Ex Vivo Evaluation of Red Blood Cell Adhesion and Whole Blood Thrombosis in Pyruvate Kinase Deficiency

Introduction Pyruvate Kinase Deficiency (PKD) is an inherited glycolytic enzymopathy that is characterized by a life-long chronic hemolytic anemia with severe comorbidities. Hypercoagulability due to increased platelet activity caused by nitric oxide sequestration by cell free hemoglobin has been well-described not just in PKD, but in other hemolytic anemias as well, such as e.g., sickle cell disease (SCD). Hypercoagulability is often accompanied by a cascade of pathophysiological events leading to cell oxidative damage, endothelial activation, and changes in both cell stability and adhesive properties. Increased red blood cell (RBC) adhesion and hypercoagulability may impair microvascular blood flow. Despite these well-recognized rheological changes that are similar to those that occur in other hemolytic anemias, the relationship between baseline erythrocyte adhesion and thrombosis potential have not been well-studied in PKD. Methods 10 PKD subjects and 5 healthy controls were recruited under the IRB-approved protocol from Wayne State University. Flow adhesion of whole blood to vascular cell adhesion molecule-1 (FA-WB-VCAM) was performed by flowing whole blood (1:1 dilution) through a microfluidic channel for 3 minutes (1 dyne/cm 2 shear stress, 1.67Hz pulse frequency). Flow adhesion avidity of the whole blood sample to VCAM-1 (FAAv-WB-VCAM), representing the strength of the RBC-VCAM-1 adhesive interactions, was assessed by quantifying adhesion following sequential increase in shear (5, 10, 20 dyne/cm 2). Thrombin generation assay was conducted using platelet poor plasma with and without thrombomodulin and microparticles (MP) as previously published [1]. Clotting time - reported as lag time (LT), time to peak (ttPeak) and peak height (velocity and amount of net thrombin production), and endogenous thrombin potential (ETP), representing number of substrates potentially convertible by thrombin, were measured. Significance was at p < 0.05. Results FA-WB-VCAM at baseline sample hematocrit was significantly elevated (Figure 1) in PKD subjects (808±377 cells/mm², n=10) compared to healthy controls (6±4 cells/mm², n=4) and even to our previously reported steady state levels in sickle cell samples (290±50 cells/mm² [2]. Thrombin generation profiles were similar between PKD subjects and healthy controls with the exception of the thrombin generation index (PPP+TP/PPP)*100ETP that was significantly (p