Use of a thrombopoietin receptor agonist in von Willebrand disease type 2B (p.V1316M) with severe thrombocytopenia and intracranial hemorrhage.

We present here a 63-year old woman with a long history of immune thrombocytopenia. She was hospitalized for a traumatic intracranial hemorrhage with thrombocytopenia. Following inefficient treatment of four platelet transfusions, immunoglobulins, and corticosteroids, we initiated treatment with a thrombopoietin (TPO) receptor agonist (eltrombopag 25 mg/d) with a good efficacy. Her mother and sister also had chronic thrombocytopenia. Clinical history, hemostasis results, and gene analysis revealed von Willebrand disease (VWD) type 2B with the mutation (c.3946G>A; p.V1316M), which combines a von Willebrand factor defect with severe thrombocytopenia, as well as a thrombocytopathy. The efficacy of TPO receptor agonists appears to counterbalance, at least to some extent, the thrombocytopathy associated with this mutation. As such, the use of TPO receptor agonists could represent an alternative therapeutic approach in cases of VWD type 2B with severe thrombocytopenia.

Platelet function analyser (PFA-100) results and von Willebrand factor deficiency: a 16-year 'real-world' experience.

The platelet function analyser (PFA-100) is a biological tool designed to explore primary haemostasis. This system has thus been widely demonstrated as reliable in detecting von Willebrand factor (VWF) deficiency. However, most studies were based on patients benefitting from regular medical care and accurate diagnosis, and it would seem probable that the results were somewhat optimistic, and do not reflect its performances in 'real-world' situations. We have chosen to study the reliability of PFA-100 for screening VWF ristocetin cofactor (VWF:RCo) deficiency. We retrospectively analysed the results (n = 6431) of 4027 patients referred to our centre between October 1997 and June 2013 and in whom PFA-Epi, PFA-ADP, and VWF:RCo activity had been evaluated. We studied the influence of blood group on the results and the performances of each method in a subgroup of 213 patients with genetically confirmed von Willebrand disease. We have shown that the PFA-100 system, in our experience, constitutes an excellent screening test for detecting VWF:RCo deficiency, whatever the clinical situation, in 'real-world' conditions. The negative predictive value (NPV), the positive predictive value, the sensitivity and the specificity were respectively: 0.98, 0.51, 0.98 and 0.40. When values adjusted for blood group are used, NPV and sensitivity are inferior to those using normal values which have not been adjusted for blood group. We have shown the PFA-100 method to be more efficient in screening for VWF deficiency than the VWF:RCo technique.

Diagnosis and management challenges in patients with mild haemophilia A and discrepant FVIII measurements.

Thirty per cent of patients with mild haemophilia A (MHA) present markedly different FVIII: C level when assayed by one-stage clotting and two-stage chromogenic assays. It is, therefore, a real clinical challenge to predict the individual bleeding risk of these patients. The aim of the present work was to study the relationship between the bleeding tendency of these patients with the results of a panel of phenotypic and genotypic tools. Thirty-six patients with MHA were included in this multicentre prospective clinical study. The severity of bleeding symptoms was evaluated using the ISTH/SSC score. FVIII:C levels were measured using an activated partial thromboplastin time-based one-stage FVIII assay (FVIII: C1) and three commercial chromogenic kits (FVIII:CR). FVIII antigen levels, thrombin generation measurement and FVIII gene mutation analysis were also performed. Our results showed that a one-stage FVIII: C assay cannot rule out the diagnosis of MHA, a combined use of FVIII:C1 with a FVIII:CR is suitable for detecting MHA. We observed that FVIII:CR results better reflected the clinical bleeding tendency of patients compared to FVIII:C1. We also observed a relationship between thrombin generation (TG) capacity and FVIII:CR of these patients. FVIII gene mutation analysis showed mutations previously reported in MHA patients with discrepant FVIII:C measurements, but with no predictive value of the individual bleeding phenotype of patients. Overall, we observed a relationship between chromogenic FVIII:C results, TG assay and bleeding tendency of patients with discrepant FVIII:C measurements, while FVIII:C1 was not well correlated with clinical bleeding phenotype in this particular population.

