Willebrand disease

Objectives: We hypothesized that higher plasma levels of von Willebrand factor (vWF) and reduced ristocetin cofactor activity (RCA)/vWF ratios at baseline and follow-up would be associated with an increased risk of death in patients with pulmonary arterial hypertension (PAH).

Background: Endothelial injury and dysfunction cause increased vWF levels in patients with cardiovascular disease. Increased vWF is associated with a higher risk of death in patients with coronary artery disease, congestive heart failure, and ARDS. In PAH, vWF is elevated and functions abnormally, resulting in reduced platelet binding. The ability of vWF to bind platelets is reflected by RCA.

Methods: We performed a retrospective cohort study of 66 PAH patients who underwent initial evaluation at our center from January 1994 to June 2002. The primary outcome was death or lung transplantation.

Results: vWF level was directly associated with Factor VIII activity, RCA, fibrinogen, and prothrombin time (p < 0.01). vWF levels at baseline and follow-up were associated with the risk of death (hazard ratio [HR] per 50% increase, 1.4; 95% confidence interval [CI], 1.1 to 1.8 [p = 0.02]; and HR per 50% increase, 1.5; 95% CI, 1.1 to 2.0 [p = 0.011]; n = 48, respectively). RCA/vWF ratios were not associated with outcome. These results were unaffected by adjustment for demographics, baseline hemodynamics, laboratory results, or PAH therapy in multivariate analyses.

Conclusions: Increased vWF levels at baseline and follow-up are associated with worse survival in patients with PAH; however, RCA/vWF ratios are not. The hemostatic effects of vWF do not account for these Findings. This suggests that endothelial injury and dysfunction may persist despite treatment and may impact on disease course. Therapies that target endothelial injury may therefore improve outcomes for patients with PAH.

Key words: cohort study; endothelial dysfunction; pulmonary hypertension; ristocetin cofactor; survival; von Willebrand factor

Abbreviations: CI = confidence interval; HR = hazard ratio; PAH = pulmonary arterial hypertension; RCA = ristocetin co-factor activity; vWF = von Willebrand factor

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Pulmonary arterial hypertension (PAH) is characterized by disordered endothelial cell and smooth-muscle proliferation in the small muscular pulmonary arteries. Endothelial dysfunction and injury have become important features of the pathobiology of this disease.1 Two components of endothelial dysfunction, eicosanoid imbalance and elevated endothelin-1, have provided the rationale for the clinical development of prostacyclin analogues and endothelin-1 receptor antagonists as important therapeutic interventions for PAH. (2-7)

In addition to regulating vasoactivity and cellular mitogenesis, the endothelium also modulates local hemostasis, von Willebrand factor (vWF) is a multimeric glycoprotein that is stored in the Weibel-Palade bodies of vascular endothelium and, to a lesser extent, in [alpha] granules of platelets. Released after endothelial perturbation, functional vWF multimers bind to exposed collagen in areas of vascular injury and initiate platelet plug formation. The ristocetin cofactor activity (RCA)/vWF ratio reflects the relative platelet-binding activity of vWF. vWF also contributes to thrombogenesis by stabilizing Factor VIII and preserving its function, vWF levels are elevated in PAH, and RCA/vWF ratios are reduced. We hypothesized that a higher vWF level and a lower RCA/vWF ratio at baseline would predict an increased risk of death in patients with PAH. We also hypothesized that persistent abnormalities in these measures despite treatment would be associated with poor outcomes.

METHODS AND MATERIALS

Study Design

We performed a retrospective cohort study of all consecutive adult patients with PAH who underwent initial evaluation from January 1994 to June 2002 with follow-up through June 2003 at the New York Presbyterian Pulmonary Hypertension Center. Details of the cohort have been published elsewhere. (8) The cohort included patients > 16 years of age with PAH (idiopathic, familial, or associated with anorexigen use) evaluated during the study period. Patients routinely underwent laboratory testing and cardiac catheterization at baseline evaluation. Acute vasoreactivity with short-acting vasodilators was defined as a decrease in mean pulmonary artery pressure of [greater than or equal to] 20% without a decrease in cardiac index. (9)

The primary combined end point was death or lung transplantation. A patient was censored as alive at the last medical contact recorded in the computerized clinical database through June 2003. Choice of therapy was consistent with consensus 'algorithms. (10) Although not all therapies were available throughout the study period, we adjusted for these differences in the analysis. The study was approved by the Columbia University Institutional Review Board.

