C. albicans strain SC5314 (from the China Medical Fungus Culture Collection Center) was grown for 12 h at 30°C in yeast extract-peptone-dextrose (YPD) broth. C. albicans was then collected by centrifugation at 2000×g for 5 min. Following centrifugation, C. albicans was resuspended in 30 mL of phosphate-buffered saline (PBS) and washed twice. Yeast cells were enumerated using a hemocytometer and then diluted with PBS to the desired working concentrations. Before C. albicans stimulated platelets, C. albicans was washed with PBS to remove their supernatant. In some experiments, after being washed and diluted with PBS, C. albicans was heat killed immediately in an 85°C water bath for 10 min. Except when otherwise specified, live microorganisms were used in the experiments. FUN-1 (Thermo Fisher, Waltham, MA, USA, F7030) staining of C. albicans was used for flow cytometry. The staining solution was added at a dilution of 1:1000 (v/v) to C. albicans and incubated for 30 min, followed by washing with PBS and then diluting to the desired working concentrations.

Platelet-rich plasma (PRP) was prepared as concentrates by thrombocytapheresis with an Amicus cell separator (Baxter) by the Department of Blood Transfusion (Jiangsu Province Blood Center). Platelet counts were performed with a Sysmex poch-80i hematologic analyzer (Sysmex Corporation). For washed platelet suspensions, PRP was washed twice with CGS buffer (0.123 M NaCl, 0.033 M D-glucose, 0.013 M trisodium citrate, pH 6.5). Platelets were collected by centrifugation at 1100×g for 5 min. Then platelets were resuspended in Medium 199 (Gibco, NY, USA, 12340030) and kept at room temperature until the experiment was performed. Except when otherwise specified, PRP was used in the experiments. To obtain platelet-poor plasma (PPP), PRP was centrifuged at 1500×g for 5 min to remove platelets.

PRP was incubated with heat-killed (HK) C. albicans spores for 6 h (1 h, 3 h, or 6 h in the western blotting experiments) or live C. albicans spores for 2 h at a multiplicity of infection (MOI) of 0.01 at 37°C. PRP incubated with equal volume of PBS was used as control. For the signaling pathway inhibition experiments, platelets were pretreated with the PI3K inhibitor LY294002 (Selleckchem, Houston, TX, USA, S1105, 90 μM) and Akt inhibitor MK2206 (Selleckchem, S1078, 30 μM) or vehicle control at 37°C for 2 h and then were incubated with live C. albicans at 37°C for 2 h. For the receptor inhibition experiments, platelets were costimulated with C. albicans after pretreatment with the GP IIb/IIIa-specific antagonist tirofiban (Selleckchem, S8594, 100 or 200 μg/mL), anti-TLR2 neutralizing antibody (In vivoGen, San Diego, CA, USA, maba2-htlr2, 1, 5, or 20 μg/mL), anti-TLR4 neutralizing antibody (In vivoGen, mabg-htlr4, 1, 5, or 20 μg/mL) or vehicle control (In vivoGen, maba2-ctrl, mabg1-ctrlm, 1, 5, or 20 μg/mL, respectively) at 37°C for 45 min. For the GP IIb/IIIa receptor blocking experiments, 2 × 10⁷ washed platelets were pretreated with the anti-GP IIb/IIIa antibody (BioLegend, San Diego, CA, USA, 359804, clone A2A9/6) at 12.5 μg/mL or vehicle control (BioLegend, 401507) at 37°C for 30 min and then incubated with live C. albicans at 37°C for 2 h. For the agonist experiments, platelets were stimulated with different concentrations of the TLR2 agonist Pam3CSK4 (In vivoGen, tlrl-pms, 2, 10, or 25 μg/mL), FSL-1 (In vivoGen, tlrl-fsl, 0.1, 0.5, or 1 μg/mL), and the TLR4 agonist LPS (Sigma-Aldrich, St. Louis, MO, USA, L2880, 1, 5, or 25 μg/mL) at 37°C for 6 h.

