Introduction:Streptococcus pyogenes (group A streptococcus, GAS) is an exclusively human pathogen. It causes a wide spectrum of diseases, ranging from mild infections such as pharyngitis to severe life-threatening conditions such as streptococcal toxic shock syndrome (STSS). Thrombocytopenia is a common feature of STSS and is associated with severe outcome. GAS produce a plethora of virulence factors, including streptolysin S (SLS), which has lytic as well as immunomodulatory properties. However, its role in platelet activation remains unclear. Methods: Washed human platelets were infected with GAS wild-type and SLS-deficient mutant (ΔsagA) strains. Platelet activation was assessed by measuring degranulation (CD62P expression). The role of calcium influx and the involvement of purinergic type 2 receptors (P2R) in platelet activation by GAS were assessed using chemical antagonists and calcium chelators. Results: GAS activate human platelets via SLS-mediated calcium influx, marked by increased surface expression of CD62P. IVIG treatment improved platelet viability in wild-type infections but failed to prevent SLS-mediated activation. Blocking of P2 receptors via suramin or NF449 as well as the use of calcium chelators reduced SLS-mediated platelet activation. Conclusion: This study identified SLS as an M-protein and consequently a serotype-independent activator of human platelets. While IVIG partially improved platelet viability in GAS infections, its inability to prevent excessive platelet activation underscores the need for additional treatment options in severe GAS infections.

Infections caused by an exclusively human pathogen S. pyogenes (group A streptococci, GAS) can lead to a broad spectrum of diseases, ranging from mild cases such as pharyngitis and impetigo to serious invasive infections [1]. GAS are a leading cause of monomicrobial necrotizing skin and soft tissue infections (NSTIs), which are often complicated by streptococcal toxic shock syndrome (STSS) [2]. STSS is particularly associated with excessive systemic hyper-inflammation, leading to multi-organ failure [3]. Such a severe presentation of GAS infections is attributed to both host as well as bacterial factors [4]. The secreted virulence factors, including superantigens, host protein-degrading proteases, and cytolysins are believed to play a crucial role in the severity of GAS infections [5].

GAS secrete two cytolysins, cholesterol-dependent streptolysin O (SLO) and streptolysin S (SLS). In contrast to SLO, SLS is a small (2.7 kDa) non-immunogenic peptide [6, 7]. The SLS-associated gene (sag) locus comprises nine genes (sagABCDEFGHI), of which the sagA gene encodes the premature form of SLS. After processing and release, the mature SLS acts as a receptor-dependent cytolysin [8]. The peptide is responsible for the β-hemolytic phenotype of GAS [8], through targeting the anion channel Band 3 on red blood cells and thereby inducing osmotic imbalance that leads to cell lysis [9]. In addition to its lytic function, SLS also exhibits modulatory actions. It directly activates nociceptors on neurons, leading to the release of calcitonin gene-related peptide into infected tissues [10]. Such actions induce severe pain, which is a hallmark of NSTIs [11]. In addition, SLS was shown to impact signaling in keratinocytes by triggering programmed cell death and inflammatory cascades [12].

Platelets are the second most abundant blood cell type in circulation. Despite their role in hemostasis, several immunomodulatory functions are ascribed [13]. In GAS infections, platelet activation through M-protein-fibrinogen and IgG interaction triggers thrombus formation [14], leading to increased consumption of platelets and coagulation factors, which in turn results in coagulopathy [15, 16]. It was shown that M1, M3, and M5 GAS serotypes utilize such mechanism, with M1 being the most efficient [14]. Furthermore, it is reported that STSS caused by GAS [17, 18] as well as by closely related Streptococcus dysgalactiae subspecies equisimilis [19] is often associated with reduced platelet counts. Low platelet counts are linked to the poor outcome of invasive streptococcal infections [20].

Platelet activation is achieved via the interaction of a diverse range of ligands with different classes of surface receptors. Thrombin is a potent trigger of platelet activation and is recognized by the protease-activated receptors (PAR) 1 and 4 [21, 22]. Upon binding of a ligand, PARs further contribute to platelet function by promoting degranulation and inducing platelet procoagulant activity [23]. The released granule content, which includes thrombin, ADP, and thromboxane among others, leads to further amplification of platelet activation [24]. Another class of platelet receptors are purinergic type 2 receptors (P2Rs) [25]. The P2Rs are classified into two subclasses, P2X and P2Y receptors. While P2X receptors act as ion channels and are activated by extracellular ATP, P2Y receptors are mostly reactive to ADP [26]. To date, three different P2Rs are well described in human platelets: P2X1, P2Y1, and P2Y12. All of them are directly or indirectly involved in Ca2+ signaling [27‒29]. It was shown that P2X1R activation amplifies platelet aggregation mediated by thrombin and ADP-activated P2Y1R [30, 31]. Furthermore, P2X1 and P2Y12 appear to promote platelet-mediated bactericidal effects in staphylococcal infections [32], thus contributing to immune responses against pathogens.

It is already known that GAS are potent platelet activators via the M1-protein and fibrinogen-IgG axis [16, 33]. However, investigations on other specific and particularly serotype-independent mechanisms of GAS-platelet interaction are scarce. Here, we show that SLS activates human platelets by mediating calcium ion influx.

Platelet Preparation

Washed human platelets were prepared as described previously [34]. In brief, platelet-rich plasma (PRP) was prepared from ACD-A anticoagulated whole blood from a defined set of healthy volunteers, who were not taking any antiplatelet or non-steroidal anti-inflammatory drugs. The PRP was washed two times in Tyrode’s buffer containing 2.5 U/mL apyrase, 1 U/mL hirudin, 0.35% BSA, and 0.1% glucose (pH 6.3). Lastly, the platelet pellet was suspended in a bicarbonate-based suspension buffer supplemented with 2.12 mm MgCl2, 1.96 mm CaCl2, 0.35% BSA, and 0.1% glucose (pH 7.2). The platelet count was adjusted to 300,000 platelets/µL.

Bacterial Strains

The following GAS strains were used: 5448 [35], 5448Δslo [36], and 5448ΔsagA [37]. All strains were cultivated in Todd-Hewitt broth (Carl Roth) supplemented with 1.5% (w/v) yeast extract (Carl Roth) at 37°C.

Platelet Infections, Erythrocytes, and PBMC Stimulations

All experiments were carried out in Tyrode’s buffer containing Ca2+ and Mg2+. The platelets (7.5 × 106) were infected with 5448, 5448Δslo, and 5448ΔsagA at a multiplicity of infections (MOIs) of 0.01/0.1/1.