Laboratory Analysis

vWF levels were measured using the Laurell rocket method (11,12) from 1994 until 1997, and the immunoturbidimetric method (STA-Liatest; Diagnostica Stago; Asnibres sur Seine, France) from 1997 until 2002. vWF is expressed as the percentage of normal control (normal < 150%). The Ristocetin Cofactor Assay Kit (Helena Laboratories; Beaumont, TX) was used to measure the agglutination of formalin-fixed normal platelets by ristocetin after the addition of test plasma. Results range from 20 to 120% activity. The normal RCA/vWF ratio is > 0.80. Factor VIII activity was measured by the ability of the sample to correct the activated partial thromboplastin time of factor VIII-deficient plasma (IL ACL 3000; Instrumentation Laboratory; Lexington, MA).

Statistical Analysis

Continuous variables were summarized by the mean [+ or -] SD or median (range). Categorical variables were summarized by frequencies with 95% confidence interval (CIs). Spearman [rho] was used for correlations with the Bonferroni correction for each variable of interest (ie, vWF and RCA/vWF ratio). Survival analysis was performed using the Kaplan-Meier estimator and the log-rank test. We used Cox proportional hazards models to analyze the association of each variable of interest with survival utilizing time-varying covariates. (13) This statistical method allows the analysis of repeated measurements of vWF and controls for the use of different therapies over the course of the study period.

Multivariate models were constructed using vWF and the RCA/vWF ratio as the primary risk factors of interest. Potential confounders were added to the models to assess the impact on the effect estimates. A change of > 20% in the coefficient of the risk factor was considered to indicate confounding. All analyses were performed with available data. The proportional hazards assumption and the presence of influential subjects were assessed, p Values < 0.05 were considered statistically significant. Analyses were performed using statistical software (Stata 7.0; Stata Corporation; College Station, TX).

RESULTS

The initial cohort was composed of 84 patients with newly diagnosed PAH. Eighteen patients who had missing data for vWF, RCA, Factor VIII, and/or right-heart catheterization were excluded. Patients with missing data were not significantly different from those in the final cohort (n = 66) in terms of the date of evaluation, demographics, etiology of PAH, baseline hemodynamics, or survival.

The characteristics of the final cohort are shown in Table 1. Twenty-nine patients (44%) were treated with IV epoprostenol, 12 patients (18%) received subcutaneous treprostinil, 21 patients (32%) received oral bosentan, and 26 patients (39%) received calcium-channel blockers. Most patients (90%) were treated with more than one therapy simultaneously or sequentially. The median follow-up was 833 days (range, 4 to 3,365 days). Eighteen patients died, and 1 patient underwent lung transplantation. Transplant-free survival was 91% (95% CI, 80 to 96%) at 1 year and 62% (95% CI, 43 to 76%) at 5 years.

Thirty patients (45%) had abnormally high (> 150%) vWF levels, and 43 patients (65%) had abnormally low (< 0.80) RCA/vWF ratios at baseline. vWF directly correlated with RCA ([rho] = 0.55, p < 0.001), Factor VIII activity ([rho] = 0.75, p < 0.001), fibrinogen ([rho] = 0.40, p = 0.01) In = 65], and prothrombin time ([rho] = 0.44, p = 0.005) [n = 65]. The RCA/vWF ratio was inversely associated with Factor VIII activity ([rho] = -0.48, p < 0.002) and prothrombin time ([rho] = -0.46, p < 0.002) [n = 65]. There were no associations between vWF or RCA/vWF ratio and age, gender, ethnicity, race, body mass index, baseline hemodynamics, or other laboratory results (data not shown).

Survival Analysis

A higher vWF level at baseline predicted an increased risk of death (Table 2). The hazard ratio (HR) for vWF was unchanged regardless of adjustment for multiple other variables, demonstrating lack of confounding by these factors. As no acutely vasoreactive patients died or underwent transplantation, we were unable to adjust for acute vasoreactivity. However, stratified analysis of only patients who were nonreactive (n = 50) [or who did not have acute vasodilators administered, n = 6] also showed an association between vWF and the risk of death (HR per 50% increase, 1.32; 95% CI, 1.02 to 1.71; p = 0.036), minimizing the possibility of confounding by acute vasoreactivity. As Factor VIII may mediate the effect of vWF on survival via thrombogenesis, this factor should not be considered a confounder. Nonetheless, the HR for vWF after adjustment for Factor VIII was 1.93 (95% CI, 1.29 to 2.87; p = 0.001).

Patients with baseline vWF levels > 220% (a cutoff used in a previous study (14)) had a dramatically increased risk of death (HR, 3.97; 95% CI, 1.52 to 10.4; p = 0.005) [Fig 1]. This association persisted despite adjustment for sex, race, ethnicity, baseline hemodynamics, other laboratory values, and treatment (data not shown). Outcomes were not associated with the baseline RCA/vWF ratio (HR per 0.10 decrease, 1.10; 95% CI, 0.95 to 1.27; p = 0.19), baseline RCA (HR per 10% increase, 1.09; 95% CI, 0.92 to 1.30; p = 0.33), or baseline Factor VIII level (HR per 50% increase, 1.03; 95% CI, 0.81 to 1.32; p = 0.79).

