Upregulation of Thrombo-Inflammatory Pathways May Contribute to Increased Thrombotic Risk in Polycythemia Vera and Essential Thrombocythemia
Upregulation of Thrombo-Inflammatory Pathways May Contribute to Increased Thrombotic Risk in Polycythemia Vera and Essential Thrombocythemia
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血栓炎症途径的上调可能导致真性红细胞增多症和原发性血小板增多症的血栓风险增加
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发表时间：
2016
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通讯作者：
J. Prchal
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作者：
R. Gangaraju;Soo Jin Kim;Jihyun Song;S. Swierczek;P. Thiagarajan;J. Prchal
血栓形成是真性红细胞增多症（PV）和原发性血小板增多症（ET）患者发病和死亡的主要原因。已知的血栓形成风险因素包括：年龄> 60岁、血栓形成史、高白细胞计数和JAK 2 V617 F阳性。一些研究未能显示血小板计数增加和血栓形成之间的相关性，尽管已经显示了多种血细胞谱系的定性异常。然而，PV/ET中血栓形成的机制基础仍然未知。为了更好地了解PV和ET血栓形成的病理生理学，我们研究了这些患者粒细胞和血小板中选定的血栓形成、炎症和缺氧诱导因子（HIF）通路基因的转录水平。
选择用于研究的基因包括：编码组织因子（TF）的F3基因; P-选择素（SELP）;丝氨酸蛋白酶抑制剂肽酶抑制剂进化枝E成员1（SERPINE 1，编码纤溶酶原激活物抑制剂I，PAI 1）;血小板反应蛋白1（THBS 1）;白细胞介素1受体相关激酶1（IRAK 1）;白细胞介素1受体辅助蛋白（IL 1 RAP）。我们还询问了这些HIF调节基因：血管内皮生长因子A（VEGFA）和溶质载体家族2（SLC 2A 1，编码葡萄糖转运蛋白1）。使用TaqMan表达测定（Applied Biosystems，CA）通过QT-RT PCR测定粒细胞和血小板中这些选择的基因的转录水平。
33例PV患者（均为JAK 2 V617 F阳性）和12例ET患者（7例JAK 2 V617 F，3例钙网蛋白突变和1例三阴性）纳入研究。其中女性25例，男性20例，平均年龄60.7岁。其中，23例患者有血栓形成病史（15例女性和8例男性）。15例患者有腹部血栓形成，8例有其他血栓形成，包括卒中、深静脉血栓形成、肺栓塞、脑静脉窦血栓形成或微血管系统中的动脉凝块。在粒细胞中，我们观察到PV/ET患者中所有这些基因的表达均高于正常对照。与未发生血栓形成的患者相比，发生血栓形成的PV和ET患者中SELP、THBS 1和IRAK 1的转录水平升高（图1）。在血小板中，除了IRAK 1表达较低外，PV/ET患者中所有检测基因的转录水平均高于对照组。血栓形成患者的F3、SERPINE 1、IL 1 RAP、VEGFA和SLC 2A 1的转录物高于无血栓形成的患者（图2）。我们现在正在比较TF活性与其F3转录和治疗的影响与羟基脲和聚乙二醇干扰素对这些基因的表达。
TF是一种主要的凝血启动因子，我们通过QT-RT PCR发现PV/ET患者中F3转录本的水平较高。我们还发现，在另一组PV患者中，通过无偏RNA测序，粒细胞中F3转录本增加。然而，单核细胞是TF的主要来源（Osterud，Thromb Res.2010），实际上，我们评估的对照单核细胞具有比粒细胞或血小板更高的F3转录物。与对照组不同，PV/ET患者的粒细胞和血小板可能转录F3。在正在进行的分析中，正在评价PV/ET单核细胞中F3的表达水平和TF活性。
我们还通过RNA Seq分析了不同PV患者队列（9例有血栓形成，16例无血栓形成）的整个转录组。在这个队列中，粒细胞分析显示2，025个基因过表达，2，137个基因下调。功能注释结果见表。在伴有血栓形成的PV患者中，644个基因表达上调，573个基因表达下调。其中约10%的上调基因与炎症和凝血途径相关，包括SERPINE 1。检测到SELP、THBS 1和IL 1 RAP的选择性剪接变体。在PV中发现884个基因存在选择性剪接变异体异常表达，在伴血栓的PV患者中发现35个基因。血小板分析正在进行中。
总之，在PV和ET患者的粒细胞和血小板中，血栓炎性基因和HIF调节基因的转录物增加，这可能在血栓形成风险增加中起作用。这些基因的转录本在粒细胞和血小板中不同，反映了它们的转录具有组织特异性的事实。基因表达的差异可以识别血栓形成风险较高的患者和可能的治疗靶点。
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没有相关的利益冲突需要申报。
[1]：待定：是
Thrombosis is a major cause of morbidity and mortality in polycythemia vera (PV) and essential thrombocythemia (ET) patients. Known risk factors for thrombosis include: age > 60 years, history of thrombosis, high leukocyte count and JAK2 V617F positivity. Several studies have failed to show correlation between increased platelet count and thrombosis, though qualitative abnormalities in multiple blood cell lineages have been shown. The mechanistic basis of thrombosis in PV/ET, however remains unknown. To better understand the pathophysiology of thrombosis in PV and ET, we studied transcript levels of selected thrombotic, inflammatory and hypoxia inducible factor (HIF) pathway genes in granulocytes and platelets of these patients.
