Plasma Levels Of vWF Research Articles

Study on the changes of thrombosis-associated factors in patients with coronary heart disease of turbidity-phlegm blocking syndrome

To preliminarily explore the relationship between thrombosis and its associated factors in patients with coronary heart disease (CHD) of turbidity-phlegm blocking syndrome (TPB), and to study its acting mechanism. Plasma levels of thrombosis-associated factors, including Von Willebrand factor: (vWF), D-dimer, and fibrinogen (Fg), in 85 patients of CHD with TPB, 93 with CHD of non-TPD, and 89 healthy persons were detected and compared. Levels of the three factors were increased in all the CHD: patients, and were higher in TPB patients than in non-TPB patients (P<0.01 or P<0.05). The TPB: syndrome in CHD patients was closely related to the blood coagulation-fibrinolytic system; they might be in pre-thrombosis state, and the plasma levels of vWF, D-dimer, and Fg could be taken as the objective indices for thrombosis differentiation of TPB syndrome in CHD patients.

Effects of endothelin-1 and phenylephrine on plasma levels of von Willebrand factor and protein S

Endothelial dysfunction is considered a major factor in the pathogenesis of a wide array of diseases and often leads to increased production of, or increased responsiveness to endogenous vasoconstrictive mediators, such as endothelin-1 (ET-1) or adrenergic agonists. Based on previous studies in animals and in-vitro, we hypothesized that ET-1 and alpha adrenergic stimulation by phenylephrine (PE) may alter plasma levels of von Willebrand factor-Ag (vWF) and protein S in humans. Ten healthy men were studied in a prospective randomized double blind trial: we investigated the effects of infusions of ET-1 and PE on plasma levels of vWF-Ag and protein S. Aditionally, we examined the effects of these vasoconstrictors on thrombomodulin, tissue plasminogen activator and on protein C. ET-1 increased plasma levels of vWF-Ag by 19% (CI: 9-36%; p = 0.008) and increased plasma levels of protein-C by 7% (CI: 0.2 -12 %; p = 0.028), even at doses which produced no increase in blood pressure. Plasma levels of protein S and TPA were not significantly affected by ET-1. After PE-infusion we observed a relative increase in plasma levels of protein S by 20% (CI: 5-28%; p = 0.036) and of TPA by 14 % (CI: 2-39%; p = 0.036). Also, platelet counts increased by 36% (CI: 12-53%; p = 0.028) which correlated (r 2 = 0.86; p = 0.014) with an increase in leukocytes by 49 % (CI: 18-84%; p = 0.028). Plasma levels of vWF and protein C were not affected by PE and neither drug had a significant effect on plasma levels of thrombomodulin. In conclusion, our results support the study hypothesis, that ET-1 and PE increase the plasma levels of vWF and protein S, respectively. ET-1 may induce a procoagulant status in various disease states by increasing vWF-Ag levels.

Baseline von Willebrand factor plasma levels and no-reflow phenomenon after primary percutaneous coronary intervention for ST segment elevation myocardial infarction

No-reflow phenomenon is associated with a poor prognosis and its underlying mechanisms are still poorly understood. von Willebrand Factor (vWF) is a central molecule in haemostasis which plays an important role in acute coronary syndromes. However its possible role in no-reflow has not been assessed prior to this study. Quantitative baseline vWF plasma antigen was measured by immunoturbidometric assay in 54 consecutive patients with a first ST segment elevation acute myocardial infarction, treated by primary percutaneous coronary intervention within 12 h of symptom onset. Definitions of no-reflow were (1) angiographic: final TIMI flow ≤2 or final TIMI flow 3 with a myocardial blush grade <2; (2) electrocardiographic: lack of ST segment resolution (≤50% reduction of ST segment elevation at 90 min). Angiographic and electrocardiographic no-reflow was observed in 32 (59%) and 30 (56%) patients, respectively (only 9 patients had both type of no-reflow). Plasma levels of vWF were significantly higher in patients with angiographic no-reflow but not in those with electrocardiographic no-reflow. Also, vWF was the most powerful independent predictors of angiographic no-reflow (OR 3.8, 95% CI 1.1-12.9, p=0.033). Our results provide new insights into no-reflow pathophysiology with appealing therapeutic implications.

