Clinical, biological, and genetic features in an afibrinogenemia patient series in Algeria.

INTRODUCTION: The incidence of afibrinogenemia had not been previously reported in Algeria. Afibrinogenemia patients are prone to both haemorrhagic and thrombotic complications. Predictive markers of thrombosis in afibrinogenemia patients are not existent. AIMS AND METHODS: Clinical and biological data from 46 afibrinogenemia patients are reported. Biological investigations included routine tests, genetics analysis and thrombin generation. RESULTS: FGA mutations (four novel and four previously described) and FGB mutations (seven mutations; five novels) were homozygous in all but one family as a result of 28 consanguineous marriages out of 30 discrete families. Incidence of afibrinogenemia in Algeria is at least 3 per million births. Umbilical bleeding was reported in 39/46 cases and was the main discovery circumstance. We also report post trauma or post-surgery (3/46) bleeding and spontaneous deep vein thrombosis (DVT) in adulthood (1/46), as discovery circumstances. The median age (10.5-year-old) of the population reported here explains why there are few hemarthrosis and obstetrical or gynaecological complications in this series. Thrombotic events were reported in seven patients (four spontaneous). Endogenous Thrombin Potential was significantly increased in thrombosis-prone patients compared to afibrinogenemic patients with and without personal or familial history (1118 vs. 744 and 817 nM IIa × min, respectively). CONCLUSION: The incidence of afibrinogenemia in Algeria is the consequence of consanguineous marriage in families carrying private mutations. The thrombin generation test (TGT) could identify, among afibrinogenemic patients, those presenting a thrombotic risk.

Real-world use of idarucizumab for dabigatran reversal in three cases of serious bleeding.

Bleeding is a rare complication of direct oral anticoagulant potentially associated with high mortality rates. Biological monitoring is necessary for more than 24 h after idarucizumab antidote therapy in case of bleeding with dabigatran therapy.

Osteoprotegerin regulates cancer cell migration through SDF-1/CXCR4 axis and promotes tumour development by increasing neovascularization.

We previously reported that OPG is involved in ischemic tissue neovascularization through the secretion of SDF-1 by pretreated-OPG endothelial colony-forming cells (ECFCs). As the vascularization is one of the key factor influencing the tumour growth and cancer cell dissemination, we investigated whether OPG was able to modulate the invasion of human MNNG-HOS osteosarcoma and DU145 prostate cancer cell lines in vitro and in vivo. Cell motility was analysed in vitro by using Boyden chambers. Human GFP-labelled MMNG-HOS cells were inoculated in immunodeficient mice and the tumour nodules formed were then injected with OPG and/or FGF-2, AMD3100 or 0.9% NaCl (control group). Tumour growth was manually followed and angiogenesis was assessed by immunohistochemistry. In vitro, SDF-1 released by OPG-pretreated ECFCs markedly attracted both MNNG-HOS and DU145 cells and induced spontaneous migration of cancer cells. In vivo, tumour volumes were significantly increased in OPG-treated group compared to the control group and OPG potentiated the effect of FGF-2. Concomitantly, OPG alone or combined with FGF-2 increased the number of new vasculature compared to the control group. Interestingly AMD3100, an inhibitor of SDF-1, prevented the in vivo effects of OPG induced by SDF-1 This study provides experimental evidence that OPG promotes tumour development trough SDF-1/CXCR4 axis.

Low-Molecular-Weight Fucoidan Induces Endothelial Cell Migration via the PI3K/AKT Pathway and Modulates the Transcription of Genes Involved in Angiogenesis.

Low-molecular-weight fucoidan (LMWF) is a sulfated polysaccharide extracted from brown seaweed that presents antithrombotic and pro-angiogenic properties. However, its mechanism of action is not well-characterized. Here, we studied the effects of LMWF on cell signaling and whole genome expression in human umbilical vein endothelial cells and endothelial colony forming cells. We observed that LMWF and vascular endothelial growth factor had synergistic effects on cell signaling, and more interestingly that LMWF by itself, in the absence of other growth factors, was able to trigger the activation of the PI3K/AKT pathway, which plays a crucial role in angiogenesis and vasculogenesis. We also observed that the effects of LMWF on cell migration were PI3K/AKT-dependent and that LMWF modulated the expression of genes involved at different levels of the neovessel formation process, such as cell migration and cytoskeleton organization, cell mobilization and homing. This provides a better understanding of LMWF's mechanism of action and confirms that it could be an interesting therapeutic approach for vascular repair.

