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.

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.

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.

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.

Effect of intentional hemodilution on platelet survival in secondary pulmonary hypertension.

Platelet regeneration time was assessed in 13 young adults with pulmonary hypertension and polycythemia secondary to congenital heart defects who underwent isovolemic hemodilution to improve clinical status and coagulation defects. The estimated platelet half-life in patients with Eisenmenger's complex was significantly shortened in comparison with normal subjects (3.8 +/- 1.9 vs 4.8 +/- 1.0 days, p less than 0.05). Hemodilution was carried out with no adverse effects, using low molecular weight dextran solutions. Lowering hematocrit from 61 to 50 percent resulted in a significant increase in platelet half-life from 3.8 +/- 1.9 to 5.7 +/- 1.8 days (p less than 0.02), which was followed by a marked rise in platelet count from 149 +/- 31 to 209 +/- 47 x 10(9) platelets/L (p less than 0.003). Arterial oxygen tension did not change significantly. These observations indicate that high hematocrit levels may have accounted for the shortened platelet survival and thrombocytopenia in these patients. Significant hemodilution may lead to a marked improvement in platelet abnormalities in patients with Eisenmenger's complex.

Abnormal multimeric and oligomeric composition is associated with enhanced endothelial expression of von Willebrand factor in pulmonary hypertension.

Abnormalities in endothelial von Willebrand factor (vWF) structure have been reported in pulmonary hypertension. These include loss of high molecular weight plasma multimers, resulting in decreased biologic activity. If endothelial processing of vWF is altered in this disorder, abnormalities in oligomeric composition may also be expected. We examined this possibility in ten adult patients with primary pulmonary hypertension. Enhanced endothelial vWF expression in these patients was indicated by increased plasma levels of vWF antigen (vWF:Ag) (214 +/- 91 vs 99 +/- 51 percent activity in controls, p < 0.001) and intense immunoperoxidase stain of pulmonary arterial endothelium for vWF (autopsy, 1 patient). Plasma from these patients also had a decreased capacity of inducing platelet aggregation in the presence of ristocetin, relative to vWF:Ag levels (57 +/- 20 percent activity). In addition to mild loss of the largest multimers, changes in oligomeric composition of plasma vWF were observed in most patients using both agarose and polyacrylamide gel electrophoresis. These included decreased concentration of dimeric (470 kDa) vWF in most patients, variable concentration of the 860-kDa fraction, and a relative decrease in subunit (223 kDa) density in subjects with elevated vWF:Ag. These findings provide additional information on the mechanisms responsible for endothelial production of dysfunctional vWF in patients with pulmonary hypertension.

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.

Human plasma kallikrein: effect on the induced platelet aggregation.

Platelet aggregation induced by ADP, collagen and adrenaline is increased by low concentrations of human plasma kallikrein, which does not cause aggregation by itself and inhibits the aggregation induced by arachidonic acid and Thrombofax in platelet rich plasma, and the aggregation induced by thrombin in washed platelets. At higher concentrations however, plasma kallikrein inhibits the aggregation induced by all the agents tested with platelet rich plasma and also causes a decrease in the aggregation induced in whole blood.

Aggregation of platelets in whole blood from children with pulmonary hypertension.

The response of aggregation of platelets to adenosine diphosphate (7.5-120 microM) and collagen (1.25 micrograms/ml) was assessed in whole blood (impedance method) in 10 children with pulmonary hypertension (hematocrit range, 42 to 71%). The response to collagen was normal (9.08 +/- 3.47 vs. 10.36 +/- 1.86 ohms in controls, P = NS) while there was a decreased response to adenosine diphosphate (6.98 +/- 3.83 vs. 11.21 +/- 2.02 ohms, P less than 0.01), in spite of high concentrations of the inducer. Lowering the hematocrit in vitro to 40% with autologous platelet-rich plasma resulted in a rise in the platelet count from 171 +/- 63 to 225 +/- 84 x 10(9) platelets/1 (P less than 0.001) and a significant increase in the response to adenosine diphosphate from 6.98 +/- 3.83 to 9.89 +/- 3.66 ohms (P less than 0.02). As in the baseline condition, high concentrations of adenosine diphosphate were required. The response to collagen did not change significantly. The results indicate that aggregatory response of platelets is relatively preserved in these children. The decreased response to adenosine diphosphate may be a result of a low count and interference of red cells on the accretion of platelets on the electrodes. Because high concentrations of adenosine diphosphate were still required after hemodilution to achieve an aggregatory response close to normal, we speculate that leakage of endogenous adenosine diphosphate from red cells may have accounted for partial activation of the platelets, resulting in a relative refractory state to in vitro stimulation.

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.

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.

Platelet regeneration time: a statistical approach.

The determination of platelet regeneration half-time (PRT t1/2) by measuring malondialdehyde after intake of acetylsalicylic acid is a simple nonisotopic method for the estimation of platelet survival. There is no available information concerning the populational distribution of PRT t1/2. Consequently, there is controversy about the utilization of parametric or nonparametric statistical tests in studies of PRT. In the present study, we demonstrate the closeness of the fit of log PRT t1/2 to the normal (Gaussian) distribution.

Plasma anticoagulant system in patients with pulmonary hypertension.

Adults with pulmonary hypertension and polycythemia (N = 22) have low levels of plasma antithrombin III (84 +/- 18 vs 98 +/- 13% for controls, N = 35, P less than 0.005) and protein C (66 +/- 21 vs 125 +/- 30%, N = 8, P less than 0.0002) but normal levels of total protein S. Data are reported as means +/- SD and percent normal values obtained for pooled plasma from normal healthy adults. Children with the same disorder (N = 6) also had low protein C levels (66 +/- 16 vs 85 +/- 5%, P less than 0.025). Total protein S was normal for children, but free protein S was decreased (66 +/- 13 vs 91 +/- 23%, P less than 0.025). Since the levels observed in these patients are above those reported for congenital deficiencies, the reduction in plasma levels of anticoagulant proteins may be the result of chronic intravascular coagulation. Furthermore, normal levels of plasminogen and fibrin degradation products suggest a localized disorder or an acquired decrease in fibrinolytic activity.

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 platelet aggregates indicative of in vivo platelet activation in pulmonary hypertension.

The authors investigated the existence of circulating cellular aggregates in 12 patients with moderate to severe pulmonary hypertension, using scanning electron microscopy. Peripheral venous blood was collected in the presence of 11.5 mM buffered ethylenediaminetetraacetic acid, in order to disperse freshly formed disaggregable aggregates. Irreversible aggregates represented by platelet clusters and/or platelet attachment to either leukocytes or red cells were identified in 7 patients with pulmonary hypertension. Endogenous platelet activation was further confirmed by a significant increase in plasma levels of beta-thromboglobulin in comparison with controls (33.8 +/- 14.1 vs 22.7 +/- 11.5 ng/mL respectively, p < 0.025). The presence of irreversible aggregates in the blood stream strongly suggests that cell-cell interactions actually occur in vivo in these patients. If so, therapeutic measures aimed at preventing in situ thrombosis and its consequences may be beneficial in this disorder.

[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.

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.