Skip to main content
NCBI home page
Research and Practice in Thrombosis and Haemostasis logoLink to Research and Practice in Thrombosis and Haemostasis
. 2026 Feb 13;10(2):103395. doi: 10.1016/j.rpth.2026.103395

Platelet desialylation, apoptosis, and T-lymphocyte-mediated immune dysregulation: unveiling the pathways of platelet clearance in platelet transfusion refractoriness

Yan Zhou 1,∗, Zhoulin Zhong 1, Huihui Mo 1, Liyang Liang 1, Changshan Su 1, Ying Chen 1, Fang Lu 1, Yuchen Huang 1, Guoguang Wu 1
PMCID: PMC12969729  PMID: 41810335

Abstract

Background

Platelet transfusion refractoriness (PTR) is a clinical challenge that can be classified into nonimmunologic and immunologic types, the latter resulting from both platelet alloimmunization and autoimmunity. Recent studies have indicated that platelet clearance can occur without detectable alloantibodies, through mechanisms such as desialylation, apoptosis, and T-lymphocyte dysfunction. In our previous study, platelet desialylation was prevalent in PTR. However, the interplay among desialylation, apoptosis, alloantibodies, and T-lymphocyte dysfunction in platelet clearance remains unclear.

Objectives

To elucidate the underlying mechanisms of PTR, focusing on the complex interactions among T-lymphocytes, desialylation, and apoptosis.

Methods

RCA-I and Neu1 expression on platelets was measured in patients with PTR and in healthy donors. The capacity of sera from patients with PTR categorized as anti-HLA positive, anti-CD36 positive, or with no detectable antibodies and their corresponding IgG fractions to induce platelet desialylation and apoptosis was assessed. The effects of Fc gamma receptor inhibitors on platelet desialylation and apoptosis were evaluated. T-lymphocyte populations and cytokine levels were investigated.

Results

RCA-I and Neu1 expression on platelets did not differ significantly between patients with and without detectable antibodies (P > .05). Patients with high desialylation exhibited lower platelet counts (P < .05), independent of platelet alloantibodies. Sera from patients induced desialylation (P < .05), whereas sera containing anti-CD36 antibodies resulted in apoptosis (P < .05). Sera and IgG fractions induced desialylation and apoptosis without significant differences in vitro (P > .05). In PTR, CD4+ T-cell count decreased, CD8+ T-cell count increased, serum interleukin-10 levels increased, and transforming growth factor-β 1 levels decreased.

Conclusion

Platelet desialylation, apoptosis, and T-lymphocyte-mediated immune dysregulation jointly contribute to PTR pathogenesis.

Keywords: alloantibody, apoptosis, desialylation, platelet transfusion refractoriness (PTR), T-lymphocytes

Essentials

  • •

    Platelet transfusion refractoriness poses a challenge in clinical transfusion.

  • •

    We studied how desialylation, apoptosis, and T-lymphocytes interplay in patients with PTR.

  • •

    High platelet desialylation is linked to lower platelet counts, regardless of alloantibodies.

  • •

    Anti-CD36 antibodies induced apoptosis/desialylation, with T-cell imbalance/cytokine shifts.

1. Introduction

Platelet transfusion refractoriness (PTR) is characterized by a persistent, insufficient posttransfusion platelet count increment following 2 or more consecutive transfusions of appropriate doses of ABO-compatible platelets [1]. The reported incidence of PTR is approximately 14% to 57.7%, presenting a significant clinical challenge [[2], [3], [4]]. With the increasing volume and frequency of platelet transfusions, PTR has become a major complication of platelet transfusion therapy in patients with hematologic disorders.

Historically, PTR has been broadly classified into 2 main types: nonimmune and immune. Nonimmune PTR can result from various clinical conditions, such as fever, infection, splenomegaly, and disseminated intravascular coagulation [5]. In contrast, immune PTR is related to platelet alloimmunization. Current paradigm suggests that clearance of opsonized platelets through the reticuloendothelial system via Fcγ-receptors (FcγR) results in thrombocytopenia and bleeding disorders [[6], [7], [8]].

In routine platelet cross-matching practice, we have repeatedly observed that, even when alloantibodies are undetectable and potential confounding factors, such as infection or fever, have been excluded, platelet transfusion therapy remains ineffective, suggesting that traditional nonimmune mechanisms alone do not fully account for platelet clearance. Additionally, even with matched platelets, some patients with PTR fail to achieve significant increments in platelet counts, suggesting that mechanisms independent of alloantibodies may also play a role.