Fucoidan Stimulates Monocyte Migration via ERK/p38 Signaling Pathways and MMP9 Secretion.

Critical limb ischemia (CLI) induces the secretion of paracrine signals, leading to monocyte recruitment and thereby contributing to the initiation of angiogenesis and tissue healing. We have previously demonstrated that fucoidan, an antithrombotic polysaccharide, promotes the formation of new blood vessels in a mouse model of hindlimb ischemia. We examined the effect of fucoidan on the capacity of peripheral blood monocytes to adhere and migrate. Monocytes negatively isolated with magnetic beads from peripheral blood of healthy donors were treated with fucoidan. Fucoidan induced a 1.5-fold increase in monocyte adhesion to gelatin (p < 0.05) and a five-fold increase in chemotaxis in Boyden chambers (p < 0.05). Fucoidan also enhanced migration 2.5-fold in a transmigration assay (p < 0.05). MMP9 activity in monocyte supernatants was significantly enhanced by fucoidan (p < 0.05). Finally, Western blot analysis of fucoidan-treated monocytes showed upregulation of ERK/p38 phosphorylation. Inhibition of ERK/p38 phosphorylation abrogated fucoidan enhancement of migration (p < 0.01). Fucoidan displays striking biological effects, notably promoting monocyte adhesion and migration. These effects involve the ERK and p38 pathways, and increased MMP9 activity. Fucoidan could improve critical limb ischemia by promoting monocyte recruitment.

[Thrombopathy induced by selective serotonin reuptake inhibitors: a case report]. / Thrombopathie induite par les inhibiteurs sélectifs de la recapture de la sérotonine: à propos d'un cas.

We report the case of a 20 years old man presenting two episodes of hematemesis. The patient was known to suffer from depression and was treated with sertraline (Zoloft®) for 6 months at the dosage of 100 mg/day. No endoscopic and radiological abnormality was found. The haemostasis report was normal. Exploration of platelet function was in favor of an abnormality of platelet secretion as evidenced by low epinephrine-induced aggregation, a slightly reduced aggregation for following agonists: TRAP, ADP 5 µM and 10 µM, and a longer latency to collagen. The diagnosis of an acquired platelet disorder was strongly suspected and in particular an acquired platelet disorder induced by selective serotonin reuptake inhibitors that is known to interfere with platelet activation pathways involving serotonin.

Tie2-dependent deletion of α6 integrin subunit in mice reduces tumor growth and angiogenesis.

The α6 integrin subunit (α6) has been implicated in cancer cell migration and in the progression of several malignancies, but its role in tumor angiogenesis is unclear. In mice, anti-α6 blocking antibodies reduce tumor angiogenesis, whereas Tie1-dependent α6 gene deletion enhances neovessel formation in melanoma and lung carcinoma. To clarify the discrepancy in these results we used the cre-lox system to generate a mouse line, α6fl/fl­Tie2Cre(+), with α6 gene deletion specifically in Tie2-lineage cells: endothelial cells, pericytes, subsets of hematopoietic stem cells, and Tie2-expressing monocytes/macrophages (TEMs), known for their proangiogenic properties. Loss of α6 expression in α6fl/fl­Tie2Cre(+) mice reduced tumor growth in a murine B16F10 melanoma model. Immunohistological analysis of the tumors showed that Tie2-dependent α6 gene deletion was associated with reduced tumor vascularization and with reduced infiltration of proangiogenic Tie2-expressing macrophages. These findings demonstrate that α6 integrin subunit plays a major role in tumor angiogenesis and TEM infiltration. Targeting α6 could be used as a strategy to reduce tumor growth.

The in vitro and in vivo effects of a low-molecular-weight fucoidan on the osteogenic capacity of human adipose-derived stromal cells.

