Von Willbrand Factor Research Articles

Evaluation of von willbrand factor in severely affected COVID-19 patients

Evaluation of von willbrand factor in severely affected COVID-19 patients

PB0825 Effect of von Willbrand Factor Concentrate on von Willebrand Factor Parameters during ECMO

PB0825 Effect of von Willbrand Factor Concentrate on von Willebrand Factor Parameters during ECMO

Determination of von willebrand factor antigen in patients with undiagnosed menorrhagia

This prospective study is about screening of von willbrand antigen in patients with undiagnosed menorrhagia, because the underlying cause may be haematological disorder in 50% of cases. To estimate the incidence of von willbrand disease in patients with undiagnosed menorrhagia. During the period of 2 months from February to april 2005, total of 50 patients were included in this study that were referred to Al-yarmok teaching hospital. The age range of these patients was 18-45 year. Full history and physical examination done, ultrasound examination to exclude local causes of menorrhagia, thyroid function done. 30 healthy volunteer age matched were included as control group. For each patient and control group the following tests were done: Bleeding time, Prothrombin time, Activated partial thromboplastin time, Factor 8 level, Von willbrand factor antigen level.Results1.There was no obvious or statistically significant difference in mean bleeding time and activated partial thromboplastine time observed between healthy controls and patients with menorrhagia.2.The mean Von Willebrand factor antigen (%) was significantly lower in patients with menorrhagia compared with healthy controls.3.The mean Von Willebrand factor antigen was slightly lower at the older age group compared to younger age group.4.The proportion of patients with prolonged activated partial thromboplastine time was obviously higher among those with abnormally low Von Willebrand factor antigen compared to those with acceptable level.5.The relative frequency of cases with abnormally low factor VIII activity was higher among subjects with other bleeding manifestations compared to those with no manifestations. 6.The relative frequency of cases with abnormally low factor VIII activity was higher among subjects with positive family history of bleeding tendency. 7.The relative frequency of cases with abnormally low Von Willebrand factor was obviously higher among subjects with other bleeding manifestations compared to those with no bleeding manifestations, also it was higher among cases with positive family history of bleeding tendency and those with past history of severe bleeding episodes.Conclusions: The prevalence of Von Willebrand disease is increased in women with menorrhagia. So it is reasonable to believe that Von Willebrand disease is the underlying cause for meborrhagia in a small but significant proportion of women. This study revealed that 6% of women with menorrhagia had Von Willebrand disease.

Assessment of vascular function in complete glycaemic spectrum

ABSTRACT Purpose: The present study was conceived to delineate the point of vascular dysfunction along the glycemic spectrum (normoglycemic individuals with no family history of diabetes, normoglycemic individuals with family history of diabetes, prediabetic individuals, and diabetic individuals). Materials and Methods: In this cross-sectional comparative study, we enrolled 252 participants of both gender in the age group of 30–50 years. They were classified based on their family history of diabetes and glycemic status into four groups along the glycemic spectrum as mentioned above. We measured flow-mediated dilation (FMD) from brachial artery and vascular function biomarkers such as enthothelin-1 (ET-1), von Willbrand Factor (vWF), Vascular Endothelial Growth Factor (VEGF) to assess the vascular function. The comparison of data between groups were done using One Way ANOVA/Kruskal–Wallis followed by post-hoc analysis using LSD/Mann–Whitney U Test depending on the normality of the data. Spearman correlation was done between vascular function and plasma glucose levels to identify its relationship. Linear regression was carried out to identify the factors influencing the FMD across the glycemic spectrum. Results: We observed that vascular function negatively correlated with blood glucose levels. However, endothelin-1 and vWF derangement was there even in normoglycemic first degree relatives of diabetes (FDRD) and the derangement increased in prediabetes and diabetes. Physiological dysfunction in terms of decreased flow-mediated dilation starts from prediabetes only. VEGF derangement is found only in diabetic individuals. Conclusion: Vascular dysfunction is found even in normoglycemic FDRD and the derangement increased and compounded with the advancement of disease.

