A focus on dominant negative variants in a series of 170 heterozygous FXI-deficient patients.

INTRODUCTION: Dominant-negative effects have been described for 10 F11 variants in the literature. AIM: The current study aimed at identifying putative dominant-negative F11 variants. MATERIAL AND METHODS: This research consisted in a retrospective analysis of routine laboratory data. RESULTS: In a series of 170 patients with moderate/mild factor XI (FXI) deficiencies, we identified heterozygous carriers of previously reported dominant-negative variants (p.Ser243Phe, p.Cys416Tyr, and p.Gly418Val) with FXI activities inconsistent with a dominant-negative effect. Our findings also do not support a dominant-negative effect of p.Gly418Ala. We also identified a set of patients carrying heterozygous variants, among which five out of 11 are novel, with FXI activities suggesting a dominant-negative effect (p.His53Tyr, p.Cys110Gly, p.Cys140Tyr, p.Glu245Lys, p.Trp246Cys, p.Glu315Lys, p.Ile421Thr, p.Trp425Cys, p.Glu565Lys, p.Thr593Met, and p.Trp617Ter). However, for all but two of these variants, individuals with close to half normal FXI coagulant activity (FXI:C) were identified, indicating an inconstant dominant effect. CONCLUSION: Our data show that for some F11 variants recognized has having dominant-negative effects, such effects actually do not occur in many individuals. The present data suggest that for these patients, the intracellular quality control mechanisms eliminate the variant monomeric polypeptide before homodimer assembly, thereby allowing only the wild-type homodimer to assemble and resulting in half normal activities. In contrast, in patients with markedly decreased activities, some mutant polypeptides might escape this first quality control. In turn, assembly of heterodimeric molecules as well as mutant homodimers would result in activities closer to 1:4 of FXI:C normal range.

Incidence of deep venous thrombosis in COVID-19 critically ill patients treated with intermediate-dose of heparin for thromboprophylaxis: The COVIDOP-DVT observational study.

INTRODUCTION: The high prevalence of deep vein thrombosis (DVT) in patients admitted to intensive care unit (ICU) for COVID-19-related acute respiratory distress syndrome (ARDS) would justify systematic screening of these patients or higher therapeutic dose of heparin for thromboprophylaxis. MATERIAL AND METHOD: We performed a systematic echo-Doppler of the lower limb proximal veins during the first 48 h (visit 1) and from 7 to 9 days after visit 1 (visit 2) in consecutive patients admitted to the ICU of a university-affiliated tertiary hospital for severe proven COVID-19 during the second wave. All patients received intermediate-dose heparin (IDH). The primary objective was to determine DVT incidence on venous Doppler ultrasound. Secondary objectives were to determine whether the presence of DVT modifies the anticoagulation regimen, the incidence of major bleeding according to International Society on Thrombosis and Haemostasis (ISTH) criteria, and the mortality rate of patients with and without DVT. RESULTS: We included 48 patients (30 [62.5%] men) with a median age of 63 years [IQR, 54-70]. The prevalence of proximal deep vein thrombosis was 4.2% (2/48). In these two patients, after DVT diagnosis, anticoagulation was changed from intermediate to curative dose. Two patients (4.2%) had a major bleeding complication according to ISTH criteria. Among the 48 patients, 9 (18.8%) died before hospital discharge. No DVT or pulmonary embolism was diagnosed in these deceased patients during their hospital stay. CONCLUSION: In critically ill patients with COVID-19, management with IDH results in a low incidence of DVT. Although our study is not designed to demonstrate any difference in outcome, our results do not suggest any signal of harm when using intermediate-dose heparin (IDH) COVID-19 with a frequency of major bleeding complications less than 5%.

Role of oculocerebrorenal syndrome of Lowe (OCRL) protein in megakaryocyte maturation, platelet production and functions: a study in patients with Lowe syndrome.