Human adipose-derived stromal cells (hASCs) may hold potential for bone tissue engineering. Osteogenic differentiation of these cells is crucial to bone formation. Low-molecular-weight fucoidan (LMWF) is a sulfated polysaccharide that potentiates several growth factors, including pro-angiogenic growth factors. To investigate whether hASC preconditioning with LMWF promoted bone repair, we compared the effects of LMWF and low-molecular-weight heparin on hASC phenotype and osteogenic differentiation. LMWF did not modify the stem-cell phenotype of hASCs but enhanced their osteogenic differentiation (formation of calcium deposits, increased activity and expression of alkaline phosphatase, and increased expression of osteopontin and runt-related transcription factor 2). However, when hASCs were exposed to LMWF before their adhesion to biphasic calcium phosphate particles and implantation in a bone-growth mouse model, no bone formation was apparent after 5 or 8 weeks, probably due to cell death. In conclusion, LMWF may hold promise for enhancing the osteogenic differentiation of hASCs before their implantation. However, concomitant vascularization would be required to enhance bone formation.

[Accreditation of automated complete blood count by the LH750 Analyzer (Beckman Coulter) in Georges Pompidou Hospital (Paris, France)]. / Vérification de méthode en vue de l'accréditation : exemple de l'hémogramme automatisé sur deux analyseurs LH750 Beckman-Coulter à l'Hôpital européen Georges Pompidou.

Preliminary evaluation of quantitative clinical laboratory measurements is a prerequisite for the accreditation of clinical laboratories, according to the French Committee of Accreditation guidelines following the European reference Standard EN ISO 15189. Numerous papers have been published regarding biochemistry and immunology. However, data are lacking for automated complete blood count accreditation. We report here our experience at Hôpital européen Georges Pompidou hematology laboratory and present the performance characteristics of two mirrored LH750 Beckman-Coulter analysers, including precision, accuracy and uncertainty of measurement.

Evaluation of recombinant activated factor VII, prothrombin complex concentrate, and fibrinogen concentrate to reverse apixaban in a rabbit model of bleeding and thrombosis.

BACKGROUND: As all anticoagulants, apixaban exposes to a bleeding risk, thus an effective way to reverse its effects is needed. Objectives were to study efficacy and safety of recombinant activated factor VII (rFVIIa), prothrombin complex concentrate (PCC), and fibrinogen concentrate (Fib) to reverse apixaban in a rabbit model of bleeding and thrombosis. METHODS: After a dose-ranging study to assess the minimal amount of apixaban increasing bleeding, 63 anaesthetized rabbits were randomized into 5 groups: control (saline), apixaban (apixaban and saline), rFVIIa (apixaban and rFVIIa), PCC (apixaban and PCC) and fibrinogen (apixaban and Fib). The Folts model was applied: a stenosis and an injury were carried out on the carotid artery, inducing thrombosis detected as cyclic flow reductions (CFRs) within 20 min. A number of parameters were recorded through ear immersion bleeding time (BT), clotting times (CT), thrombelastography, and thrombin generation time (TGT). Ultimately, a hepatosplenic section was performed to evaluate as primary endpoint the blood loss in 15 min. RESULTS: Apixaban increased blood loss (11.6 ± 3 g vs. 8.3 ± 3 g for control, p < 0.0003), lengthened BT, the prothrombin time (PT), thrombelastographic CT and decreased thrombin generation. Only rFVIIa reduced BT yet failed to improve blood loss. PCC and rFVIIa both shortened the PT, CT in thrombelastographic, and lag time in TGT. Fib improved clot firmness, enhanced thrombin generation but increased bleeding. Regarding safety, neither rFVIIa, PCC, nor Fib increased CFRs. CONCLUSION: rFVIIa, PCC, and Fib failed to reverse apixaban-induced bleeding. They only improved several laboratory parameters.

Mechanistic study of the proangiogenic effect of osteoprotegerin.

