Outi Laine

Enhanced thrombin formation and fibrinolysis during acute Puumala hantavirus infection

INTRODUCTION Nephropathia epidemica (NE) is a viral hemorrhagic fever with renal syndrome associated with thrombocytopenia and mild bleeding. We assessed activation of coagulation and fibrinolysis during the acute phase of NE. MATERIALS AND METHODS 19 hospital-treated patients were involved. Plasma levels of D-dimer, prothrombin fragments 1+2 (F1+2), activated partial thromboplastin time (APTT), prothrombin time (PT%), thrombin time (TT), fibrinogen, antithrombin (AT), protein S free antigen (PS), protein C (PC) and complete blood count (CBC) were measured three times during the acute phase and once at 32-54 days after the onset of fever (recovery phase). Laboratory abnormalities were evaluated by the disseminated intravascular coagulation (DIC) scoring advocated by the International Society of Thrombosis and Haemostasis (ISTH). RESULTS APTT was prolonged and D-dimer and F1+2 increased during the acute phase of NE. AT, PC and PS decreased, and TT was shortened, all implying increased thrombin generation. Acutely F1+2 was 3.4-fold and D-dimer even 24-fold higher compared with the recovery phase (median 726 vs 213 pmol/l, and median 4.8 vs 0.2mg/l, respectively, p<0.001 for both). Platelet count correlated with AT, PC, and PS (r=0.73, r=0.81, and r=0.71, respectively, p<0.001 for all) as well as with fibrinogen (r=0.72, p<0.001). Only five patients fulfilled the ISTH diagnosis of DIC. CONCLUSIONS During acute NE thrombocytopenia was associated with decreased natural anticoagulants, shortened thrombin time and enhanced fibrinolysis. Augmented thrombin formation and fibrinolysis characterize this hantavirus infection.

The pathogenesis of nephropathia epidemica: new knowledge and unanswered questions.

Puumala virus (PUUV) causes an acute hemorrhagic fever with renal syndrome (HFRS), a zoonosis also called nephropathia epidemica (NE). The reservoir host of PUUV is the bank vole (Myodes glareolus). Herein we review the main clinical manifestations of NE, acute kidney injury, increased vascular permeability, coagulation abnormalities as well as pulmonary, cardiac, central nervous system and ocular manifestations of the disease. Several biomarkers of disease severity have recently been discovered: interleukin-6, pentraxin-3, C-reactive protein, indoleamine 2,3-dioxygenase, cell-free DNA, soluble urokinase-type plasminogen activator, GATA-3 and Mac-2 binding protein. The role of cytokines, vascular endothelial growth hormone, complement, bradykinin, cellular immune response and other mechanisms in the pathogenesis of NE as well as host genetic factors will be discussed. Finally therapeutic aspects and directions for further research will be handled.

Complement activation in Puumala hantavirus infection correlates with disease severity

Abstract Introduction. Hantaviruses are important human pathogens that cause clinical diseases characterized by renal and cardiopulmonary manifestations. Their pathogenesis is currently poorly understood. We have studied the role of the complement system in the pathogenesis of Puumala (PUUV) hantavirus infection. Material and methods. We studied the activation of complement by measuring the terminal complement complex SC5b-9 and complement component C3 and C4 levels in patients with acute PUUV infection. Several laboratory parameters and clinical findings reflecting the severity of PUUV-HFRS were evaluated with regard to complement activation. Results. The levels of SC5b-9 were significantly increased and C3 decreased in the acute stage as compared to the levels at full recovery (P < 0.001). We found that SC5b-9 levels were higher in patients with chest X-ray abnormalities than in patients with a normal X-ray during the acute stage (P = 0.028). Furthermore, SC5b-9 and C3 levels showed significant correlation with several clinical and laboratory parameters that reflect the severity of the acute PUUV infection. Conclusions. We showed that the complement system becomes activated via the alternative pathway in the acute stage of PUUV infection and the level of activation correlates with disease severity. The results further suggest that complement activation may contribute to the pathogenesis of acute PUUV infection.

