Markers of Blood Cell Activation and Complement Activation in Factor VIII and von Willebrand Factor Concentrates

Abstract

SummaryBackground: Preparations of commercially available clotting factor VIII are complex protein mixtures. Most of them contain either von Willebrand factor or human serum albumin as stabilizers. The aim of the study was to quantify further proteins in twelve concentrates either of recombinant origin or derived from human plasma. Methods: Proteins were separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Some proteins were quantified by ELISA. Results: Recombinant clotting factor preparations showed fewer protein spots in the 2D-PAGE, than plasma-derived preparations. Proteins identified in some of the plasma-derived concentrates included up to 90 ng/IU of the anaphylatoxin C3a, up to 40 ng/IU of the platelet a-granule protein thrombospondin-1, up to 0.85 ng/IU of the platelet agranule protein platelet factor 4, 3.5 ng/IU myeloperoxidase secreted by leukocytes and up to 0.05 ng/IU of the leukocyte-secreted protein a-defensin. The protein content differed between concentrates from different manufacturers. Conclusion: The origin of the plasma used to prepare the factor concentrates might influence the protein impurities in these products. It is unknown whether the impurities observed have long-term consequences for chronic inflammatory conditions. Schlüsselwörter

Components in Plasma-Derived Factor VIII, But Not in Recombinant Factor VIII Downregulate Anti-Inflammatory Surface Marker CD163 in Human Macrophages through Release of CXCL4 (Platelet Factor 4)

“…As we reported earlier, several plasma-derived FVIII products contain substances that influence platelet activation status and/or inflammation that were absent in recombinant FVIII [21]. Among them was alpha-defensin, a neutrophil cationic peptide, which besides exerting antimicrobial and proinflammatory functions, acts as a potent platelet agonist [20].…”

“…Previously, we identified up to 0.85 ng/IU of the platelet alpha granule protein CXCL4 as well as platelet-activating substances such as myeloperoxidase and alpha-defensin [20] in some plasma-derived FVIII concentrates [21]. Therefore, we tested the direct effect of FVIII products on differentiation of human macrophages.…”

Cellular Stress Induced by Plasma-Derived Factor VIII Products

“…Three FVIII products were evaluated for their in vitro effects on cell stress sensors: a pdFVIII product with a moderate level of impurities (product 1, Haemoctin®SDH; Biotest Pharma GmbH, Dreieich, Germany), a pdFVIII product with a relatively high level of impurities (product 2, Octanate®; Octapharma GmbH, Langenfeld, Germany) [4,6], and an rFVIII product formulated with sucrose (rFVIII-FS, KOGENATE®; Bayer Pharma AG, Berlin, Germany).…”

“…However, other impurities that could affect patient health may be present in pdFVIII products. In an earlier study [4], we identified hematocyte-derived proteins and proteins of the complement activation pathway in several pdFVIII products of varying purities. Proteins isolated in some of the pdFVIII products tested included anaphylatoxin C3a, the platelet activation markers thrombospondin-1 and platelet factor 4, and leukocyte-secreted myeloperoxidase and α-defensin [4].…”

“…Proteins isolated in some of the pdFVIII products tested included anaphylatoxin C3a, the platelet activation markers thrombospondin-1 and platelet factor 4, and leukocyte-secreted myeloperoxidase and α-defensin [4]. Although the concentrations of these impurities were low, the presence of these proteins clearly indicates that some of the blood cells in the blood donations used as starting material for the purification process had been activated [4]. …”