Lowe syndrome (LS) is an oculocerebrorenal syndrome of Lowe (OCRL1) genetic disorder resulting in a defect of the OCRL protein, a phosphatidylinositol-4,5-bisphosphate 5-phosphatase containing various domains including a Rho GTPase-activating protein (RhoGAP) homology domain catalytically inactive. We previously reported surgery-associated bleeding in patients with LS, suggestive of platelet dysfunction, accompanied with a mild thrombocytopenia in several patients. To decipher the role of OCRL in platelet functions and in megakaryocyte (MK) maturation, we conducted a case-control study on 15 patients with LS (NCT01314560). While all had a drastically reduced expression of OCRL, this deficiency did not affect platelet aggregability, but resulted in delayed thrombus formation on collagen under flow conditions, defective platelet spreading on fibrinogen and impaired clot retraction. We evidenced alterations of the myosin light chain phosphorylation (P-MLC), with defective Rac1 activity and, inversely, elevated active RhoA. Altered cytoskeleton dynamics was also observed in cultured patient MKs showing deficient proplatelet extension with increased P-MLC that was confirmed using control MKs transfected with OCRL-specific small interfering(si)RNA (siOCRL). Patients with LS also had an increased proportion of circulating barbell-shaped proplatelets. Our present study establishes that a deficiency of the OCRL protein results in a defective actomyosin cytoskeleton reorganisation in both MKs and platelets, altering both thrombopoiesis and some platelet responses to activation necessary to ensure haemostasis.

Efficacy and safety of a recombinant Von Willebrand Factor treatment in patients with inherited Von Willebrand Disease requiring surgical procedures.

INTRODUCTION: Von Willebrand Disease is a common inherited haemorrhagic disorder due to a deficiency of Von Willebrand Factor (VWF). In case of surgical procedures in patients who are not responsive or have contraindications to desmopressin, replacement therapy with VWF concentrates is indicated. Until recently, only plasma-derived VWF concentrates were available. A new recombinant VWF (rVWF) concentrate that contains no Factor VIII (FVIII) but a high amount of high molecular weight VWF multimers has been available in France since 2018. AIM: Describe real-world experience of using rVWF in surgical procedures. METHODS: Sixty-three surgeries for 55 patients were retrospectively analysed in 7 French haemostasis centres. RESULTS: During minor surgeries, the median (range) number of infusions was 1 (1-8) with a preoperative loading dose of 35 (19-56) rVWF IU/kg and a total median dose of 37.5 IU (12-288). During major surgeries, the median (range) number of infusions was only 3 (1-14) with a median preoperative loading dose of 36 IU (12-51) rVWF IU/kg, and a total median dose of 108 IU (22-340) rVWF IU/kg. The overall clinical efficacy was qualified as excellent/good in 61 of the procedures (97%), moderate in 1 (1.5%) and poor in 1 (1.5%). There was no accumulation of VWF or FVIII during postoperative monitoring. No thromboembolic events, anti-VWF antibodies or adverse events were reported. CONCLUSION: This French 'real-world' experience shows that a few infusions and low doses of rVWF provided effective prevention of bleeding in major and minor surgeries in inherited VWD, with no clinically significant safety concerns.

Rapid screening of COVID-19 patients using white blood cell scattergrams, a study on 381 patients.

Complementary tools are warranted to increase the sensitivity of the initial testing for COVID-19. We identified a specific 'sandglass' aspect on the white blood cell scattergram of COVID-19 patients reflecting the presence of circulating plasmacytoid lymphocytes. Patients were dichotomized as COVID-19-positive or -negative based on reverse transcriptase polymerase chain reaction (RT-PCR) and chest computed tomography (CT) scan results. Sensitivity and specificity of the 'sandglass' aspect were 85·9% and 83·5% respectively. The positive predictive value was 94·3%. Our findings provide a non-invasive and simple tool to quickly categorize symptomatic patients as either COVID-19-probable or -improbable especially when RT-PCR and/or chest CT are not rapidly available.

Multicentre pharmacokinetic evaluation of rFVIII-Fc (efmoroctocog alfa) in a real life and comparison with non-extended half-life FVIII concentrates.