Complement and coagulation cascades activation is the main pathophysiological pathway in early-onset severe preeclampsia revealed by maternal proteomics

Preeclampsia is a pregnancy-specific multisystem disorder and a leading cause of maternal and perinatal morbidity and mortality. The exact pathogenesis of this multifactorial disease remains poorly defined. We applied proteomics analysis on maternal blood samples collected from 14 singleton pregnancies with early-onset severe preeclampsia and 6 uncomplicated pregnancies to investigate the pathophysiological pathways involved in this specific subgroup of preeclampsia. Maternal blood was drawn at diagnosis for cases and at matched gestational age for controls. LC–MS/MS proteomics analysis was conducted, and data were analyzed by multivariate and univariate statistical approaches with the identification of differential pathways by exploring the global human protein–protein interaction network. The unsupervised multivariate analysis (the principal component analysis) showed a clear difference between preeclamptic and uncomplicated pregnancies. The supervised multivariate analysis using orthogonal partial least square discriminant analysis resulted in a model with goodness of fit (R2X = 0.99, p < 0.001) and a strong predictive ability (Q2Y = 0.8, p < 0.001). By univariate analysis, we found 17 proteins statistically different after 5% FDR correction (q-value < 0.05). Pathway enrichment analysis revealed 5 significantly enriched pathways whereby the activation of the complement and coagulation cascades was on top (p = 3.17e−07). To validate these results, we assessed the deposits of C5b-9 complement complex and on endothelial cells that were exposed to activated plasma from an independent set of 4 cases of early-onset severe preeclampsia and 4 uncomplicated pregnancies. C5b-9 and Von Willbrand factor deposits were significantly higher in early-onset severe preeclampsia. Future studies are warranted to investigate potential therapeutic targets for early-onset severe preeclampsia within the complement and coagulation pathway.

Role of Plasma Von Willebrand Factor-Antigen in predicting the presence of Esophageal Varices and occurrence of its bleeding in cirrhotic patients

Background and study aim: Baveno VI consensus recommended the use of non- invasive predictors of EV to avoid unnecessary endoscopies. Von Willbrand factor (VWF) and VITRO score, (VWF/ platelet count), are both correlated to liver cirrhosis and fibrosis. The aim of this study was to evaluate the role of VWF and VITRO score as predictors of esophageal varices and their bleeding. Patients and Methods: Seventy seven patients were included in this study. They were randomly selected from cirrhotic patients admitted to endoscopy unit for the first time. They were allocated into two groups;group I:patients who have esophageal varices,group II:patients with no esophageal varices. Results: VWF Ag and VITRO score were significantly higher in the varices group (group I). VWF Ag level was 169.3±20.2 in group I vs 146.8±35.5 µg/dL in group II p<0.001. VITRO score was 2.2±1.1 in group I vs 1.6±0.7 µg/108 platelet p=0.05. We found that at cut off value of 153% VWF can predict the presence of EV with sensitivity 88.1% and specificity of 61.1% and AUC= 0.66 p=0.04. VITRO score can predict the presence of varices with sensitivity of 69.5% and specificity of 50% at a cut off value 1.5 AUC=0.065 P=0.05. Conclusion: VWF and VITRO score rise significantly in patients with esophageal varices. Both markers can be reliable in prediction of the presence of EV’s. VWF Ag can be reliable marker in prediction of risky and bleeding varices.

The Role of Chromogenic and Immunoturbidimetry Assays in Diagnostic of Thrombotic State in Myeloproliferative Disorders

Polycythaemia vera (PV) is a monoclonal myeloproliferative disorder characterized by a haemostatic imbalance resulting in increased risk for thrombotic events. In PV there is an important affection of the vascular endothelium, so we analized in our study three markers of endothelium disfunction- von Willbrand factor (vWF), Protein C (PC) and the inhibitor plasminogen activator type-1. (PAI-1) The study comprised 40 patients and the objective of the study was to investigate the level of the mentioned parameters in two groups of patients-the group of polycytemic patients without cardiovascular diseases (PV) and the group of polycitemic patients with cardiovascular pathology associated (PV+CVD). The levels of the studied parameters were found significantly modified in the PV group (p[0.05); for the PV+CVD group the level of vWF was higher (p[0.001), but the levels of the other parameters, didn�t show a significant statistic difference compared with the first group (p]0.05). In accordance with the presented results, the conclusions of our study was that in PV the risk of thrombotic accidents is related by the increase of the vWF level, as the vascular endothelium plays a key role in this pathology.

