Decreased circulating thrombomodulin is improved by tadalafil therapy in hypoxemic patients with advanced pulmonary arterial hypertension.

INTRODUCTION: Advanced pulmonary arterial hypertension (PAH) in patients with congenital cardiac communications and right-to-left shunting (Eisenmenger syndrome - PAH-ES) is associated with hypoxemia and decreased circulating levels of thrombomodulin (TM), probably reflecting decreased endothelial TM production. The combination of these two factors has been shown to induce fibrin deposition, with increased risk of thrombosis, a well known complication in this syndrome. PATIENTS AND METHODS: We tested the hypothesis that vasodilator therapy with the phosphodiesterase-5 inhibitor tadalafil, an approved drug for management of PAH could improve endothelial dysfunction markers, in particular plasma TM, in addition to improving the physical capacity (expected effect of pulmonary vasodilatation) in PAH-ES patients. This was a prospective observational study of treatment-naïve patients subjected to specific PAH therapy. Fifteen patients aged 12 to 51years (median 30years) were treated for 6months with a single daily dose of 40mg oral tadalafil. The physical capacity (distance walked during the 6-min walk test - 6MWD), systemic oxygen saturation and laboratory parameters were measured at baseline, and 90days and 180days of treatment. RESULTS: Plasma TM, which was decreased at baseline compared to controls (p<0.001) increased at 90 and 180days (p=0.003), and this was directly related (r=0.57, p=0.026) to improvement of oxygen saturation (p=0.008). Heightened baseline tissue-type plasminogen activator decreased during treatment (p=0.010), while heightened von Willebrand factor antigen remained unchanged. The 6MWD improved significantly (p<0.001). CONCLUSION: Tadalafil therapy improved circulating TM and tissue-type plasminogen activator, in addition to improving the physical capacity and oxygen saturation in PAH-ES patients.

Plasma von Willebrand factor as a predictor of survival in pulmonary arterial hypertension associated with congenital heart disease

Biomarkers have been identified for pulmonary arterial hypertension, but are less well defined for specific etiologies such as congenital heart disease-associated pulmonary arterial hypertension (CHDPAH). We measured plasma levels of eight microvascular dysfunction markers in CHDPAH, and tested for associations with survival. A cohort of 46 inoperable CHDPAH patients (age 15.0 to 60.2 years, median 33.5 years, female:male 29:17) was prospectively followed for 0.7 to 4.0 years (median 3.6 years). Plasma levels of von Willebrand factor antigen (VWF:Ag), tissue plasminogen activator (t-PA) and its inhibitor (PAI-1), P-selectin, reactive C-protein, tumor necrosis factor alpha, and interleukin-6 and -10 were measured at baseline, and at 30, 90, and 180 days in all subjects. Levels of six of the eight proteins were significantly increased in patients versus controls (13 to 106 percent increase, P < 0.003). Interleukin-10 level was 2.06 times normal (P = 0.0003; Th2 cytokine response). Increased levels of four proteins (t-PA, PAI-1, P-selectin, and interleukin-6) correlated with disease severity indices (P < 0.05). Seven patients died during follow-up. An average VWF:Ag (mean of four determinations) above the level corresponding to the 95th percentile of controls (139 U/dL) was independently associated with a high risk of death (hazard ratio = 6.56, 95 percentCI = 1.46 to 29.4, P = 0.014). Thus, in CHDPAH, microvascular dysfunction appears to involve Th2 inflammatory response. Of the biomarkers studied, plasma vWF:Ag was independently associated with survival.

Plasma von Willebrand factor as a predictor of survival in pulmonary arterial hypertension associated with congenital heart disease.

