The haemorrhagic disorders form only a small, although often most alarming part of the diseases inflicted upon mankind, and in spite of the enthusiasm of the disproportionally great number of workers investigating the problem of the coagulation of the blood, this problem until quite recently could only be looked upon as being mainly of academic physiological interest. With the advent and daily increasing use of the highly dangerous anti-coagulant therapy for thrombosis and embolism, demanding at least a general understanding of the mechanism of blood clotting, every aspect of this mechanism has become of practical clinical importance, and no serious effort to clarify the question can be considered wasted. It is peculiar that the last phase of coagulation, the contraction of the clot, which is by no means the least spectacular, has received rather scanty attention compared to the enormous amount of work that has been expended on the earlier stages. No agreement exists with regard to the mechanism or purpose of clot retraction; the factors controlling it are to a large-extent unknown, and even the fundamental question of the part played by the platelets is undecided.

Les accidents vasculaires cérébraux (AVC) ischémiques, l’infarctus du myocarde (IDM) et l’embolie pulmonaire (EP) sont la première cause de mortalité et de morbidité dans le monde. Leur prise en charge est limitée à la recanalisation des vaisseaux occlus par le thrombus. La thrombolyse intraveineuse par le rt-PA (l’activateur tissulaire du plasminogène recombinant) est le seul traitement médicamenteux utilisé dans ces situations d’urgences vasculaires. En dépit de ces bénéfices, cette thérapie s’entrave de plusieurs effets délétères principalement d’un risque d’hémorragie cérébrale. Augmenter le rendement fibrinolytique du rt-PA via sa vectorisation sans augmenter ses effets délétères serait d’un grand intérêt. Le Fucoidane, un polysaccharide anionique extrait principalement des algues brunes, ayant une affinité de l’ordre du nanomolaire à la P-sélectine, a démontré un ciblage spécifique du thrombus in vivo. Dans notre travail, nous avons développé une molécule bipolaire associant le Fucoidane comme vecteur au thrombus, aminé par des lysines en N–terminales pour lier le rt-PA exogène. Le potentiel de vectorisation du t-PA par le Fucoidane aminé ainsi que la preuve du concept ont été validé par notre travail. Nous avons montré sur des modèles de thrombi in vitro une accélération de lyse des thrombi, et in vivo une augmentation de l’efficacité thrombolytique du t-PA, couplé au Fucoidan aminé, par injection périphérique. / Acute ischemic stroke (AIS), acute myocardial infarction (AMI), and pulmonary embolism (PE) are the main cause of mortality and morbidity worldwide. Thrombolysis by intravenous injection (IV) of recombinant tissue plasminogen activator (rt-PA) remains the most common non-interventional treatment to recanalize vessels occluded. However, Its use is limited by significant drawbacks, including bleeding complications. Recent studies showed that Fucoidan (sulfated polysaccharides extracted from brown algae) target in vivo intraluminal thrombus.In this study, we have developed a bipolar construct associating Fucoidan, able to target the thrombus, and a NH2 exposing lysine platform able to bind to t-PA. We hypothesize that this construct was able to vectorize t-PA to the thrombus, would increase its fibrinolytic efficacy and avoid its deleterious effects. In vitro assays, t-PA complexed to amino Fucoidan has shown its over thrombolytic superiority as compared to tPA alone. In vivo mesenteric, arterial and deep vena cava thrombosis and thrombolysis, vascular reperfusion was significantly increased in mice treated with amio Fucoidan complexed to t-PA as compared in mice treated by t-PA alone. This data demonstate that amino Fucoidan is an excellent vector of t-PA to the thrombus.

A substrate was developed for measuring the milk clotting strength of rennet preparations on an automatic clot-timer . The substrate contained 8 percent pasteurized skim milk solids, 1 percent chloroform, 0.3 percent 200 bloom gelatin, 0.03 M added CaCl2 and was buffered to pH 6.6 with 0.057 M cacodylic acid and 0.042 M triethanolamine. The substrate was shelf-stable for 18 days at room temperatures. It was found that rennet preparations could be standardized to within 1 percent of each other, in terms of milk clotting strength, by use of the substrate on the automatic clot-timer. The method appears to have advantages over conventional rennet standardizing procedures. The research included studies on the effect of chloroform, nonfat dry milk and CaCl2 concentrations; heat and ionizing radiation on the substrate coagulation time.

