Predictors of the rise in vWF after ST elevation myocardial infarction: implications for treatment strategies and clinical outcome

An ENTIRE-TIMI 23 substudy

Kausik K. Ray*, David A. Morrow, C. Michael Gibson, Sabina Murphy, Elliott M. Antman and Eugene Braunwald for the ENTIRE-TIMI 23 Study Group

The TIMI Study Group and Cardiovascular Division, Department of Medicine, Brigham & Women's Hospital/Harvard Medical School, Boston, MA, USA

Received 15 July 2004; revised 14 November 2004; accepted 25 November 2004; online publish-ahead-of-print 26 January 2005.

* Corresponding author: TIMI Study Group, 350 Longwood Avenue, First Floor, Boston, MA 02115, USA. Tel: +1 617 525 6865; fax: +1 617 734 7329. E-mail address: kkray{at}partners.org

See page 421 for the editorial comment on this article (doi:10.1093/eurheartj/ehi125)


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Conclusion
 Acknowledgements
 References
 
Aims Prior studies suggest that acute coronary syndromes (ACSs) are associated with endothelial activation and that this is of prognostic significance. We hypothesized that endothelial activation, as measured by a rise in von Willebrand Factor ({Delta}vWF), was influenced by the thrombolysis in myocardial infarction flow grade (TFG), the corrected TIMI frame count (CTFC) and the choice of anticoagulant therapy after fibrinolysis in ST elevation myocardial infarction (STEMI).

Methods and results Data were drawn from the enoxaparin and tenecteplase tissue plasminogen activator (TNK-tpa) with or without GPIIb/IIIa inhibitor as the reperfusion strategy in the STEMI trial (ENTIRE-TIMI 23). Three hundred and fourteen patients had serial measurements of vWF (baseline and 48–72 h) and angiographic data available. TFG<3 (P=0.0042) or CTFC≥40 at 60 min (P=0.0035) were associated with a higher {Delta}vWF. {Delta}vWF ≥75th percentile was associated with a higher incidence of death or myocardial infarction (MI) at 30 days, compared with <75th percentile (11.2 vs. 4.1%, P=0.027). Enoxaparin independently reduced the {Delta}vWF (P=0.019) and also the composite of death or MI (OR 0.33, 95% CI 0.12–0.91, P=0.03) compared with unfractionated heparin.

Conclusion In STEMI treated by fibrinolysis, coronary flow at 60 min and choice of adjunctive anticoagulant appear to be independent determinants of {Delta}vWF. Enoxaparin is independently associated with a reduction in {Delta}vWF and a reduction in death or MI. The clinical benefits of enoxaparin as an adjunctive treatment in STEMI may be mediated in part by a reduction in vWF release.

Key Words: vWF • TIMI flow grade • Corrected TIMI frame count • ST elevation myocardial infarction • Endothelial activation


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Conclusion
 Acknowledgements
 References
 
Von Willebrand factor (vWF) is stored within platelets, megakaryocytes, and endothelial cells. It is released from platelets following activation by a number of agents including thrombin. In animal and clinical transplant models of von Willebrand's disease, where either endothelial or platelet stores of vWF were replaced, the existing data suggest that the soluble form of vWF found in plasma may predominantly be of endothelial origin,13 hence plasma vWF has been used as a surrogate marker of endothelial activation.4,5 Acute coronary syndromes (ACSs) are associated with a rise in markers of endothelial activation6 and high levels may predict adverse events.7,8 At a cellular level, endothelial activation occurs in response to cytokines and thrombin (both abundant in ACS), resulting in a number of phenotypic changes. These include the expression of adhesion molecules and tissue factor, the down-regulation of thrombomodulin, and the release of vWF from intracellular stores.911

vWF levels rise in ACS, and in the setting of ST elevation myocardial infraction (STEMI) become elevated at 24 h and peak at 48–72 h before returning to baseline at around day 14.8,12 A greater rise in {Delta}vWF in patients with non-ST elevation ACS13 or with STEMI has been shown to predict adverse events.8 It is, however, uncertain which factors independently influence the rise in vWF. Potential determinants of {Delta}vWF include the choice of anticoagulant used as the adjunctive treatment to fibrinolysis and the coronary blood flow. In non-ST elevation ACS, low molecular weight heparin (LMWH) reduces the rise in vWF and also improves prognosis.13 In small studies, the patency of the infarct-related artery 90 min after fibrinolysis was associated with differences in the rise in vWF at 24–72 h.14