Role of endothelial dysfunction in relation to prothrombogenesis in polycythemia vera

Background: The morbidity and mortality in polycythemia vera (PV) are closely associated with cardiovascular diseases burden and clonal evolution. These complications were primarily attributed to abnormal rheology consequent to the raised hematocrit, leukocytosis, and thrombocytosis, and in vivo activation of leukocytes, thrombocytes, and endothelial cells. It has been established that damage of endothelium determines the release in circulation of specific markers including thrombomodulin (TM), selectins, and von Willbrand factor (vWF) which are released and favor the formation of cellular aggregates. Objectives: The objective of this study is to evaluate the pathophysiological role of endothelial dysfunction (ED) in relation to increased risk of thrombosis in PV patients. Patients and Methods: In a case–control study, 53 patients enrolled in this study from Al-Imamain Al-Khadimiyan Medical City, and the National Center for Hematology Diseases and Researches. They comprised of thirty patients with PV with mean age of − 54.87 ± 13.44 years-and another twenty-three patients with secondary polycythemia, whose mean age was 40.13 ± 12.21 years. Another thirty aged- and sex-matched, non-smokers healthy volunteers comprised 16 males and 14 females were also studied, their mean age was = 52.1 ± 11.16 years. JAK2 mutation was assessed for PV group while Serum erythropoietin (Epo), vWF and TM were determined for all patient and control group. Results: TM was significantly different among the three studied groups (P Conclusion: ED is one of mechanisms that contribute in prothrombogenesis in PV patient.

Study of A Disintegrin and Metalloproteinase with ThromboSpondin Type 1 Repeats 13 (ADAMTS 13) in Children with Idiopathic Nephrotic Syndrome

Background: Nephrotic syndrome (NS) is a well-known risk factor for arterial or venous thromboembolism (TE). There is higher risk of TE in steroid-resistant NS (SRNS) than in steroid-sensitive NS (SSNS). Objective: The aim of this study was to investigate serum level of von Willbrand factor cleaving protease activity which is known as ADAMTS in children with idiopathic nephrotic Syndrome(INS) and its relation to clinical and laboratory parameters. Patients and methods: This study was conducted on 120 children with INS including 40 SSNS, 40 SRNS, 40 healthy controls. All subjects are investigated by CBC, 24 hours collected urine analysis for urine volume, urinary proteins, total Serum protein and serum albumin,total serum cholesterol,prothrombi time (PT), partial thromboblastin time(PTT) and Serum ADAMTS 13 activity. Results: There was highly significant decrease in serum ADAMTS 13 activity in SSNS and SRNS groups when compared to control group while there was no significant difference in serum ADAMTS 13 activity between SSNS and SRNS groups. Conclusion: Serum ADAMTS 13 activity is a biomarker for endothelial dysfunction and hypercoagulable state. The decreased ADAMTS13 activity in different extent of nephrotic patients (SSNS and SRNS) may be one of the pathogenesis of thrombosis as a common complication of NS. Recommendation: Regular follow-up of nephrotic patients and estimation of serum ADAMTS 13 level as its decresed level is a risk factors of thrombosis.

