文件可供查阅。

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Supplementary information

补充信息

Transparent Peer Review file

透明同行评审文件

Supplementary information

补充信息

Description of Additional Supplementary File

附加补充文件的描述

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Sakata, A., Tatsumi, K., Matsumoto, N.

坂田，A.，龙崎，K.，松本，N.