Institute for Clinical and Experimental Trauma Immunology, University Hospital of Ulm, Ulm, Germany.
Department of Transfusion Medicine and Hemostaseology, University Hospital of Erlangen, Friedrich-Alexander University Erlangen-Nürnberg (FAU), Erlangen, Germany.
PLoS One. 2023 Jan 12;18(1):e0280069. doi: 10.1371/journal.pone.0280069. eCollection 2023.
Surface compatibility with blood is critical both for scientific investigations on hemostasis and clinical applications. Regarding in vitro and ex vivo investigations, minimal alteration in physiological hemostasis is of particular importance to draw reliable conclusions on the human coagulation system. At the same time, artificial coagulation activation must be avoided, which is relevant for the patient, for example to prevent stent graft occlusion. The aim was to evaluate the advantages and disadvantages of antithrombotic and antifouling surface coatings in the context of their suitability for ex vivo incubation and the study of coagulation properties.
We investigated the impact of different protocols for surface coating of synthetic material and different anticoagulants on hemostasis and platelet activation in ex vivo human whole blood. Blood samples from healthy donors were incubated in coated microtubes on a rotating wheel at 37°C. Two protocols for surface coating were analyzed for hemostatic parameters and metabolic status, a heparin-based coating (CHC, Corline Heparin Conjugate) without further anticoagulation and a passivating coating (MPC, 2-methacryloyloxethyl phosphorylcholine) with added anticoagulants (enoxaparin, ENOX; or fondaparinux, FPX). Employing the MPC-based coating, the anticoagulants enoxaparin and fondaparinux were compared regarding their differential effects on plasmatic coagulation by thrombelastometry and on platelet activation by flowcytometry and platelet function assays.
Using the CHC coating, significant coagulation cascade activation was observed, whereas parameters remained mostly unchanged with MPC-based protocols. Extended incubation caused significantly elevated levels of the soluble membrane attack complex. Neither ENOX nor FPX caused a relevant impairment of platelet function or activation capacity and thrombelastometric parameters remained unchanged with both protocols. For translational purposes, we additionally modeled endotoxemia with the MPC-based protocols by incubating with lipopolysaccharide plus/minus thrombin. While coagulation parameters remained unchanged, elevated Interleukin 8 and Matrix Metalloproteinase 9 demonstrated preserved immune cell responsiveness.
The MPC-based protocols demonstrated better hemocompatibility compared to CHC, and ENOX and FPX proved useful for additional anticoagulation. Furthermore, this simple-to-use whole blood model may be useful for experimental analyses of the early coagulatory and immunological response without decalcification.
血液表面的兼容性对于止血的科学研究和临床应用都至关重要。 对于体外和离体研究， 对生理止血的最小改变对于得出关于人体凝血系统的可靠结论尤为重要。 同时， 必须避免人为的凝血激活， 这对于患者很重要， 例如为了防止支架移植物闭塞。 目的是评估抗血栓和抗污表面涂层在适合于体外孵育和凝血特性研究方面的优缺点。
我们研究了不同的合成材料表面涂层方案和不同的抗凝剂对体外人全血止血和血小板激活的影响。 将来自健康供体的血液样本在 37°C 下在旋转轮上孵育于涂覆的微管中。 分析了两种用于止血参数和代谢状态的表面涂层方案： 一种基于肝素的涂层（CHC， 胶原肝素缀合物） ， 没有进一步的抗凝剂， 一种钝化涂层（MPC， 2-甲基丙烯酰氧乙基磷酸胆碱） ， 添加了抗凝剂（依诺肝素， ENOX； 或磺达肝癸钠， FPX） 。 在使用基于 MPC 的涂层时， 通过血栓弹性描记术比较了依诺肝素和磺达肝癸钠对血浆凝血的差异作用， 通过流式细胞术和血小板功能测定比较了它们对血小板激活的差异作用。
使用 CHC 涂层， 观察到明显的凝血级联激活， 而基于 MPC 的方案则保持了大部分参数不变。 延长孵育会导致可溶性膜攻击复合物的水平显著升高。 依诺肝素和磺达肝癸钠均不会对血小板功能或激活能力造成显著损害， 两种方案的血栓弹性描记术参数均保持不变。 为了进行转化研究， 我们还通过与脂多糖加/不加凝血酶孵育， 在用基于 MPC 的方案模拟内毒素血症。 虽然凝血参数保持不变，但升高的白细胞介素 8 和基质金属蛋白酶 9 表明免疫细胞的反应性仍然保留。
与 CHC 相比，基于 MPC 的方案显示出更好的血液相容性，依诺肝素和磺达肝癸钠可用于进一步抗凝。 此外，这种简单易用的全血模型可能有助于在不脱钙的情况下对早期凝血和免疫反应进行实验分析。