Many pathogen infections in nature are accompanied by injury and subsequent coagulation. Despite the contribution of hemolymph coagulation to wound sealing, little is known about its immune function. Based on the molecular knowledge of Drosophila innate immunity, this thesis investigated the immune function of clot both in vitro and in vivo, the immune relevant genes involved in a natural infection model, involving entomopathgenic nematodes (EPN) and the factors leading to crystal cell activation. Transglutaminase (TG) and its substrate Fondue (Fon) have been identified as bona fide clot components in Drosophila larvae. By knocking down TG or Fon via RNAi, we observed an increased susceptibility to EPN in larvae. In addition, this increased susceptibility was associated with an impaired ability of hemolymph clots to entrap bacteria. Immunostaining revealed that both clot components (Fon and TG) were able to target microbial surfaces. All these data suggest an immune function for the Drosophila hemolymph clot. Strikingly, similar results were obtained when we ran parallel experiments with human FXIIIa, an ortholog of Drosophila TG, indicating a functional conservation. We also found evidence for the regulation on both clot and immunity by eicosanoids in Drosophila larvae. The combination of EPN infection with the Drosophila model system allowed us to discover an immune function for TEP3 and Glutactin. However the molecular mechanism underlying the involvement of these two proteins in this particular host-pathogen interaction remains to be elucidated. Prophenoloxidase, the proform of enzyme involved in hardening the clot matrix, has been shown to be released by rupture of crystal cells. This cell rupture is dependent on activation of the JNK pathway, Rho GTPases and Eiger. Our work further identified the cytoskeletal component, Moesin, and the cytoskeletal regulator Rac2 as mediators of cell rupture. Despite the possible role of caspases in crystal cell activation, such cell rupture was turned out to be different from apoptosis. The implication of Rab5 in this process indicated that proper endocytosis is required for cell activation and subsequent melanization. Our findings furthered not only our understanding of the release of proPO via cell rupture but also our knowledge on different paths of immune cell activation. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 1: In press. Paper 4: Manuscript.<strong></strong></p>

Diss. (sammanfattning) Linköping : Linköpings universitet, 2008. / Härtill 5 uppsatser. Includes bibliographical references.

Elevated plasma concentrations of lipoprotein(a) (Lp(a)) are associated with increased risk of atherothrombotic disease. Lp(a) is a unique lipoprotein consisting of a low density lipoprotein-like moiety covalently linked to apolipoprotein(a) (apo(a)), a homologue of the fibrinolytic proenzyme plasminogen. Apo(a) is extremely heterogeneous in size with small isoforms being independently associated with increased cardiovascular risk. Several in vitro and in vivo studies have shown that Lp(a)/apo(a) can inhibit tissue-type plasminogen activator (tPA)-mediated plasminogen activation on fibrin surfaces, although the mechanism of inhibition by apo(a) remains controversial. Essential to fibrin clot lysis are a number of plasmin-dependent positive feedback reactions that enhance the efficiency of plasminogen activation, including the plasmin-mediated conversion of Glu1-plasminogen to Lys78-plasminogen. Additionally, abnormal fibrin clot structures have been associated with both an increased risk of cardiovascular disease and elevated Lp(a) levels. Similarly, oxidized phospholipids have been implicated in the development of cardiovascular disease, and are not only preferentially carried by Lp(a) in the plasma but have also been shown to covalently-modify both apo(a) and plasminogen. In this thesis, we built upon the understanding of the role of apo(a) in plasminogen activation on the fibrin/degraded fibrin surface by determining that: (i) apo(a) inhibits plasmin-mediated Glu1-plasminogen to Lys78-plasminogen conversion and identifying the critical domains in apo(a) responsible for this effect, (ii) apo(a) isoform size does not affect either the inhibition of tPA-mediated plasminogen activation or the inhibition of plasmin-mediated Glu1-plasminogen to Lys78-plasminogen conversion, (iii) apo(a) modifies fibrin clot structure to form more dense clots with thinner fibers and reduced permeability, modifications that enhance the ability of apo(a) to inhibit tPA-mediated plasminogen activation and (iv) the phosphorus content of apo(a) affects its ability to inhibit tPA-mediated plasminogen activation and the phosphorus content of plasminogen affects its ability to be activated by tPA. By understanding these individual reactions, each of which has the potential to affect the broader fibrin clot lysis process, we have expanded our understanding of the overall effect of Lp(a)/apo(a) in the inhibition of plasminogen activation on the fibrin/degraded fibrin surface and thus broadened our understanding of how Lp(a)/apo(a) may mediate the inhibition of thrombolysis in vivo. / Thesis (Ph.D, Biochemistry) -- Queen's University, 2011-12-14 08:26:54.99

Diss. (sammanfattning) Linköping : Linköpings universitet, 2008. / Härtill 5 uppsatser.