REVASCULARIZATION OF ACELLULAR RAT LUNG SCAFFOLDS USING THE ENDOTHELIAL COLONY FORMING CELLS

s S157 revascularization of acellular lung scaffolds using ECFCs as a critical first step in engineering truly functional grafts. OBJECTIVES: 1) Compare decellularization Methods to achieve optimal decellularization with maintained vascular perfusion. 2) Regenerate vascular endothelium using ECFCs. METHODS: Acellular rat lung scaffolds were generated by static incubation and perfusions Methods of decellularization. For static incubation, lungs were submerged into Triton-X 100 (0.1%) and sodium deoxycholate (2%) based decellularization buffer. For perfusion decellularization, isolated rat lungs were perfused using CHAPS buffer (8mM CHAPS, 1M NaCl, 25mM EDTA in 1X PBS) or SDS-Triton-X buffer (0.1% SDS followed by 1% Triton-X). Decellularization of lungs was evaluated by tissue histology, total DNA measurement and immuno-blotting. For revascularization, ECFCs were generated from human peripheral blood or rat bone marrow derived mononuclear cells and characterized for ECFCmarkers. Further, we also studied initial engraftment of human ECFCs in the rat lung scaffolds. RESULTS: Static incubation method of decellularization generated acellular lung scaffolds with no evidence of intact cells; however, presence of intracellular protein (GAPDH) was evident. Vascular perfusion could not be established in these scaffolds. On the other side, perfusion decellularization using CHAPS buffer produced acellular lung scaffolds with maintained extracellular structure. Moreover, perfusion decellularization with CHAPS buffer consistently demonstrated complete absence of cells, below detectable DNA remnants and intracellular protein, with more optimal vascular perfusion. ECFCs were successfully derived from human peripheral blood and rat bone-marrow mononuclear cells. ECFCs were positive for endothelial specific markers such as Ac-LDL uptake, lectin binding and expressed CD31, von Willbrand Factor (vWF) and VEGFR2. Moreover, these cells did not express pan-leukocyte marker CD45. Initial engraftment study of human ECFCs in rat lung scaffolds showed engraftment of human ECFCs in vascular compartment of the lung scaffolds. CONCLUSION: We successfully prepared acellular lung scaffolds and identified ECFC, which can be utilized to generate truly functional grafts. HSF, CIHR 204 GENERATION AND UTILIZATION OF HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS-DERIVED-IPS FOR STUDYING ENDOTHELIAL GENE REGULATION M Nakhaei-Nejad, AG Murray, N Jahroudi

Human Primary Lung Endothelial Cells in Culture

Pulmonary endothelial functions are critical to maintain the low pressure of the pulmonary circulation and effective diffusion capacity of the lung. To investigate pulmonary endothelial cell biology in healthy or diseased lungs, we developed methods to harvest and culture pure populations of primary pulmonary arterial endothelial cells and microvascular endothelial cells from human lung explanted at time of transplantation or from donor lungs not used in transplantation. The purity and characteristics of cultured endothelial cells is ascertained by morphologic criteria using phase contrast and electron microscopy; phenotypic expression profile for endothelial specific proteins such as endothelial nitric oxide synthase, platelet/endothelial cell adhesion molecule, and von Willbrand factor; and endothelial function assays such as Dil-acetylated low-density lipoprotein uptake and tube formation. This detailed method provides researchers with the ability to establish cells for molecular, genetic, and biochemical investigation of human pulmonary vascular diseases.

Nicotine improves the functional activity of late endothelial progenitor cells via nicotinic acetylcholine receptors

The aim of this study is to investigate whether nicotinic acetylcholine receptors (nAChRs) are involved in the modulation of functional activity of late endothelial progenitor cells (EPCs) induced by nicotine. Total mononuclear cells (MNCs) were isolated from human umbilical cord blood by Ficoll density gradient centrifugation, and then the cells were plated on fibronectin-coated culture plates. Late EPCs were positive for 1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine-labeled acetylated low-density lipoprotein (DiI-acLDL) uptake and fluorescein-isothiocyanate-conjugated Ulex europaeus agglutinin lectin (UEA-1) binding. Expression of von Willbrand factor (vWF), kinase insert domain receptor (KDR), and α7 nAChR was detected by indirect immunofluorescence staining. Late EPCs of 3-5 passages were treated for 32h with either vehicle or nicotine with or without pre-incubation of nAChR antagonism, mecamylamine, or α-bungarotoxin. The viability, migration, and in vitro vasculogenesis activity of late EPCs were assayed with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, modified Boyden chamber assay, and in vitro angiogenesis assay, respectively. Late EPCs adhesion assay was performed by replating cells on fibronectin-coated plates, and then adherent cells were counted. Incubation with 10nmol/L nicotine enhanced viable, migratory, adhesive, and in vitro vasculogenesis capacity of late EPCs. The effect of nicotine on late EPCs can be attenuated by mecamylamine or α-bungarotoxin. In conclusion, nicotine improves the functional activity of late EPCs via nAChRs.