Management of bleeding in severe factor V deficiency with a factor V inhibitor.

Factor V (FV) inhibitor arises rarely after using fresh frozen plasma (FFP) to treat inherited FV deficiency and is often a real therapeutic challenge. Here, we report a patient with a severe FV deficiency who developed such an inhibitor and was then treated with recombinant activated FVII (rFVIIa) and platelet concentrates (PC). Monitoring was assessed by thrombin generation assay (TGA). PC were more effective than rFVIIa in treating bleeding, but there was no correlation between the TGA results and clinical efficacy.

Validation of the first commercial ELISA for type 2N von Willebrand's disease diagnosis.

Type 2N von Willebrand's disease (VWD) is characterized by a factor VIII (FVIII) deficiency and a low FVIII/VWF ratio related to a markedly decreased affinity of von Willebrand factor (VWF) to FVIII. Type 2N VWD is diagnosed using assays allowing the measurement of plasma VWF capacity to bind FVIII (VWF:FVIIIB). These assays, crucial in order to distinguish type 2N VWD patients from mild haemophiliacs A and haemophilia A carriers, remain exclusively homemade and limited to laboratories possessing a high level of expertise in VWD. We evaluated the first commercial ELISA (Asserachrom® VWF:FVIIIB; Stago) comparated to a reference method in a multicentric study involving 205 subjects: 60 healthy volunteers, 37 haemophiliacs A, 17 haemophilia A carriers, 37 patients with type 2N VWD, 9 heterozygous carriers for a 2N mutation and 45 patients with miscellaneous other types of VWD (all previously characterized). A diluted plasma sample adjusted to 10 IU dL(-1) of VWF:Ag was incubated with a rabbit antihuman VWF polyclonal antibody. After removing the endogenous FVIII, recombinant FVIII (rFVIII) was added and bound rFVIII was quantified using a peroxydase-conjugated mouse antihuman FVIII monoclonal antibody. The intra-assay and inter-assay reproducibility was satisfactory. In all subgroups, both methods were well correlated. All type 2N VWD patients exhibited a markedly decreased VWF:FVIIIB (lower than 15%) and all heterozygous 2N carriers had a moderately decreased VWF:FVIIIB (between 30% and 65%). All controls (healthy subjects, haemophiliacs A and haemophilia A carriers) had a normal VWF:FVIIIB (higher than 80%) except one healthy volunteer and three haemophiliacs who exhibited a moderately decreased VWF:FVIIIB suggesting a heterozygous status for a 2N mutation. In conclusion, the Asserachrom® VWF:FVIIIB is easy to perform, standardized and accurate for type 2N VWD diagnosis with a 100% sensitivity and specificity.

The use of the new ReFacto AF Laboratory Standard allows reliable measurement of FVIII:C levels in ReFacto AF mock plasma samples by a one-stage clotting assay.

Factor VIII coagulant (FVIII:C) levels measured in patients receiving ReFacto® (B-domain-deleted recombinant FVIII) using chromogenic substrate assay (CSA) and one-stage clotting assay (OSA) have frequently shown discrepancies, and the use of the ReFacto Laboratory Standard (RLS) has therefore been recommended to minimize these differences. The potency of ReFacto AF®, the albumin-free successor of ReFacto®, is determined using CSA for the titration of vials, and a new standard (RLS-AF) was developed to measure its biological efficacy using OSA. This multicentre study therefore evaluated the efficacy of this new RLS in minimizing differences between OSA and CSA when measuring FVIII:C levels in plasma. Mock plasma samples were prepared by diluting ReFacto AF® in FVIII-deficient plasma to obtain four concentrations ranging from 15 to 90 IU dL⁻¹ . FVIII:C levels were then measured in six laboratories on four separate days using three different procedures, i.e. OSA with a plasma standard (PS) as reference, OSA with RLS-AF and CSA with PS. The inter-centre standard deviation ranged from 1.4 to 5.5 IU dL⁻¹. However, FVIII:C levels measured with OSA were closer to the expected values when RLS-AF was used. In addition, the uncertainty of measurement, reflecting the inter-method discrepancy was greatly reduced when RLS-AF was employed in OSA (15%) in place of PS (33%). This study demonstrates that the OSA performed with RLS-AF to establish calibration curves provides a valuable alternative to CSA to measure FVIII:C in ReFacto-AF-treated patients.