Un certain nombre d'essais cliniques contrôlés, randomisés et publiés récemment en littérature a démontré que la thrombectomie mécanique, offerte aux patients présentant un AVC ischémique aigu, est liée à une meilleure évolution clinique en comparaison au traitement standard de fibrinolyse intraveineuse. Les stents retriever ont été reconnus dans ces essais comme les dispositifs les plus efficaces pour la thrombectomie intracrânienne. Actuellement, toutes les industries produisant des dispositifs neuro-interventionnels lancent sur le marché un nombre croissant de stents retriever. Chaque nouveau dispositif proposé est censé avoir une particularité permettant de meilleures performances par rapport aux dispositifs déjà disponibles sur le marché. Néanmoins, aucune étude clinique n’a démontré, jusqu'à présent, la supériorité en termes de résultats anatomiques et cliniques d'un stent retriever donné. En outre, le mécanisme d'interaction entre les stents retriever et le thrombus n'a pas été évalué jusqu'ici de façon exhaustive. Dans la présente étude, nous avons analysé expérimentalement les performances de tous les stents retriever disponibles sur le marché français jusqu'à juin 2015. Le but de cette étude était d'identifier toutes les caractéristiques des dispositifs fonctionnels à la capture du thrombus. Chaque dispositif a été évalué par des tests mécaniques et fonctionnels : les tests mécaniques ont été effectués afin d'étudier la force radiale des dispositifs. L'objectif était d'évaluer la force radiale exercée par le stent dans deux conditions spécifiques : lors du déploiement et pendant le retrait.Les tests fonctionnels ont visé à évaluer visuellement la capacité du stent à rester en apposition sur la paroi des vaisseaux et à maintenir le thrombus à l'intérieur de ses mailles au cours du retrait. Nous avons évalué l'interaction des dispositifs avec thrombus de taille et de caractéristiques différentes que nous avons générées en utilisant du sang humain afin d'obtenir deux types de caillot : un souple « de type rouge » composé par tous les éléments du sang et un dur « de type blanc» qui a été principalement composé de plasma riche en plaquettes. Ces essais ont été effectués en utilisant un modèle vasculaire rigide reproduisant la circulation cérébrale antérieure. Deux neuro-interventionnels ayant une expérience dans les procédures de thrombectomie ont effectué les tests fonctionnels. Chaque expérience a été filmée et deux auteurs par la suite ont effectué une analyse visuelle des résultats.Les essais mécaniques ont montré un comportement différent en termes de variation de pression radiale au cours du retrait pour chaque stent. Une pression radiale constante pendant le retrait est liée à une cohésion constante sur la paroi artérielle pendant le retrait, avec un taux plus important de retrait du caillot. Tous les stents retriever glissent sur le caillot blanc de grande taille (diamètre>6 mm) ayant un très bas taux d’efficacité en termes de retrait. / A number of randomized controlled trials recently appeared in literature demonstrated that early mechanical thrombectomy offered to patients presenting with acute ischemic stroke is related to improved functional outcome in comparison to standard care intravenous fibrinolysis. Stent retrievers have been recognized in these trials as the most effective devices for intracranial thrombectomy. Currently, all industries producing neuro-interventional devices are launching into the market an increasing number of stent-based retriever tools. Each new device proposed for clinical use is supposed to have peculiar features allowing better performances in comparison to devices already available for clinical practice. Nevertheless, none clinical study has demonstrated so far the superiority, in terms of anatomical and clinical results, of a given stent retriever device. Furthermore, the mechanism of interaction between stent retrievers and thrombi has not exhaustively evaluated so far. In the present study we experimentally analyzed performances of all stent retrievers available into the French market up to Juin 2015. The aim of this study was to identify any device feature that was functional to the thrombus removal.Stent retrievers were evaluated by mechanical and functional test: mechanical tests were performed in order to investigate devices radial force, the aim was to evaluate the radial force exerted by the stent in two specific conditions: upon deployment and during the retrieval.Functional tests were aimed to visually evaluate the stent retriever’s ability in remaining in close apposition to the vessels wall and to maintain the thrombus engaged within its struts during the retrieval. We evaluated the interaction of the devices with thrombi of different features and sizes that we generated using human blood in order to obtain two types of clot: one softer “red type” that was composed by all elements of the whole blood and one stiffer “white type” that was mainly composed by platelet-rich plasma. Such tests were conducted using a rigid 3D printed vascular model reproducing the brain anterior circulation. Two neuro-interventionalists with experience in thrombectomy procedures performed functional tests, each experiment was filmed and two authors thereafter conducted visual analysis of the results.Mechanical tests showed different behavior in terms of radial pressure variation during retrieval for each stent. Constant radial pressure during retrieval was related to constant cohesion over the vessel wall during retrieval and higher rate of clot removal efficacy. All stent retrievers slide over the clot failing in clot removal when interact with white large thrombi (diameter>6 mm).

