Laura Martos1, Luis Andrés Ramón1, Julia Oto1, Álvaro Fernández-Pardo1, Santiago Bonanad1,2, Ana Rosa Cid1,2, Andras Gruber3, John H Griffin4, Francisco España1, Silvia Navarro1, Pilar Medina1. 1. Grupo de Investigación en Hemostasia, Trombosis, Arteriosclerosis y Biología Vascular, Instituto de Investigación Sanitaria La Fe (IIS La Fe), Hospital Universitario y Politécnico La Fe, Valencia, Spain. 2. Unidad de Trombosis y Hemostasia, Servicio de Hematología, Hospital Universitario y Politécnico La Fe, Valencia, Spain. 3. Department of Biomedical Engineering, Oregon Health and Science University, Portland, Oregon, United States. 4. Department of Molecular Medicine, The Scripps Research Institute, La Jolla, California, United States.
Abstract
BACKGROUND: Activated protein C (APC) is a major regulator of thrombin formation. Two major plasma inhibitors form complexes with APC, protein C inhibitor (PCI) and α1-antitrypsin (α1AT), and these complexes have been quantified by specific enzyme-linked immunosorbent assays (ELISAs). Also, complexes of APC with α2-macroglobulin (α2M) have been observed by immunoblotting. Here, we report an ELISA for APC:α2M complexes in plasma. METHODS: Plasma samples were pre-treated with dithiothreitol and then with iodoacetamide. The detection range of the newly developed APC:α2M assay was 0.031 to 8.0 ng/mL of complexed APC. Following infusions of APC in humans and baboons, complexes of APC with α2M, PCI and α1AT were quantified. These complexes as well as circulating APC were also measured in 121 patients with a history of venous thromboembolism (VTE) and 119 matched controls. RESULTS: In all the in vivo experiments, α2M was a significant APC inhibitor. The VTE case-control study showed that VTE patients had significantly lower APC:α2M and APC levels than the controls (p < 0.001). Individuals in the lowest quartile of APC:α2M or the lowest quartile of APC had approximately four times more VTE risk than those in the highest quartile of APC:α2M or of APC. The risk increased for individuals with low levels of both parameters. CONCLUSION: The APC:α2M assay reported here may be useful to help monitor the in vivo fate of APC in plasma. In addition, our results show that a low APC:α2M level is associated with increased VTE risk. Schattauer GmbH Stuttgart.
BACKGROUND:Activated protein C (APC) is a major regulator of thrombin formation. Two major plasma inhibitors form complexes with APC, protein C inhibitor (PCI) and α1-antitrypsin (α1AT), and these complexes have been quantified by specific enzyme-linked immunosorbent assays (ELISAs). Also, complexes of APC with α2-macroglobulin (α2M) have been observed by immunoblotting. Here, we report an ELISA for APC:α2M complexes in plasma. METHODS: Plasma samples were pre-treated with dithiothreitol and then with iodoacetamide. The detection range of the newly developed APC:α2M assay was 0.031 to 8.0 ng/mL of complexed APC. Following infusions of APC in humans and baboons, complexes of APC with α2M, PCI and α1AT were quantified. These complexes as well as circulating APC were also measured in 121 patients with a history of venous thromboembolism (VTE) and 119 matched controls. RESULTS: In all the in vivo experiments, α2M was a significant APC inhibitor. The VTE case-control study showed that VTEpatients had significantly lower APC:α2M and APC levels than the controls (p < 0.001). Individuals in the lowest quartile of APC:α2M or the lowest quartile of APC had approximately four times more VTE risk than those in the highest quartile of APC:α2M or of APC. The risk increased for individuals with low levels of both parameters. CONCLUSION: The APC:α2M assay reported here may be useful to help monitor the in vivo fate of APC in plasma. In addition, our results show that a low APC:α2M level is associated with increased VTE risk. Schattauer GmbH Stuttgart.
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