A doença pulmonar obstrutiva crônica é uma doença que leva à obstrução pulmonar geralmente irreversível e está intimamente relacionada com o hábito de fumar. Ao longo dos anos, ocorre destruição dos septos alveolares com a degradação das fibras elásticas e depósito do colágeno que compõe estes septos. Muito tem se discutido sobre a existência de inflamação sistêmica no paciente com DPOC e sobre as suas possíveis manifestações extra-pulmonares . O processo de aterosclerose pode fazer parte deste espectro inflamatório a partir da presença de dano endotelial. O fator de Von Willebrand é um marcador de dano endotelial e pode ser dosado de forma quantitativa e qualitativa. Este trabalho demonstra uma diferença estatisticamente significativa, qualitativa e quantitativamente, entre os níveis de fator de Von Willebrand em tabagistas e em pacientes com DPOC, quando comparados ao grupo controle. Ao analisarmos os pacientes com DPOC dividindo-os em subgrupos considerando quatro classificações distintas: GOLD 2006 (Anexo A), GOLD 2011 (Anexo B), grau de sintomatologia a partir da escala de dispneia MRC modificada (Anexo C) e número de exacerbações no último ano. Observamos uma diferença estatisticamente significativa, em relação ao nível qualitativo do fator de von Willebrand, apenas quando comparamos pacientes com DPOC sintomáticos e não sintomáticos. Demonstramos ainda uma correlação inversa entre o percentual predito de volume expiratório forçado no primeiro segundo (VEF1%) com os níveis qualitativos de fator de von Willebrand. Desta forma, o fator de von Willebrand está aumentado no paciente com DPOC, sendo um possível marcador sérico de sintomatologia relacionado a esta doença. Apesar de não se conseguir definir gravidade dos pacientes com DPOC pelo GOLD, o fator de von Willebrand estabelece uma correlação inversa com os níveis de VEF1%, sugerindo algum tipo de participação na progressão da doença. / Chronic obstructive pulmonary disease (COPD) is an entity that leads to pulmonary obstruction generally irreversible. This is closely related to the smoking habit and leads to alveolar septal destruction with elastic fibres degradation and collagen fibres degradation. There are many argues about the presence of systemic inflammation in the COPD patients. The atherosclerosis process can be part of this inflammation spectrum from the presence of endothelial damage. The von Willebrand factor is a marker of endothelial damage and may be measured in a quantitative and qualitative form. This study demonstrates a significant statistically difference in both qualitative and quantitative form of von Willebrand factor in smokers and COPD patients compared to the control group. When analyzing subgroups in COPD patients considering four different classifications: GOLD 2006, GOLD 2011, degree of symptoms from the MRC dyspnea scale and number of exacerbations in the last year. We observed a significant statistically difference in the level of qualitative von Willebrand factor only in the subgroup of the degree of symptoms. Inverse correlation was observed with the percent of forced expiratory volume in one second (FEV1%) and the qualitative von Willebrand factor. Thus, the von Willebrand factor is increased in COPD patient and could be a possible serum marker of the presence of symptoms in this patient. We cannot identify the severity of COPD patients in GOLD classification using von Willebrand factor, but we established a inverse correlation between qualitative von Willebrand factor and FEV1%, what may suggest a participation in the progression of the disease.

The hamster estrous cycle lasts four days and is considered to be a physiological model for angiogenesis. Angiogenesis is the formation of new capillaries from preexisting vessels, and it occurs extensively during corpus luteum formation in the estrous cycle. Von Willebrand Factor (vWF) is a glycoprotein that is secreted uniquely in endothelial cells and megakaryocytes. It is frequently used as an endothelial cell marker and it is able to detect vessels within tissues when it is used in immunohistochemical staining techniques. This study explores von Willebrand Factor expression within Golden Hamster ovarian tissue. In particular, this study uses cage control and antiorthostatic cage suspension tissue. Antiorthostatic cage suspension is a model developed to mimic and study the physiological effects caused by microgravity, such as that experienced in space flight. It is hypothesized that simulated microgravity caused by antiorthostatic cage suspension would result in lower levels of vasculature and expression of vWF within ovarian tissue. Due to financial considerations, conclusive data was not obtained due to a lack of statistics. However, our study indicates that vasculature and vWF expression may be increased in antiorthostatic cage suspension tissue.

