研究目的：本研究旨在解析哺乳动物胚胎期血小板的形态学特征、多组学分子表达谱及功能优势，揭示其与成年期血小板的发育差异。通过探索胚胎期血小板独特的生物学特性及其潜在应用价值，填补血小板发育生物学空白，并以发育时期为切入点，探索血小板异质性并鉴定具有胚胎期功能优势的血小板亚群。

研究方法：

1. 通过透射电子显微镜观察胚胎期与成年期血小板超微结构；

2. 优化血小板纯化流程，结合转录组与蛋白组测序技术对比胚胎期与成年期血小板分子特征；

3. 利用体外成纤维细胞划痕实验、体内皮肤创伤修复模型验证血小板发育支持与促修复能力；

4. 利用形态学观察、单细胞测序、流式高通量筛查探索血小板异质性，鉴定具有胚胎期分子特征与功能优势的血小板亚群，并评估其临床转化潜力。

研究结果：

1. 形态特征：与成年期相比，胚胎期血小板具有较大的平均体积、流动性更强的胞膜、更小的α颗粒、更稀少的致密颗粒、体积更大的线粒体及含量更为丰富的核糖体。

2. 分子特征：1) 转录组特征：能量生成、蛋白合成、脂质代谢等维持血小板自身稳态的相关基因在胚胎期血小板中表达上调；生长因子、形态发育、心肺等实体器官发育、神经发育、行为认知、学习记忆等大量发育支持相关基因的表达也在胚胎期血小板中显著上调；止凝血与免疫调控基因的表达在胚胎期较成年期显著下调。2) 蛋白组特征：与转录组趋势一致，能量生成、蛋白合成与发育支持相关蛋白在胚胎期血小板中表达显著上调；同时止凝血与免疫调控相关蛋白的表达较成年期显著下调。

3. 功能优势：1) 与成年期相比，胚胎期血小板促进成纤维细胞增殖的能力显著增强，添加胚胎期血小板后成纤维细胞划痕愈合效率提高近20%。2) 与成年期相比，胚胎期血小板具有更强的促修复能力。在皮肤全层损伤模型中，涂抹胚胎期血小板后，糖尿病小鼠创面愈合比例在创伤后第六天可达到80%，显著优于成年期血小板在同期60%的愈合比例。

4. 异质性解析：1) 电镜下细胞器观察表明胚胎期血小板存在结构异质性，部分血小板中核糖体与线粒体含量异常丰富。2) 单细胞测序提示胚胎期血小板并非均质性群体，其分子特征存在异质性，且能量生成、蛋白合成、发育支持相关基因集中高表达于同一亚群。3) 表面标志分子CD59a可筛选出一群“类胚胎”血小板亚群，其比例在胚胎期约为80%，成年期约为15%。该亚群血小板线粒体功能活跃，蛋白合成与发育支持相关分子及促修复效应因子表达水平较高，且促修复能力显著优于CD59a阴性群体，表现出更类似于胚胎期血小板的特征与功能。4) 小鼠CD59a在人源样本中的同源蛋白CD59同样能够筛选出一群促修复能力更强的血小板，表明该血小板亚群具有临床转化潜力。

研究结论：本研究首次揭示了胚胎期血小板的转录组与蛋白组分子表达谱，基于其形态特征与多组学分子特征发现了胚胎期血小板具有较强的能量生成、蛋白合成、促增殖及促修复能力。并以血小板“发育特异性”为切入点鉴定到CD59a阳性血小板是一群具有胚胎期分子特征与功能优势的血小板亚群，且该亚群在创伤修复中表现出的显著优势具有人鼠保守性，为难愈合性创伤治疗提供了更为精准高效的细胞治疗新思路。

Objective: This study aims to analyze the morphological characteristics, multi-omics molecular profiles, and functional advantages of mammalian embryonic platelets, highlighting their developmental differences from adult platelets. By investigating the unique biological properties of embryonic platelets, this study seeks to fill the gap in platelet developmental biology. Furthermore, using the embryonic stage as a focal point, it explores platelet heterogeneity and identifies platelet subpopulations with embryonic-specific characteristics and advantageous functions.

