Coagulation activation during air travel is not initiated via the extrinsic pathway

Recently, it has been firmly established that air travel is associated with a 2- to 4-fold increased risk of venous thrombosis (Kuipers et al, 2007). The mechanisms underlying air travel-related venous thrombosis are incompletely understood but probably include immobilization and hypoxia (Cannegieter, 2012). However, a recent study showed that neither immobilization nor hypoxia individually affect coagulation activation, and thus a combination of factors appears to explain air travel-related thrombosis (Venemans-Jellema et al, 2014). Individuals with genetic or acquired hypercoagulability appear to have a particularly increased risk of venous thrombosis after air travel (Cannegieter et al, 2006). We previously demonstrated coagulation activation after air travel in some, but not all, healthy individuals (Schreijer et al, 2006). Seventeen percent of these subjects had increased thrombin-antithrombin (TAT) complexes after an 8-h- flight, but not after an 8-h movie marathon, or an 8-h daily life routine. It has not been established how activation of the coagulation system occurs in these subjects. Knowledge on the initiating trigger of coagulation activation after air travel may give a clue on the mechanism of air travel-related thrombosis. It has been suggested that tissue factor-bearing microparticles are the initiating trigger in venous thrombosis in general (Giesen et al, 1999; Furie & Furie, 2008; Manly et al, 2011). We therefore hypothesized that coagulation activation after air travel proceeds via the tissue factor (TF) pathway. Here we used two distinct assays that assess plasma levels of activated (but not zymogen) factor VII (FVIIa) as markers for extrinsic coagulation activation. As TF-mediated initiation of coagulation starts with the conversion of FVII to FVIIa, we assume that increased plasma levels of FVIIa reflect TF-mediated coagulation activation. Additionally, we measured activity and antigen levels of FVII. Recruitment of volunteers and the procedures of the study were described earlier (Schreijer et al, 2006). Briefly, 71 healthy volunteers were followed for 8 h of air travel and, as two control periods, during an 8-h movie marathon, and 8 h of daily life. FVII activity was determined on an automated coagulation analyser (Behring Coagulation System, Siemens Healthcare Diagnostics, Marburg, Germany) with reagents and protocols from the manufacturer. Plasma FVIIa activity was measured using a commercially available kit according to manufacturer's instructions (STACLOT VIIa-rTF, Diagnostica Stago, Asnieres, France). The plasma FVII and FVIIa antigen levels were measured using in-house developed enzyme-linked immunosorbent assays (ELISA), as described earlier (Hyseni et al, 2013). Plasma activity and antigen levels of FVII were correlated as shown in Fig 1A [r = 0·373 (95% confidence interval (CI) 0·301–0·441)]. However, FVII antigen levels of some individuals were discordant with the activity levels. Plasma activity and antigen levels of FVIIa were correlated as shown in Fig 1B [r = 0·265 (95%CI 0·188–0·339)]. The correlation between the assays was poor at low levels of FVIIa, which may be related to the sensitivity of the antigen assay, as the low antigen levels were close to the detection limit of the assay. Subsequently, we analysed plasma levels of FVII and FVIIa activity and antigen in samples taken of healthy volunteers before, during, and after 8-h flight, 8-h movie marathon and an 8-h daily life routine. Figure 1C shows the median FVII and FVIIa activity levels before, during, and after each exposure. Table 1A shows the median absolute individual changes after each exposure. Plasma FVII activity levels did not change during or after exposure to the flight, movie marathon or daily life routine. The plasma FVIIa activity levels were higher after the flight, movie marathon and daily life routine compared to the FVIIa levels before and during the exposures, and the absolute individual changes were similar for all exposures. Taken together, these results do not suggest activation of zymogen FVII during or after air travel. Figure 1D shows the FVII and FVIIa antigen levels. In line with the results of the activity assays, FVII antigen levels were equivalent throughout all three exposures. In contrast with the results of the activity assays, FVIIa antigen levels dropped during all three exposures. FVIIa antigen levels were slightly lower compared to baseline values after the cinema and flight, but slightly higher than baseline after the daily life routine. The reason for the clear discrepancy between the FVIIa activity and antigen tests is, at present, unclear. One explanation would be that the antigen test recognizes both 'active' FVIIa and FVIIa in complex with inhibitors. However, we have demonstrated that the assay recognizes both free FVIIa and FVIIa in complex with TF, but not the FVIIa-antithrombin complex (data not shown). Although the pattern of FVIIa activity and antigen levels over time is inconsistent, the results of both assays indicate that coagulation activation during air travel does not result from activation of the extrinsic pathway. Finally, we analysed FVII and FVIIa activity and antigen levels in those individuals who showed an appreciable increase in TAT complex levels after the flight compared with those who did not (Schreijer et al, 2006). FVII and FVIIa activity and antigen levels did not differ between those individuals with an activated clotting system after the flight and those without evidence of coagulation activation (Table 1B). Taken together, the results of this study, using two distinct assays for FVII and FVIIa, do not provide evidence for activation of the extrinsic pathway of coagulation during air travel. We hypothesized that air travel-related activation of coagulation would proceed via TF-bearing microparticles analogous to the proposed role of TF in the development of venous thrombosis. Nevertheless, previous studies performed in the cohort described in this paper may be consistent with the alternative scenario, i.e., that the intrinsic pathway is responsible for air travel-related activation of coagulation (Schreijer et al, 2010). These previous studies showed higher plasma soluble P-selectin and plasminogen activator inhibitor type 1 levels in those individuals with elevated TAT levels after the flight. These findings may reflect increased platelet activation in the individuals with activated coagulation after air travel. Platelet activation results in the release of polyphosphates, which, among other procoagulant effects, activate the intrinsic pathway of coagulation (Müller et al, 2009). We thank Tesy Merkx (UMC Utrecht, The Netherlands) for expert technical assistance. This study was supported by an unrestricted educational grant from Novo Nordisk. We have no conflict of interest to report. A.M. Schut participated in the design of the present study, helped to coordinate the study, performed experiments, analysed and interpreted data and wrote the manuscript. A. Venemans-Jellema performed data analysis, interpreted data and revised the manuscript. J. C. M. Meijers supervised some of the experiments, interpreted data and revised the manuscript. S. Middeldorp and F.R. Rosendaal participated in the study design, interpreted data and revised the manuscript. P. G. de Groot and M. Roest supervised some of the experiments, interpreted data and revised the manuscript. T. Lisman participated in the design and supervision of the present study, interpreted data and wrote the report. S.C. Cannegieter participated in study design, supervised data analysis, interpreted data and revised the manuscript.