Atorvastatin Improves Endothelial Function and Cardiac Performance in Patients with Dilated Cardiomyopathy: The Role of Inflammation

Statins have been demonstrated to significantly affect the prognosis and outcome of patients with cardiac diseases. Several studies have suggested pleiotropic effects of the statins in these patients. The present study was designed to examine the effects of atorvastatin on inflammation, endothelial function, cardiac performance and exercise tolerance in patients with idiopathic dilated cardiomyopathy (IDCM). Sixty-four patients with IDCM were divided randomly into an atorvastatin treatment group (atorvastatin 10 mg/d orally) and a placebo control group. Before and 12 weeks after the treatment, circulating soluble intercellular adhesion molecule-1 (sICAM-1), Von Willebrand factor (vWF) and C-reactive protein (CRP) levels were detected using enzyme-linked immunosorbent assay (ELISA); Flow-mediated dilatation (FMD) of the brachial artery was measured, and left ventricular ejection fraction (LVEF) and the 6-min walk test (6MWT) evaluated. After atorvastatin treatment, LVEF increased from 34.5 +/- 5.7% to 41.4 +/- 4.5% (P < 0.05), and from 32.8 +/- 4.0% to 36.9 +/- 5.2% (P < 0.05) in the placebo group. Also, the distances covered in the 6MWT increased from 358 +/- 61 m to 431 +/- 66 m in the atorvastatin group, and from 351 +/- 70 m to 382 +/- 74 m in the placebo group (both p < 0.05 vs. baseline). The increases in LVEF and 6MWT distances were significantly greater in the atorvastatin than in the placebo group. sICAM-1, CRP and vWF levels decreased and FMD increased significantly in the atorvastatin group, but not in the control group. Correlation analysis showed that the baseline sICAM-1 level was positively correlated with plasma CRP and vWF levels (r = 0.554 and 0.628, respectively); FMD was inversely correlated with serum sICAM-1 and plasma vWF levels (r = -0.579 and -0.590, respectively) and positively correlated with LVEF and distance attained in 6MWT (r = 0.536 and 0.522, respectively). Twelve weeks of treatment with atorvastatin significantly decreased serum sICAM-1, CRP and vWF levels, and improved the FMD, LVEF and 6MWT outcomes. Inhibition of inflammation, alleviating endothelium damage and endothelial dysfunction might comprise part of the underlying mechanisms leading to the improvement of LV function and exercise tolerance in patients with IDCM.

Modifier genes for disorders of thrombosis and hemostasis.

Most inherited hemostatic disorders exhibit incomplete penetrance and variable expressivity, which can be because of genetic or environmental interactions. This wide phenotypic variability for a given disease can be partly explained by modifier gene interactions. Modifier gene interactions have been described for VWD, TTP and venous thrombosis associated with the factor V Leiden mutation. We have exploited advances in mouse genetics in an effort to identify novel genetic loci that may serve as candidate genetic modifiers for bleeding and thrombosis in humans. We have identified several loci affecting plasma VWF levels and have identified and characterized mouse models of ADAMTS13 deficiency and Factor V Leiden that could be useful for identifying novel genes contributing to thrombosis risk in humans.

Abstract: S5-20 ENDOTHELIAL MARKERS AS PREDICTORS OF 5-YEAR CARDIOVASCULAR MORTALITY IN PATIENTS ON CHRONIC HAEMODIALYSIS

S5-20 Citation: Atherosclerosis Supplement 2009, Vol. 10, Issue 2 ENDOTHELIAL MARKERS AS PREDICTORS OF 5-YEAR CARDIOVASCULAR MORTALITY IN PATIENTS ON CHRONIC HAEMODIALYSIS D Kirmizis, M Pangalou, G Niavis, A Tsiandoulas, M Protopappa, K Barboutis Alpha-Nephrodynamic Dialysis Unit, Serres; Laboratory of Haematology, Serres General Hospital, Serres; Department of Nephrology, Serres General Hospital, Serres The objective of this follow-up study was to determine the significance of established endothelial markers, ie plasma fibrinogen (Fb), von Willebrand factor (vWF) and D-dimer (Dd), as predictors of 5-year cardiovascular (CV) mortality in pts on chronic hemodialysis (HD). Methods: In 75 HD pts (43 males, mean age 63, range 40-77 yrs) we studied the 5-yr CV mortality in relation to Fb, vWF and Dd. Fb was measured by Clauss method and vWf and Dd by latex-enhanced immunoassay. Serum CRP was measured by nephelometry. Cut-off values with the optimal sensitivity and specificity for CV morbidity were calculated with the use of AUC, LR and ROC curves for each variable. Survival analysis was made by the Kaplan-Meier (K-M) method, with the use of Breslow test and Cox regression analysis. Results: Overall 30 of the 68 pts who completed the study (44%) died from CV events. Pts with Dd 335 mg/L had a shorter survival compared to patients with Dd <335 mg/L (median survival time: 28.6 vs 46.8 mts respectively, P<0.001). Plasma Fb 333mg/L and vWF 201%, respectively were not found to be of any predictive CV significance. In Cox regression analysis logDd levels were a strong predictor of CV death, as much as serum logCRP and history of CVD. Age, sex, HD duration, arterial hypertension and plasma Fb and vWF levels failed to show any predictive CV significance. Conclusion: In HD patients, Dd predict the 5-yr CV survival and might represent a marker of atheromatous process. Funding: Any funding sources of commercial nature for the work being presented are disclosed.

