Evaluation of coagulation activation after Rhinovirus infection in patients with asthma and healthy control subjects: an observational study

It is increasingly recognized that inflammation and haemostasis are interdependent and closely linked processes that can stimulate each other [1, 2]. Many chronic inflammatory diseases, including inflammatory bowel diseases [3, 4], rheumatic arthritis [5, 6], COPD [7‐9], and sarcoidosis [10], are associated with increased coagulability of blood. This procoagulant state has also been observed in the airways of patients with stable asthma as reflected by increased levels of tissue factor (TF), thrombin-antithrombin complexes (TATc) and the plasminogen activator inhibitor-1 (PAI-1), as well as decreased levels of the natural anticoagulant protein C [11]. Local activation of coagulation may be clinically relevant for asthmatic individuals, because we have recently shown that the risk of pulmonary embolism (PE) is increased in severe asthma [12]. Recently a second study on pulmonary embolism and asthma showed not only that there is an increased risk to develop PE but that PE was associated with disease exacerbations [13].

In addition to chronic inflammatory conditions of the lung, viral and bacterial infections have been shown to induce pulmonary coagulation as well [14]. For example, elderly patients with proven respiratory viral infection had activated coagulation, as shown by increased levels of von Willebrand factor (vWF), plasmin-α2-antiplasmin complexes (PAPc), D-dimer and endogenous thrombin potential (ETP) [15]. In addition, respiratory infections were a risk factor for venous thromboembolic events in a large cohort from the general population [16].

Asthma exacerbations are most often caused by respiratory viruses [17, 18], in particular rhinovirus [19]. Whether rhinovirus induced airway inflammation synergistically affects the haemostatic balance in patients with asthma is as yet unknown.

We hypothesized that rhinovirus infection enhances coagulation and reduces fibrinolysis in patients with asthma to a larger extent than in healthy control subjects. Therefore, the aim of the present study was to determine the effect of experimental rhinovirus infection on coagulation (TATc, TF-activity of microparticles, D-dimer and ETP), endothelial activation (vWF) and fibrinolysis (PAPc and PAI-1) in peripheral blood and bronchoalveolar lavage (BAL) fluid of patients with mild allergic asthma and healthy control subjects. The second aim was to assess the relationship between coagulation and fibrinolytic parameters and markers of airway inflammation in BAL fluid.

In total 28 volunteers (14 patients with mild asthma and 14 healthy controls) were included in the study. Out of the 28 patients infected with RV16, 13 patients with mild asthma and 11 healthy control subjects had a positive PCR at day 6 (either in BAL fluid or in nasal swabs or both) and/or a serologic conversion for RV16 at day 42. Two BAL fluid samples were excluded from the analysis as these samples contained >15% bronchial and squamous epithelial cells. Therefore disruption of the epithelial barrier and subsequent contamination of BAL fluid with plasma cannot be excluded for these samples. Clinical and virologic characteristics of patients and controls are shown in Table 1.

This study shows that rhinovirus infection in patients with mild asthma, but not in healthy subjects, increases procoagulant activity in the pulmonary environment as evidenced by increased microparticle-associated TF activity (FGT) in BAL fluid. Moreover, fold changes in eosinophil cationic protein and myeloperoxidase were both associated with the fold changes in FGT and TATc levels in BAL fluid after viral infection. This suggests that rhinovirus infection increases the local procoagulant activity in the airways through both eosinophilic and neutrophilic airway inflammation. In addition to local procoagulant activity, RV16-load showed linear correlations with markers of fibrinolysis and haemostatic activity in plasma.

This is the first study investigating the effect of experimental virus infection on activation of coagulation in patients with asthma. We observed an increase in the intrinsic capability to coagulate in BAL fluid, which is associated with eosinophilic and neutrophilic inflammation. This confirms previous studies showing that inflammatory and allergic asthma exacerbations induce coagulation in the airways as reflected by increased levels of TATc, TF activity and factor Xa [24‐34]. Both fibrin and thrombin have been implicated in airway hyperresponsiveness and allergic airway inflammation [35], which may be the result of inactivation of surfactant and/or the formation of sputumplugs [36].

