Red cell extracellular vesicles and coagulation activation pathways.

PURPOSE OF REVIEW: Packed red blood cells (PRBCs) are the most commonly transfused blood products. Preparation of PRBCs requires blood collection from donors, processing, and storage prior to transfusion to recipients. Stored red blood cells (RBCs) undergo structural and metabolic changes collectively known as the storage lesion. RBC extracellular vesicles (sREVs) are released in PRBC units during storage, and are transfused along with intact RBCs into recipients. For several decades, extracellular vesicles have been the focus of intense research, leading to the discovery of a wide variety of endogenous biological properties that may impact numerous physiologic and/or pathologic pathways. RECENT FINDINGS: This study reviews the characteristics of extracellular vesicles present in PRBC units and the impact of prestorage and pretransfusion processing, as well as storage conditions, on their generation. Importantly, we discuss recently described interactions of sREVs with coagulation pathways and related interplay with inflammatory pathways in vitro and in vivo using animal models. SUMMARY: Extracellular vesicles present in stored PRBC units are capable of activating coagulation pathways. However, it remains unclear whether this affects clinical outcomes in recipients of PRBC units. Further understanding of these pathways and their relationship to any adverse outcomes may yield novel strategies to mitigate complications of blood transfusion.

Passenger Lymphocyte Syndrome and Autoimmune Hypothyroidism Following Hematopoietic Stem Cell Transplantation.

We present the case of a 24-year-old male, who received a minor ABO-incompatible allogeneic hematopoietic stem cell transplant (HSCT, blood group O+ ⟶ A+) from an HLA-matched unrelated female donor, as consolidation therapy for relapsed precursor-B-cell acute lymphoblastic leukemia. The donor had a known history of Hashimoto's thyroiditis before HSCT. At day +10 posttransplant, the patient developed severe hemolysis, which required emergent red blood cell exchange. Additionally, about a year posttransplant, he had circulating antithyroglobulin antibodies, decreased free-T4 (fT4) and increased serum thyroid-stimulating hormone (TSH). The potential causes of the posttransplant hemolytic episode and hypothyroidism are discussed. While the hemolysis was worsened by the transfusion of A red blood cells (RBCs) in the context of passenger lymphocyte syndrome, the thyroid dysfunction might be explained by an autoimmune disease transferred from the donor. The case highlights the possibility of several non-relapse-related complications of HSCT occurring in the same patient. It is critical that such adverse outcomes are distinguished from classical graft-versus-host disease (GVHD) for adequate recipient counseling, posttransplant screening, and prompt treatment.

Elevated Plasma Fibrinogen Is Associated With Excessive Inflammation and Disease Severity in COVID-19 Patients.

Background: The coronavirus disease-19 (COVID-19) is characterized with intense inflammatory response, cardiac involvement, and coagulopathy. Fibrinogen, as a biomarker for inflammation, cardiovascular disease, and coagulation, has not been fully investigated yet. The aim of this study was to assess the clinical application of fibrinogen in COVID-19 patients. Methods: We retrospectively analyzed the demographic and laboratory characteristics of 119 COVID-19 patients in the University of Alabama of Birmingham Medical Center. Correlations of fibrinogen on admission with intensive care unit (ICU) admission, disease severity, and laboratory parameters were analyzed. Results: Among the 119 COVID-19 patients, 77.3% (92/119) had severe disease, and 59.5% (71/119) patients were admitted to the ICU. Elevated fibrinogen was detected in 67.2% (80/119) of the patients. Fibrinogen levels were significantly associated with inflammatory markers and disease severity, but not with cardiac injury biomarker high sensitivity troponin I. Patients with severe disease had increased fibrinogen levels upon admission compared to patients with non-severe disease (P = 0.001). Fibrinogen level at 528.0 mg/dl was the optimal cutoff to predict disease severity, with a sensitivity and specificity of 66.7% and 70.3% (area undty -60er the curve [AUC] 0.72, P = 0.0006). Conclusions: Fibrinogen is commonly elevated in COVID-19 patients, especially in those with severe disease. Elevated fibrinogen correlates with excessive inflammation, disease severity, and ICU admission in COVID-19 patients.