Phase I evaluation of orally-administered CYT997, a novel cytotoxic vascular-disrupting agent, in patients with advanced cancer

3568 Background: CYT997 is a novel, small-molecule vascular disrupting agent which binds tubulin and inhibits microtubule assembly. The compound demonstrates potent antitumour and vascular-disrupting activity in preclinical models. A phase I study of CYT997 given by intravenous infusion showed favourable safety and tolerability, pharmacokinetics and preliminary evidence of pharmacodynamic activity. Unlike most other VDAs, CYT997 is orally available and a phase I dose-ranging study with oral capsule dosing is underway. Methods: CYT997 was administered orally every 2 weeks to patients with advanced solid tumours. Doses were escalated using an accelerated phase I design to cohort 6 and thereafter with a standard 3+3 design. Pharmacodynamic effects on tumour vasculature were assessed with DCE-MRI scans, circulating endothelial cell (CEC) assays and von Willebrand factor (vWF) plasma levels. Results: 21 patients (M/F: 16/5; median age 63, range 48–77) have been treated on study. A total of 56 cycles of CYT997 have been administered (median 2/patient, range 1–7) over 8 dose levels (15 - 164 mg/m2). Doses up to 118 mg/m2 were well tolerated. However, dose-limiting toxicities were observed at 164 mg/m2, consisting of grade 3 hypoxia in one patient and grade 3–4 asthenia in two patients. The grade 3 hypoxia was reversible. PK profiles revealed favourable oral absorption which was generally dose-linear. DCE-MRI assessments indicating significant changes in tumour Ktrans values consistent with vascular disruption were observed in 6 of 10 currently evaluated patients. No objective tumour responses were seen; however, disease stabilisation for six weeks or greater was observed in 13 patients. vWf and CEC analysis is currently ongoing. Conclusions: Orally-administered CYT997 is well tolerated at doses associated with vascular targeting activity. Moreover, its good oral bioavailability suggests the potential for novel administration schedules, including metronomic dosing. [Table: see text]

Exercise-induced shear stress is associated with changes in plasma von Willebrand factor in older humans

Shear stress is the frictional force of blood against the endothelium, a stimulus for endothelial activation and the release of von Willebrand factor (vWF). This study tested the hypothesis that the increase in shear stress associated with exercise correlates with plasma vWF. Young (n = 14, 25.7 +/- 5.4 years) and older (n = 13, 65.6 +/- 10.7 years) individuals participated in 30 min of dynamic handgrip exercise at a moderate intensity. Brachial artery diameter and blood flow were measured using ultrasound Doppler and blood samples were collected before, immediately after, and following 30 min of recovery from exercise with plasma levels of vWF. Plasma levels of vWF increased (P < 0.05) by 6 +/- 2% in young individuals and 4 +/- 1% in older individuals immediately after exercise. The change in plasma vWF was linearly correlated with the increase in shear stress during exercise in older individuals (post-exercise: r = 0.78, 30 min recovery: r = 0.77, P < 0.01), but no association was found in the young individuals. These changes in plasma levels of vWF in humans suggest that aging influences endothelial activation and hemostasis.

Effects of Losartan on Fibrinolytic Parameters and von Willebrand Factor in Chinese Subjects with Hypertension: A Comparative Study versus Atenolol

To compare the effects of losartan and atenolol on plasma fibrinolytic parameters and von Willebrand factor (vWF), Chinese subjects with mild-to-moderate hypertension were randomized to receive losartan (50 mg/day; n = 30) or atenolol (50 mg/day; n = 30) for 8 weeks. If target blood pressure (< 140/90 mmHg) was not achieved at week 4, hydrochlorothiazide (12.5 mg/day) was also administered. Plasma levels of tissue plasminogen activator (tPA), plasminogen activator inhibitor-1 (PAI-1) and vWF were determined at baseline and after treatment. Between-group baseline characteristics and blood pressure decrease were comparable. Losartan significantly reduced plasma PAI-1 and vWF and PAI-1/tPA ratio. Atenolol significantly increased plasma tPA, but PAI-1, vWF and PAI-1/tPA ratio were unchanged. In conclusion, losartan, but not atenolol improved the fibrinolytic system and reduced plasma vWF levels in Chinese hypertensives.