[Appropriate laboratory investigation in women with menorrhagia]. / Bonne pratique et valeur diagnostique de la biologie : hémostase-hématologie.

Inherited bleeding disorders are potentially causes of menorrhagia and must be investigated if no specific cause is identified. The reported prevalence of inherited bleeding disorders is high in women with menorrhagia compared to the general population. The most frequent disorders reported are von Willebrand's disease and Factor XI deficiency. Menorrhagia is, also, a frequent finding in women with congenital bleeding disorders. Morever, menorrhagia represents the major cause of iron-deficiency anemia among women of reproductive age. Primary evaluation for an underlying disorder of hemostasis in a woman wih menorrhagia is a focused history for familiy and personal history of bleeding symptoms and a complete blood cell count. This will rule out thrombocytopenic bleeding and also assesses for the degree, if any, of anemia. Those women with a positive screen and normal platelet count should be evaluated with laboratory investigation including prothrombin time, activated partial thromboplastin time, factor VIII, VWF ristocetin cofactor and antigen. If initial hemostasis testing above is normal, then further hemostasis testing can be considered, especially in terms of platelet aggregation, in a multidisciplinary clinic.

Prospective evaluation of the '4Ts' score and particle gel immunoassay specific to heparin/PF4 for the diagnosis of heparin-induced thrombocytopenia.

BACKGROUND: Heparin-induced thrombocytopenia (HIT) is a severe disease that is often difficult to diagnose. A clinical scoring system, the '4Ts' score, has been proposed to estimate its probability before laboratory testing, and a particle gel immunoassay (H/PF4 PaGIA) has also been developed for rapid detection of HIT antibodies. AIM: To evaluate the performance of both methods when HIT is suspected clinically. METHODS: Two hundred thirteen consecutive patients were included in four centers. The probability of HIT was evaluated using the 4Ts score blind to antibody test results. HIT was confirmed only when the serotonin release assay (SRA) was positive. RESULTS: The risk of HIT was evaluated by the 4Ts score as low (LowR), intermediate (IR) or high (HR) in 34.7%, 60.6% and 4.7% of patients, respectively. The negative predictive value (NPV) of the 4Ts score was 100%, as the SRA was negative in all LowR patients. PaGIA was negative in 176 patients without HIT (99.4%, NPV) and the negative likelihood ratio (LR-) was 0.05. PaGIA was positive in 37 patients, including 21 with HIT (positive predictive value = 56.8%), with a positive LR of 11.4. A negative PaGIA result decreased the probability of HIT in IR patients from 10.9% before assay to 0.6%, whereas a positive result did not substantially increase the likelihood for HIT. CONCLUSION: The use of the 4Ts score with PaGIA appears to be a reliable strategy to rule out HIT.

Fibrinogen angers with a new deletion (gamma GVYYQ 346-350) causes hypofibrinogenemia with hepatic storage.

