Division of Cardiovascular Surgery, Hospital of the University of Pennsylvania, Philadelphia, Pennsylvania.
MD/PhD Program, Washington University in St. Louis, St. Louis, Missouri.
Ann Thorac Surg. 2018 Mar;105(3):807-814. doi: 10.1016/j.athoracsur.2017.05.060. Epub 2017 Sep 21.
Recent data suggest that hemolysis contributes to left ventricular assist device (LVAD) thrombosis, but the mechanism is unknown. In a clinical study, we measured plasma free hemoglobin (pfHgb) and the incidence of LVAD thrombosis. In an in vitro study, we examined biophysical relationships between shear stress, pfHgb and von Willebrand factor (vWF) metabolism toward understanding mechanisms of LVAD thrombosis.
In the clinical study, blood samples were obtained from continuous-flow LVAD patients (n = 30). Plasma free hemoglobin was measured via enzyme-linked immunosorbent assay. Plasma lactate dehydrogenase (LDH) was measured with a fluorimetric assay. In the in vitro study, to investigate mechanism, human plasma (n = 10) was exposed to LVAD-like shear stress (175 dyne/cm) with and without free hemoglobin (30 mg/dL). ADAMTS-13 (the vWF protease) activity was quantified with Förster resonance energy transfer. vWF size was quantified with immunoblotting. vWF clotting function was quantified with an enzyme-linked immunosorbent assay.
In the clinical study, LVAD support caused subclinical hemolysis. In all patients, LDH increased significantly from 213 ± 9 U/L to 366 ± 31 U/L at 10 days of support (p < 0.0001) and remained significantly elevated at 280 ± 18 U/L at 1 month of support (p < 0.01). In 21 patients that did not develop LVAD thrombosis, pfHgb increased early but decreased over time (pre-LVAD: 5.2 ± 0.8 mg/dL; 1 week: 19.8 ± 4.4 mg/dL, p < 0.01; 3 months: 9.3 ± 2.2 mg/dL, p = 0.07). In 9 patients that developed LVAD thrombosis, pfHgb was significantly elevated versus patients without thrombosis before (p < 0.001) and after 3 months (p < 0.05) of support (pre-LVAD: 20.2 ± 6.3 mg/dL; 1 week: 17.3 ± 3.7 mg/dL; 3 months: 21.5 ± 7.8 mg/dL). Similarly, after 3 months, patients that did not develop LVAD thrombosis had an LDH of 271 ± 28 U/L, whereas patients that later developed LVAD thrombosis had a significantly higher LDH of 625 ± 210 U/L (p = 0.02). In the in vitro study, shear stress degraded vWF similarly to an LVAD. Free hemoglobin inhibited ADAMTS-13 activity during shear stress (633 ± 27 ng/mL to 565 ± 24 ng/mL; p < 0.001). vWF was thereby protected from degradation, 4 vWF fragments decreased significantly (p ≤ 0.05), and vWF clotting function increased (1.15 ± 0.09 U/mL to 1.29 ± 0.09 U/mL, p = 0.06).
These are the first data to demonstrate mechanistic relationships between subclinical hemolysis and a procoagulant state during continuous-flow LVAD support. Patients with high pfHgb and LDH were more likely to develop LVAD thrombosis. In vitro experiments demonstrated that free hemoglobin inhibited ADAMTS-13, protected vWF from degradation, increased vWF clotting function, and created a procoagulant state. As such, pfHgb may be a clinical target to prevent LVAD thrombosis.
最近的数据表明，溶血有助于左心室辅助装置（LVAD）血栓形成，但机制尚不清楚。在一项临床研究中，我们测量了血浆游离血红蛋白（pfHgb）和 LVAD 血栓形成的发生率。在一项体外研究中，我们研究了剪切应力、pfHgb 和血管性血友病因子（vWF）代谢之间的生物物理关系，以了解 LVAD 血栓形成的机制。
在临床研究中，从连续流动 LVAD 患者（n=30）中采集血样。通过酶联免疫吸附试验测量血浆游离血红蛋白。通过荧光法测量血浆乳酸脱氢酶（LDH）。在体外研究中，为了研究机制，将人血浆（n=10）暴露于类似于 LVAD 的剪切应力（175 达因/平方厘米），同时有无游离血红蛋白（30 毫克/分升）。用荧光共振能量转移法定量 ADAMTS-13（vWF 蛋白酶）活性。用免疫印迹法定量 vWF 大小。用酶联免疫吸附试验定量 vWF 凝血功能。
在临床研究中，LVAD 支持导致亚临床溶血。在所有患者中，LDH 从 213±9U/L 显著增加到 10 天支持时的 366±31U/L（p<0.0001），并且在 1 个月支持时仍显著升高至 280±18U/L（p<0.01）。在 21 名未发生 LVAD 血栓形成的患者中，pfHgb 早期升高，但随时间降低（LVAD 前：5.2±0.8mg/dL；1 周：19.8±4.4mg/dL，p<0.01；3 个月：9.3±2.2mg/dL，p=0.07）。在 9 名发生 LVAD 血栓形成的患者中，pfHgb 明显高于未发生血栓形成的患者（p<0.001）和支持后 3 个月（p<0.05）（LVAD 前：20.2±6.3mg/dL；1 周：17.3±3.7mg/dL；3 个月：21.5±7.8mg/dL）。同样，在 3 个月后，未发生 LVAD 血栓形成的患者的 LDH 为 271±28U/L，而随后发生 LVAD 血栓形成的患者的 LDH 明显更高，为 625±210U/L（p=0.02）。在体外研究中，剪切应力类似于 LVAD 降解 vWF。游离血红蛋白抑制剪切应力下的 ADAMTS-13 活性（633±27ng/mL 至 565±24ng/mL；p<0.001）。因此，vWF 受到保护，不会降解，4 个 vWF 片段显著减少（p≤0.05），vWF 凝血功能增加（1.15±0.09U/mL 至 1.29±0.09U/mL，p=0.06）。
这些是第一个证明亚临床溶血与连续流动 LVAD 支持期间促凝状态之间存在机制关系的研究数据。pfHgb 和 LDH 较高的患者更有可能发生 LVAD 血栓形成。体外实验表明，游离血红蛋白抑制了 ADAMTS-13，保护了 vWF 免受降解，增加了 vWF 的凝血功能，并产生了促凝状态。因此，pfHgb 可能是预防 LVAD 血栓形成的临床靶点。
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