VTL C3A Cells Secrete Factors Involved in Coagulation Homeostasis
Authors: Riley Susan L , Mendez Dayrin F , Bedard Patricia W , Landeen Lee K 
Published: November 2023

Introduction

Liver 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).2

The 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.

Methods

Spent 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.

Results

All 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.

Conclusions

This 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)

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