Division of Immunohematology and Transfusion Medicine.
Department of Hematology; Hospital Papa Giovanni XXIII, Bergamo, Italy.
Thromb Res. 2016 Apr;140 Suppl 1:S183. doi: 10.1016/S0049-3848(16)30152-9. Epub 2016 Apr 8.
The myeloproliferative neoplasms ET and PV are characterized by a high incidence of both arterial and venous thrombosis, and/or microcirculatory disturbances. Three somatic mutations, i.e. JAK2-V617F, Calreticulin (CalR) and MPL, commonly found in these diseases, correlate with different thrombotic risk levels.
To analyze the influence of JAK2-V617F, CalR and MPL mutations on PLT adhesion, evaluated by a dynamic method under flow conditions in a group of patients with ET and PV.
86 patients, i.e. 51 ET (19 M/32 F; age range 32-86 years) and 35PV (22 M/13 F; 41-83 yrs.), and 24 healthy controls (13 M/11 F; 28-61 yrs.) were enrolled upon informed consent. For the adhesion assay, peripheral venous whole blood was perfused over collagen for 4' at a 1,000 s-1 shear rate. PLTs were then stained with an anti-P-selectin-FITC antibody to evaluate PLT activation, and annexin V-AlexaFluor647 to detect procoagulant phosphatidylserine expression. Then, images of adherent PLTs in random fields were taken using phase contrast and fluorescence imaging by EVOS® fluorescence microscope. Results are mean±SEM of the % area covered by PLTs, or as the % of adherent PLTs positive for P-selectin or phosphatidylserine. Main hematological parameters and mutational status were recorded.
PLT adhesion was significantly (p<0.01) greater in ET (44.6±1.6%) and PV patients (49.0±1.9%) compared to controls (37.9±1.7%). In ET, PLT adhesion was highest in JAK2-V617F mutation carriers (n=23), followed by CalR-positive (n=16) and triple negative subjects (n=9), and lowest in the MPL-positive patients (n=3). In PV, no difference in PLT adhesion was observed between JAK2-V617F heterozygous and homozygous subjects. P-selectin expression by adherent PLTs was not statistically different between patients and controls. Differently, phosphatidylserine expression on adherent PLTs was significantly reduced (p<0.01) in both ET and PV compared to healthy subjects. In ET patients, a significant (p<0.05) correlation was found between PLT adhesion and PLT count in JAK2-V617F and CalR-positive mutation carriers. Multivariate regression analysis adjusted for age and sex, confirmed PLT count as a significant determinant of PLT adhesion in JAK2-V617F positive patients only.
ET and PV platelets show an increased adhesion to collagen in vitro, particularly in those carrying the JAK2-V617F mutation. A prospective study is ongoing to evaluate the predictive value of our PLT thrombus formation dynamic model for the thrombotic risk in ET and PV patients.
Project funded by "AIRC-IG2013" grant Nr. 14505 from the "Italian Association for Cancer Research" (A.I.R.C.).
骨髓增殖性肿瘤 ET 和 PV 的特征是动脉和静脉血栓形成和/或微循环障碍的发生率均较高。三种体细胞突变，即 JAK2-V617F、钙网蛋白（CalR）和 MPL，常见于这些疾病，与不同的血栓形成风险水平相关。
分析 JAK2-V617F、CalR 和 MPL 突变对 ET 和 PV 患者群体中在流动条件下通过动态方法评估的血小板黏附的影响。
在知情同意的情况下，纳入了 86 名患者，即 51 名 ET（19 名男性/32 名女性；年龄范围 32-86 岁）和 35 名 PV（22 名男性/13 名女性；41-83 岁），以及 24 名健康对照者（13 名男性/11 名女性；28-61 岁）。为了进行黏附测定，将外周静脉全血以 1000 s-1 的剪切速率在胶原蛋白上灌注 4'。然后，用抗 P-选择素-FITC 抗体对血小板进行染色，以评估血小板的激活，并用 annexin V-AlexaFluor647 检测促凝血性磷脂酰丝氨酸表达。然后，使用相差和荧光成像通过 EVOS®荧光显微镜拍摄随机视野中黏附血小板的图像。结果表示为黏附血小板的百分比区域±SEM，或以 P-选择素或磷脂酰丝氨酸阳性的黏附血小板的百分比表示。记录主要的血液学参数和突变状态。
ET（44.6±1.6%）和 PV 患者（49.0±1.9%）的血小板黏附率显著高于对照组（37.9±1.7%）（p<0.01）。在 ET 中，JAK2-V617F 突变携带者（n=23）的血小板黏附率最高，其次是 CalR 阳性者（n=16）和三重阴性者（n=9），而 MPL 阳性患者（n=3）的血小板黏附率最低。在 PV 中，JAK2-V617F 杂合子和纯合子患者之间的血小板黏附率无差异。与对照组相比，患者的黏附血小板上的 P-选择素表达没有统计学差异。不同的是，与健康对照组相比，ET 和 PV 中黏附血小板上的磷脂酰丝氨酸表达明显降低（p<0.01）。在 ET 患者中，JAK2-V617F 和 CalR 阳性突变携带者中，血小板黏附与血小板计数之间存在显著相关性（p<0.05）。调整年龄和性别后的多元回归分析证实，仅在 JAK2-V617F 阳性患者中，血小板计数是血小板黏附的显著决定因素。
ET 和 PV 血小板在体外对胶原蛋白的黏附增加，特别是在携带 JAK2-V617F 突变的患者中。目前正在进行一项前瞻性研究，以评估我们的血小板血栓形成动态模型对 ET 和 PV 患者血栓形成风险的预测价值。
本项目由“意大利癌症研究协会”（AIRC）的“IG2013”项目资助，编号为 14505。
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