The force-induced activation of adhesive proteins such as von Willebrand Factor (VWF), which experience high hydrodynamic forces, is essential in initiating platelet adhesion. The importance of the mechanical force induced functional change is manifested in the multimeric VWF’s crucial role in blood coagulation, when high fluid shear stress activates pVWF multimers to bind platelets. Here we showed that a pathological level of high shear flow exposure of pVWF multimers results in domain conformational changes, and the subsequent shifts in the unfolding force allow us to use force as a marker to track the dynamic states of multimeric VWF. We found that shear-activated pVWF multimers (spVWF) are more resistant to mechanical unfolding than non-sheared pVWF multimers, as indicated in the higher peak unfolding force. These results provide insight into the mechanism of shear-induced activation of pVWF multimers.

Von Willebrand factor (VWF) is a multimeric glycoprotein that critically supports platelet aggregation in hemostasis. Disordered VWF function causes both thrombotic and bleeding disorders, and genetic defects in VWF are responsible for von Willebrand’s disease (VWD), the most common inherited bleeding disorder in humans. Very large VWF multimers exhibit the greatest thrombogenic activity, which is attenuated by ADAMTS13 cleavage in the A2 domain. A2 cleavage is regulated by mechanical force, and pathologically high shear forces are known to enhance proteolysis and cause bleeding in patients. Enhanced cleavage is also described in patients with VWD 2A mutations. In contrast, VWF A2 is stabilized against cleavage by a calcium binding site within A2. Single molecule studies have demonstrated that mechanical unfolding is required for A2 cleavage to expose the scissile bond. In this dissertation, we aim to better understand the mechanosensitivity of A2 cleavage by characterizing the force sensitivity of A2 unfolding and refolding. We first characterized the interaction between VWF A2 and calcium using bulk isothermal calorimetry and thermal denaturation assays. In parallel, we used single molecule optical tweezers to characterize A2 unfolding and refolding. Calcium was found to bind A2 with high affinity, stabilize A2 against thermal denaturation, and enhance domain refolding. In contrast, we found that VWD 2A mutations destabilize the A2 domain against thermal denaturation. R1597W, the most common VWD 2A mutation, lies within the calcium binding loop and exhibited diminished calcium stabilization against thermal denaturation. Using optical tweezers, we found that R1597W also diminished A2 refolding. R1597W refolding in the presence of calcium was similar to that of wild-type A2 in the absence of calcium, suggesting that loss of calcium stabilization contributes to the disease mechanism of R1597W. Other VWD 2A mutations lying outside the calcium binding loop also destabilized A2, but retained calcium mediated stabilization. These studies provide a better understanding of VWD 2A pathophysiology and offer structural insights into A2 unfolding and refolding pathways. By exploring the role of mechanical force in regulating VWF cleavage, this work moves towards a better understanding of how hydrodynamic forces within the vasculature regulate VWF function in hemostasis and thrombosis.

Von Willebrand factor (VWF) promotes platelet adhesion and aggregation at sites of vascular damage. This function is directly related to the multimer size of VWF. The VWF-specific metalloprotease ADAMTS13 decreases VWF multimer size by cleaving at Y1605-M1606 in the VWF A2 domain. This thesis examined the sensitivity of ADAMTS13 cleavage to mutagenesis of the full-length multimerized VWF substrate, and a small VWF A2 domain fragment, VWF115. The ADAMTS13 cleavage site at Y1605-M1606 was mutated with the most severe loss of cleavage observed in Y1605A/M1606A. In addition, 4 single nucleotide polymorphisms were examined, with D1472H, Q1571H, P1601T proteins all showing increased resistance to cleavage. In contrast, G1643S has enhanced cleavage in the full-length VWF substrate but shows cleavage resistance in VWF115. Three von Willebrand disease mutations were also examined. In patients, R1597W has enhanced ADAMTS13 cleavage and a loss of high molecular weight multimers, while R1205H has enhanced protein clearance resulting in very low VWF levels and Y1584C patients have moderately low VWF levels. R1597W has enhanced cleavage of full-length VWF, while a slight cleavage increase is observed in VWF115 for Y1584C, and no change is seen with R1205H. The VWF mutations R1597W, Y1605A/M1606A, R1205H and Y1584C were further examined in the VWF knockout mouse using recombinant