Platelets play an essential role in haemostasis by adhering to the damaged vessel wall and forming a platelet plug to arrest bleeding. Although platelets are traditionally thought of as pro-coagulant, they possess the ability to harbour functional proteins that are key to fibrinolysis, the breakdown of the blood clot, on their surface. They are therefore substantially well equipped to regulate local fibrinolysis. This thesis aims to further define the role of platelets in fibrinolysis, in particular platelet-derived plasminogen activator inhibitor 1 (PAI-1) and plasminogen. PAI-1 is the principal physiological inhibitor of tissue-type plasminogen activator (tPA), and plasminogen is the zymogen for plasmin. In Chapter 3, we show that platelet-derived PAI-1 is released from platelet α-granules by an αIIbβ3 and fibrin dependent mechanism. We found that a significant portion of α-granular PAI1 is retained on the surface of highly activated PS-positive platelets, and activity analysis revealed the majority of PAI-1 on the platelet surface was in its active form. The functional role of platelet PAI-1 was investigated by analysis of tPA-mediated lysis of Chandler model thrombi. Our data revealed a striking dependence for platelet PAI-1 in stabilising platelet-rich thrombi against degradation. Chapter 4 characterises the expression of a novel transmembrane receptor, Plg-RKT, on the surface of human and mouse platelets. This revealed that plasminogen and Plg-RKT augment one another's binding to the platelet surface. Furthermore, analysis of plasminogen binding to the platelet surface revealed two distinct binding sites: 1) via Plg-RKT and 2) via a fibrin and αIIbβ3 dependent mechanism. Finally, Chapter 5 of this thesis discusses the optimisation of a system that monitors thrombus formation and fibrinolysis under flow. Use of this model will help to further elucidate the complex role that platelets play in controlling the balance between coagulation and fibrinolysis.

