Open Forum Infectious Diseases BRIEF REPORT Hantavirus infection activates intravascular coagulation path- Differential Regulation of PAI-1 ways in the acute stage of HCPS and HFRS [4–6]. At the same in Hantavirus Cardiopulmonary time, however, PUUV has been suggested to cause enhanced Syndrome and Hemorrhagic Fever b fi rinolysis , that is, excessive solubilization of blood clots, which we recently showed to be characterized by elevated ac- With Renal Syndrome tivity of circulating tissue plasminogen activator (tPA), whereas 1 2 3 4 4 Carla Bellomo, Miša Korva, Anna Papa, Satu Mäkelä, Jukka Mustonen, 2 5 1 5 its main physiological inhibitor, plasminogen activator in- Tatjana Avšič-Županc, Antti Vaheri, Valeria P. Martinez, and Tomas Strandin 1 hibitor–1 (PAI-1), remains unaltered . In this study, we Laboratorio Nacional de Referencia para Hantavirus, Servicio Biología Molecular, Departamento Virología—INEI-ANLIS “Dr C. G. Malbrán,” Buenos Aires, Argentina; Institute assessed the levels of tPA and PAI-1 in HCPS and HFRS caused of Microbiology and Immunology, Faculty of Medicine University of Ljubljana, Ljubljana, by ANDV and DOBV, respectively, to better understand the role Slovenia; Department of Microbiology, Medical School, Aristotle University of Thessaloniki, Thessaloniki, Greece; Department of Internal Medicine, Tampere University Hospital and hemostatic regulation in hantavirus pathogenesis in general. Faculty of Medicine and Life Sciences, University of Tampere, Tampere, Finland; Department of Virology, Medicum, Faculty of Medicine, University of Helsinki, Helsinki, Finland. METHODS The study protocol was conducted in accordance with the eth- We analyzed the levels of circulating tissue plasminogen acti- ical standards laid down by the Declaration of Helsinki and was vator (tPA) and plasminogen activator inhibitor (PAI)–1 in acute hantavirus cardiopulmonary syndrome (HCPS) and approved by the Ethical Committee in Research of ANLIS “Dr hemorrhagic fever with renal syndrome (HFRS). The levels of C. Malbrán,” Slovenian National Medical Ethics Committee, tPA commonly increased in both diseases, whereas PAI-1 corre- and the Ethics Committee of the Tampere University Hospital lated with disease severity in HCPS but not in HFRS. for serologically confirmed ANDV, DOBV, and PUUV cases, Keywords. hantavirus; HCPS; HFRS; PAI-1; tPA. respectively. The study cohort consisted of patient samples that were collected ≤10 days after the beginning of the disease, that is, after onset of fever. Severe HCPS was classified, for example, due to pulmonary failure and need of mechanical ventilation, Hantaviruses cause two diseases: hantavirus cardiopulmonary whereas signs of lower respiratory compromise were considered syndrome (HCPS) and hemorrhagic fever with renal syndrome mild HCPS. Non-HCPS cases with pulmonary complications (HFRS). The hallmark of both diseases is increased vascular were included as reference. The DOBV patients were classified permeability [1, 2]; however, the most ae ff cted organ generally as severe or mild, for example, based on the need for dialysis. varies from the lungs in HCPS to the kidneys in HFRS. The type Additional information on patient demographics and disease of hantavirus disease is determined by the causative species. classification criteria can be found in Supplementary Table 1 Andes (ANDV) and Sin Nombre (SNV) hantaviruses cause and the Supplementary Methods, respectively. HCPS with mortality that can reach 40% in South and North Protein levels of tPA and PAI-1 were quantitated from patient America, respectively. Puumala hantavirus (PUUV) causes plasma or serum by enzyme-linked immunosorbent assays, a mild HFRS, also called nephropathia epidemica (NE; mor- similar to methods described previously . tality ~0.1%), mainly in Northern Europe and Russia, whereas Dobrava (DOBV) and Hantaan (HTNV) viruses cause severe RESULTS HFRS (mortality 1%–5%) in the Balkans and East Asia, respect- The levels of circulating tPA were significantly elevated above ively . control in both severe and mild acute HCPS but not in symp- tomatic non-HCPS cases (Figure 1A). Furthermore, tPA was significantly increased in severe as compared with mild HCPS. Its main physiological inhibitor, PAI-1, was also found to be Received 5 December 2017; editorial decision 9 January 2018; accepted 16 January 2018. increased above control in severe HCPS. However, in contrast Correspondence: T. Strandin, PhD, Department of Virology, Faculty of Medicine, University of to tPA, PAI-1 was not elevated in the mild HCPS or non-HCPS Helsinki, Haartmaninkatu 3, FIN-00014 Helsinki, Finland (firstname.lastname@example.org). groups (Figure 1B). Thus, simultaneously elevated circulating Open Forum Infectious Diseases © The Author(s) 2018. Published by Oxford University Press on behalf of Infectious Diseases levels of tPA and PAI-1 distinguish severe HCPS from mild Society of America. This is an Open Access article distributed under the