The goal of this study was to determine the factors that independently influence the change in vWF after STEMI. Specifically we aimed to correlate coronary artery flow at 60 min after fibrinolysis using the TIMI flow grade (TFG) and corrected TIMI frame count (CTFC) with functional changes in vWF in patients with STEMI enrolled in the ENTIRE-TIMI 23 study,15 and also study the effect of a LMWH (enoxaparin) on vWF release. We hypothesized that a worse TFG and CTFC after fibrinolytic therapy would be associated with a higher {Delta}vWF (possibly reflecting greater endothelial activation) in the peri-infarct period and that a putatively more potent inhibitor of thrombin generation (enoxaparin) would reduce the rise in vWF compared with unfractionated heparin.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Conclusion
 Acknowledgements
 References
 
Study population
The ENTIRE-TIMI 23 trial evaluated a LMWH (enoxaparin) vs. unfractionated heparin (UFH) with full-dose tenecteplase (TNK) and half dose TNK plus abciximab in STEMI between February 2000 and September 2001.15 The study enrolled 483 patients within 6 h of the onset of symptoms,15 and of these, 314 had evaluable angiograms as well as blood samples taken at enrolment prior to fibrinolysis and during follow-up at 48–72 h after fibrinolysis. The clinical endpoints of death and MI were recorded at 30 days as part of the ENTIRE-TIMI 23 study protocol and adjudicated by a clinical events committee.

Patients excluded from this analysis either had uninterpretable angiograms or did not have blood samples collected at both pre-specified times to allow core lab analysis. Those excluded from these analyses (primarily because serial blood samples were not collected) had similar demographic characteristics to the analysis population and importantly had no differences in TFG, CTFC, ST-segment resolution, or treatment randomization (data not shown). The full protocol design of ENTIRE-TIMI 23 has been previously described.15

Measurement of vWF
We measured only the biologically active form of soluble vWF as vWF co-factor activity using the Dade Behring ristocetin co-factor assay, with a lower limit of detection of 10% activity and an inter-assay coefficient of variation of 8–10.3%. In the presence of ristocetin, vWF causes agglutination of stabilized platelets contained in the reagent. The vWF co-factor activity was measured by a change in optical density and reported as percentage of normal. For the purposes of our analyses {Delta}vWF was measured by subtracting baseline vWF co-factor activity levels from follow-up levels after fibrinolysis.

Angiographic and ECG measurements
The TFG and CTFC were determined as previously described at 60 min after fibrinolytic therapy.16,17 CTFC was also assessed both as a continuous and as a categorical variable with a cut-off of <40 frames, as this correlates with an open artery.18 TIMI myocardial perfusion grade (TMPG) was also determined as previously described.19 In addition, 181 patients had evaluable 12-lead electrocardiograms (ECGs) at study entry and 60 min after the onset of fibrinolysis.

Statistical analysis
Continuous variables are reported as medians (in figures) and median and interquartile ranges (in tables). The Wilcoxon rank-sum (for two-way comparisons) test or the Kruskal–Wallis test (for >2-way comparisons) was used for the analysis of categorical variables (Table 1). The relationship between continuous variables and vWF was assessed by linear regression (Table 2). The {chi}2 test was used for the analysis of categorical variables when sample size was ≥5 for all cells in a table. Two-sided testing was used for all analyses at the P<0.05 level and there was no adjustment for multiple testing. A graph of the residuals against the fitted values was examined and no non-linear pattern was observed. Heteroskedasticity was formally evaluated using the Breusch–Pagan/Cook–Weisberg test and the assumption was not violated (P=0.2789). As the data were not significantly different from normal, a multivariable linear regression model was used to assess the association between {Delta}vWF and its univariate predictors including baseline clinical characteristics, angiographic characteristics, and treatment. Only univariate predictors at a level of P<0.1 were included in the model. All analyses were performed using Stata version 7.0.20