Methods: 

1. Ultrastructural Analysis: Transmission electron microscopy (TEM) was used to examine the ultrastructure of embryonic platelets.

2. Molecular Characterization: An optimized platelet purification protocol was established, followed by transcriptomic and proteomic sequencing to compare the molecular characteristics of embryonic and adult platelets.

       3. Functional Evaluation: The fibroblast scratch assay and             cutaneous wound healing model were conducted to assess             the development-supporting and wound-healing capacities           of embryonic platelets.

4. Heterogeneity Exploration：Morphological classification, single-cell sequencing, and high-throughput flow cytometry screening were used to investigate platelet heterogeneity. Specialized subpopulation with embryonic molecular characteristics and enhanced functions were identified, and their potential for clinical translation was evaluated.

Results: 

1. Morphological Characteristics: 

Compared with adult platelets, embryonic platelets exhibited larger average volumes, more variable cell contours, smaller α-granules, fewer dense granules, larger mitochondria, and a higher abundance of ribosomes.

2. Molecular Characteristics:

a.Transcriptomic Profile: Genes related to energy production, protein synthesis, and lipid metabolism, which are essential for maintaining platelet homeostasis, were upregulated in embryonic platelets. Genes associated with growth factors, morphogenesis, organ development (e.g., heart and lung), neurodevelopment, cognition, learning, and memory were significantly upregulated in embryonic platelets. Conversely, genes related to hemostasis, coagulation, and immune regulation were significantly downregulated in embryonic platelets compared to adult platelets. b.Proteomic Profile: Consistent with transcriptomic findings, proteins associated with energy production, protein synthesis, and developmental support were significantly upregulated in embryonic platelets, whereas those involved in hemostasis, coagulation, and immune regulation were downregulated compared to adult platelets.

3. Functional Advantages:

a.Compared with adult platelets, embryonic platelets exhibited significantly enhanced ability to promote fibroblast proliferation, increasing fibroblast scratch closure rate by 20%.

b.Embryonic platelets demonstrated stronger pro-repair capabilities than adult platelets. In the diabetic mice, the application of embryonic platelets resulted in 80% wound closure by day 6 post-injury, significantly higher than the 60% closure rate observed with adult platelets.

4. Heterogeneity Analysis:

a.TEM analysis revealed structural heterogeneity among embryonic platelets, with some exhibiting an exceptionally high abundance of ribosomes and mitochondria.

b.Single-cell sequencing indicated that embryonic platelets are not a homogeneous population, as their molecular characteristics exhibit heterogeneity, with energy production, translation, and developmental support genes being highly expressed in a specific subset.

c.The surface marker CD59a identified an embryonic-like subpopulation, constituting approximately 80% of embryonic platelets and 15% of adult platelets. This subset possesses active mitochondria, higher expression of protein synthesis and developmental support molecules. Furthermore, its pro-repair capacity is significantly superior to that of the CD59a-negative population, displaying characteristics and functions more closely resembling those of embryonic platelets.

d.The homologous protein CD59 in human samples, corresponding to the mouse CD59a surface protein, was also able to isolate a subpopulation of platelets with enhanced pro-repair capacity, indicating its potential for clinical translation.

Conclusion: This study is the first to reveal the transcriptomic and proteomic molecular profiles of embryonic platelets. Based on their morphological and multi-omics molecular characteristics, it was found that embryonic platelets exhibit enhanced energy production, protein synthesis, pro-proliferative, and pro-repair capabilities. By leveraging the developmental specificity of platelets, CD59a-positive platelets were identified as a subpopulation with embryonic molecular features and functional advantages. This subpopulation demonstrated significant advantages in wound healing, exhibiting conserved characteristics in both human and mouse platelets, providing a more precise and efficient approach for cell-based therapy in clinic.