Genes selected for the study include: F3 gene encoding tissue factor (TF); P-selectin ( SELP ); serpin peptidase inhibitor clade E member 1 ( SERPINE1 , encoding plasminogen activator inhibitor I, PAI1); thrombospondin 1 (THBS1 ); interleukin 1 receptor associated kinase 1 ( IRAK1 ); interleukin 1 receptor accessory protein ( IL1RAP) . We also interrogated these HIF regulated genes: vascular endothelial growth factor A ( VEGFA ) and solute carrier family 2 ( SLC2A1 , encoding glucose transporter 1). Transcript level of these selected genes in granulocytes and platelets was determined by QT-RT PCR using TaqMan Expression Assays (Applied Biosystems, CA).
Thirty-three patients with PV (all JAK2 V617F positive), and 12 ET patients (7 JAK2 V617F , 3 calreticulin mutated and 1 triple negative) were included in the study. There were 25 females and 20 males with a mean age of 60.7 years. Of these, 23 patients had a history of thrombosis (15 females and 8 males). Fifteen patients had abdominal and 8 had other thromboses, including stroke, deep vein thrombosis, pulmonary embolism, cerebral venous sinus thrombosis or arterial clots in microvasculature. In granulocytes, we observed a higher expression of all these genes in PV/ET patients compared to normal controls. Transcript levels of SELP, THBS1 and IRAK1 were elevated in PV and ET patients with thrombosis, compared to those who did not have thrombosis (Figure 1). In platelets, all the tested genes had higher transcript levels in PV/ET patients compared to controls, except IRAK1, which had lower expression. Thrombotic patients had higher transcripts of F3, SERPINE1, IL1RAP, VEGFA and SLC2A1 than those without thrombosis(Figure 2). We are now comparing TF activity with its F3 transcript and the effects of therapy with hydroxyurea and pegylated interferon on the expression of these genes.
TF is a principal initiator of coagulation and we found high levels of F3 transcripts in PV/ET patients by QT-RT PCR. We also found, in a different cohort of PV patients, increased transcripts of F3 in granulocytesby unbiased RNA sequencing. However, monocytes are the principal source of TF (Osterud, Thromb Res. 2010), and indeed, control monocytes we evaluated had higher transcripts of F3 than granulocytes or platelets. Unlike controls, it is possible that granulocytes and platelets may transcribe F3 in PV/ET patients. In ongoing analysis, the expression levels of F3, and TF activity in PV/ET monocytes is being evaluated.
We also analyzed the whole transcriptome in a different cohort of PV patients (9 with and 16 without thrombosis) by RNA Seq. In this cohort, granulocyte analysis revealed that 2,025 genes were overexpressed and 2,137 genes downregulated. Functional annotation results are shown in the Table. In PV patients with thrombosis, 644 genes were upregulated and 573 genes downregulated. About 10 % of these upregulated genes are associated with inflammation and coagulation pathways, including SERPINE1 .Alternative splicing variants of SELP , THBS1 and IL1RAP were detected. Alternative splicing variants with aberrant expression were found in 884 genes in PV, and 35 genes in PV patients with thrombosis. Analysis of platelets is ongoing.
In conclusion, transcripts of thrombo-inflammatory and HIF -regulated genes are increased in granulocytes and platelets in PV and ET patients, which may play a role in increased thrombotic risk. The transcripts of these genes differ in granulocytes and platelets, reflecting the fact that their transcription is tissue specific. The differences in gene expression may identify patients with higher risk for thrombosis and possible therapeutic targets.
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Disclosures No relevant conflicts of interest to declare.
[1]: pending:yes