Pro-haemostatic effect of DDAVP is partially derived through non-classical (CD14dim /CD16++ ) monocytes residing the spleen.

The splenic endothelial Weibel-palade bodies are one of the most important candidate organelles to release von Willebrand factor upon stimulation with desmopressin. However, the presence of functional desmopressin-specific receptor has not yet been demonstrated on endothelial cells. Experimental evidences are in favour of an indirect pro-haemostatic effect of desmopressin, but the exact mediator and its cellular origin are largely elusive. Here, we report partially hampered desmopressin response in a splenectomised severe haemophilia A/Beta Thalassemia patient without any genetic variant relevant to his incomplete desmopressin response. To further investigate the role of the spleen in this phenomenon, the release of VWF from desmopressin-treated human splenic endothelial cells was assessed in vitro. As a result, desmopressin induced the release of VWF from endothelial cells when the cells were co-cultured with non-classical (CD14dim /CD16++ ), but not other subtypes of monocytes or PBMCs. This in vitro study which resembles close proximity of endothelial cells of sinusoids to monocyte reservoir reside in parenchyma of subcapsular red pulp of the spleen sheds a light upon the role of this highly vascularized VWF-producing organ in driving indirect effect of desmopressin.

Coagulation Factors Accumulate During Normothermic Liver Machine Perfusion Regardless of Donor Type and Severity of Ischemic Injury.

BACKGROUND: Coagulation factors may inform on liver function during normothermic machine perfusion (NMP). We investigated whether graft ischemic injury impairs the accumulation of anticoagulation factors during NMP of porcine and human livers. METHODS: Dynamics of FV, FVII, FVIII, FIX, and FX during NMP and their correlation with graft injury was investigated in porcine livers with minimal (no warm ischemia, n = 5) or severe injury (60 min warm ischemia, n = 5). Next, FV, FVIII, FIX, fibrinogen, and antithrombin were measured in 35 matched human liver NMPs from the COPE trial. Correlation of these factors with outcomes was explored. Livers were categorized in to 4 groups depending on donor type and posttransplant peak aspartate aminotransferase (AST) as surrogate of minimal (peak < 500 IU/L) or moderate injury (peak > 1000 IU/L). RESULTS: Factor concentrations increased significantly during NMP regardless of severity of injury. In porcine livers, factor concentrations were 2- to 6-fold lower in severely injured grafts (all P < 0.05). All factors negatively correlated with AST (coefficient range: from -0.50 to -0.93; all P < 0.05) and lactate (range: from -0.51 to -0.67; all P < 0.05). In human livers, no difference in factor accumulation rates and no correlation with other markers were observed. One graft with primary nonfunction had low rate of factor accumulation. CONCLUSIONS: Anticoagulation factors accumulate during NMP regardless of donor type and severity of injury. In pigs, severe ischemic injury resulted in significantly lower factor concentrations. In human livers with life-sustaining function, they do not correlate with hepatic injury. Whether low concentrations predict nonfunction in high-risk livers with severe injury requires further investigation.

Antielastin B-cell and T-cell immunity in patients with chronic obstructive pulmonary disease.

RATIONALE: Antielastin autoimmunity has been hypothesised to drive disease progression in chronic obstructive pulmonary disease (COPD). The proposed mechanism is currently disputed by conflicting data. The authors aimed to explore antibody responses against elastin in a large and extensively characterised COPD population and to assess elastin-specific peripheral T-cell reactivity in a representative subgroup. METHODS: Antielastin antibodies were analysed with indirect ELISA on the plasma of 320 patients with COPD (Global Initiative for Chronic Obstructive Lung Disease 1-4) and 143 smoking controls. In a second group of 40 patients with COPD and smoking controls, T-cell responses against extracellular matrix (elastin, collagen I and collagen V) were determined with enzyme-linked immunosorbent spot (EliSpot) (interferon γ (IFNγ) and interleukin-2) on peripheral blood mononuclear cells and compared with the responses of 11 never-smoking controls. RESULTS: Antielastin antibody titres were not elevated in patients with COPD compared with smoking controls and even decreased significantly with increasing severity of COPD (p<0.001). Lower antielastin antibody titres were also found in a subgroup of patients with CT-proven emphysema. Elastin-specific INFγ-mediated T helper 1 responses could not be revealed in smoking subjects with and without COPD. Collagen I-mediated T-cell responses were also absent, which contrasted with a significant increased anticollagen V response in the smoking controls and patients with COPD compared with the never smokers (p=0.008). Collagen V-mediated T-cell responses could not discriminate between patients with COPD and smoking controls. CONCLUSION: A systemic immune response against elastin could not be identified in patients with COPD. By contrast, collagen V-mediated autoimmunity was increased in the subgroup of smokers and may potentially contribute to the pathogenesis of COPD.

Activation of human endothelial cells from specific vascular beds induces the release of a FVIII storage pool.

