Mechanistic Investigation of Coagulation Activation in Childhood Acute Lymphoblastic Leukemia

Abstract

Thrombosis is a well-known complication in children with acute lymphoblastic leukemia (ALL) with a reported frequency of up to 36.7%, resulting in significant morbidity and mortality. Children with ALL were found to have persistent increased thrombin generation following diagnosis, yet, the pathogenesis and impact of therapy on thrombin activation are still unknown. Athale et al. previously demonstrated an association between the presence of peripheral lymphoblasts with increased levels of Von Willebrand Factor and parameters of thrombin generation. The objective of this study is to confirm the clinical observation in an in vitro co-culture model and to explore the effect of lymphoblasts on hemostasis. Human umbilical vein endothelial cells were co-cultured with peripheral-blood derived lymphoblasts in reduced serum media. Biomarkers of endothelial activation and the expression of endothelial products involved in hemostasis were measured by enzyme-linked immunosorbent assays. Endothelial health was evaluated by endothelial permeability assay and LDH cytotoxicity assay. Thrombin generation on the endothelial surface was monitored with a thrombin substrate. The clotting time of plasma mixed with supernatants derived from lymphoblasts was measured by a plasma recalcification assay. Compared to unstimulated endothelium, the presence of leukemic lymphoblasts significantly increased biomarkers of endothelial activation including VWF and adhesion molecules. In addition, the expression of endothelial products involved in hemostasis was altered towards a prothrombotic phenotype. On endothelium cocultured with leukemic blasts, plasma clotting time was faster, and increased thrombin generation was recorded. When mixed with plasma, secretants from leukemic lymphoblasts increased parameters of thrombin generation. In conclusion, we confirmed the clinical observation that peripheral blasts are capable of activating endothelium, leading to the elevation of VWF; and may cause the prothrombotic state seen in children with acute lymphoblastic leukemia.