Biomarkers have been identified for pulmonary arterial hypertension, but are less well defined for specific etiologies such as congenital heart disease-associated pulmonary arterial hypertension (CHDPAH). We measured plasma levels of eight microvascular dysfunction markers in CHDPAH, and tested for associations with survival. A cohort of 46 inoperable CHDPAH patients (age 15.0 to 60.2 years, median 33.5 years, female:male 29:17) was prospectively followed for 0.7 to 4.0 years (median 3.6 years). Plasma levels of von Willebrand factor antigen (VWF:Ag), tissue plasminogen activator (t-PA) and its inhibitor (PAI-1), P-selectin, reactive C-protein, tumor necrosis factor alpha, and interleukin-6 and -10 were measured at baseline, and at 30, 90, and 180 days in all subjects. Levels of six of the eight proteins were significantly increased in patients versus controls (13 to 106% increase, P < 0.003). Interleukin-10 level was 2.06 times normal (P = 0.0003; Th2 cytokine response). Increased levels of four proteins (t-PA, PAI-1, P-selectin, and interleukin-6) correlated with disease severity indices (P < 0.05). Seven patients died during follow-up. An average VWF:Ag (mean of four determinations) above the level corresponding to the 95th percentile of controls (139 U/dL) was independently associated with a high risk of death (hazard ratio = 6.56, 95%CI = 1.46 to 29.4, P = 0.014). Thus, in CHDPAH, microvascular dysfunction appears to involve Th2 inflammatory response. Of the biomarkers studied, plasma vWF:Ag was independently associated with survival.

Rosuvastatin and vascular dysfunction markers in pulmonary arterial hypertension: a placebo-controlled study.

We investigated whether chronic rosuvastatin administration could improve the abnormalities of the circulating levels of vascular dysfunction markers in pulmonary arterial hypertension (PAH). Sixty patients, aged 13 to 60 years, with idiopathic (N = 14) or congenital heart disease-associated PAH (N = 46) were equally but randomly assigned to rosuvastatin treatment (10 mg a day, orally) or placebo for 6 months in a blind fashion. Plasma levels of P-selectin, tissue-plasminogen activator and its inhibitor as well as von Willebrand factor antigen were measured by enzyme-linked immunoassay before and after 1, 3, and 6 months of treatment. Baseline levels of biomarkers were elevated (68, 16, 45 and 46% increase relative to controls, for P-selectin, von Willebrand factor antigen, tissue-plasminogen activator and its inhibitor, respectively; P < 0.001). P-selectin values at baseline, 1, 3, and 6 months were 39.9 +/- 18.5, 37.6 +/- 14.6, 34.8 +/- 14.6, and 35.4 +/- 13.9 ng/mL, respectively, for the rosuvastatin group and 45.7 +/- 26.8, 48.0 +/- 26.9, 48.1 +/- 25.7, and 45.7 +/- 25.6 ng/mL for the placebo group. The P-selectin level was lower in the rosuvastatin group compared with placebo throughout treatment (P = 0.037, general linear model). A trend was observed towards a decrease in tissue-plasminogen activator in the statin group (16% reduction, P = 0.094), with no significant changes in the other markers. Since P-selectin is crucial in inflammation and thrombosis, its reduction by rosuvastatin is potentially relevant in the pathophysiological scenario of PAH.

Rosuvastatin and vascular dysfunction markers in pulmonary arterial hypertension: a placebo-controlled study

We investigated whether chronic rosuvastatin administration could improve the abnormalities of the circulating levels of vascular dysfunction markers in pulmonary arterial hypertension (PAH). Sixty patients, aged 13 to 60 years, with idiopathic (N = 14) or congenital heart disease-associated PAH (N = 46) were equally but randomly assigned to rosuvastatin treatment (10 mg a day, orally) or placebo for 6 months in a blind fashion. Plasma levels of P-selectin, tissue-plasminogen activator and its inhibitor as well as von Willebrand factor antigen were measured by enzyme-linked immunoassay before and after 1, 3, and 6 months of treatment. Baseline levels of biomarkers were elevated (68, 16, 45 and 46 percent increase relative to controls, for P-selectin, von Willebrand factor antigen, tissue-plasminogen activator and its inhibitor, respectively; P < 0.001). P-selectin values at baseline, 1, 3, and 6 months were 39.9 ± 18.5, 37.6 ± 14.6, 34.8 ± 14.6, and 35.4 ± 13.9 ng/mL, respectively, for the rosuvastatin group and 45.7 ± 26.8, 48.0 ± 26.9, 48.1 ± 25.7, and 45.7 ± 25.6 ng/mL for the placebo group. The P-selectin level was lower in the rosuvastatin group compared with placebo throughout treatment (P = 0.037, general linear model). A trend was observed towards a decrease in tissue-plasminogen activator in the statin group (16 percent reduction, P = 0.094), with no significant changes in the other markers. Since P-selectin is crucial in inflammation and thrombosis, its reduction by rosuvastatin is potentially relevant in the pathophysiological scenario of PAH.

A mathematical framework for group analysis of von Willebrand factor multimeric composition following luminography.