Human monocytic leukemia cell line, THP-1, was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA, TIB-202). Peripheral blood mononuclear cells (PBMCs) were isolated from 3 healthy volunteers (3 biological replicates) by Lymphoprep (STEMCELL Technologies, Vancouver, BC, Canada). To isolate primary human monocytes, magnetic activated cell sorting with paramagnetic CD14-beads (Miltenyi Biotec, Paris, France) was used to separate CD14⁺ monocytes according to the manufacturer’s instructions. THP-1 cells and primary human monocytes were cultured in complete medium [RPMI 1640 (Gibco) containing 10% fetal bovine serum (Gibco), 100 U/mL penicillin (Gibco), and 100 mg/mL streptomycin (Gibco)] in a humidified incubator with 5% CO₂ at 37°C. For stimulation experiments, THP-1 cells and primary human monocytes were seeded in 6-well plates at a density of 1 × 10⁶ cells/well and then cocultured with C. albicans (3:1, fungi to monocytes) in the presence of PRP (50:1, platelets to monocytes) or an equal volume of PPP at 37°C for 2 h. For inhibitor experiments, PRP was pretreated with tirofiban (200 μg/mL) or the antiplatelet agent cilostazol (Selleckchem, S1294, 200 μM) for 45 min before costimulation with THP-1 cells and C. albicans.

To analyze platelet activation, cultured platelets were centrifuged at 1200×g for 5 min at 4°C and fixed with 1% paraformaldehyde on ice for 30 min. Then, platelets were washed with PBS once and diluted to 10⁶/100 μL. Platelets were stained with 1:200-diluted CD41a-phycoerythrin (PE)-Cy7 (Thermo Fisher, 25-0419-42, clone HIP8) and 1:150-diluted P-selectin-APC (Thermo Fisher, 17-0626-82, clone Psel.K02.3) or 1:150-diluted immunoglobulin isotype control of P-selectin-APC (Thermo Fisher, 17-4714-82, clone P3.6.2.8.1), followed by incubation for 30 min at room temperature. After washing, the samples were assessed immediately on a flow cytometer (BD FACSVers). The expression of GP IIb/IIIa (also known as CD41/CD61) on platelets was identified using CD41a-PE-Cy7. To assess the phagocytosis of C. albicans by THP-1 cells or primary human monocytes, cultured cells were collected into flow tubes and centrifuged at 300×g for 5 min, followed by washing with ice-cold PBS to clear nonphagocytosed C. albicans and platelets. Then, samples were assessed on a flow cytometer. Untreated THP-1 cells or primary human monocytes were used as a negative control, and pure FUN-1-C. albicans was used as a positive control, with gating according to forward and side scatter. Phagocytosis of C. albicans-FUN-1 by THP-1 cells or primary human monocytes was assessed based on the fluorescence of the FITC channel.

PRP from a single donor was paired to be treated with either C. albicans or PBS. After costimulation of platelets and C. albicans, platelets were centrifuged at 1500×g for 5 min at 4°C. Cell supernatant was collected and stored immediately at -80°C until cytokines were measured. The levels of PF4 (RayBiotech, Norcross, GA, USA), CCL5 (R&D Systems, Minneapolis, MN, USA), β-defensin-2 (Arigo Biolaboratories, Hsinchu, China), tumor necrosis factor (TNF) -α (R&D Systems), interleukin (IL) -1β (R&D Systems), and CXCL8 (R&D Systems) were measured using commercial ELISA kits according to the manufacturer’s instructions.