The use of enhanced half-life (EHL) FVIII has improved the quality of prophylaxis in haemophilia A, but with a benefit that may vary from one patient to another. We analysed the pharmacokinetic data obtained with efmoroctocog alfa (rFVIII-Fc) in 114 patients and, in 47 cases, compared them to those previously measured with non-EHL FVIII. The in vivo recovery (IVR) of rFVIII-Fc measured with one stage clotting assay (OSA) and chromogenic assay (CSA) was 2.2 and 2.8 IU/mL per IU/kg, respectively. The median half-life (T1/2 ) of rFVIII-Fc was 14.5 hours whatever the FVIII:C assay used, but variable and correlated with preinfusion VWF:Ag levels (r = .76). Both IVR and T1/2 were lower in patients under 12 years old (2.4 IU/mL per IU/kg and 11.1 hours, respectively; CSA). PK study of rFVIII-Fc vs non-EHL FVIII showed a T1/2 ratio of 1.4 in favour of rFVIII-Fc, regardless of the patient's age. However the relative increase in T1/2 with rFVIII-Fc was lower than 30% in one-third of patients evaluated, particularly when the previous FVIII administered was a BHK-derived product. This study therefore suggests that analysis of individual PK profile in response to a specific FVIII concentrate is potentially useful before a switch in haemophilia A patients.

Factor VIII and IX assays for post-infusion monitoring in hemophilia patients: Guidelines from the French BIMHO group (GFHT).

Replacement therapy with plasma-derived or recombinant FVIII and FIX (pdFVIII/pdFIX or rFVIII/rFIX) concentrates is the standard of treatment in patients with haemophilia A and B, respectively. Measurement of factor VIII (FVIII:C) or factor IX (FIX:C) levels can be done by one-stage clotting assay (OSA) or chromogenic substrate assay (CSA). The French study group on the Biology of Hemorrhagic Diseases (a collaborative group of the GFHT and MHEMO network) presents a literature review and proposals for the monitoring of FVIII:C and FIX:C levels in treated haemophilia A and B patients, respectively. The use of CSA is recommended for the monitoring of patients treated with pdFVIII or rFVIII including extended half-life (EHL) rFVIII. Except for rFVIII-Fc, great caution is required when measuring FVIII:C levels by OSA in patients substituted by EHL-rFVIII. The OSA is recommended for the monitoring of patients treated with pdFIX or rFIX. Large discordances in the FIX:C levels measured for extended half-life rFIX (EHL-rFIX), depending on the method and reagents used, must lead to great attention when OSA is used for measuring FIX:C levels in patients substituted by EHL-rFIX. Data of most of recent studies, obtained with spiked plasmas, deserve to be confirmed in plasma samples of treated patients.

Bleeding risk of surgery and its prevention in patients with inherited platelet disorders.

Excessive bleeding at surgery is a feared complication in patients with inherited platelet disorders. However, very few studies have evaluated the frequency of surgical bleeding in these hemorrhagic disorders. We performed a worldwide, multicentric, retrospective study to assess the bleeding complications of surgery, the preventive and therapeutic approaches adopted, and their efficacy in patients with inherited platelet disorders: the Surgery in Platelet disorders And Therapeutic Approach (SPATA) study. We rated the outcome of 829 surgical procedures carried out in 423 patients with well-defined forms of inherited platelet disorders: 238 inherited platelet function disorders and 185 inherited platelet number disorders. Frequency of surgical bleeding was high in patients with inherited platelet disorders (19.7%), with a significantly higher bleeding incidence in inherited platelet function disorders (24.8%) than in inherited platelet number disorders (13.4%). The frequency of bleeding varied according to the type of inherited platelet disorder, with biallelic Bernard Soulier syndrome having the highest occurrence (44.4%). Frequency of bleeding was predicted by a pre-operative World Health Organization bleeding score of 2 or higher. Some types of surgery were associated with a higher bleeding incidence, like cardiovascular and urological surgery. The use of pre-operative pro-hemostatic treatments was associated with a lower bleeding frequency in patients with inherited platelet function disorders but not in inherited platelet number disorders. Desmopressin, alone or with antifibrinolytic agents, was the preventive treatment associated with the lowest bleedings. Platelet transfusions were used more frequently in patients at higher bleeding risk. Surgical bleeding risk in inherited platelet disorders is substantial, especially in inherited platelet function disorders, and bleeding history, type of disorder, type of surgery and female sex are associated with higher bleeding frequency. Prophylactic pre-operative pro-hemostatic treatments appear to be required and are associated with a lower bleeding incidence.

