Thrombin-activatable Fibrinolysis Inhibitor (TAFI) is a 60 kDa plasma protein that can be activated to the enzyme, TAFIa, by thrombin, plasmin or trypsin. TAFIa is a carboxypeptidase B-like enzyme that attenuates fibrinolysis. Thrombomodulin (TM) is a cofactor which increases the overall efficiency of thrombin-mediated TAFI activation by 1250-fold. Thus, the thrombin-TM complex is believed to be the physiological TAFI activator. The minimal structure of TM required for efficient TAFI activation contains the EGF-like domains 3 through 6. New structure models have postulated that the C-loop of TM EGF-like domain 3 has a negatively charged molecular surface that could interact with several positively charged surface patches on TAFI. In this study, we constructed recombinant TAFI variants to assess whether the selected positively charged residues on TAFI complement the negative electrostatic potential of the TM EGF-like domain 3, thereby promoting the TAFI-TM interaction in the formation of the ternary thrombin/TM/TAFI complex. TAFI has exclusive triple lysine residues on its activation peptide. When they are substituted by alanine residues (K42/43/44A), compared to the wild-type, the catalytic efficiencies for TAFI activation by thrombin in the presence and absence of TM decreased by factors of 9 and 3.5, respectively. Other derivatives of TAFI with alanine point mutations at positions K133, K211, K212, and R220, which together represent one positively charged surface patch of TAFI, showed decreased catalytic efficiencies for TAFI activation by thrombin-TM complex from 2.4 to 2.9-fold. A second positive surface patch includes residues K240 and R275. Alanine mutations of these two residues caused decreased catalytic efficiencies by 1.7 and 2.1-fold, respectively. Together, our data show that no single mutation completely eliminates TM dependence in TAFI activation by thrombin, but each mutated residue contributes in the formation of the ternary thrombin/TM/TAFI complex. In addition, all TAFIa derivatives had half lives (8.1 ± 0.6 min) comparable to that of wild-type TAFIa (8.4 ± 0.3 min) at 37 ºC, suggesting that these residues are not involved in TAFIa inactivation by conformational instability. / Thesis (Master, Biochemistry) -- Queen's University, 2008-01-31 15:10:19.209

Thesis (M.Med.(Haematology))--University of the Witwatersrand, Faculty of Health Sciences, 2003.

Dogs with pituitary-dependent hyperadrenocorticism (PDH) are at increased risk of thromboembolic disease (TED); however the pathogenesis of thrombosis in these patients is poorly characterized. Thromboelastography (TEG®) is a whole blood hemostatic test that has recently been shown to be capable of detecting hypercoagulability in veterinary patients. A modification of TEG, PlateletMappingTM (TEG-PM) measures platelet response to the agonists arachidonic acid (MAAA) and adenosine diphosphate (MAADP), and compares this to fibrin clot strength in the absence of platelet activation (MAfibrin). This prospective study evaluated dogs with PDH for hypercoagulability using TEG-PM as well as conventional plasma-based coagulation tests (prothrombin time [PT], activated partial thromboplastin time [aPTT], fibrinogen concentration). Hemostatic testing was performed in 40 healthy dogs, 19 dogs with untreated PDH, 16 of the dogs with PDH after 3 months’ treatment and 15 dogs after 6 months’ treatment. Systolic blood pressure (SBP) was also measured in all the dogs with PDH before and during treatment. In addition, urine protein to creatinine ratio (UPCR) and antithrombin activity [AT] were measured in some dogs with PDH. PT was significantly decreased in the dogs with PDH compared to controls, however all of the dogs with PDH had results within the reference interval. Dogs with PDH were hyperfibrinogenemic compared to healthy dogs; fibrinogen concentrations reduced with treatment of PDH but remained significantly elevated. AT activity in the PDH dogs was not significantly decreased despite the majority of dogs tested having significant proteinuria. Approximately half of the dogs with untreated PDH were hypertensive, and blood pressure did not change significantly following resolution of hypercortisolemia. Serum cholesterol was increased in dogs with untreated PDH but normalized following control of PDH. TEG-PM revealed decreased κ, increased α-angle and increased MAthrombin in dogs with PDH in comparison to healthy dogs. Platelet response to AA was significantly increased in dogs with untreated PDH. Following treatment of PDH, the majority of TEG-PM parameters (with the exception of MAthrombin) did not change significantly. In conclusion, dogs with PDH had evidence of hypercoagulability and hypertension, which persisted despite medical treatment of PDH. These factors may explain the association between hyperadrenocorticism and TED. / OVC Pet Trust Fund, Vetoquinol

