Evidence is provided herein for a new pathway of platelet regulation by NF-κB. Our studies demonstrate that primary human megakaryocytic cells, the Meg-01 cell line, and platelets contain nearly all known NF-κB family members including c-Rel, p105/p50, p100/p52, I-κ-Bs and IKKs. Moreover, expression levels and modification of specific NF-κB proteins was dependent on cell type and sensitive to agonist stimulation. For example, RelA (p65) and RelB were highly expressed in unstimulated Meg-01 cells. In contrast, normal platelets expressed low levels of RelB and human primary megakaryocytic cells required agonist stimulation to produce detectable amounts. We also observed activation-specific changes in the levels of some proteins (e.g.
RelA and p50) and phosphorylated protein (IKK-γ) in both megakaryocytes and platelets. mRNA transcripts for several NF-κB family members have been reported in human platelets, including I-κB-α[3
]. These observations support the concept that NF-κB proteins are important in platelet function.
Platelets are known to contain other transcription factors such as PPARγ and its binding partner, RXR[9
]. Both proteins regulate platelet function by nongenomic mechanisms, including RXR interaction with Gq[24
] and PPARγ ligand dampening of platelet activation[9
]. Additionally, PPARγ and RXR are released in platelet microparticles and elicit a PPARγ ligand-dependent transcellular response in a monocytic cell line[10
]. Further studies are in progress to determine whether NF-κB subunits, like PPARγ, are released during platelet activation.
A role for NF-κB in platelet physiology is also supported by data from the Bloodomics Consortium that identified a transcript in platelets encoding a novel gene COMMD7 (copper metabolism gene MURR1 domain containing 7) that correlated with both platelet function and coronary artery disease risk. COMMD7 is a member of the COMM domain protein family involved in the negative regulation of NF-κB activity (Goodall and Ouwehand, unpublished observations, 2009). A Western blot probed with an anti-COMMD7 antibody revealed the presence of the CommD7 protein in platelet lysates (data not shown).
In further support of NF-κB function in platelets, we demonstrate that inhibitors of NF-κB have profound effects on platelet signaling pathways involved in shape change and spreading, modulation of clot retraction, and thrombus stability under flow conditions. The Rho GTPase, Rac1, which is ubiquitously expressed in all mammalian cells, stimulates actin polymerization and lamellae formation in platelets[17
]. Here, we show that NF-κB inhibition attenuates lamellae formation, an essential function during vascular wound healing. Intriguingly, the Rho GTPase family was previously reported to be specifically involved in regulating NF-κB activity in nucleated cells, including a role in cytoskeletal organization of critical cellular functions[25
]. A significant finding herein was that BAY treatment of fully spread platelets reversed lamellipodia formation, reinstating platelets to a spherical morphology. These new data support a dynamic role for NF-κB in the regulation of platelet cytoskeletal architecture.
Our data also show that NF-κB inhibition significantly hampered clot retraction, although platelets were still able to aggregate as measured by aggregometry at low levels of shear. Our aggregation data are in contrast to the findings of Malaver et. al
, who showed that BAY-11-7082 inhibited platelet aggregation[13
]. These differences may be attributed to the concentration of the platelet agonist used in PRP (0.8 U/mL thrombin vs 0.05 U/mL thrombin, Malaver et. al
). We found by our investigative methods that thrombin addition stimulated clot formation in PRP, but platelets failed to form a tightly condensed clot in a dose-dependent manner. Furthermore, flow adhesion assays indicated that NF-κB-inhibited platelets failed to form stable thrombi on a collagen matrix at physiological levels of shear stress. Although platelets were recruited to the matrix, the aggregates that did form were less compact compared to untreated or vehicle-treated platelets, and emboli were copiously released. It was previously demonstrated that inhibition of signaling events that support platelet activation following integrin activation and platelet aggregation are necessary for clot retraction and for promoting thrombus growth and stability. Our data support a role for NF-κB inhibitors interfering with outside-in signaling via the fibrinogen-binding integrin, αIIb
]. Initial fibrinogen binding mediates aggregation (inside-out signaling), which subsequently triggers outside-in responses that stabilize aggregates and initiates platelet spreading, vesicle secretion and clot retraction. These observations support a central role for platelet NF-κB during vascular injury, and suggest that dysregulation of NF-κB function may contribute to vascular disease processes.
