Le rôle du fibrinogène dans les coagulopathies par hémorragie a fait récemment l'objet de travaux importants, la plupart hors du champ obstétrical. L'adaptation de la coagulation et du fibrinogène au cours de la grossesse est méconnue même si sa mise en jeu paraît indispensable à l'hémostase utérine lors de la délivrance. Nous avons donc étudié les modifications gestationnelles du fibrinogène et analysé leurs impacts sur la coagulation et l'hémorragie de la délivrance (HDD). Nous avons montré que le fibrinogène augmentait progressivement pendant la grossesse pour atteindre [3,5-6,5 g/L] (5ème-95ème p.) au 3ème trimestre. L'étude en thromboélastométrie (TEM) a révélé une élévation progressive du "potentiel coagulant" et de la fermeté du caillot chez la femme enceinte. Nous avons ensuite analysé le lien entre le taux initial de fibrinogène lors d'une HDD et le risque d'aggravation (Etude PITHAGORE 6). Le taux de fibrinogène était le meilleur marqueur du risque d'évolution grave. Des seuils de fibrinogène inférieurs à 2 et 3 g/L étaient associés à un risque accru d'aggravation par rapport aux femmes avec un taux > 3 g/L (respectivement OR=11,99 ; IC95% [2,56-56,06] et OR=1.90; IC95% [1,16-3,09]. Enfin l'étude en TEM a montré que les paramètres précoces CA5- et CA15-FIBTEM étaient étroitement corrélés aux taux de fibrinogène lors des HDD permettant l'optimisation du monitorage de la coagulation. Nos résultats nous conduisent à proposer deux scores de coagulopathie obstétricale prenant en compte les spécificités de la grossesse. Des essais seront nécessaires pour valider la pertinence de ces scores et pour évaluer le bénéfice de la compensation précoce en fibrinogène dans les HDD / The role of fibrinogen in haemorrhage-induced coagulopathies has recently been the subject of important work, most of it outside the field of obstetrics. The changes in coagulation and fibrinogen during pregnancy are poorly understood, even though its involvement is essential for uterine haemostasis during the afterbirth. We thus studied the course of fibrinogen levels during pregnancy and analysed their effects on coagulation and postpartum (third-stage) haemorrhage (PPH). We showed that fibrinogen increases progressively during pregnancy, reaching [3.5-6.5 g/L] (5th-95th p.) during the 3rd trimester. The thromboelastometry (TEM) study revealed a progressive increase in the coagulant potential and firmness of clots in pregnant women. We then analysed the association between the initial fibrinogen level during PPH and the risk of aggravation (in the PITHAGORE 6 study). A woman's fibrinogen level was the best marker of the risk that her condition would worsen. Thresholds below 2 and 3 g/L were associated with higher risks of aggravation than in women with fibrinogen concentrations >3g/L (respectively OR=11.99 ; 95% CI [2.56-56.06] and OR=1.90; 95% CI [1.16-3.09]. Finally the TEM study showed that FIBTEM assessment of the early indicators, clot amplitude at 5 and 15 minutes (CA5 and CA15), was closely correlated with fibrinogen levels during PPH and thus helped to optimise coagulation monitoring. Our results lead us to suggest two obstetric coagulopathy scores that take the specificities of pregnancy into account. Trials will be necessary to validate their relevance and to assess the benefits of early fibrinogen replacement in PPH

Measurements of relaxin and acute-phase proteins have not been validated for use in canine serum as a method of pregnancy diagnosis. This means that handling and anesthesia is still necessary to check the pregnancy status of most non-domestic canines. Therefore, the intention of this study was to determine whether relaxin and/or acute-phase proteins could be detected in the urine and/or feces of the domestic dog in order to evaluate the potential for a noninvasive pregnancy test in canines. Blood, urine and feces were collected from 18 domestic dogs and assayed for the presence of relaxin, fibrinogen, alpha-1 acid glycoprotein, and ceruloplasmin. Urinary relaxin appeared to be significant for detecting pregnancy of 30 Days or more in the domestic dog. Additionally, further research might shed light on the presence of relaxin in the feces and fibrinogen and AGP in the urine of the domestic dog and their significance for pregnancy diagnosis.

No description available.

No description available.