El etamsilato es un fármaco ampliamente utilizado en medicina veterinaria y humana para el control de la hemostasia en diversos procesos patológicos, intervenciones quirúrgicas y manipulaciones obstétricas. Se ha descrito que su mecanismo de acción se basa en la interacción endotelio-plaquetas a nivel de la hemostasia primaria y presumiblemente mediado por la prostaciclina. Sin embargo, a pesar de su amplio uso en bóvidos, no existen trabajos científicos publicados que avalen su seguridad y eficacia en esta especie.Al objeto de sentar unas bases experimentales que avalaran su utilización en la clínica del vacuno se han llevado a cabo tres estudios, cuyos resultados analizados conjuntamente deberían permitir el uso del etamsilato sobre la base de criterios científicos.En primer lugar, para conocer la evolución de las concentraciones plasmáticas de etamsilato a lo largo del tiempo, se realizó un estudio farmacocinético a dosis única tras su administración intravenosa (IV) e intramuscular (IM) en terneros. Para ello se escogió una dosis de 7,5 mg/kg por ser la más representativa del rango de dosis utilizadas habitualmente en clínica.En segundo lugar, a fin de describir un margen de seguridad mínimo, se realizó un estudio de tolerancia local, tras la administración de 7,5 mg/kg por vía IM y otro de tolerancia general, tras la administración IV de dosis reiteradas. Para el estudio de la tolerancia general se escogió como referencia la dosis máxima de las utilizadas en la práctica habitual (12,5 mg/kg) y se duplicó para forzar la aparición de cualquier signo de intolerancia a nivel clínico, hemostático, hematológico o bioquímico. Asimismo, y con el mismo objetivo, se administró el producto durante 10 días consecutivos, por ser el doble de la máxima pauta recomendada.Finalmente, para avalar la eficacia del etamsilato en el control de la hemostasia se realizó un estudio en el que se midieron los efectos de una dosis única de etamsilato sobre el tiempo de sangría (TS). Se escogió el TS por ser un test estandarizado y que ya se había utilizado con éxito para la demostración de la eficacia del etamsilato en diversas especies. Se utilizó la dosis más alta de las recomendadas (12,5 mg/kg) para facilitar la detección de los efectos del producto, en una única administración. El estudio se realizó siguiendo un modelo cruzado de modo que cada animal actuó como su propio control tomando como referencia tanto su nivel basal como los efectos de la administración de un placebo.Las concentraciones plasmáticas del etamsilato tras su administración IV mostraron una distribución limitada, evidenciada por un Volumen de Distribución (Vd) de 0,44±0,10 L/kg, una eliminación rápida, con un aclaramiento (Cl) de 0,21±0,04 L/h.kg y una Semivida de eliminación (T1/2) de 1,49±0,16 h. Tras su administración IM se observó una Biodisponibilidad (F) muy elevada y superior al 98%. Las concentraciones plasmáticas alcanzadas por ambas vías fueron substancialmente menores que las descritas en el hombre tras la administración de dosis equivalentes, a pesar de tener una T1/2 similar. El estudio de tolerancia reveló una ausencia de efectos tanto a nivel local, medida por las variaciones en la enzima creatinfosfokinasa (CK), como general, valorada por la sintomatología clínica y diversos parámetros sanguíneos (hematológicos, bioquímicos y hemostáticos). El estudio de eficacia mostró un descenso significativo del TS del 16% respecto a sus valores basales dos horas después de la administración IV de etamsilato. El porcentaje de disminución del TS fue mayor en aquellos animales que tenían el TS basal más largo, mostrando una correlación estadísticamente significativa. Sin embargo, el porcentaje medio de reducción fue sensiblemente menor a los descritos para otras especies animales o para el hombre y no alcanzó diferencias estadísticamente significativas respecto al grupo control. Se concluye que el etamsilato es un fármaco útil para su uso como antihemorrágico en la práctica de la clínica de vacuno, siendo muy seguro para esta especie, tanto tras su administración IV como IM, sin producir ningún tipo de efectos adversos y mostrando una eficacia moderada a la dosis recomendada. Su administración IM permite mantener unos niveles plasmáticos más persistentes con una elevada biodisponibilidad y buena tolerancia local. Sin embargo, es necesario realizar ulteriores