A Novel C1-Esterase Inhibitor Oxygenator Coating Prevents FXII Activation in Human Blood

Figures

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  Figure 1. Schematic representation of the contact activation system. The contact of the blood with the artificial surface (polymethylpentene membrane) leads to the activation of factor (F) XII to FXIIa. FXIIa can then activate four different downstream pathways: (i) the coagulation pathway by cleaving the zymogen FXI to FXIa, eventually leading to thrombin activation and fibrin formation, (ii) the complement system by activation of the C1qrs-complex subunits C1r and C1s, (iii) the fibrinolytic pathway by plasma kallikrein activating plasmin to eventually degrade fibrin, (iv) the inflammatory kallikrein-kinin signaling pathway by plasma kallikrein cleaving high-molecular-weight kininogen liberating the pro-inflammatory molecule bradykinin [15]. Heparin and C1-INH are both able to inhibit one or more of these pathways. While heparin only inhibits coagulation, C1-INH positively affects coagulation, the complement system, fibrin degradation, and inflammation [16,24].

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  Figure 2. Schematic representation of the generated coatings. (a) C1-INH coating, (b) heparin coating, (c) the combined coating of C1-INH and heparin.

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  Figure 3. Detection of bioactive (a) C1-INH or (b) heparin amounts on PMP membranes. Significantly increased amounts of C1-INH were detected compared to the uncoated samples. However, only heparin-coating resulted in significantly increased immobilization of heparin compared to untreated samples. The results are shown as mean ± scanning electron microscopy (SEM). Statistical analysis was performed using one-way ANOVA with Bonferroni’s multiple comparison post hoc test. n = 5; **** p < 0.0001, *** p < 0.001.

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  Figure 4. Comparative analyses of the blood cell number before (baseline) and after dynamic incubation revealed no significant loss of leukocytes and platelets after incubation with either coated or uncoated membranes. Shown are mean numbers of red blood cells (RBCs) (a), white blood cells (WBCs) (b), and platelets (c) per microliter heparinized whole blood ± SEM. Statistical analysis was performed using one-way ANOVA with Bonferroni’s multiple comparison post hoc test (a,c), or Kruskal–Wallis test with Dunn′s multiple comparison post hoc test (b). n = 5.

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  Figure 5. C1-INH coating prevented FXII activation, while heparin coating prevented TAT formation. Hemocompatibility analysis of differently coated PMP membranes: blood markers for the contact phase system (FXIIA-like activity) (a), coagulation (TAT) (b), activation of neutrophils (PMN-elastase) (c), complement system (C3a) (d), and platelet activation (β-thromboglobulin) (e). Statistical analysis was performed using one-way ANOVA with Bonferroni’s multiple comparison post hoc test. The mean value of each column was compared with the mean value of each other column. n = 5; * p < 0.05.

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  Figure 6. Fluorescence microscopy revealed reduced cell adhesion on single C1-INH and heparin as well as combined coatings. Mean cell counts ± SEM of DAPI-stained membranes show the significant reduction of nucleated cells on the surface of coated membranes (a), proportional reduction of mean adhered nucleated cells visualize the effect in direct comparison of the different coatings (b). Statistical analysis was performed using one-way ANOVA with Bonferroni’s multiple comparison post hoc test. The mean value of each column was compared with the mean value of each other column. n = 5; **** p < 0.0001, *** p < 0.001, ** p < 0.01.

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  Figure 7. SEM images show an obvious decrease in platelet aggregation and fibrin formation. This effect is particularly noticeable with the combined coatings of heparin and C1-INH. For each type of coating, images were taken with a 100×, 500×, and 1000× magnification. For a visual comparison of the effects, images of an uncoated PMP membrane without blood contact are also shown (last column).