Cultured platelets were washed twice with ice-cold PBS and then lysed with RIPA buffer (CWBIO, Beijing, China) containing protease and phosphatase inhibitors (Roche, Mannheim, Germany) on ice for 20 min. After centrifugation (12,000×g, 4°C for 5 min), the supernatant was collected. The protein concentration was evaluated using a Pierce BCA Protein Assay Kit (Thermo Fisher, 23227). Then, SDS (Generay Biotech, Shanghai, China) and RIPA buffer were added to the solution, followed by boiling for 5 min. Antibodies against p-Stat3 (1:1000, Cell Signaling Technology, Danvers, MA, USA, 9145), Stat3 (1:1000, Cell Signaling Technology, 4904), p-AKT (1:1000, Cell Signaling Technology, 4060), AKT (1:1000, Cell Signaling Technology, 4691), p-ERK1/2 (1:1000, Cell Signaling Technology, 4377), ERK1/2 (1:1000, Cell Signaling Technology, 4695), p-p38 (1:1000, Cell Signaling Technology, 4511), p38 (1:1000, Cell Signaling Technology, 9212), and GAPDH (1:1000, Cell Signaling Technology, 5174) were used for immunoblot analysis according to the manufacturers’ protocols. Proteins were separated by 10% SDS-polyacrylamide gels (Beyotime, Shanghai, China) and visualized by the ECL system. After detection of phosphorylated proteins, the membrane was stripped (Thermo Fisher, 46430) for 15 min on a rotator before blotting for total protein. Quantification was performed with ImageJ software.

Total RNA was extracted from cultured cells using RNAiso Plus reagent (Takara, Tokyo, Japan). For each well, approximately 8 µg of RNA was yielded. Complementary DNA was synthesized using 500 ng with Prime Script RT Master Mix (Takara). Quantitative PCR (qPCR) was carried out with iTaq Universal SYBR Green Supermix (Bio-Rad, Hercules, CA, USA) and 7300 System SDS Software (version 1.4.0, Applied Biosystems). The relative expression of target genes was calculated through the 2-ΔΔCT method relative to the reference gene (GAPDH), and then, the fold change relative to the reference sample was calculated. The following primers were used:

GAPDH (human) forward: 5′-GTCTCCTCTGACTTCAACAGCG-3′, reverse: 5′-ACCACCCTGTTGCTGTAGCCAA-3′; IL-1β (human) forward: 5′-CCACAGACCTTCCAGGAGAATG-3′, reverse: 5′-GTGCAGTTCAGTGATCGTACAGG-3′; IL-6 (human) forward: 5′-TTCGGCAAATGTAGCATG-3′, reverse: 5′-AATAGTGTCCTAACGCTCATAC-3′; TNF-α (human) forward: 5′-CTCTTCTGCCTGCTGCACTTTG-3′, reverse: 5′-ATGGGCTACAGGCTTGTCACTC-3′; and CXCL8 (human) forward: 5′-TTTTGCCAAGGAGTGCTAAAG-3′, reverse: 5′-AACCCTCTGCACCCAGTTTTC-3′.

The statistical tests used are specified in the figure legends. Numeric data were analyzed using one-way analysis of variance (ANOVA). Two groups were compared by a two-tailed Student’s t test. The significance of the data was assessed using GraphPad Prism version 8.0 (GraphPad Software, San Diego, CA, USA). A P-value less than 0.05 was considered statistically significant.

To investigate whether C. albicans could induce platelet activation, we incubated live C. albicans and HK C. albicans with human platelets and evaluated the expression of P-selectin (also known as CD62P) on platelets, which translocates from α-granules to the platelet surface membrane following platelet activation (Blair and Flaumenhaft, 2009). We found that both live C. albicans and HK C. albicans significantly increased the expression of P-selectin on platelets ( Figures 1A–D ). To exclude the influence of experimental settings on platelet activation, we also incubated Candida parapsilosis (C. parapsilosis) and Cutibacterium acnes (C. acnes) with platelets. We found that C. acnes could not increased the expression of P-selectin on platelets. Only HK C. parapsilosis but live C. parapsilosis could increase the expression of P-selectin. The expression of P-selectin on HK C. parapsilosis-treated platelets was less than that on HK C. albicans-treated platelets ( Supplemental Figure S1 ). Generally, activated platelets secrete a variety of chemokines, including PF4 and CCL5. PF4 is also regarded as a marker of platelet activation (Gremmel et al., 2016). We found that both live C. albicans and HK C. albicans significantly elevated the levels of PF4 and CCL5 in the coculture suspension ( Figure 1E ).

