Trauma-induced coagulopathy (TIC) is a recently described condition which traditionally has been diagnosed by the common coagulation tests (CCTs) such as prothrombin time/international normalized ratio (PT/INR), activated partial thromboplastin time (aPTT), platelet count, and fibrinogen levels. The varying sensitivity and specificity of these CCTs have led trauma coagulation researchers and clinicians to use Viscoelastic Tests (VET) such as Thromboelastography (TEG) to provide Targeted Thromboelastographic Hemostatic and Adjunctive Therapy (TTHAT) in a goal directed fashion to those trauma patients in need of hemostatic resuscitation. This review describes the utility of VETs, in particular, TEG, to provide TTHAT in trauma and acquired non-trauma-induced coagulopathy.

Acute Upper Gastrointestinal Bleeding (AUGIB) accounts for 7000 deaths in the UK annually and is the single leading indication for transfusion of blood components. A large UK audit in 2007 reported high case fatality and rates of further bleeding. Since many deaths are determined by pre-existing co-morbidity, strategies to improve outcome should be targeted at preventable deaths and therefore focus upon improved control of haemorrhage and prevention of further bleeding, which are investigated in this thesis. Data for the analyses presented originate from the UK national audit of AUGIB, a laboratory study and a cross sectional survey. Five broad themes were investigated including service provision and timing of endoscopy, the use of transcatheter arterial embolisation (TAE) or surgery for refractory bleeding, the impact of coagulopathy on outcome, management of acute variceal haemorrhage (AVH) and haemostatic derangements after AVH, and the use of red blood cells (RBCs). Although there was no evidence of a “weekend effect” for mortality, earlier endoscopy (<12 hours) was associated with improved control of haemorrhage in higher risk patients compared to later endoscopy (>24 hours). TAE was an effective and safe modality for refractory bleeding, but the high post-surgical mortality (29%) raises questions about the appropriateness of case selection for surgery. Coagulopathy after non-variceal haemorrhage was associated with a 5-fold increase in risk-adjusted mortality. Further bleeding after AVH was strikingly high (26%) with notable deficiencies in the use of vasopressors, antibiotics and endotherapy. Global assessments of coagulation demonstrated that thrombin generation after AVH was normal, but clot strength was poor with excessive fibrinolysis. Platelets, fibrinogen and antifibrinolytics improved haemostasis ex vivo but coagulation factor transfusion had no effect. RBC transfusion practice is variable. This work on AUGIB provides new data highlighting areas of sub-optimal care, and informs both current practice and research questions for new interventional trials.

No description available.

Haemorrhage is a leading cause of trauma morbidity and mortality, with many deaths potentially preventable. Hyperfibrinolysis is a central characteristic of trauma-induced coagulopathy (TIC) which develops rapidly and is associated with poor outcomes. Tranexamic acid (TXA) improves survival in trauma haemorrhage but its uptake worldwide remains variable, in part because its effects on the coagulation system during trauma haemorrhage have not been described. Further uncertainty regarding patient selection for TXA therapy has emerged following the description of an early viscoelastic haemostatic assay (VHA) diagnosed hypofibrinolytic phenotype in whom TXA may potentiate thrombotic complications. The patient characteristics and mechanisms leading to this apparent hypofibrinolytic phenotype are poorly understood. Over 900 trauma patients prospectively recruited to a multicentre observational cohort study had blood drawn within 2-hours of injury for VHA and fibrinolysis plasma protein analysis. Patients were categorised according to VHA maximum lysis (ML) and D-dimer (DD) levels. Patients with MLLOW exhibited heterogeneity in clinical and injury characteristics and outcomes. Those who died were severely injured, with a high incidence of traumatic brain injury and a 7-fold higher D-dimer. Patients with MLLOW+DDHIGH had a hyperfibrinolytic biomarker profile, with the fibrinolytic mediator S100A10 identified as a potential driver of fibrinolysis, which can ex-vivo artificially reduce ML. Empiric TXA could benefit this occult hyperfibrinolytic phenotype. Over two subsequent observational studies, the effects of TXA on the coagulation system during trauma haemorrhage and the effect of TXA infusion and timing of treatment on thrombotic events were investigated. Early empiric TXA avoided VHA-hyperfibrinolysis and provided a degree of protection from TIC. Whilst univariate analysis suggested increased thromboses with later TXA treatment in patients receiving TXA bolus+infusion, neither the TXA infusion nor time to bolus were associated with thrombotic events after multivariate analysis. A single TXA bolus may provide a lower effective therapeutic dose with reduced complications.

