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

Contribution of in vitro thrombin generation in the study of coagulation abnormalities in sickle cell disease

La drépanocytose est associée à un état d’hypercoagulabilité qui se manifeste sur le plan clinique par un risque augmenté de thromboses artérielles et veineuses. L’exploration de la coagulation chez les patients drépanocytaires montrait surtout une activation de la coagulation et des altérations des acteurs pro- et anticoagulants du système hémostatique. Les tests de coagulation globale de routine tels que l’aPTT et le PT sont peu sensibles aux états d’hypercoagulabilité. La fonction hémostatique globale des patients drépanocytaires était donc peu connue. Le test de génération de thrombine est un test de coagulation globale, sensible aux états d’hypo- et d’hypercoagulabilité, facile à réaliser de nos jours avec une bonne reproductibilité. Nous l’avons utilisé pour démontrer que la coagulation globale des enfants drépanocytaires était caractérisée par une accélération des réactions de formation de la thrombine et par une augmentation du potentiel de thrombine endogène. Nous avons par la suite montré que les taux élevés de microparticules pro-coagulantes et du facteur VIII chez les enfants drépanocytaires seraient les facteurs déterminant l’accélération des réactions de formation de thrombine tandis que la réduction de l’activité anticoagulante du système protéine C / protéine S serait le facteur déterminant l’augmentation du potentiel de thrombine endogène. Les marqueurs de l’hémolyse corrélaient significativement avec ces facteurs ainsi qu’avec les paramètres de génération de thrombine, suggérant que l’hémolyse serait le mécanisme pathologique à la base de l’augmentation du potentiel de génération de thrombine chez les enfants drépanocytaires. Les paramètres de génération de thrombine n’étaient pas significativement différents entre l’état de stabilité clinique et l’état de crise vaso-occlusive. Chez les enfants hétérozygotes composites, ces paramètres avaient des valeurs intermédiaires entre celles des enfants contrôles et celles des enfants drépanocytaires homozygotes. Près de 40 % des enfants drépanocytaires homozygotes avaient un potentiel hémostatique supérieur à la moyenne + 2DS des enfants contrôles du même âge. Ces enfants présentant une génération de thrombine élevée se distinguaient des autres par leur plus jeune âge, une plus grande intensité de l’hémolyse, une plus courte durée de traitement par l’hydroxyurée et des vélocités du flux sanguin au doppler transcrânien plus élevées. Ces données suggèrent davantage un lien entre le potentiel de génération de thrombine et la vasculopathie cérébrale chez les enfants drépanocytaires. L’analyse de 4 enfants ayant reçu une greffe de cellules souches hématopoïétiques montrait une tendance à la réduction du potentiel de génération de thrombine et des autres altérations de la coagulation trois mois après la greffe. Le test de génération de thrombine permet une meilleure exploration de la coagulation globale des enfants drépanocytaires. Sa réalisation sur sang total permettrait une analyse plus globale en intégrant la participation des éléments figurés du sang particulièrement les globules rouges./Sickle cell disease is associated with a hypercoagulable state that express clinically by an increased risk of arterial and venous thrombosis. The exploration of coagulation in sickle cell patients showed mainly activation of coagulation and alterations pro-and anticoagulants actors of the hemostatic system. Routine global testing of coagulation such as the prothrombin time and the activated partial thromboplastin time are insensitive to hypercoagulable states. The overall hemostatic function in sickle cell disease was so little known. The thrombin generation test is a test of overall coagulation. It is sensitive to both hypo- and hypercoagulable states. It is easy to perform nowadays with good reproducibility. We used it to demonstrate that the overall coagulation of sickle cell disease children was characterized by an acceleration of the reactions of thrombin formation and an increase of the endogenous thrombin potential. We have subsequently shown that high levels of procoagulant microparticles and high levels of factor VIII in children with sickle cell disease are the factors determining the acceleration of reactions leading to thrombin formation. Our results also showed that the reduced activity of the protein C / S anticoagulant pathway is a determining factor of the increased endogenous thrombin potential in sickle cell children. Markers of hemolysis correlated significantly with these factors as well as the parameters of thrombin generation, suggesting that hemolysis is probably the pathological mechanism underlying the increased potential for thrombin generation in children with sickle cell disease. Nearly 40% of children with homozygous sickle cell disease had their hemostatic potential above the mean + 2SD that of controls children of the same age. These children with high thrombin generation differed from others by their younger age, greater intensity of hemolysis, a shorter duration of treatment with hydroxyurea. They also had higher velocity of blood flow using transcranial Doppler. These data further suggest a potential link between thrombin generation and cerebral vasculopathy in children with sickle cell disease. Analysis of four children who received hematopoietic stem cells transplantation showed a tendency towards a reversibility of thrombin generation and other alterations of coagulation three months after the transplant. Thrombin generation assay allows a better exploration of the global coagulation of sickle cell disease children. Its realization on whole blood would be a more comprehensive analysis as it would integrate the participation of blood cells particularly red blood cells.Doctorat en Sciences biomédicales et pharmaceutiquesinfo:eu-repo/semantics/nonPublishe