Euglobulin clot lysis time reveals a high frequency of fibrinolytic activation in trauma.

Activation of the fibrinolytic system plays a central role in the host response to trauma. There is significant heterogeneity in the degree of fibrinolysis activation at baseline that is usually assessed by whole blood thromboelastography (TEG). Few studies have focused on plasma markers of fibrinolysis that could add novel insights into the frequency and mechanisms of fibrinolytic activation in trauma. Global fibrinolysis in plasma was assessed using a modified euglobulin clot lysis time (ECLT) assay in 171 major trauma patients and compared to commonly assessed analytes of fibrinolysis. The median ECLT in trauma patients was significantly shorter at 8.5 h (IQR, 1.3-19.5) compared to 19.9 h (9.8-22.6) in healthy controls (p < 0.0001). ECLT values ≤2.5th percentile of the reference range were present in 83 (48.5%) of trauma patients, suggesting increased fibrinolytic activation. Shortened ECLT values were associated with elevated plasmin-antiplasmin (PAP) complexes and free tissue plasminogen activator (tPA) levels in plasma. Sixteen (9.2%) individuals met the primary outcome for massive transfusion, here defined as the critical administration threshold (CAT) of 3 units of packed red cells in any 60-minute period within the first 24 h. In a univariate screen, plasma biomarkers associated with CAT included D-dimer (p < 0.001), PAP (p < 0.05), free tPA (p < 0.05) and ECLT (p < 0.05). We conclude that fibrinolytic activation, measured by ECLT, is present in a high proportion of trauma patients at presentation. The shortened ECLT is partially driven by high tPA levels and is associated with high levels of circulating PAP complexes. Further studies are needed to determine whether ECLT is an independent predictor of trauma outcomes.

Plasmin-mediated cleavage of high-molecular-weight kininogen contributes to acetaminophen-induced acute liver failure.

Acetaminophen (APAP)-induced liver injury is associated with activation of coagulation and fibrinolysis. In mice, both tissue factor-dependent thrombin generation and plasmin activity have been shown to promote liver injury after APAP overdose. However, the contribution of the contact and intrinsic coagulation pathways has not been investigated in this model. Mice deficient in individual factors of the contact (factor XII [FXII] and prekallikrein) or intrinsic coagulation (FXI) pathway were administered a hepatotoxic dose of 400 mg/kg of APAP. Neither FXII, FXI, nor prekallikrein deficiency mitigated coagulation activation or hepatocellular injury. Interestingly, despite the lack of significant changes to APAP-induced coagulation activation, markers of liver injury and inflammation were significantly reduced in APAP-challenged high-molecular-weight kininogen-deficient (HK-/-) mice. Protective effects of HK deficiency were not reproduced by inhibition of bradykinin-mediated signaling, whereas reconstitution of circulating levels of HK in HK-/- mice restored hepatotoxicity. Fibrinolysis activation was observed in mice after APAP administration. Western blotting, enzyme-linked immunosorbent assay, and mass spectrometry analysis showed that plasmin efficiently cleaves HK into multiple fragments in buffer or plasma. Importantly, plasminogen deficiency attenuated APAP-induced liver injury and prevented HK cleavage in the injured liver. Finally, enhanced plasmin generation and HK cleavage, in the absence of contact pathway activation, were observed in plasma of patients with acute liver failure due to APAP overdose. In summary, extrinsic but not intrinsic pathway activation drives the thromboinflammatory pathology associated with APAP-induced liver injury in mice. Furthermore, plasmin-mediated cleavage of HK contributes to hepatotoxicity in APAP-challenged mice independently of thrombin generation or bradykinin signaling.

Protease: Serpin complexes to assess contact system and intrinsic pathway activation.