The inflammatory cytokines TNF-α, IL-1β, and CXCL8 and the antimicrobial peptide β-defensin-2 play an important role in resisting C. albicans and have been reported to be released by activated platelets (Lindemann et al., 2001; Aslam et al., 2006; Tohidnezhad et al., 2012; Yeaman, 2014; Netea et al., 2015; Jarva et al., 2018; Fusco et al., 2021). Therefore, we further determined whether C. albicans enhanced the secretion of these proteins by platelets. We found that both live C. albicans and HK C. albicans significantly elevated the secretion of low concentrations of CXCL8 and TNF-α from platelets ( Figure 1E ). HK C. albicans elevated the secretion of β-defensin-2, while live C. albicans did not ( Figure 1E ). The level of IL-1β, which was secreted at a relatively low concentration, did not vary in HK C. albicans-treated or live C. albicans-treated platelets ( Figure 1E ).

Platelet activation requires intracellular signal transduction initiated by pathogen- and damage-associated molecular pattern molecules, adhesion proteins and soluble agonists (Gremmel et al., 2016; Estevez and Du, 2017). Next, we investigated the mechanism of C. albicans-induced platelet activation. Stat3, AKT, ERK1/2 and p38 phosphorylation have been shown to be involved in platelet signal transduction when platelets are activated (Rex et al., 2009; Zhou et al., 2013; Guidetti et al., 2015). Therefore, we detected the phosphorylation of these proteins to further explore the signaling pathway mediating C. albicans-induced platelet activation. We found that AKT phosphorylation on Ser473 and Stat3 phosphorylation on Tyr705 were elevated in HK C. albicans-treated platelets ( Figures 2A, B ). However, only AKT Ser473 phosphorylation levels were significantly elevated in live C. albicans-treated platelets ( Figures 2C, D ).

The PI3K-Akt signaling pathway has been deeply investigated in platelet activation (Guidetti et al., 2015). Therefore, we hypothesized that the PI3K-Akt pathway mediated C. albicans-induced platelet activation. To test this hypothesis, platelets were treated with the PI3K inhibitor LY294002 and Akt inhibitor MK2206 before costimulation with C. albicans. LY294002 and MK2206 markedly reduced AKT Ser473 phosphorylation levels ( Figure 2E ) and P-selectin expression on platelets ( Figure 2F ). These data suggest that C. albicans induces platelet activation through a PI3K-AKT signaling pathway.

As mentioned above, platelets express pattern recognition receptors, such as TLR2 and TLR4, and these receptors recognize a variety of microbes and hence facilitate platelet activation, platelet-neutrophil aggregation, and leukocyte phagocytosis (Schattner, 2019; Gautam et al., 2020). To explore whether TLR2 and TLR4 mediated C. albicans-induced platelet activation, we treated platelets with anti-TLR2 or anti-TLR4 neutralizing antibodies before costimulation with C. albicans. We found that blocking TLR2 or TLR4 did not suppress the expression of P-selectin ( Supplemental Figure S2A ). In addition, platelet stimulation with the TLR2 agonists Pam3CSK4 and FSL-1 and the TLR4 agonist LPS did not elevate the expression of P-selectin ( Supplemental Figure S2B ) or secretion of PF4 ( Supplemental Figure S2C ) as C. albicans did. These data suggest that C. albicans-mediated platelet activation is independent of the TLR2 and TLR4 receptors.

GP IIb/IIIa is capable of mediating platelet activation and PI3K-Akt pathway activation when recognizing specific pathogens or nonpathogen ligands (Guidetti et al., 2015; Gautam et al., 2020). Therefore, we further explored whether GP IIb/IIIa mediated C. albicans-induced platelet activation. Surprisingly, we found that the expression of GP IIb/IIIa determined by flow cytometry was reduced on C. albicans-treated platelets ( Figures 3A, B ). We speculated that C. albicans bound to the GP IIb/IIIa receptor and thus reduced its detectable amounts. We also found that the binding of FITC-coupled antibody PAC-1, which bound only to the activated form of GP IIb/IIIa, did not vary on C. albicans-treated and control platelets ( Supplemental Figure S3 ).