[FIGURE 1 OMITTED]

Forty-eight patients from the cohort had vWF assessments repeated after initiation of therapy. The median follow-up time in this subset was 1,305 days (range, 147 to 3,365 days) with repeat assays assessed on average 2.9 [+ or -] 1.0 times ([+ or -] SD) per patient. Therapies used were similar to those in the entire cohort. Patients who were reassessed had a better survival compared to those who were not (p = 0.02), whereas age, gender, and baseline hemodynamics were similar between the groups (p > 0.10).

Elevated vWF levels at baseline and follow-up were associated with an increased risk of death in bivariate and multivariate analyses (Table 3). Stratified analysis of patients who did not show acute reactivity to vasodilators (n = 33) [or who were not tested, n = 6] showed similar results (HR, 1.41; 95% CI, 1.06 to 1.87; p = 0.0017). Adjustment for calcium-channel blocker use could not be performed, since no patients treated with calcium-channel blockers died during the study period. A vWF level > 220% over time was associated with a significantly increased risk of death (HR, 4.71; 95% CI, 1.14 to 19.39; p = 0.032) [Fig 2], which was not affected by adjustment for other variables (data not shown). The HR for vWF over time after adjustment for Factor VIII activity was 1.40 (95% CI, 0.99 to 1.98; p = 0.06).

[FIGURE 2 OMITTED]

Although a persistently low RCA/vWF ratio over time may have been associated with an increased risk of death in bivariate analysis (Table 4), this was not statistically significant and may have been confounded by other factors. Neither RCA (HR per 10% increase, 1.09; 95% CI, 0.88 to 1.35; p = 0.45) nor Factor VIII activity (HR per 50% increase, 1.29; 95% CI, 0.98 to 1.69; p = 0.07) over time were associated with the risk of death.

We repeated the survival analysis including only patients with idiopathic PAH (n = 51) or idiopathic and familial PAH (n = 62); the results were unchanged. Adjustment for the type of vWF assay used or the date of baseline evaluation did not significantly alter the conclusions. Both vWF levels and the RCA/vWF ratios met the assumption of proportional hazards. There were no particularly influential subjects.

DISCUSSION

We have shown for the first time a significant association between increased vWF and worse outcome in PAH (idiopathic, familial, or related to anorexigen use). This relationship persisted (ie, the HRs were unchanged or increased) after adjustment for multiple potential confounding variables and variations in assumptions. Although a reduced RCA/ vWF ratio over time may be associated with a higher risk of death, this finding was not statistically significant and may have been confounded, as the HR significantly decreased after adjustment for certain factors (eg, erythrocyte sedimentation rate and fibrinogen). We and others (15-18) have previously shown that patients with PAH have increased circulating levels of vWF with reduced platelet binding. Lopes et al (18-20) attributed the reduced biological activity of vWF in PAH to hyposialylated subunits with loss of the normal multimeric structure and an increase in smaller molecules. High-molecular-weight vWF multimers bind more tightly to platelet receptors and increase signaling through crosslinking. (21-23)

We have also previously shown a significant decrease in vWF levels and increase in RCA/vWF ratios in a small group of PAH patients treated with IV epoprostenol for 1 year. (15) Veyradier et al (24) demonstrated a decrease in vWF levels to near normal, partial restoration of the high- to low-molecular-weight multimer ratio, and a decrease in proteolytic fragments after treatment with IV epoprostenol for 30 days. These studies demonstrate the responsiveness of endothelial injury and vWF release to IV epoprostenol therapy in select patients. This may be due to a direct effect of epoprostenol on the pulmonary or systemic endothelium or an epiphenomenon of improved cardiopulmonary hemodynamics.

Lopes et al (14,25,26) have shown that patients with idiopathic PAH (n = 11) have higher vWF levels than patients with other etiologies of PAH (eg, PAH related to congenital heart disease) or patients with pulmonary hypertension associated with schistosomiasis. However, these studies (14,25,26) did not show statistically significant associations between vWF levels and survival in the patients with idiopathic PAH.

vWF is a strong and independent risk factor for coronary artery disease and venous thromboembolism in healthy subjects and for cardiovascular events in patients with known coronary artery disease. (27-30) Several studies (28,31-33) have shown associations between increased vWF and the risk of death in healthy subjects and patients with coronary artery disease, congestive heart failure, or ARDS. We have now shown that increased vWF levels predict the risk of death in PAH as well.

The systemic inflammatory milieu, hypercoagulable state, and/or endothelial injury may explain these findings. Systemic inflammation with cytokine up-regulation and increased neutrophil reactivity is present in patients with PAH. (34-40) As vWF is an acute-phase reactant, increased levels in PAH may merely reflect worse systemic inflammation. However, this is unlikely in our cohort, as adjustment for other markers of systemic inflammation, such as fibrinogen, erythrocyte sedimentation rate, and platelet count, did not change the results. In addition, unlike vWF, these variables were not associated with survival (data not shown). Shear stress increases proteolysis of vWF. (41) While worse hemodynamics and increased shear stress in PAH could account for the relationship between vWF and outcome, adjustment for hemodynamic parameters at baseline did not significantly change the results.