To assess SLS and SLO expression in Tyrode’s buffer, the same amount of bacteria was incubated in Tyrode’s buffer for 30 min, supernatants were collected and erythrocytes or PBMCs (1.0 × 105) were incubated with the supernatants at 1:4 dilution for 30 min or 24 h, respectively. PBMCs were isolated from buffy coats by Lymphoprep (Axis-Shields) gradient centrifugation as previously described [38, 39].

SLO and SLS hemolytic activity was measured by use of the conventional erythrocyte lysis activity assay as previously published [40] and the activities were related to a Triton-X100 lysis control. Tyrode’s buffer was used as a negative control.

Cytolytic activities were assessed via flow cytometry [39]. Briefly, dead cells were labeled using the Zombie Aqua Fixable Viability Kit (BioLegend). Unspecific binding of immunoglobulins was blocked by using Human TruStain FcX (BioLegend) according to the manufacturer’s instructions. Incubations of cells with titrated amounts of CD45 (BV605, HI30) monoclonal antibody were carried out for 30 min at 4°C in the dark. Cells were washed between each staining step and fixed using the Cyto-Fast Fix/Perm Buffer Set (BioLegend).

Furthermore, bacterial supernatants were separated by 12% SDS-PAGE and transferred to a PVDF membrane. As molecular mass markers, pre-stained protein standards (Bio-Rad) were used. The membranes were blocked with 5% skim milk and incubated with primary antibodies (1 mg/mL IVIG), washed and then incubated with secondary antibodies (anti-human IgG horseradish peroxidase linked Fab fragment; GE Healthcare). Positive binding was detected by Super Signal West Femto maximum sensitivity substrate (Thermo-Scientific) [41].

Platelet Activation Assay

The washed human platelets were infected as described above. In a subset of experiments, platelets were subsequently stimulated with 20 µm thrombin receptor activator peptide (TRAP)-6 5 min prior to the end of infections. The platelets were stained with FITC-labeled mouse anti-human CD42a and PE-Cy5-labeled mouse anti-human CD62P (both BD Biosciences) for 10 min at RT. Platelets were fixed with 2% paraformaldehyde for 30 min. The activation of platelets was assessed by measuring CD62P expression of CD42a-positive cells using a FACSCalibur or FACSAria III flow cytometer (Becton Dickinson) and CellQuestPro 6.0 or FACS Diva software, respectively. Data were analyzed using FCS Express 7 software (De Novo Software). The gating strategy for selected infectious conditions is shown in online supplementary Figure 1 (for all online suppl. material, see https://doi.org/10.1159/000544951).

All experiments performed with IVIG (98% IgG; Privigen, CSL Behring) included prior platelet incubation with monoclonal antibody IV.3 (1:15, 45 min, 37°C) to avoid FcγRIIa-dependent activation. The bacteria were subjected to 1 mg × mL−1 IVIG treatment for 30 min before infection.

Initially, platelet viability was assessed using the RealTime-Glo Cell Viability Assay kit (Promega) for over 120 min. Reagents were prepared according to the manufacturer’s instructions. Human washed platelets were infected with GAS, as described above. The luminescence was measured 1 min after the plate was mixed and shaken (300 rpm for 3 s); measurements were taken every 180 s thereafter. PBS and TritonX-100 (Sigma-Aldrich) were used as the negative and positive controls, respectively. All experiments were conducted in duplicates.

Inhibition of Platelet Activation

All infections in inhibition studies were performed in the presence of IVIG. Prior to infections, platelets were pre-incubated with different agents for 30 min. The following chemicals were used: ethylenediaminetetraacetic acid (EDTA) or ethylene glycol bis(β-aminoethylether) tetraacetic acid (EGTA) to chelate extracellular Ca2+; general P2R antagonist suramin (Sigma-Aldrich); specific P2X1R antagonist NF449 (Cayman Chemicals); TPRC6 receptor antagonist BI749327 (Focus Biomolecules).

Calcium Mobilization Assay

Ca2+ mobilization was measured using Fluo-4 AM as previously described with minor modifications [42]. In brief, platelets were washed and suspended in Ca2+ and Mg2+ free PBS (pH 7.4) containing 5 µm Fluo-4 AM (Molecular Probes). The suspensions were incubated for 10 min at 37°C. Next, CaCl2 was added to a final concentration of 2 mm and platelets were transferred to a 96-well F-bottom plate to measure baseline fluorescence at 520 nm for 1 min using a FLUOstar Omega fluorometer (BMC LabTech). Subsequently, bacteria (MOI 1) and the respective controls (PBS and TRAP-6) were added to platelets. The fluorescence was recorded every 30 s for 15 min. The amount of free intracellular Ca2+ induced by GAS infections was normalized to PBS control at the respective measurement.

Statistics

If not otherwise indicated, statistically significant differences were determined using the Kruskal-Wallis test with Dunn’s posttest. Statistics were performed using GraphPad Prism version 7 (GraphPad software). A p value <0.05 was considered significant.

GAS Activate Platelets via SLS

To assess the viability and activation of human platelets in response to GAS infections, cell suspensions were infected with the wild-type strain 5448 at different MOIs. Initial viability analyses included a luminescence-based approach. However, the bacteria incorporated the substrate (online suppl. Fig. 2), and therefore, the viability was assessed based on the increasing/decreasing frequencies of CD42a+ cells within the total GAS-platelet population instead (Fig. 1a). These analyses revealed that GAS kill and also activate dose-dependent human platelets (Fig. 1a, b). While MOI 0.01 had no effect on both processes, high-dose infections (MOI 1) almost completely killed the platelets (Fig. 1a). The remaining negligible viable population was fully activated in high-dose infections (Fig. 1b). To determine whether additional activation could be achieved, TRAP-6 was added to infections 5 min prior to the termination of the experiment. The frequencies of CD62P+ cells increased when low MOIs (0.01 and 0.1) were used. The platelets remained unresponsive to additional TRAP-6 stimulation at MOI 1 (Fig. 1b).

Fig. 1.

Infection-dose-dependent killing of platelets by S. pyogenes. Washed human platelets were infected with GAS 5448 wild-type strain at MOI 0.01, 0.1, or 1.0 and platelet viability (a) and activation (b) were assessed. Platelet viability (a) and activation (b) were measured via flow cytometry using FITC-labeled CD42a and PE-Cy5-labeled CD62P antibodies, respectively. TRAP-6 (20 µm) and PBS were used as positive and negative controls, respectively. Alternatively, 5 min prior to the end of infection, TRAP-6 (20 µm) was added. Each dot in a, b represents one independent experiment with cells from one donor (n = 5). Horizontal lines depict median values. The level of significance was determined using the Kruskal-Wallis test with Dunn’s posttest. RLU, relative luminescence units.