Immuno-inflammatory predictors of stroke at follow-up in patients with chronic non-valvular atrial fibrillation (NVAF)

The aim of the present study was to determine the rates of stroke in patients with chronic NVAF (non-valvular atrial fibrillation), evaluating the relationship between plasma levels of inflammatory variables at admission and the occurrence of stroke during a 3-year follow-up. A total of 373 consecutive patients with chronic NVAF were enrolled. Blood samples were drawn within 72 h of admission, and we evaluated plasma levels of IL (interleukin)-1beta, TNF-alpha (tumour necrosis factor-alpha), IL-6, IL-10, E-selectin, P-selectin, ICAM-1 (intercellular adhesion molecule-1), VCAM-1 (vascular cell adhesion molecule-1) and vWF (von Willebrand Factor). Subsequent patient events (stroke at follow-up) were monitored over a 3 year period. By multivariate analysis, only age, hypertension and high levels of IL-6, TNF-alpha and vWF remained significant predictors of a higher risk of experiencing ischaemic stroke at follow-up. Moreover, plasma values of TNF-alpha, IL-6 and vWF had a significant area under the ROC (receiver operating characteristic) curve. In conclusion, baseline plasma levels of TNF-alpha, IL-6 and vWF are predictors of new-onset ischaemic stroke at follow-up in patients with chronic NVAF.

Effect of ginkgo leaf extract on vascular endothelial function in patients with early stage diabetic nephropathy

To explore the effect of ginkgo leaf extract (GLE) on vascular endothelial function (VEF) in patients with early stage diabetic nephropathy (DN). Sixty-four patients were randomized equally by a randomzing digital table into two groups, the treated group and the control group. They were all treated for 8 weeks with conventional therapy for diabetes, but GLE tablets were given to the treated group additionally. Changes in VEF were estimated before and after treatment by ultrasonic examination of the brachial artery. In the meantime, changes in plasma levels of the von Willebrand factor (vWF), nitric oxide (NO) and endothelin-1 (ET-1) were observed as well. The brachial arterial endothelium dependent dilating function in the treated group increased from 4.91+/-2.31% before treatment to 6.78+/-3.89% after treatment (P<0.05), while the level of vWF decreased from 182.05+/-64.13% to 128.56+/-48.98%, and that of NO increased from 50.16+/-24.64 micromol/L to 70.65+/-28.71 micromol/L (P<0.01). However, these indexes were not significantly changed in the control group after treatment (P>0.05). GLE could decrease the plasma level of vWF, raise the plasma NO level and improve the endothelium dependent vascular dilating function in DN patients.

Variations in the ratio between von Willebrand factor and its cleaving protease during systemic inflammation and association with severity and prognosis of organ failure

Von Willebrand factor (VWF) and related parameters as well as the protease activity regulating its biological activity were measured in plasma of healthy controls and patients with different cause and severity of systemic inflammation to examine the efficacy of the measures to detect highly prothrombotic states including thrombotic microangiopathy (TMA), one of the sequelae of sepsis. Plasma levels of VWF increased with increasing severity of systemic inflammation, probably due to activation of the endothelium. In parallel, the proteolytic activity of VWF inactivating protease, ADAMTS13, stepwise declined with the severity of inflammation, emphasizing the role of VWF-triggered platelet aggregation on the endothelium subsequently followed by development of TMA. As a consequence, the ratio of VWF antigen level and ADAMTS13 activity was significantly higher in patients with inflammation and sepsis, suggesting that this ratio might be more useful for the diagnosis of highly prothrombotic states including TMA than VWF multimer analysis alone. These findings suggest that ADAMTS13, VWF and related parameters, even in a combined approach, might be useful for the diagnosis and the therapeutic monitoring of patients with sepsis associated thrombotic microangiopathy.

ADAMTS13 is a Risk Factor for MI, Stroke and Vascular Death in the Oxford Vascular Study.

