PHILEOS (haemoPHILia and ostEoporOSis) Study: protocol of a multicentre prospective case-control study.

INTRODUCTION: Two meta-analyses showed lower bone mineral density (BMD) in patients with haemophilia (haemophilia type and severity were often not specified) compared with healthy controls. This finding could be related to reduced mobility and sedentary lifestyle, and/or hepatitis C or HIV infection. The aim of this study is to determine osteoporosis prevalence in patients with haemophilia classified in function of the disease type (A or B) and severity, and to evaluate the potential role of regular prophylactic factor replacement (early vs delayed initiation) in preserving or restoring BMD. METHODS AND ANALYSIS: The haemoPHILia and ostEoporOSis Study is a prospective, controlled, multicentre study that will include patients in France (13 haemophilia treatment centres), Belgium (1 centre) and Romania (1 centre). In total, 240 patients with haemophilia and 240 matched healthy controls will be recruited (1:1). The primary objective is to determine osteoporosis prevalence in patients with severe haemophilia A and B (HA and HB) without prophylaxis, compared with healthy controls. Secondary outcomes include: prevalence of osteoporosis and osteopenia in patients with mild, moderate and severe HA or HB with prophylaxis (grouped in function of their age at prophylaxis initiation), compared with healthy subjects; BMD in patients with HA and HB of comparable severity; correlation between BMD and basal factor VIII/IX levels and thrombin potential; and quantification of plasmatic markers of bone remodelling (formation and resorption) in patients with haemophilia. ETHICS AND DISSEMINATION: The protocol was approved by the French Ethics Committee and by the French National Agency for Medicines and Health Products Safety (number: 2019-A03358-49). The results of this study will be actively disseminated through scientific publications and conference presentations. TRIAL REGISTRATION NUMBER: NCT04384341.

Tissue factor pathway inhibitor is the main determinant of thrombin generation in haemophilic patients.

The thrombin generation (TG) assay evaluates haemostatic balance, which is influenced by the levels of many coagulation factors and inhibitors. Our objective was to identify the determinant factors of TG in haemophilia A (HA) and haemophilia B (HB) patients and to compare them to those in healthy controls. Coagulation factor and inhibitor levels, and TG, were measured in platelet-poor plasma from 40 patients with HA, 32 patients with HB and 40 healthy subjects. Data were analysed using multiple regression models. In HA patients, factor VIII was a positive determinant of endogenous thrombin potential (ETP) and peak, whereas tissue factor pathway inhibitor (TFPI) and factor V were negative determinants of ETP and peak. In HB patients, FIX was a positive determinant of ETP and peak, FVII being a positive determinant of peak. Antithrombin and protein S (PS) were negative determinants of ETP while FX was a negative determinant of peak. Above all, in HB patients, TFPI was a negative determinant of ETP and peak. In healthy subjects, FVIII was a positive determinant of ETP and peak, whereas FX and protein S were negative determinants of these parameters. TFPI was not a negative determinant of either peak or ETP. In haemophilic patients, the determinant factors of TG are all implicated in FXa generation and inhibition, the crucial determinant factor being TFPI whatever the type of haemophilia, A or B. These findings contribute to the rationale that recently place TFPI as a target for innovative therapies of haemophilia.

Acetylsalicylic acid differentially limits the activation and expression of cell death markers in human platelets exposed to Staphylococcus aureus strains.

Beyond their hemostatic functions, platelets alter their inflammatory response according to the bacterial stimulus. Staphylococcus aureus is associated with exacerbated inflammation and thrombocytopenia, which is associated with poor prognosis during sepsis. Acetylsalicylic acid and statins prevent platelet aggregation and decrease the mortality rate during sepsis. Therefore, we assessed whether these two molecules could reduce in vitro platelet activation and the inflammatory response to S. aureus. Platelets were exposed to clinical strains of S. aureus in the presence or absence of acetylsalicylic acid or fluvastatin. Platelet activation, aggregation, and release of soluble sCD62P, sCD40 Ligand, RANTES and GROα were assessed. Platelet cell death was evaluated by analyzing the mitochondrial membrane potential, phosphatidylserine exposure, platelet microparticle release and caspase-3 activation. All S. aureus strains induced platelet activation but not aggregation and decreased the platelet count, the expression of cell death markers and the release of RANTES and GROα. Acetylsalicylic acid but not fluvastatin limited platelet activation and inflammatory factor release and restored the platelet count by protecting platelets from Staphylococcus-induced expression of cell death markers. This study demonstrates that acetylsalicylic acid limits S. aureus-induced effects on platelets by reducing cell death, revealing new strategies to reduce the platelet contribution to bacteremia-associated inflammation.