京都大学 / 新制・課程博士 / 博士(人間健康科学) / 甲第24292号 / 人健博第107号 / 新制||人健||8(附属図書館) / 京都大学大学院医学研究科人間健康科学系専攻 / (主査)教授 藤井 康友, 教授 岡 昌吾, 教授 滝田 順子 / 学位規則第4条第1項該当 / Doctor of Human Health Sciences / Kyoto University / DFAM

No description available.

Background Haemodilution and hypothermia induce coagulopathy separately, but their combined effect on coagulation has not been widely studied. Fibrinogen concentrate can correct dilutional coagulopathy and has an additional effect when combined with factor XIII concentrate. However, their effect on dilutional coagulopathy concomitant with hypothermia has not been studied previously. Free oscillation rheometry – FOR (Reorox®) – is a novel viscoelastic haemostatic assay that has not been studied in this context before. Methods Blood from 10 healthy volunteers was diluted by 33% with hydroxyethyl starch or Ringer’s acetate solutions. Effects of fibrinogen added in vitro with and without factor XIII were studied at 33°C and 37°C. Coagulation velocity (coagulation time) and clot strength (elasticity) were assessed with FOR. Coagulation was initiated in vitro with thromboplastin alone, or thromboplastin plus a platelet inhibitor. Results Hydroxyethyl starch increased the coagulation time and decreased clot strength significantly more than Ringer’s acetate solution, both in the presence and absence of a platelet inhibitor. There was a significant interaction between haemodilution with hydroxyethyl starch and hypothermia, resulting in increased coagulation time. After addition of fibrinogen, coagulation time shortened and elasticity increased, with the exception of fibrinogen-dependent clot strength (i.e., elasticity in the presence of a platelet inhibitor) after hydroxyethyl starch haemodilution. Factor XIII had an additional effect with fibrinogen on fibrinogen-dependent clot strength in blood diluted with Ringer’s acetate solution. Hypothermia did not influence any of the coagulation factor effects. Conclusions Both haemodilution and mild hypothermia impaired coagulation. Coagulopathy was more pronounced after haemodilution with hydroxyethyl starch than with Ringer’s acetate. Addition of fibrinogen with factor XIII was unable to reverse hydroxyethyl starch induced clot instability, but improved coagulation in blood diluted with Ringer’s acetate solution. Fibrinogen improved coagulation irrespective of hypothermia. / <p>Funding Agencies|Region Skane (Sweden)||CSL Beehring||</p>