Higher vWF levels may cause in situ thrombosis in PAH. vWF contributes to clot formation in two ways. vWF binds to exposed subendothelial collagen at sites of vascular injury and subsequently to platelet Ib-IX-V and IIb-IIIa receptors, recruiting and activating platelets, vWF also stabilizes and activates Factor VIII. However, our findings do not support a thrombogenic mechanism for vWF in PAH. Neither absolute nor relative RCA (which reflect potential for platelet aggregation) were associated with survival. Adjustment for Factor VIII levels in our analysis did not change our results, making it less likely that Factor VIII mediates the increased risk of death conferred by elevated vWF.

Circulating vWF most likely reflects the severity of endothelial injury and dysfunction, which may independently impact on event-free survival. A recent study (42) in congestive heart failure has shown associations with other measures of endothelial perturbation, such as flow-mediated dilatation and circulating endothelial cells. The strong association between baseline vWF and outcome, despite treatment with prostacyclin analogues or the endothelin-1 receptor antagonist bosentan, suggests that there are factors beyond eicosanoid metabolism and endothelin-1 at the time of PAH diagnosis that are integral to disease progression. As in atherosclerotic heart disease and congestive heart failure, specific therapies that target endothelial dysfunction by distinct pathways may be important adjunctive therapy for this disease. Results from studies in animal models of PAH treated with 3-hydroxy-3-methylglutaryl co-enzyme A reductase inhibitors support such an approach. (43-45) Evidence of improvement in endothelial dysfunction with angiotensin-converting enzyme inhibitors and 3hydroxy-3-methylglutaryl co-enzyme A reductase inhibitors provides a rationale for conducting randomized clinical trials of these agents in patients with PAH. (46-48) If validated, vWF could be useful as a surrogate end point in such studies. (49-50)

Limitations

The original vWF assay used in the early years of this cohort was subsequently replaced by a more current technique. Despite this change, the reference values for vWF were standardized normals, and adjustment for the different methods utilized did not affect the results. In addition, such a change would be expected to introduce measurement variability, if anything, biasing the results to the null. These data do not elucidate the source of vWF in PAH. While platelet degranulation is possible, evidence from many other diseases makes an endothelial source much more likely. While there were some missing data, we found no significant differences between patients in the final cohort and those who were excluded, making selection bias unlikely. Patients who had multiple assessments over time not surprisingly had a better survival than those who did not. This likely reflects the lower severity of illness of those who lived long enough to be reassessed. While assessments were not performed at standard times or in relation to changes in therapy, we used time-varying analyses to account for this variability.

CONCLUSION

Endothelial injury is important in the pathobiology of PAH. Increased vWF levels at baseline and follow-up are strong predictors of outcome despite controlling for other known determinants of survival. Current therapies may not fully address this aspect of disease activity. Future work should focus on the implications of and targeted treatments for endothelial dysfunction in PAH.

ACKNOWLEDGMENT: We thank the personnel of the Special Coagulation Laboratory at the New York Presbyterian Hospital for their assistance.

This work was supported by National Institutes of Health grant HL67771 and the Florence and Herbert Irving Clinical Research Career Award.

Manuscript received December 21, 2004; revision accepted April 13, 2005.

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Steven M. Kawut, MD, MS, FCCP; Evelyn M. Horn, MD; Ketevan K Berekashvili, MD; Allison C. Widlitz, PA, MS; Erika B. Rosenzweig, MD; and Robyn J. Barst, MD, FCCP

* From the Departments of Medicine (Drs. Kawut and Horn) and Pediatrics (Ms. Widlitz and Drs. Rosenzweig and Barst), College of Physicians and Surgeons, and the Department of Epidemiology (Dr. Berekashvili), Joseph L. Mailman School of Public Health, Columbia University, New York, NY.

Dr. Kawut has received funding from Actelion Pharmaceuticals, Ltd. and Pfizer, Inc. Drs. Horn, Rosenzweig, and Barst, and Ms. Widlitz have received funding from Actelion Pharmaceuticals, Ltd., Pfizer, Inc., United Therapeutics Corporation, Encysive Pharmaceuticals, Inc., INO Therapeutics, Inc., CoTherix, Inc., Myogen, Inc., and Medtronic, Inc.

Correspondence to: Steven Kawut, MD, MS, FCCP, Division of Pulmonary, Allergy, and Critical Care Medicine, PH 8E, Room 101, 622 W 168 St, New York, NY 10032; e-mail: sk2097@columbia.edu

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