The increase in procoagulant activity in patients with asthma after rhinovirus infection can be explained by several mechanisms. Firstly, coagulant TF-exposing microparticles are likely to play a key role in the local activation of coagulation in the airways of asthmatic individuals, since these microparticles have been shown to be indispensable to initiate coagulation through the extrinsic pathway [22]. Besides activation of coagulation, microparticles have been shown to be mediators of inflammation during infection [6, 37].

TF-exposing microparticles derived from eosinophils and neutrophils have been described in related eosinophilic diseases, like Churg Strauss vasculitis and bullous pemphigoid [38‐40]. However, eosinophils as the potential source of coagulant TF-exposing microparticles remain controversial, since TF mRNA is below the detection limit in both resting and stimulated eosinophils [41, 42]. Monocytes may be able to transfer TF to microparticles originating from eosinophils and neutrophils. This may explain the associations we observed in BAL-fluid between FGT and TATc with ECP and MPO and fits with the observation by Bouwman et al. [25] that virus-infected monocytes show a higher TF expression.

This study extends the role of increased procoagulant activity as described in previous studies to infectious aetiologies of asthma exacerbation [31, 32]. For example, we observed a decreased fibrinolysis in plasma of patients with mild asthma after RV16 infection, while markers of fibrinolysis were undetectable in BAL fluid. In addition, we observed linear correlations between RV16-load in BAL-fluid and systemic PAI-1 and ETP and an inverse correlation with systemic PAPc were found. Two possible explanations could be provided for these differences in plasma. First, IFN-γ treatment has been shown to decrease levels of PAI-1 in healthy controls [43, 44]. Since interferon production in patients with asthma has been shown to be impaired [45‐47] this might explain the increased levels of PAI-1 in the present study. Although systemic fibrinolysis is impaired in patients with mild asthma, TATc levels in plasma were higher in healthy control subjects, while the systemic fold changes from baseline in TATc, ETP, vWF and PAI-1 are increased in the mild asthmatic group. A possible explanation may be that there is no suitable trigger at baseline leading to decreased TATc levels in plasma of patients with mild allergic asthma. Whether there are compensatory mechanisms leading to lower TATc levels in plasma of asthmatic individuals as compared to healthy individuals is as yet unknown and may be subject for further research. It is important to note that after infection with a mild rhinovirus infection the changes from baseline in plasma are larger in the mild asthma group than in the healthy group and illustrates the increased capability for coagulation.

Secondly, the linear correlations between RV16-load in BAL-fluid and systemic PAI-1 and ETP and inverse correlation with systemic PAPc observed for patients with mild asthma point towards endothelial activation by RV16. vWF and PAI-1 are stored in endothelial cells [48]. Quiescent endothelial cells are generally driving the haemostatic balance in favour of fibrinolysis through activated protein C generation and production of tissue-type plasminogen activator. Upon a stimulus, for example an infection, this balance is changed into a procoagulant state by release of amongst others vWF and expression of PAI-1 [48, 49]. Our current study points towards activation of endothelial cells by rhinovirus, although it is unclear whether the activation is directly or indirectly. Endothelial cells express intercellular adhesion molecule 1 (ICAM-1), which is not only an important molecule for leukocyte adhesion [48], it is also the receptor for rhinovirus to infect human cells [50]. However, the linear correlation between rhinovirus in the airway lumen and procoagulant activity in plasma points towards indirect activation of endothelial cells by rhinovirus, for example through an inflammatory mediators produced by airway epithelial cells [50]. Either way, activation of the endothelium results in an increase of PAI-1 and vWF. The increase in PAI-1 may explain the decrease in PAPc [51], while the increase in ETP might be explained by the increased levels of factor VIII [3, 52] commonly associated with an increase in vWF and other pro-coagulants [53]. Both impaired IFN production and endothelial activation may play a role in the increased systemic haemostatic activity found in patients with mild asthma. Our results are in line with previous studies about the activation of haemostasis in healthy controls in vitro (viral infection of endothelial cells and monocytes) [54, 55] and in vivo (elderly patients with acute respiratory tract infections) [56].