Normal haemostasis requires a balance between ultra-high MW VWF synthesis/release and its degradation by ADAMTS13. Although a severe acquired/inherited deficiency of ADAMTS13 occurs in TTP, as VWF is an established risk factor for arterial thrombotic disease it has been recently hypothesized that intermediate to low levels of ADAMTS13 could also be an important risk indicator. A new rapid ADAMTS13 Technozym® ELISA activity assay (Technoclone, Vienna, Austria) was therefore used to measure ADAMTS13 activity within plasma samples from a cohort of 521 patients within the Oxford Vascular Study (OXVASC). OXVASC is a large population based study of all patients with acute vascular events (TIA, stroke, acute coronary syndromes and acute peripheral vascular events) and includes a substudy of various risk factors (including platelet function, aspirin responsiveness and classical coagulation markers) for thrombosis. The ADAMTS13 normal range was 68.96 – 144.36% (n = 20, mean = 106.7%, 95% CI = 95.8–115.5%). Within 521 OXVASC samples, the mean ADAMTS13 level was found to be significantly lower (p < 0.0001, Mann-Whitney test) at 72.34% (95% CI = 70.33–74.35%). The minimum and maximum values obtained were 29.17% and 201.86% respectively. Although plasma VWF levels were significantly elevated, they were not inversely proportional to ADAMTS13 activity (r = 0.093). Furthermore low ADAMTS13 levels were not significantly associated with aspirin non-responsiveness (p = 0.0759) as defined by the Collagen/Epinephrine cartridge on the PFA-100, although VWF levels were significantly higher in non-responsive patients (p = <0.0001). Increased VWF levels were predictive of the combined outcome of stroke, MI or vascular death (HR per decile increase 1.12, 95% CI 1.06–1.19, p = 0.0002), whereas lower ADAMTS13 levels were associated with increased risk (HR per decile decrease 0.91 95% CI, 0.85–0.97, p = 0.004). In conclusion, low ADAMTS13 activity was not associated with high VWF levels and there was no association with aspirin non-responsiveness in a high shear dependent assay (PFA-100). However, low levels of ADAMTS13 were predictive of recurrent major vascular events. ADAMTS13 may therefore have some potential as a risk factor in patients with arterial disease.

Severe Plasmodium Falciparum Malaria Is Associated with Circulating Ultra-Large Von Willebrand Multimers and ADAMTS13 Inhibition

Cytoadhesion of Plasmodium falciparum-infected erythrocytes (IE) to activated endothelial cell (EC) surfaces plays a key role in the pathophysiology of cerebral malaria. Moreover, recent evidence suggests that platelet adhesion and aggregation are critical in facilitating this cytoadhesion process. We have recently reported marked increased plasma VWF and VWF propeptide levels in severe P. falciparum infection, indicative of acute EC perturbation. Furthermore, plasma VWF:Ag levels in patients with malaria inversely correlated with platelet count, and plasma VWF propeptide levels correlated with other established biochemical markers of malaria severity, including plasma lactate. Nevertheless, it remains unclear whether VWF plays any direct role in mediating IE cytoadhesion, or whether the increased plasma levels of both VWF and propeptide merely serve as a marker of acute EC activation.To address this question, we collected plasma samples from a cohort of African children presenting with cerebral malaria (CM), or non-cerebral severe malaria (SM). In children with CM and SM, plasma VWF:Ag levels were significantly elevated (medians 3.1 and 3.4 IU/ml) as before. VWF collagen binding (VWF:CB) levels were also markedly increased (medians 7.6 and 7.0 IU/ml). Furthermore, the relative rise in VWF:CB was much greater, so that the ratio of CB:Ag was consistently increased. VWF multimer analysis demonstrated abnormal circulating ULVWF multimers in children with CM and SM, consistent with acute, regulated VWF secretion. To characterize mechanisms responsible for the markedly increased VWF:CB and circulating ULVWF in malarial plasma, we investigated ADAMTS13 antigen and activity levels in CM and SM plasma. Plasma ADAMTS13 activity levels (FRETS-VWF73 assay) and antigen levels were both significantly reduced (medians = 0.63 U/ml and 0.56 U/ml; p< 0.001) in children with CM and SM compared to controls. Classical mixing studies of malaria and normal plasma demonstrated no evidence of immediate ADAMTS13 inhibition. However, ADAMTS13 activity in normal plasma was significantly reduced (~60%) following 30 min incubation with malarial plasma (75%:25% mix). This inhibitory ability of malaria plasma was further confirmed by spiking malarial plasmas with recombinant human ADAMTS13. Significant time-dependent inhibition of FRETS-VWF73 activity was again observed in the plasmas of children with severe P. falciparum, but not in normal control plasmas. Potential inhibitors of ADAMTS13 in vivo include interleukin-6 (IL-6), thrombospondin-1 (TSP-1), thrombin and plasmin, free plasma haemoglobin, and reduced FVIII levels. Although plasma IL-6 levels were significantly elevated in children with either CM (mean 240 pg/ml; p<0.001) or SM (mean 217 pg/ml; p=0.01) compared to normal controls, levels did not approach those previously reported necessary to inhibit ADAMTS13 activity. Disseminated intravascular coagulation (DIC) is associated with enhanced thrombin generation, and consumption of FVIII, both of which can inhibit VWF proteolysis by ADAMTS13. However, in children with CM or SM respectively, we observed significantly increased plasma FVIII:C levels. Although intravascular haemolysis is also a recognised complication of malarial infection, we observed only minor increased plasma haemoglobin concentrations, again well below that previously described to significantly inhibit ADAMTS13 activity. Finally, in contrast to the increased plasma levels of IL-6 and FVIII:C, we found that plasma TSP-1 levels were not significantly elevated in children with either CM or SM compared to pooled normal plasma.In conclusion, we demonstrate that the presence of ULVWF multimers in the plasma of children with severe P. falciparum malaria is the result ofacute EC activation and release of ULVWF from WP bodies;significantly reduced plasma ADAMTS13 antigen levelsa circulating but unidentified inhibitor of human ADAMTS13 activity.In view of the critical role played by VWF in mediating platelet adhesion/aggregation, and the accumulating evidence suggesting that platelet adhesion/aggregation also facilitate cytoadhesion of IE, we propose a novel role for ULVWF multimers in the pathophysiology of severe P. falciparum malaria.