La Malaltia de von Willebrand (VWD) és la coagulopatia congènita més freqüent a la població general. Consisteix en una diàtesi hemorràgica causada per una deficiència qualitativa i/o quantitativa del factor de von Willebrand (VWF) que es transmet amb caràcter autosòmic dominant o, menys freqüentment, recessiu. El VWF és una glicoproteïna adhesiva present en plaquetes, cèl•lules endotelials i megacariòcits que té diferents funcions donat que participa en l’hemostàsia primària i col•labora al mateix temps en la secundària. És un mediador de l’adhesió de les plaquetes al subendotel•li en el lloc de la lesió vascular i transporta al FVIII, al que protegeix de la degradació proteolítica prematura. El gen del VWF (VWF) s’extén unes 178 kilobases en el genoma i conté un total de 52 exons, sent un dels gens més grans i complexos descrits en humans. Addicionalment existeixen una sèrie de factors que dificulten de manera considerable la caracterització molecular de la VWD i que han fet que la seqüenciació directa no s’hagi considerat el mètode de referència per al seu diagnòstic. En primer lloc, el VWF és un gen altament polimòrfic i, fins al moment, s’hi han descrit 102 SNPs (Build 132) en regió codificant, el que pot dificultar la identificació de les mutacions i, en segon lloc, existeix un pseudogèn parcial al cromosoma 22 d’aproximadament 30 kb molt homòleg (>96%) als exons 23-34 del VWF. Amb l’objectiu de facilitar l’anàlisi genètic de la VWD, s’ha dissenyat un procediment simplificat basat en la seqüenciació completa del gen, que s’ha utilitzat per identificar la mutació en un total de 40 famílies i demostra la seva validesa com a mètode rutinari de diagnòstic molecular. Amb l’aplicació d’aquest mètode s’han identificat un total de 58 mutacions (41 diferents), 19 de las quals no s’havien descrit prèviament a la literatura. Entre els diferents tipus de mutació responsables de la VWD, aquelles que modifiquen la regió codificant del gen tenen un clar efecte deleteri, però les conseqüències de les mutacions que afecten potencialment l’splicing (PSSM) són menys evidents. Amb l’objectiu d’estudiar l’efecte d’aquestes mutacions s’ha desenvolupat un mètode per a la seqüenciació completa del cDNA del VWF en leucòcits i plaquetes que ens ha permès revelar l’efecte de diverses PSSM. L’aparició de les plataformes de seqüenciació de nova generació (NGS), que són fins 200 vegades més ràpides i econòmiques que la seqüenciació tradicional, ha plantejat nous reptes en el diagnòstic molecular de les malalties hereditàries. Per això s’ha desenvolupat una nova estratègia d’amplificació del gen en un total de 14 PCRs llargues i hem adaptat el procediment desenvolupat prèviament per a la seqüenciació completa del VWF a les noves plataformes de NGS. Aquestes estratègies permetran l’anàlisi simultània d’un gran nombre de mostres de pacients i familiars de manera més ràpida i econòmica que per seqüenciació tradicional. Amb l’objectiu de recopilar tota la informació generada a partir del diagnòstic molecular dels pacients amb VWD i fer-la accessible, hem dissenyat un nou apartat dins d’Hemobase (registre de mutacions per a les Hemofílies A i B) dedicat a la VWD (www.vwf.hemobase.com). Aquesta pàgina d’accés lliure per Internet, conté un registre de les mutacions identificades en pacients amb VWD després de la seqüenciació directa del VWF. El registre permet realitzar cerques, relacionar qualsevol mutació amb la base de dades internacional i accedir directament a les publicacions corresponents. Es pretén que l’estudi molecular dels pacients permeti una millor comprensió dels mecanismes implicats en la fisiopatologia de la malaltia i ofereixi una visió més àmplia de l’epidemiologia molecular a la nostra població. / Von Willebrand Disease (VWD) is the most frequent congenital coagulopathy in the general population. It has been proved to be particularly complex due to a series of factors that make difficult the molecular diagnostic of the disease: the von Willebrand Factor gene (VWF) is large and complex; it is very polymorphic; there is a partial pseudogene in chromosome 22 highly homologous (>96%) to a region of the VWF; and the existence of other genes implied in the disease cannot be discarded. All this set of difficulties causes that the molecular study of the VWD remains confined to basic investigation and the application to the clinical routine has been considerably delayed. With the aim to facilitate the genetic study of the VWD, we designed and optimized a procedure for direct sequencing of the VWF, that allowed us to study 40 families identifying 58 mutations (41 different), 19 of which were new. Among the different types of mutation that cause VWD, those affecting the coding region have frequently a clear deleterious effect; however, the consequences of the potential splice site mutations (PSSM) are less predictable. A method for the complete sequencing of the VWF cDNA in leukocytes and platelets has been developed allowing the elucidation of the effect of several PSSM studied. Next Generation Sequencing (NGS) platforms are faster and cheaper that the traditional sequencing. In order to take advantage of this new technology, we have developed an optimized strategy for the amplification of the VWF by LR-PCRs and we have tailored the previously developed short PCR procedure. The objective is to achieve high performance in the molecular characterization of VWD patients and relatives and to establish the basis for a large-scale molecular study approach. Finally, a database of the mutations identified, responsible for the pathology (www.vwf.hemobase.com), has been established in order to correlate molecular and clinical parameters. The development of suitable tools for the molecular diagnosis of VWD will significantly facilitate the clinical diagnosis and will guide the clinician towards the better therapeutic option.