Effects of osteopontin on functional activity of late endothelial progenitor cells

The aim of this study is to investigate the effect of osteopontin (OPN) on functional activity of late endothelial progenitor cells (EPCs). Total mononuclear cells (MNCs) were isolated from human umbilical cord blood by Ficoll density gradient centrifugation, and then the cells were plated on fibronectin-coated culture plates. Late EPCs were positive for both 1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine-labeled acetylated low-density lipoprotein (DiI-acLDL) and fluorescein-isothiocyanate-conjugated Ulex europaeus agglutinin lectin (UEA-1). Expression of von Willbrand factor (vWF) and kinase insert domain receptor (KDR) were detected by indirect immunofluorescence staining. Late EPCs of 3-5 passages were treated for 24 h with OPN (to make a series of final concentration: 0.005 µg/ml, 0.01 µg/ml, 0.05 µg/ml, 0.5 µg/ml, 2.5 µg/ml), or vehicle control. The proliferation, migration, and in vitro vasculogenesis activity of late EPCs were assayed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, modified Boyden chamber assay and an in vitro angiogenesis assay, respectively. Late EPCs adhesion assay was performed by replating cells on fibronectin-coated plates, and then adherent cells were counted. Incubation with OPN dose-dependently inhibited the proliferative, adhesive, and in vitro vasculogenesis capacity and increased migratory activity of late EPCs.

Proteolytic fragmentation and sugar chains of plasma ADAMTS13 purified by a conformation-dependent monoclonal antibody

ADAMTS13 is a metalloproteinase that specifically cleaves unusually large von Willbrand factor multimers under high-shear stress. Deficiency of ADAMTS13 activity induces a life-threatening generalized disease, thrombotic thrombocytopenic purpura. We established a simple and efficient method to purify plasma ADAMTS13 (pADAMTS13) from cryosupernatant using an anti-ADAMTS13 monoclonal antibody (A10) that recognizes a conformational epitope within the disintegrin-like domain. Using the purified pADAMTS13, the amino acid residues involved in cleavage by thrombin, plasmin and leucocyte elastase were determined, and the carbohydrate moieties of this enzyme was analysed by lectin blots. Purified pADAMTS13 had a specific activity of 300 U/mg (25,057-fold purification) and the pI was 5.1-5.5. Cleavage sites of the purified pADAMTS13 by three proteases were identified; thrombin cleaved the four peptidyl bonds between Arg257-Ala258, Arg459-Ser460, Arg888-Thr889 and Arg1176-Arg1177, plasmin cleaved the three peptidyl bonds between Arg257-Ala258, Arg888-Thr889 and Arg1176-Arg1177, and elastase cleaved the two peptidyl bonds between Ile380-Ala381 and Thr874-Ser875. Lectin blot analysis indicated the presence of non-reducing terminal α2-6 and α2-3-linked sialic acid residues with penultimate β-galactose residues on the N- and O-linked sugar chains of pADAMTS13, suggesting that pADAMTS13 is cleared from the circulation via the hepatic asialoglycoprotein receptor like other plasma glycoproteins.

Thrombotic thrombocytopenic purpura associated with syaphylococcus aureus endocarditis in a diabetic with furunculosis

Thrombotic thrombocytopenic purpura (TTP) is an uncommon and potentially fatal syndrome which is due to either congenital or acquired deficiency of the ultra large von Willbrand's factor (ULVWF) cleavage protease. It is characterized by mechanical micro angiopathic hemolytic anemia, and organ dysfunctions.