Mounting evidence suggests that a variety of disease states are pathophysiologically related to activation of the contact system in vivo. The plasma contact system is composed of a cascade of serine proteases initiated by surface activation of factor XII, which can then proceed through a procoagulant pathway by activating the intrinsic coagulation factor XI, or a proinflammatory pathway by activating prekallikrein. Serpins are the primary endogenous inhibitors of the contact system, which irreversibly inhibit their respective protease(s), forming a stable complex. We modified an existing assay strategy for detecting these complexes in plasma using ELISAs and determined the effect of preanalytical variation caused by anticoagulant selection and processing time. The assays were sensitive and specific to inherited deficiency of individual contact factors. We conclude that these assays are robust and represent a relatively simple approach to the assessment of contact factor activation in plasma samples.

Quantification of citrullinated histones: Development of an improved assay to reliably quantify nucleosomal H3Cit in human plasma.

BACKGROUND: Recent data propose a diagnostic and prognostic capacity for citrullinated histone H3 (H3Cit), a marker of neutrophil extracellular traps (NETs), in pathologic conditions such as cancer and thrombosis. However, current research is hampered by lack of standardized assays. OBJECTIVES: We aimed to develop an assay to reliably quantify nucleosomal H3Cit in human plasma. METHODS: We assessed the common practice of in vitro enzymatically modified histone H3 as calibration standards and the specificity of available intrapeptidyl citrulline antibodies. Based on our findings, we developed and validated a novel assay to quantify nucleosomal H3Cit in human plasma. RESULTS: We show that enzymatically citrullinated H3 proteins are compromised by high enzyme-dependent lot variability as well as instability in plasma. We furthermore demonstrate that the majority of commercially available antibodies against intrapeptidyl citrulline display poor specificity for their reported target when tested against a panel of semi-synthetic nucleosomes containing distinct histone H3 citrullinations. Finally, we present a novel assay utilizing highly specific monoclonal antibodies and semi-synthetic nucleosomes containing citrulline in place of arginine at histone H3, arginine residues 2, 8, and 17 (H3R2,8,17Cit) as calibration standards. Rigorous validation of this assay shows its capacity to accurately and reliably quantify nucleosomal H3Cit levels in human plasma with clear elevations in cancer patients compared to healthy individuals. CONCLUSIONS: Our novel approach using defined nucleosome controls enables reliable quantification of H3Cit in human plasma. This assay will be broadly applicable to study the role of histone citrullination in disease and its utility as a biomarker.

Development and application of global assays of hyper- and hypofibrinolysis.

Numerous methods for evaluation of global fibrinolytic activity in whole blood or plasma have been proposed, with the majority based on tissue-type plasminogen activator (t-PA) addition to initiate fibrinolysis. We propose that such an approach is useful to reveal hypofibrinolysis, but t-PA concentrations should be kept to a minimum. In this paper, we describe a low-concentration t-PA plasma turbidity assay to evaluate several congenital factor deficiencies, including plasminogen activator inhibitor-1 (PAI-1) and plasminogen deficiency, as well as hemophilia A and B. In addition, we demonstrate a threshold dependency on endogenous PAI-1 levels. To assess endogenous hyperfibrinolysis, we suggest that assays that avoid t-PA addition are preferable, with assays based on euglobulin fractionation remaining a viable choice. We describe a euglobulin fraction clot lysis time (ECLT) assay with spectrophotometric readout and other modifications, and evaluate it as a tool to measure hyperfibrinolysis in inherited clotting factor deficiency states. We demonstrate that the ECLT is predominantly driven by residual amounts of PAI-1, t-PA, and α2-antiplasmin. These assays should be further evaluated for the detection of hypo- or hyperfibrinolysis in acquired thrombotic or hemorrhagic disorders.

Red blood cell microvesicles activate the contact system, leading to factor IX activation via 2 independent pathways.