Fig. 1.

Infection-dose-dependent killing of platelets by S. pyogenes. Washed human platelets were infected with GAS 5448 wild-type strain at MOI 0.01, 0.1, or 1.0 and platelet viability (a) and activation (b) were assessed. Platelet viability (a) and activation (b) were measured via flow cytometry using FITC-labeled CD42a and PE-Cy5-labeled CD62P antibodies, respectively. TRAP-6 (20 µm) and PBS were used as positive and negative controls, respectively. Alternatively, 5 min prior to the end of infection, TRAP-6 (20 µm) was added. Each dot in a, b represents one independent experiment with cells from one donor (n = 5). Horizontal lines depict median values. The level of significance was determined using the Kruskal-Wallis test with Dunn’s posttest. RLU, relative luminescence units.

Close modal

To potentially improve platelet viability, isogenic mutants lacking single streptolysins SLO (5448Δslo) or SLS (5448ΔsagA) were used in the next set of experiments. First approach included MOI 0.1 infections for up to 120 min. The analyses revealed that (i) the majority of platelets remained viable (online suppl. Fig. 3a), (ii) only wild-type infections activated up to 40% of the platelet population (online suppl. Fig. 3b), and (iii) no bactericidal effects were observed (online suppl. Fig. 3c). In the second approach, the infectious dose was increased to MOI 1 and infection time was reduced to 30 min. Again, the viability dropped in all infectious conditions (Fig. 2a). In contrast, while a high proportion of the remaining platelets were activated in 5448 and 5448Δslo infections, the 5448 ΔsagA mutant failed to activate the cells (Fig. 2b). In congruence with the results above, all bacterial strains remained viable (Fig. 2c).

Fig. 2.

SLS activates platelets. Washed human platelets were infected with GAS strains at MOI 1 for 30 min and platelet viability (a) and activation (b) were assessed via flow cytometry using FITC-labeled CD42a and PE-Cy5-labeled CD62P antibodies, respectively. TRAP-6 (20 µm) and PBS were used as positive and negative controls, respectively. Each dot in a and b represents one independent experiment with cells from one donor (n = 10). c CFU of the indicated GAS strains after 30 and 60 min of platelet infection at MOI 1. Each dot represents one independent experiment with washed platelets from one donor (n = 5). Horizontal lines depict median values. The level of significance was determined using the Kruskal-Wallis test with Dunn’s posttest. CFU, colony-forming unit.

Fig. 2.

SLS activates platelets. Washed human platelets were infected with GAS strains at MOI 1 for 30 min and platelet viability (a) and activation (b) were assessed via flow cytometry using FITC-labeled CD42a and PE-Cy5-labeled CD62P antibodies, respectively. TRAP-6 (20 µm) and PBS were used as positive and negative controls, respectively. Each dot in a and b represents one independent experiment with cells from one donor (n = 10). c CFU of the indicated GAS strains after 30 and 60 min of platelet infection at MOI 1. Each dot represents one independent experiment with washed platelets from one donor (n = 5). Horizontal lines depict median values. The level of significance was determined using the Kruskal-Wallis test with Dunn’s posttest. CFU, colony-forming unit.

Close modal

To confirm that both streptolysins are released by the bacteria under chosen conditions (MOI 1), the bacteria was incubated in Tyrode’s buffer for 30 min and the supernatants were either directly subjected to Western blot analyses or hemolytic and cytolytic assays (online suppl. Fig. 4). While protein analysis showed that the bacteria release certain factors, they remained inconclusive in terms of SLO (online suppl. Fig. 4a), potentially due to the release of a limited amount, hemolysis as well as the cytolysis assays indirectly confirmed that both factors, SLS and SLO, are released (online suppl. Fig. 4b, c). Based on these observations, all subsequent infection experiments were performed with MOI 1 for 30 min.

IVIG Neutralizes the Cytotoxic Effect of GAS but Does Not Prevent SLS-Mediated Activation

Since the use of single streptolysin mutants did not improve platelet viability, IVIG, a commonly used adjunctive therapeutic agent in streptococcal NSTIs and STSS [43], which neutralizes a plethora of proteinous streptococcal virulence factors including toxins, was used in all subsequent experiments. Initially, two approaches were tested: (i) platelets were infected with GAS and IVIG was added 5 min post infections (Fig. 3a) or (ii) GAS were incubated with IVIG prior to platelet infections (Fig. 4a). Both approaches improved platelet viability, characterized by increased frequencies of CD42+ cells (Fig. 3, 4b). However, the second approach was slightly more effective (Fig. 4). In contrast, IVIG treatment did not neutralize 5448 wild-type as well as 5448Δslo-mediated platelet activation (Fig. 3, 4c). High frequencies of CD62P+ cells were observed. In congruence with the results above, 5448ΔsagA mutant strain completely failed to activate human platelets (Fig. 3, 4c). Nonetheless, the addition of TRAP-6 to 5448ΔsagA infections activated platelets confirming their viability and responsiveness. Based on these results, all subsequent experiments were performed with 5448 wild-type and 5448ΔsagA mutant strains in the presence of IVIG.

Fig. 3.

Platelet viability is improved by the addition of IVIG 5 min post infection. a Schematic overview of the experimental setup. Platelet viability (b) and activation (c) were measured via flow cytometry using FITC-labeled CD42a and PE-Cy5-labeled CD62P antibodies, respectively. TRAP-6 (20 µm) and PBS were used as positive and negative controls, respectively. Alternatively, 5 min prior to the end of infection, TRAP-6 (20 µm) was added. Each dot in b, c represents one independent experiment with cells from one donor (n = 5). Horizontal lines depict median values. The level of significance was determined using the Kruskal-Wallis test with Dunn’s posttest.

Fig. 3.

Platelet viability is improved by the addition of IVIG 5 min post infection. a Schematic overview of the experimental setup. Platelet viability (b) and activation (c) were measured via flow cytometry using FITC-labeled CD42a and PE-Cy5-labeled CD62P antibodies, respectively. TRAP-6 (20 µm) and PBS were used as positive and negative controls, respectively. Alternatively, 5 min prior to the end of infection, TRAP-6 (20 µm) was added. Each dot in b, c represents one independent experiment with cells from one donor (n = 5). Horizontal lines depict median values. The level of significance was determined using the Kruskal-Wallis test with Dunn’s posttest.