INTRODUCTION: This study reports a family with chronically abnormal blood liver function tests (LFT) and congenital hypofibrinogenemia. The proposita had cirrhosis initially related to alcohol abuse and chronic viral hepatitis C (HCV), but abnormal LFT persisted even when alcohol intake was stopped and despite HCV treatment was efficient based on serum RNA negative testing. RESULTS: Needle biopsy specimens of the proposita and her brother showed eosinophilic intra-cytoplasmic inclusions that reacted strongly with fibrinogen antisera on direct immunofluorescence. Electron microscopic examination showed that the rough endoplasmic reticulum was filled with inclusions that consisted of densely packed, curved tubular structures arranged in a fingerprint-like pattern. Coagulation studies revealed low functional and antigenic fibrinogen concentrations suggestive of hypofibrinogenemia. Amplification and DNA sequencing showed a heterozygous deletion of the a7690 to g7704 nucleotides of the gamma chain gene in the 3'end of exon 8 (g 7690_7704del14; Genbank access M10014); this deletion encompassed the splicing site at position 7703 and predicts in a new putative consensus splicing sequence (AATGgtatgtt). RNA was extracted from a liver specimen from the proposita's brother. The cDNA obtained by reverse transcription polymerase chain reaction confirmed the usage of a newly generated donor site at position 7688 of the genomic sequence resulting in an in-frame heterozygous 5 amino acid deletion (GVYYQ 346-350; p.G372_Q376del) and that this mutation is responsible for a new splicing site at position 7688 of the genomic sequence. CONCLUSION: we suggest that the molecular defect in fibrinogen Angers results in an impaired assembly and causes defective secretion and hepatic storage of fibrinogen.

Agreement between factor XIII activity and antigen assays in measurement of factor XIII: A French multicenter study of 147 human plasma samples.

INTRODUCTION: Factor XIII (FXIII) deficiency is a rare hemorrhagic disorder whose early diagnosis is crucial for appropriate treatment and prophylactic supplementation in cases of severe deficiency. International guidelines recommend a quantitative FXIII activity assay as first-line screening test. FXIII antigen measurement may be performed to establish the subtype of FXIII deficiency (FXIIID) when activity is decreased. METHODS: The aim of this multicenter study was to evaluate the analytical and diagnostic levels of performance of a new latex immunoassay, K-Assay® FXIII reagent from Stago, for first-line measurement of FXIII antigen. Results were compared to those obtained with the Berichrom® FXIII chromogenic assay for measurement of FXIII activity. Of the 147 patient plasma samples, 138 were selected for analysis. RESULTS: The accuracy was very good, with intercenter reproducibility close to 7%. Five groups were defined on FXIII activity level (<5% (n = 5), 5%-30% (n = 23), 30%-60% (n = 17), 60%-120% (n = 69), above 120% (n = 24)), without statistical differences between activity and antigen levels (P value >0.05). Correlation of the K-Assay® with the Berichrom® FXIII activity results was excellent (r = 0.919). Good agreement was established by the Bland and Altman method, with a bias of +9.4% on all samples, and of -1.4% for FXIII levels lower than 30%. One patient with afibrinogenemia showed low levels of Berichrom® FXIII activity but normal antigen level and clot solubility as expected. CONCLUSIONS: The measurement of FXIII antigen using the K-Assay® is a reliable first-line tool for detection of FXIII deficiency when an activity assay is not available.

Does the presence of von Willebrand factor in FVIII-deficient plasma influences the measurement of FVIII inhibitor titres in haemophilia A patients?

INTRODUCTION: Reliable measurement of FVIII inhibitor is critical in the follow-up of haemophilia A patients. We performed a multicentre study to evaluate whether the presence of von Willebrand factor (VWF) in FVIII-deficient plasma (FVIII-DP) influences FVIII inhibitor titres. METHODS: Six French haematology laboratories participated in this study. Three samples with varying FVIII inhibitor titres (1, 5 and 15 BU/mL) and one sample without any detectable FVIII inhibitor were tested using four different procedures for FVIII inhibitor assay. The Nijmegen method and a modified assay with imidazole were performed using FVIII-DP with and without VWF in the control mixture and as substrate plasma in the FVIII one stage assay (OSA). Each mixture (reference and test) was incubated for two hours at 37 °C with buffered normal pool plasma. RESULTS: Higher inhibitor titres were measured in 5 and 15 BU/mL samples when assays were performed with the Nijmegen method and FVIII-DP without VWF. When samples were diluted in imidazole buffer, similar inhibitor titres, close to expected values, were measured whether VWF was present in the FVIII-DP or not. The data obtained were also more accurate when residual FVIII activity levels between 40% and 60% were used to calculate inhibitor titres, despite a linear type I reaction kinetics. CONCLUSION: These results support the hypothesis that reliable FVIII inhibitor titres can be measured without the use of FVIII-DP containing VWF when an imidazole-modified assay is used.