investigaciones para confirmar su eficacia en bóvidos a dosis y tiempos de determinación previamente validados. / Ethamsylate is a widely used drug in veterinary and human medicine for the control of hemostasis in several pathologic processes, surgical procedures and obstetric manoeuvres. It has been described that its mechanism of action is based on the interaction endothelium-platelet at the primary hemostasis level and, presumably mediated by prostacyclin. However, despite its widespread use in bovine, there are no published scientific reports supporting its safety and efficacy in this species.In order to obtain some experimental basis for its use in bovine practice three studies have been carried out, the results of which, analysed as a whole, should allow the use of ethamsylate upon scientific criteria.Firstly, in order to know the evolution of the plasmatic concentrations of ethamsylate versus time, a single dose pharmacokinetic study after its intravenous (IV) and intramuscular (IM) administration to calves was performed. For this purpose a dose of 7,5 mg/kg was chosen as the most representative of the dose range normally used in practice.Secondly, in order to describe a minimum safety margin, a local tolerance study after its administration of 7,5 mg/kg IM and another of general tolerance, after the IV repeated dosing, were performed. For the general tolerance study the maximum dose normally used in practice (12,5 mg/kg) was chosen as a reference and was duplicated in order to force the appearance of any sign of intolerance in the clincal examination or in the haemostatic, haematological or biochemical parameters studied. Also, and with the same objective, the product was administered during 10 consecutive days, since this is twice the maximum recommended treatment duration.Finally, in order to endorse the efficacy of ethamsylate in the control of hemostasis, a study measuring the effects of a single ethamsylate dose on bleeding time (BT), was carried out. BT was chosen since it is a standardised test and it had previously been successfully used for the demonstration of the efficacy of ethamsylate in several species. The maximum recommended dose (12,5 mg/kg) was selected in order to facilitate the detection of the effects of the product after a single administration. The study was performed following a cross-over design so each animal acted as its own control taking as references its basal level as well as the effects of the administration of a placebo.The plasmatic concentrations of ethamsylate after its IV administration showed a limited distribution, substantiated by a Volume of Distribution (Vd) of 0,44±0,10 L/kg, a rapid elimination, with a Clearance (Cl) of 0,21±0,04 L/h.kg and an elimination Half Life (T1/2) of 1,49±0,16 h. After its IM administration, a very high Bioavailability (F), over 98%, was observed. The plasmatic concentrations reached by both routes were substantially lower than those described in man after the administration of equivalent doses, despite having a similar T1/2. The tolerance study revealed an absence of effects both at local level, measured by the variations of the enzyme Creatinphosphokinase (CK), and at general level, assessed by the clinical symptomatology and several blood parameters (haematological, biochemical and hemostatic).The efficacy study showed a significant shortening of the BT of 16% respective to its basal values two hours after the IV administration of ethamsylate. The percentage of reduction of the BT was higher in those animals having the longest basal BT, showing a statistically significant correlation. However, the average percentage of reduction was sensitively lower than those described for other animal species or for humans and it did not reach statistically significant differences with respect to the control group.It is concluded that ethamsylate is a useful drug for its use as antihemorragic in bovine clinical practice, being very safe for this species after its IV or IM administration, without inducing any kind of adverse effects and having a moderate efficacy at the recommended dose. Its IM administration maintains more persistent plasmatic levels, with a high bioavailability and good local tolerance. However, it is necessary to carry out further research in order to confirm its efficacy in bovine at previously validated doses and timings.

