Despite increased understanding of the genomic landscape of Myeloproliferative Neoplasms (MPNs), the pathological mechanisms underlying abnormal megakaryocyte (Mk)-stromal crosstalk and fibrotic progression in MPNs remain unclear. We conducted mass spectrometry-based proteomics on mice with Romiplostim-dependent myelofibrosis to reveal alterations in signaling pathways and protein changes in Mks, platelets, and bone marrow (BM) cells. The chemokine Platelet Factor 4 (PF4)/Cxcl4 was up-regulated in all proteomes and increased in plasma and BM fluids of fibrotic mice. High TPO concentrations sustained in vitro PF4 synthesis and secretion in cultured Mks, while Ruxolitinib restrains the abnormal PF4 expression in vivo. We discovered that PF4 is rapidly internalized by stromal cells through surface glycosaminoglycans (GAGs) to promote myofibroblast differentiation. Cxcl4 gene silencing in Mks mitigated the profibrotic phenotype of stromal cells in TPO-saturated co-culture conditions. Consistently, extensive stromal PF4 uptake and altered GAGs deposition were detected in Romiplostim-treated, JAK2^V617F mice and BM biopsies of MPN patients. BM PF4 levels and Mk/platelet CXCL4 expression were elevated in patients, exclusively in overt fibrosis. Finally, pharmacological inhibition of GAGs ameliorated in vivo fibrosis in Romiplostim-treated mice. Thus, our findings highlight the critical role of PF4 in the fibrosis progression of MPNs and substantiate the potential therapeutic strategy of neutralizing PF4-GAGs interaction.

尽管人们对骨髓增生性肿瘤（MPN）的基因组景观有了更多的了解，但 MPN 中异常巨核细胞（Mk）-基质串扰和纤维化进展的病理机制仍不清楚。我们对患有 Romiplostim 依赖性骨髓纤维化的小鼠进行了基于质谱的蛋白质组学，以揭示 Mks、血小板和骨髓 (BM) 细胞中信号通路的改变和蛋白质变化。趋化因子血小板因子 4 (PF4)/Cxcl4 在所有蛋白质组中均上调，并在纤维化小鼠的血浆和骨髓液中增加。高TPO浓度维持体外培养的Mks中PF4的合成和分泌，而鲁索替尼抑制体内PF4的异常表达。我们发现PF4通过表面糖胺聚糖（GAG）被基质细胞快速内化，以促进肌成纤维细胞分化。 Mks 中的Cxcl4基因沉默减轻了 TPO 饱和共培养条件下基质细胞的促纤维化表型。一致地，在 Romiplostim 治疗的 JAK2 ^V617F小鼠和 MPN 患者的 BM 活检中检测到广泛的基质 PF4 摄取和改变的 GAG 沉积。患者的 BM PF4 水平和 Mk/血小板CXCL4表达升高，仅在明显纤维化的情况下。最后，GAG 的药理抑制可改善 Romiplostim 治疗小鼠的体内纤维化。因此，我们的研究结果强调了 PF4 在 MPN 纤维化进展中的关键作用，并证实了中和 PF4-GAG 相互作用的潜在治疗策略。