Introduction: Haemostasis is the arrest of bleeding. In recent years, in-vitro studies have suggested that secondary haemostasis (blood coagulation) is subject to activation by reactive oxygen species (ROS). It is known that patients who suffer from vascular disease are typically hypoxaemic and in the case of peripheral occlusive artery disease (POAD), physical exercise is used to improve symptom free mobility in the affected limbs. Hypoxia and physical exercise are two potent independent and synergistic initiators of ROS. We identified a clear need for in-vivo analysis of this novel area of research. Aims: There were two main aims of this research. 1. To explore the in-vivo influences of inspiratory hypoxia and physical exercise on biomarkers of haemostasis; and in doing so and subsequently carry out a randomised double blind placebo control trial to explore the interaction between oxidative stress (ROS accumulation) and haemostasis. Hypothesis: It was hypothesised that hypoxia and exercise would be independently and synergistically associated with an increase in oxidative stress, resulting in coagulation activation. We hypothesised that intervention with free radical reaction-chain breaking antioxidant vitamins would attenuate oxidative stress and thus attenuate the activation of coagulation. Methods: study 1 - Healthy males were subjected to six hours of normobaric hypoxia (12% inspired oxygen) and then a physical exercise challenge to exhaustion (cycling ramp-test). Citrated plasma was collected pre hypoxic exposure, post six hours of exposure, then immediately post exercise and analysed for routine clinical markers of coagulation (aPTT, PT, TT and fibrinogen) and analysed with and without correction for plasma volume shift. Data were analysed using a one-factor repeated measures ANOVA incorporating one within (condition: time point) subjects factor. Following a significant main effect and interaction, paired samples t-tests were employed to make post hoc comparisons at each level of the within-subjects factor. Study! - Healthy males were subjected to a double blind, randomised, placebo controlled intervention with vitamin C (a water soluble) and vitamin E (a lipid soluble), two ROS-scavenging, chain breaking antioxidants. The intervention lasted eight weeks to insure membrane enrichment with antioxidants. The methods of study one were repeated but with a pre-intervention time point added and the addition of two extra markers of thrombin generation (PF1+2 and T-AT). Data were analysed using a two-factor mixed ANOVA incorporating one between (group: antioxidant intervention vs. placebo control) and one within (condition: time point) subjects factor. Following a significant main effect and interaction, a paired samples t-test was used to make post hoc comparisons at each level of the within-subjects factor, with the alpha level Bonferroni corrected for multiple comparisons Between-group comparisons were assessed using independent samples t-tcsts applied to each level of the between-subjects factor. Results: Study 1 - Hypoxia was not associated with activation of coagulation. Physical exercise increased the activity of contact factor coagulation pathway activation. Study 2 - The intervention increased thrombin generation in the antioxidant group. This was met with an antagonistic antithrombin activation. Hypoxia did not impact the placebo group, but normalised the thrombin generation of the antioxidant group. Physical exercise increased contact factor pathway (CFP) as per study 1, but thrombin generation was unaltered. Hypoxia suppressed fibrinolysis post exercise, which is known to be activated in normoxic exercise. Discussion: hi study one, hypoxia alone did not activate coagulation. We hypothesised that this could be tentative evidence of a ROS concentration threshold for activation since once exercise was superimposed the accumulation of ROS activated the CFP. Correction for changes in plasma volume nullified the increased activity of the CFP. Corrections for shifts in plasma volume are routinely ignored in the literature and this was a novel finding. Study 2 was the first intervention of its kind. The increase in thrombin generation pre-post intervention with antioxidants suggests compelling evidence of in-vivo regulation of coagulation by ROS. But the direction of change was completely contrary to the original hypothesis. The confirmation that hypoxia does not activate coagulation is important, especially given the controversy surrounding long-haul flight deep vein thrombosis. Interestingly, exercise did not increase thrombin generation, despite the increase in the CFP. These findings suggest haemostasis is indeed subject to control by the body's redox state invivo via an as of yet, unknown mechanism.

