The Role of Complement in Coagulation Activation Induced by Alginate Microspheres in Human Whole Blood
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Diabetes mellitus (DM) is serious disease that affects millions of people worldwide.Type 1 Diabetes (T1D) is an autoimmune disease, caused by destruction of the insulin producing beta cells (ß-cells) of the pancreas. Alginate encapsulation of pancreatic islets is a potential treatment of TID that attempts to restore the normal glucose homeostasis and provides a way to overcome the need for treatment with insulin and other drugs. Alginate is polysaccharides that is harvested from brown algae and can be used for microencapsulation of ß-cells of the pancreas and other cell therapies. However, alginate as immobilization materials is vulnerable and provokes inflammatory reactions, which eventually lead to fibrosis and destruction of the encapsulated cells. The aim of this present study was to examine the biocompatibility of alginate polycation (poly-L-lysine) (AP), alginate polycation (poly?L-lysine) alginate (APA), Trondheim alginate microcapsule (TAM) and poly (methylene?co-guanidine) complexes with sodium alginate and cellulose sulphate microcapsules (PMCG). Alginate capsule-induced activation of coagulation, TF mRNA, Tissue factor (TF) surface expression and functional activity in plasma microparticles and cytokine release in fresh human whole blood were examined. The human whole blood model makes it possible to study the underlying mechanisms of biomaterial induced inflammation in vitro. By using the highly specific thrombin inhibitor, lepirudin, as anticoagulant which does not affect complement activation, the role of complement in alginate microcapsule-induced inflammation could be examined. The role of complement in alginate microcapsule-induced coagulation activation and release of cytokines were examined by using two specific complement inhibitors, the C3 convertase inhibitor compstatin and the anti-C5 monoclonal antibody eculizumab. Coagulation activation was measured by analysing prothrombin factor1+2 levels (PTF1.2) in plasma. The TF surface expression on monocytes and TF mRNA levels were analysed, by flow cytometry and real time quantitative PCR (qPCR), respectively. The TF functional activity in plasma microparticles was measured in cell ?free plasma using a commercially available ELISA kit. The terminal complement complex (sTCC) was measured by ELISA. Multiplex technology was utilized to analyze 27 different cytokines in plasma.TAM microcapsules did not enhance coagulation activation, complement activation, or Tissue factor (TF) expression, indicating a minimal inflammatory potential. Therefore, TAM microcapsule seems to be the most suited alginate microcapsule for the in vivo applications. In contrast, PMCG microcapsules significantly enhanced coagulation activation, TF mRNA levels, TF surface expression on monocytes, and TF functional activity in plasma microparticles. PMCG capsules enhanced the complement activation measured by the formation of sTCC in plasma. AP and APA microcapsules significantly increased complement activation and enhanced the coagulation activation measured as PTF1.2 levels, TF surface expression, TF mRNA and TF functional activity in plasma microparticles.The study suggests that the PMCG- and glass-induced PTF1.2 levels are most likely due to activation of the intrinsic coagulation pathway. However, TF is the most potent trigger of the coagulation system. The PMCG2-induced TF expression suggests that PMCG2 microcapsules also activate coagulation at least partly through TF up-regulation. The PMCG2-induced TF up-regulation was partly complement dependent. Therefore, the extrinsic coagulation pathway is possibly involved in the PMCG2-induced coagulation activation. This study suggests that the AP- and APA microcapsule-induced coagulation activation probably was due to TF up-regulation, leading to the activation of the extrinsic coagulation pathway. By using the specific C3 convertase inhibitor compstatin and the anti-C5 monoclonal antibody eculizumab, this study demonstrates that the AP and APA-induced coagulation activation were highly dependent on complement. In contrast, the PMCG1 microcapsule-induced coagulation activation was not dependent on complement. In conclusion, complement including C3 and C5 plays a role in alginate microcapsule-induced coagulation activation.