Close modal
Fig. 4.

Platelet viability is further improved by the addition of IVIG prior to infections. a Schematic overview of the experimental setup. Platelet viability (b) and activation (c) were measured via flow cytometry using FITC-labeled CD42a and PE-Cy5-labeled CD62P antibodies, respectively. TRAP-6 (20 µm) and PBS were used as positive and negative controls, respectively. Alternatively, 5 min prior to the end of infection, TRAP-6 (20 µm) was added. Each dot in b, c represents one independent experiment with cells from one donor (n = 5). Horizontal lines depict median values. The level of significance was determined using the Kruskal-Wallis test with Dunn’s posttest.

Fig. 4.

Platelet viability is further improved by the addition of IVIG prior to infections. a Schematic overview of the experimental setup. Platelet viability (b) and activation (c) were measured via flow cytometry using FITC-labeled CD42a and PE-Cy5-labeled CD62P antibodies, respectively. TRAP-6 (20 µm) and PBS were used as positive and negative controls, respectively. Alternatively, 5 min prior to the end of infection, TRAP-6 (20 µm) was added. Each dot in b, c represents one independent experiment with cells from one donor (n = 5). Horizontal lines depict median values. The level of significance was determined using the Kruskal-Wallis test with Dunn’s posttest.

Close modal

SLS Induces Continuous Ca2+ Influx in Human Platelets

As Ca2+ signaling plays a key role in platelet activation [44], the effect of SLS on Ca2+ mobilization in platelet infections was assessed. Intracellular Ca2+ (iCa2+) dynamics were monitored using a Fluo-4 AM assay. Infections with 5448 resulted in a significant continuous increase of iCa2+ as compared to the PBS control (Fig. 5a). In contrast, while TRAP-6 induced high levels of iCa2+ within a short time, only minor iCa2+ accumulation was noted in 5448ΔsagA infections (Fig. 5a).

Fig. 5.

SLS induces Ca2+ influx to activate platelets. a Kinetics of Ca2+ mobilization in platelets in response to GAS infections. After 60 s baseline measurement, controls and IVIG-treated bacteria (MOI 1) were added to the platelets (↓). Curves represent the median values of 5 independent experiments with five donors (n = 5). The level of significance was determined by comparing endpoint measurements using the Kruskal-Wallis test with Dunn’s posttest. b Schematic overview of the experimental setup. EDTA- (c) or EGTA-treated (d) washed platelets were infected with bacteria. Platelet activation was measured via flow cytometry using PE-Cy5-labeled CD62P antibody. TRAP-6 (20 µm) and PBS were used as positive and negative controls, respectively. Alternatively, 5 min prior to the end of infection, TRAP-6 (20 µm) was added. Left panels and right panels in c and d show representative histograms and a summary of the data, respectively. Each dot in c and d represents one independent experiment with cells from one donor (b, n = 6; c, n = 7). Horizontal lines depict median values. The level of significance was determined using the Kruskal-Wallis test with Dunn’s posttest.

Fig. 5.

SLS induces Ca2+ influx to activate platelets. a Kinetics of Ca2+ mobilization in platelets in response to GAS infections. After 60 s baseline measurement, controls and IVIG-treated bacteria (MOI 1) were added to the platelets (↓). Curves represent the median values of 5 independent experiments with five donors (n = 5). The level of significance was determined by comparing endpoint measurements using the Kruskal-Wallis test with Dunn’s posttest. b Schematic overview of the experimental setup. EDTA- (c) or EGTA-treated (d) washed platelets were infected with bacteria. Platelet activation was measured via flow cytometry using PE-Cy5-labeled CD62P antibody. TRAP-6 (20 µm) and PBS were used as positive and negative controls, respectively. Alternatively, 5 min prior to the end of infection, TRAP-6 (20 µm) was added. Left panels and right panels in c and d show representative histograms and a summary of the data, respectively. Each dot in c and d represents one independent experiment with cells from one donor (b, n = 6; c, n = 7). Horizontal lines depict median values. The level of significance was determined using the Kruskal-Wallis test with Dunn’s posttest.

Close modal

Next, Ca2+ influx was inhibited by chelating the extracellular source. Two reagents were used, EDTA and EGTA (Fig. 5b–d). High concentrations of EDTA (10 mm) completely abolished wild-type mediated platelet activation (Fig. 5c). Next, EGTA, which has a higher affinity for Ca2+, was used. A dose-dependent reduction of platelet activation was observed in 5448 infections (Fig. 5d). In both scenarios, (i) no activation in 5448ΔsagA infections was observed, (ii) TRAP-6 responsiveness by platelets remained unaffected by the chelating agents, and (iii) both agents had a minor effect on additional TRAP-6 responsiveness of infected platelets (Fig. 5c, d). These analyses revealed that SLS targets platelets via direct activation of Ca2+ influx, which is a thrombin receptor-independent pathway.

SLS Activates Platelets via P2 Receptors

P2X1R and TRPC6 are receptor-operated calcium entry channels in platelets [44, 45] and potential targets of SLS. To explore whether these receptors are targeted by SLS, chemical antagonists were used in a series of experiments (Fig. 6, 7). The specific antagonist for TRPC6, BI749327, did not reduce platelet activation in 5448 infections (Fig. 6). In contrast, a general P2R antagonist, suramin, as well as NF449, a suramin analog that is highly selective for P2X1R [46, 47], dose-dependently reduced platelet activation by the wild-type strain 5448 (Fig. 7). In control experiments, (i) the highest dosages of the inhibitors had no activating effects, (ii) the platelets remained responsive to additional TRAP-6 stimulations, and (iii) 5448ΔsagA did not activate human platelets (Fig. 6, 7).

Fig. 6.

Inhibition of TRPC6 does not affect platelet activation in GAS infections. a Schematic overview of the experimental setup. b Platelet activation was measured via flow cytometry using PE-Cy5-labeled CD62P antibody. TRAP-6 (20 µm) and PBS were used as positive and negative controls, respectively. Alternatively, 5 min prior to the end of infection, TRAP-6 (20 µm) was added. Each dot in b represents one independent experiment with cells from one donor (n ≥ 4). Horizontal lines depict median values. The level of significance was determined using the Kruskal-Wallis test with Dunn’s posttest.

Fig. 6.

