Influenza-induced thrombocytopenia is dependent on the subtype and sialoglycan receptor and increases with virus pathogenicity

Abstract

Thrombocytopenia is a common complication of influenza virus infection, and its severity predicts the clinical outcome of critically ill patients. The underlying cause(s) remain incompletely understood. In this study, in patients with an influenza A/H1N1 virus infection, viral load and platelet count correlated inversely during the acute infection phase. We confirmed this finding in a ferret model of influenza virus infection. In these animals, platelet count decreased with the degree of virus pathogenicity varying from 0% in animals infected with the influenza A/H3N2 virus, to 22% in those with the pandemic influenza A/H1N1 virus, up to 62% in animals with a highly pathogenic A/H5N1 virus infection. This thrombocytopenia is associated with virus-containing platelets that circulate in the blood. Uptake of influenza virus particles by platelets requires binding to sialoglycans and results in the removal of sialic acids by the virus neuraminidase, a trigger for hepatic clearance of platelets. We propose the clearance of influenza virus by platelets as a paradigm. These insights clarify the pathophysiology of influenza virus infection and show how severe respiratory infections, including COVID-19, may propagate thrombocytopenia and/or thromboembolic complications.

Graphical abstract

Figure 1.

Influenza-induced and subtype-dependent thrombocytopenia in humans and ferrets. (A) The blood platelet count and viral RNA load were inversely correlated in 2009 influenza A/H1N1 virus–infected patients (n = 34). Pearson’s r = −0.45; 95% CI, −0.68 to −0.14. (B) Experimental setup: ferrets inoculated with seasonal A/H3N2 (n = 24), pandemic A/H1N1 (n = 24), or A/H5N1 (n = 20) influenza virus with increasing disease severity in humans and ferrets. Arrows: the virus replication sites in the URT and LRT of both humans and ferrets with similar α2,3- and α2,6-sialoglycan receptor distributions. (C) An inverse correlation is shown between platelet count and viral loads (PCR) in throat swabs of A/H5N1 virus–infected ferrets (n = 20). Pearson’s r = −0.69; 95% CI, −0.88 to −0.33. (D) Platelet counts and viral loads (PCR) were inversely correlated in nasal swabs of A/H5N1 virus-infected ferrets (n = 20). Pearson’s r = −0.49; 95% CI, −0.78 to −.03. (E) There was no significant correlation in A/H3N2 (n = 24) and A/H1N1 (n = 24) virus-infected animals. (F) Platelet counts at about the peak of virus infection (days 2-4; n = 12). Error bars, SD; Student t test (unpaired). (G) Top: mean lung virus titer in ferrets (n = 4). Error bars, SD. Bottom: blood platelet count. Mean (n = 4) platelet count. Error bars, SD; Student t test (unpaired). (C-G) Dotted lines indicate the lower limit of normal platelet levels in the ferret (729 × 10⁹/L ± 360 × 10⁹/L). *P < .05; **P < .01; ***P < .001, ****P < .0001.

Figure 2.

Immunohistopathology and platelet electron microscopy of ferrets infected with different influenza viruses. (A-D) Disease severity scoring of ferrets infected with A/H3N2 (blue), A/H1N1 (green), and A/H5N1 (red) virus at postinfection day 4. Median (n = 4) histopathology score (range 0-3). Error bars, SD. (E-F) Median (n = 4) histopathology score (range, 0-1). (G-I) Virus titer in homogenized organs (heart, liver, and spleen) at days 0.5, 1, and 4. Mean (n = 4). Error bars, SD. Dotted line represents the lower limit of detection. Adapted from Van den Brand et al with permission. (J-K) Virus particle internalization (∼120 nm; red arrows) by platelets isolated from the blood drawn from a 2009 A/H1N1 virus-infected ferret. Red arrows: viruses. TEM original magnification ×20 000 (J); ×50 000 (K). The bars represent 250 nm. (L) Internalization of A/PR/8/34 virus by human platelets at 1, 5, and 20 minutes of incubation. The bars bar represent 250 nm.

Figure 3.

Influenza virus subtypes that bind to platelet sialoglycans. (A) Fixed, washed human platelets attached to a BLI sensor and visualized by light microscopy (original magnification ×20). Red arrow: single platelet. (B) Determination of mean virus particle count by NTA. Total peak values are presented (n = 5). A/H3N2 (5.4 × 10¹⁰ ± 3.6 × 10⁹ virus particles per milliliter); A/H1N1 (3.6 × 10¹⁰ ± 9.4 × 10⁸ virus particles per milliliter); and A/H5N1 (2.2 × 10¹⁰ ± 1.8 × 10⁹ virus particles per milliliter). (C) A highly concentrated A/PR/8/34 (PR8; 1 × 10¹² virus particles per milliliter) virus was used for a full titration on fixed platelets in the presence of OSC (100 µM). Mean (n = 2); error bars, SD. The K_D value for PR8 (9.5 ± 1.0 pM) was determined from a Langmuir model. The dotted line represents the plateau value (b = 17.89 nm). (D) Enlargement of the dotted box in panel C. Estimated plateau values (b) are 3.91 nm (95% CI, 3.41-4.53 nm) for A/H3N2, 10.6 nm (95% CI, 9.33-12.13 nm) for A/H1N1, and 12.7 nm (95% CI, 11.43-14.20 nm) for A/H5N1. (E) Virus binding to α2,6-sialoglycan density gradients ranging from 0 to 11.75 pmol/cm². Obtained threshold glycan densities are given above each curve. (F) The same as in panel E, but for the α2,3-sialoglycan density gradient. (G) Virus association and dissociation to BLI-sensors functionalized with α2,3-sialoglycans in the presence and absence of OSC (100 µM).

Figure 4.

Virus phagocytosis by platelets is dependent on the presence of SAs. (A) Virus uptake over time by human platelets. Original magnification ×25 000. Red arrows: internalized viruses. (B) Quantification of membrane-bound and internalized viruses in ultrathin-sliced platelet sections (n = 100; 50 nm) fixed after 1, 5, 10, 20, 30, and 60 minutes of incubation with influenza A/PR/8/34 virus. Error bars, standard error of the mean. (C) Temperature-dependent virus uptake. Mean (n = 5); error bars, SD. (D) Platelet desialylation with exogenous NA abolishes virus uptake. Control (+) and sialidase-treated (−) platelets. Mean (n = 3); error bars, SD. (E) Virus uptake is independent of DC-SIGN. Uptake in the absence (−) or presence (+) of αDC-SIGN receptor blocking antibody. Mean (n = 4); error bars, SD. ****P < .0001, n.s., nonsignificant, by Student t test.

Figure 5.

Platelet activation and aggregation upon binding of an influenza virus. (A) Virus NA-dependent desialylation of platelets from human platelets determined by lectin (ECL-FITC) binding. Mean (n = 6); error bars, SD; Tukey’s multiple-comparisons test, *P < .0001; OSC (100 µM). (B) Virus activation of platelets in human plasma. Positive control (ADP; 10 µM). Final virus concentration 50 pM. OSC 100 µM. Mean (6 donors); error bars, SD; Tukey’s multiple-comparisons test, *P < .05; **P < .01. (C) Dose-dependent activation of platelets in plasma by influenza A/PR/8/34 virus. Error bars, SD; Tukey’s multiple-comparisons test, ****P < .0001. (D) Aggregation of platelets by influenza virus subtypes in 3 donors. Positive control (ADP; 10 μM). Final virus concentration (50 pM).