Platelet ligands and Adamts13 during puumala hantavirus infection and associated thrombocytopenia

We aimed here to elucidate the role of adhesive platelet ligands and endothelial involvement during the acute phase of Puumala hantavirus (PUUV) infection. Nineteen hospital-treated patients with serologically confirmed diagnosis of acute PUUV infection were included. Patient charts were reviewed for clinical and basic laboratory data. Plasma levels of von Willebrand factor antigen (VWF : Ag), ristocetin cofactor (VWF : RCo), factor VIII (FVIII : C) and a disintegrin and metalloproteinase with a thrombospondin type 1 domain 13 (ADAMTS13) activities as well as fibrinogen and fibronectin were measured three times acutely and once during the recovery phase. VWF : Ag and VWF : RCo were nearly three-fold higher acutely compared with recovery (median 252 vs. 88%, and mean 267 vs. 98%, respectively; P < 0.001 for both), whereas FVIII : C was only slightly elevated (median 118 vs. 88%, P = 0.002) and remarkably failed to show association with VWF in the acute phase. ADAMTS13 activity and fibronectin concentration were lower in the acute compared with the recovery phase (median 56 vs. 63%, P = 0.003, and median 221 vs. 330 &mgr;mol/l, P = 0.001, respectively). Fibrinogen raised acutely (mean 5.0 vs. 3.3 g/l, P < 0.001), negatively correlating with the platelet count (r = −0.468, P = 0.043). Markedly upregulated fibrinogen and VWF together with decreased levels of ADAMTS13 activity and fibronectin were observed during acute PUUV infection. VWF and FVIII : C did not associate during the acute phase, whereas thrombocytopenia correlated negatively with fibrinogen. These findings imply several rearranged interactions between platelets and their ligands.

Polymorphisms of PAI-1 and platelet GP Ia may associate with impairment of renal function and thrombocytopenia in Puumala hantavirus infection

INTRODUCTION Puumala virus (PUUV) infection is a viral hemorrhagic fever with renal syndrome (HFRS) characterized by thrombocytopenia and acute impairment of renal function. We aimed to assess whether genetic polymorphisms of platelet antigens together with those of von Willebrand factor (VWF) and plasminogen activator inhibitor (PAI-1) correlate with disease severity. Patients and methods 172 consecutive hospital-treated patients with serologically confirmed acute PUUV infection were included. Platelet glycoprotein (GP) IIIa T>C (rs5918), GP Ia T>C (rs1126643), GP Ib C>T (rs6065), GP VI T>C (rs1613662), VWF A>G (rs1063856) and PAI-1 A>G (rs2227631) were genotyped. The associations of the rarer alleles with variables reflecting the severity of the disease were analyzed. RESULTS PAI-1G-carriers had higher maximum creatinine level compared with the non-carriers (median 213 μmol/l, range 60-1499 μmol/l vs. median 122 μmol/l, range 51-1156 μmol/l, p = 0.01). The GG-genotypes had higher creatinine levels than GA- and AA-genotypes (medians 249 μmol/l, 204 μmol/l and 122 μmol/l, respectively, p = 0.03). Polymorphisms of GP VI and VWF associated with lower creatinine levels during PUUV infection. The minor C-allele of GP Ia associated with lower platelet counts (median 44 × 10(9)/l, range 20-90 × 10(9)/l vs median 64 × 10(9)/l, range 3-238 × 10(9)/l; p = 0.02). CONCLUSIONS Polymorphism of PAI-1, a major regulator of fibrinolysis, has an adverse impact on the outcome of kidney function in PUUV-HFRS. Platelet collagen receptor GP Ia polymorphism associates with lower platelet count.

Headache and low platelets in a patient with acute leukemia

Department of Internal Medicine, Tampere University Hospital, Tampere, Finland Department of Virology, Haartman Institute, University of Helsinki, Helsinki, Finland Department of Virology, Helsinki University Hospital Laboratory (HUSLAB), Helsinki, Finland Department of Basic Veterinary Sciences, Faculty of Veterinary Medicine, University of Helsinki, Helsinki, Finland Finnish Red Cross Blood Service, Helsinki, Finland

Interferons Induce STAT1–Dependent Expression of Tissue Plasminogen Activator, a Pathogenicity Factor in Puumala Hantavirus Disease

Hantaviruses are zoonotic viruses that show various degrees of vasculopathy in humans. In this study, we analyzed the regulation of 2 fibrinolytic parameters, tissue plasminogen activator (tPA) and its physiological inhibitor, plasminogen activator inhibitor 1 (PAI-1), in Puumala hantavirus (PUUV)-infected patients and in human microvascular endothelial cells. We detected strong upregulation of tPA in the acute phase of illness and in PUUV-infected macaques and found the tPA level to positively correlate with disease severity. The median levels of PAI-1 during the acute stage did not differ from those during the recovery phase. In concordance, hantaviruses induced tPA but not PAI-1 in microvascular endothelial cells, and the induction was demonstrated to be dependent on type I interferon. Importantly, type I and II interferons directly upregulated tPA through signal transducer and activator of transcription 1 (STAT1), which regulated tPA gene expression via a STAT1-responsive enhancer element. These results suggest that tPA may be a general factor in the immunological response to viruses.

