Microfluidic focal thrombosis model for measuring murine platelet deposition and stability: PAR4 signaling enhances shear-resistance of platelet aggregates
Microfluidic focal thrombosis model for measuring murine platelet deposition and stability: PAR4 signaling enhances shear-resistance of platelet aggregates
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DOI：
10.1111/j.1538-7836.2008.03188.x
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发表时间：
2008-12-01
影响因子：
10.4
通讯作者：
Diamond, S. L.
中科院分区：
文献类型：
--
作者：
Neeves, K. B.;Maloney, S. F.;Diamond, S. L.
背景：流动室允许离体研究血小板在受控壁剪切应力下对限定表面的反应。然而，大多数检测需要 1-10 mL 的血液，不太适合小鼠全血实验。目的：使用 100 μL 全血和受控流动暴露来测量小鼠血小板沉积和对血栓前刺激焦点区域的稳定性。方法：使用微流体方法在 100 μm x 100 μm 贴片中对酸溶性胶原进行图案化，并创建体积为 150 nL 的流道。在收集到 PPACK 和荧光抗小鼠 CD41 mAb 中 1 分钟内，将来自正常小鼠或缺乏整合素 α(2) 亚基的小鼠的全血在图案化胶原上灌注 5 分钟。在静脉和动脉壁剪切率下测量血小板积累。 5 分钟后，通过“剪切步进”至 8000 s(-1) 测量血栓稳定性。结果：野生型鼠血小板以双相剪切依赖性方式在胶原上粘附和聚集，100至400 s(-1)时沉积增加，但1000 s(-1)时沉积减少。缺乏功能性α(2)β(1)整合素的血小板对图案化胶原蛋白的粘附严重减弱。当受到剪切增强挑战时，那些确实粘附的整合素α(2)缺陷的血小板被从表面去除。 PAR4 激动剂 (AYPGKF) 对血栓进行 5 分钟处理，增强了剪切增强过程中聚集体的稳定性。结论：PAR4 信号传导通过独立于其他凝血酶依赖性途径（例如纤维蛋白形成）的机制增强聚集稳定性。
Background: Flow chambers allow the ex vivo study of platelet response to defined surfaces at controlled wall shear stresses. However, most assays require 1-10 mL of blood and are poorly suited for murine whole blood experiments. Objective: To measure murine platelet deposition and stability in response to focal zones of prothrombotic stimuli using 100 mu L of whole blood and controlled flow exposure. Methods: Microfluidic methods were used for patterning acid-soluble collagen in 100 mu m x 100 mu m patches and creating flow channels with a volume of 150 nL. Within 1 min of collection into PPACK and fluorescent anti-mouse CD41 mAb, whole blood from normal mice or from mice deficient in the integrin alpha(2) subunit was perfused for 5 min over the patterned collagen. Platelet accumulation was measured at venous and arterial wall shear rates. After 5 min, thrombus stability was measured with a 'shear step-up' to 8000 s(-1). Results: Wild-type murine platelets adhered and aggregated on collagen in a biphasic shear-dependent manner with increased deposition from 100 to 400 s(-1), but decreased deposition at 1000 s(-1). Adhesion to patterned collagen was severely diminished for platelets lacking a functional alpha(2)beta(1) integrin. Those integrin alpha(2)-deficient platelets that did adhere were removed from the surface when challenged to shear step-up. PAR4 agonist (AYPGKF) treatment of the thrombus at 5 min enhanced aggregate stability during the shear step-up. Conclusions: PAR4 signaling enhances aggregate stability by mechanisms independent of other thrombin-dependent pathways such as fibrin formation.