Multiple interacting mechanisms control the formation and dissolution of clots to maintain blood in a state of delicate balance. In addition to a myriad of biochemical reactions, rheological factors also play a crucial role in modulating the response of blood to external stimuli. The broad stimuli for clot formation were laid out, more than a century ago, in, what is now referred to as, Virchow’s triad. To date, a comprehensive model for clot formation and dissolution, that takes into account the biochemical, medical and rheological factors, has not been put into place, the existing models emphasizing either one or the other of the factors. In this dissertation, a model is developed for clot formation and dissolution that incorporates many of the relevant crucial factors that have a bearing on the problem. The model, though just a first step towards understanding a complex phenomenon goes further than previous models in integrating the biochemical, medical and rheological factors that come into play. The model is tested in some simple flow situations as part of an attempt to elucidate Virchow’s triad. Extensions to the model, along with detailed numerical studies, will hopefully aid in a clearer understanding of the phenomenon, and in making relevant clinical correlations.

Thesis (M.D.S.)--University of Hong Kong, 2000. / Includes bibliographical references (leaves 138-164).

Thesis (M.D.S.)--University of Hong Kong, 2000. / Includes bibliographical references (leaves 138-164). Also available in print.

Master of Science / Department of Grain Science and Industry / X. Susan Sun / One of the leading causes of death following traumatic injury is exsanguination. The body addresses bleeding through the process of hemostasis which includes the formation of a fibrin mesh structure that holds a blood clot together. During traumatic injury, hemostasis may be unable to stop excess bleeding. Fibrin based hemostatic agents have been developed, however, these studies often use fibrin obtained from biological sources, which poses risk of infection. A novel amphiphilic peptide (h9e) has been studied to form three dimensional nanofibers networks. In this research, we studied the ability to form a synthetically produced, fibrin-mimic, hemostatic material from the h9e peptide sequence. The objective of this study was to determine the blood gelation strength of the h9e peptide necessary to arrest bleeding in the Wistar Rat model. Commercial mouse blood was used for blood gelation in vitro studies. Dynamic rheometer was used to determine the gelation kinetics at varied h9e peptide concentrations ranging from 1-5% wt. By directly mixing the h9e peptide with blood, we observed that the blood gelation strength right after mixing increased as the h9e peptide weight % concentration increased, from 67 to 1086 Pascals in the peptide concentration from 1 to 5%, respectively. After 24 hours, final gelation strength of all concentrations with commercial mouse blood was lower than the instantaneous strength but consistent throughout testing. Similar testing was conducted using commercial Wistar Rat blood with weight % concentrations of 1, 3, and 5% of h9e peptide. The gelation strength was 500, 1665, and 1914 Pascals, respectively. We also determined the gelation strength of Wistar Rat blood components, such as red blood cells, serum, and plasma with 1% h9e peptide. We observed the gelation response induced with individual blood components; however, the strength is weaker than whole blood. In vivo, we applied the cut-tail method by dipping the cut-tail of Wistar Rats into the h9e peptide solutions for 10 seconds and then took it out for blood lost collection. We observed that h9e peptide solution at 1, 3, and 5% weight concentrations can all generate hemostatic function. The h9e peptide solution at 5% weight concentration (1914 Pa) was able to outperform a commercial hemostatic material (Moore Medical CELOX* Hemostatic Granules), significantly reducing both bleeding time and blood lost: h9e peptide at 5% had a bleeding time of 94 sec and 0.75 mL blood lost, while the Celox hemostatic granules had a bleeding time of 225 sec and 1.5 mL blood lost. Transmission Electron Microscopy and Spinning Disk Confocal Microscope imaging indicated a blood component reinforced, web-like, h9e nanofiber structure similar to the structure formed by fibrin in a blood clot. This study showed that h9e peptide has the potential to be used to induce hemostasis.

No description available.

Factor XI (FXI) is the zymogen of the coagulation protease factor XIa (FXIa) that contributes to thrombin generation through the intrinsic pathway. FXI is activated by the contact pathway protease, factor XIIa (FXIIa), in a high molecular weight kininogen-dependent manner. It is also known to be activated by thrombin in a positive feedback reaction, however, the mechanism of this activation is not yet completely understood. Therefore, our objectives were to identify the role of polyanions in the thrombin mediated FXI activation, and the role of the thrombin exosites in the activation. To study this activation, we assessed the activation of FXI by thrombin in the presence and absence of the polyanions, dextran sulfate and polyphosphate (polyP). We utilized surface plasmon resonance to determine whether FXI and thrombin bind to the polyanions, and how the exosites effect thrombin’s ability to bind using thrombin exosite 1 and 2 variants. To investigate the role of the exosites in FXI activation, we analyzed the activation of FXI by the thrombin variants. In addition, we explored how inhibiting the thrombin exosites using DNA aptamers affects thrombin’s ability to bind to polyanions and activate FXI. We found that polyanions are required as a cofactor for the activation of FXI by thrombin, and stimulate the activation in a concentration dependent fashion, suggesting a template mechanism. Our findings also show that exosite 1 and 2 are required for thrombin to bind to polyanions, however, exosite 2 plays the predominant role in FXI activation. Our aptamer data showed that either exosite can be targeted to inhibit FXI activation. These findings enrich our understanding of the mechanism of FXI activation by thrombin and provides further insight on how to attenuate the activation for potential antithrombotic therapies. / Thesis / Master of Science (MSc)