Assessment of haemostasis in patients with cirrhosis: Relevance of the ROTEM tests?: A prospective, cross-sectional study.

BACKGROUND: In patients with cirrhosis, decreased rotational thromboelastometry (ROTEM) parameters suggest hypocoagulability secondary to liver dysfunction. However, observed normal or increased thrombin generation suggests preserved haemostasis and/or a procoagulant state. The correlated levels of both coagulation factors and inhibitors also support preserved haemostasis. OBJECTIVE: The objective of this study is to investigate the correlation between three specific approaches of haemostasis (ROTEM, thrombin generation and coagulation factors/inhibitors) on the same plasma sample from patients with cirrhosis. DESIGN: A prospective, observational study. SETTING: Single university hospital. PARTICIPANTS: Forty patients with cirrhosis. INTERVENTION: Measurement of the following factors: model for end-stage liver disease (MELD) scores; ROTEM maximum clot firmness (ROTEM-MCF) in EXTEM, INTEM, FIBTEM assays; fibrinogen; factors V and VIII; von Willebrand factor; protein C; protein S; antithrombin; and the thrombin generation test (TGT) enabling the calculation of endogenous thrombin potential without and with thrombomodulin, and the ratio of endogenous thrombin potential with-to-without thrombomodulin (regarded as an index of hypercoagulability). RESULTS: ROTEM-MCF values were distributed within the normal and hypocoagulation ranges; were correlated to variations in factor V, fibrinogen, protein C and S and antithrombin; and were inversely correlated to MELD scores (ρ > 0.5; P < 0.05). Levels of von Willebrand factor were above normal and were not correlated with any other factor levels. After addition of thrombomodulin, endogenous thrombin potential values were distributed within or above normal values. Factor V variation was correlated to the ratio of endogenous thrombin potential with-to-without thrombomodulin. CONCLUSION: ROTEM indicated hypocoagulability correlated to liver dysfunction. In contrast, the TGT indicated a preserved or even increased coagulation profile (which was supported by the correlation between coagulant factors and inhibitors) and a potential for hypercoagulability inversely correlated to the degree of liver dysfunction. ROTEM may not be appropriate for haemostasis assessment in patients with liver cirrhosis and could lead to the unnecessary transfusion of fresh frozen plasma. TRIAL REGISTRATION: S.C. 3024 - ID RCB: 2012-A01728-35.

Commercial human frozen plasmas for local, cross-site and long-term comparability of coagulation analysers.

Laboratory compliance implies to correlate instruments for coagulation parameter with a wide range of measure using patient samples or commercialized sets of frozen plasmas. The aim of this study was to evaluate the intra, inter-reproducibility and long-term stability of ExpertCor Routine (ECR) plasma sets (Stago) on different parameters. The study was realized in two laboratories on four different instruments. Inter-site and intra-site correlation of ECR sets for PT, aPTT, Fibrinogen, INR, factor V (FV) UFH and LMWH anti-Xa and intra-reproducibility of DDimer (DDI), factor VIII (FVIII:C) and antithrombin (AT) assays were tested. To evaluate ECR long-term stability, samples were tested until 180 after delivery in one laboratory. Intra-site evaluation correlation coefficients is around 1. All predefined criteria to fulfil good comparability between inter-site instruments are met with Passing slopes between 0.9 and 1.1 and intercepts ranging from -0.62 to 2.83%. Long-term stability evaluation does not show any deviation over 180 days for aPTT, fibrinogen, DDI, UFH, LMWH but a drift for FV with STA-NeoPTimal reagent. On contrary, AT and FVIII:C are not stable. PT in second has an excellent stability unlike PT in percentage. Our study validates the use of ECR sets for correlation between instruments and inter-sites agreement, as for parameters claimed on the products than for factor V and FVIII:C. The evaluation of stability confirming the possible extension of use for 180 days after delivery except for FVIII:C and AT. These plasmas sets are an excellent alternative to local plasma patient use to perform instrument comparison.

Factors Influencing Anti-Xa Assays: A Multicenter Prospective Study in Critically Ill and Noncritically Ill Patients Receiving Unfractionated Heparin.