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Table 1 The association of categorical baseline characteristics and treatments with {Delta}vWF co-factor activity
 

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Table 2 The association between baseline characteristics and {Delta}vWF co-factor activity
 

    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Conclusion
 Acknowledgements
 References
 
Relationship between baseline vWF co-factor activity and coronary flow
No association was found between baseline vWF prior to fibrinolysis and coronary flow at 60 min after fibrinolysis using either the TFG or the CTFC. Median vWF for patients with TFG=3 vs. TFG<3 were 206% of normal [interquartile range (IQR) 157–282] vs. 210% of normal (IQR 152–260), P=0.17 and the median vWF for patients with CTFC<40 vs. ≥40 frames was 205% of normal (IQR 156–290) vs. 210% of normal (IQR 152–260), P=0.11. There was no association between baseline vWF and peak cardiac specific creatinine kinase (CKMB) at 48 h. No correlation was seen between baseline vWF and death or MI at 30 days.

Relationship between coronary flow, ST resolution, and {Delta}vWF co-factor activity
Median vWF for the entire cohort at baseline was 205% of normal (IQR 153–267) and rose to 217% (IQR 170–278), P=0.008 at 48–72 h. Poor coronary artery flow at 60 min, as assessed by a TFG<3 or a CTFC≥40, was associated with a higher {Delta}vWF (Figure 1). The median {Delta}vWF was significantly lower in patients with TFG=3 [8.5 (IQR –67 to 59) P=0.0042] and with CTFC <40 frames [7 (IQR –67 to 54) P=0.0035]. When CTFC was assessed as a continuous variable vs. {Delta}vWF, a statistically significant correlation was observed (R2=0.03, P=0.004). ST resolution of ≥70% at 60 min was similarly associated with a significantly lower {Delta}vWF compared with that in patients without 70% resolution, P=0.015 (Figure 1). No difference was seen between the TMPG and {Delta}vWF.



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Figure 1 Showing the change in vWF from baseline (median and IQR) in patients with: slow flow (TFG<3 or CTFC≥40) vs. fast flow (TFG=3 or CTFC<40); and in patients with ST segment resolution <70% vs. ≥70% resolution at 60 min. {Delta}vWF co-factor activity is measured as a percentage of normal. Slow flow was associated with higher {Delta}vWF co-factor activity (P=0.0042 for TFG<3 and 0.0035 for CTFC≥40). ST resolution ≥70% at 60 min was associated with a lower {Delta}vWF (P=0.015).

 
Baseline characteristics, treatment, and {Delta}vWF co-factor activity
Baseline characteristics, treatments, and their association with {Delta}vWF co-factor activity are shown in Table 1 (categorical variables) and Table 2 (continuous variables). The characteristics associated with high {Delta}vWF co-factor activity were symptom duration prior to fibrinolyis (P<0.0001), prior treatment with a beta-blocker (P=0.006), higher admission pulse (P=0.0001), and systolic blood pressure (P=0.001). Treatment with enoxaparin was associated with a reduction in {Delta}vWF co-factor activity compared with UFH (P=0.019). Diabetes, Killip class II–IV on admission, history of smoking, hypertension, prior statin use, use of abciximab, or full dose TNK were not associated with significant differences in {Delta}vWF. In a multivariable model (Table 3) correcting for significant univariate associations, longer duration of symptoms and higher admission pulse were independently associated with higher {Delta}vWF (95% CI 7.0–26.6 and 0.7–2.2, respectively), whereas TFG=3 and enoxaparin treatment were independently associated with a lower {Delta}vWF (95% CI –52.5 to –3.1 and –55.5 to –5.5, respectively).