The objective of the present study was to establish a method for quantitative analysis of von Willebrand factor (vWF) multimeric composition using a mathematical framework based on curve fitting. Plasma vWF multimers from 15 healthy subjects and 13 patients with advanced pulmonary vascular disease were analyzed by Western immunoblotting followed by luminography. Quantitative analysis of luminographs was carried out by calculating the relative densities of low, intermediate and high molecular weight fractions using laser densitometry. For each densitometric peak (representing a given fraction of vWF multimers) a mean area value was obtained using data from all group subjects (patients and normal individuals) and plotted against the distance between the peak and IgM (950 kDa). Curves were constructed for each group using nonlinear fitting. Results indicated that highly accurate curves could be obtained for healthy controls and patients, with respective coefficients of determination (r2) of 0.9898 and 0.9778. Differences were observed between patients and normal subjects regarding curve shape, coefficients and the region of highest protein concentration. We conclude that the method provides accurate quantitative information on the composition of vWF multimers and may be useful for comparisons between groups and possibly treatments.

A mathematical framework for group analysis of von Willebrand factor multimeric composition following luminography

The objective of the present study was to establish a method for quantitative analysis of von Willebrand factor (vWF) multimeric composition using a mathematical framework based on curve fitting. Plasma vWF multimers from 15 healthy subjects and 13 patients with advanced pulmonary vascular disease were analyzed by Western immunoblotting followed by luminography. Quantitative analysis of luminographs was carried out by calculating the relative densities of low, intermediate and high molecular weight fractions using laser densitometry. For each densitometric peak (representing a given fraction of vWF multimers) a mean area value was obtained using data from all group subjects (patients and normal individuals) and plotted against the distance between the peak and IgM (950 kDa). Curves were constructed for each group using nonlinear fitting. Results indicated that highly accurate curves could be obtained for healthy controls and patients, with respective coefficients of determination (r²) of 0.9898 and 0.9778. Differences were observed between patients and normal subjects regarding curve shape, coefficients and the region of highest protein concentration. We conclude that the method provides accurate quantitative information on the composition of vWF multimers and may be useful for comparisons between groups and possibly treatments

Differential effects of enzymatic treatments on the storage and secretion of von Willebrand factor by human endothelial cells.

Enzymatic treatment used for passaging of endothelial cells may induce release of von Willebrand factor (vWF). Decreased ability to replenish intracellular stores results in decreased secretion of vWF in later passages of cells. Since both trypsin and pancreatin complex have been used for passaging endothelial cells, we analyzed the effects of successive passaging with these two enzyme preparations on the storage and secretion of vWF by human umbilical vein endothelial cells (HUVECs). Measurements were performed after the second to fifth passages. Cytoplasmic vWF was analyzed by indirect immunofluorescence and secreted vWF was measured in the supernatant of cultured HUVECs by ELISA. In trypsin-passaged cells, secreted vWF decreased progressively from passages 2 to 5. Respective concentrations were 355.0 +/- 30.4, 201.0 +/- 84.5, 150.0 +/- 1.4 and 120.5 +/- 38.9 ng vWF/10(5) cells. Comparatively, pancreatin-passaged cells secreted even less vWF protein (P = .001) at passages 4 and 5 (108.5 +/- 12.0 and 100.0 +/- 4.2 ng/10(5) cells, respectively) and had less vWF-positive cytoplasmic granules per cell. Thus, in experiments involving measurements of endothelial vWF, the use of low passage cells is recommendable and passaging with a pure trypsin preparation appears to be more appropriate.

Decreased sialic acid content of plasma von Willebrand factor in precapillary pulmonary hypertension.

In pulmonary hypertension, defective von Willebrand factor protein (vWF) lacking large multimers is present in circulation. This is associated with evidence of chronic endogenous platelet activation. Since asialo vWF has been shown to promote platelet activation and aggregation, we decided to investigate possible changes in the sialic acid content of plasma vWF in patients with precapillary pulmonary hypertension. vWF-associated sialic acid was measured indirectly as a wheat germ agglutinin-reactive substance (WGA-RS, Western blotting), and directly, as a thiobarbituric acid-reactive substance (TBA-RS, spectrophotometric reading). In the sixteen patients we studied (ages 8-45 yr), circulating vWF concentration was 2.18 times normal (p <0.001). However, patient vWF subunit contained 19% (WGA-RS) to 24% (TBA-RS) less sialic acid than the normal protein (p <0.05 for both determinations). In five patients, vWF-associated sialic acid was below 50% normal. We conclude that circulating vWF is hyposialylated in precapillary pulmonary hypertension and speculate that this might influence its interaction with platelets in vivo in these patients.