Au cours de l’infection par le virus Ebola (EBOV), plusieurs glycoprotéines solubles sont massivement libérées à partir de cellules infectées mais le rôle précis de ces protéines virales n’a pas encore été identifié. Nous émettons l'hypothèse que l'altération de l’hémostase et du système et vasculaires observée au cours de l'infection à virus Ebola pourrait être, au moins en partie causée par ces glycoprotéines solubles. Ainsi pour la première fois, nous avons identifié les cibles cellulaires de la protéine soluble « shed GP » d’Ebola et nous avons démontré que sa partie glycosylé peut activer les cellules dendritiques et les macrophages non infectés, induisant, par le récepteur TLR4, la sécrétion de cytokines pro-inflammatoires. Nous démontrons aussi que l'activité de la shed GP est neutralisé lors de l'addition de la MBL, une protéine connue pour interagir avec certains motifs glycosylés présents à la surface de différents micro-organismes. Nous avons également montré que la shed GP active la perméabilité des HUVEC de façon directe et indirecte, via la libération de cytokines. En conclusion, cette étude suggère que la shed GP peut être l'un des principaux facteurs responsables de la stimulation précoce des cellules immunitaires qui produisent alors de grandes quantités de cytokines pro-inflammatoires, des éléments qui, combinés avec la réplication massive du virus et les dommages cellulaires induits par le virus, peuvent conduire à un syndrome de type choc septique et une mortalité élevée. / During Ebola virus (EBOV) infection several soluble glycoproteins are released in high amounts from infected cells but as yet still no clear role has been identified for these viral proteins. We hypothesized that the impairment of coagulation and vascular systems observed during EBOV infection could be, at least in part, due to these soluble glycoproteins.Here, for the first time we identify the cellular targets of EBOV soluble protein shed GP and provide evidence that through its glycosylation, shed GP can activate non-infected dendritic cells and macrophages, inducing, through TLR4, the secretion of pro-inflammatory cytokines. We also demonstrate that shed GP activity is negated upon addition of Mannose-Binding sera Lectin (MBL), a molecule known to interact with sugar arrays present on the surface of different microorganisms. We have also revealed that shed GP activates permeability of HUVECs both directly and indirectly through cytokine release. Overall, this study suggests that shed GP may be one of the principal factors responsible for the early stimulation of immune cells that then produce high amounts of proinflammatory cytokines that, combined with massive virus replication and virus-induced cell damage, can lead to a septic shock-like syndrome and high mortality.

Ce travail porte sur le rôle des microparticules procoagulantes (MPs) générées par les cellules vasculaires en réponse à un état septique. Après une introduction rappelant la structure et les propriétés des microparticules et la réponse del’hôte à un agent pathogène, en particulier en terme d’activation de la coagulation, nous rapportons nos travauxexpérimentaux et cliniques. Le premier travail a été réalisé sur un modèle cellulaire de vésiculation induite par le LPS. Il nous a permis de caractériser le transfert du complexe CD14/TLR4 à différents types cellulaires in-vitro dépourvus du récepteur au LPS. Ainsi, les MPs monocytaires pourraient avoir un rôle d’amplification de la réponse inflammatoire mais aussi dans la réponse anti-inflammatoire secondaire en participant à l’apoptose lymphocytaire. Le second travail aété réalisé chez l’animal. Après induction d’un choc septique, nous avons observé une amélioration hémodynamique enréponse à la perfusion de protéine C activée associée à une modulation du phénotype des MPs. Réinjectées à des ratsnaïfs, les MPs issues des rats septiques traités par protéine C activée développaient une moindre vasoplégie. Enfin, nous avons réalisé une étude prospective sur 100 patients en choc septique. Nous avons ainsi pu caractériser la présence d’une concentration élevée de microparticules procoagulantes, avec une variation phénotypique en présence de coagulation intravasculaire disséminée (CIVD) : réduction du contingent plaquettaire au profit des MPs d’origine leucocytaires qui deviennent prépondérantes et témoignent d’une activation leucocytaire accrue, et surtout une activation des cellules endothéliales avec génération de MPs porteuses d’endogline (CD105). En analyse multivariée,CD105+-MPs étaient fortement associée à la CIVD et pourraient constituer un marqueur précoce de l’atteinte endothéliale au cours du choc septique. / This work focused on procoagulant microparticles shed after vascular cells stress during sepsis. The first part gives an overview on MPs and host response during pathogen challenge. The first lab experimental work confirms direct and functional transfer of CD14/TLR4 LPS sensor by MPs shed to target cells after monocytic THP-1 challenge by LPS.CD14-MPs amplify LPS-induced apoptosis in monocytes but also prompted lymphocyte apoptosis and could play a role in secondary anti-inflammatory response. Then, septic shock was induced in rats after caecal ligature and puncture.Activated protein C (APC) infusion improved haemodynamic parameters and alter septic microparticular content. Infused in naïve rats, APC-treated MPs were associated with reduced hypotension and inflammatory response, confirming cytoprotective effect of both APC and APC-induced MPs. Finally, we performed a clinical prospective study in 3 medical ICU in France. Patients referred for septic shock had an increased level of circulating procoagulant MPs regardless disseminated intravascular coagulopathy (DIC) diagnosis. Nevertheless, DIC patients evidenced a specific pattern with lower platelet-MPs, increased leucocyte-MPs and specific endothelial cells activation with endoglin (CD105) shedding. In multiple logistic regression analysis, CD105-MPs were strongly associated with DIC and were evidenced before DIC diagnosis according to routine laboratory assays.