Storage lesion-induced, red cell-derived microvesicles (RBC-MVs) propagate coagulation by supporting the assembly of the prothrombinase complex. It has also been reported that RBC-MVs initiate coagulation via the intrinsic pathway. To elucidate the mechanism(s) of RBC-MV-induced coagulation activation, the ability of storage lesion-induced RBC-MVs to activate each zymogen of the intrinsic pathway was assessed in a buffer system. Simultaneously, the thrombin generation (TG) assay was used to assess their ability to initiate coagulation in plasma. RBC-MVs directly activated factor XII (FXII) or prekallikrein, but not FXI or FIX. RBC-MVs initiated TG in normal pooled plasma and in FXII- or FXI-deficient plasma, but not in FIX-deficient plasma, suggesting an alternate pathway that bypasses both FXII and FXI. Interestingly, RBC-MVs generated FIXa in a prekallikrein-dependent manner. Similarly, purified kallikrein activated FIX in buffer and initiated TG in normal pooled plasma, as well as FXII- or FXI-deficient plasma, but not FIX-deficient plasma. Dual inhibition of FXIIa by corn trypsin inhibitor and kallikrein by soybean trypsin inhibitor was necessary for abolishing RBC-MV-induced TG in normal pooled plasma, whereas kallikrein inhibition alone was sufficient to abolish TG in FXII- or FXI-deficient plasma. Heating RBC-MVs at 60°C for 15 minutes or pretreatment with trypsin abolished TG, suggesting the presence of MV-associated proteins that are essential for contact activation. In summary, RBC-MVs activate both FXII and prekallikrein, leading to FIX activation by 2 independent pathways: the classic FXIIa-FXI-FIX pathway and direct kallikrein activation of FIX. These data suggest novel mechanisms by which RBC transfusion mediates inflammatory and/or thrombotic outcomes.

Neutrophils and neutrophil extracellular traps enhance venous thrombosis in mice bearing human pancreatic tumors.

Pancreatic cancer is associated with a high incidence of venous thromboembolism. Neutrophils have been shown to contribute to thrombosis in part by releasing neutrophil extracellular traps (NET). A recent study showed that increased plasma levels of the NET biomarker, citrullinated histone H3 (H3Cit), are associated with venous thromboembolism in patients with pancreatic and lung cancer but not in those with other types of cancer, including breast cancer. In this study, we examined the contribution of neutrophils and NET to venous thrombosis in nude mice bearing human pancreatic tumors. We found that tumor-bearing mice had increased circulating neutrophil counts and levels of granulocyte-colony stimulating factor, neutrophil elastase, H3Cit and cell-free DNA compared with controls. In addition, thrombi from tumor-bearing mice contained increased levels of the neutrophil marker Ly6G, as well as higher levels of H3Cit and cell-free DNA. Thrombi from tumor-bearing mice also had denser fibrin with thinner fibers consistent with increased thrombin generation. Importantly, either neutrophil depletion or administration of DNase I reduced the thrombus size in tumor-bearing but not in control mice. Our results, together with clinical data, suggest that neutrophils and NET contribute to venous thrombosis in patients with pancreatic cancer.

Is There Any Improvement of the Coagulation Imbalance in Sickle Cell Disease after Hematopoietic Stem Cell Transplantation?

Several components of the clotting system are modified towards hypercoagulability in sickle cell disease (SCD). To date, hematopoietic stem cell transplantation (HSCT) is the only validated curative treatment of SCD. Here, we investigated the changes in the hemostatic potential of SCD children who've received a successful HSCT. Seventeen children with severe SCD were enrolled in the study. Thrombin generation (TG) was performed on citrated platelet-poor plasma, obtained before and 3, 6, 9, 12 and 15 months after HSCT. TG was triggered using 1 pM tissue factor and 4 µM phospholipids with or without thrombomodulin (TM). Before the HSCT, SCD children showed a higher endogenous thrombin potential (ETP), higher peak, higher velocity and shorter time-to-peak of TG than the normal controls (NC). ETP did not significantly change following the HSCT. However, the peak, velocity and time-to-peak of TG reversed to normal ranges from 3 months post-HSCT and remained so up to 15 months post-HSCT. The reduction of ETP after the addition of thrombomodulin (RETP) was dramatically reduced in SCD children before HSCT as compared with the NC. A partial reversal of RETP was observed from 3 months through 15 months post-HSCT. No statistical difference was observed for patient age or donor hemoglobinopathy status. In summary, successful HSCT improves the kinetics of TG but not the total thrombin capacity in SCD children.