We detail for the first time the uniquely altered fibrin polymerization of homophenotypic Aα R16H dysfibrinogen. By polymerase chain reaction amplification and DNA sequencing, our new proposita's genotype consisted of a G>A transition encoding for Aα R16H, and an 11 kb Aα gene deletion. High-performance liquid chromatography disclosed fibrinopeptide A release approximately six times slower than its fibrinopeptide B. Turbidimetric analyses revealed unimpaired fibrin repolymerization, and abnormal thrombin-induced polymerization (1-7 μmol/l fibrinogen, > 96% coagulable), consisting of a prolonged lag time, slow rate, and abnormal clot turbidity maxima, all varying with thrombin concentration. For example, at 0.2-3 U/ml, the resulting turbidity maxima ranged from lower to higher than normal control values. By scanning electron microscopy, clots formed by 0.3 and 3 thrombin U/ml displayed mean fibril diameters 42 and 254% of the respective control values (n = 400). Virtually no such differences from control values were demonstrable, however, when clots formed in the presence of high ionic strength (μ = 0.30) or of monoclonal antiβ(15-42)IgG. The latter also prolonged the thrombin clotting time approximately three-fold. Additionally, thrombin-induced clots displayed decreased elastic moduli, with G′ values of clots induced by 0.3, 0.7 and 3 thrombin U/ml corresponding to 11, 34, and 45% of control values. The results are consistent with increased des-BB fibrin monomer generation preceding and during polymerization. This limited the inherent gelation delay, decreased the clot stiffness, and enabled a progressively coarser, rather than finer, network induced by increasing thrombin concentrations. We hypothesize that during normal polymerization these constitutive des-BB fibrin monomer properties attenuate their des-AA fibrin counterparts.

Coagulopathy, a condition in which blood coagulation is impaired, can be inherited or result from a variety of conditions including severe trauma, illness or surgery. Perioperative monitoring of a patient’s coagulation status is important to identify coagulopathic patients. Thromboelastography or TEG remains the gold standard for whole blood coagulation monitoring. However, TEG suffers from certain well-documented drawbacks such as contact containment and manipulation of the blood sample, large and uncontrolled strain, and the inability to distinguish the contribution of elasticity and viscosity during blood coagulation. We developed a non-contact blood rheometer which uses a single drop of blood to measure its viscoelastic properties. Small sample size (typically 5-15 μL), low shear strain (linear viscoelasticity), and non-contact manipulation and containment of samples make this technique unique for real-time monitoring of blood coagulation. In the first part of this work, we addressed the development of the technique, benchmarking the results against known material properties standards. We observed large amplitude oscillations of the levitated drop results in multiple resonance modes and excessive dissipation. We suggested upper bound limits for drop oscillation amplitudes required to satisfy the Lamb theoretical expressions for drop frequency and damping. In the second part, we applied our technique to study sickle-cell disease. Our technique showed that the shape oscillation of blood drops was able to assess an abnormally increased viscosity in sickle cell patients when compared with normal controls over a range of hematocrit. Furthermore, the technique was sensitive enough to detect viscosity changes induced by hydroxyurea treatment. The third part of this work focused on blood coagulation monitoring. The technique showed sensitivity to coagulation parameters, such as platelet count, calcium ion concentration, and hematocrit. A comparison of the results with TEG showed coagulation started sooner in the levitation technique, but with a lower rate and lower maximum stiffness. Thus, the technique developed can be used as a monitoring tool to assess blood mechanical properties sensitively enough to be of use in clinical diagnostic settings. / 2020-06-04T00:00:00Z