NF-κB Links TLR2 and PAR1 to Soluble Immunomodulator Factor Secretion in Human Platelets.

The primary toll-like receptor (TLR)-mediated immune cell response pathway common for all TLRs is MyD88-dependent activation of NF-κB, a seminal transcription factor for many chemokines and cytokines. Remarkably, anucleate platelets express the NF-κB machinery, whose role in platelets remains poorly understood. Here, we investigated the contribution of NF-κB in the release of cytokines and serotonin by human platelets, following selective stimulation of TLR2 and protease activated receptor 1 (PAR1), a classical and non-classical pattern-recognition receptor, respectively, able to participate to the innate immune system. We discovered that platelet PAR1 activation drives the process of NF-κB phosphorylation, in contrast to TLR2 activation, which induces a slower phosphorylation process. Conversely, platelet PAR1 and TLR2 activation induces similar ERK1/2, p38, and AKT phosphorylation. Moreover, we found that engagement of platelet TLR2 with its ligand, Pam3CSK4, significantly increases the release of sCD62P, RANTES, and sCD40L; this effect was attenuated by incubating platelets with a blocking anti-TLR2 antibody. This effect appeared selective since no modulation of serotonin secretion was observed following platelet TLR2 activation. Platelet release of sCD62P, RANTES, and sCD40L following TLR2 or PAR1 triggering was abolished in the presence of the NF-κB inhibitor Bay11-7082, while serotonin release following PAR1 activation was significantly decreased. These new findings support the concept that NF-κB is an important player in platelet immunoregulations and functions.

Clinical Studies of Ex Vivo Expansion to Accelerate Engraftment After Umbilical Cord Blood Transplantation: A Systematic Review.

Cell dose limits greater use of umbilical cord blood (UCB) in hematopoietic cell transplantation. The clinical benefits of ex vivo expansion need clarity to understand its potential impact. A systematic search of studies addressing UCB ex vivo expansion was conducted. Fifteen clinical studies (349 transplanted patients) and 13 registered trials were identified. The co-infusion of an expanded unit and a second unmanipulated unit (8 studies), the fractional expansion of 12% to 60% of a single unit (5 studies), and the infusion of a single expanded unit (2 studies) were reported. More recently, published studies and 12 of 13 ongoing trials involve the use of novel small molecules in addition to traditional cytokine cocktails. Higher total cell number was closely associated with faster neutrophil engraftment. Compared with historical controls, neutrophil engraftment was significantly accelerated in more recent studies using small molecules or mesenchymal stromal cells (MSC) co-culture, and in some cases, platelet recovery was also statistically improved. Recent studies using nicotinamide and StemRegenin-1 reported long-term chimerism of the expanded unit. No significant improvement in survival or other transplant-related outcomes was demonstrated for any of the strategies. Ex vivo expansion of UCB can accelerate initial neutrophil engraftment after transplant. More recent studies suggest that long-term engraftment of ex vivo expanded cord blood units is achievable. Results of larger randomized controlled trials are needed to understand the impact on patient outcomes and health care costs.

Regenerative Therapy and Immune Modulation Using Umbilical Cord Blood-Derived Cells.

Since the first cord blood transplantation in 1988, umbilical cord blood has become an important option as a source of cells for hematopoietic transplantation. Beyond its role in regenerating the blood and immune systems to treat blood diseases and inherited metabolic disorders, the role of nonhematopoietic progenitor cells in cord blood has led to new and emerging uses of umbilical cord blood in regenerative therapy and immune modulation. In this review, we provide an update on the clinical and preclinical studies using cord blood-derived cells such as mesenchymal stromal cells, endothelial-like progenitor cells, and others. We also provide insight on the use of cord blood cells as vehicles for the delivery of therapeutic agents through gene therapy and microvesicle-associated strategies. Moreover, cord blood can provide essential reagents for regenerative applications. Clinical activity using cord blood cells is increasing rapidly and this review aims to provide an important update on the tremendous potential within this fast-moving field.