Evaluation of three APTT reagents in a routine laboratory: toward a compromise.

Our current activated partial thromboplastin time (APTT) reagent (PTT-A) is often prolonged for unexplained reasons.Journal ArticleSCOPUS: ar.jinfo:eu-repo/semantics/publishe

Test de génération de thrombine: importance d'établir les valeurs de référence en fonction de l'âge et des concentrations en facteur tissulaire avant l'implémentation au laboratoire.

The calibrated and automated thrombinography (CAT) developed by H.C. Hemker is a simple and reproducible technique that can be potentially used in coagulation laboratories. This test is able to record the complete thrombin generation in vitro, giving an interesting approach in the evaluation of the haemostatic potential at the individual level. We aimed to implement this test in our laboratory to follow patients with haemorrhagic or thrombotic pathologies. Haemorrhagic and thrombotic disorders are incompletely explored by the coagulation tests used presently in routine labs. These tests don't indeed reflect the real haemostatic phenotype of the patient neither the individual response to haemostatic treatments. Furthermore, they don't have any predictive value for the occurrence of haemorrhage and/or thrombosis. We report here reference values we established in a population of children and adults in pre-analytical conditions easily applicable in coagulation labs. Platelet poor plasma is prepared by a double centrifugation and analyzed immediately or frozen at -80 degrees C for delayed analysis.English AbstractJournal ArticleSCOPUS: ar.jinfo:eu-repo/semantics/publishe

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.info:eu-repo/semantics/publishe

Evaluation of the procoagulant activity of endogenous phospholipids in the platelet-free plasma of children with sickle cell disease using functional assays.

The mechanisms of hypercoagulability in sickle cell disease (SCD) are poorly understood.Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe

Transient leukemia in a newborn without Down syndrome: case report and review of the literature.

Transient neonatal leukemia occurs almost exclusively in Down syndrome babies. We report here the unusual case of a newborn without Down syndrome who presented neonatal transient leukemia and who achieved spontaneously complete remission. Trisomy 21 and GATA1 mutation were both present in leukemic cells. While close follow-up is advised since true leukemia may develop later, the patient is still in remission for 2.5 years. We performed a literature review of 15 other similar cases. Conclusion: Our case of transient leukemia without Down syndrome and the literature review highlight the important role of trisomy 21 and GATA1 mutation in the development of transient neonatal leukemia.JOURNAL ARTICLESCOPUS: ar.jSCOPUS: ar.jinfo:eu-repo/semantics/publishe

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 alpha(2)-antiplasmin. These assays should be further evaluated for the detection of hypo- or hyperfibrinolysis in acquired thrombotic or hemorrhagic disorders