Platelets and megakaryocytes contain functional nuclear factor-kappaB

Abstract

Objective: To investigate the presence and role of NF-kappaB proteins in megakaryocytes and platelets. The nuclear factor-kappaB (NF-kappaB) transcription factor family is well known for its role in eliciting inflammation and promoting cell survival. We discovered that human megakaryocytes and platelets express the majority of NF-kappaB family members, including the regulatory inhibitor-kappaB (I-kappaB) and I-kappa kinase (IKK) molecules.

Methods and results: Anucleate platelets exposed to NF-kappaB inhibitors demonstrated impaired fundamental functions involved in repairing vascular injury and thrombus formation. Specifically, NF-kappaB inhibition diminished lamellapodia formation, decreased clot retraction times, and reduced thrombus stability. Moreover, inhibition of I-kappaB-alpha phosphorylation (BAY-11-7082) reverted fully spread platelets back to a spheroid morphology. Addition of recombinant IKK-beta or I-kappaB-alpha protein to BAY inhibitor-treated platelets partially restored platelet spreading in I-kappaB-alpha inhibited platelets, and addition of active IKK-beta increased endogenous I-kappaB-alpha phosphorylation levels.

Conclusions: These novel findings support a crucial and nonclassical role for the NF-kappaB family in modulating platelet function and reveal that platelets are sensitive to NF-kappaB inhibitors. As NF-kappaB inhibitors are being developed as antiinflammatory and anticancer agents, they may have unintended effects on platelets. On the basis of these data, NF-kappaB is also identified as a new target to dampen unwanted platelet activation.

Figure 1

Megakaryocytes and Platelets contain NF-κB family members. Western blots for NF-κB family members were performed using Meg-01, primary human megakaryocytic cells (PHM) and platelet lysates (10 μg/lane). PHMs were activated with PMA (50 ng/mL) and harvested at 4 and 24 hours. Platelets were activated with thrombin (Thr) (0.8 U/mL) collagen (Col) (10 μg/mL) or Untreated (UT).

Figure 2

NF-κB family members, p65 and p50 retain the ability to bind DNA. Platelets were rigorously purified and lysates analyzed for RelA (p65) and p50 DNA-binding activity using a NF-κB activity assay. DNA bound p65 or p50 was detected using a p65 or p50 antibody, respectively. The results are graphed as the OD value at 450 nm. A non-specific binding negative control, reagent blank and positive controls confirmed specific binding of platelet-derived proteins (not shown). The results are representative of three individual healthy donors.

Figure 3

NF-κB inhibitors impair platelet spreading. Purified human platelets (2×10⁷/mL) were left untreated (UT) or incubated with NF-κB inhibitors, SC-514 (10 μM) or BAY-11-7082 (1 μM). The platelets were spread on fibrinogen. After washing, the coverslips were (A) fixed in 2.5% glutaraldehyde and processed for imaging by scanning electron microscopy (SEM) (B) simultaneously extracted in Triton X-100 and glutaraldehyde [40] and processed for SEM (C) fixed in 4% PFA and stained for actin fillaments (Phalloidin). Results are representative of at least three experiments. Platelet spreading was quantified by counting and surface area. (D) Data represents counts of 6-10 fields of 80-100 Platelets/field. At least three individual donors were used. Bars = S.E. Platelet spreading for untreated versus inhibitor-treated platelets was significantly different for each condition *p<0.0002; **p<0.01; ***p<0.0002. (E) Platelet surface area was determined from a representative donor. The percent (%) platelet surface area is calculated from equivalent fields and normalized to 426 platelets/treatment.

Figure 4

Fully spread platelets convert to spheroid morphology upon BAY inhibitor treatment. Platelets were first spread on fibrinogen, and subsequently treated with increasing doses of BAY inhibitor. (A) DIC images are shown for vehicle (VEH) and BAY (5 μM). (B) Graphical representation of BAY inhibitor dose response. At least three individual donors were used. Bars = S.E. *p<0.005.

Figure 5

Addition of recombinant IKK-β or I-κB-α partially restores platelet spreading following BAY-11-7082 inhibition. Purified human platelets (2×10⁷/mL) were treated with vehicle or BAY-11-7082 (1 μM). Recombinant IKK-β or I-κB-α GST-tagged proteins were added following platelet spreading (45 minutes). After thorough washing, platelets were (A) removed from coverslips, lysed and western blots performed using anti-I-κB-α, anti-IKK-β or anti-GST antibodies, (B) fixed in 2.5% glutaraldehyde and imaged by differential imaging contrast (DIC) microscopy, (C) quantified by counting. Bars = S.E. BAY-treated versus BAY and IKK-β-treated platelets was significantly different *p<0.004, (D) removed from coverslips, lysed and a western blot performed using an anti-phospho I-κB-α antibody. All results are representative of at least three experiments.

Figure 6

BAY-11-7082 hinders fibrin clot retraction and thrombus stability under flow. PRP (clot retraction) or whole blood (flow studies) was dosed with BAY inhibitor. (A) Following incubation with no inhibitor, Bay-11-7082 or DMSO vehicle, thrombin was added, and clot formation and retraction was monitored over time. Retracted clots were quantified as described in Methods. Results are representative of at least three experiments. Bars = S.E. BAY100 and BAY200 treatments were significantly different from vehicle-treated platelets *p<0.05. The BAY50 results approach conventional statistical significance (p=0.06), reflecting a less potent effect compared to higher BAY doses, confirming a dose response relationship between BAY concentration and platelet function. (B) Control and inhibitor-treated human whole blood were perfused through collagen coated glass capillaries. Flow direction and time (t<60s) are indicated by arrows in the margins. Thrombus stability was monitored over time using live imaging microscopy. The image series on the left (top to bottom) shows stably formed thrombi in vehicle-treated platelets over time. In contrast, the image series on the right is a representative demonstration of unstable thrombi in BAY-treated platelets, indicated by release of emboli (circle).