Open Access

The multidrug resistance protein 4 (MRP4) is highly expressed in platelets and several lines of evidence point to an impact on platelet function. MRP4 represents a transporter for cyclic nucleotides as well as for certain lipid mediators. The aim of the present study was to comprehensively characterize the effect of a short-time specific pharmacological inhibition of MRP4 on signaling pathways in platelets. Transport assays in isolated membrane vesicles showed a concentrationdependent inhibition of MRP4-mediated transport of cyclic nucleotides, thromboxane (Tx)B2 and fluorescein (FITC)- labeled sphingosine-1-phosphate (S1P) by the selective MRP4 inhibitor Ceefourin-1. In ex vivo aggregometry studies in human platelets, Ceefourin-1 significantly inhibited platelet aggregation by about 30-50% when ADP or collagen was used as activating agents, respectively. Ceefourin-1 significantly lowered the ADP-induced activation of integrin aIIbb3, indicated by binding of FITC-fibrinogen (about 50% reduction at 50 mM Ceefourin-1), and reduced calcium influx. Furthermore, pre-incubation with Ceefourin-1 significantly increased PGE1- and cinaciguat-induced vasodilatorstimulated phosphoprotein (VASP) phosphorylation, indicating increased cytosolic cAMP as well as cGMP concentrations, respectively. The release of TxB2 from activated human platelets was also attenuated. Finally, selective MRP4 inhibition significantly reduced both the total area covered by thrombi and the average thrombus size by about 40% in a flow chamber model. In conclusion, selective MRP4 inhibition causes reduced platelet adhesion and thrombus formation under flow conditions. This finding is mechanistically supported by inhibition of integrin aIIbb3 activation, elevated VASP phosphorylation and reduced calcium influx, based on inhibited cyclic nucleotide and thromboxane transport as well as possible further mechanisms.

Abstract Background Heparin induced thrombocytopenia (HIT) is likely a misdirected bacterial host defense mechanism. Platelet factor 4 (PF4) binds to polyanions on bacterial surfaces exposing neo‐epitopes to which HIT antibodies bind. Platelets are activated by the resulting immune complexes via FcγRIIA, release bactericidal substances, and kill Gram‐negative Escherichia coli. Objectives To assess the role of PF4, anti‐PF4/H antibodies and FcγRIIa in killing of Gram‐positive bacteria by platelets. Methods Binding of PF4 to protein‐A deficient Staphylococcus aureus (SA113Δspa) and non‐encapsulated Streptococcus pneumoniae (D39Δcps) and its conformational change were assessed by flow cytometry using monoclonal (KKO,5B9) and patient derived anti‐PF4/H antibodies. Killing of bacteria was quantified by counting colony forming units (cfu) after incubation with platelets or platelet releasate. Using flow cytometry, platelet activation (CD62P‐expression, PAC‐1 binding) and phosphatidylserine (PS)‐exposure were analyzed. Results Monoclonal and patient‐derived anti‐PF4/H antibodies bound in the presence of PF4 to both S. aureus and S. pneumoniae (1.6‐fold increased fluorescence signal for human anti‐PF4/H antibodies to 24.0‐fold increase for KKO). Staphylococcus aureus (5.5 × 104cfu/mL) was efficiently killed by platelets (2.7 × 104cfu/mL) or their releasate (2.9 × 104cfu/mL). Killing was not further enhanced by PF4 or anti‐PF4/H antibodies. Blocking FcγRIIa had no impact on killing of S. aureus by platelets. In contrast, S. pneumoniae was not killed by platelets or releasate. Instead, after incubation with pneumococci platelets were unresponsive to TRAP‐6 stimulation and exposed high levels of PS. Conclusions Anti‐PF4/H antibodies seem to have only a minor role for direct killing of Gram‐positive bacteria by platelets. Staphylococcus aureus is killed by platelets or platelet releasate. In contrast, S. pneumoniae affects platelet viability.

Platelet adhesion and spreading at the sites of vascular injury is vital to hemostasis. As an integral part of the innate immune system, platelets interact with opsonized bacterial pathogens through FcγRIIA and contribute to host defense. As mechanoscavangers, platelets actively migrate and capture bacteria via cytoskeleton-rich, dynamic structures, such as filopodia and lamellipodia. However, the role of human platelet FcγRIIA in cytoskeleton-dependent interaction with opsonized bacteria is not well understood. To decipher this, we used a reductionist approach with well-defined micropatterns functionalized with immunoglobulins mimicking immune complexes at planar interfaces and bacteriamimetic microbeads. By specifically blocking of FcγRIIA and selective disruption of the platelet cytoskeleton, we show that both functional FcγRIIA and cytoskeleton are necessary for human platelet adhesion and haptotaxis. The direct link between FcγRIIA and the cytoskeleton is further explored by single-particle tracking. We then demonstrate the relevance of cytoskeleton-dependent differential mobilities of FcγRIIA on bacteria opsonized with the chemokine platelet factor 4 (PF4) and patient-derived anti-PF4/polyanion IgG. Our data suggest that efficient capture of opsonized bacteria during host-defense is governed by mobility dynamics of FcγRIIA on filopodia and lamellipodia, and the cytoskeleton plays an essential role in platelet morphodynamics at biological interfaces that display immune complexes.

Cold storage of platelet concentrates (PC) has become attractive due to the reduced risk of bacterial proliferation, but in vivo circulation time of cold-stored platelets is reduced. Ca2+ release from storage organelles and higher activity of Ca2+ pumps at temperatures < 15 °C triggers cytoskeleton changes. This is suppressed by Mg2+ addition, avoiding a shift in Ca2+ hemostasis and cytoskeletal alterations. We report on the impact of 2–10 mM Mg2+ on cytoskeleton alterations of platelets from PC stored at room temperature (RT) or 4 °C in additive solution (PAS), 30% plasma. Deformation of platelets was assessed by real-time deformability cytometry (RT-DC), a method for biomechanical cell characterization. Deformation was strongly affected by storage at 4 °C and preserved by Mg2+ addition ≥ 4 mM Mg2+ (mean ± SD of median deformation 4 °C vs. 4 °C + 10 mM Mg2+ 0.073 ± 0.021 vs. 0.118 ± 0.023, p < 0.01; n = 6, day 7). These results were confirmed by immunofluorescence microscopy, showing that Mg2+  ≥ 4 mM prevents 4 °C storage induced cytoskeletal structure lesion. Standard in vitro platelet function tests showed minor differences between RT and cold-stored platelets. Hypotonic shock response was not significantly different between RT stored (56.38 ± 29.36%) and cold-stored platelets with (55.22 ± 11.16%) or without magnesium (45.65 ± 11.59%; p = 0.042, all n = 6, day 1). CD62P expression and platelet aggregation response were similar between RT and 4 °C stored platelets, with minor changes in the presence of higher Mg2+ concentrations. In conclusion, increasing Mg2+ up to 10 mM in PAS counteracts 4 °C storage lesions in platelets, maintains platelet cytoskeletal integrity and biomechanical properties comparable to RT stored platelets.