Red blood cells modulate structure and dynamics of venous clot formation in sickle cell disease.

Sickle cell disease (SCD) is associated with chronic activation of coagulation and an increased risk of venous thromboembolism. Erythrocyte sickling, the primary pathologic event in SCD, results in dramatic morphological changes in red blood cells (RBCs) because of polymerization of the abnormal hemoglobin. We used a mouse model of SCD and blood samples from sickle patients to determine if these changes affect the structure, properties, and dynamics of sickle clot formation. Sickling of RBCs and a significant increase in fibrin deposition were observed in venous thrombi formed in sickle mice. During ex vivo clot contraction, the number of RBCs extruded from sickle whole blood clots was significantly reduced compared with the number released from sickle cell trait and nonsickle clots in both mice and humans. Entrapment of sickled RBCs was largely factor XIIIa-independent and entirely mediated by the platelet-free cellular fraction of sickle blood. Inhibition of phosphatidylserine, but not administration of antisickling compounds, increased the number of RBCs released from sickle clots. Interestingly, whole blood, but not plasma clots from SCD patients, was more resistant to fibrinolysis, indicating that the cellular fraction of blood mediates resistance to tissue plasminogen activator. Sickle trait whole blood clots demonstrated an intermediate phenotype in response to tissue plasminogen activator. RBC exchange in SCD patients had a long-lasting effect on normalizing whole blood clot contraction. Furthermore, RBC exchange transiently reversed resistance of whole blood sickle clots to fibrinolysis, in part by decreasing platelet-derived PAI-1. These properties of sickle clots may explain the increased risk of venous thromboembolism observed in SCD.

Neutrophils: back in the thrombosis spotlight.

Reactive and clonal neutrophil expansion has been associated with thrombosis, suggesting that neutrophils play a role in this process. However, although there is no doubt that activated monocytes trigger coagulation in a tissue factor-dependent manner, it remains uncertain whether stimulated neutrophils can also directly activate coagulation. After more than a decade of debate, it is now largely accepted that normal human neutrophils do not synthetize tissue factor, the initiator of the extrinsic pathway of coagulation. However, neutrophils may passively acquire tissue factor from monocytes. Recently, the contact system, which initiates coagulation via the intrinsic pathway, has been implicated in the pathogenesis of thrombosis. After the recent description of neutrophil extracellular trap (NET) release by activated neutrophils, some animal models of thrombosis have demonstrated that coagulation may be enhanced by direct NET-dependent activation of the contact system. However, there is currently no consensus on how to assess or quantify NETosis in vivo, and other experimental animal models have failed to demonstrate a role for neutrophils in thrombogenesis. Nevertheless, it is likely that NETs can serve to localize other circulating coagulation components and can also promote vessel occlusion independent of fibrin formation. This article provides a critical appraisal of the possible roles of neutrophils in thrombosis and highlights some existing knowledge gaps regarding the procoagulant activities of neutrophil-derived extracellular chromatin and its molecular components. A better understanding of these mechanisms could guide future approaches to prevent and/or treat thrombosis.

Different profile of thrombin generation in children with acute lymphoblastic leukaemia treated with native or pegylated asparaginase: A cohort study.

BACKGROUND: Asparaginase (Asp) and corticosteroid (CS) treatment in patients with acute lymphoblastic leukaemia (ALL) is associated with an increased risk of thrombotic events. OBJECTIVE: Characterization of global haemostatic phenotypes of patients with ALL during Asp therapy. PROCEDURE: Thrombin generation (TG) was monitored in platelet-poor plasma of 56 children treated for a B lineage ALL (36 with native, 20 with PEG Asp) using 1 pM tissue factor and 4 µM phospholipids, with and without thrombomodulin. Protein C activity (PC), free protein S (PS), antithrombin (AT) and fibrinogen levels were also measured. RESULTS: Elevated endogenous thrombin potential (ETP) and peak of TG were noted at diagnosis, throughout the Induction phase and Late Intensification but was significantly less for PEG than for native Asp (P < 0.001), while age, sex, type of corticosteroid during Induction and molecular response had no significant effect. The reduction of ETP after addition of thrombomodulin was significantly lower in ALL children compared with that in controls, suggesting impairment in PS/PC pathway. Three patients experienced thrombosis: two treated with native and one with PEG Asp. The two patients with native Asp had, at the time of thrombosis, a prothrombotic profile. CONCLUSIONS: Treatment with Asp, in combination with CS, enhances TG in children with ALL, more significantly with native than PEG Asp, which is present early at diagnosis, persists during Induction and reappears during Late Intensification. This is consistent with the high incidence of thrombotic events described during these phases of therapy. The less pronounced effect of PEG Asp remains to be elucidated.