Platelets and infections - complex interactions with bacteria.

Platelets can be considered sentinels of vascular system due to their high number in the circulation and to the range of functional immunoreceptors they express. Platelets express a wide range of potential bacterial receptors, including complement receptors, FcγRII, Toll-like receptors but also integrins conventionally described in the hemostatic response, such as GPIIb-IIIa or GPIb. Bacteria bind these receptors either directly, or indirectly via fibrinogen, fibronectin, the first complement C1q, the von Willebrand Factor, etc. The fate of platelet-bound bacteria is questioned. Several studies reported the ability of activated platelets to internalize bacteria such as Staphylococcus aureus or Porphyromonas gingivalis, though there is no clue on what happens thereafter. Are they sheltered from the immune system in the cytoplasm of platelets or are they lysed? Indeed, while the presence of phagolysosome has not been demonstrated in platelets, they contain antimicrobial peptides that were shown to be efficient on S. aureus. Besides, the fact that bacteria can bind to platelets via receptors involved in hemostasis suggests that they may induce aggregation; this has indeed been described for Streptococcus sanguinis, S. epidermidis, or C. pneumoniae. On the other hand, platelets are able to display an inflammatory response to an infectious triggering. We, and others, have shown that platelet release soluble immunomodulatory factors upon stimulation by bacterial components. Moreover, interactions between bacteria and platelets are not limited to only these two partners. Indeed, platelets are also essential for the formation of neutrophil extracellular traps by neutrophils, resulting in bacterial clearance by trapping bacteria and concentrating antibacterial factors but in enhancing thrombosis. In conclusion, the platelet-bacteria interplay is a complex game; its fine analysis is complicated by the fact that the inflammatory component adds to the aggregation response.

The Inflammatory Role of Platelets via Their TLRs and Siglec Receptors.

Platelets are non-nucleated cells that play central roles in the processes of hemostasis, innate immunity, and inflammation; however, several reports show that these distinct functions are more closely linked than initially thought. Platelets express numerous receptors and contain hundreds of secretory products. These receptors and secretory products are instrumental to the platelet functional responses. The capacity of platelets to secrete copious amounts of cytokines, chemokines, and related molecules appears intimately related to the role of the platelet in inflammation. Platelets exhibit non-self-infectious danger detection molecules on their surfaces, including those belonging to the "toll-like receptor" family, as well as pathogen sensors of other natures (Ig- or complement receptors, etc.). These receptors permit platelets to both bind infectious agents and deliver differential signals leading to the secretion of cytokines/chemokines, under the control of specific intracellular regulatory pathways. In contrast, dysfunctional receptors or dysregulation of the intracellular pathway may increase the susceptibility to pathological inflammation. Physiological vs. pathological inflammation is tightly controlled by the sensors of danger expressed in resting, as well as in activated, platelets. These sensors, referred to as pathogen recognition receptors, primarily sense danger signals termed pathogen associated molecular patterns. As platelets are found in inflamed tissues and are involved in auto-immune disorders, it is possible that they can also be stimulated by internal pathogens. In such cases, platelets can also sense danger signals using damage associated molecular patterns (DAMPs). Some of the most significant DAMP family members are the alarmins, to which the Siglec family of molecules belongs. This review examines the role of platelets in anti-infection immunity via their TLRs and Siglec receptors.

LPS stimulation of purified human platelets is partly dependent on plasma soluble CD14 to secrete their main secreted product, soluble-CD40-Ligand.

