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Background: Hyperhomocysteinemia (HHcy) is an established risk factor for thrombotic diseases yet the underlying mechanism remain unclear. In this study we investigated the effect of HHcy on endothelial cell-platelet interaction and its role in thrombosis. Methods and Results: We used a novel mouse model of HHcy (plasma homocysteine, Hcy 80 micromolar) in which a Zn2+ inducible human cystathionine beta-synthase (CBS) transgene was introduced to circumvent the neonatal lethality of the Cbs gene deficiency (Tg-hCBS Cbs-/- mice). Hcy-lowering therapy was performed by giving ZnSO4 water to induce human CBS transgene expression in adult mice. Thrombus formation was examined by photo dye-induced cremaster microvasculature thrombosis using intravital microscopy, in which endothelium was preserved, and by FeCl3-induced carotid artery thrombosis, which denudated the endothelium. HHcy accelerated cremaster arteriolar thrombosis and decreased blood flow cessation time from 41.8 min in control mice to 30.5 min in TghCBS Cbs-/- mice. Venular blood flow cessation time was slightly decreased from 5.6 to 5.0 min. Hcy-lowering therapy reduced Hcy level from 80 micromolar to 6.8 micromolar after 2 weeks of ZnSO4 water and prolonged arteriolar blood cessation time from 30.5 to 37.8 min. Interestingly, FeCl3-induced carotid artery thrombosis did not change the occlusion time. Hcy did not potentiate the aggregation and secretion function in washed human platelets from healthy donor treated with Hcy (50, 100 micromolar) or from Tg-hCBS Cbs-/- mice. However, inter-cellular adhesion molecule 1 (ICAM-1) levels, but not vascular adhesion molecule 1 (VCAM-1), were increased in cremaster tissues from Tg-hCBS Cbs-/- mice by western blot. In cultured human umbilical vein ECs (HUVEC), Hcy (100 micromolar, 24h) promoted human platelet adhesion by 200% in static adhesion assay. Using western blot, FACS and RT-PCR, we found that Hcy increased protein and mRNA levels of ICAM-1, but not that of VCAM-1, in HUVEC. ICAM-1 blocking antibody partially reversed Hcy increased platelets adhesion to HUVEC. Hcy induced ICAM-1 expression and reduced DNA methylation on ICAM-1 promoter, which were mimicked by DNA methyltransferase inhibitor azacytidine, and histone deacetylase inhibitors sodium butyrate and trichostatin A. Hcy treatment also increased intracellular Hcy, Sadenosylhomocysteine (SAH) accumulation and decreased SAM/SAH ratio in HUVECs. Hcy decreased methyl CpG binding protein 2 (MeCP2) binding and increased acetylated histone H3 (AcH3) binding to ICAM-1 core promoter region using chromatin immunoprecipitation. Pyrosequencing of ICAM-1 core promoter and adjacent region shows a decreased DNA methylation by Hcy treatment. In high methionine diet-induce HHcy in WT and Icam-/- mice, Icam-/- mice fed with HM diet only show moderately accelerated venular and barely accelerated arteriolar occlusion time compared with WT mice with CT diet using photo dye-induced thrombosis model. Conclusion: HHcy accelerates arteriolar thrombosis and increases EC-platelet interaction via ICAM-1 induction partially through DNA hypomethylation. / Pharmacology