In vitro activation of coagulation by human neutrophil DNA and histone proteins but not neutrophil extracellular traps.

NETosis is a physiologic process in which neutrophils release their nuclear material in the form of neutrophil extracellular traps (NETs). NETs have been reported to directly promote thrombosis in animal models. Although the effects of purified NET components including DNA, histone proteins, and neutrophil enzymes on coagulation have been characterized, the mechanism by which intact NETs promote thrombosis is largely unknown. In this study, human neutrophils were stimulated to produce NETs in platelet-free plasma (PFP) or in buffer using phorbol myristate actetate or calcium ionophore. DNA and histone proteins were also separately purified from normal human neutrophils and used to reconstitute chromatin using a salt-gradient dialysis method. Neutrophil stimulation resulted in robust NET release. In recalcified PFP, purified DNA triggered contact-dependent thrombin generation (TG) and amplified TG initiated by low concentrations of tissue factor. Similarly, in a buffer milieu, DNA initiated the contact pathway and amplified thrombin-dependent factor XI activation. Recombinant human histones H3 and H4 triggered TG in recalcified human plasma in a platelet-dependent manner. In contrast, neither intact NETs, reconstituted chromatin, individual nucleosome particles, nor octameric core histones reproduced any of these procoagulant effects. We conclude that unlike DNA or individual histone proteins, human intact NETs do not directly initiate or amplify coagulation in vitro. This difference is likely explained by the complex histone-histone and histone-DNA interactions within the nucleosome unit and higher-order supercoiled chromatin leading to neutralization of the negative charges on polyanionic DNA and modification of the binding properties of individual histone proteins.

Coagulation abnormalities of sickle cell disease: Relationship with clinical outcomes and the effect of disease modifying therapies.

Sickle cell disease (SCD) is a hypercoagulable state. Patients exhibit increased platelet activation, high plasma levels of markers of thrombin generation, depletion of natural anticoagulant proteins, abnormal activation of the fibrinolytic system, and increased tissue factor expression, even in the non-crisis "steady state." Furthermore, SCD is characterized by an increased risk of thrombotic complications. The pathogenesis of coagulation activation in SCD appears to be multi-factorial, with contributions from ischemia-reperfusion injury and inflammation, hemolysis and nitric oxide deficiency, and increased sickle RBC phosphatidylserine expression. Recent studies in animal models suggest that activation of coagulation may contribute to the pathogenesis of SCD, but the data on the contribution of coagulation and platelet activation to SCD-related complications in humans are limited. Clinical trials of new generations of anticoagulants and antiplatelet agents, using a variety of clinical endpoints are warranted.

Effective tranexamic acid concentration for 95% inhibition of tissue-type plasminogen activator induced hyperfibrinolysis in children with congenital heart disease: A prospective, controlled, in-vitro study.

