特发性血小板减少性紫癜的诊治进展

由于诊断特发性血小板减少性紫癜(ITP)的标准差异较大,使临床试验结果难以互比,也给临床实践带来不便.为解决此问题,ITP国际工作组于2007年召开会议,就ITP的命名、定义、分期以及严重度分级、预后和疗效标准等达成一些新共识.随着新的治疗药物问世,ITP治疗也取得显著进步.利妥昔单抗是一种抗CD20单克隆抗体,可清除能产生自身抗体的B淋巴细胞,因其疗效高和安全性好,现已在ITP中广为应用.最近研究表明,血小板生成减少可能与ITP发生有关.因此,新的血小板生成刺激剂,如血小板生成素受体激动剂Romiplostim和Eltrombopag已成为治疗ITP的新选择.     

A novel method for automated assessment of megakaryocyte differentiation and proplatelet formation

Transfusion of platelet concentrates represents an important treatment for various bleeding complications. However, the short half-life and frequent contaminations with bacteria restrict the availability of platelet concentrates and raise a clear demand for platelets generated ex vivo. Therefore, in vitro platelet generation from megakaryocytes represents an important research topic. A vital step for this process represents accurate analysis of thrombopoiesis and proplatelet formation, which is usually conducted manually. We aimed to develop a novel method for automated classification and analysis of proplatelet-forming megakaryocytes in vitro. After fluorescent labelling of surface and nucleus, MKs were automatically categorized and analysed with a novel pipeline of the open source software CellProfiler. Our new workflow is able to detect and quantify four subtypes of megakaryocytes undergoing thrombopoiesis: proplatelet-forming, spreading, pseudopodia-forming and terminally differentiated, anucleated megakaryocytes. Furthermore, we were able to characterize the inhibitory effect of dasatinib on thrombopoiesis in more detail. Our new workflow enabled rapid, unbiased, quantitative and qualitative in-depth analysis of proplatelet formation based on morphological characteristics. Clinicians and basic researchers alike will benefit from this novel technique that allows reliable and unbiased quantification of proplatelet formation. It thereby provides a valuable tool for the development of methods to generate platelets ex vivo and to detect effects of drugs on megakaryocyte differentiation.     

Proplatelet formation deficit and megakaryocyte death contribute to thrombocytopenia in Myh9 knockout mice

See also Ravid K. The value of a native milieu: mutated non‐muscle myosin IIA does lead to thrombocytopenia. This issue, pp 2241–2. Summary. Background: Inactivation of the mouse Myh9 gene (Myh9Δ) or its mutation in MYH9‐related diseases leads to macrothrombocytopenia. Paradoxically, previous studies using in vitro differentiated megakaryocytes showed an increased capacity for proplatelet formation when myosin was absent or inhibited. Methods: To explore the origin of the thrombocytopenia induced by myosin deficiency, we studied proplatelet formation using bone marrow explants of wild‐type (WT) and Myh9Δ mouse where megakaryocytes have matured in their native environment. Results and discussion: A dramatic decrease in the number and complexity of proplatelets was observed in megakaryocytes from Myh9Δ mice, while inhibition of myosin activity by blebbistatin increased proplatelet formation from WT mature megakaryocytes. Moreover, Myh9Δ megakaryocytes had a smaller size than the WT cells. These data indicate that myosin deficiency acts negatively on proplatelet formation, probably by impairing in situ megakaryocyte maturation, while myosin activity is dispensable at the latest stage of proplatelet formation. In addition, ultrastructural examination of Myh9Δ bone marrow revealed an increased proportion of megakaryocytes exhibiting signs of non‐apoptotic cell death as compared with the WT mice. Conclusion: These data indicate that thrombocytopenia in Myh9Δ mice results from defective development of megakaryocyte size, impaired proplatelet formation and increased cell death.     