Pentoxifylline inhibits the expression of tissue factor mRNA in endotoxin-activated human monocytes.

Tissue factor (TF) is a transmembrane glycoprotein which, in association with factor VII(a), is the main activator of coagulation. In illnesses such as Gram-negative endotoxemia, circulating monocytes synthesize and express substantial TF activity, resulting in extensive disseminated intravascular coagulation. We investigated the way in which TF is suppressed by pentoxifylline (PTX), and found that PTX down-regulates immunologic TF expression and specific mRNA production in response to LPS. Since TF mRNA stability is not altered, this effect appears to take place at the transcriptional level.

Interleukin-10 and pentoxifylline inhibit C-reactive protein-induced tissue factor gene expression in peripheral human blood monocytes.

Fibrin deposition is an integral feature of the inflammatory response. In response to C-reactive protein (CRP), an acute-phase reactant, blood monocytes synthesize and express tissue factor (TF), the main initiator of blood coagulation. We report the inhibitory effect of interleukin 10 (IL-10) and that of pentoxifylline, a methyl xanthine derivative, on monocyte expression of TF activity, TF protein and TF mRNA in response to CRP. These agents may be of use in diseases where a TF-induced prothrombotic state is detrimental.

Interleukin-10 inhibits endotoxin-induced tissue factor mRNA production by human monocytes.

In Gram-negative septic shock, human monocytes synthesize and express on their cytoplasmic membrane tissue factor (TF), a potent activator of the coagulation cascades. The role of TF in triggering disseminated intravascular coagulation (DIC) in these patients appears to be clear. We report the suppressive effect of interleukin-10 (IL-10) on endotoxin-induced TF activity and antigen levels, and on the expression of TF mRNA levels in human monocytes. These results emphasize the potential therapeutic value of this cytokine in septic shock, a condition still associated with a high mortality rate.

Increased monocyte procoagulant activity independent of the lupus anticoagulant in patients with systemic lupus erythematosus.

Monocytes can play a role in the activation of coagulation via increased procoagulant activity (PCA). We investigated the level of monocyte PCA in 19 patients with systemic lupus erythematosus (SLE), given the high rate of thrombotic events in this condition. Nine of these subjects also presented the lupus anticoagulant (LA). The PCA generated by patient monocytes was significantly higher than control values and was identified as tissue factor-like. Moreover, the number of monocytes with membrane-associated D dimer, a parameter which we have shown to be correlated with the PCA expressed in vitro by endotoxin-activated monocytes, was also significantly increased. Serum from both groups of patients (i.e. SLE and SLE + LA) stimulated the generation of PCA by control monocytes. By contrast, purified IgG from both patient groups had the same effect as control IgG on PCA generation by control monocytes. The nature of the stimulating agent in the serum was not identified. In conclusion, increased monocyte PCA may account for the increased incidence of thrombosis in SLE patients, although other, superimposed, factors would appear to exist in SLE + LA patients, given the higher incidence of thrombosis in this subgroup.

Protein tyrosine kinase activation is required for LPS and PMA induction of tissue factor mRNA in human blood monocytes.