The Mechanisms for Platelet Hyperactivity Induced by Hypergravity.

Abstract 5059Many gravity change-related thrombotic and hemorrhagic diseases or fatalities have been reported to occur in human beings, such as crewmen in space, roller coaster riders, and aircrew subjected to high-G training. Because platelets, as anucleated blood cell, are the primary players in both thrombosis and hemostasis, function change of platelets in altered gravity conditions might offer some insights into the mechanisms leading to these gravity change-related diseases. We have demonstrated that the platelet functions are inhibited in microgravity environments, and activated under high-G conditions, which reveals a novel mechanism for gravity change-related hemorrhagic and thrombotic diseases. The mechanisms, however, remain to be further investigated. Here, firstly we investigated the effect of hypergravity (acceleration centrifugation) on platelet functions. Platelet rich plasma (PRP) or washed platelets were exposed to hypergravity at 8 G for different time points (2 minutes, 10 minutes, 15 minutes, 20 minutes), and then platelet aggregation was measured at each time point. No platelet aggregation was induced by 8 G alone, whereas ristocetin or collagen-induced platelet aggregation was time-dependently increased by hypergravity stimulus. The effect was maximal at 15 minutes after hypergravity treatment. Spreading assay shows that the number of platelet adhesion to immobilized fibrinogen and the mean platelet area spreading on von Willbrand factor (VWF) matrix were obviously increased by hypergravity. To further explore the effect of hypergravity on platelet activity state, the markers of platelet activations (integrin αaIIbβ3 activation, P-selectin surface expression) were assessed by flow cytometry. αaIIbβ3 was activated, in part, in 8 G-exposed platelets as detected by PAC-1 binding, but no significant difference was observed in P-selectin surface expression compared with 1 G controls. Next, we investigated the effects of hypergravity stimulus on actin distribution and intracellular Ca2+ ([Ca2+]i) in isolated human platelets. Hypergravity at 8 G induced actin cytoskeleton reorganization in platelets, and the release of Ca2+ from internal stores, but not Ca2+ influx via mechanosensitive Ca2+ channels in the plasma membrane. Pretreatment of platelets with intercellular Ca2+ chelator BAPTA/AM had no effect on hypergravity-induced changes in cytoskeleton, but significantly reduced platelet aggregation induced by hypergravity/ristocetin. In addition, using two different platelet activation inhibitors acetylsalicylic acid (aspirin) and tirofiban, which are widely used as antithrombotic drugs, we assessed the effects of the drugs on hypergravity-induced platelet activation in mice. Treatment of mice with aspirin or tirofiban significantly reduced hypergravity-induced P-selectin expression on platelet surface, prolonged tail bleeding time in vivo and reduced death rate in hypergravity-treated mice. Thus, our results indicate that hypergravity leads to human platelet hyperactivity, but fails to incur essential platelet activation events. Actin cytoskeleton reorganization and [Ca2+]i play important roles in regulation of platelet functions under hypergravity condition. The reorganization of actin cytoskeleton may be upstream or independent of [Ca2+]i, suggesting that hypergravity would affect various intercellular signals leading to platelet hyperactivity. In addition, antithrombotic agents such as aspirin and tirofiban have the therapeutic potential for hypergravity-induced thrombotic diseases. DisclosuresNo relevant conflicts of interest to declare.