A Reliable Marker of Endothelial Cell Dysfunction and Vascular Damage: Legend or Reality?

Abstract Endothelial dysfunction is considered to be a pre-clinical stage of atherosclerosis and represents an initial step of plaque progression. The aim of this study is to investigate whether the overproduction of biochemical markers is related to vascular damage. We studied 72 patients split into three groups: 20 patients had no vessels significant disease; 18 patients had multi-vessels disease; 34 patients had multi-vessels disease and stenosis &amp;gt;50%. Patients with known coronary artery disease, stroke, hypertension, diabetes and/or dyslipidemia were excluded from the study. We measured plasma levels of vWF (a marker of endothelial stress), VEGF (a marker of neointimal proliferation), TF and E-selectin (a marker of endothelial activation), all were measured by ELISA, and we investigated if their increased levels are related to the endothelial damage (table 1). Table 1. No disease Multi vessels Multi vessels disease+stenosis vWF (IU/dl) 84–102 128–154 146–182 VEGF (pg/ml) 28–124 120–180 160–240 TF (pg/ml) 16–98 80–220 230–370 E-Selectin (ng/ml) 30–74 72–96 94–128 There is a significant relation between vWF, VEGF, TF and E-Selectin and vessels disease severity; patients with no vessels disease had an average thickness of 0.48 mm, (IMT: 0.6–1.18mm), patients with multi vessels disease had a median atheroma score of 1.12, (IMT:1.18–2.42mm), patients with multi vessels disease and stenosis had a mediam atheroma score of 2.40, (IMT:&amp;gt;2.42mm), indeed we did not find any significant relation between increased arterial stiffness and laminar shear-stress. Healthy endothelium exerts a favourable and athero-protective effects by a numbers of factors. Table 2. Favorable and Atheroprotective Effects of the Healthy Endothelium Promotion of vasodilation Antioxidant effects Anti-inflammatory effects Inhibition of leukocyte adhesion and migration Inhibition of smooth muscle cell proliferation and migration Inhibition of platelet aggregation and adhesion Anticoagulant effects Profibrinolytic effects Table 3. Risk factors and predictors of endothelium dysfunction Recruiment of inflammatory cells SMC proliferation Neointimal formation Oxidative stress Activation of procoagulant activities Activation of plateolet adhesion and aggregation Stasis of blood flow In conclusion, we demonstrated that high levels of vWF, VEGF, TF, E-Selectin may play a critical role as for the development and the progression of atherosclerosis, even if further and wider studies are needed to confirm this statement.

Protein Phosphatase 2B Inhibition Promotes the Secretion of Von Willebrand Factor from Endothelial Cells

Ultra-large von Willebrand factor (ULVWF) multimers secreted from the Weibel-Palade bodies (WPBs) of endothelial cells plays a crucial role in the development of thrombotic microangiopathies. Secretion of WPB contents is regulated, in part, by the phosphorylation of vesicle trafficking proteins that constitutes the endothelial exocytotic machinery. In comparison to agonist-induced, protein kinase-dependent signaling pathways that regulate the exocytosis of WPB contents, a role for protein phosphatases in regulating endothelial exocytosis is currently undefined. In this study, we show that inhibition of serine/threonine protein phosphatase 2B (PP2B) activity by cyclosporine A (CsA), tacrolimus (FK506) or a cell-permeable PP2B autoinhibitory (AI) peptide promotes the secretion of hyper-adhesive ULVWF from human umbilical vein endothelial cells (HUVECs) in the absence of any other endothelial cell-stimulating agent. PP2B inhibitor-induced secretion and anchorage of ULVWF strings from HUVECs induce the tethering of adhesive platelets. In support of a role for PP2B in VWF secretion, we demonstrated that the catalytic subunit of PP2B expressed as a GST fusion protein interacts with the vesicle trafficking protein, Munc18c. Serine phosphorylation of Munc18c, which promotes granule exocytosis in other secretory cells, was significantly increased in CsA-treated HUVECs, suggesting that this process may be involved in CsA-mediated WPB exocytosis and secretion of VWF. Furthermore, plasma VWF antigen levels were also enhanced in CsA-treated mice, and siRNA-mediated knockdown of PP2Bb in HUVECs significantly enhanced VWF secretion. These observations suggest that CsA promotes VWF release, at least in part by inhibition of PP2B activity. These observations are compatible with the clinically observed association of CsA treatment and increased plasma VWF levels in humans, as well as the association between prolonged exposure to CsA and thrombotic microangiopathy in a subset of exposed patients.