Severe Puumala virus infection in a patient with a lymphoproliferative disease treated with icatibant

Abstract Early identification of patients at risk of a severe course of hantaviral disease and lack of effective medication represent a global challenge in the treatment of this emerging infection. We describe a 67-year-old female patient with a history of chronic lymphoproliferative disease involving the spleen and an extremely severe acute Puumala hantavirus infection. She was treated with the bradykinin receptor antagonist icatibant and recovered. She is the second patient with a spleen abnormality and severe Puumala infection treated with icatibant in our hospital. We suggest that patients with spleen abnormalities may be more susceptible to severe hantavirus disease. The activation of the kinin-kallikrein system and the formation of bradykinin in hantavirus-infected endothelial cells indicate that the role of bradykinin receptor antagonist icatibant in the treatment of hantavirus disease is worth studying.

Hantavirus infection-induced thrombocytopenia triggers increased production but associates with impaired aggregation of platelets except for collagen

INTRODUCTION We evaluated the mechanisms of thrombocytopenia encountered in hantavirus disease by studying platelet production together with platelet aggregation and deposition to collagen surface. PATIENTS AND METHODS The study group consisted of 31 prospectively recruited, consecutive, hospitalized patients having acute Puumala hantavirus infection. Blood samples were collected acutely and at the control visit and subjected to analysis in Sysmex® XE-5000 to capture mean platelet volume (MPV) and immature platelet fraction (IPF%). Platelet aggregation under low shear rate conditions was assessed with impedance aggregometry Multiplate®, whereas platelet function analyzer (PFA)-100® was applied under blood flow of high shear forces. RESULTS IPF% was 3.1-fold higher acutely compared with the control (median 7.4%, range 2.0-23.8% vs. median 2.4%, range 1.4%-5.2%, p<0.001) tightly associating with the low platelet count (r=-0.76, p<0.001). Accordingly, acute MPV was high (median 11.4f l, range 9.4-13.1 fl vs. median 10.5 fl, range 9.0-12.0 fl, p=0.003). Acute platelet aggregation in Multiplate® was decreased to all agonists compared with the later control (p<0.05 for all agonists). Aggregation capacity associated with thrombocytopenia (for all agonists r ≥ 0.81, p<0.001), but impaired aggregation occurred also among patients with a nearly normal platelet count. Triggered by collagen, 20% of values were below reference range, while 73% of responses were low with thrombin receptor activating peptide. Significantly, under high shear platelet deposition to collagen surface was normal despite thrombocytopenia. CONCLUSIONS During acute hantavirus disease, platelet aggregation is impaired especially when induced with thrombin. Platelet adhesive mechanisms on collagen are intact despite thrombocytopenia while thrombopoiesis is active.

Spleen enlargement is a common finding in acute Puumala hantavirus infection and it does not associate with thrombocytopenia.

Abstract The pathogenesis of thrombocytopenia in Puumala hantavirus (PUUV) infection is probably multifactorial. We aimed to evaluate the possible spleen enlargement during acute PUUV infection, and to determine its association with thrombocytopenia and disease severity. Magnetic resonance imaging (MRI) of the spleen was performed in 20 patients with acute PUUV infection. MRI was repeated 5–8 months later. The change in spleen length was compared with markers describing the severity of the disease. In all patients, the spleen length was increased in the acute phase compared with the control phase (median 129 mm vs 111 mm, p < 0.001). The change correlated with maximum C-reactive protein value (r = 0.513, p = 0.021) and inversely with maximum leukocyte count (r = –0.471, p = 0.036), but not with maximum serum creatinine level or minimum platelet count. Enlarged spleen, evaluated by MRI, was shown to be a common finding during acute PUUV infection. However, it does not associate with thrombocytopenia and acute kidney injury.