Alginate microbeads are coagulation compatible, while alginate microcapsules activate coagulation secondary to complement or directly through FXII

Abstract

Alginate microspheres are presently under evaluation for future cell-based therapy. Their ability to induce harmful host reactions needs to be identified for developing the most suitable devices and efficient prevention strategies. We used a lepirudin based human whole blood model to investigate the coagulation potentials of alginate-based microspheres: alginate microbeads (Ca/Ba Beads), alginate poly-l-lysine microcapsules (APA and AP microcapsules) and sodium alginate-sodium cellulose sulfate-poly(methylene-co-cyanoguanidine) microcapsules (PMCG microcapsules). Coagulation activation measured by prothrombin fragments 1+2 (PTF1.2) was rapidly and markedly induced by the PMCG microcapsules, delayed and lower induced by the APA and AP microcapsules, and not induced by the Ca/Ba Beads. Monocytes tissue factor (TF) expression was similarly activated by the microcapsules, whereas not by the Ca/Ba Beads. PMCG microcapsules-induced PTF1.2 was abolished by FXII inhibition (corn trypsin inhibitor), thus pointing to activation through the contact pathway. PTF1.2 induced by the AP and APA microcapsules was inhibited by anti-TF antibody, pointing to a TF driven coagulation. The TF induced coagulation was inhibited by the complement inhibitors compstatin (C3 inhibition) and eculizumab (C5 inhibition), revealing a complement-coagulation cross-talk. This is the first study on the coagulation potentials of alginate microspheres, and identifies differences in activation potential, pathways and possible intervention points.

Statement of significance: Alginate microcapsules are prospective candidate materials for cell encapsulation therapy. The material surface must be free of host cell adhesion to ensure free diffusion of nutrition and oxygen to the encapsulated cells. Coagulation activation is one gateway to cellular overgrowth through deposition of fibrin. Herein we used a physiologically relevant whole blood model to investigate the coagulation potential of alginate microcapsules and microbeads. The coagulation potentials and the pathways of activation were depending on the surface properties of the materials. Activation of the complement system could also be involved, thus emphasizing a complement-coagulation cross-talk. Our findings points to complement and coagulation inhibition as intervention point for preventing host reactions, and enhance functional cell-encapsulation devices.

Keywords: Alginate microcapsules; Coagulation; Complement; Cross-talk; Factor XII; Tissue factor.

Figure 1

Effect of alginate microspheres (alginate microbeads and microcapsules) on coagulation (PTF1.2) and complement (TCC) responses in the human whole blood model. A) Time-dependent effect on PTF1.2 (mean±SEM, N=4 donors), B) PTF1.2 at 240 min (N=11–14 donors), C) Time effect on TCC (mean±SEM, N=4 donors), D) TCC after 240 min (N=12–14). The data in B and D are given as box whisker plots with median, 25th and 75th percentiles and min/max values, and the level of significance shown as * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001.

Figure 2

Effect of alginate microbeads (Ca/Ba Beads) and microcapsules (APA, AP, PMCG1, PMCG2) on monocyte TF expression in human whole blood. A) Monocyte TF expression (% positive cells) after 240 min incubation (box whisker plots giving median, 25th and 75th percentiles and min/max values of N=8), B) TF mRNA synthesis with time (relative quantitation (RQ) to baseline, mean ±SEM of N=3). The level of significance is *** P<0.001, **** P<0.0001.

Figure 3

Complement inhibition at the level of C3 (compstatin, C) or C5 (eculizumab, E) abolish the TCC induction. A) APA microcapsules (N=7, N=5 for Ctr), B) AP microcapsules (N=7, N=5 for Ctr), PMCG1 microcapsules (N=5) and PMCG2 microcapsules (N=5). The x-axis abbreviations reflect the responses by the given microcapsule under incubation with either NaCl, compstatin (C), eculizumab (E) or the control peptide (Ctr). Graphs are given as box whisker plots with median, 25th and 75th percentiles and min/max values. The level of significance is **** P<0.0001.

Figure 4

Effect of complement inhibition at the level of C3 (compstatin, C) and C5 (eculizumab, E) on the PTF1.2 induction. A) APA microcapsules (N=9, N=5 for Ctr), B) AP microcapsules (N=9, N=5 for Ctr), C) PMCG1 microcapsules (N=5) and D) PMCG2 microcapsules (N=5). The x-axis abbreviations reflect the responses by the given microcapsule under incubation with either NaCl, compstatin (C), eculizumab (E) or the control peptide (Ctr). Graphs are given as box whisker plots with median, 25th and 75th percentiles and min/max values. The level of significance was at * P<0.05, **** P<0.0001 whereas the non-significant differences are given as n.s.

Figure 5

Effect of complement inhibition at the level of C3 (compstatin, C) and C5 (eculizumab, E) on tissue factor (TF) mRNA from human whole blood. A) APA microcapsules (N=4), B) AP microcapsules (N=4), PMCG1 microcapsules (N=4) and PMCG2 microcapsules (N=4). The x-axis abbreviations reflect the responses by the given microcapsule under incubation with either NaCl, compstatin (C), eculizumab (E) or the control peptide (Ctr). Data are relative to the saline control (without microspheres) and given as box whisker plots with median, 25th and 75th percentiles and min/max values. The level of significance are * P<0.05, ** P<0.01, *** P<0.001 whereas non-significant differences are given as n.s.

Figure 6

Effect of TF inhibition on microcapsule-induced coagulation (PTF1.2) in human whole blood after 240 min incubation. The microcapsules were either incubated with NaCl only, inhibitory TF antibody ( TF) or its respective isotype control antibody (Ctr, mouse IgG1). A) APA microcapsules (N=8), B) AP microcapsules (N=8), C) PMCG1 microcapsules (N=6), D) PMCG2 microcapsules (N=6). Data are given as box whisker plots with median, 25th and 75th percentiles and min/max values. The level of significance was ** P<0.01, whereas non-significant differences are given as n.s.

Figure 7

Effect of FXII inhibition on microcapsules induced coagulation (PTF1.2) in human whole blood after 30 min. The microcapsules were either incubated with NaCl only, the factor XII inhibitor CTI or albumin (Ctr). A) PMCG1 microcapsules (N=7), B) PMCG2 microcapsules (N=7), C) Glass (N=4) and D) Ca/Ba Beads (N=4). Data are given as box whisker plots with median, 25th and 75th percentiles and min/max values. The level of significance was **<0.01 and *** P<0.001, **** P<0.0001, whereas non-significant differences are given as n.s.