Further investigation revealed that blood treated with NF-κB inhibitors coagulated normally, as evidenced by normal prothrombin, partial thromboplastin and euglobulin clot lysis times, indicating that inhibitor effects were platelet specific. Interestingly, Ono et al. recently demonstrated a fibrin-independent platelet contractile mechanism essential for the initial stages of hemostasis and in promoting thrombus contraction [28
]. It will be interesting to discover whether NF-κB plays a role in this regulation.
Of particular importance to our hypothesis was the finding that inhibition of IKK-β activity was attenuated by addition of active recombinant IKK-β protein. Our data show that the effects on platelet function following BAY inhibitor treatment were overcome by addition of active recombinant IKK-β protein. With that in mind, we also investigated a specific target of IKK-β phosphorylation, I-κB-α, and discovered that rhIKK-β addition increased I-κB-α phosphorylation levels. Moreover, addition of exogenous rhI-κB-α protein restored platelet spreading, supporting that BAY-11-7082 is targeting NF-κB driven mechanisms.
We very recently initiated studies to comprehensively determine where NF-κB proteins reside (e.g. granules, cytoskeleton, adhesion contacts, mitochondria) and to identify the types of NF-κB protein interactions in both untreated and BAY inhibitor-treated platelets. Our immunofluorescent pilot data indicated that both IKK-β and I-κB-α are centrally located in the unspread platelet. Interestingly, in spreading platelets, IKK-β remains centralized, while I-κB-α begins to appear throughout the lamellipodial region (data not shown).
Although the underlying mechanism(s) of NF-κB function in platelets require(s) further analysis, our overall findings provide a strong rationale that NF-κB proteins have an important role in human platelet physiology. The expanding versatility of platelet regulation leads us to envision possible mechanisms. Firstly, our data and others[13
] indicate that RelA and p50 retain the ability to bind DNA. Although platelets lack nuclear DNA, it is plausible that a novel mechanism of mitochondrial gene regulation may function in platelets. It has been reported that the NF-κB subunits RelA and p50, as well as I-κB-α play an important role in regulating mitochondrial mRNAs in other cell types[29
]. Secondly, it is conceivable that NF-κB proteins regulate post-transcriptional gene expression in platelets by directly binding RNA in a signal-dependent manner. In fact, we have demonstrated a novel function for glucocorticoid receptor whereby this transcription factor binds directly to monocyte chemoattractant protein (MCP) -1 mRNA, decreasing its stability in arterial smooth muscle cells[31
]. Our final point is that platelet Rel/NF-κB/I-κB proteins could elicit effects through direct protein/protein interactions. For example, PPARγ exerts some of its anti-inflammatory effects by directly binding nuclear-localized NF-κB complexes to facilitate nuclear exit and sequestration of NF-κB in the cytoplasm[16
]. Also consistent with our data is the fact that Bcl-3, an I-κB-α family member[32
], specifically interacts with a Src-family tyrosine kinase, Fyn, and is involved in cytoskeletal regulation[6
] and inhibition of clot retraction in platelets[14
]. Interestingly, Fyn is an upstream regulator of the Rho family of GTPases[33
] and associates with αIIb
to regulate platelet adhesion and spreading[34
Our data support the idea that transcription factors, such as NF-κB have important roles in platelet physiology. It is well-documented that dysregulation of NF-κB function contributes to the development of many human diseases, including chronic inflammatory diseases and cancer. Approaches to specifically inhibit the NF-κB pathway are under active development as therapeutic interventions. Thus, it is important to now recognize that platelets and megakaryocytes contain nearly all NF-κB family members, and that these cells could be affected by NF-κB inhibitory drugs. This new understanding of a NF-κB-platelet connection reveals a novel target, namely NF-κB, for dampening platelet activation. The impact of influencing NF-κB function in platelets and megakaryocytes could lead to a host of new therapeutics for platelets.