Fibrinogen concentrates are used widely as a sealant during and after surgery to reduce blood loss. Commercially available fibrin sealants are made from pooled human blood, which carries the risk of blood-borne diseases, and are expensive. These concerns have brought to focus the need for autologous fibrinogen concentrates. This need has been addressed by utilizing a unique approach in which fibrinogen is precipitated from plasma with protamine. The physical properties of fibrin sealant prepared from fibrinogen precipitated with protamine were evaluated. The optimal precipitation conditions included a plasma protamine concentration of 10 mg/mL at room temperature. Under these conditions 96% ± 4% of the fibrinogen present in the plasma was precipitated and 98% ± 0.9% of the precipitated fibrinogen was clottable. In addition, it was shown that almost 50% of the factor XIII in the plasma was also precipitated along with the fibrinogen. The tensile and adhesion strengths and kinetics of fibrin sealant prepared from protamine-fibrinogen concentrate were evaluated. Tensile strength and adhesion strength both increased with increasing fibrinogen concentration. Addition of calcium chloride significantly increased the tensile and adhesion strengths. The addition of aprotinin and ε-aminocaproic acid (used to inhibit natural fibrinolysis) to the fibrinogen concentrate was shown to have no effect on the mechanical properties of the sealant. Kinetic experiments showed that the clotting time decreased as the thrombin and fibrinogen concentrations were increased. A rat model with controlled renal incisions was employed to evaluate the hemostatic efficacy of the fibrin sealant made from the protamine-fibrinogen concentrate. The fibrin sealant significantly reduced the blood loss and bleeding time when compared with controls (no sealant, plasma, and a commercial product). The sealant also significantly reduced blood loss and bleeding time in rats that were anticoagulated with heparin. A mathematical model based on tensile strength and adhesion strength was developed to predict the bleeding time in the animal wound. Model predictions showed that the ability of the fibrin sealant to reduce bleeding time, and therefore blood loss, was limited by the adhesion strength.

Corrosion and metal release mechanisms of CoCrMo alloys are at human biological conditions not fully understood. The main objective of this master thesis was to investigate whether the Vroman effect influences the extent of metal release from CoCrMo alloy in mixed protein solutions. The project focuses on the corrosion properties and release of cobalt (Co), chromium (Cr) and molybdenum (Mo) from a CoCrMo alloy into simulated physiological solutions of pH 7.2-7.4 in the presence of proteins. The metal release study was performed in phosphate buffered saline (PBS) for 4 and 24 h at 37 °C with and without different concentration of proteins (bovine serum albumin-BSA and fibrinogen-Fbn from bovine plasma). In order to investigate whether any Vroman effect could affect the extent of released metals in solutions, sequential tests were performed by sampling after 1, 4, 6 and 24 h in solutions that were partially replenished after 5 h. Significant metal-induced protein aggregation and precipitation were observed in solutions of physiologically-relevant protein concentrations (40 g/L BSA and 2.67 g/L Fbn). Cr was strongly enriched in the surface oxide of CoCrMo after exposure in all solutions. This was for all solutions accompanied by metal release processes dominated by Co. Based on electrochemical investigations, the electrochemical activity did not increase, but rather decreased, in protein-containing solutions as compared to PBS alone. This could possibly be explained by blocking of cathodic areas as a result of protein adsorption.

No description available.

No description available.

No description available.