terms of the Creative HCPS and symptomatic non-HCPS cases. Strikingly, when Commons Attribution-NonCommercial-NoDerivs licence (http://creativecommons.org/licenses/ by-nc-nd/4.0/), which permits non-commercial reproduction and distribution of the work, in any taking into account all HCPS cases in our cohort, a robust cor- medium, provided the original work is not altered or transformed in any way, and that the work relation between fatality and PAI-1 (r = .514; P = .001), but not is properly cited. For commercial re-use, please contact email@example.com DOI: 10.1093/ofid/ofy021 tPA (r = .262; P = .1), was observed. BRIEF REPORT • OFID • 1 Downloaded from https://academic.oup.com/ofid/article-abstract/5/2/ofy021/4812611 by Ed 'DeepDyve' Gillespie user on 16 March 2018 A B ** ** ** 60 150 40 100 20 50 0 0 Severe Mild Non-HCPS Controls Severe Mild Non-HCPS Controls C D tPAPAI-1 ** 180 ** ** Mild Non-fatal FatalP DOBV UUV Controls Devere EF tPAPAI-1 ** * DOBV PUUV Controls Mild Severe DOBV-HFRS Figure 1. Levels of tissue plasminogen activator (tPA) and plasminogen activator inhibitor (PAI)–1 in acute hantavirus cardiopulmonary syndrome (HCPS) and hemorrhagic fever with renal syndrome (HFRS). Box plots of the levels of tPA (A) and PAI-1 (B) in acute (≤10 days after onset of disease) serum samples of severe (n = 18) and mild (n = 22) Andes (ANDV)-caused HCPS, non-HCPS cases (n = 5 for tPA and n = 12 for PAI-1) with similar symptoms to HCPS, and healthy volunteers (n = 10) as controls. (C) The levels of tPA and PAI-1 in early acute (≤5 days after onset of disease) serum samples of fatal (n = 7), severe but nonfatal (n = 4), and mild (n = 15) ANDV-caused HCPS. The levels of tPA (D) and PAI-1 (E) in acute (≤10 days after onset of disease) plasma samples of Dobrava (DOBV)- (n = 7) and Puumala hantavirus (PUUV)–caused (n = 16) HFRS in addition to healthy volunteers (n = 15) as controls. (F) The levels of tPA and PAI-1 in acute plasma samples of mild (n = 4) and severe (n = 3) DOBV-caused HFRS. Box plots illustrate the median value (center horizontal line), interquartile range (the lower and upper quartiles), and the highest and lowest values (whiskers) that are not outliers. Distributions ** * across groups were compared by the Mann-Whitney U test. Statistically highly significant difference at P < .01. Statistically significant difference at P < .05. To get further insight into the upregulation kinetics and and found either statistically significantly or close to signif- the role of tPA and PAI-1 in HCPS disease severity, we fur- icantly elevated levels of PAI-1 in fatal HCPS as compared ther classified severe HCPS to fatal and nonfatal cases at with mild (P = .001) or severe but nonfatal (P = .073) cases, early acute stage (≤5 days after onset of fever) (Figure 1C) respectively. tPA levels did not differ between groups. These 2 • OFID • BRIEF REPORT Downloaded from https://academic.oup.com/ofid/article-abstract/5/2/ofy021/4812611 by Ed 'DeepDyve' Gillespie user on 16 March 2018 ng/ml PAI-1 ng/ml tPA ng/ml PAI-1 ng/ml ng/ml tPA ng/ml findings suggest a direct role for PAI-1, but not tPA, in the are one of the major physiological sources of PAI-1 . This fatality of HCPS. ae ff cts baseline PAI-1 levels, as indicated by the increased con- The levels of tPA and PAI-1 were also compared in the centration of PAI-1 in the serum of healthy volunteers (com- plasma of acute DOBV- and PUUV-caused HFRS patients. pare Figure 1B and E). On the other hand, this effect is probably As seen in Figure 1D, the median levels of tPA were increased negligible in HCPS patient serum due to the oen s ft evere significantly above control in HFRS irrespective of the causa- thrombocytopenia associated with the disease , and thus the tive virus, but were further elevated by DOBV as compared difference between PAI-1 levels in HCPS patients and controls with PUUV. While PAI-1 was not elevated in PUUV-HFRS in circulation is most likely higher than estimated in this study. (Figure 1E), it was significantly increased above control in In fact, thrombocytopenia might explain the slightly lower DOBV-HFRS. This finding suggests a marked difference in level of PAI-1 in mild HCPS vs healthy controls (Figure 1B). regulation of PAI-1 based on the etiology and/or severity of Another potential caveat in our study is the fact that all se- HFRS. Interestingly, however, when categorizing DOBV-HFRS vere HCPS cases were, by definition, subjected to mechanical to mild vs severe forms of the disease based mainly on the ventilation, which has the potential to increase the circulating extent of kidney failure and need for hemodialysis, PAI-1 was levels of PAI-1 . However, while we are not aware whether significantly lower in severe DOBV-HFRS, whereas no change the analyzed samples were acquired before or aer m ft echanical between groups was observed for tPA. ventilation, we did not observe any changes in tPA or PAI-1 es Th e results indicate marked differences between the