VWF protein infusion and hydrodynamic delivery of VWF cDNA to determine the effects these mutations produce on VWF antigen levels, multimer structure, secretion, clearance and function in a thrombotic injury model. All four mutations had different pathogenic mechanisms. R1597W showed accelerated clearance with loss of multimer structure, and greatly increased time to thrombotic occlusion. Y1605A/M1606A showed accelerated clearance with normal or supranormal multimer structure, a loss of thrombotic occlusion but increased platelet accumulation. Y1584C showed no change in protein clearance, with decreased VWF antigen level, reduced multimer structure, and reduced thrombotic potential. R1205H demonstrated a synthetic defect in vitro and in vivo increased clearance with a decrease in VWF antigen levels and normal multimer structure and a variable thrombotic potential. These results validate the use of the genetically-modified VWF knockout mouse model for evaluating the pathogenic mechanisms of putative VWF mutations. / Thesis (Ph.D, Pathology & Molecular Medicine) -- Queen's University, 2010-07-28 10:24:40.654

A doença pulmonar obstrutiva crônica é uma doença que leva à obstrução pulmonar geralmente irreversível e está intimamente relacionada com o hábito de fumar. Ao longo dos anos, ocorre destruição dos septos alveolares com a degradação das fibras elásticas e depósito do colágeno que compõe estes septos. Muito tem se discutido sobre a existência de inflamação sistêmica no paciente com DPOC e sobre as suas possíveis manifestações extra-pulmonares . O processo de aterosclerose pode fazer parte deste espectro inflamatório a partir da presença de dano endotelial. O fator de Von Willebrand é um marcador de dano endotelial e pode ser dosado de forma quantitativa e qualitativa. Este trabalho demonstra uma diferença estatisticamente significativa, qualitativa e quantitativamente, entre os níveis de fator de Von Willebrand em tabagistas e em pacientes com DPOC, quando comparados ao grupo controle. Ao analisarmos os pacientes com DPOC dividindo-os em subgrupos considerando quatro classificações distintas: GOLD 2006 (Anexo A), GOLD 2011 (Anexo B), grau de sintomatologia a partir da escala de dispneia MRC modificada (Anexo C) e número de exacerbações no último ano. Observamos uma diferença estatisticamente significativa, em relação ao nível qualitativo do fator de von Willebrand, apenas quando comparamos pacientes com DPOC sintomáticos e não sintomáticos. Demonstramos ainda uma correlação inversa entre o percentual predito de volume expiratório forçado no primeiro segundo (VEF1%) com os níveis qualitativos de fator de von Willebrand. Desta forma, o fator de von Willebrand está aumentado no paciente com DPOC, sendo um possível marcador sérico de sintomatologia relacionado a esta doença. Apesar de não se conseguir definir gravidade dos pacientes com DPOC pelo GOLD, o fator de von Willebrand estabelece uma correlação inversa com os níveis de VEF1%, sugerindo algum tipo de participação na progressão da doença. / Chronic obstructive pulmonary disease (COPD) is an entity that leads to pulmonary obstruction generally irreversible. This is closely related to the smoking habit and leads to alveolar septal destruction with elastic fibres degradation and collagen fibres degradation. There are many argues about the presence of systemic inflammation in the COPD patients. The atherosclerosis process can be part of this inflammation spectrum from the presence of endothelial damage. The von Willebrand factor is a marker of endothelial damage and may be measured in a quantitative and qualitative form. This study demonstrates a significant statistically difference in both qualitative and quantitative form of von Willebrand factor in smokers and COPD patients compared to the control group. When analyzing subgroups in COPD patients considering four different classifications: GOLD 2006, GOLD 2011, degree of symptoms from the MRC dyspnea scale and number of exacerbations in the last year. We observed a significant statistically difference in the level of qualitative von Willebrand factor only in the subgroup of the degree of symptoms. Inverse correlation was observed with the percent of forced expiratory volume in one second (FEV1%) and the qualitative von Willebrand factor. Thus, the von Willebrand factor is increased in COPD patient and could be a possible serum marker of the presence of symptoms in this patient. We cannot identify the severity of COPD patients in GOLD classification using von Willebrand factor, but we established a inverse correlation between qualitative von Willebrand factor and FEV1%, what may suggest a participation in the progression of the disease.