The effect of short-term aerobic training on coagulation and fibrinolytic factors in sedentary healthy postmenopausal women

ObjectiveThe purpose of this study was to assess the effect of short-term aerobic training on the fibrinolytic and coagulative factors in postmenopausal women. Study designTwenty volunteer sedentary healthy postmenopausal women (48–53 years), who entered the menopause naturally, were divided randomly into two groups: training (n=10) and control (n=10). Methods and main outcome measuresTraining consisted of 10 sessions of submaximal aerobic cycling, 35min for each session (5min warm-up, 25min aerobic training with 70% HRmax, 5min active and 15min passive recovery), 3 times a week. Coagulation and fibrinolytic factors were assessed both before and after aerobic program in both groups. ResultsFibrinogen, von Willbrand factor (vWF-Ag) antigen, plasminogen activator inhibitor-1 activity (PAI-1:Ac) and antigen (PAI-1:Ag) showed significant reduction after 10 sessions in the training group (P<0.05). Also after training, prothrombin time (PT), partial thromboplastin time (PTT), tissue plasminogen activator activity (tPA:Ac) and antigen (tPA:Ag) increased (P<0.05). ConclusionIt was concluded that fibrinolytic activity on postmenopausal women could be improved by a 3-week regular submaximal training program. These changes on the hemostatic factors suggest that short-term aerobic training may prevent the decline in fibrinolytic function observed in sedentary postmenopausal women.

Efecto de la hipergravedad en la función plaquetaria

Thrombotic diseases or fatalities have been reported to occasionally occur under conditions of hypergravity although the mechanism is still unclear. To investigate the effect of hypergravity on platelets that are the primary players in thrombus formation, platelet rich plasma (PRP) or washed platelets were exposed to hypergravity at 8 G for 15 minutes. No platelet aggregation was induced by 8 G alone, whereas ristocetin or collagen-induced platelet aggregation was significantly increased. The number of platelets adherent to immobilized fibrinogen and the area of platelets spreading on von Willbrand factor (VWF) matrix were increased simultaneously. Flow cytometry assay indicated that integrin alphaIIbbeta3 was partially activated in 8 G-exposed platelets, but there was no significant difference in P-selectin surface expression between platelets treated with 8 G and 1 G control. The results indicate that hypergravity leads to human platelet hyperactivity, but fails to incur essential platelet activation events, suggesting a novel mechanism for thrombotic diseases occurring under hypergravitional conditions.

DRAGON, a GPI‐anchored membrane protein, inhibits BMP signaling in C2C12 myoblasts

Bone morphogenetic proteins (BMPs) induce osteoblastic differentiation of myoblasts via binding to cell surface receptors. Repulsive guidance molecules (RGMs) have been identified as BMP co-receptors. We report here that DRAGON/RGMb, a member of the RGM family, suppressed BMP signaling in C2C12 myoblasts via a novel mechanism. All RGMs were expressed in C2C12 cells that were differentiated into myocytes and osteoblastic cells, but RGMc was not detected in immature cells. In C2C12 cells, only DRAGON suppressed ALP and Id1 promoter activities induced by BMP-4 or by constitutively activated BMP type I receptors. This inhibition by DRAGON was dependent on the secretory form of the von Willbrand factor type D domain. DRAGON even suppressed BMP signaling induced by constitutively activated Smad1. Over-expression of neogenin did not alter the inhibitory capacity of DRAGON. Taken together, these findings indicate that DRAGON may be an inhibitor of BMP signaling in C2C12 myoblasts. We also suggest that a novel molecule(s) expressed on the cell membrane may mediate the signal transduction of DRAGON in order to suppress BMP signaling in C2C12 myoblasts.

Establishment of vascular endothelial cell lines from the aortas of wild Japanese serows (Capricornis crispus)

Endothelial cell lines were established from the aortas of wild Japanese serows (Capricornis crispus) by transfection of a simian virus 40 (SV40) large T antigen gene. The cloned cell lines, designed SeET (Japanese serow endothelial-SV40T) cells, express SV40T antigen and retain cobblestone-like morphology. Although von Willbrand Factor (vWF) is expressed in the cells, the expression rate and the quantity are lower than in serow primary endothelial cells. The SeET cells exhibit positive uptake of acetylated low density lipoprotein and dose-dependent cell proliferation upon exposure to vascular endothelial growth factor. These results suggest that these SeET cells have preserved endothelial phenotypes and able to function with decreased expression of vWF. The SeET cell line will be a valuable tool for in vitro studies on the physiological properties of endothelial cells and for the propagation of viruses and parasites of Japanese serows.