VTL C3A Cells Secrete Factors Involved in Coagulation Homeostasis

Abstract

IntroductionLiver disease patients have complex coagulation disorders, sometimes associated with bleeding and sometimes with thrombosis.1 Factors typically reduced in these patients include Prothrombin, Factors V, VII, IX, X, XI, XIII, Antithrombin III, Protein C, Plasminogen, alpha‐2‐Antiplasmin, alpha‐2‐Macroglobulin, and Thrombin‐activatable Fibrinolysis Inhibitor. However, these may be offset by elevated levels of Factor VIII, Tissue Factor, Tissue Plasminogen Activator, and Plasminogen Activator Inhibitor‐1 (PAI‐1).2The ELAD System is an investigational human hepatic cell‐based liver treatment comprised of four metabolically‐active ELAD C3A cell cartridges with ancillary delivery device components and cell support circuitry intended to continuously treat subjects with liver failure secondary to acute hepatocellular insult and alcohol use.The purpose of this study was to evaluate VTL C3A cell‐secreted coagulation factors to better understand how they may affect subjects during treatment.MethodsSpent medium collected during manufacture of ELAD cartridges (~500 × 109 total cells/4 cartridges) was assayed using multiplex (Myriad) or singleplex ELISAs (Assaypro, Abcam, and Molecular Innovations) for comparison to reported concentrations in plasma of normal subjects. A pilot number of acute alcoholic hepatitis (AAH) subject (n=4) plasma samples were measured by ELISA multiplex.ResultsAll factors evaluated, except for Factors VIII, IX, and Protein S, were measureable in ELAD cartridge spent medium above media controls (Table 1). PAI‐1 and Tissue Factor Pathway Inhibitor (TFPI) were approximately 15‐fold and 3‐fold higher, respectively, whereas all other measured coagulation factors were lower, compared with normal plasma concentrations.AAH subject plasma levels were altered at baseline compared to normal plasma levels (data not shown). During the 5‐day ELAD treatment period, AAH subject plasma levels (on average) decreased ~50% for Fibrinogen, increased ~24% for Factor VII, and increased ~21% for PAI‐1 relative to baseline. The modest increase in AAH subject plasma levels for PAI‐1 was unexpected given the substantial amounts of PAI‐1 that ELAD cartridges secrete into the manufacturing spent medium. Potential explanations for this observation may include alterations in the expression of VTL C3A cell‐secreted factors upon exposure to pro‐inflammatory factors within the AAH subject plasma, or undetermined pharmacokinetic properties (i.e. absorption, distribution, metabolism, elimination) in this patient population.ConclusionsThis study provides supporting evidence that VTL C3A cells produce a wide array of coagulation, anti‐coagulation, and anti‐fibrinolysis factors typical of hepatocytes and certain factors (e.g. PAI‐1 and TFPI) that are typically produced by endothelial cells. Given that VTL C3A cells were originally derived from a human hepatoblastoma, their capacity to produce these and other non‐hepatocyte factors may offer unique advantages as a cell‐based liver therapy over primary human hepatocytes or non‐cell‐based therapies. Continuing efforts are underway to better understand the temporal responses during treatment of liver failure subjects. Factors involved in coagulation and fibrinolysis, as measured in spent media collected during the routine manufacture of ELAD C3A cell cartridges. Factor Role in Coagulation Plasma Conc. Change in Liver Disease1,2 Normal Plasma Conc. Average ELAD Conc. above Media Control Fold of Normal Plasma Conc. Plasminogen Activator Inhibitor‐1 (PAI‐1) AF increased 90 ng/mL 1334 ng/mL 15 Tissue Factor Pathway Inhibitor (TFPI) AC no change 75 ng/mL 241 ng/mL 3.2 Prothrombin C decreased 100 μg/mL 16.3 μg/mL 0.16 Tissue Factor (TF) C increased 138 pg/mL 16 pg/mL 0.12 Protein C AC decreased 4.0 μg/mL 0.21 μg/mL 0.05 Antithrombin III (ATIII) AC decreased 150 μg/mL 2.6 μg/mL 0.02 Factor VII C decreased 600 ng/mL 9.1 ng/mL 0.015 Factor X C decreased 8.50 μg/mL 0.027 μg/mL 0.003 Alpha 2‐Macroglobulin (A2M) AF decreased 1.7 mg/mL 0.004 mg/mL 0.002 Factor XII C no change 29 μg/mL 0.05 μg/mL 0.002 Fibrinogen C no change 4.5 mg/mL 0.003 mg/mL 0.001 Factor V C decreased 10 μg/mL 0.005 μg/mL 0.0005 Factor IX C decreased 5.0 μg/mL ND μg/mL ND Protein S AC decreased 24 μg/mL BLQ μg/mL BLQ Factor VIII C increased 342 ng/mL BLQ ng/mL BLQ AF (anti‐fibrinolysis), AC (anti‐coagulation), C (coagulation), ND (not detected above media control), BLQ (below limit of quantitation) Saner FH. Digestion 2013; 88:135–144 Caldwell SH. Hepatology 2006; 44:1039–1046