15-deoxy-Δ12,14 prostaglandin J2-induced heme oxygenase-1 in megakaryocytes regulates thrombopoiesis

Summary.  Background:  Platelet production is an intricate process that is poorly understood. Recently, we demonstrated that the natural peroxisome proliferator-activated receptor gamma (PPARγ) ligand, 15-deoxy-Δ^12,14 prostaglandin J₂ (15d-PGJ₂), augments platelet numbers by increasing platelet release from megakaryocytes through the induction of reactive oxygen species (ROS). 15d-PGJ₂ can exert effects independent of PPARγ, such as increasing oxidative stress. Heme oxygenase-1 (HO-1) is a potent antioxidant and may influence platelet production. Objectives:  To further investigate the influence of 15d-PGJ₂ on megakaryocytes and to understand whether HO-1 plays a role in platelet production. Methods:  Meg-01 cells (a primary megakaryoblastic cell line) and primary human megakaryocytes derived from cord blood were used to examine the effects of 15d-PGJ₂ on HO-1 expression in megakaryocytes and their daughter platelets. The role of HO-1 activity in thrombopoiesis was studied using established in vitro models of platelet production. Results and conclusions:  15d-PGJ₂ potently induced HO-1 protein expression in Meg-01 cells and primary human megakaryocytes. The platelets produced from these megakaryocytes also expressed elevated levels of HO-1. 15d-PGJ₂-induced HO-1 was independent of PPARγ, but could be replicated using other electrophilic prostaglandins, suggesting that the electrophilic properties of 15d-PGJ₂ were important for HO-1 induction. Interestingly, inhibiting HO-1 activity enhanced ROS generation and augmented 15d-PGJ₂-induced platelet production, which could be attenuated by antioxidants. These new data reveal that HO-1 negatively regulates thrombopoiesis by inhibiting ROS.     

Reference intervals for mean platelet volume and immature platelet fraction determined on a sysmex XE5000 hematology analyzer

Background New parameters describing the platelet population of the blood are mean platelet volume (MPV), which is a crude estimate of thrombocyte reactivity, and immature platelet fraction (IPF), which reflects megakaryopoietic activity. This study aimed to define reference intervals for MPV and IPF and to investigate whether separate reference intervals according to smoking status, age or sex are necessary.

Methods Blood samples were obtained from subjects participating in The Danish General Suburban Population Study. MPV and IPF measurements were performed by the use of the Sysmex XE-5000 hematology analyzer. Reference intervals were established by a non-parametric method.

Results In total, 1674 apparently healthy individuals (910 females and 764 males) were included. No significant age, sex or smoking status difference was observed. The reference interval was 9.6–13.1 fL for MPV and 1.3–9.0% for IPF, respectively.

Conclusion We have generated reference intervals for MPV and IPF in a large, adult Danish population and found those parameters remarkably stable across age, sex and smoking status.     

Impaired proplatelet formation in immune thrombocytopenia: a novel mechanism contributing to decreased platelet count

The pathophysiological mechanisms contributing to the decreased platelet count in immune thrombocytopenia (ITP) are not entirely understood. Here, we investigated the key step of proplatelet formation (PPF) by studying the effect of ITP plasma in thrombopoiesis. Normal cord blood‐derived mature megakaryocytes were cultured in the presence of recalcified plasma from ITP patients, and PPF was evaluated by microscopic analysis. Patient samples induced a dose‐dependent inhibition in PPF, as well as decreased complexity of proplatelet architecture. Although slightly increased, plasma‐induced megakaryocyte apoptosis was not related to PPF impairment. Purified IgG reproduced the inhibitory effect, while platelet‐adsorbed plasma induced its reversion, suggesting the involvement of auto‐antibodies in the inhibition of thrombopoiesis. Impaired PPF, induced by ITP plasmas bearing anti‐GPIIb‐IIIa antibodies, was related to their ability to interfere with the normal function of this integrin, as assessed by megakaryocyte PAC‐1 binding and β3 integrin phosphorylation while the presence of anti‐glycoprotein Ia‐IIa auto‐antibodies was associated with loss of normal inhibition of PPF induced by type I collagen. In conclusion, abnormal thrombopoiesis comprising decreased PPF and morphological changes in proplatelet structure are induced by patient samples, unveiling new mechanisms contributing to decreased platelet count in ITP.