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Protocol
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医学

血管性血友病因子-血小板串形成的流室模型研究血管血友病因子的病理生理学

Published: August 14, 2017
doi:
10.3791/55917
, , , ,
1Department of Pathology and Molecular Medicine,Queen’s University, 2Department of Medicine,Queen’s University

Summary

本文介绍了一种方法来评估内皮血管性血友病因子释放和随后的血小板捕获在流体剪切应激反应的炎症刺激使用的体外流室系统。

Abstract

血管性血友病因子 (VWF) 是一种 multimeric 糖蛋白凝血因子, 介导血小板黏附和聚集在内皮损伤的部位, 并携带因子 VIII 在循环。VWF 是由内皮细胞合成的, 或者被释放组成到血浆中, 或者被储存在专门细胞器中, 称为 Weibel-Palade 体 (WPBs), 以满足止血的挑战而按需释放。促和促炎刺激能迅速诱导 WPB 胞和 VWF 释放。内皮细胞释放的大部分 VWF 在血浆中循环;然而, VWF 的一部分被锚定在内皮细胞表面。在生理剪切条件下, 内皮锚 VWF 可与血小板结合, 形成 VWF-血小板柱, 可能代表血栓形成的病灶。流室系统可用于视觉观察的释放 VWF 从内皮细胞和随后的血小板捕获的方式, 是重现性和相关的病理生理 VWF 介导的血栓形成。利用这种方法, 内皮细胞培养在流室, 并随后刺激与促诱导 WPB 胞。洗过的血小板在活化的内皮细胞上进行灌注。血小板被激活, 并随后绑定到细长的 VWF 弦的流体流向。利用胞外组蛋白作为促和促炎症刺激, 我们观察到, 与未处理的内皮细胞相比, VWF-血小板的形成与组织治疗的内皮细胞的增加有关。本议定书描述了定量的, 视觉的和 real-time 的评估, 活化的 VWF-血小板相互作用模型的血栓和止血。

Introduction

血栓形成是导致全球死亡的主要原因1 , 可以在反应 todysregulated 血小板活化和凝血酶生成两 veinsand 动脉。血浆水平 VWF 是血液凝固的一个关键的调节器, 藉以低水平 (< 50%) 导致被称为血管性血友病病 (VWD)2和高水平 (> 150%) 的出血性疾病与增加的风险静脉的3和动脉4血栓形成。

VWF 是巨和内皮细胞合成的 multimeric 糖蛋白, 分别保存在血小板α颗粒和 WPBs 中。在止血的挑战, VWF 可以释放从内皮 WPBs 到绳循环血小板的活化内皮细胞5或暴露的胶原蛋白在血管壁上的6。VWF 对内皮细胞的锚定是由 p-选择素7和整合因子αvβ38所介导的。随后释放的血小板α颗粒存储可以进一步增加局部 VWF 浓度, 稳定血小板与血小板的相互作用, 为血小板堵塞的形成, 需要支架的传播凝血叶栅和纤维蛋白沉积.VWF 的血小板结合活动受其 multimeric 结构的调节, 高分子重量体具有较大的止血活性9,10。在循环中, VWF 也充当凝血因子 VIII. 的载体。

流体剪切应力是 VWF 生理的重要调节因子。在没有剪切应力的情况下, VWF 存在于球状形态中, 为血小板糖蛋白 Ib 黏附力的粘附域11。当剪切应力存在时, 金属、整合和金属与凝血酶母模 (ADAMTS13) 的劈裂部位被暴露。ADAMTS13 克里夫斯裸和血小板修饰的 VWF 弦来调节 multimer 的大小, 从而减少其止血活性12。

VWF 是一种急性相蛋白, 许多刺激, 包括缺氧13, 感染的14, 和促炎细胞因子, 已被证明是调解 VWF 释放内皮细胞。类似于其他炎症因子, 胞外组蛋白也被证明在小鼠体内诱发全身 VWF 释放15,16和血小板活化,体外17,18, 19. 这被证明取决于组蛋白亚型, 因为赖氨酸和精氨酸含量的差异可能影响功能15。我们的研究目的是建立一个流室模型来研究富含赖氨酸 (HK) 和精氨酸 (HR) 组蛋白亚型和促对内皮 VWF 释放和 real-time 血小板捕获的影响, 潜在的早期事件炎症引起的血栓。

这种流室方法重述在体内的内胶原蛋白, 内皮细胞, VWF 和血小板之间的相互作用, 在一个体外系统, 是可视化, 重现性和可量化的。它允许对调节 VWF-血小板相互作用的通路的所有方面进行 real-time 评估, 包括 WPB 分泌、血小板活化和 VWF 蛋白水解。在受控剪应力条件下的 VWF 的研究已被用来评价 VWD 突变 , 影响 VWF 释放和血小板结合功能20, WPB 生理学21, VWF ADAMTS135。我们使用这种方法来量化 VWF-血小板串形成的结果, 炎症刺激: 胞外组蛋白。

Protocol

这些研究得到了加拿大皇后大学研究伦理学委员会的批准。 1. 内皮细胞刺激 胶原蛋白涂层一个6良好的组织培养板。 提前24小时, 在37° c 的情况下, 用1毫升胶原蛋白缓冲液 (50 µg/mL 鼠尾胶原 1, 0.02 米冰醋酸) 涂上6层组织培养板。 用2毫升的汉克平衡盐溶液 (HBSS) 冲洗两次井。注: 板材可以用锡纸包裹, 并存放在4° c 的密闭塑料袋中。 <…