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Table 3 Linear regression analysis of the univariate predictors of {Delta}vWF co-factor activity expressed as a percentage of normal; 95% confidence intervals for a {Delta}vWF are shown
 
Relationship between {Delta}vWF co-factor activity, myocardial necrosis, and clinical events
The relationship between peak CKMB isoenzyme within the first 48 h after fibrinolysis and {Delta}vWF suggested a threshold effect. Patients with CKMB ≥75th percentile had a subsequent higher {Delta}vWF [median 31.5, IQR –25.5 to 84) vs. patients with CKMB <75th percentile (13.5, IQR –59.5 to 59), P=0.04. Using the combined clinical endpoint of death or MI at 30 days, there was an 11.2% event rate among patients with {Delta}vWF≥65.5 corresponding to the 75th percentile (fourth quartile) vs. 4.1% for those with {Delta}vWF < 65.5 (Figure 2), which was statistically significant (OR 2.02, 95% CI 1.2–3.3, P=0.027). There was no association between admission with pulmonary oedema by day 30 and {Delta}vWF.



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Figure 2 Rates of death or MI for patients with {Delta}vWF co-factor activity ≥75th percentile (≥65.5) vs. <75th percentile (<65.5), (P=0.027 {chi}2).

 
Relationship between enoxaparin, reperfusion, and clinical outcome
There was no statistical difference between enoxaparin and UFH in the proportion of patients that achieved TFG=3 (52.6 vs. 49.5%), TMPG=3 (51.7 vs. 52.4%), or ≥70% ST resolution (19.4 vs. 21.1%) at 60 min. Use of enoxaparin was associated with a lower frequency of death and MI at 30 days (2.9 vs. 12%, P=0.001). In a multivariable model that adjusted for clinical characteristics, abciximab use, {Delta}vWF quartiles, and the presence or absence of TFG=3 at 60 min, the use of enoxaparin was the only independent predictor of death or MI at 30 days (OR 0.33, 95% CI 0.12–0.91, P=0.03). A {Delta}vWF ≥75th percentile tended directionally towards worse outcome but this did not achieve statistical significance in the model (OR 1.7, 95% CI 0.49–6, P=0.4).


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Conclusion
 Acknowledgements
 References
 
This study demonstrates that, following fibrinolytic therapy, the use of a LMWH (enoxaparin) as an adjunctive treatment to fibrinolysis is independently associated with a reduction in the rise in vWF compared with unfractionated heparin and also a reduction in death or MI at 30 days. The use of enoxaparin was not associated with any change in coronary flow at 60 min after fibrinolysis or with any other marker of reperfusion. We therefore hypothesize that some of the potential clinical benefits of enoxaparin in STEMI may be mediated by a reduction in endothelial activation, as suggested by a lower {Delta}vWF at 48–72 h. Additionally, we demonstrate that reduced coronary artery flow at 60 min (TFG<3) is independently associated with a higher {Delta}vWF at 48–72 h. Better early TFG and CTFC are known to predict a more favourable outcome following fibrinolysis and much of this benefit is thought to be related to myocardial preservation.21,22 Our data suggest that a reduction in vWF, and hence plausibly endothelial activation, may also play an important role by which better early reperfusion reduces subsequent clinical events.23

vWF is released from both activated platelets and endothelial cells in response to several stimuli including the presence of thrombin. In the setting of STEMI, the rise in vWF could represent the release from both these stores and it is unclear which makes the greater contribution to plasma levels. In bone marrow and lung transplant models of von Willebrand's disease that manipulate either the endothelial or platelet source of vWF, it has been suggested that vWF in plasma is predominantly of endothelial origin13 and hence it has been used as a surrogate marker of endothelial activation.4,5 In conditions associated with endothelial activation, vWF levels in plasma track well with other soluble markers of endothelial activation although the individual kinetics of different markers may vary.2427 Within endothelial cells, vWF is stored inside the Weibel-Palade bodies as large molecular weight multimers and, in vitro, vWF is released in response to a number of stimuli including thrombin, histamine, adrenaline, and hypoxia.4,28,29 Endothelial activation results in the release of the most potent biologically active high molecular weight forms of vWF.29 In STEMI the rise in vWF antigen levels is accompanied by a simultaneous rise in the high molecular weight multimers,12 possibly supporting an endothelial process. Thus, in ACS, the measurement of biologically active vWF based on the measurement of high molecular weight vWF multimers (ristocetin co-factor assay) has given comparable results to the measurement of vWF antigen.30

