Acute respiratory tract infections in elderly patients increase systemic levels of hemostatic proteins

Acute respiratory tract infections are associated with a temporarily increased risk of acute ischemic heart disease and venous thrombosis [1,2]. The precise underlying mechanism of this association is unclear, but infection-induced systemic inflammation with transient coagulation activation, and endothelial cell perturbation,may contribute to this [3,4]. Endothelial cell perturbation and coagulation activation are associatedwith an increased risk of future ischemic heart disease [5–8]. Several cross-sectional reports showed increased levels of hemostatic proteins during symptoms of acute respiratory tract infection [9–13]; however, these findings are difficult to interpret, as the diagnosis was not unequivocally established. Whether viruses infecting the respiratory tract indeed perturb endothelial cells and thereby increase the risk of acute ischemic heart disease remains to be proven. Therefore, we determined the effect of naturally occurring acute respiratory tract infection on hemostatic proteins in a prospective study. We included 255 different subjects from a general practise, mean age 67 years (± 14 years). Eligible subjects were at risk for influenza, as defined by the criteria to participate in the national influenza vaccination campaign. The Institutional Review Board approved the study, and all subjects provided informed consent. At the time of inclusion, a medical history, including influenzavaccinationstatus,wasobtainedandablood sample was collected; immediately thereafter, the follow-up period started. Blood samples and nose swabs were obtained when the predefined criteria of flu-like symptoms were met.We defined flu-like disease as the acute onset of fever (body temperature > 37.8 C) plus at least two of the following flu-like symptoms: headache, myalgia, sore throat, and cough. All specimens were obtained in the first 3 days of flu-like symptoms. In the convalescent phase, 14 days after the beginning of symptoms, the specimen collection was repeated. Blood was collected in empty vacuum tubes and in tubes containing 0.106 Mtrisodiumcitrate.All sampleswere taken in themorning and processed within 60 min: the tubes were centrifuged for 15 min at 2000 · g, and plasma was pooled and then centrifuged again at 2000 · g for 5 min. Subsequently, plasma was frozen and stored in aliquots of 0.5 mLat)80 C.Prothrombin fragment 1 + 2 (F1+2) and plasmin–a2-antiplasmin (PAP) complexes were measured by enzyme-linked immunosorbent assay (ELISA) from Dade Behring (Marburg, Germany), plasminogen activator inhibitor type I (PAI-1) antigen was measured by an ELISA from Hyphen BioMed (Andresy, France), and von Willebrand factor (VWF) antigen was measured by an ELISA from Dako (Glostrup, Denmark). Results for VWF are presented as percentages of normal pool plasma. The presence of antibodies against respiratory pathogens was established using ELISAs for influenza A virus, influenza B virus, parainfluenza virus type 1 and type 2, respiratory syncytial virus (RSV), adenovirus, and specific IgM and IgG against Mycoplasma pneumoniae. For the detection of IgA antibodies, in-house assays were used; for thedetectionof IgGor IgMantibodies, testspurchased from Serion/Viron (Wurzburg, Germany) were used. We considered respiratory tract infection to be proven when there was a 4-fold elevation between the sample taken on the first day of flu-like symptoms and the sample taken in the convalescent phase 2 weeks later. In addition, samples were tested for RSV, influenza viruses typeAand typeB, parainfluenza viruses 1, 2, 3 and 4, adenovirus, enterovirus, rhinovirus and human metapneumovirus (HMPV) by routine diagnostic immunofluorescence assays 48 h after inoculation. Total RNA and DNAwas isolated with the total nucleic acid isolation kit (Roche Applied Science,Almere, theNetherlands), andamplificationofHMPV, human coronavirusNL63 and influenza viruswas performed as described previously [14–17]. Amplification of RSV type A and type B, rhinovirus, coronavirus C43 and coronavirus 229E was performed using TaqMan EZ RT-PCR reagents (Applied Biosystems, Nieuwerkerk a/d IJssel, The Netherlands). Detection was performed on an ABI7700 detection system. Allmaterialswere spikedwithan internal control, consistingofa fixed amount of phocine distemper virus to monitor for inhibition and/or loss ofmaterial.Data are presented asmedian ± interquartile range (IQR). Levels of hemostatic proteins betweenbaselineand theacutephaseof thedisease, andbetween Correspondence: Tymen T. Keller, Department of Vascular Medicine, Room F4-121, Academic Medical Centre, Meibergdreef 9, 1105 AZ, Amsterdam, the Netherlands. Tel. : +31 20 566 2377; fax: +31 20 696 8833; e-mail: t.t. keller@amc.nl