Multimerin 1 (human: MMRN1, mouse: Mmrn1) is a large homopolymeric glycoprotein that is synthesized and stored by platelets and endothelial cells until activation-induced release. MMRN1 is able to support platelet adhesion through mechanisms involving von Willebrand factor (VWF) and glycoprotein (GP)Ibα, and β3 integrins on activated platelets, and it enhances platelet adhesion to fibrillar collagen, potentially by binding to putative MMRN1-specific GPAGPOGPX (where O is hydroxyproline and X is valine or glutamine) motifs in fibrillar collagens. Using mice with and without selective Mmrn1 deficiency, the goals of this thesis were: 1) further characterize the ability of Mmrn1 to enhance platelet adhesion to collagen, 2) explore the role of fluid shear stress in the ability of Mmrn1 to enhance platelet adhesion, and 3) test the specificity of the GPAGPOGPX motif for Mmrn1 and the ability of GPAGPOGPX to support or enhance platelet adhesion. Mmrn1-deficient (Mmrn1-/-) mouse platelets showed impaired aggregate formation on fibrillar collagen surfaces under high (1500 s-1) and low (300 s-1) shear flow compared to wild-type (Mmrn1+/+) mouse platelets, which was due to reduced initial adhesion and a slower rate of platelet accumulation onto collagen surfaces. Similarly, Mmrn1-/- platelets formed smaller aggregates on immobilized recombinant (r)Vwf surfaces compared to wild-type platelets, and Mmrn1-/- platelets had impaired adhesion and aggregate formation on immobilized murine fibrinogen, but not fibrin, when platelets were pre-activated to release Mmrn1. Type I fibrillar collagen was found to contain a variant of the GPAGPOGPX motif (GPAGPOGPI), and GPAGPOGPX motifs supported adhesion of wild-type, but not Mmrn1-/-, platelets. When presented with the VWF-binding GPRGQOGVMGFO motif and the integrin α2β1-binding GFOGER motif present in fibrillar collagens, the GPAGPOGPX motifs synergistically enhanced platelet adhesion. These findings expand upon the known adhesive functions of platelet multimerin 1 and update knowledge of the motifs that support platelet adhesion to fibrillar collagens. / Dissertation / Doctor of Philosophy (Medical Science)

A 53 year-old male presented with a one-day history of a swollen arm and dull, aching pain in the right upper extremity. The patient reported commencing exercising daily over the prior week with a modified, oscillating dumbbell; commonly referred to as a Shake Weight. Imaging revealed an occlusive thrombus in the right axillary, proximal brachial and basilic veins. The patient was treated with a 24-hour tPA infusion followed by mechanical thrombectomy, balloon angioplasty, and stent placement for a residual thrombus and stenosis. The patient was discharged the following day on warfarin and aspirin. This is the first report of effort-induced thrombosis of the upper extremity following the use of a modified, oscillating dumbbell. Due to the growing popularity of modified dumbbells and the possible risk for axillary vein thrombosis, consideration should be made to caution consumers of this potential complication.

INTRODUÇÃO: Um grande número de estudos epidemiológicos têm demonstrado que o fibrinogênio é fator de risco consistente e independente para doença cardiovascular. O fibrinogênio, além de ser um determinante de trombose arterial foi considerado fator de risco de trombose venosa. Foram avaliados os níveis plasmáticos do fibrinogênio em indivíduos que apresentaram algum tipo de trombose não influenciada por reação de fase aguda ou resposta inflamatória. MÉTODOS: Neste estudo de caso-controle realizado entre julho de 2003 a abril de 2005 foram incluídos 39 pacientes, entre 25 e 65 anos, com diagnóstico objetivamente confirmado de trombose, sem antecedentes de neoplasia e colagenose. O tempo mínimo entre a evento e a coleta da amostra de sangue foi de 6 meses. O grupo controle foi constituído de doadores e funcionários voluntários do Hemocentro Regional de Juiz de Fora. A concentração plasmática de fibrinogênio e a medida da Proteína C Reativa foram realizados nos dois grupos. RESULTADOS:Os níveis médios de fibrinogênio foram significativamente maiores nos pacientes ( 316)que no grupo controle (259), p=0,0002. a média de idade foi 48,3 para os pacientes e 45,5 para o controle. A aplicação do teste qui quadrado demonstrou que não houve diferenças significativas nos grupos de pacientes e controles (30,8% e 27%, respectivamente) em relação ao tabagismo(pvalor = 0,72). A frequência de hipertensão foi significativamente maior no grupo de pacientes (28,2%) que no controle (5,4%) (p-valor=0,008). O teste t para a diferença dos níveis médios de fibrinogênio entre os grupos de trombose venosa e arterial não apresentou resultado estatisticamente significante (p-valor = 0,69). CONCLUSÃO: Com base nos dados deste estudo, os níveis de fibrinogênio estão relacionados com trombose, independente se arterial ou venosa. / INTRODUCTION: A great number of prospective epidemiologic studies have reported that fibrinogen is consistently and independently risk factor for the cardiovascular disease. The fibrinogen, a determinant of arterial thrombosis, was also considered a risk factor for the venous thrombosis. It was valued the fibrinogen plasmatic level in patients that had showed some kind of thrombosis event without influence by acute phase reactions or ongoing inflamatory responses. METHODS: In this cases-control study, fulfilled between july 2003 and april 2005, was included 39 patients, among 25 e 65 ears, with confirmed diagnosis of thrombosis and none neoplasis and collagenosis antecedent. Six months was the minimum time between event and blood sample collect. The control group was composed by blood donor and voluntary employee of the Hemocentro Regional de Juiz de Fora. The fibrinogen plasmatic concentration and the C-reactive proteins measure was made in both groups. RESULTS: The medium levels of fibrinogen were significantly higher in patients (316) than the control group (259), p=0,0002. The age average was 48,3 for the patients and 45,5 for the control. The “qui-quadrado" test application proved there wasn’t any significatives differences in both groups, patients (30,8%) and control (27%), in the relation with smoking (p-value = 0,72). The frequency of arterial hypertension was significantly higher in patient group (28,2%) than the control group (5,4%) (p-value = 0,008). The t-test for the differences of the fibrinogen average levels between venous and arterial thrombosis didn’t present any significant statistic result. CONCLUSION: Established in this research, the higher levels of fibrinogen are associated with thrombosis, independently if arterial or venous.