Endothelial cell dysfunction correlates differentially with survival in primary and secondary pulmonary hypertension.

BACKGROUND: Plasma von Willebrand factor antigen (vWF:Ag) has been used as a marker of endothelial perturbation in a number of vascular disorders. In this study, vWF:Ag was determined as an attempt to evaluate the severity of endothelial cell dysfunction in primary pulmonary hypertension (PPH) and congenital heart disease-associated pulmonary hypertension (CHD-PH) comparatively and to determine its impact on short-term survival. METHODS AND RESULTS: Clinical, hemodynamic, and biochemical data were obtained from 11 patients with PPH and 24 with CHD-PH. Patient groups were similar in terms of age and pulmonary artery pressure. vWF:Ag was measured by electroimmunodiffusion. Patients were followed up for 1 year and at that time, data collected at the beginning of the study were subjected to univariate and multivariate analyses. vWF:Ag was increased in patients (normal reference value 87% +/- 23% activity, mean +/- SD), with higher levels in the PPH group (231% +/- 89%) in comparison with the CHD-PH group (127% +/- 68%) (P <.001). Multivariate analysis showed that survival was influenced by the underlying cause of pulmonary hypertension and vWF:Ag levels but not by patient age, sex, or pulmonary artery pressure. Seven of 10 nonsurvivors but only 4 of 25 survivors had PPH (P =.007). vWF:Ag was 255% +/- 90% in the nonsurvivor group and 121% +/- 54% in the survivors (P <.001). CONCLUSIONS: Our findings suggest that short-term survival is related to the severity of endothelial cell dysfunction in pulmonary hypertension. In addition, exceedingly high vWF:Ag levels in PPH might reflect a particular pattern of endothelial cell dysfunction that could be associated with decreased short-term life expectancy in this disorder compared with secondary forms of pulmonary hypertension.

Cytoskeletal organization and incorporation of beta 3 integrin in thrombin-stimulated platelets: effect of acetylsalicylic acid.

Platelet stimulation by agonists is followed by changes in cytoskeletal organization that includes actin polymerization and association of the membrane skeleton (which is connected with the integrin alpha IIb beta 3) with the underlying cytoplasmic actin filaments. The effect of orally administered acetylsalicylic acid to healthy volunteers on incorporation of contractile protein and beta 3 integrin into the cytoskeletal core of thrombin-stimulated platelets was studied. Stimulation was followed by increased contractile protein and beta 3 incorporation into the cytoskeleton. Acetylsalicylic acid intake resulted in decreased incorporation of myosin and actin (32% and 20%, respectively), and a decrease (36%) in the association of beta 3 integrin with the cytoskeletal elements was evident. In conclusion, we have shown that acetylsalicylic acid, besides the known inhibitory effect on thromboxane synthesis, promotes changes in the cytoskeletal organization of thrombin-stimulated platelets that could limit thrombus formation.

Circulating von Willebrand factor antigen as a predictor of short-term prognosis in pulmonary hypertension.