Osteoprotegerin (OPG), a soluble tumour necrosis factor receptor superfamily member, inhibits RANKL-mediated osteoclastogenesis. We have previously reported that OPG enhances the proangiogenic properties of endothelial colony-forming cells (ECFCs) in vitro, and promotes vasculogenesis in vivo. Here we investigated how OPG promotes neovascularisation. Proteomic experiments showed that OPG pretreatment affected ECFCs protein expression in two ways, 23 spots being down-regulated and 6 upregulated. These spots corresponded to proteins involved in cell motility, adhesion, signal transduction and apoptosis. In keeping with these proteomic results, we found that OPG induced ECFCs adhesion to activated endothelium in shear stress conditions, promoting intermediate but not focal adhesion to fibronectin and collagen. Treatment with OPG induced a reorganization of the ECFCs cytoskeleton, with the emergence of cell protrusions characteristic of a migratory phenotype. These effects correlated with decreased FAK phosphorylation and enhanced integrin αVß3 expression. OPG drastically reduced caspase-3/7 activities and maintained ECFCs viability after 48 h of treatment. All these effects were significantly attenuated by ECFCs incubation with the CXCR4 antagonist AMD-3100, and by prior heparan sulphate proteoglycan disruption. The proangiogenic properties of OPG appeared to be mediated by the proteoglycan syndecan-1, although OPG 1-194 lacking its heparin-binding domain still had pro-vasculogenic effects in vitro and in vivo. These results suggest that OPG may interact with ECFCs by binding to HSPGs/syndecan-1, thereby induce an anti-adhesive effect and promoting ECFCs migration through a SDF-1/CXCR4 dependent pathway.

A motif within the N-terminal domain of TSP-1 specifically promotes the proangiogenic activity of endothelial colony-forming cells.

Thrombospondin-1 (TSP-1) gives rise to fragments that have both pro- and anti-angiogenic effects in vitro and in vivo. The TSP-HepI peptide (2.3 kDa), located in the N-terminal domain of TSP-1, has proangiogenic effects on endothelial cells. We have previously shown that TSP-1 itself exhibits a dual effect on endothelial colony-forming cells (ECFC) by enhancing their adhesion through its TSP-HepI fragment while reducing their proliferation and differentiation into vascular tubes (tubulogenesis) in vitro. This effect is likely mediated through CD47 binding to the TSP-1 C-terminal domain. Here we investigated the effect of TSP-HepI peptide on the angiogenic properties of ECFC in vitro and in vivo. TSP-HepI peptide potentiated FGF-2-induced neovascularisation by enhancing ECFC chemotaxis and tubulogenesis in a Matrigel plug assay. ECFC exposure to 20 µg/mL of TSP-HepI peptide for 18 h enhanced cell migration (p < 0.001 versus VEGF exposure), upregulated alpha 6-integrin expression, and enhanced their cell adhesion to activated endothelium under physiological shear stress conditions at levels comparable to those of SDF-1α. The adhesion enhancement appeared to be mediated by the heparan sulfate proteoglycan (HSPG) syndecan-4, as ECFC adhesion was significantly reduced by a syndecan-4-neutralising antibody. ECFC migration and tubulogenesis were stimulated neither by a TSP-HepI peptide with a modified heparin-binding site (S/TSP-HepI) nor when the glycosaminoglycans (GAGs) moieties were removed from the ECFC surface by enzymatic treatment. Ex vivo TSP-HepI priming could potentially serve to enhance the effectiveness of therapeutic neovascularisation with ECFC.

Tie2-dependent knockout of α6 integrin subunit in mice reduces post-ischaemic angiogenesis.

AIMS: Integrins α6ß1 and α6ß4 are receptors for laminins, the main components of the basement membrane underlying the endothelial cells. In vitro, α6 integrin subunit (α6) expression at the surface of endothelial cells and their progenitors (EPCs) is up-regulated by pro-angiogenic growth factors and is crucial for adhesion, migration, and pseudotube formation. We investigated the role for α6 in post-ischaemic vascular repair in vivo. METHODS AND RESULTS: We used the cre-lox system to generate a mouse line with specific α6 gene deletion in Tie2-lineage cells. In a model of hind-limb ischaemia, Tie2-dependent α6 deletion reduced neovessel formation and reperfusion of the ischaemic limb. Concerning the role for α6 in post-ischaemic vasculogenesis, we showed previously that α6 was required for EPC recruitment at the site of ischaemia. Here, we found that α6 deletion also reduced EPC mobilization from the bone marrow after ischaemia. Examination of the ischaemic muscles showed that Tie2-dependent α6 deletion decreased the recruitment of pro-angiogenic Tie2-expressing macrophages. In the Matrigel plug assay, fibroblast growth factor-2-induced vascularization was diminished in mice lacking endothelial α6. To specifically investigate the role for α6 in angiogenesis, aortic rings were embedded in Matrigel or collagen and cultured ex vivo. In Matrigel, neovessel outgrowth from rings lacking α6 was strongly diminished, whereas no genotype-dependent difference occurred for rings in collagen. CONCLUSION: α6 plays a major role in both post-ischaemic angiogenesis and vasculogenesis.