Inhibition of TRPC6 does not affect platelet activation in GAS infections. a Schematic overview of the experimental setup. b Platelet activation was measured via flow cytometry using PE-Cy5-labeled CD62P antibody. TRAP-6 (20 µm) and PBS were used as positive and negative controls, respectively. Alternatively, 5 min prior to the end of infection, TRAP-6 (20 µm) was added. Each dot in b represents one independent experiment with cells from one donor (n ≥ 4). Horizontal lines depict median values. The level of significance was determined using the Kruskal-Wallis test with Dunn’s posttest.

Close modal
Fig. 7.

Inhibition of P2R leads to reduced SLS-mediated platelet activation. a Schematic overview of the experimental setup. Suramin- (b) or NF449-treated (c) platelets were infected with bacteria. Platelet activation was measured via flow cytometry using PE-Cy5-labeled CD62P antibody. TRAP-6 (20 µm) and PBS were used as positive and negative controls, respectively. Alternatively, 5 min prior to the end of infection, TRAP-6 (20 µm) was added. Left panels and right panels in b, c show representative histograms and summary of the data, respectively. Each dot in b, c represents one independent experiment with cells from one donor (b, n = 6; c, n = 7). Horizontal lines depict median values. The level of significance was determined using the Kruskal-Wallis test with Dunn’s posttest.

Fig. 7.

Inhibition of P2R leads to reduced SLS-mediated platelet activation. a Schematic overview of the experimental setup. Suramin- (b) or NF449-treated (c) platelets were infected with bacteria. Platelet activation was measured via flow cytometry using PE-Cy5-labeled CD62P antibody. TRAP-6 (20 µm) and PBS were used as positive and negative controls, respectively. Alternatively, 5 min prior to the end of infection, TRAP-6 (20 µm) was added. Left panels and right panels in b, c show representative histograms and summary of the data, respectively. Each dot in b, c represents one independent experiment with cells from one donor (b, n = 6; c, n = 7). Horizontal lines depict median values. The level of significance was determined using the Kruskal-Wallis test with Dunn’s posttest.

Close modal

Severe GAS infections are often associated with coagulopathy and low platelet counts [17‒19]. While GAS are known to activate platelets in a serotype-dependent fashion, the general activating mechanisms remain elusive. Here, we show that GAS induce platelet activation through SLS-mediated P2R activation and subsequent calcium influx.

Our results are in congruence with other studies that confirm the ability of GAS to quickly activate human platelets [14, 16, 33, 48]. To study rather general and M-protein-independent mechanisms, we used washed platelets instead of PRP [16]. Indeed, this approach revealed that GAS activate human platelets without utilizing the previously described M-protein-fibrinogen-IgG axis [16]. However, since both streptolysins were active in wild-type infections, a tremendous lytic effect was observed. Single knock-outs of each streptolysin did not improve platelet viability. However, the SLS mutant strain completely failed to activate the remaining viable platelet population. To neutralize SLO and other streptococcal virulence factors, we used IVIG in all infectious conditions. IVIG is commonly used as an adjunct therapy in severe GAS infections [43, 49, 50] to neutralize bacterial toxins, modulate the immune response, and improve pathogen clearance [51]. In experimental GAS infections, IVIG has been shown to completely neutralize SLO and other streptococcal virulence factors except for SLS [52]. Consequently, the use of IVIG neutralized SLO-mediated killing of platelets but did not prevent SLS-triggered activation in our experimental setup. However, IVIG did not completely neutralize the killing of platelets in 5448ΔsagA infections, suggesting that other streptococcal potentially non-immunogenic factors or even host factors might still induce a partial killing [53, 54]. In general, IVIG administration, combined with antibiotic treatment, has been shown to increase platelet counts in patients, thus positively impacting the outcomes [55, 56]. Furthermore, studies using murine models have also indicated potential benefits, particularly in NSTIs and STSS [52].

Platelet activation is achieved through calcium mobilization. Upon activation, calcium is released from intracellular stores and/or enters the cell via receptor-operated calcium entry and SOCE [44]. Furthermore, direct stimulation of ligand-gated ion channels and consequent Ca2+ influx result in platelet activation, which can be further amplified [57]. SLS of S. anginosus was recently shown to induce Ca2+ influx in HSC-2 cells [58]. Given the partially conserved nature of SLS on the streptococcal genome, we hypothesized that SLS might have similar activating effects on platelets. We demonstrate that SLS deficiency resulted in reduced Ca2+ influx during platelet infection. Inhibition of Ca2+ influx with chelators significantly decreased SLS-dependent effects in wild-type infections, consistent with the findings in S. anginosus infections of HSC-2 cells [58]. It is reported that staphylococcal α-hemolysin (Hla) also induces Ca2+ influx and thereby activates platelets. In this case, the influx of Ca2+ is linked to the formation of small pores in the platelet membrane, which subsequently leads to cell death [59]. In contrast, we show that platelet activation mediated by SLS is not linked to lysis. Currently, only a limited number of SLS receptors on human cells are known. These include the anion transporters Band 3 on erythrocytes, NBCn1 on keratinocytes, and nociceptors on neurons [9, 10, 60]. In addition to these interactions, we have identified a family of SLS-interacting partners belonging to the P2R family. The general P2R antagonist, suramin, as well as the P2X1R-specific antagonist NF449 significantly reduced SLS-mediated platelet activation in GAS infections. However, NF449 is a high-affinity antagonist for P2X1R that generally acts within the picomolar range [46]. In this study, micromolar concentration of NF449, which also antagonizes P2Y1R as well as P2Y12R [61], reduced platelet activation in wild-type infections. Thus, SLS-mediated platelet activation cannot be specifically linked to one particular receptor but rather to the P2R family. The non-immunogenic nature, size, as well as natural posttranslational processing of the peptide, limited the ability to conduct further studies on SLS-P2R interactions.

In conclusion, this study highlights that GAS-mediated platelet activation is a multifactorial process. By examining platelet activation beyond the well-studied M-protein-fibrinogen-IgG interaction, we show that SLS triggers calcium influx through P2 family receptors. However, the contribution of the different P2Rs to SLS-mediated platelet activation remains to be elucidated.

We would like to thank Constantin Klein for expert technical assistance.

The use of whole blood and washed platelets from healthy adults was approved by the Ethics Committee of the University Medicine Greifswald (BB 044/18). Buffy coats obtained from healthy blood donors were anonymously provided by the blood bank at the University Medicine Greifswald. The use of PBMCs was approved by the Ethical Research Committee at the University Medicine Greifswald (Ref. No. BB 014/14). All volunteers gave written informed consent in accordance with the Declaration of Helsinki. All experiments were carried out in accordance with the approved guidelines.