BACKGROUND: The presence of dextran sulfate (DS) in reagents and the type of blood collection tube (citrate/citrated-theophylline-adenosine-dipyridamole [CTAD]) can lead to discrepancies between unfractionated heparin (UFH) anti-Xa levels. OBJECTIVES: To evaluate the extent of the effect (1) of different reagents containing or not containing DS and (2) of the blood collection tubes, on UFH anti-Xa levels, in various clinical situations (NCT04700670). METHODS: We prospectively included patients from eight centers: group (G)1, cardiopulmonary bypass (CPB) after heparin neutralization (n = 39); G2, cardiothoracic intensive care unit (ICU) after CPB (n = 35); G3, medical ICU (n = 53); G4, other medical inpatients (n = 38). Blood was collected into citrated and CTAD tubes. Chromogenic anti-Xa assays were centrally performed, using seven reagent/analyzer combinations including two without DS. The association between anti-Xa levels and covariates was tested using a linear mixed-effects model. RESULTS: We analyzed 4,546 anti-Xa values from 165 patients. Median anti-Xa levels were systematically higher with reagents containing DS, whatever the patient group, with the greatest effect observed in G1 (0.32 vs. 0.05 IU/mL). Anti-Xa levels were slightly higher in CTAD than in citrate samples, irrespective of the assay. The model showed: (1) a significant dextran-patient group interaction (p < 0.0001), the effect of DS on anti-Xa levels varying from 30.9% in G4 to 296% in G1, and (2) a significant effect of CTAD, varying between patient groups (p = 0.0302). CONCLUSION: The variability of anti-Xa levels with a great overestimation of the values, using a reagent containing DS, can lead to different treatment decisions, especially after heparin neutralization by protamine. Clinical consequences of these differences remain to be demonstrated.

Obstetrical complications in hereditary fibrinogen disorders: the Fibrinogest study.

BACKGROUND: Women with hereditary fibrinogen disorders (HFDs) seem to be at an increased risk of adverse obstetrical outcomes, but epidemiologic data are limited. OBJECTIVES: We aimed to determine the prevalence of pregnancy complications; the modalities and management of delivery; and the postpartum events in women with hypofibrinogenemia, dysfibrinogenemia, and hypodysfibrinogenemia. METHODS: We conducted a retrospective and prospective multicentric international study. RESULTS: A total of 425 pregnancies were investigated from 159 women (49, 95, and 15 cases of hypofibrinogenemia, dysfibrinogenemia, and hypodysfibrinogenemia, respectively). Overall, only 55 (12.9%) pregnancies resulted in an early miscarriage, 3 (0.7%) resulted in a late miscarriage, and 4 (0.9%) resulted in an intrauterine fetal death. The prevalence of live birth was similar among the types of HFDs (P = .31). Obstetrical complications were observed in 54 (17.3%) live birth pregnancies, including vaginal bleeding (14, 4.4%), retroplacental hematoma (13, 4.1%), and thrombosis (4, 1.3%). Most deliveries were spontaneous (218, 74.1%) with a vaginal noninstrumental delivery (195, 63.3%). A neuraxial anesthesia was performed in 116 (40.4%) pregnancies, whereas general or no anesthesia was performed in 71 (16.6%) and 129 (44.9%) pregnancies, respectively. A fibrinogen infusion was administered in 28 (8.9%) deliveries. Postpartum hemorrhages were observed in 62 (19.9%) pregnancies. Postpartum venous thrombotic events occurred in 5 (1.6%) pregnancies. Women with hypofibrinogenemia were at an increased risk of bleeding during the pregnancy (P = .04). CONCLUSION: Compared with European epidemiologic data, we did not observe a greater frequency of miscarriage, while retroplacental hematoma, postpartum hemorrhage, and thrombosis were more frequent. Delivery was often performed without locoregional anesthesia. Our findings highlight the urgent need for guidance on the management of pregnancy in HFDs.

[Home treatment of low-risk pulmonary embolism patients : Efficacy and safety of an outpatient program including the general practitioner]. / Prise en charge ambulatoire des embolies pulmonaires à risque faible ; expérience d'une filière centrée sur le lien ville-hôpital.