PK Comparability of VWF/FVIII Concentrates after Repeated Dosing

Introduction: Appropriate correction of hemostasis in VWD is essential not only to stop acute perioperative bleeding, but also to promote postoperative wound healing. In order to prevent a drop of the VWF plasma level below the hemostatically necessary concentration, repeated infusions of VWF containing concentrates are required. Plasma derived VWF/FVIII concentrates vary, however, in several aspects such as the VWF/FVIII ratio. As therapy of VWD primarily aims at correcting functional VWF level, the question arises, whether VWF/FVIII concentrates differ with regard to the plasma level of FVIII resulting from repeated concentrate infusions.Methods: The goal of the present study was to compare VWF and FVIII levels after repeated administration of 150 IU (VWF:RCo)/kg (7 doses at 4-hour intervals). Two plasma derived VWF/FVIII concentrates, Humate-P®/Haemate® P and Wilate®, were selected for the comparison. The concentrates were administered as i.v. bolus to CHB rabbits (n=6/group). Blood samples were collected for close monitoring of the resulting substitution levels. The VWF and FVIII levels were quantified using commercially available ELISA assays.Results: Pharmacokinetic analysis (AUC, Cmax, half-life, MRT, clearance) demonstrated that the time course of VWF plasma levels were similar. The ratio of the VWF AUC of Humate-P®(Haemate® P)/Wilate® was 0.98 and the maximal VWF plasma concentration of Humate-P®/Haemate® P was slightly higher, at a ratio of 1,16. In contrast, the FVIII levels continuously accumulated upon treatment with Wilate® leading to trough levels, which were about 1,6 fold higher compared to Humate-P®/Haemate® P (Figure). Accordingly, also the peak FVIII level was 1,7 fold higher upon treatment with Wilate®, while the half-life of FVIII did not differ substantially between the two concentrates. The FVIII trough level of Wilate® together with its higher peak level resulted in an AUC, which was 2 fold higher (p<0.0001) than observed for Humate-P®/Haemate® P.Conclusion: Against the background of Wilate® containing a two fold higher FVIII concentration compared to Humate-P®/Haemate® P, such differential effects appear plausible. At nominally equal VWF:RCof repeated doses, the associated AUC with respect to FVIII:Ag of Humate-P®/Haemate® P was significantly lower, as compared to Wilate®. This is obviously due to the lower FVIII content in the Humate-P®/Haemate® P product. Therefore, a lower accumulation of FVIII trough levels was observed for Humate-P®/Haemate® P as compared to Wilate®. [Display omitted]

Abstract 2009: ALX-0081 a Novel Anti-Thrombotic: Results of a Single-Dose Phase 1 Study in Healthy Volunteers and Further Development in Patients with Stable Angina Undergoing PCI

Background : ALX-0081 is a bivalent Nanobody ® based on the variable domain of naturally occurring heavy-chain only antibodies. It binds with high affinity to the A1 domain of von Willebrand Factor (vWF) and thereby blocks the interactions between platelets and vascular collagen. It selectively prevents thrombus formation under high shear stress conditions. Aim : Test ALX-0081 single IV infusions (60 minutes) dosed from 0.5mg to 12mg total in 40 male healthy volunteers in double-blind, randomized, placebo controlled study and assess pharmacokinetic (PK), pharmacodynamic (PD), safety and immunogenicity. Results : ALX-0081 displayed non-linear pharmacokinetic properties, following a 2 compartment model. Ristocetin induced platelet aggregation (RIPA) was analyzed as marker for PD effect with full inhibition (defined as measured levels dropping &lt;10%) observed at ALX-0081 concentrations of ~ 400ng/ml. All subjects dosed ≥ 2mg achieved full RIPA inhibition at 1h post-dosing for maximum of 12h. ALX-0081 treatment was well tolerated and safe, no signs of bleeding were reported and no immunogenic response was detected. Target related mild and transient reductions of vWF and FVIII plasma levels were observed and all events were fully reversible. Phase Ib study design : double-blind, randomized, placebo controlled, multiple ascending dose study. ALX-0081 added to standard anti-thrombotic regimen (ASA, clopidogrel, UFH) in patients with stable angina undergoing elective PCI. Single-dose escalation will be followed by multiple dosing (up to 4 doses in 24h). Dose escalation will be guided by safety and efficacy marker. Endpoints: safety, pharmacological profile, biomarker (RIPA, RICO and ACT) and early clinical outcome (MACE, IMR, molecular marker). Conclusion : ALX-0081 can be administered safely over a wide range of dose-regimen. First results of the phase Ib study in stable angina patients will be presented.