Tissue factor (TF) is a transmembrane glycoprotein which assembles with factor VIIa on cell surfaces to form a proteolytically active cofactor-enzyme complex; the TF/VIIa complex initiates the coagulation protease cascade. In response to bacterial lipopolysaccharide (LPS) and phorbol-12 myristate 13-acetate (PMA), monocytes synthesize and express TF on their surface. However, the mechanisms by which LPS and PMA activate TF synthesis by human blood monocytes are not fully understood. As it has been established that LPS and PMA activate protein tyrosine kinase (PTK) in monocytes, we studied the role of PTK in LPS and PMA induction of TF by human blood monocytes. Both LPS- and PMA-induced TF activity was inhibited in a concentration-dependent manner by the protein tyrosine kinase-specific inhibitors herbimycin A and genistein. TF antigen determination confirmed that LPS- and PMA-induced cell surface TF protein levels decreased in parallel to TF functional activity under herbimycin A and genistein treatment. Northern blot analysis of total RNA from LPS- and PMA-stimulated monocytes showed a concentration-dependent decrease in TF mRNA levels in response to herbimycin A and genistein. The rate of decay of LPS-induced TF mRNA, evaluated after the arrest of transcription by actinomycin D was not affected by genistein and herbimycin A, suggesting that the inhibitory effects occur at least partly at the transcriptional level. We conclude that LPS- and PMA-induced TF production by human monocytes is dependent on tyrosine kinase activation.

Endotoxin-induced tissue factor in human monocytes is dependent upon protein kinase C activation.

Tissue factor (TF) is a transmembrane receptor which, in association with factors VII and VIIa, activates factor IX and X, thereby activating the coagulation protease cascades. In response to bacterial lipopolysaccharide (LPS) monocytes transcribe, synthesize and express TF on their surface. We investigated whether LPS-induced TF in human monocytes is mediated by protein kinase C (PKC) activation. The PKC agonists phorbol 12-myristate 13-acetate (PMA) and phorbol 12, 13 dibutyrate (PdBu) were both potent inducers of TF in human monocytes, whereas 4 alpha-12, 13 didecanoate (4 alpha-Pdd) had no such effect. Both LPS- and PMA-induced TF activity were inhibited, in a concentration dependent manner, by three different PKC inhibitors: H7, staurosporine and calphostin C. TF antigen determination confirmed that LPS-induced cell-surface TF protein levels decreased in parallel to TF functional activity under staurosporine treatment. Moreover, Northern blot analysis of total RNA from LPS- or PMA-stimulated monocytes showed a concentration-dependent decrease in TF mRNA levels in response to H7 and staurosporine. The decay rate of LPS-induced TF mRNA evaluated after the arrest of transcription by actinomycin D was not affected by the addition of staurosporine, suggesting that its inhibitory effect occurred at a transcriptional level. We conclude that LPS-induced production of TF and its mRNA by human monocytes are dependent on PKC activation.

Effect of phosphopyridoxylation on thrombin interaction with platelet glycoprotein Ib.

The purpose of this study was to determine the effect of chemical modification of lysyl residues on thrombin interaction with platelet membrane proteins. Modification of lysyl residues by pyridoxal-5'-phosphate affected two different sites on thrombin and resulted in a greatly decreased binding to platelets. Using a crosslinking bifunctional reagent [bis(sulphosuccinimidyl) suberate (BS3)], we show that modified thrombin retained the ability to form high molecular mass (greater than or equal to 400 kDa) complexes with yet unidentified platelet proteins and to bind to platelet protease nexin I, but had lost the ability to bind to platelet glycoprotein Ib (GPIb). As previously reported by others, heparin protected one of the two sites from phosphopyridoxylation. In contrast modified thrombin, heparin-protected modified thrombin retained the ability to bind to GPIb, indicating that the lysyl residue(s) protected by heparin from the modification are essential for GPIb binding. While unprotected modified thrombin failed to bind hirudin, heparin-protected modified thrombin retained its ability to bind the carboxy-terminal hirudin peptide H54-65. Tritium-labelling of the modified lysyl residues and degradation of modified thrombins by CNBr or trypsin confirmed that the lysyl residue(s) protected by heparin and essential for GPIb binding are located in the thrombin binding domain for the carboxyl-terminal tail of hirudin, within the sequence 18-73 of the thrombin B chain.