Evaluation of prothrombin complex concentrate and recombinant activated factor VII to reverse rivaroxaban in a rabbit model.

BACKGROUND: As a potent anticoagulant agent, rivaroxaban exposes a risk of bleeding. An effective way to reverse its effects is needed. Objectives were to study efficacy and safety of recombinant activated factor VII (rFVIIa) and prothrombin complex concentrate (PCC) to reverse the anticoagulant effect of an overdose of rivaroxaban in a rabbit model of bleeding and thrombosis. METHODS: First, a dose-ranging study assessed the minimal rivaroxaban dose that increased bleeding. Then, 48 anesthetized and ventilated rabbits were randomized into four groups: control (saline), rivaroxaban (rivaroxaban and saline), rFVIIa (rivaroxaban and rFVIIa), and PCC (rivaroxaban and PCC). The Folts model was applied: a stenosis and an injury were carried out on the carotid artery, inducing thrombosis, detected as cyclic flow reductions, which were recorded over 20 min. Then the following were measured: ear immersion bleeding time, clotting times, anti-Xa activity, thrombelastometric parameters, and thrombin generation test. Ultimately, a hepatosplenic section was performed and the total amount of blood loss after 15 min was evaluated as primary endpoint. RESULTS: Rivaroxaban increased blood loss (17 g [8-32] vs. 7 g [5-18] for control (median [range]), P = 0.0004), ear bleeding time, clotting times, thrombelastographic clotting time, and decreased thrombin generation. In contrast, rFVIIa decreased ear bleeding time (92 s [65-115] vs. 140 s [75-190], P < 0.02), but without efficacy on blood loss. PCC and rFVIIa decreased activated partial thromboplastin time as well as thrombelastographic clotting time. Regarding safety, neither rFVIIa nor PCC increased cyclic flow reductions. CONCLUSION: rFVIIa and PCC partially improved laboratory parameters, but did not reverse rivaroxaban induced-bleeding.

Marine polysaccharides: a source of bioactive molecules for cell therapy and tissue engineering.

The therapeutic potential of natural bioactive compounds such as polysaccharides, especially glycosaminoglycans, is now well documented, and this activity combined with natural biodiversity will allow the development of a new generation of therapeutics. Advances in our understanding of the biosynthesis, structure and function of complex glycans from mammalian origin have shown the crucial role of this class of molecules to modulate disease processes and the importance of a deeper knowledge of structure-activity relationships. Marine environment offers a tremendous biodiversity and original polysaccharides have been discovered presenting a great chemical diversity that is largely species specific. The study of the biological properties of the polysaccharides from marine eukaryotes and marine prokaryotes revealed that the polysaccharides from the marine environment could provide a valid alternative to traditional polysaccharides such as glycosaminoglycans. Marine polysaccharides present a real potential for natural product drug discovery and for the delivery of new marine derived products for therapeutic applications.

[Antiphospholipid syndrome diagnosis: an update]. / Diagnostic du syndrome des antiphospholipides : actualités.

The antiphospholipid syndrome (APS) is characterized by arterial and/or venous thrombosis and pregnancy morbidity in association with the persistent presence of autoantibodies called antiphospholipid antibodies (APAs). APAs are a heterogeneous group of circulating autoantibodies that can be detected either by phospholipid-dependent coagulation test for lupus anticoagulant (LA) or ELISA test for anticardiolipin and anti-ß2GPI antibodies. In 2006, the revised criteria for the diagnosis of APS introduce the anti-ß2GPI antibodies as a new biological criterion and highlight the necessity to increase the interval between two positive APA test from 6 to 12 weeks. However, despite these updated criteria, the diagnosis of APS remains challenging and we proposed here to make an overview of the latest evolution in the diagnosis of this syndrome.