A thrombopoietin receptor agonist to rescue an unusual platelet transfusion-induced reaction in a p.V1316M-associated von Willebrand disease type 2B patient

International audienceThis report describes the first case of splenic injury in a patient with p.V1316M-associated von Willebrand disease type 2B (VWD2B) with chronic thrombocytopenia, successfully treated with nonoperative management including von Willebrand factor (VWF) replacement therapy, and platelet transfusions relayed by a thrombopoietin receptor agonist (TPO-RA, Eltrombopag). Eltrombopag was initially introduced to rescue an unusual post-platelet-transfusion reaction exacerbating the thrombocytopenia. In-depth analysis of the dramatic platelet count drop and VWF measurements timeline ruled out an allo-immune reaction and supported an alternative hypothesis of a sudden platelet clearance as a consequence of stress-induced release of abnormal VWF. One year later, a second life-threatening bleeding episode required urgent surgery successfully managed with VWF replacement therapy and platelet transfusions. Eltrombopag was further introduced in the post-surgery period to allow bleeding-free and platelet-transfusion-free successful recovery. Treatment decisions are particularly challenging in patients with VWD2B, and this case highlights how such decisions can benefit from understanding the molecular origin of platelet count fluctuations observed in these patients. Here, we successfully used a new therapeutic approach combining VWF-replacement therapy and initial platelet-transfusion relayed by TPO-RA to optimize patient management. Plain language summary A combination of von Willebrand factor replacement and thrombopoietin receptor agonist in thrombocytopenic patients with von Willebrand disease type 2B: a new therapy approach to optimize patient management? Therapeutic management of patients with von Willebrand disease type 2B are particularly challenging in case of severe thrombocytopenia. Treatment includes von Willebrands factor replacement therapy and iterative platelet transfusions. We describe the first case of splenic injury in a patient with p.V1316M-associated von Willebrand disease type 2B successfully treated with nonoperative management including von Willebrand factor replacement therapy and platelet transfusions relayed by a thrombopoietin receptor agonist. We showed that the unusual post-platelet-transfusion reaction associated with a dramatic platelet count drop was a consequence of stress-induced release of abnormal von Willebrand factor. The combination of von Willebrand factor replacement therapy and thrombopoietin receptor agonist may offer a new therapeutic approach to optimize patient management

Red blood cell Gardos channel (KCNN4): the essential determinant of erythrocyte dehydration in hereditary xerocytosis

International audienceNo abstrac

A factor VIII–nanobody fusion protein forming an ultrastable complex with VWF: effect on clearance and antibody formation

International audienceVon Willebrand factor (VWF) modulates factor VIII (FVIII) clearance and the anti-FVIII immune response. Despite the high affinity that defines the FVIII/VWF interaction, association/dissociation kinetics dictates 2% to 5% FVIII being present as free protein. To avoid free FVIII when studying the FVIII-VWF complex in vivo, we designed a FVIII-nanobody fusion protein, with the nanobody part being directed against VWF. This fusion protein, designated FVIII-KB013bv, had a 25-fold higher affinity compared with B-domainless FVIII (BDD-FVIII) for VWF. In vitro analysis revealed full cofactor activity in 1-stage clotting and chromogenic assays (activity/antigen ratio 1.0 ± 0.3 and 1.1 ± 0.3, respectively). In vivo, FVIII-013bv displayed a twofold increased mean residence time compared with BDD-FVIII (3.0 hours vs 1.6 hours). In a tail clip–bleeding assay performed 24 hours after FVIII infusion, blood loss was significantly reduced in mice receiving FVIII-KB013bv vs BDD-FVIII (15 ± 7 μL vs 194 ± 146 μL; P = .0043). Unexpectedly, when examining anti-FVIII antibody formation in FVIII-deficient mice, the immune-response toward FVIII-KB013bv was significantly reduced compared with BDD-FVIII (1/8 vs 14/16 mice produced anti-FVIII antibodies after treatment with FVIII-KB013bv and BDD-FVIII, respectively). Our data show that a stabilized interaction between FVIII and VWF is associated with a prolonged survival of FVIII and a reduced immune response against FVIII

Rapid Gardos Hereditary Xerocytosis Diagnosis in 8 Families Using Reticulocyte Indices

International audienceGardos channelopathy (Gardos-HX) or type 2 stomatocytosis/xerocytosis is a hereditary hemolytic anemia due to mutations in the KCNN4 gene. It is rarer than inherited type 1 xerocytosis due to PIEZO1 mutations (Piezo1-HX) and its diagnosis is difficult given the absence of a specific clinical or biological phenotype. We report here that this diagnosis can be sped up using red blood cell (RBC) indices performed on an ADVIA 2120 (Siemens(R)) analyzer, which measures reticulocyte mean corpuscular volume (rMCV) and mean corpuscular hemoglobin concentration (rMCHC). We studied reticulocyte indices in 3 new and 12 described patients (8 families) with Gardos-HX, 11 subjects presented the recurrent p.Arg352His mutation, 4 cases (two families) carried a private KCNN4 mutation. They were compared to 79 described patients (49 families) with Piezo1-HX. Surprisingly, in Gardos-HX cases, rMCV revealed to be smaller than MCV and rMCHC higher than MCHC, in contrast with normal or Piezo1-HX RBC. Consequently, Delta MCV (rMCV-MCV) was -0.9 +/- 5 fL vs. 19.8 +/- 3 fL (p < 0.001) in Gardos compared with Piezo1-HX and Delta MCHC (rMCHC-MCHC) was 18.7 +/- 13 vs. -50 +/- 8.7 g/L (p < 0.001). A threshold of 8.6 fL for Delta MCV and -5.5 g/L for Delta MCHC could discriminate between Gardos and Piezo1-HX with 100% sensitivity and specificity, regardless of age, mutation or splenectomy status. Consequently, we showed that reticulocytes indices are useful to suggest Gardos-HX on blood count results, allowing to rapidly target these patients for gene analysis. In addition, these parameters may prove useful as a `functional tool' in interpreting new KCNN4 variants