Von Willebrand Factor, Red Cell Fragmentation, and Disease Activity in Systemic Lupus Erythematosus

This study sought to determine whether the plasma levels of Von Willebrand factor (vWf) and the degree of red blood cell (RBC) fragmentation on peripheral smear correlate with disease activity in systemic lupus erythematosus (SLE). Forty consecutive patients who fulfilled the criteria for SLE were studied prospectively for 1 year. Patients were categorized according to the SLE Disease Activity Index (SLEDAI) as either active (>2) or inactive disease and followed up monthly (active) or quarterly (inactive). At each visit, patients were examined fully and had complete blood count, tests on antibodies to double-stranded DNA, C3, and C4 levels, and urinalysis. Citrated plasma was analyzed for vWf antigen by standard enzyme-linked immunosorbent assay. A Wright's stained blood smear was obtained and schistocytes were quantitated on blood smear. The number of schistocytes per 500 RBCs was determined and a schistocyte index (SI) was calculated. At baseline, vWf correlated with SLEDAI (r = 0.64, p < 0.01), SI correlated with SLEDAI (r = 0.62, p < 0.01), and vWf and SI correlated with each other (r = 0.41, p = 0.01). There was an inverse correlation between baseline C3 levels and vWf (r = 0.49, p = 0.0013) and C3 levels and SI (r = 0.40, p = 0.01). Over time, there was also a correlation of SLEDAI with vWf (r = 0.53, p = 0.002) and SI (r = 0.57;p = 0.002). The relation of vWf with SI approached but did not reach statistical significance (r = 0.37, p = 0.06). We found that the plasma levels of vWf and the degree of RBC fragmentation correlate with lupus disease activity over time. Therefore, inflammation in SLE may be associated with endothelial injury.

Increased serum heat-shock protein 70 levels reflect systemic inflammation, oxidative stress and hepatocellular injury in preeclampsia

It has been previously reported that serum levels of 70-kDa heat-shock protein (Hsp70) are elevated in preeclampsia. The aim of the present study was to examine whether increased serum Hsp70 levels are related to clinical characteristics and standard laboratory parameters of preeclamptic patients, as well as to markers of inflammation (C-reactive protein), endothelial activation (von Willebrand factor antigen) or endothelial injury (fibronectin), trophoblast debris (cell-free fetal DNA) and oxidative stress (malondialdehyde). Sixty-seven preeclamptic patients and 70 normotensive, healthy pregnant women were involved in this case-control study. Serum Hsp70 levels were measured with enzyme-linked immunosorbent assay (ELISA). Standard laboratory parameters (clinical chemistry) and C-reactive protein (CRP) levels were determined by an autoanalyzer using the manufacturer's kits. Plasma von Willebrand factor antigen (VWF:Ag) levels were quantified by ELISA, and plasma fibronectin concentration by nephelometry. The amount of cell-free fetal DNA in maternal plasma was determined by quantitative real-time polymerase chain reaction analysis of the sex-determining region Y gene. Plasma malondialdehyde levels were measured by the thiobarbituric acid-based colorimetric assay. Serum Hsp70 levels were increased in preeclampsia. Furthermore, serum levels of blood urea nitrogen, creatinine, bilirubin and CRP, serum alanine aminotransferase and lactate dehydrogenase (LDH) activities, as well as plasma levels of VWF:Ag, fibronectin, cell-free fetal DNA and malondialdehyde were also significantly higher in preeclamptic patients than in normotensive, healthy pregnant women. In preeclamptic patients, serum Hsp70 levels showed significant correlations with serum CRP levels (Spearman R = 0.32, p = 0.010), serum aspartate aminotransferase (R = 0.32, p = 0.008) and LDH activities (R = 0.50, p < 0.001), as well as with plasma malondialdehyde levels (R = 0.25, p = 0.043). However, there was no other relationship between serum Hsp70 levels and clinical characteristics (age, parity, body mass index, blood pressure, gestational age, fetal birth weight) and laboratory parameters of preeclamptic patients, including markers of endothelial activation or injury and trophoblast debris. In conclusion, increased serum Hsp70 levels seem to reflect systemic inflammation, oxidative stress and hepatocellular injury in preeclampsia. Nevertheless, further studies are required to determine whether circulating Hsp70 plays a causative role in the pathogenesis of the disease.