Orientador: Joyce Annichino-Bizzacchi / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-13T01:42:43Z (GMT). No. of bitstreams: 1 Bittar_LuisFernando_M.pdf: 1309049 bytes, checksum: 8d7bbcbc4a659300d128bc58336cbb88 (MD5) Previous issue date: 2009 / Resumo: Níveis elevados de fator VIII (FVIII) são um fator de risco independente e prevalente para trombose venosa profunda (TVP), e tem influência do FvW. A ADAMTS13 é responsável pela modulação do tamanho molecular do FvW, clivando os multímeros de altíssimo peso molecular. Alterações moleculares no gene da ADAMTS13 têm correlação com sua atividade. Neste estudo avaliamos a prevalência do polimorfismo A4751G no gene do FvW (região de ligação com a ADAMTS13), os polimorfismos C1797T e C1852G e a mutação C4006T no gene da ADAMTS13 em 435 pacientes com TVP (156M/279F; idade mediana=37) e 580 controles (170M/410F, idade mediana=35). Investigamos a relação entre os genótipos e a dosagem de FVIII e FvW no plasma e o risco de TVP. A dosagem de FVIII:C foi realizada por método coagulométrico de um estágio, e as dosagens de FVIII:Ag e FvW:Ag por método imunoenzimático. As alterações moleculares foram determinadas por PCR e digestão com enzimas específicas, ou SSCP e sequenciamento para confirmação. Pacientes com TVP mostraram níveis significantemente aumentados de FVIII:C (203.7 UI/dl vs. 127 UI/dl; p<0.001), FVIII:Ag (109.6 UI/dl vs. 82.4 UI/dl; p<0.001) e FvW:Ag (154.2 UI/dl vs. 108 UI/dl; p<0.001) quando comparados com o grupo controle. Não houve diferença significativa entre os grupos na prevalência das alterações moleculares estudadas. Os indivíduos com genótipo AG (FvW A4751G) apresentavam níveis significativamente reduzidos de FVIII:C (p=0.04). Embora também tenha demonstrado uma discreta associação com níveis diminuídos de FvW:Ag, esta não foi estatisticamente significativa (p= 0.07). Os indivíduos com genótipo CG (ADAMTS13 C1852G) apresentavam níveis significativamente aumentados de FVIII:Ag (p=0.05) e FvW:Ag (p= 0.01). Apesar da relação com diminuição do FVIII o polimorfismo A4751G não mostrou um efeito protetor para TVP. O polimorfismo ADAMTS13 C1852G está associado à diminuição desta metaloprotease, e sua associação com níveis aumentados de FVIII:Ag e FvW:Ag neste estudo favorece essa hipótese. / Abstract: Elevated levels of factor VIII (FVIII) are a prevalent and independent risk factor for deep venous thrombosis (DVT), and are affected by von Willebrand factor (vWF) levels. ADAMTS13 is responsible for the modulation of the molecular size of vWF, cleaving the ultra large multimers. Mutations and polymorphisms in the ADAMTS13 gene are related with its activity. This study evaluated the prevalence of polymorphism A4751G in the vWF gene, polymorphisms C1797T, C1852G and the mutation C4006T in the ADAMTS13 gene in 435 patients with DVT and 580 healthy controls. Subsequently, we investigated the relationship between the genotypes and plasma levels of FVIII and vWF and DVT risk. FVIII:C was measured by a one-stage clotting method, and FVIII:Ag and vWF:Ag were measured by chromogenic method. The molecular changes were determined by restriction endonucleases or single strand conformation polymorphism followed by sequencing. Statistical test:U Mann-Whitney, = 0.05. Patients with DVT had higher plasma levels of FVIII:C (mean 203,7 UI/dl vs. 127 UI/dl; p<0.001), FVIII:Ag (mean 109,6 UI/dl vs. 82,4 UI/dl; p<0.001) and vWF:Ag (154,2 UI/dl vs. 108 UI/dl; p<0.001) when compared to controls. We observed no statistical difference in the prevalence of all molecular changes studied between patients and controls. A4751G heterozygotes had significantly reduced levels of FVIII:C (p=0,04). Althought there was a slight association with reduced levels of vWF: Ag, this association was not statistically significant (p= 0,07). C1852G heterozygotes had significantly elevated levels of FVIII:Ag (p=0.05) and vWF:Ag (p= 0,01). Despite the relative decline of FVIII:C with the A4751G polymorphism there was no protective effect for DVT. The C1852G polymorphism is associated with a reduction of ADAMTS13, and its association with increased levels of FVIII: Ag and vWF: Ag observed in this study supports this hypothesis. / Mestrado / Biologia Estrutural, Celular, Molecular e do Desenvolvimento / Mestre em Fisiopatologia Médica