A computational model of thrombus initiation and aggrandizement was proposed. The model separated the thrombotic process into three mechanisms, including shear enhanced diffusivity, platelet margination, and platelet adhesion. The model indicates that transport mechanisms may be the rate limiting condition of thrombus formation at physiological shear rates and that at higher shear rates; platelet binding becomes the rate limiting condition. Additionally a wall shear rate of 20000 s-1 and above should be considered as a new criterion for prophylactic treatment of an atherosclerotic lesion.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 6 uppsatser.

INTRODUÇÃO: Um grande número de estudos epidemiológicos têm demonstrado que o fibrinogênio é fator de risco consistente e independente para doença cardiovascular. O fibrinogênio, além de ser um determinante de trombose arterial foi considerado fator de risco de trombose venosa. Foram avaliados os níveis plasmáticos do fibrinogênio em indivíduos que apresentaram algum tipo de trombose não influenciada por reação de fase aguda ou resposta inflamatória. MÉTODOS: Neste estudo de caso-controle realizado entre julho de 2003 a abril de 2005 foram incluídos 39 pacientes, entre 25 e 65 anos, com diagnóstico objetivamente confirmado de trombose, sem antecedentes de neoplasia e colagenose. O tempo mínimo entre a evento e a coleta da amostra de sangue foi de 6 meses. O grupo controle foi constituído de doadores e funcionários voluntários do Hemocentro Regional de Juiz de Fora. A concentração plasmática de fibrinogênio e a medida da Proteína C Reativa foram realizados nos dois grupos. RESULTADOS:Os níveis médios de fibrinogênio foram significativamente maiores nos pacientes ( 316)que no grupo controle (259), p=0,0002. a média de idade foi 48,3 para os pacientes e 45,5 para o controle. A aplicação do teste qui quadrado demonstrou que não houve diferenças significativas nos grupos de pacientes e controles (30,8% e 27%, respectivamente) em relação ao tabagismo(pvalor = 0,72). A frequência de hipertensão foi significativamente maior no grupo de pacientes (28,2%) que no controle (5,4%) (p-valor=0,008). O teste t para a diferença dos níveis médios de fibrinogênio entre os grupos de trombose venosa e arterial não apresentou resultado estatisticamente significante (p-valor = 0,69). CONCLUSÃO: Com base nos dados deste estudo, os níveis de fibrinogênio estão relacionados com trombose, independente se arterial ou venosa. / INTRODUCTION: A great number of prospective epidemiologic studies have reported that fibrinogen is consistently and independently risk factor for the cardiovascular disease. The fibrinogen, a determinant of arterial thrombosis, was also considered a risk factor for the venous thrombosis. It was valued the fibrinogen plasmatic level in patients that had showed some kind of thrombosis event without influence by acute phase reactions or ongoing inflamatory responses. METHODS: In this cases-control study, fulfilled between july 2003 and april 2005, was included 39 patients, among 25 e 65 ears, with confirmed diagnosis of thrombosis and none neoplasis and collagenosis antecedent. Six months was the minimum time between event and blood sample collect. The control group was composed by blood donor and voluntary employee of the Hemocentro Regional de Juiz de Fora. The fibrinogen plasmatic concentration and the C-reactive proteins measure was made in both groups. RESULTS: The medium levels of fibrinogen were significantly higher in patients (316) than the control group (259), p=0,0002. The age average was 48,3 for the patients and 45,5 for the control. The “qui-quadrado” test application proved there wasn’t any significatives differences in both groups, patients (30,8%) and control (27%), in the relation with smoking (p-value = 0,72). The frequency of arterial hypertension was significantly higher in patient group (28,2%) than the control group (5,4%) (p-value = 0,008). The t-test for the differences of the fibrinogen average levels between venous and arterial thrombosis didn’t present any significant statistic result. CONCLUSION: Established in this research, the higher levels of fibrinogen are associated with thrombosis, independently if arterial or venous.

