Cremer Signe E, Catalfamo James L, Goggs Robert, Seemann Stefan E, Kristensen Annemarie T, Szklanna Paulina B, Maguire Patricia B, Brooks Marjory B
Department of Veterinary Clinical Sciences University of Copenhagen Copenhagen Denmark.
Department of Population Medicine and Diagnostic Sciences Cornell University Ithaca NY USA.
Res Pract Thromb Haemost. 2020 Dec 3;5(1):55-68. doi: 10.1002/rth2.12450. eCollection 2021 Jan.
Domestic dogs represent a translational animal model to study naturally occurring human disease. Proteomics has emerged as a promising tool for characterizing human platelet pathophysiology; thus a detailed characterization of the core canine activated platelet secretome (CAPS) will enhance utilization of the canine model. The objectives of this study were development of a robust, high throughput, label-free approach for proteomic identification and quantification of the canine platelet (i) thrombin releasate proteins, and (ii) the protein subgroup that constitutes CAPS.
Platelets were isolated from 10 healthy dogs and stimulated with 50 nmol/L of γ-thrombin or saline. Proteins were in-solution trypsin-digested and analyzed by nano-liquid chromatography-tandem spectrometry. Core releasate proteins were defined as those present in 10 of 10 dogs, and CAPS defined as releasate proteins with a significantly higher abundance in stimulated versus saline controls (corrected < .05).
A total of 2865 proteins were identified; 1126 releasate proteins were present in all dogs, 650 were defined as CAPS. Among the differences from human platelets were a canine lack of platelet factor 4 and vascular endothelial growth factor C, and a 10- to 20-fold lower concentration of proteins such as haptoglobin, alpha-2 macroglobulin, von Willebrand factor, and amyloid-beta A4. Twenty-eight CAPS proteins, including cytokines, adhesion molecules, granule proteins, and calcium regulatory proteins have not previously been attributed to human platelets.
CAPS proteins represent a robust characterization of a large animal platelet secretome and a novel tool to model platelet physiology, pathophysiology, and to identify translational biomarkers of platelet-mediated disease.
家犬是用于研究自然发生的人类疾病的一种转化动物模型。蛋白质组学已成为表征人类血小板病理生理学的一种有前景的工具；因此，对犬类活化血小板分泌组核心成分（CAPS）进行详细表征将提高犬类模型的利用率。本研究的目的是开发一种强大的、高通量的、无标记的方法，用于蛋白质组学鉴定和定量犬类血小板中的（i）凝血酶释放蛋白，以及（ii）构成CAPS的蛋白质亚组。
从10只健康犬中分离血小板，并用50 nmol/L的γ-凝血酶或生理盐水刺激。蛋白质经溶液内胰蛋白酶消化后，通过纳升液相色谱-串联质谱分析。核心释放蛋白定义为在10只犬中的10只中都存在的蛋白，CAPS定义为与生理盐水对照相比，在刺激组中丰度显著更高的释放蛋白（校正后 < 0.05）。
共鉴定出2865种蛋白质；所有犬中均存在1126种释放蛋白，650种被定义为CAPS。与人类血小板的差异包括犬类缺乏血小板因子4和血管内皮生长因子C，以及触珠蛋白、α-2巨球蛋白、血管性血友病因子和淀粉样β蛋白A4等蛋白质的浓度低10至20倍。28种CAPS蛋白，包括细胞因子、黏附分子、颗粒蛋白和钙调节蛋白，以前未被归因于人类血小板。
CAPS蛋白代表了对大型动物血小板分泌组的有力表征，以及一种用于模拟血小板生理、病理生理和识别血小板介导疾病的转化生物标志物的新型工具。