severe levels in non-HCPS cases receiving similar treatment as com- forms of HCPS and HFRS in the regulation of PAI-1. Further pared with HCPS cases (Figure 1A and B). Furthermore, by corroborating this difference, tPA was found to strongly correl- comparing fatal with nonfatal mechanically ventilated HCPS ate with PAI-1 in HCPS (r = .471; P = .002) but not in DOBV- cases (Figure 1C), we observed a difference in PAI-1 levels, HFRS (r = .402; P = .371). No correlation was observed between suggesting that ventilation per se is not a significant factor in tPA or PAI-1 with platelet counts in HCPS or patient age in the regulation of PAI-1 in HCPS. This is also in concordance HCPS or HFRS (data not shown). with another study indicating that PAI-1 levels were increased in fatal vs nonfatal SNV-HCPS cases receiving mechanical ven- DISCUSSION tilation and/or extracorporeal membrane oxygenation . All severe DOBV-HFRS cases received hemodialysis, which also The results of this study show for the first time that circu- potentially ae ff cts hemostasis . However, all DOBV samples lating levels of tPA are increased in acute HCPS and HFRS were acquired before hemodialysis, eliminating the influence of caused by ANDV and DOBV, respectively. In addition, higher this factor in the observed results. tPA levels were found in DOBV- vs PUUV-caused HFRS and Taken together, it is possible that the observed increase in in severe vs mild HCPS, indicating a possible role for tPA tPA/PAI-1 ratio accounts for elevated hemorrhagic compli- in hantavirus pathogenesis. This is in accordance with our cations and kidney failure in severe as compared with mild previous study, where tPA was found to positively correlate DOBV-HFRS. This is in line with our previous findings, where with hemorrhages in PUUV-HFRS . However, no signifi- tPA, in the concurrent absence of neutralizing PAI-1, correlated cant differences in tPA levels were observed between severe with hemorrhages in PUUV-HFRS . Analogously, the highly vs mild DOBV-HFRS or fatal vs nonfatal severe HCPS, indi- elevated levels of PAI-1 could also explain the absence of hem- cating that additional factors are necessary to fully explain orrhages in severe HCPS , which is in striking contrast to virulence in both diseases. severe HFRS cases . Minor hemorrhages were reported in In contrast to tPA, its main physiological inhibitor, PAI-1, 20% of our HCPS patients, but no correlations between bleed- was only increased in severe HCPS and generally in DOBV- ing and disease severity, tPA, or PAI-1 were found (data not HFRS. Furthermore, PAI-1 levels were nearly statistically sig- shown). Further studies on the role of tPA and PAI-1 in hanta- nificantly elevated in fatal vs nonfatal severe HCPS, indicating virus diseases are certainly warranted. a possible role for this protein in the mortality of HCPS. This latter finding is in accordance with a previous report indicat- Supplementary Data ing higher plasma PAI-1 levels in fatal SNV-caused HCPS as Supplementary materials are available at Open Forum Infectious Diseases compared with nonfatal cases . However, seemingly contra- online. Consisting of data provided by the authors to benefit the reader, dicting the possible role of PAI-1 in the pathogenesis of HFRS, the posted materials are not copyedited and are the sole responsibility of it was reduced in severe vs mild DOBV-HFRS (Figure 1F) and the authors, so questions or comments should be addressed to the corre- sponding author. not elevated at all in PUUV-HFRS (Figure 1E) . Due to differences in blood sampling (serum vs plasma), we Acknowledgments were not capable of reliably comparing the quantity of PAI-1 Financial support. This work is financially supported by the Academy between HCPS and HFRS cases. Platelets, which are activated of Finland (grant 1275597 to T.S.), Sigrid Juselius Foundation (to J.M. and during blood clotting in the preparation of serum samples, A.V.), and Magnus Ehrnrooth Foundation (to A.V.). BRIEF REPORT • OFID • 3 Downloaded from https://academic.oup.com/ofid/article-abstract/5/2/ofy021/4812611 by Ed 'DeepDyve' Gillespie user on 16 March 2018 7. Strandin T, Hepojoki J, Laine O, et al. Interferons induce STAT1-dependent Potential conifl cts of interest. All authors: no reported conflicts of expression of tissue plasminogen activator, a pathogenicity factor in Puumala interest. All authors have submitted the ICMJE Form for Disclosure of hantavirus disease. J Infect Dis 2016; 213:1632–41. Potential Conflicts of Interest. Conflicts that the editors consider relevant to 8. Braaten JV, Handt S, Jerome WG, et al. 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Am J Pathol 1995; 146:552–79. 4 • OFID • BRIEF REPORT Downloaded from https://academic.oup.com/ofid/article-abstract/5/2/ofy021/4812611 by Ed 'DeepDyve' Gillespie user on 16 March 2018
Open Forum Infectious Diseases – Oxford University Press
Published: Feb 1, 2018
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