The strength of our study is the experimental design, which provided standardized exposure and comparable time points of post-infection sampling. In addition, the patients were free of steroids, which may affect inflammatory and coagulant outcomes. However, we cannot exclude the influence of potential confounding factors in the present outcomes. First, the male/female ratio was different between the two groups, with more female subjects in the control group than in the group of patients with asthma. Since many women used oral contraceptives, this might have influenced coagulation activity [57, 58]. However, we do not think that this factor biased our results since oral contraceptive users were equally distributed between the two groups and fibrinolysis was only impaired in patients with asthma.

Secondly, we did not observe significant changes for some relevant coagulation parameters in BAL fluid and plasma. This might relate to the mild nature of the experimental rhinovirus infection (i.e. low infectious dose and mild rhinovirus strain). We used this method to infect volunteers because it most closely resembles the natural course of rhinovirus infection. In addition, the procedure has been proven to be safe in patients with mild asthma and resulted in increase in common cold symptoms in both asthmatic and healthy individuals. The effects on coagulation are likely more pronounced after a rhinovirus-induced exacerbation in patients with moderate to severe asthma or in more severe disease exacerbations (i.e. influenza).

Another explanation for the non significant changes of coagulant parameters in BAL fluid and plasma could be the use of short-acting beta-2-agonists. Although it is suggested that beta-2 agonists may have a small anti-inflammatory effect [59, 60], we believe this has not influenced our results as both groups of volunteers used salbutamol during the spirometry test just before the bronchoscopy with BAL.

Thirdly, the study was limited by the fact that some haemostatic parameters were below the detection limit in BAL fluid which could be explained by the timing of the second bronchoscopy and/or the dilution involved with bronchoalveolar lavage. This is a common problem often encountered and difficult to circumvent in this type of studies due to the dilution involved with bronchoalveolar lavage. Still, our data provides evidence of increased procoagulant activity in the airways of patients with asthma after viral infection, which correlates with inflammation, and thus matches with our hypothesis.

And finally, one could argue that a larger study population would have shown more procoagulant activity and systemic procoagulant changes. Based on our present findings, we calculated that we would need more than 1000 volunteers to reach statistical significance, which is neither ethical nor feasible. Evaluation of coagulation after rhinovirus infection in patients with more severe asthma would be more relevant as these patients are more at risk for venous thromboembolism [12].

Although this study is observational in nature, the findings of the present study may have important clinical implications. Patients with asthma have an increased risk of pulmonary embolism [12, 13] which is associated with asthma exacerbations [13]. Our results indicate for the first time that even a mild rhinovirus infection in patients with mild asthma induces procoagulant activity in the airways and a possible systemic increase in haemostatic activity. One could hypothesize that patients with severe asthma have more procoagulant activity and could therefore be at increased risk to develop acute thromboembolic events due to rhinovirus infections. In this perspective, it would be required to further investigate a potential causal relationship between coagulation and eosinophilic inflammation in asthmatic airways and to further explore the nature of TF-bearing microparticles in patients with asthma. Furthermore, follow-up studies should focus on activation of haemostasis in patients with moderate to severe asthma as well as procoagulant changes induced by influenza and respiratory syncytial virus (RSV), as both influenza and RSV are frequently isolated in patients that are hospitalized for their asthma exacerbation [18].

In conclusion we have provided evidence of local procoagulant activity after rhinovirus infection in the airways of patients with asthma, which is associated with the intensity of eosinophilic and neutrophilic airway inflammation. As rhinovirus infection is a very common trigger of asthma exacerbations the clinical consequences of this induction of procoagulant activity deserve further investigation, in particular in patients with severe asthma who have already an increased risk of myocardial infarction, stroke and venous thromboembolism [12, 61].