Umeå universitets logga

Thrombocytopenia is a cardinal symptom of hantavirus-induced diseases including Puumala virus (PUUV)-induced hemorrhagic fever with renal syndrome (HFRS), which is associated with impaired platelet function, bleeding manifestations and augmented thrombotic risk. However, the underlying mechanisms causing thrombocytopenia and platelet hypo-responsiveness are unknown. Thus, we investigated the direct and indirect impact of PUUV on platelet production, function and degradation. Analysis of PUUV-HFRS patient blood revealed that platelet hypo-responsiveness in PUUV infection was cell-intrinsic and accompanied by reduced platelet-leukocyte aggregates (PLAs) and upregulation of monocyte tissue factor (TF), whereas platelet vasodilator-stimulated phosphoprotein (VASP) phosphorylation was comparable to healthy controls. Plasma CXCL4 levels followed platelet count dynamics throughout disease course. PUUV activated both neutrophils and monocytes in vitro, but platelet desialylation, degranulation and GPIIb/IIIa activation as well as PLA formation and endothelial adhesion under flow remained unaltered in the presence of PUUV. Further, MEG-01 megakaryocytes infected with PUUV displayed unaltered polyploidization, expression of surface receptors and platelet production. However, infection of endothelial cells with PUUV significantly increased platelet sequestration. Our data thus demonstrate that although platelet production, activation or degradation are not directly modulated, PUUV indirectly fosters thrombocytopenia by sequestration of platelets to infected endothelium. Upregulation of immunothrombotic processes in PUUV-HFRS may further contribute to platelet dysfunction and consumption. Given the pathophysiologic similarities of hantavirus infections, our findings thus provide important insights into the mechanisms underlying thrombocytopenia and highlight immune-mediated coagulopathy as potential therapeutic target.

Besides their primary role in haemostasis, platelets are actively involved in immune responses as they respond to various inflammatory stimuli, including microbial infection. Further, platelets contain intracellular IgG, but their physiologic function remains unknown. Thus, we aimed to elucidate the function of platelet-derived IgGs and their effect on viral infections. Human and murine platelets contained IgG which were released upon shear stress. However, IgG loss did not correlate with P-Selectin exposure or CXCL4 release and α-granule deficient (Nbeal2-/-) platelets failed to show reduced IgG content and release, indicating an extragranular IgG storage site within platelets. While platelet IgG could derive from megakaryocytes that have taken up IgG from the bone marrow microenvironment, naïve platelets also took up IgG directly from plasma in vitro and in vivo. Murine platelets from anti-IAV IgG seropositive mice reduced IAV infection in vitro and in vivo more efficiently than plasma containing comparable IgG levels. Further, human platelets from anti-CMV IgG seropositive but not seronegative donors also potently neutralized in vitro CMV-infection of HUVEC under microvascular shear stress. Our data indicate that IgG storage in platelets may not be restricted to α-granules. Further, our results show that platelets have the potential to mediate potent IgG-mediated antiviral effects both in vitro and in vivo directly at foci of infection. This indicates that platelet-derived IgG may represent a yet unexplored mechanism for focused serological immunity.

Background: Platelet activation and subsequent thrombus formation is a well‐defined process to maintain vascular integrity upon tissue damage. However, platelets are also activated by an array of inflammatory stimuli, including microbial infection. Further, circulating platelets contain intracellular IgG that are released upon activation.

Aim: We aimed to elucidate the physiologic function of platelet‐derived IgGs and their effect on viral infections.

Methods: IgG levels, subclass and light chain distributions were quantified by ELISA. For neutralization assays, CMV‐infected HUVECs were perfused with platelets or plasma of anti‐CMV IgG seropositive or seronegative donors before quantification of infection by IF or qPCR. IgG content of neonatal Fc‐receptor (FcRn)‐deficient or wild‐type murine megakaryocytes (MK) was measured by flow cytometry.

Results: Human platelets can store and release anti‐IAV and anti‐CMV IgG. Platelets from anti‐CMV IgG seropositive but not seronegative donors potently neutralized in vitro CMV‐infection under microvascular shear stress. In spite of containing approximately 100‐fold less IgG, platelets were equally efficient at neutralization as plasma from the same donor. Platelets were not enriched for a specific IgG subclass, nor for a specific kappa or lambda light chain. As MKs contain FcRn, sequestration of IgG might occur in the shared microenvironment of MKs and plasma cells. Indeed, MK FcRn was partially responsible for IgG uptake and may thus rescue IgG from degradation after endocytosis.

Conclusion: Our data show that platelets have the potential to mediate potent IgG‐mediated antiviral effects directly at foci of infection, indicating that platelet activation may represent a novel mechanism for focused serological immunity.