PB1033 - Tissue factor (TF) in peripheral blood mononuclear cells (PBMCs) is regulated by PACMA-31 through transcriptional and posttranscriptional mechanisms

Tuesday, July 12, 2022

6:30 PM – 7:30 PM

Location: Exhibition

Presentation Format: In-Person

Presenting Author(s)

Lennart Beckmann

Physician
Zentrum für Onkologie, UKE, Hamburg, Germany
Hamburg, Hamburg, Germany

Co-Author(s)

Jonathan Mäder

Medical student
UKE, Hamburg, Germany
Hamburg, Hamburg, Germany
MV

Minna Voigtländer

Specialist Physician
II. Medizinische Klinik und Poliklinik, Zentrum für Onkologie, UKE, Hamburg, Germany
Hamburg, Hamburg, Germany
FK

Felix Klingler

Physician
II. Medizinische Klinik und Poliklinik, Zentrum für Onkologie, UKE, Hamburg, Germany
Hamburg, Hamburg, Germany
AS

Anita Schulenkorf

Research technician
II. Medizinische Klinik und Poliklinik, Zentrum für Onkologie, UKE, Hamburg, Germany
Hamburg, Hamburg, Germany
CL

Carina Lehr

Research technician
II. Medizinische Klinik und Poliklinik, Zentrum für Onkologie, UKE, Hamburg, Germany
Hamburg, Hamburg, Germany
CB

Carsten Bokemeyer

Head of Department
II. Medizinische Klinik und Poliklinik, Zentrum für Onkologie, UKE, Hamburg, Germany
Hamburg, Hamburg, Germany
WR

Wolfram Ruf

Scientific Director
University Medicine of the Johannes Gutenberg University Mainz, Center for Thrombosis and Hemostasis (CTH)
Mainz, Rheinland-Pfalz, Germany
CR

Christina C. Rolling

Specialist physician
II. Medizinische Klinik und Poliklinik, Zentrum für Onkologie, UKE, Hamburg, Germany
Hamburg, Hamburg, Germany
FL

Florian Langer

Medical Specialist in Internal Medicine
Universitätsklinikum Eppendorf, II. Medizinische Klinik und Poliklinik, Hubertus Wald Tumorzentrum, Universitäres Cancer Center Hamburg (UCCH), Hamburg, Germany
Hamburg, Hamburg, Germany

Background: Thiol isomerase inhibitors, bacitracin and rutin, inhibit monocyte TF production under inflammatory conditions. Whether PACMA-31, a more specific antagonist of protein disulfide isomerase with distinct pharmacologic properties, has the same antithrombotic effects, is unclear.

Aims: To investigate the effect of PACMA-31 on the monocyte thrombo-inflammatory response.

Methods: Isolated PBMCs or citrate-anticoagulated whole blood were stimulated with lipopolysaccharide (LPS) and PACMA-31 or DMSO vehicle. In some experiments, stimulation was carried out in the presence of the protease-activated receptor 2 (PAR2) inhibitor ENMD547 or TF antibody 10H10. Cells were analyzed for TF antigen (flow cytometry, ELISA), procoagulant activity (PCA) (single-stage clotting or Xa generation assay), and mRNA (RT-PCR). Release of IL-6 and TNFα was measured by ELISA.

Results: PACMA-31 (25 µM) alone had no effect, but co-incubation with LPS amplified monocyte TF production in whole blood. The effect was at least partially regulated on the mRNA level and could not be explained by increased phosphatidylserine membrane exposure. In contrast, equal PACMA-31 concentrations were cytotoxic in purified PBMCs, and thus prevented LPS-induced TF production. Titration of PACMA-31 to 1 µM, however, recapitulated the potentiating effect with increased monocyte TF production and IL-6 and TNFα release from LPS-stimulated PBMCs. Western blot analysis revealed enhanced activation of the IκB-NFκB-signaling pathway. The stimulating effect of PACMA-31 was prevented by co-incubation with ENMD547, but not by TF antibody 10H10. In sharp contrast, short-term incubation of LPS-stimulated PBMCs with high PACMA-31 concentrations (25 µM) was non-cytotoxic and had no effect on TF antigen levels, but significantly reduced surface-located TF PCA.

Conclusion(s): Regulation of monocyte TF by PACMA-31 is time- and concentration-dependent, involving transcriptional and posttranscriptional mechanisms. While short-term incubation with high PACMA-31 concentrations converts preformed monocyte TF into its cryptic, non-coagulant form, long-term incubation with low PACMA-31 concentrations amplifies monocyte TF production by enhancing LPS-dependent PAR2 signaling.