BACKGROUND: Platelets are instrumental to primary haemostasis; in addition, as they are central to endothelium vascular repair, they play a role in physiological inflammation. Platelets have also been demonstrated to be key players in innate immunity and inflammation, expressing Toll-like receptors (TLRs) to sense microbial infection and initiate inflammatory responses. They are equipped to decipher distinct signals, to use alternate pathways of signalling through a complete signalosome, despite their lack of a nucleus, and to adjust the innate immune response appropriately for pathogens exhibiting different types of 'danger' signals. Previous work has described the two main LPS isoforms-TLR4 activation pathways in purified platelets. However, the precise mechanism of TLR4 signalling in platelets is not completely unravelled, especially how this signalling may occur since platelets do not express CD14, the TLR4 pathophysiological companion for LPS sensing. Thus, we investigated from what source the CD14 molecules required for TLR4 signalling in platelets could come. RESULTS: Here we show that CD14, required for optimal response to LPS stimulation, is obtained from plasma, but used with restrictive regulation. These data add to the body of evidence that platelets are closer to regulatory cells than to first line defenders. The readout of our experiments is the canonical secreted cytokine-like protein, soluble (s)CD40L, a molecule that is central in physiology and pathology and that is abundantly secreted by platelets from the alpha-granules upon stimulation. CONCLUSIONS: We show that sCD14 from plasma contributes to LPS/TLR4 signalling in platelets to allow significant release of soluble CD40L, thereby elucidating the mechanism of LPS-induced platelet responses and providing new insights for reducing LPS toxicity in the circulation.

Immune-reactive soluble OX40 ligand, soluble CD40 ligand, and interleukin-27 are simultaneously oversecreted in platelet components associated with acute transfusion reactions.

BACKGROUND: Leukoreduction of labile blood components dramatically decreases the frequency of minor, intermediate, and severe adverse events (AEs), referred to as acute transfusion reactions (ATRs), especially after transfusion of platelet components (PCs). The pathophysiology of AEs may result from accumulation of soluble, secreted, platelet (PLT) factors with proinflammatory functions stored in PCs. Thus, several cosynergizing factors associated with PLT accumulation in PCs may contribute to clinically reported ATRs with inflammatory symptoms. STUDY DESIGN AND METHODS: We screened for 65 PLT-associated secretory products in PCs that caused ATRs and identified PLT molecules associated with ATRs and inflammation. A functional in vitro study using PC supernatants assayed on reporting immune cells was performed to indicate relevance. RESULTS: Among 10,600 apheresis PCs, 30 caused inflammatory ATRs and contained significantly elevated levels of soluble CD40 ligand (sCD40L), interleukin (IL)-27, and soluble OX40 ligand (sOX40L). Normal PLTs secreted IL-27 and sOX40L at bioactive concentrations upon thrombin stimulation and were up regulated in association with ATRs, similar to sCD40L. Other secreted products were identified but not investigated further as their positivity was not consistent. CONCLUSIONS: This study demonstrates the putative participation of PLT-derived sOX40L, IL-27, and sCD40L, which accumulate in PC supernatants, with inflammatory-type ATRs. Further studies are required to determine the clinical significance of these findings to forecast preventive measures whenever possible.

Human platelets can discriminate between various bacterial LPS isoforms via TLR4 signaling and differential cytokine secretion.

Platelets are currently acknowledged as cells of innate immunity and inflammation and play a complex role in sepsis. We examined whether different types of LPS have different effects on the release of soluble signaling/effective molecules from platelets. We used platelet-rich plasma from healthy volunteers and LPS from two strains of gram-negative bacteria with disparate LPS structures. We combined LPS-stimulated platelet supernatants with reporter cells and measured the PBMC cytokine secretion profiles. Upon stimulation of platelets with both Escherichia coli O111 and Salmonella minnesota LPS, the platelet LPS::TLR4 interaction activated pathways to trigger the production of a large number of molecules. The different platelet supernatants caused differential PBMC secretion of IL-6, TNFα, and IL-8. Our data demonstrate that platelets have the capacity to sense external signals differentially through a single type of pathogen recognition receptor and adjust the innate immune response appropriately for pathogens exhibiting different types of 'danger' signals.

Altered release of regulated upon activation, normal T-cell expressed and secreted protein from human, normal platelets: contribution of distinct HIV-1MN gp41 peptides.

Platelets can bind HIV and, in turn, be altered in with respect to and function during HIV progression. This study examines the secretion of normal platelets after exposure to recombinant HIV-1MN gp120 or gp41 peptides. There was a modest but significant decrease in regulated upon activation, normal T-cell expressed and secreted protein production in the presence of two out of 10 peptides, which was restored by monoclonal antibodies to gp41. Our data provide novel information on possible primary interactions between platelets and HIV env proteins.