Representative Results

为了直接评估组蛋白对内皮细胞 VWF 释放的影响, 我们将汇合 BOECs 暴露在含有 PMA (阳性对照)、呃、HR 和 HK 2 h 的无血清培养基中。我们发现, 在治疗的内皮细胞 (图 1) 中, 香港诱导的 VWF 蛋白 (VWF: Ag) 增加了2倍。有趣的是, 当 BOECs 被刺激与 UH 和 HR, 有较少 VWF: Ag 在媒介比在未处理的情况发现了。我们假设, 由于组蛋白可以直接绑定到 VWF30</s…

Discussion

虽然 VWF-血小板串的生理相关性仍然存在争议, 由于它们在 VWF 裂解蛋白酶 ADAMTS13 中的迅速溶解, 它们可以作为一个可量化的体外模型, 通过 VWF 的方法在现场进行血小板招募。在组蛋白水平增加的情况下, 血栓可能形成于5。此外, 在缺乏 ADAMTS13 活动的病理-如血栓性血小板减少紫癜 (或炎症环境-ADAMTS13 活动是抑制-VWF-血小板字符串可以观察到在体内32 …

Disclosures

The authors have nothing to disclose.

Acknowledgements

艾莉森州是来自加拿大卫生研究院 (研究院) 的弗雷德里克班廷和查尔斯最佳加拿大研究生奖学金获得者。劳拉· l · Swystun 是研究院奖学金获得者。戴维 Lillicrap 是一个加拿大研究椅子分子止血的接受者。这项研究部分由研究院的营运补助金 (MOP-97849) 资助。

Materials

Calf-thymus unfractionated histones (UH) Worthington Biochemical HLY Reconstituted in serum-reduced media (5 mg/mL)
Calf-thymus lysine-rich histones (HK) Sigma-Aldrich H5505 Reconstituted in serum-reduced media (5 mg/mL)
Calf-thymus arginine-rich histones (HR) Sigma-Aldrich H4830 Reconstituted in serum-reduced media (5 mg/mL)
Phorbol 12-myristate 13-acetate (PMA) Sigma-Aldrich P8139 Reconstituted in DMSO (20 mM)
Histamine Sigma-Aldrich H7125-1G Reconstituted in water (50 mg/mL)
3,3' Dihexyloxacarbocyanine Iodide (DiOC6) Invitrogen D273 Reconstituted in methanol (20 mM)
Rabbit Anti-VWF Coating Antibody DAKO A0082 For VWF ELISA
Rabbit Anti-VWF Detection Antibody, HRP conjugated DAKO P0026 For VWF ELISA and histone-VWF binding assay
Nunc MaxiSorp flat-bottom 96-well microplates eBioscience 44-2404-21 For histone-VWF binding assay
Immulon 4 HBX Flat Bottom Microtiter 96-Well Plates Thermo Scientific 3855 For VWF ELISA
Humate-P CSL Behring N/A Plasma-derived human von Willebrand factor/factor VIII complex
Normal Reference Plasma Precision BioLogic CCNRP-05 For VWF ELISA standard curve
O-Phenylenediamine dihydrochloride (OPD) reagent Sigma-Aldrich P8287 Equivalent product available through ThermoFisher Scientific (Catalogue Number: 34006)
EGM-2 BulletKit Lonza CC-3162 For culturing and initial seeding of BOEC
Hank's Balanced Salt Solution (HBSS) ThermoFisher Scientific 14025092
Rat-tail Collagen Type 1 Corning 354236
Gibco Opti-MEM I Reduced Serum Media ThermoFisher Scientific 31985070 For endothelial cell stimulations
METAMORPH Microscopy Automation and Image Analysis Software Molecular Devices N/A
BD Vacutainer Blood Collection Tubes, No Additive BD Biosciences 366703
µ-Slide III 0.1 (flow chambers) Ibidi This product has been discontinued. We suggest using µ-Slide VI 0.1 (#80661) or 0.4 (# 80601) and recalculating flow rate and platelet volume needed to maintain a shear stress of 4.45 dyn/cm2
Silicone Tubing 1.6 mm ID: 5 m, sterilized Ibidi 10842
Luer Lock Connector Female: natural Polypropylene, sterilized Ibidi 10825
Elbow Luer Connector Male: white Polypropylene, sterilized Ibidi 10802
Blunted 18G Needle BD Biosciences 305180
20 mL syringes BD Biosciences 302830
Syringe Pump New Era Pump Systems Inc. NE-1600 Multi-PhaserTM N/A
Quorum WaveFX- 4X1 spinning disk microscope Quorum Technologies N/A
Image Processing Software ImageJ N/A
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Tags

Von Willebrand FactorVWF-platelet String FormationFlow Chamber ModelCollagenEndothelial CellsPlateletsSecretagoguesIn VitroMicroscopySyringe Pump

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Michels, A., Swystun, L. L., Mewburn, J., Albánez, S., Lillicrap, D. Investigating von Willebrand Factor Pathophysiology Using a Flow Chamber Model of von Willebrand Factor-platelet String Formation. J. Vis. Exp. (126), e55917, doi:10.3791/55917 (2017).
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