OC 66.4 - Platelet factor 4 (PF4) enhances bacterial uptake and clearance by the endothelium, contributing to improved outcomes in sepsis

Tuesday, June 27, 2023

15:30 – 15:45 ET

Room: 524

Session Available As: On-Demand

Presenting Author(s)

Anh T. Ngo, PhD

Postdoc
Division of Hematology, Children's Hospital of Philadelphia, Philadelphia, PA
Philadelphia, Pennsylvania, United States

Co-Author(s)

Veronica Bochenek

Research Technician
Children's Hospital of Philadelphia
Philadelphia, Pennsylvania, United States
NL

Nate Levine

Research Technician
Children's Hospital of Philadelphia
Philadelphia, Pennsylvania, United States
AS

Amrita Sarkar

Research Associate
Division of Hematology, Children's Hospital of Philadelphia, Philadelphia, PA
Philadelphia, Pennsylvania, United States
LR

Lubica Rauova, MD, PhD

Associate Professor
Division of Hematology, Children's Hospital of Philadelphia, Philadelphia, PA
Philadelphia, Pennsylvania, United States
MK

M. Anna Kowalska

Scientist III
Children's Hospital of Philadelphia
Philadelphia, Pennsylvania, United States
MP

Mortimer Poncz

Professor
Division of Hematology, Children's Hospital of Philadelphia, Philadelphia, PA
Philadelphia, Pennsylvania, United States
Kandace Gollomp

Assistant Professor
Division of Hematology, Children's Hospital of Philadelphia, Philadelphia, PA
Philadelphia, Pennsylvania, United States

Background: Sepsis is a life-threatening response to infection. We previously showed that the cationic chemokine platelet factor 4 (PF4) limits bacterial dissemination by binding to the polyanionic bacterial wall, enhancing pathogen capture by neutrophil extracellular traps (NETs). We now address how PF4 modulates interactions between bacteria and the endothelium.

Aims: To test whether PF4 binding to the bacterial wall modulates bacterial uptake and clearance by vascular cells.

Methods: Heat-inactivated Staphylococcus (S.) aureus conjugated to pHrodo, a dye that only fluoresces following cellular internalization, was pre-incubated with PF4 (0-100g/mL) and then exposed to human umbilical vein endothelial cells (HUVECs) in the absence or presence of tumor necrosis factor  (TNF) to simulate inflammation. Bacteria fluorescent signal was imaged 16 hr post-exposure. HUVECs were stained for von Willebrand factor (VWF) release to indicate endothelial activation.

C57Bl6 mice were injected with S. aureus pre-incubated with saline or PF4. Plasma samples were obtained at 15 minutes, 6 hours and 24 hours post-infusion. Thrombin anti-thrombin (TAT), citrullinated histones (CitH3) and VWF levels were measured as indicators of intravascular inflammation.

Results: PF4 coating enhanced bacterial uptake by HUVECs in a bell-shaped distribution, peaking at 20-50µg/mL PF4. TNF-treated HUVECs more readily internalized PF4-coated bacteria at 1-20 µg/mL PF4 (Figure 1). Although PF4 promoted S. aureus uptake, it limited HUVEC VWF secretion.

Bacteria infusion into C57Bl6 mice increased TAT, VWF and CitH3 levels, with all markers returning to baseline by 24 hours. PF4 alleviated elevated TAT and CitH3, while there was a slight increase in circulating VWF compared to animals given uncoated-bacteria (Figure 2). There was no difference in blood counts and body temperatures between treatment groups.

Conclusion(s): PF4 enhances bacteria internalization while preventing bacteria-induced endothelial dysfunction, NETosis and coagulopathy. These studies support that PF4 is protective in bacterial sepsis, meriting further translational studies of a PF4-based therapeutic.

Learning Objectives:

define the role of platelet factor 4 in modulating neutrophil extracellular trap's anti-microbial activity
define the role of platelet factor 4 in modulating endothelial cells' uptake and killing of bacteria
define the role of platelet factor 4 in protection against leukopenia, thrombocytopenia and coagulopathy, while enhancing bacterial clearance in polymicrobial sepsis