Introduction

Platelets are abundant, yet short-lived, blood cells whose number is second only to red blood cells within circulation. Platelet function is multifaceted, comprising important physiological processes such as hemostasis, vascular integrity, and immunity [1,2,3,4,5,6]. The lifespan of human platelets is 8–10 days, while in mice it is only 4–5 days. The number of platelets in the blood is controlled through the balance of their rate of production in the bone marrow and lung with their clearance in peripheral compartments that can include the spleen and liver. To maintain a stable platelet count, approximately 1011 human platelets are produced and removed daily from circulation while the dysregulation of this homeostatic process can contribute to multiple pathological conditions such as sepsis and immune thrombocytopenia [4, 7,8,9,33], with modifications. Briefly, mouse blood was collected from the retrobulbar venous plexus via a glass capillary and added to a tube containing 3.2% (w/v) sodium citrate. Blood was then diluted 1:1 with Tyrode’s buffer (129 mM NaCl, 2.8 mM KCl, 0.8 mM MgCl2, 0.8 mM KH2PO4, 8.9 mM NaHCO3, 10 mM Hepes, 5.6 mM glucose, pH 7.4) and centrifuged at 50 g for 10 min at room temperature. The supernatant was transferred into a new tube and centrifuged at 180 g for 10 min to obtain the platelet pellet, followed by two washes with Tyrode’s buffer containing 0.5 μM prostacyclin (Sigma).

Flow cytometry

Flow cytometry was performed following our published methods [9, 32, 33]. For lectin binding analysis, platelet staining was performed in Tyrode’s buffer with 2 μg/ml biotinylated Maackia Amurensis lectin II (MAL II, detecting α2,3 sialic acid, #b-1265, Vector Laboratories) and 2 μg/ml fluorescein-labeled Ricinus Communis agglutinin I (RCA I-FITC, recognizing desialylated galactose, #FL-1081, Vector Laboratories) for 20 min at room temperature. For biotinylated lectin, platelets were then stained with 2 μg/ml PE-streptavidin (#405203, BioLegend) for 20 min at room temperature, followed by a wash with Tyrode’s buffer. Samples incubated with PE-streptavidin only were used as negative controls. For P-selectin analysis, washed platelets stimulated with thrombin (0.3 U/ml for 5 min, Sigma) were used as positive controls. To examine platelet-neutrophil interactions, 20 μl mouse blood was collected from facial vein and added to a tube containing 3.2% sodium citrate. The cells were subsequently stained with CD41-Percp-cy5.5 and Ly6G-PE. To analyze EGFP expression on megakaryocytes and platelets from ROSAmTmG;Pf4Cre mice, peripheral blood was stained with CD41-Percp-Cy5.5 for platelet analysis. For megakaryocytes, bone marrow cells were harvested from femurs of ROSAmTmG;Pf4Cre mice and then stained with CD41-Percp-Cy5.5 and then with Hoechst33342. All samples were analyzed using a FACSCelesta (BD Bioscience).

Quantitative reverse transcription PCR (qRT-PCR)

Total RNA was extracted from WT, Clec4f+/-, Clec4f−/− liver tissues using Trizol (Sigma). RNA concentration was measured via Nanodrop (Thermo Fisher Scientific). Complementary DNA (cDNA) was synthesized using the M-MLV reverse transcriptase (Qiagen). Expression of Clec4f transcripts was analyzed using a forward primer: 5′-TCCACCTGCTTTCAGCCTTCA-3′ and a reverse primer: 5′-AGAAGACTGCCATCTGGGTCTC-3′, using a CFX96 instrument (Bio-Rad) with Fast SYBR Green Master Mix (Fisher Scientific).

Western blot

Freshly isolated mouse livers were lysed with a lysis buffer containing 50 mM Tris pH 7.4, 150 mM NaCl, 1% Triton x-100, 1% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, 0.1% SDS with protease inhibitors (1:100; Thermo Fisher Scientific). Protein samples were separated by SDS-polyacrylamide gel and transferred to a PVDF membrane. The membrane was incubated with primary goat anti-mouse CLEC4F (R&D Systems) followed by HRP-conjugated donkey anti-goat IgG (Abcam).

Platelet desialylation, count, and survival

For in vitro desialylation of platelets, washed platelets were obtained as described above. 2 × 108 platelets in Tyrode’s buffer were treated with the Arthrobacter ureafaciens-, or Clostridium perfringens-derived neuraminidase (25 mU/ml) at 37 °C for 20 min. Desialylation was confirmed by binding to RCA I-FITC and biotinylated MAL II. Platelets were labeled with 0.5 μM of CellTracker Deep Red for 20 min at 37 °C in the dark, followed by two washes with Tyrode’s buffer. Labeled platelets were then transfused into WT or Clec4f−/− recipient mice through intravenous injections.

For in vivo desialylation, and the labeling of desialylated platelets, recipient mice were intravenously injected with neuraminidase (50 mU/mouse). Before and after neuraminidase injection, 40 μl blood was collected from the cheek vein at various time points. To determine platelet count and platelet galactose exposure in the circulation, 10 μl whole blood was diluted 1:20 in FACS buffer, then incubated with PE anti-mouse CD41 (2 μg/ml) and RCA I-FITC (2 μg/ml) for 20 min at room temperature in the dark. The percentage of platelets was determined via flow cytometry with PE anti-mouse CD41 at a low speed for 20 s. Percentage of platelet RCA I binding was defined as PE anti-mouse CD41 and RCA I-FITC positive cells per 10,000 platelets analyzed. After 120 min, recipients were sacrificed and intravascularly perfused. The liver, spleen, and other organs were then collected for imaging analysis.