Background: Venous thromboembolism (VTE), comprising the conditions of deep vein thrombosis (DVT) and pulmonary embolism (PE), is a common acute cardiovascular event associated with increased long-term morbidity, functional disability, all-cause mortality, and high rates of recurrence. Major advances in identification, prophylaxis, and treatment over the past 3-decades have likely changed its clinical epidemiology. However, there are little published data describing contemporary, population-based, trends in VTE prevention and management. Objectives: To examine recent trends in the epidemiology of clinically recognized VTE and assess the risk of recurrence after a first acute episode of VTE. Methods: We used population-based surveillance to monitor trends in acute VTE among residents of the Worcester, Massachusetts, metropolitan statistical area (WMSA) from 1985 through 2009, including in-hospital and ambulatory settings. Results: Among 5,025 WMSA residents diagnosed with acute PE and/or lower-extremity DVT between 1985 and 2009 (mean age = 65 years), 46% were men and 95% were white. Age- and sex-adjusted annual event rates (per 100, 000) of clinically recognized acute first-time and recurrent VTE was 142 overall, increasing from 112 in 1985/86 to 168 in 2009, due primarily to increases in PE occurrence. During this period, non-invasive diagnostic VTE testing increased, vi while treatment shifted from the in-hospital (chiefly with warfarin and unfractionated heparin) to out-patient setting (chiefly with low-molecular-weight heparins and newer anticoagulants). Among those with community-presenting first-time VTE, subsequent 3-year cumulative event rates of key outcomes decreased from 1999 to 2009, including all-cause mortality (41% to 26%), major bleeding episodes (12% to 6%), and recurrent VTE (17% to 9%). Active-cancer (with or without chemotherapy), a hypercoagulable state, varicose vein stripping, and Inferior vena cava filter placement were independent predictors of recurrence during short- (3-month) and long-term (3-year) follow-up after a first acute episode of VTE. We developed risk score calculators for VTE recurrence based on a 3-month prognostic model for all patients and separately for patients without active cancer. Conclusions: Despite advances in identification, prophylaxis, and treatment between 1985 and 2009, the disease burden from VTE in residents of central Massachusetts remains high, with increasing annual events. Declines in the frequency of major adverse outcomes between 1999 and 2009 were reassuring. Still, mortality, major bleeding, and recurrence rates remained high, suggesting opportunities for improved prevention and treatment. Clinicians may be able to use the identified predictors of recurrence and risk score calculators to estimate the risk of VTE recurrence and tailor outpatient treatments to individual patients.

Hypercoagulable states occur when the process of hemostasis is disturbed. It manifests as a deep vein thrombosis or a pulmonary embolism. It is also one of the most common cardiovascular diseases. There are two factors- genetic and acquired. Inherent factors involve coagulation inhibitor deficiency, prothrombin gene mutation and activated protein C resistance. Acquired factors include antiphospolipid syndrome, pregnancy, overweight etc. The analytic part of the thesis is focused on the effect of a low molecular weight heparin. It was shown in many theses that the LMWH could have a negative impact on a parturition complications or a week of parturition, a birth weight. The results of statistical analysis show that there is a coherence between the week of parturition and the type of parturition. What is more, the anticoagulation drug therapy shows a positive effect on the week of parturion. The data used in the thesis originate in laboratory information management system of Clinical Haematology ward in the hospital Nemocnice České Budějovice a.s.