In the context of STEMI studies, it has been shown that vWF levels are not altered in the first hour after fibrinolysis, but become elevated at 24 h peaking at around 48–72 h and subsequently reverting to normal at day 14.12 In vitro data suggest that the rise in vWF occurs as a result of release from intracellular stores rather than from increased transcription, although the latter cannot be excluded in vivo.28,31,32 It is unclear which endothelial bed may be the possible origin of the increased release of vWF. Despite the greater contribution of the pulmonary circulation to the total endothelial surface, the association between vWF and coronary flow and the lack of association between vWF and presenting Killip class, or subsequent heart failure, may favour the coronary circulation, as suggested by other studies.29

Elevated absolute levels of vWF in stable coronary disease33 as well as a rise in vWF during acute coronary events predict future cardiovascular risk, suggesting that heightened endothelial activation may influence prognosis.8,13 In our study baseline vWF did not predict differences in reperfusion (TFG, CTFC, or ST resolution) or clinical events, suggesting that endothelial activation did not significantly contribute to changes in early coronary flow. vWF levels subsequently rose at 48–72 h and showed an independent correlation with coronary flow at 60 min, suggesting that the rise in vWF was the consequence rather than the cause of impaired early coronary perfusion. These data are in keeping with earlier smaller studies that showed that an occluded coronary artery 90 min after fibrinolysis was associated with a higher vWF at 24 h compared with a patent artery, but no difference was observed in baseline vWF between patient groups.14 We did not measure vWF at 60 min after fibrinolysis and therefore cannot be certain that there was no correlation between vWF and TFG at 60 min, or that the rise in vWF observed at 24 h did not occur exclusively between baseline and 60 min after fibrinolysis. However, existing data suggest that there is no significant difference in vWF between baseline and 60 min after fibrinolysis.12

Fibrinolytic therapy increases activation of the coagulation system (hence thrombin generation),34 and this may contribute to thrombin-mediated release of vWF from endothelial cells or platelets. The rise in vWF in plasma is modifiable by anticoagulant treatment and, in particular, LMWHs such as enoxaparin, which are possibly more potent inhibitors of thrombin generation, reduce the rise in vWF more than UFH does in non-ST elevation ACS.3538 The present study is the first to show that a LMWH significantly reduces the rise in vWF in the setting of STEMI, and it suggests that anticoagulants with powerful antithrombin activity may attenuate endothelial or platelet release of vWF when administered with fibrinolytic agents. While an increase in vWF (high {Delta}vWF) may be a useful marker of endothelial or platelet activation in the context of fibrinolytic therapy, vWF could provide an indirect assessment of thrombin inhibition by different adjunctive anticoagulants. While we did not measure thrombin levels in this study, there is evidence that LMWH is a more potent inhibitor of thrombin generation than UFH in non-ST elevation ACS35 and we hypothesize that this is also the case in STEMI.

In this analysis, as in the main ENTIRE-TIMI 23 study, enoxaparin reduced the combined clinical endpoints of death and MI at 30 days compared with UFH, without any significant influence on the TFG, CTFC, or ST resolution at 60 min, suggesting that the clinical benefits are not determined by early effects on reperfusion.15 This may indicate an alternative mechanism such as a reduction in subsequent endothelial activation. The ability of enoxaparin to blunt the rise in vWF and reduce clinical events has previously been demonstrated in non-ST elevation ACS,13 but not in STEMI. Clinical studies, such as HART II,39 have also failed to show early improvements in TFG with LMWH, but suggested a non-significant trend towards better coronary artery patency at days 5–7. Similarly ASSENT-PLUS40 did not show a greater percentage of TFG=3 at days 4–7 but did show less TFG 0–1 with LMWH. Although we did not perform angiography at day 4–7, our analyses, when taken together with the ASSENT-PLUS data, lead us to speculate that endothelial activation could plausibly play a role in the aetiology of late thrombotic re-occlusion. A highly activated endothelium may bind more platelets and inflammatory cells at sites of reduced endothelial integrity, resulting in thrombus formation. In this regard, vWF may not only be a marker of endothelial activation but may also play a pathological role in late thrombotic occlusion.