Orientador: Margareth Castro Ozelo / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-15T13:42:22Z (GMT). No. of bitstreams: 1 Damian_GuilhermeBenedini_M.pdf: 1979799 bytes, checksum: 8b7242b6fe37a1f5f52bc6f26ca9d829 (MD5) Previous issue date: 2010 / Resumo: A doença de von Willebrand (DVW) é a doença hemorrágica hereditária mais freqüente. Dentre os subtipos relacionados à DVW, o tipo 2N apresenta exclusivamente diminuição da afinidade do fator de von Willebrand (FVW) ao fator VIII (FVIII). Como conseqüência, esses pacientes apresentam redução do FVIII plasmático. Por essa razão, comumente esses casos são diagnosticados erroneamente como portadores de hemofilia A. Em um recente estudo brasileiro, foi observado que cerca de 10% dos casos de hemofílicos A leve sem antecedente familiar prévio para a doença, na verdade tratavam-se de DVW tipo 2N (SIMON & ROISENBERG, 2004). Para o diagnóstico de certeza da DVW tipo 2N, a avaliação molecular é o método mais utilizado, uma vez que os testes laboratoriais confirmatórios não apresentam boa reprodutividade. Esse estudo tem como objetivos fazer a investigação molecular de casos previamente diagnosticados ou com suspeita de DVW tipo 2N baseados na história clínica, familiar e dados laboratoriais. Além disso, pacientes diagnosticados com hemofilia A moderada e leve, foram investigados para a presença das quatro mutações mais freqüentes relacionadas à DVW tipo 2N. Esse estudo avaliou oito casos não relacionados de paciente com suspeita ou diagnóstico de DVW tipo 2N, acompanhados no Hemocentro da UNICAMP (Campinas, SP) e no Hemocentro do Espírito Santo (Vitória, ES). Os oito casos foram investigados para mutações presentes na região que corresponde ao local do sítio de ligação do FVIII ao FVW, localizada entre os éxons 18 a 27 do gene do FVW. Apenas um caso foi conclusivo, com a presença da mutação em homozigose R816W no gene do FVW Essa mutação corresponde à 11% dos casos de DVW tipo 2N. Os outros sete casos foram exaustivamente investigados para mutações nessa região, incluindo o seqüenciamento do DNA genômico e codificante da região entre éxon 18 a 27 do gene do FVW e não foram conclusivos. Esses mesmos casos foram investigados através do seqüenciamento do gene do FVIII, para avaliar a presença de mutação associada à hemofilia A. Em um dos casos do sexo feminino, foi possível a identificação de uma mutação em homozigose G265 +1 G>T no íntron 3 do gene do FVIII. Trata-se de uma mutação não descrita no local de um sítio doador de splice. Foram avaliados ainda 67 pacientes não relacionados com diagnóstico de hemofilia A leve ou moderada para as quatro mutações mais freqüentes relacionadas à DVW tipo 2N. Em nenhum desses casos essas mutações estavam presente, nem mesmo em heterozigose. Em conclusão, na avaliação de oito casos não relacionados com suspeita de DVW tipo 2N, em apenas um caso esse diagnóstico foi confirmado. Em outro caso do sexo feminino foi confirmado o diagnóstico de hemofilia A grave/moderada. Ao contrário do que foi observado em outra população brasileira estuda, entre os pacientes diagnosticados com hemofilia A leve e moderada aqui estudos, as quatro mutações mais freqüentes na região do sítio de ligação do FVIII ao FVW estavam ausentes em todos os casos. O diagnóstico de certeza entre a DVW tipo 2N e hemofilia A é de extrema importância para o tratamento correto desses casos e para o aconselhamento genético. A investigação molecular continua sendo a melhor maneira para a diferenciação desses casos / Abstract: The von Willebrand disease (VWD) is the most frequently hemorrhagic disease. Among the different types of VWD, the type 2N VWD is characterized by a markedly decreased affinity of von Willebrand factor (VWF) to factor VIII (FVIII). As consequence, the clearance of FVIII is accelerated and these patients while maintaining the concentration of VWF and its ability to normal platelet aggregation have reduced plasma FVIII. Frequently these cases are misdiagnosed as carriers of hemophilia A (HA). In a recent Brazilian study, it was observed that about 10% of cases of mild hemophilia A with no family history, it was actually type 2N VWD (SIMON & ROISENBERG, 2004). The molecular investigation