Histology, immunostaining, and microscopy

For immunofluorescent staining, tissues were fixed in 4% paraformaldehyde (PFA) at 4 °C overnight, washed in PBS, soaked in 20% sucrose at 4 °C overnight, embedded in 50% tissue freezing medium/50% OCT. Thick cryosections (20–50 μm) were used for immunostaining. In some experiments, tissues were fixed in 10% formalin for 24 h, washed in PBS, embedded in paraffin and cut into 5 μm thick sections. For immunostaining, the paraffin sections were deparaffinized, hydrated, and treated with an antigen unmasking solution (Vector) for 20 min. Sections were first blocked with 3% BSA, 3% goat serum, 3% donkey serum, and 0.3% Triton x-100 in PBS, and then stained with primary antibodies at 4 °C overnight, followed by staining with fluorescently conjugated secondary antibodies for 1 h. Antibodies used include goat anti-mouse CLEC4F (#AF2784, R&D Systems), rat anti-mouse CLEC4F (#MAB2784, R&D Systems), rat anti-mouse F4/80 (#123102, BioLegend), rabbit anti-mouse ASGR1 (# PA5-32030, Invitrogen), Alexa Fluor 488 conjugated rat anti-mouse F4/80 (#53-4801-80, Invitrogen), PE conjugated rat anti-mouse CD41 (#133906, BioLegend), and DyLight 488, Alexa Fluor 555, or DyLight 649 conjugated secondary antibodies (Jackson ImmunoResearch). Finally, sections were mounted with FluoromountTM aqueous mounting medium with DAPI (Sigma). A confocal microscope (Zeiss 710 Microscope System) was used for imaging. Volume images from the confocal image datasets were further processed with IMARIS software (Bitplane AG), and some images were presented as maximum intensity projections of the z-stacks or for three-dimensional views. The total number of bound platelets in the liver was counted using 10× or 20× magnification confocal images in a blinded manner. To determine whether platelets were associated on the surface or internalized inside of the Kupffer cell, 40× 3D z-stacks images were reconstructed and rotated to view the platelet location through various angles, and with orthogonal projection images of z-stacks.

Desialylation, Kupffer cell isolation, and immunofluorescent staining

For desialylation of platelets in vivo, mice were injected with neuraminidase (50 mU/mouse) as described above. For specific inhibition of the macrophage galactose lectin (MGL) receptor, mice were intravenously injected with anti-MGL1/2 antibody (2 μg/g body weight, #AF4297, R&D Systems) 20 min prior to neuraminidase treatment. In some experiments, 2 × 108 platelets from ROSAmTmG;Pf4Cre mice were transfused to either WT or Clec4f−/− recipient mice, followed immediately by neuraminidase injection (50 mU/mouse). After 120 min, recipient mice were sacrificed and immediately perfused by in situ liver perfusion with 15 ml Hank’s balanced salt solution (HBSS, 1.7 ml/min, 37 °C) through the inferior vena cava, and then by 10 ml 0.1% (w/v) type IV collagenase (Worthington Biochemical Corp.) buffered in HBSS (1.7 ml/min, 37 °C). The liver was subsequently collected and transferred to a petri dish containing 10 ml cold RPMI 1640 (Hyclone) buffer. Cell suspension was prepared by gentle pipetting and then filtered through a 100 μm cell strainer on ice. Non-parenchymal cells were further separated by centrifugation for 5 min at 50 g at room temperature. The cells were then seeded onto 0.2% gelatin (Sigma) coated cover slips in RPMI 1640 supplemented with 10% fetal bovine serum (Hyclone) and 100 U/ml penicillin/streptomycin (Sigma) in a 24-well plate, and incubated for 2 h in a 5% CO2 atmosphere at 37 °C. Nonadherent cells were removed by gently washing with HBSS and the adherent cells were fixed with 2% PFA for 20 min at room temperature. After blocking with 1% BSA in PBS-T, the cells were incubated with primary antibodies at 4 °C overnight and then with fluorescently-conjugated secondary antibodies for 1 h. DAPI was used for counterstaining. Immunofluorescent staining was performed and the cells were analyzed using the Zeiss 710 Microscope System described above.

Kupffer cell depletion

Mice were injected intravenously with clodronate 200 μl (#F70101C-A, FormuMax) per 20–25 g body weight 2 days before being treated with neuraminidase (50 mU/mouse). The mice were then sacrificed and perfused with PBS and 4% PFA 2 h after sialidase administration. The liver was collected for imaging analysis of Kupffer cells and platelets.

Transmission electron microscopy (TEM)

Mouse livers were fixed by intravascular perfusion with a mixture of 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M HCl–sodium cacodylate buffer, pH 7.2, followed up by immersion in the same fixative for 1 h. The remaining procedures followed our published methods [32, 33]. Ultrathin sections were stained with uranyl acetate and lead citrate, before being examined with a Hitachi H-7600 electron microscope equipped with a 4 megapixel digital monochrome camera and AMT-EM image acquisition software (Advanced Microscopy Techniques).

Statistical analysis

All experiments were performed at least three times. Data are presented as mean ± SD or mean ± SEM. For parametric data, unpaired Student’s t test or ANOVA was used. For non-parametric data, the Mann–Whitney U Test was used. P < 0.05 was defined as statistical significance.