STUDY OBJECTIVE: To determine the potential value of plasma von Willebrand factor antigenic activity (vWF:Ag) and other commonly measured clinical variables for predicting which patients with precapillary pulmonary hypertension would be unlikely to survive 1 year. DESIGN: Prospective clinical study. The data obtained at the beginning of the study were analyzed at the end of the first year of follow-up. PATIENTS AND METHODS: Forty patients aged 1.2 to 45 years (median, 24 years) entered the study. Eleven patients had primary pulmonary hypertension, and in the remaining ones, pulmonary vascular disease was associated with antiphospholipid syndrome (n = 1), collagen vascular disease (n = 1), schistosomiasis (n = 3), or congenital heart defects (Eisenmenger's syndrome) (n = 24). Plasma vWF:Ag was determined by electroimmunodiffusion, and the results were expressed as the percentage of activity. RESULTS: Seven of 11 patients with primary pulmonary hypertension but only 4 of 29 patients with secondary pulmonary hypertension died during the follow-up period (p < 0.005). Initial vWF:Ag values were significantly higher in the nonsurvivor group in comparison with the survivors (256.6+/-85.3% and 132.0+/-59.3% activity, respectively; p < 0.0001). The likelihood of fatal outcome as a function of plasma vWF:Ag levels was estimated for primary and secondary pulmonary hypertensive patients using logistic regression analysis. Decreased life expectancy was significantly related to high vWF:Ag levels and an established diagnosis of primary pulmonary hypertension. A plasma vWF:Ag of >240% (p = 0.003) was 54% sensitive and 93% specific for identifying patients who were unlikely to survive 1 year, with an overall predictive value of 75%. No other variables correlated significantly with survival. CONCLUSION: Plasma vWF:Ag seems to be a useful biochemical index for predicting short-term prognosis in patients with pulmonary hypertension. In contrast to hemodynamic and histopathological predictors of survival, vWF:Ag does not require invasive techniques to be determined. In light of the possibility of false-negative results, serial measurements should be performed over time in patients with vWF:Ag of <240%. This observation proved helpful in two patients in this study.

Abnormalities in circulating von Willebrand factor and survival in pulmonary hypertension.

BACKGROUND: Changes in circulating von Willebrand factor (vWF) have been widely used for evaluating the severity of endothelial dysfunction in vascular disorders. In pulmonary hypertension, quantitative and structural abnormalities in circulating von Willebrand factor have been identified. We therefore hypothesized that these abnormalities could have prognostic implications. PATIENTS AND METHODS: We studied 30 consecutive medically treated patients with primary (n = 11) or secondary precapillary pulmonary hypertension associated with congenital heart disease (n = 16) or schistosomiasis (n = 3). Plasma antigenic activity of vWF (vWF:Ag) was measured by electroimmunodiffusion. The relative concentration of low molecular weight vWF multimers (vWF:LMW/Total) was determined by Western immunoblotting. Results of initial evaluation were analyzed at the end of the first and third years of follow-up. RESULTS: Baseline vWF:Ag activity (P <0.0002) and the vWF: LMW/Total ratio (P <0.005) were higher in patients who died during the first year than in survivors. All patients with vWF:Ag activity >250% or a vWF:LMW/Total ratio >70% died in the first year. All 7 patients with vWF:Ag activity <100% were alive at the end of 3 years of follow-up. A vWF:LMW/Total ratio >68% was 67% sensitive and 95% specific for 1-year mortality, with an overall predictive value of 80%. Both vWF:Ag levels and mortality were greater in the patients with primary pulmonary hypertension than in patients with secondary pulmonary hypertension. CONCLUSION: Patients with pulmonary hypertension who have abnormalities in circulating vWF have reduced 1-year survival. This might affect decisions such as patient assignment to lung transplantation.

[Von Willebrand factor and pulmonary endothelial dysfunction. Prognostic implications]. / Fator von Willebrand e disfunção endotelial pulmonar. Implicações prognósticas.

PURPOSE: To analyze quantitative and structural changes in circulating von Willebrand factor (vWF) in 40 precapillary pulmonary hypertensive patients, as an attempt to identify possible correlations between endothelial cell dysfunction and patient short-term (one year) survival. METHODS: Plasma antigenic activity of vWF (vWF:Ag) was analyzed by immunoelectrophoresis. The relative concentration of vWF low molecular weight multimers (LMWM%) and the composition of vWF subunit were determined by densitometric analysis of Western blots. RESULTS: vWF:Ag was importantly increased in patients in comparison with normals (p < 0.001). Patients also had increased LMWM% (p < 0.001) and increased degradation of vWF main subunit (p < 0.05). At the beginning of the study, nonsurvivors (N = 11) had higher vWF:Ag (p < 0.001) and LMWM% (p < 0.005) values in comparison with survivors. LMWM% was selected by logistic regression analysis as a predictor of death during the first year of follow-up (p < 0.05). CONCLUSION: Marked changes in circulating vWF likely reflect extensive pulmonary vascular endothelial cell dysfunction and are associated with poor short-term prognosis in pulmonary hypertension.

