To uncover the role of Vps34, the sole class III phosphoinositide 3-kinase (PI3K), in megakaryocytes (MKs) and platelets, we created a mouse model with Vps34 deletion in the MK/platelet lineage (Pf4-Cre/Vps34lox/lox). Deletion of Vps34 in MKs led to the loss of its regulator protein, Vps15, and was associated with microthrombocytopenia and platelet granule abnormalities. Although Vps34 deficiency did not affect MK polyploidisation or proplatelet formation, it dampened MK granule biogenesis and directional migration toward an SDF1α gradient, leading to ectopic platelet release within the bone marrow. In MKs, the level of phosphatidylinositol 3-monophosphate (PI3P) was significantly reduced by Vps34 deletion, resulting in endocytic/trafficking defects. In platelets, the basal level of PI3P was only slightly affected by Vps34 loss, whereas the stimulation-dependent pool of PI3P was significantly decreased. Accordingly, a significant increase in the specific activity of Vps34 lipid kinase was observed after acute platelet stimulation. Similar to Vps34-deficient platelets, ex vivo treatment of wild-type mouse or human platelets with the Vps34-specific inhibitors, SAR405 and VPS34-IN1, induced abnormal secretion and affected thrombus growth at arterial shear rate, indicating a role for Vps34 kinase activity in platelet activation, independent from its role in MKs. In vivo, Vps34 deficiency had no impact on tail bleeding time, but significantly reduced platelet prothrombotic capacity after carotid injury. This study uncovers a dual role for Vps34 as a regulator of platelet production by MKs and as an unexpected regulator of platelet activation and arterial thrombus formation dynamics.

中文翻译：

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III 类 PI3K、Vps34 在血小板生成和血栓生长中的双重作用

为了揭示 Vps34，唯一的 III 类磷酸肌醇 3-激酶 (PI3K)，在巨核细胞 (MKs) 和血小板中的作用，我们创建了一个小鼠模型，在 MK/血小板谱系 (Pf4-Cre/Vps34lox/lox) 中有 Vps34 缺失. MKs 中 Vps34 的缺失导致其调节蛋白 Vps15 的丢失，并与微血小板减少症和血小板颗粒异常有关。尽管 Vps34 缺乏不影响 MK 多倍体化或血小板前体形成，但它抑制了 MK 颗粒生物发生和向 SDF1α 梯度的定向迁移，导致骨髓内异位血小板释放。在 MK 中，由于 Vps34 缺失，磷脂酰肌醇 3-单磷酸 (PI3P) 的水平显着降低，导致内吞/运输缺陷。在血小板中，PI3P 的基础水平仅受 Vps34 损失的轻微影响，而 PI3P 的刺激依赖性池显着减少。因此，在急性血小板刺激后观察到 Vps34 脂质激酶的比活性显着增加。与缺乏 Vps34 的血小板相似，用 Vps34 特异性抑制剂 SAR405 和 VPS34-IN1 对野生型小鼠或人血小板进行体外处理，在动脉剪切速率下诱导异常分泌并影响血栓生长，表明 Vps34 激酶活性的作用在血小板活化中，独立于其在 MKs 中的作用。在体内，Vps34 缺乏对尾部出血时间没有影响，但在颈动脉损伤后显着降低血小板促血栓形成能力。