Platelet Factor 4 (PF4) Binding to Neutrophil Extracellular Traps (NETs) and Von Willebrand Factor (vWF) Improves Bacterial Capture and Killing in an Electrostatic Charge-Dependent Manner

Introduction:
Sepsis, a dysregulated response to infection, remains a leading cause of mortality with limited targeted therapeutics. Platelets are increasingly recognized as integral to the immune response, and thrombocytopenia is associated with increased mortality in sepsis. Therefore, we sought to elucidate how platelets contribute to the anti-bacterial response to develop therapeutics that can restore platelet host-defense functions. In the setting of infection, platelets release high concentrations of PF4, a positively-charged chemokine with high affinity for polyanions, including polymers in the bacterial cell wall, von Willebrand factor (vWF), and NET DNA. While PF4 does not directly kill bacteria, we have shown that PF4 improves outcomes in the murine sepsis models by enhancing bacterial capture by NETs. In this study we seek to define how PF4 tethers bacteria to NETs, determine if PF4 similarly promotes bacterial capture by vWF, and explore whether these interactions modulate bacterial killing.

Method:
To generate NET-lined microfluidic channels, human neutrophils were isolated by density gradient centrifugation and plated on Bioflux microfluidic channels coated with fibronectin. The channels were infused with phorbol 12-myristate 13-acetate (PMA, 100 nM), to induce NETosis, and the resulting NETs were fluorescently labeled with SYTOX orange (Invitrogen). vWF-coated channels were prepared by incubating channels with recombinant vWF (Vonvendi, 50µg/ml) after which they were subjected to flow at 20 dynes/cm² to promote physiologic factor multimerization. Both NET- and vWF-lined channels were infused with PF4 (0-100µg/ml) suspended in HBSS and microscopy was used to quantify PF4 binding and changes in NET morphology. Initial bacterial adhesion studies were conducted with heat-inactivated bacterial bioparticles including Alexa Fluor 488-conjugated Escherichia (E) coli (K-12 strain) or Staphylococcus (S) aureus (Invitrogen). Experiments were repeated with live green fluorescent protein (GFP) expressing E coli strains, including K12 E coli and the colistin-resistant PmrA53 E coli mutant that has reduced negative surface charge. To assess the effect of PF4 on bacterial-retention, NET-lined channels were infused with DNase I (Biolegend) and VWF-channels were infused with recombinant ADAMTS13 (Abcam) and N-acetylcysteine (NAC, Sigma-Aldrich). Mean fluorescent intensity (MFI) was recorded over time to quantify bacterial adhesion and results were corroborated with colony forming unit (CFU) assays of channel eluate. Bacterial killing was measured by a fluorescent plate assay in which live GFP-expressing bacteria were incubated with isolated NETs (10 µg/ml) and/or vWF (0-20 µg/ml) ± PF4 (0-100 µg/ml). Change in MFI was measured over 6 hours. Results were confirmed with the CFU assay.

Results:
We found that at concentrations as low as 5 µg/ml, PF4 was able to physically compact NETs and increase resistance to DNase I digestion. PF4 similarly bound to vWF to induce resistance to ADAMTS13 cleavage, but not to NAC. PF4 markedly enhanced both NET and vWF capture of E. coli and S. aureus bioparticles as well as live E. coli. Moreover, PF4-binding increased NET and vWF retention of bacteria during DNase I and ADAMTS13 infusions, respectively. However, PF4 binding did not improve NET or vWF capture of the less-negatively charged PmrA53 mutant. In the fluorescent plate bacterial killing assay, VWF ± PF4 did not kill bacteria, while NETs alone reduced K12 growth by 30-50%. Although PF4 alone exerted no bactericidal effects, when combined with NETs, PF4 20 µg/ml enhanced NET-bacterial killing by 50%. PmrA53 remained resistant to NET-mediated killing, even following the addition of PF4 up to 100 µg/ml.

Conclusions:
These in vitro studies strengthen our hypothesis that PF4 tethers bacteria to NETs through electrostatic interactions, enhancing bacterial killing. PF4 also tethers bacteria to vWF, and while this does not directly promote bacterial killing, it may accelerate bacterial clearance. These results suggest that treatment with PF4 may act synergistically with antibiotics to improve bacterial clearance, with the most pronounced benefit potentially in thrombocytopenic patients.