Fator von Willebrand e disfunçäo endotelial pulmonar. Implicaçöes prognósticas / von Willebrand factor and pulmonary vascular endothelial cell dysfunction. Prognostic implications

OBJETIVO - Avaliar alteraçöes quantitativas e estruturais do fator von Willebrand (fvW) circulante em 40 pacientes com hipertensäo pulmonar pré-capilar e verificar possíveis implicaçöes prognósticas dos resultados iniciais, em um ano de seguimento. MÉTODOS - A atividade antigênica plasmática do fator von Willebrand (vWF:Ag) foi analisada por imunoeletroforese. A concentraçäo de multímeros de baixo peso molecular em relaçäo...

Decreased biological activity is related to abnormal multimeric structure of von Willebrand factor in pulmonary hypertension.

We evaluated the correlation between decreased biological activity and abnormalities in the multimeric structure of plasma von Willebrand factor (vWF) in 27 pulmonary hypertensive patients (median age, 21 years). The biological activity of vWF was measured by the ristocetin cofactor assay and its multimeric structure was assessed by Western immunoblotting after SDS-agarose gel electrophoresis. In spite of high antigenic activity of vWF in plasma (139 +/- 65 vs 91 +/- 27% in controls, P = 0.003), the biological activity expressed as a percent of the control value was decreased in pulmonary hypertensive patients (60-88% activity, 95% CI for the mean). High molecular weight multimers (biologically active forms) were absent in patients and there was a significant increase in the concentration of low molecular weight polymers in comparison with normals (56 +/- 12 and 35 +/- 12% of total multimer density, respectively, P < 0.001). Multimeric abnormalities were positively correlated with plasma vWF levels (r = 0.51, P = 0.007) and negatively correlated with vWF biological activity (r = -0.54, P = 0.004). Thus, decreased biological function is related to abnormalities in the multimeric structure of vWF, possibly reflecting extensive endothelial dysfunction in pulmonary hypertension.

Decreased biological activity is related to abnormal multimeric structure of von Willebrand factor in pulmonary hypertension

We evaluated the correlation between decreased biological activity and abnormalities in the multimeric structure of plasma von Willebrand factor (vWF) in 27 pulmonary hypertensive patients (median age, 21 years). The biological activity of vWF was measured by the ristocetin cofactor assay and its multimeric structure was assessed by Western immunoblotting after SDS-agarose gel electrophoresis. In spite of high antigenic activity of vWF in plasma (139 ñ 65 vs 91 ñ 27 per cent in controls, P=0.003), the biological activity expressed as a percent of the control value was decreased in pulmonary hypertensive patients (60-88 per cent activity, 95 per cent CI for the mean). High molecular weight multimers (biologically active forms) were absent in patients and there was a significant increase in the concentration of low molecular weight polymers in comparison with normals (56 ñ 12 and 35 ñ 12 per cent of total multimer density, respectively, P<0.001). Multimeric abnormalities were positively correlated with plasma vWF levels (r=0.51, P=0.0007) and negatively correlated with vWF biological activity (r=-0.54, P=0.004). Thus, decreased biological function is related to abnormalities in the multimeric structure of vWF, possibility reflecting extensive endothelial dysfunction in pulmonary hypertension.

Abnormal degradation of von Willebrand factor main subunit in pulmonary hypertension.

We wished to investigate whether the abnormalities in multimeric structure and biological function of von Willebrand factor (vWF) observed in pulmonary hypertensive patients could be related to increased proteolytic degradation. We therefore analysed plasma vWF subunit composition in 24 pulmonary hypertensive patients, aged 1.2-45 yrs. After immunoisolation, vWF was subjected to 5.5% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western immunoblotting, vWF intact subunit (225 kDa) and four proteolytic fragments (189, 176, 150 and 140 kDa) were visualized by peroxidase staining and analysed in a laser densitometer. In pulmonary hypertensive patients, the relative density of the intact subunit was decreased, and this was associated with an increase in the 176 kDa proteolytic fragment. The mean densities of the other fragments were not significantly changed, but in some patients the 150 and 140 kDa polypeptides were markedly increased. Abnormalities in multimeric structure of vWF (loss of high molecular weight multimers and increase in low molecular weight forms in comparison with controls), were associated with a significant decrease in biological activity (62-92% activity, 95% confidence interval (CI) for the mean). Total proteolytic fragment density correlated positively with multimeric abnormalities (rs = 0.45), and negatively with biological activity of vWF (rs = -0.49). Thus, in pulmonary hypertension, multimeric abnormalities and decreased biological activity are related to proteolytic degradation of vWF main subunit, possibly reflecting extensive endothelial dysfunction.