Histidine-rich glycoprotein (HRG) is a protein present in plasma at ~ 2 μM, but whose physiologic function is unclear. HRG is a multi-domain protein that contains a unique histidine-rich core that interacts with zinc and hydrogen ions to modulate ligand binding. Due to its modular structure and capacity to sense local changes in zinc and pH, HRG binds several ligands including complement proteins, phospholipids, DNA, fibrin(ogen), heparin, factor (F) XIIa and plasmin. Thus, it is hypothesized that HRG functions as an accessory or adapter protein that bridges different ligands together. Despite the array of ligands and potential involvement in immunity, angiogenesis, coagulation and fibrinolysis, no clear role for HRG has emerged. Congenital HRG deficiency in humans has been associated with a variable phenotype; some investigators report increased susceptibility to thrombosis while others do not. However, studies in HRG-deficient mice reveal that HRG attenuates coagulation. Coagulation is initiated via the intrinsic (or contact) and extrinsic (or tissue factor) pathways and culminates in the generation of thrombin. Thrombin catalyzes the conversion of fibrinogen into a fibrin meshwork that reinforces the platelet plug at sites of vascular injury. There are two circulating isoforms of fibrinogen that differ with respect to their γ-chains. Bulk fibrinogen is composed of a pair of γA-chains, and is designated γA/γA-fibrinogen, whereas a minor variant contains a γA-chain and a γʹ-chain, and is designated γA/γʹ-fibrinogen. The γʹ-chain contains an anionic 20-amino acid residue extension at its COOH-terminus, which provides an accessory binding site for thrombin. Thrombin possesses an anion binding pocket termed exosite II that flanks the active site and mediates its interaction with the γʹ-chain of fibrinogen. Exosite II is an evolutionary feature that is unique to thrombin, as this region is not observed on the prototypic serine protease trypsin or on other defibrinogenating enzymes from snake venom such as batroxobin. Although the physiologic function of the thrombin-γʹ-chain interaction is unclear, it is proposed that this interaction modulates thrombin’s activity when it is bound to fibrin clots. Consistent with this, we show that γA/γʹ-fibrin attenuates thrombin’s capacity to promote clot expansion compared with thrombin bound to γA/γA-fibrin clots, thereby demonstrating that γA/γʹ-fibrin attenuates thrombin’s activity. In the presence of physiologic concentrations of zinc, HRG binds the γʹ-chain of fibrino(gen) and competes with thrombin for binding, thereby suggesting that HRG is a unique modulator of thrombin activity on fibrin clots. Platelets store zinc and HRG in their α-granules and release both components when they undergo activation at sites of injury, which localizes HRG in the vicinity of fibrin-bound thrombin. Consistent with the role of HRG in modulating coagulation, we also show that HRG attenuates contact activation of coagulation, but has no impact on clotting initiated by the extrinsic pathway. The intrinsic pathway is initiated when FXII is activated by polyanions such as RNA and DNA, which are released into the blood after cellular activation, injury or death. FXIIa activates FXI, thereby propagating coagulation and leading to thrombin generation and fibrin formation. Recently, studies using rodent, rabbit and non-human primate models of thrombosis have shown that knock down of FXII or FXI with antisense oligonucleotides or blocking FXIIa or FXIa activity with inhibitors attenuates thrombosis, while having a minimal impact on hemostasis. With increasing evidence that the intrinsic pathway plays an important role in thrombosis, FXII and FXI have emerged as prominent targets for new anticoagulants. However, little is known about how the intrinsic pathway is regulated, so as to prevent uncontrolled clotting. HRG attenuates the intrinsic pathway by binding both FXIIa and the contact activators, RNA and DNA. By binding nucleic acids, HRG is localized to the site of contact activation, where it is poised to inhibit FXIIa. HRG binds to an allosteric region on FXIIa and attenuates its capacity to feedback activate FXII and to activate FXI, thereby inhibiting the initiating steps of contact activation. In addition, HRG attenuates the cofactor role of RNA and DNA in thrombin activation of FXI, which is an important feedback step. With the capacity to modulate multiple steps in the intrinsic pathway, HRG likely serves as a dynamic regulator of contact activation. We tested our hypothesis that HRG is a novel inhibitor of the intrinsic pathway in a murine model of FeCl3-induced arterial injury. HRG-deficient mice exhibit accelerated thrombosis compared with wild type controls, an effect that was abolished by repletion with human HRG. Therefore, these studies indicate that HRG deficiency induces a prothrombotic phenotype. Consistent with the role of HRG as a modulator of the intrinsic pathway, we show that thrombosis after the FeCl3-induced arterial injury is attenuated by administration of RNase, but not DNase, or by knock down of FXII, but not FVII. Therefore, these studies show that thrombosis in this model is induced by RNA and occurs in a FXII-dependent manner. Furthermore, blood loss after tail tip amputation is similar in HRG-deficient and wild type mice, demonstrating that HRG does not modulate hemostasis. Therefore, these studies suggest that HRG is a dynamic regulator of the intrinsic pathway, and acts as a molecular brake to limit procoagulant stimuli. The observations that HRG binds fibrin(ogen), FXIIa and nucleic acids and modulates the thrombin-γʹ-interaction and intrinsic pathway of coagulation, suggest that HRG is a key regulator of coagulation. HRG, the contact system and fibrin are also important in the innate immune response, demonstrating that the interaction of HRG with these factors may provide a unique link between coagulation and immunity. Since immune cells and the coagulation system contribute to both deep vein thrombosis and sepsis, further characterization of the role of HRG in these conditions will contribute to a better understanding of the pathophysiological role of HRG, and may identify novel therapeutic directions. / Thesis / Doctor of Philosophy (PhD)