is the best method to confirm the diagnosis of type 2N VWD once the confirmatory test, VWF:FVIII binding assay (VWF:FVIIIB) it is not reproducible. This study aims investigate the molecular diagnosis of cases previously diagnosed or with suspect of type 2N VWD, based on clinical and family history, and laboratorial data. Furthermore, patients diagnosed with mild or moderate hemophilia A, were investigated for the presence of the four most frequent mutations related to type 2N VWD. The study evaluated eight unrelated cases with suspect or diagnosis of type 2N VWD followed at Hemocentro UNICAMP (Campinas, SP) and Hemocentro do Espírito Santo (Vitória, ES). The eight cases were investigated for mutations in the region that corresponds to the location of the binding site of FVIII to VWF, located between exons 18 to 27 of the VWF gene. Only one case was conclusive, with the presence of the R816W mutation in the VWF gene. This mutation corresponds to 11% of type 2N VWD. The other seven cases were repeatedly investigated for mutations in this region, including the sequencing of genomic DNA and the coding region of exon 18 to 27 of the VWF gene and were not conclusive. These same cases were investigated by sequencing of the FVIII gene to assess the presence of mutations associated with hemophilia A. In one female case, it was possible to determine the presence in homozigose of a mutation G265 +1 G>T, a donor splicing region in the intron 3 of the FVIII gene. This mutation was not previously described. In addition, we evaluated 67 unrelated patients with diagnosis of mild or moderate hemophilia A for the four most frequent mutations related to type 2N VWD. None of these cases showed the presence of these mutations, even in heterozygosis. In conclusion, the evaluation of eight unrelated cases for type 2N VWD showed that just one case this diagnosis was confirmed. Different to what was observed in another Brazilian population studied, among mild and moderate hemophilia A followed at the Hemocentro UNICAMP, the four mutations in the binding site of FVIII to VWF were absent in all the cases. The confirmatory diagnosis between type 2N VWD and hemophilia A is extremely important for the correct treatment and the appropriate genetic counselling. The molecular investigation remains the best way to differentiate these cases / Mestrado / Clinica Medica / Mestre em Clinica Medica

No description available.

No / Background: The molecular regulation of endothelial nitric oxide synthase (eNOS) in blood platelets and the signalling events induced by platelet-derived NO are poorly defined. In particular, the ability of von Willebrand factor (VWF) to stimulate cyclic guanosine monophosphate (cGMP) formation in platelets has produced conflicting data. Objectives: To determine the mechanisms leading to eNOS activation and clarify the downstream signaling pathways activated by platelet-derived NO in response to VWF. Methods: We used three independent markers of NO signaling, [3H] l-citrulline production, cGMP accrual and immunoblotting of vasodilator¿stimulated phosphoprotein (VASP) to examine the NO signaling cascade in response to VWF. Results: VWF increased NO synthesis and bioavailability, as evidenced by increased [3H] l-citrulline production and cGMP accrual, respectively. VWF-induced eNOS activation was GPIb-IX-dependent and independent of integrin ¿IIbß3. cGMP formation in response to VWF required Ca2+ mobilization, Src family kinases, phosphatidylinositol 3-kinase and phospholipase C, but not protein kinase C. This suggests that a cross-talk between the signaling mechanisms regulates platelet activation and NO synthesis. VWF-induced cGMP accrual was completely blocked by apyrase and indomethacin, demonstrating an essential role for platelet-derived ADP and thromboxane A2 (TxA2). Elevated cGMP levels led to increased VASP phosphorylation at serine239 that was both protein kinase G (PKG)- and protein kinase A (PKA)-dependent. Conclusions: We demonstrate that VWF activates eNOS through a specific Ca2+-dependent GPIb receptor-signaling cascade that relies on the generation of platelet-derived ADP and TxA2. Furthermore, we provide the first evidence to suggest that platelet derived-NO/cGMP activates PKA in addition to PKG.