El manejo quirúrgico de pacientes bajo tratamiento anticoagulante oral ha estado por mucho tiempo determinado por la suspensión del fármaco 2 días antes del acto quirúrgico. La intención de este trabajo fue la de dejar en el pasado dicha metodología, manteniendo la terapia anticoagulante, realizando un examen de laboratorio previo para obtener el nivel de anticoagulación en que se encuentra el paciente y una vez realizada la exodoncia (no complicada), otorgar hemostasia con medios locales mecánicos. Según los resultados obtenidos, aparentemente sería un procedimiento seguro, tomando en cuenta que el único riesgo que se toma es el de un sangramiento post-operatorio, el cual puede ser controlado. Lo anterior es además corroborado por la literatura existente y permite al operador no enfrentar un posible evento Tromboembólico que pudiese ocurrir al suspender el tratamiento anticoagulante oral.

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Bc. Ladislava Cablíková Supervisors: Ass. Prof. Mojca Božič-Mijovski, Ph.D., prof. PharmDr. Petr Pávek, Ph.D., RNDr. Jana Nekvindová, Ph.D. Thesis title: The Impact of Apixaban on Overall Hemostatic Potential Disorders at certain levels of the complicated haemostatic system can lead to either bleeding or excessive blood coagulation. These pathological conditions are treated with anticoagulants, which aim to correct excessive coagulation. However, traditional anticoagulant therapy has many limitations, which initiated efforts to develop oral anticoagulants with a better profile. These new-generation anticoagulants are called DOAC - Direct Oral AntiCoagulans. Apixaban, as one of xabans, has predictable pharmacokinetics and pharmacodynamics and therefore does not require a routine laboratory monitoring of the treatment effect. Nevertheless, it still requires evaluation in urgent clinical situations. Standard coagulation screening assays, e.g., PT (prothrombin test) and APTT (activated partial thromboplastin test), do not fully reflect the actual status of the drug. Therefore, researchers aim is to find a relatively simple and fast hemostatic assay that would correlate with the actual condition...

Two groups of New Zealand white rabbits were injected with homogenates from Tumorous-head (Tuh) and Wild-type (WT) Drosophila melanogaster. A third group was used as a saline injected control. Blood collected in both acute and chronic studies was subjected to various hematological and post mortem studies. The Tuh injected group showed a five-fold increase in thrombocytes (blood platelets) over the controls and four-fold increase over the wild-type group. Reduced clotting times were noted from acute to chronic studies in both tumorous and wild-type studies; however, the magnitude of change between the two groups was insignificant. Investigations involving electrophoretic banding patterns, differential blood cell counts, and comparative hematocrits, provided less significant results. The author concludes that the reduced clotting times reported in tumorous-head injected rabbits represent a decrease in bleeding time. This was caused by the more effective plugging of the damaged vessel by the increased number of platelets.

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 95-101).

Factor VII, the initiator of the extrinsic coagulation cascade, circulates in human plasma mainly in its zymogen form, Factor VII and in small amounts in its activated form, Factor VIIa. However, the mechanism of initial generation of Factor VIIa is not known despite intensive research using currently available model systems. Earlier findings suggested serine proteases Factor VII activating protease, and hepsin play a role in activating Factor VII, however, it has remained controversial. In this work I estimated the levels of Factor VIIa and Factor VII for the first time in adult zebrafish plasma and also reevaluated the role of the above two serine proteases in activating Factor VII in vivo using zebrafish as a model system. Knockdown of factor VII activating protease did not reduce Factor VIIa levels while hepsin knockdown reduced Factor VIIa levels. After identifying role of hepsin in Factor VII activation in zebrafish, I wanted to identify novel serine proteases playing a role in Factor VII activation. However, a large scale knockdown of all serine proteases in zebrafish genome using available knockdown techniques is prohibitively expensive. Hence, I developed an inexpensive gene knockdown method which was validated with IIb gene knockdown, and knockdown all serine proteases in zebrafish genome. On performing the genetic screen I identified 2 novel genes, hepatocytes growth factor like and prostasin involved in Factor VII activation.

published_or_final_version / Dentistry / Master / Master of Dental Surgery