In univariate analyses, our study showed that impaired ST resolution at 60 min, higher peak CKMB mass, and longer symptom duration were associated with higher {Delta}vWF, which further supports a link between reperfusion and endothelial activation. Our study also demonstrates that patients with {Delta}vWF ≥75th percentile have a two-fold increased risk of death and MI at 30 days compared with patients with lower {Delta}vWF. The latter finding is in keeping with the observations of Collet et al.8 who observed that patients who had subsequent adverse clinical events had higher vWF levels at 24 h after STEMI, but included patients with greater symptom duration and patients who had not received fibrinolysis. In contrast to their study, we did not observe a relationship between the rise in vWF and subsequent heart failure.

In a multivariable analysis, increased symptom duration and heart rate, TFG<3, and use of UFH were independently associated with elevated {Delta}vWF. Possible mechanisms for the associations between heart rate and endothelial activation include increased haemodynamic stress.12 A longer duration of symptoms may increase the amount of myonecrosis, leading to inflammation, which could then drive endothelial activation. Plausibly, increased endothelial activation may form another mechanistic link between increased symptom duration and adverse clinical outcomes in STEMI. Although high CKMB mass (48–72 h) was associated with higher {Delta}vWF we did not observe any association between ejection fraction or TMPG at 60 min and {Delta}vWF. Oxidative stress due to reperfusion is unlikely to explain our observations as an open artery is associated with greater reperfusion injury and increased free radical generation41 and hence it would be expected to induce a greater rise in {Delta}vWF, in contrast to our findings.

In summary, the present study provides support for the observation that a rise in vWF is associated with adverse cardiac events in STEMI, and further demonstrates novel associations between symptom duration, coronary flow, and the subsequent rise in vWF. We hypothesize that the change in vWF represents endothelial activation and may mechanistically link early improvements in coronary flow with the subsequent reduction in clinical events observed in STEMI trials. We also demonstrate for the first time that enoxaparin, in combination with fibrinolytic therapy, is independently associated with a reduction in vWF, justifying the enoxaparin effect on death or MI as previously reported by our group.15 We postulate that earlier treatment of STEMI and novel strategies that improve early reperfusion or inhibit subsequent thrombin generation may result in a greater reduction in endothelial activation, which could translate into a reduction in late clinical events. The latter is analogous to observations made in non-ST elevation ACS.

Limitations
This is an exploratory analysis and is designed to be hypothesis generating. We measured vWF at two time points and additional measurements may have been helpful to establish causality. The use of additional, more specific, endothelial markers and the measurement of thrombin would have further supported our observations. Despite these limitations, our observations appear to be broadly consistent with the published literature while presenting some novel observations.


    Conclusion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Conclusion
 Acknowledgements
 References
 
Higher CTFC or TFG<3 at 60 min and longer duration of symptoms are independently associated with higher {Delta}vWF after fibrinolysis. High {Delta}vWF is associated with death or MI at 30 days. Enoxaparin independently reduces {Delta}vWF and death or MI compared with UFH in STEMI. This may reflect a reduction in endothelial activation in patients treated with enoxaparin and be clinically relevant in studies comparing enoxaparin with UFH in STEMI, such as the ongoing EXTRACT-TIMI 25 study.


    Acknowledgements
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Conclusion
 Acknowledgements
 References
 
The ENTIRE-TIMI 23 study was funded by Aventis Pharma and Genentech. K.K.R. is funded by a British Heart Foundation International Fellowship.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Conclusion
 Acknowledgements
 References
 

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