Platelet count reduction during in vitro membrane oxygenation affects platelet activation, neutrophil extracellular trap formation and clot stability, but does not prevent clotting

Abstract

Introduction: Due to improved technology and increased application the mortality during extracorporeal membrane oxygenation (ECMO) is constantly declining. Nevertheless, complications including haemorrhage or thrombus formation remain frequent. Local mitigation of coagulation within an ECMO system to prevent thrombus formation on ECMO components and optimizing systemic anticoagulation is an approach to reduce clotting and bleeding complications at once. Foreign surfaces of ECMO systems, activate platelets (PLTs), which besides their major role in coagulation, can trigger the formation of neutrophil extracellular traps (NETs) contributing to robust thrombus formation. The impact of a reduced PLT count on PLT activation and NET formation is of paramount importance and worth investigating.

Methods: In this study platelet poor (PLT^-) and native (PLT⁺) heparinized human blood was circulated in two identical in vitro test circuits for ECMO devices for 6 hours. PLT reduction was achieved by a centrifugation protocol prior to the experiments. To achieve native coagulation characteristics within the test circuits, the initial heparin dose was antagonized by continuous protamine administration.

Results: The PLT^- group showed significantly lower platelet activation, basal NET formation and limited clot stability measured via thromboelastometry. Fluorescent and scanning electron microscope imaging showed differences in clot composition. Both groups showed equal clot formation within the circuit.

Conclusions: This study demonstrated that the reduction of PLTs within an ECMO system is associated with limited PLT activation and NET formation, which reduces clot stability but is not sufficient to inhibit clot formation per se.

Keywords: clot stability; extracorporeal membrane oxygenation (ECMO); in vitro test circuit; neutrophil extracellular trap (NET) formation; platelet activation.

Figure 1.

Flow chart. White arrow: Overview of blood preparation and experimental groups prior to circulation. Blue arrow: Points in time for blood sampling and infusions during in vitro circulation in the test circuit.

Figure 2.

Display of: (a) platelets (PLTs), (b) platelet activation (CD62+/CD61+), (c) neutrophils and (d) neutrophil extracellular trap formation (NET formation) over time. Baseline (BL). *p < 0.05. **p < 0.01.***p < 0.001.

Figure 3.

Display of: (a) haemoglobin (HGB), (b) free haemoglobin (fHGB), (c) pH and (d) pO₂ over time. Baseline (BL).

Figure 4.

Display of: (a) Mean clot firmness of extrinsic coagulation pathway (MCF EXTEM) and (b) time until first signs (an increase of pump’s power to maintain flow) of coagulation within the test circuit (Time to clot). (c) Table summarizing experiments with (clot) and without (c/o clot) clotting within the test circuits in the experimental groups: platelet poor whole blood (PLT^–) and whole blood (PLT⁺). Images of pump heads including clots. Representative fluorescence microscopy images of histological clot sections excised from the pump heads (blue: platelets anti-CD41; green: erythrocytes anti-TER119; red: histones in NETs anti-H2A). Baseline (BL). **p < 0.01.

Figure 5.

Representative scanning electron microscopy images of polymethylpentene fibres of oxygenators after 360 minutes in vitro blood circulation: (A) PLT⁺ group, (B) PLT^– group. 1 100-fold magnification; 2 1000-fold magnification; 3 2500-fold magnification; 4 25,000-fold magnification. PMP: Polymethylpentene fibres of the oxygenator membrane; Arrow: Fibrin deposit; Filled arrow: Neutrophil granulocyte; § Neutrophil extracellular traps; * Activated, flat platelets; FF: Fibrin fibres.