BACKGROUND: Although recent studies have assessed tranexamic acid (TXA) pharmacokinetics in different subgroups, the effective concentration of TXA required to completely inhibit fibrinolysis remains to be determined. OBJECTIVE: An in-vitro determination of the effective TXA concentration needed for 95% inhibition (EC95) of tissue-type plasminogen activator (t-PA) activated fibrinolysis, using an experimental model designed for thromboelastometry (ROTEM). DESIGN: A prospective interventional study. SETTING: Department of Anaesthesiology, Queen Fabiola Children's University Hospital and Laboratory of Haematology and Haemostasis, Brugmann University Hospital. Patients were enrolled between June 2013 and October 2014. PATIENTS AND VOLUNTEERS: Twenty children, aged between 1 and 10 years, undergoing elective cardiac catheterisation were included (10 with cyanotic and 10 with noncyanotic diseases). Exclusion criteria were child requiring a procedure in a moribund state. Ten adult volunteers were also included as controls. INTERVENTION: Citrated whole blood samples were obtained from children and volunteers. MAIN OUTCOMES MEASURES: The extrinsic coagulation pathway was activated by tissue factor using the EXTEM test on ROTEM. The degree of lysis measured 30 min (LI30) after the clotting time (CT), and clot amplitudes measured at different times were recorded at baseline, after addition of 1535 units t-PA ml(-1), and following the addition of increasing TXA concentrations in t-PA activated samples. RESULTS: The concentration-effect analysis performed with lysis index after 30 min (LI30) allowed the determination of TXA efficacy concentration 50% (EC50), and calculation of the EC95, which was significantly lower in cardiac surgery children than in adults [8.6 µg ml(-1); 95% confidence interval (95% CI) 6.9 to 14.9 versus 11.3 µg ml(-1); 95% CI 10.6 to 12.9, P < 0.001]. CONCLUSION: In this in-vitro study, we observed that the EC95 TXA concentration that completely inhibited t-PA induced hyperfibrinolysis in children with congenital heart was significantly lower than the concentration required in healthy adult volunteers. Further studies are needed to confirm that this plasma concentration can effectively inhibit fibrinolysis activation in children undergoing cardiac surgery.

Sickle cell disease and venous thromboembolism in pregnancy and the puerperium.

Recent data strongly suggest an increased risk of venous thromboembolism in subjects with sickle cell disease and to a lesser extent, sickle cell trait. However, most studies have been retrospective, case-control or cross-sectional based on data obtained from administrative databases. More data from adequately powered prospective studies that include matched controls are needed to definitely establish the link between venous thromboembolism during pregnancy and sickle hemoglobin disorders. Similarly, there remains a need for properly designed randomized control trials to establish the safety of various hormonal contraceptive methods in women with sickle cell disorders.

Reduction of the six-minute walk distance in children with sickle cell disease is correlated with silent infarct: results from a cross-sectional evaluation in a single center in Belgium.

BACKGROUND: The 6-minute walk test (6 MWT) is used in adults and children affected by a wide range of chronic diseases to evaluate their sub-maximal exercise capacity. It reflects the global response of various physiological systems in a situation simulating a daily life activity. METHODS: We analyzed factors affecting the 6 MWT in 46 Sickle Cell Disease children. Forty-two were treated with hydroxyurea (HU). Patients with normal test (>80% of the age-standardized predicted value) were compared to patients with abnormal test (≤ 80%). Baseline hematological values, clinical events, cerebrovascular disease, cardio-pulmonary parameters and disease-modifying treatment were compared according to the performance of the test. RESULTS: Among the 46 patients, 14 had an abnormal 6 MWT. In univariate analysis, both groups were similar for biological and clinical data. Six of the 14 patients with an abnormal 6 MWT had silent infarct (SI) compared to 6/32 with a normal test (P = 0.09). When excluding chronically transfused patients, 4 of the 11 patients with an abnormal 6 MWT had SI compared to 1/26 (P = 0.02). Baseline pulse oximetry was normal in both groups but slightly lower in patients with abnormal 6 MWT (P = 0.02). No patient presented exercise-induced desaturation. In multivariate analysis, the only factor associated with abnormal 6 MWT was the presence of SI (P = 0.045). CONCLUSIONS: In our cohort of 46 patients characterized by high exposure rate to HU and by the absence of severe cardiopulmonary disease, the sole factor independently associated with 6 MWT was the presence of SI. The lower exercise capacity of children with SI may reflect some subclinical neurological impairment as they do not differ by hemoglobin level or cardiopulmonary parameters.