Role of platelet factor 4 (PF4) and anti-PF4/polyanion IgG in human platelet FcγRIIA mediated anti-bacterial activity

R. Palankar, T. Kohler, N. Medvedev, J. Wesche, K. Krauel, S. Hammerschmidt, A. Greinacher (Greifswald, Germany)

Vascular Wall, Platelets and Acquired Problems
Date: 16.02.2017,
Time: 14:00 - 15:15

Objective: Platelets contribute to innate immunity by interacting directly and indirectly with bacteria. Here we addressed the role of the chemokine platelet factor 4 (PF4), anti-PF4/Heparin (H) IgG and human platelet FcγRIIA in bacterial recognition and killing.

Methods: Planar and biomimetic bacteria-like micropatterns functionalized with IgG, heat aggregated IgG (HAG) and PF4+anti-PF4/H IgG immune complexes were prepared using lithography. E. coli wild-type BW30270 and mutants KPM53 (ΔwaaC)/KPM121 (ΔwaaA) expressing truncated lipopolysaccharide (LPS) were micropatterned as live bacteria arrays. These E.coli show PF4-binding activity (WT<ΔwaaC<ΔwaaA). Platelet adhesion, morphodynamics and activation on ligand functionalized micropatterns and on bacterial killing on micropatterns were assessed in the presence or absence of FcγRIIA, αIIbβ3 blockers and cytoskeletal inhibitors. Calcium mobilization, quantitative fluorescence microscopy and platelet-bacteria co-culture experiments were performed.

Results: Platelets adhered on planar micropatterns functionalized with IgG [% area covered 60.23 % ±13.1 mean (SD)], HAG [83.41 % ±11.36] and PF4 + anti-PF4/H IgG [74.41 % ±10.2]. FcγRIIA blocking mAb IV.3 reduced (P < 0.0001) platelet adhesion on IgG [3.39% ±3.35], HAG [11.41%±3.96] and PF4 + anti-PF4/H IgG [5 % ± 2.89]. Similarly, blocking of αIIbβ3 or the use of cytochalasin D and blebbistatin reduced platelet adhesion and spreading on these micropatterns. Similar results were obtained on functionalized bacteria-like microbead arrays. In the presence of anti-PF4/H IgG, platelets were able to kill E.coli strains within two hours, directly dependent on the bacterial PF4 binding capacity (up to 65.4 ± 6.3% killing rate), which was inhibited by mAb IV.3 blocking FcγRIIA (P < 0.005).

Conclusion: The chemokine PF4 not only binds to polyanion such as heparin but it can also bind to polyanions on bacteria, thereby enabling opsonization by anti-PF4/H IgG, which in turn mediates killing of E.coli via platelet FcγRIIA. Because PF4 binds to both Gram-negative and Gram-positive bacteria, preformed anti-PF4/polyanion IgG can recognize bacterial pathogens the host may not have encountered before. Moreover, the role of PF4 and anti-PF4/Polyanion IgG in FcγRIIA mediated platelet anti-bacterial activity shows bridging of innate and adaptive immune system by platelets and may be relevant in controlling bacterial infections.