Although the liver is known to be the main site of factor VIII (FVIII) production, other organs are probably also important for the regulation of FVIII secretion. However, the study of the regulation of extrahepatic FVIII production has been hampered by the lack of definitive identification of human tissues able to secrete FVIII. Recent studies have shown that lung endothelial cells can synthesize FVIII. We therefore studied the production of FVIII by endothelial cells purified from other vascular beds. Because physiologic stress results in a rapid elevation of FVIII, we also investigated whether endothelial cells can store FVIII and secrete it after treatment with agonists. Microvascular endothelial cells from lung, heart, intestine, and skin as well as endothelial cells from pulmonary artery constitutively secreted FVIII and released it after treatment with phorbol-myristate acetate and epinephrine. By contrast, endothelial cells from the aorta, umbilical artery and umbilical vein did not constitutively secrete FVIII or release it after treatment with agonists, probably because of a lack of FVIII synthesis. Extrahepatic endothelial cells from certain vascular beds therefore appear to be an important FVIII production and storage site with the potential to regulate FVIII secretion in chronic and acute conditions.

FVIII production by human lung microvascular endothelial cells.

While extrahepatic factor VIII (FVIII) synthesis suffices for hemostasis, the extrahepatic production sites are not well defined. We therefore investigated the ability of the human lungs to produce FVIII. Lungs from heart-beating donors who were declined for transplantation were perfused and ventilated in an isolated reperfusion model for 2 hours. A progressive accumulation of FVIII and von Willebrand factor (VWF) was recorded in the perfusion medium in 3 of 4 experiments. By contrast, factor V, fibrinogen, and immunoglobulin G (IgG) levels remained constant during the perfusion period, indicating that the accumulation of FVIII and VWF was not due to diffusion from the intercellular medium into the vascular system. Purified human lung microvascular endothelial cells produced FVIII during at least 2 passages in vitro. Altogether, these data identify the lung endothelial cells as a FVIII production site in humans.

Deletion of alanine 2201 in the FVIII C2 domain results in mild hemophilia A by impairing FVIII binding to VWF and phospholipids and destroys a major FVIII antigenic determinant involved in inhibitor development.

The C2 domain of factor VIII (FVIII) mediates FVIII binding to von Willebrand factor (VWF) and phospholipids (PLs), thereby determining the stability and the activity of FVIII. A deletion of Ala2201 (Del2201) was identified in the FVIII C2 domain of 2 unrelated patients with mild hemophilia A (FVIII:C 11%-33%). This mutation prevents FVIII binding to a human monoclonal antibody recognizing the C2 domain and inhibiting FVIII binding to VWF and phospholipids. By comparison to healthy FVIII, Del2201 FVIII had a significantly reduced binding to VWF, which likely contributes to reduced FVIII levels in plasma. Del2201 FVIII interaction with phospholipids was evaluated in an FXa generation assay, using various concentrations of synthetic phospholipid vesicles mimicking an activated platelet surface. At the lowest phospholipid concentration allowing FXa generation, Del2201 FVIII activity was reduced 3-fold. This is the first report of a mutation altering FVIII binding to phospholipids and occurring in patients with hemophilia A.

CD4+ T-cell clones specific for wild-type factor VIII: a molecular mechanism responsible for a higher incidence of inhibitor formation in mild/moderate hemophilia A.

Mild/moderate hemophilia A patients carrying certain mutations in the C1 domain of factor VIII (FVIII) have a higher risk of inhibitor occurrence. To analyze the mechanisms responsible for inhibitor development in such patients, we characterized FVIII-specific CD4(+) T-cell clones derived from a mild hemophilia A patient carrying an Arg2150His substitution in the C1 domain and who presented with a high titer inhibitor toward normal but not self-FVIII. All T-cell clones recognized synthetic peptides encompassing Arg2150. The peptides were presented to the T-cell clones by DRB1*0401/DRB4*01 or DRB1*1501/DRB5*01. Interestingly, the latter haplotype was previously reported as being associated with an increased incidence of inhibitor formation. Peptide I2144-T2161 also bound to other DR molecules such as DRB1*0101 and DRB1*0701, indicating that the peptide binds to major histocompatibility complex (MHC) class II molecules expressed in more than 60% of the population. None of the T-cell clones recognized recombinant FVIII carrying the substitution Arg2150His, even when FVIII was presented by an FVIII-specific B-cell line. The mutation likely alters T-cell recognition of the mutated peptide associated to MHC molecules, because the mutated peptide bound to immunopurified DR molecules nearly as effectively as the native peptide. These observations demonstrate that T cells of this patient with mutation Arg2150His distinguish between self- and wild-type FVIII and provide a plausible mechanism for the frequent occurrence of an inhibitor in patients carrying this substitution. A similar phenomenon may occur with other mutations associated to an increased incidence of inhibitor formation.