Is there a role for the laboratory monitoring in the management of specific antidotes of direct oral anticoagulants?

International audienceGiven the growing number of patients receiving direct oral anticoagulant (DOAC), patients requiring rapid neutralization is also increasing in case of major bleedings or urgent surgery/procedures. Idarucizumab is commercialized as a specific antidote to dabigatran while andexanet alfa has gained the Food and Drug Administration and the European Medicines Agency approval as an oral anti-factor Xa inhibitors antidote. Other antidotes or hemostatic agents are still under preclinical or clinical development, the most advanced being ciraparantag. DOAC plasma levels measurement allows to appropriately select patient for antidote administration and may prevent unnecessary prescription of expensive molecules in some acute clinical settings. However, these tests might be inconclusive after some antidote administration, namely andexanet alfa and ciraparantag. The benefit of laboratory monitoring following DOAC reversal remains unclear. Here, we sought to provide an overview of the key studies evaluating the safety and efficacy of DOAC reversal using the most developed/commercialized specific antidotes, to discuss the potential role of the laboratory monitoring in the management of patients receiving DOAC specific antidotes and to highlight the areas that deserve further investigations in order to establish the exact role of laboratory monitoring in the appropriate management of DOAC specific antidotes

A specific plasminogen activator inhibitor‐1 antagonist derived from inactivated urokinase

Abstract Fibrinolysis is a process responsible for the dissolution of formed thrombi to re‐establish blood flow after thrombus formation. Plasminogen activator inhibitor‐1 (PAI‐1) inhibits urokinase‐type and tissue‐type plasminogen activator (uPA and tPA) and is the major negative regulator of fibrinolysis. Inhibition of PAI‐1 activity prevents thrombosis and accelerates fibrinolysis. However, a specific antagonist of PAI‐1 is currently unavailable for therapeutic use. We screened a panel of uPA variants with mutations at and near the active site to maximize their binding to PAI‐1 and identified a potent PAI‐1 antagonist, PAItrap. PAItrap is the serine protease domain of urokinase containing active‐site mutation (S195A) and four additional mutations (G37bR–R217L–C122A–N145Q). PAItrap inhibits human recombinant PAI‐1 with high potency (K d = 0.15 nM) and high specificity. In vitro using human plasma, PAItrap showed significant thrombolytic activity by inhibiting endogenous PAI‐1. In addition, PAItrap inhibits both human and murine PAI‐1, allowing the evaluation in murine models. In vivo, using a laser‐induced thrombosis mouse model in which thrombus formation and fibrinolysis are monitored by intravital microscopy, PAItrap reduced fibrin generation and inhibited platelet accumulation following vascular injury. Therefore, this work demonstrates the feasibility to generate PAI‐1 inhibitors using inactivated urokinase

Soluble Siglec-5 associates to PSGL-1 and displays anti-inflammatory activity

International audienceInteractions between endothelial selectins and the leukocyte counter-receptor PSGL1 mediates leukocyte recruitment to inflammation sites. PSGL1 is highly sialylated, making it a potential ligand for Siglec-5, a leukocyte-receptor that recognizes sialic acid structures. Binding assays using soluble Siglec-5 variants (sSiglec-5/C4BP and sSiglec-5/Fc) revealed a dose- and calcium-dependent binding to PSGL1. Pre-treatment of PSGL1 with sialidase reduced Siglec-5 binding by 79 ± 4%. In confocal immune-fluorescence assays, we observed that 50% of Peripheral Blood Mononuclear Cells (PBMCs) simultaneously express PSGL1 and Siglec-5. Duolink-proximity ligation analysis demonstrated that PSGL1 and Siglec-5 are in close proximity (<40 nm) in 31 ± 4% of PBMCs. In vitro perfusion assays revealed that leukocyte-rolling over E- and P-selectin was inhibited by sSiglec-5/Fc or sSiglec-5/C4BP, while adhesion onto VCAM1 was unaffected. When applied to healthy mice (0.8 mg/kg), sSiglec-5/C4BP significantly reduced the number of rolling leukocytes under basal conditions (10.9 ± 3.7 versus 23.5 ± 9.3 leukocytes/field/min for sSiglec-5/C4BP-treated and control mice, respectively; p = 0.0093). Moreover, leukocyte recruitment was inhibited over a 5-h observation period in an in vivo model of TNFalpha-induced inflammation following injection sSiglec-5/C4BP (0.8 mg/kg). Our data identify PSGL1 as a ligand for Siglec-5, and soluble Siglec-5 variants appear efficient in blocking PSGL1-mediated leukocyte rolling and the inflammatory response in general