AIM: Outpatient treatment (OT) of patients with low-risk pulmonary embolisms (PE) is recommended. A multidisciplinary OT program including the general practitioner (GP) has been implemented at Versailles hospital in 2019. The objectives of the study were to assess the feasibility, safety and acceptability of the program. MATERIAL AND METHODS: The feasibility of, and the inclusion criteria for OT were defined from a retrospective cohort study of PE patients carried out in 2018. In the prospective study, consecutive patients consulting in the emergency department between 2019 and 2021 with confirmed PE were eligible for OT if they had sPESI and HESTIA scores equal to 0, normal troponin and NT-pro-BNP levels, and no right ventricular dilation on imaging. PEs associated with COVID were excluded. The OT program included 4 appointments within 3 months, including 2 with the GP. Events (death, recurrence of PE or venous thromboembolism, bleeding, rehospitalisation) were collected at 3-month follow-up. RESULTS: In the retrospective study, 19% of the 138 PE patients seen in the emergency department were eligible for OT. No complication occurred at Day 90. In the prospective study, 313 consecutive patients with confirmed PE in the emergency department were included, 66 (21%) were eligible for OT. Overall, 43 patients (14%) received OT (39 eligible) and 27 patients eligible for OT were hospitalised (92% because of pulmonary infarction). At 3-month follow-up, there were no death, no recurrence of thromboembolism, and one patient has been early hospitalised for COVID; 3 female patients treated with rivaroxaban had minor bleeding (heavy menstrual bleeding). The satisfaction rate of general practitioner was 95%. CONCLUSIONS: This study confirms the feasibility and safety of our OT program for low-risk EP patients, centered on the general practitioner. It reduces the time spent in the emergency department, reduces hospitalisations and strengthens the city-hospital link for care.

Platelets and viruses: an ambivalent relationship.

Thrombocytopenia is a frequent complication of viral infections providing evidence that interaction of platelets with viruses is an important pathophysiological phenomenon. Multiple mechanisms are involved depending on the nature of the viruses involved. These include immunological platelet destruction, inappropriate platelet activation and consumption, and impaired megakaryopoiesis. Viruses bind platelets through specific receptors and identified ligands, which lead to mutual alterations of both the platelet host and the viral aggressor. We have shown that HIV-1 viruses are internalized specifically in platelets and megakaryocytes, where they can be either sheltered, unaltered (with potential transfer of the viruses into target organs), or come in contact with platelet secretory products leading to virus destruction and facilitated platelet clearance. In this issue, we have reviewed the various pathways that platelets use in order to interact with viruses, HIV and others. This review also shows that more work is still needed to precisely identify platelet roles in viral infections, and to answer the challenge of viral safety in platelet transfusion.

Real Life Population Pharmacokinetics Modelling of Eight Factors VIII in Patients with Severe Haemophilia A: Is It Always Relevant to Switch to an Extended Half-Life?

We retrospectively analysed the data files of 171 adults and 87 children/adolescents with severe haemophilia, except for 14 patients (moderate; minor) (1), to develop a global population pharmacokinetic (PK) model for eight factors VIII (FVIII) that could estimate individual PK parameters for targeting the desired level of FVIII activity (FVIII:C); and (2) to compare half-life (HL) in patients switching from a standard half-life (SHL) to an extended half-life (EHL) and evaluate the relevance of the switch. One-stage clotting assay for the measurement of FVIII activity (FVIII:C, IU/mL) was used for population PK modelling. The software, Monolix version 2019R1, was used for non-linear mixed-effects modelling. A linear two-compartment model best described FVIII:C. The estimated PK parameters (between-subject variability) were: 2640 mL (23.2%) for volume of central compartment (V1), 339 mL (46.8%) for volume of peripheral compartment (V2), 135 mL/h for Q (fixed random effect), and 204 mL/h (34.9%) for clearance (Cl). Weight, age, and categorical covariate EHL were found to influence Cl and only weight for V1. This model can be used for all of the FVIII cited in the study. Moreover, we demonstrated, in accordance with previous studies, that Elocta had longer half-life (EHL) than SHL (mean ratio: 1.48) as compared to Advate, Factane, Kogenate, Novoeight, and Refacto.