The authors have no conflicts of interest to declare.

This research is supported by the German Research Foundation (DFG; Grant 492903360 to N.S.). The funder had no role in the design, data collection, data analysis, and reporting of this study.

Conceptualization: A.R., J.W., and N.S. Experimental work: A.R. and K.J. Data analysis: A.R., K.J., and N.S. Volunteer inclusion: J.W. and T.T. Project administration: N.S. Supervision: J.W., T.T., and N.S. Writing – original draft: A.R. and N.S. Writing – review and editing: all authors.

All data associated with this study are presented in the manuscript and supplementary material. Further inquiries can be directed to the corresponding author.

1.
Siemens
N
,
Lütticken
R
.
Streptococcus pyogenes (“Group A Streptococcus”), a highly adapted human pathogen-potential implications of its virulence regulation for epidemiology and disease management
.
Pathogens
.
2021
;
10
(
6
):
776
.
2.
Lamagni
TL
,
Darenberg
J
,
Luca-Harari
B
,
Siljander
T
,
Efstratiou
A
,
Henriques-Normark
B
, et al
.
Epidemiology of severe Streptococcus pyogenes disease in europe
.
J Clin Microbiol
.
2008
;
46
(
7
):
2359
67
.
3.
Singer
M
,
Deutschman
CS
,
Seymour
CW
,
Shankar-Hari
M
,
Annane
D
,
Bauer
M
, et al
.
The third international consensus definitions for sepsis and septic shock (Sepsis-3)
.
JAMA
.
2016
;
315
(
8
):
801
10
.
4.
Siemens
N
,
Snäll
J
,
Svensson
M
,
Norrby-Teglund
A
.
Pathogenic mechanisms of streptococcal necrotizing soft tissue infections
.
Adv Exp Med Biol
.
2020
;
1294
:
127
50
.
5.
Shumba
P
,
Mairpady Shambat
S
,
Siemens
N
.
The role of streptococcal and staphylococcal exotoxins and proteases in human necrotizing soft tissue infections
.
Toxins
.
2019
;
11
(
6
):
332
.
6.
Robinson
JJ
.
The nonantigenicity of streptolysin S
.
J Immunol
.
1951
;
66
(
6
):
661
5
.
7.
Mitchell
DA
,
Lee
SW
,
Pence
MA
,
Markley
AL
,
Limm
JD
,
Nizet
V
, et al
.
Structural and functional dissection of the heterocyclic peptide cytotoxin streptolysin S
.
J Biol Chem
.
2009
;
284
(
19
):
13004
12
.
8.
Nizet
V
,
Beall
B
,
Bast
DJ
,
Datta
V
,
Kilburn
L
,
Low
DE
, et al
.
Genetic locus for streptolysin s production by group a streptococcus
.
Infect Immun
.
2000
;
68
(
7
):
4245
54
.
9.
Higashi
DL
,
Biais
N
,
Donahue
DL
,
Mayfield
JA
,
Tessier
CR
,
Rodriguez
K
, et al
.
Activation of band 3 mediates group A Streptococcus streptolysin S-based beta-haemolysis
.
Nat Microbiol
.
2016
;
1
:
15004
6
.
10.
Pinho-Ribeiro
FA
,
Baddal
B
,
Haarsma
R
,
O’Seaghdha
M
,
Yang
NJ
,
Blake
KJ
, et al
.
Blocking neuronal signaling to immune cells treats streptococcal invasive infection
.
Cell
.
2018
;
173
(
5
):
1083
97.e22
.
11.
Madsen
MB
,
Arnell
P
,
Hyldegaard
O
.
Necrotizing soft-tissue infections: clinical features and diagnostic aspects
.
Adv Exp Med Biol
.
2020
;
1294
:
39
52
.
12.
Flaherty
RA
,
Puricelli
JM
,
Higashi
DL
,
Park
CJ
,
Lee
SW
.
Streptolysin S promotes programmed cell death and enhances inflammatory signaling in epithelial keratinocytes during group A Streptococcus infection
.
Infect Immun
.
2015
;
83
(
10
):
4118
33
.
13.
Elzey
BD
,
Tian
J
,
Jensen
RJ
,
Swanson
AK
,
Lees
JR
,
Lentz
SR
, et al
.
Platelet-mediated modulation of adaptive immunity: a communication link between innate and adaptive immune compartments
.
Immunity
.
2003
;
19
(
1
):
9
19
.
14.
Palm
F
,
Chowdhury
S
,
Wettemark
S
,
Malmström
J
,
Happonen
L
,
Shannon
O
.
Distinct serotypes of streptococcal M proteins mediate fibrinogen-dependent platelet activation and proinflammatory effects
.
Infect Immun
.
2022
;
90
(
2
):
e0046221
21
.
15.
Stevens
DL
.
Invasive streptococcal infections
.
J Infect Chemother
.
2001
;
7
(
2
):
69
80
.
16.
Shannon
O
,
Hertzén
E
,
Norrby-Teglund
A
,
Mörgelin
M
,
Sjöbring
U
,
Björck
L
.
Severe streptococcal infection is associated with M protein-induced platelet activation and thrombus formation
.
Mol Microbiol
.
2007
;
65
(
5
):
1147
57
.
17.
JiaPathak
X-JL
,
Tong
JF
,
Su
JMF
,
Su
J-M
.
A case report of hemolytic streptococcal gangrene in the danger triangle of the face with thrombocytopenia and hepatitis
.
BMC Pediatr
.
2018
;
18
(
1
):
198
.
18.
Sakaguchi
YM
,
Murakami
K
,
Akebo
H
,
Sada
RM
,
Abe
N
,
Maeda
T
, et al
.
Successful treatment of streptococcal toxic shock syndrome complicated by primary peritonitis and bilateral empyema in a healthy young woman: identification of uncommon clone emm103 and novel sequence type 1363
.
IDCases
.
2024
;
35
:
e01927
.
19.
Shen
X
,
Liang
H
,
Wu
G
,
Chen
M
,
Li
J
.
A case report of Streptococcus dysgalactiae toxic shock syndrome complicated with symmetric peripheral gangrene
.