To further clarify the role of GP IIb/IIIa in C. albicans-induced platelet activation, we pretreated platelets with the GP IIb/IIIa-specific antagonist tirofiban before costimulation with C. albicans. Tirofiban is a small-molecule GP IIb/IIIa receptor inhibitor with high affinity and is capable of preventing the binding of fibrinogen and von Willebrand factor to activated GP IIb/IIIa. Tirofiban also reacts with the resting form of GP IIb/IIIa, so it binds to non-stimulated and stimulated platelets (Hagemeyer and Peter, 2010). We found that treatment of quiescent platelets with tirofiban blocked GP IIb/IIIa and reduced the detectable amounts of GP IIb/IIIa on platelets ( Supplemental Figure S4 ).We then found that pretreatment of platelets with tirofiban significantly suppressed the expression of P-selectin in a concentration-dependent manner ( Figures 3C–F ). In order to eliminate the interference of fibrinogen in PRP, we confirmed the results by using washed platelets ( Supplemental Figure S5 ). We also confirmed the results by pretreatment of washed platelets with blocking antibody for GP IIb/IIIa, which significantly suppressed the expression of P-selectin ( Figures 3G, H ). In addition, pretreatment of platelets with tirofiban almost completely reversed the elevated AKT Ser473 phosphorylation levels caused by C. albicans ( Figure 3I ). Taken together, these data suggest that C. albicans induces platelet activation in a GP IIb/IIIa-dependent manner.

Platelets are capable of promoting phagocytosis and affecting inflammation responses of monocytes-macrophages during bacterial infection (Weyrich et al., 1996; Rodland et al., 2010; Carestia et al., 2019), and the inflammatory cytokines such as IL-1β, IL-6 and TNF-α play a critical role in the outcome of candidiasis by recruiting activated leukocytes (Netea et al., 2015).Therefore, we further explored whether platelets had an effect on monocyte phagocytosis of C. albicans and the expression of inflammatory cytokines. We found that compared to PPP, PRP enhanced the capacity of THP-1 monocytes and primary human monocytes to phagocytose FUN-1-labeled C. albicans ( Figures 4A, B ). We observed that PRP upregulated the expression of IL-1β by THP-1 monocytes ( Figure 4C ) and the expression of IL-1β, IL-6, TNF-α and CXCL8 by primary human monocytes in the presence of C. albicans ( Figure 4D ).

We found that C. albicans induced platelet activation, and platelet aggregations were observed in the monocyte, C. albicans, and platelet coculture system ( Figure 4B , black arrow). Therefore, we further determined whether the elevated phagocytosis activity of monocytes was dependent on platelet activation. Anti-platelet agent cilostazol is a specific and strong inhibitor of PDE3 in platelets, and it diminishes intracellular calcium, causing inhibition of platelet activation and aggregation (Schror, 2002). Cilostazol significantly inhibits platelet activation in vitro, and its inhibition effect is much stronger than that of the well known anti-platelet agent aspirin (Ito et al., 2004). We found that treatment of platelets with cilostazol before coculture with THP-1 cells and C. albicans removed the elevated phagocytosis effect in PRP-treated THP-1 cells ( Figure 5A ). These data suggest that activated platelets enhance monocyte phagocytosis of C. albicans. To elucidate whether GP IIb/IIIa contributes to platelet activation and platelet-mediated phagocytosis of monocytes in this setting, we pretreated platelets with tirofiban before coculture with THP-1 cells and C. albicans. We observed that blocking GP IIb/IIIa receptors on platelets inhibited platelet aggregation ( Supplemental Figure S6 ) and removed the elevated phagocytosis effect in PRP-treated THP-1 cells ( Figure 5B ). Additionally, pretreatment of platelets with cilostazol or tirofiban removed the elevated expression of IL-1β induced in THP-1 cells treated with C. albicans ( Figure 5C ).