Elevated plasma levels and platelet-associated expression of the pro-thrombotic and pro-inflammatory protein, TNFSF14 (LIGHT), in sickle cell disease.

Chronic vascular inflammation and endothelial activation may initiate vaso-occlusion in sickle cell disease (SCD). TNFSF14 (CD258; LIGHT), a recently-identified pro-thrombotic and pro-inflammatory tumour necrosis factor (TNF)-superfamily cytokine, has a potent activating effect on endothelial cells. We evaluated whether TNFSF14 production is altered in SCD and whether platelets contribute to this production. TNFSF14 was measured in platelet-free plasma from healthy-control individuals (CON), steady-state sickle cell anaemia (SCA), SCA on hydroxycarbamide therapy (SCAHC) and haemoglobin SC (HbSC) patients. Mean plasma TNFSF14 was significantly increased in SCA, SCAHC and HbSC, compared to CON individuals. In SCA/SCAHC patients, plasma TNFSF14, showed no correlation with haematological variables, but was significantly correlated with serum lactate dehydrogenase and inflammatory markers (CD40LG , IL8 and ICAM1). Platelet-membrane TNFSF14 expression was significantly augmented on SCA platelets, and correlated with platelet activation; furthermore, measurement of platelet TNFSF14 release indicated that platelets may be a major source of circulating TNFSF14 in SCA. Interestingly, high plasma TNFSF14 was significantly associated with elevated tricuspid regurgitant velocity (≥2·5 m/s) in a population of SCA/SCAHC patients. The pro-inflammatory and atherogenic cytokine, TNFSF14, could contribute to endothelial activation and inflammation in SCA; future investigations may confirm whether this protein contributes to major clinical complications of the disease, such as pulmonary hypertension, and represents a potential therapeutic target.

Increased adhesive properties of platelets in sickle cell disease: roles for alphaIIb beta3-mediated ligand binding, diminished cAMP signalling and increased phosphodiesterase 3A activity.

Whilst high pro-coagulant activity is reported in sickle cell disease (SCD), the precise role of platelets (PLTs) in SCD inflammatory and vaso-occlusive processes is unclear. Adhesion of PLTs from healthy controls (CON), SCD individuals (SCD) and SCD patients on hydroxycarbamide (SCDHC) to fibrinogen (FB) was compared using static adhesion assays. PLT adhesion molecules and intraplatelet cyclic adenosine monophosphate (icAMP) were observed by flow cytometry and enzyme-linked immunosorbent assay. SCD-PLTs demonstrated significantly greater adhesion than CON-PLTs to FB. Participation of the alpha(IIb)beta(3)-integrin in SCD-PLT adhesion was implicated by increased alpha(IIb)beta(3) activation and data showing that an alpha(IIb)beta(3)-function-inhibiting antibody significantly diminished SCD-PLT adhesion to FB. Platelet activation was potentiated by reductions in icAMP; cAMP levels were decreased in SCD-PLTs, being comparable to those of thrombin-stimulated CON-PLTs. Furthermore, SCD-PLT adhesion to FB was significantly reduced by cilostazol, an inhibitor of cAMP-hydrolyzing phosphodiesterase 3A (PDE3A). Both alpha(IIb)beta(3)-integrin activation and icAMP correlated significantly with fetal haemoglobin in SCD. Accordingly, hydroxycarbamide therapy was associated with lower PLT adhesion and higher icAMP. SCD-PLTs may be capable of adhering to proteins encountered on the inflamed vascular wall and, potentially, participate in vaso-occlusive processes. Hydroxycarbamide and, speculatively, nitric oxide donor or cyclic-nucleotide-targeted therapies may aid in the reversal of PLT adhesive properties in SCD.

Endothelial activation by platelets from sickle cell anemia patients.

Sickle cell anemia (SCA) is associated with a hypercoagulable state. Increased platelet activation is reported in SCA and SCA platelets may present augmented adhesion to the vascular endothelium, potentially contributing to the vaso-occlusive process. We sought to observe the effects of platelets (PLTs) from healthy control (CON) individuals and SCA individuals on endothelial activation, in vitro. Human umbilical vein endothelial cells (HUVEC) were cultured, in the presence, or not, of washed PLTs from CON or steady-state SCA individuals. Supernatants were reserved for cytokine quantification, and endothelial adhesion molecules (EAM) were analyzed by flow cytometry; gene expressions of ICAM1 and genes of the NF-κB pathway were analyzed by qPCR. SCA PLTs were found to be more inflammatory, displaying increased adhesive properties, an increased production of IL-1ß and a tendency towards elevated expressions of P-selectin and activated αIIbß3. Following culture in the presence of SCA PLTs, HUVEC presented significant augmentations in the expressions of the EAM, ICAM-1 and E-selectin, as well as increased IL-8 production and increased ICAM1 and NFKB1 (encodes p50 subunit of NF-κB) gene expressions. Interestingly, transwell inserts abolished the effects of SCA PLTs on EAM expression. Furthermore, an inhibitor of the NF-κB pathway, BAY 11-7082, also prevented the induction of EAM expression on the HUVEC surface by SCA PLTs. In conclusion, we find further evidence to indicate that platelets circulate in an activated state in sickle cell disease and are capable of stimulating endothelial cell activation. This effect appears to be mediated by direct contact, or even adhesion, between the platelets and endothelial cells and via NFκB-dependent signaling. As such, activated platelets in SCD may contribute to endothelial activation and, therefore, to the vaso-occlusive process. Results provide further evidence to support the use of anti-platelet approaches in association with other therapies for SCD.