Background: Hyperthyroid cats are predisposed to thrombus formation. The mechanism for thrombogenesis is currently unknown, but could be associated with altered hemostasis as seen in hyperthyroid humans. Objective: The purpose of this study was to evaluate markers of hemostasis in hyperthyroid cats compared to healthy cats, and in hyperthyroid cats before and after treatments with radioactive iodine (RIT). Methods: Twenty-five cats with hyperthyroidism and 13 healthy euthyroid cats > 8 years of age were studied. Prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen, antithrombin (AT), D-dimers, thrombin-antithrombin complexes (TAT), von Willebrand Factor antigen (vWF:Ag), and activity of factors VIII and IX were measured. An echocardiogram was performed in all cats and healthy cats with abnormal echocardiograms were excluded. Measurements of hemostasis were evaluated again in 7 cats > 6 months after RIT and deemed to have restored euthyroid status. Results: There is a significant likelihood of being in hypercoagulable state based on hyperthyroid state (P = 0.019) and serum T4 level is significantly associated with predicating hypercoagulability (P = 0.043). Hyperthyroidism is associated with significantly higher median fibrinogen concentration (P < 0.0001), higher median AT activity (P < 0.0001), and higher median vWF:Ag level (P = 0.01) with all values decreasing significantly post-RIT. Fibrinogen and AT had a strong positive correlation with serum T4 value (r = 0.79; 95% CI 0.63 - 0.89 and r = 0.70; 95% CI 0.50 - 0.84, respectively). Presence of an abnormal echocardiogram in hyperthyroid cats was associated with a significantly higher median fibrinogen concentration (P = 0.03). Echocardiographic status did not have a significant impact on the remaining hemostatic markers in hyperthyroid cats. Conclusions: These results provide evidence of altered hemostasis and hypercoagulability in hyperthyroid cats that do not appear to be solely attributed to cardiac abnormalities. These differences of altered hemostasis resolved after radioiodine therapy, but further studies are warranted to determine if hypercoagulable state resolves. / Master of Science / In feline hyperthyroidism, there is a predisposition for thrombus formation. An alteration of hemostasis has been documented in hyperthyroid humans, but despite reports of thrombus formation in hyperthyroid cats, the underlying mechanism is currently unknown. Hyperthyroidism can lead to cardiac abnormalities that could possibly contribute thrombus formation, although thrombus formation has occurred in hyperthyroid cats without detected abnormalities. The goal of this study was to evaluate markers of hemostasis in hyperthyroid cats presenting for radioiodine therapy to evaluate for presence of hypercoagulability. Twenty-five hyperthyroid cats were evaluated with hemostasis panels and echocardiograms. The results were compared to a group of 13 healthy cats. Markers of hemostasis and echocardiograms in 7 hyperthyroid cats were also compared to results 6 months or greater post-radioiodine therapy. There was evidence of altered hemostasis and hypercoagulability in hyperthyroid cats. The alterations noted resolved after radioiodine therapy and do not appear to be solely attributed to cardiac abnormalities seen in hyperthyroid cats.

Coagulation has and continues to be one of the most important elements in medicine. Issues from a lack of hemostasis range from poorer clinical outcomes to sudden death. The evolution of treatments for hemostasis have evolved from the use of Tamponade with direct pressure and bandages, the use of materials such as cobwebs and dust, the use of heat with hot oil or heated irons, to the use of suture, glues, plasmas, staplers, and electricity. This evolution has continued to bring about the prophylactic use of technology in an effort to prevent blood loss. This change from reactive treatments to proactive continue to be on a localized or superficial basis. One of the largest opportunities to proactively reduce blood loss in surgical patients is during the resection of solid organs such as the liver, kidney, and spleen. Few options have existed to help improve hemostasis short of the complete occlusion of blood supplying the tissue such as in the Pringle Maneuver. Recent studies have begun to show that practices such as this may have a significant detrimental effect on morbidity. It has been found that by applying radio frequency electrical energy in a particular way that large amounts of tissue can be pre-coagulated prior to resection. A series of animal and human clinical work has been completed to help evolve and confirm the method and the device that was created and refined during this effort. During the course of this work fifty-three patients were treated at four institutions on three continents. Average blood loss for liver resections performed with this pre-coagulation technique using the developed device in a multicenter control trail was 3.35 ml/cm2 as compared to 6.09 ml/cm2 (p < 0.05) for resections performed using standard surgical techniques alone. Additionally, the transection time necessary was also reduced from mean value of 27 minutes (2 -- 219 minutes) to 35 minutes (5 -- 65 minutes). Patients treated included those suffering from liver cirrhosis, fatty liver disease, and post chemotherapy fibrosis. From this work the use of pre-coagulation with methods and device developed was shown to be safe and effective for reducing the amount of blood loss and transection time during liver resections.