Infect Drug Resist
.
2023
;
16
:
5977
83
.
20.
Lin
J-N
,
Chang
L-L
,
Lai
C-H
,
Lin
H-H
,
Chen
Y-H
.
Group A streptococcal necrotizing fasciitis in the emergency department
.
J Emerg Med
.
2013
;
45
(
5
):
781
8
.
21.
Vu
T-KH
,
Hung
DT
,
Wheaton
VI
,
Coughlin
SR
.
Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation
.
Cell
.
1991
;
64
(
6
):
1057
68
.
22.
XuAndersen
W-H
,
Whitmore
TE
,
Presnell
SR
,
Yee
DP
,
Ching
A
,
Gilbert
T
, et al
.
Cloning and characterization of human protease-activated receptor 4
.
Proc Natl Acad Sci
.
1998
;
95
(
12
):
6642
6
.
23.
Andersen
H
,
Greenberg
DL
,
Fujikawa
K
,
Xu
W
,
Chung
DW
,
Davie
EW
.
Protease-activated receptor 1 is the primary mediator of thrombin-stimulated platelet procoagulant activity
.
Proc Natl Acad Sci
.
1999
;
96
(
20
):
11189
93
.
24.
Yun
S-H
,
Sim
E-H
,
Goh
R-Y
,
Park
J-I
,
Han
J-Y
.
Platelet activation: the mechanisms and potential biomarkers
.
BioMed Res Int
.
2016
;
2016
(
1
):
9060143
.
25.
Burnstock
G
.
P2X ion channel receptors and inflammation
.
Purinergic Signal
.
2016
;
12
(
1
):
59
67
.
26.
Coutinho-Silva
R
,
Savio
LEB
.
Purinergic signalling in host innate immune defence against intracellular pathogens
.
Biochem Pharmacol
.
2021
;
187
:
114405
.
27.
Rolf
MG
,
Brearley
CA
,
Mahaut-Smith
MP
.
Platelet shape change evoked by selective activation of P2X1 purinoceptors with α,β-methylene ATP
.
Thromb Haemost
.
2001
;
85
(
02
):
303
8
.
28.
Hardy
AR
,
Jones
ML
,
Mundell
SJ
,
Poole
AW
.
Reciprocal cross-talk between P2Y1and P2Y12receptors at the level of calcium signaling in human platelets
.
Blood
.
2004
;
104
(
6
):
1745
52
.
29.
Ren
H
,
Teng
Y
,
Tan
B
,
Zhang
X
,
Jiang
W
,
Liu
M
, et al
.
Toll-like receptor-triggered calcium mobilization protects mice against bacterial infection through extracellular ATP release
.
Infect Immun
.
2014
;
82
(
12
):
5076
85
.
30.
Erhardt
JA
,
Toomey
JR
,
Douglas
SA
,
Johns
DG
.
P2X1 stimulation promotes thrombin receptor-mediated platelet aggregation
.
J Thromb Haemost
.
2006
;
4
(
4
):
882
90
.
31.
Jones
S
,
Evans
RJ
,
Mahaut-Smith
MP
.
Ca2+ influx through P2X1 receptors amplifies P2Y1 receptor-evoked Ca2+ signaling and ADP-evoked platelet aggregation
.
Mol Pharmacol
.
2014
;
86
(
3
):
243
51
.
32.
Trier
DA
,
Gank
KD
,
Kupferwasser
D
,
Yount
NY
,
French
WJ
,
Michelson
AD
, et al
.
Platelet antistaphylococcal responses occur through P2X1 and P2Y12 receptor-induced activation and kinocidin release
.
Infect Immun
.
2008
;
76
(
12
):
5706
13
.
33.
Shannon
O
.
The role of platelets in sepsis
.
Res Pract Thromb Haemost
.
2021
;
5
(
1
):
27
37
.
34.
Jahn
K
,
Shumba
P
,
Quach
P
,
Müsken
M
,
Wesche
J
,
Greinacher
A
, et al
.
Group B streptococcal hemolytic pigment impairs platelet function in a two-step process
.
Cells
.
2022
;
11
(
10
):
1637
.
35.
Kaul
R
,
McGeer
A
,
Low
DE
,
Green
K
,
Schwartz
B
,
Simor
AE
.
Population-based surveillance for group A streptococcal necrotizing fasciitis: clinical features, prognostic indicators, and microbiologic analysis of seventy-seven cases. Ontario Group A Streptococcal Study
.
Am J Med
.
1997
;
103
(
1
):
18
24
.
36.
Timmer
AM
,
Timmer
JC
,
Pence
MA
,
Hsu
LC
,
Ghochani
M
,
Frey
TG
, et al
.
Streptolysin O promotes group A Streptococcus immune evasion by accelerated macrophage apoptosis
.
J Biol Chem
.
2009
;
284
(
2
):
862
71
.
37.
Datta
V
,
Myskowski
SM
,
Kwinn
LA
,
Chiem
DN
,
Varki
N
,
Kansal
RG
, et al
.
Mutational analysis of the group A streptococcal operon encoding streptolysin S and its virulence role in invasive infection
.
Mol Microbiol
.
2005
;
56
(
3
):
681
95
.
38.
Shumba
P
,
Sura
T
,
Moll
K
,
Chakrakodi
B
,
Tölken
LA
,
Hoßmann
J
, et al
.
Neutrophil-derived reactive agents induce a transient SpeB negative phenotype in Streptococcus pyogenes
.
J Biomed Sci
.
2023
;
30
(
1
):
52
.
39.
Tölken
LA
,
Paulikat
AD
,
Jachmann
LH
,
Reder
A
,
Salazar
MG
,
Medina
LMP
, et al
.
Reduced interleukin-18 secretion by human monocytic cells in response to infections with hyper-virulent Streptococcus pyogenes
.
J Biomed Sci
.
2024
;
31
(
1
):
26
.
40.
Siemens
N
,
Kittang
BR
,
Chakrakodi
B
,
Oppegaard
O
,
Johansson
L
,
Bruun
T
, et al
.
Increased cytotoxicity and streptolysin O activity in group G streptococcal strains causing invasive tissue infections
.
Sci Rep
.
2015
;
5
:
16945
.
41.
Mairpady Shambat
S
,
Chen
P
,
Nguyen Hoang
AT
,
Bergsten
H
,
Vandenesch
F
,
Siemens
N
, et al
.
Modelling staphylococcal pneumonia in a human 3D lung tissue model system delineates toxin-mediated pathology
.
Dis Model Mech
.
2015
;
8
(
11
):
1413
25
.
42.
Tamang
HK
,
Stringham
EN
,
Tourdot
BE
.
Platelet functional testing via high-throughput microtiter plate-based assays
.
Curr Protoc
.
2023
;
3
(
2
):
e668
.
43.
Madsen
MB
,
Bergsten
H
,
Norrby-Teglund
A
.
Treatment of necrotizing soft tissue infections: IVIG
.
Adv Exp Med Biol
.
2020
;
1294
:
105
25
.
44.
Varga-Szabo
D
,
Braun
A
,
Nieswandt
B
.
Calcium signaling in platelets
.
J Thromb Haemost
.
2009
;
7
(
7
):
1057
66
.
45.
Jardin
I
,
Ben Amor
N
,
Bartegi
A
,
Pariente
JA
,
Salido
GM
,
Rosado
JA
.
Differential involvement of thrombin receptors in Ca2+ release from two different intracellular stores in human platelets
.
Biochem J
.
2007
;
401
(
1
):
167
74
.
46.
Hülsmann
M
,
Nickel
P
,
Kassack
M
,
Schmalzing
G
,
Lambrecht
G
,
Markwardt
F
.
NF449, a novel picomolar potency antagonist at human P2X1 receptors
.
Eur J Pharmacol
.
2003
;
470
(
1–2
):
1
7
.
47.
Kassack
MU
,
Braun
K
,
Ganso
M
,
Ullmann
H
,
Nickel
P
,
Böing
B
, et al
.
Structure–activity relationships of analogues of NF449 confirm NF449 as the most potent and selective known P2X1 receptor antagonist
.
Eur J Med Chem
.
2004
;
39
(
4
):
345
57
.
48.
Svensson
L
,
Baumgarten
M
,
Mörgelin
M
,
Shannon
O
.
Platelet activation by Streptococcus pyogenes leads to entrapment in platelet aggregates, from which bacteria subsequently escape
.
Infect Immun
.
2014
;
82
(
10
):
4307
14
.
49.
Parks
T
,
Wilson
C
,
Curtis
N
,
Norrby-Teglund
A
,
Sriskandan
S
.
Polyspecific intravenous immunoglobulin in clindamycin-treated patients with streptococcal toxic shock syndrome: a systematic review and meta-analysis
.
Clin Infect Dis
.
2018
;
67
(
9
):
1434
6
.
50.
Senda
A
,
Endo
A
,
Fushimi
K
,
Otomo
Y
.
Effectiveness of intravenous immunoglobulin therapy for invasive group A Streptococcus infection: a Japanese nationwide observational study
.
Int J Infect Dis
.
2023
;
135
:
84
90
.
51.
Chaigne
B
,
Mouthon
L
.
Mechanisms of action of intravenous immunoglobulin
.
Transfus Apher Sci
.
2017
;
56
(
1
):
45
9
.
52.
Tarnutzer
A
,
Andreoni
F
,
Keller
N
,
Zürcher
C
,
Norrby-Teglund
A
,
Schüpbach
RA
, et al
.
Human polyspecific immunoglobulin attenuates group A streptococcal virulence factor activity and reduces disease severity in a murine necrotizing fasciitis model
.
Clin Microbiol Infect
.
2019
;
25
(
4
):
512.e7
13
.
53.
Chandrasekaran
S
,
Caparon
MG
.
The NADase-negative variant of the Streptococcus pyogenes toxin NAD(+) glycohydrolase induces JNK1-mediated programmed cellular necrosis
.
mBio
.
2016
;
7
(
1
):
e02215-15
.
54.
Hancz
D
,
Westerlund
E
,
Bastiat-Sempe
B
,
Sharma
O
,
Valfridsson
C
,
Meyer
L
, et al
.
Inhibition of inflammasome-dependent interleukin 1β production by streptococcal NAD+-Glycohydrolase: evidence for extracellular activity
.
mBio
.
2017
;
8
(
4
):
e00756-17
.
55.
Cawley
MJ
,
Briggs
M
,
Haith
LR
Jr
,
Reilly
KJ
,
Guilday
RE
,
Braxton
GR
, et al
.
Intravenous immunoglobulin as adjunctive treatment for streptococcal toxic shock syndrome associated with necrotizing fasciitis: case report and review
.
Pharmacotherapy
.
1999
;
19
(
9
):
1094
8
.
56.
He
W
,
Wu
C
,
Zhong
Y
,
Li
J
,
Wang
G
,
Yu
B
, et al
.
Case report: therapeutic strategy with delayed debridement for culture-negative invasive group A streptococcal infections diagnosed by metagenomic next-generation sequencing
.
Front Public Health
.
2022
;
10
:
899077
.
57.
Erhardt
JA
,
Pillarisetti
K
,
Toomey
JR
.
Potentiation of platelet activation through the stimulation of P2X1 receptors
.
J Thromb Haemost
.
2003
;
1
(
12
):
2626
35
.
58.
Yamada
T
,
Yamamori
Y
,
Matsuda
N
,
Nagamune
H
,
Ohkura
K
,
Tomoyasu
T
, et al
.
Streptolysin S induces pronounced calcium-ion influx-dependent expression of immediate early genes encoding transcription factors
.
Sci Rep
.
2023
;
13
(
1
):
13720
.
59.
Jahn
K
,
Handtke
S
,
Palankar
R
,
Kohler
TP
,
Wesche
J
,
Wolff
M
, et al
.
α-hemolysin of Staphylococcus aureus impairs thrombus formation
.
J Thromb Haemost
.
2022
;
20
(
6
):
1464
75
.
60.
Hammers
DE
,
Donahue
DL
,
Tucker
ZD
,
Ashfeld
BL
,
Ploplis
VA
,
Castellino
FJ
, et al
.
Streptolysin S targets the sodium-bicarbonate cotransporter NBCn1 to induce inflammation and cytotoxicity in human keratinocytes during Group A Streptococcal infection
.
Front Cell Infect Microbiol
.
2022
;
12
:
1002230
.
61.
Hechler
B
,
Magnenat
S
,
Zighetti
ML
,
Kassack
MU
,
Ullmann
H
,
Cazenave
J-P
, et al
.
Inhibition of platelet functions and thrombosis through selective or nonselective inhibition of the platelet P2 receptors with increasing doses of NF449 [4,4′,4″,4″′-(Carbonylbis(imino-5,1,3-benzenetriylbis-(carbonylimino)))tetrakis-benzene-1,3-disulfonic acid octasodium salt]
.
J Pharmacol Exp Ther
.
2005
;
314
(
1
):
232
43
.