Our previous research has demonstrated that platelet desialylation is frequently observed in patients with PTR [9]. Recent studies have identified platelet desialylation and apoptosis as important pathways in platelet clearance. These mechanisms are implicated in abnormal platelet clearance in diseases such as immune thrombocytopenia (ITP) and systemic lupus erythematosus, contributing to reduced platelet counts [[10], [11], [12], [13], [14], [15], [16]]. Additionally, abnormalities in T-lymphocyte function can lead to ITP [[17], [18], [19], [20], [21], [22]]. The suppression of T-lymphocyte activation and proliferation is likely mediated by inhibitory cytokines, specifically interleukin-10 (IL-10) and transforming growth factor-β 1 (TGF-β1), which are central to this molecular mechanism [[23], [24], [25]]. However, the role of T-lymphocyte populations and associated cytokines in PTR remains unclear.

This study aimed to elucidate the underlying mechanisms of platelet clearance in PTR and to explore the potential relationships among platelet alloantibodies, T-lymphocyte dysfunction, platelet desialylation, and apoptosis.

2. Methods

2.1. Patients and controls

In total, 101 blood samples were collected from 101 patients diagnosed with PTR from various hospitals in Nanning from October 2024 to December 2024. Patients with confounding factors, such as infection or fever were excluded. Blood samples from all patients were collected 18 to 24 hours after platelet transfusion. Additionally, 14 previously frozen serum samples containing anti-CD36 antibodies were included (stored at −80 °C). These samples were obtained from patients with PTR and preserved for further analysis. Overall, 101 samples from healthy donors were randomly selected from the Nanning Blood Center. All patients and donors included in this study provided written consent to participate. The study adhered to the principles outlined in the Declaration of Helsinki and was approved by the Ethics Committee of Nanning Blood Center (Approval number: 2023-EC-08).

2.2. Antibody characterization by enzyme-linked immunosorbent assay

Platelet alloantibodies were detected using the PakPlus enzyme-linked immunosorbent assay (ELISA) kit (Immucor GTI Diagnostics, Inc) according to the manufacturer’s instructions.

2.3. Platelet desialylation and neuraminidase expression

A total of 1 × 106 platelets were isolated from blood samples of 101 patients with PTR and 101 healthy donors using density gradient centrifugation and were resuspended in HEPES–Tyrode buffer, as described by Li et al.[10]. To detect surface β-galactose expression following platelet desialylation, fluorescein isothiocyanate (FITC)-labeled Ricinus communis agglutinin I (RCA-I, 10 μg/mL; Vector Laboratories, Burlingame) was used, because it specifically binds to β-galactose residues. The platelets were incubated with FITC-labeled RCA-I for 20 minutes at room temperature (approximately 24-26 °C), platelets were washed and analyzed via FACSCanto II flow cytometry (BD Biosciences). A total of 10,000 events were analyzed using FACS Diva software (BD Biosciences) to obtain the percentage of positive cells. Similarly, neuraminidase (Neu1) expression on the platelet surface was assessed using phycoerythrin (PE)-labeled Neu1 (1:50; Santa Cruz Biotechnology).

Owing to the limited volume of available sera, platelet desialylation induced by sera was performed using pooled platelets. Briefly, pooled platelets (1 × 106) from 10 fresh O-type apheresis donors were incubated with 20 μL of sera from patients with PTR carrying different alloantibodies (including anti-HLA antibodies, anti-CD36 antibodies, or no detectable antibodies) or donor sera at 37 °C for 30 minutes, followed by washing and incubation with FITC-labeled RCA-I, as described above. In some experiments, the IgG fraction was purified using the Melon Gel IgG Spin Purification Kit (Thermo Fisher Scientific).

2.4. Platelet apoptosis

Similarly, to assess the effect of sera from patients with PTR on platelet apoptosis, pooled platelets (1 × 106) from 10 fresh O-type apheresis donors were incubated with 20 μL sera from patients with PTR carrying different alloantibodies or donor sera at 37 °C for 30 minutes. After washing, the samples were incubated with FITC-labeled Annexin V (BD Biosciences) for 20 minutes at room temperature (approximately 24-26 °C) and analyzed via flow cytometry to assess phosphatidylserine exposure on the platelet surface. In selected experiments, purified IgG fractions from patients with PTR were used.

To investigate the role of FcγR in platelet apoptosis, platelets were pretreated with anti-FcγRIIa antibody (clone IV.3; 2.5 μg/mL; BioXCell) for 10 minutes at 37 °C before exposure to patient sera.

2.5. T-lymphocyte populations in PTR

Peripheral blood mononuclear cells were extracted from whole blood using Ficoll–Hypaque (Sigma-Aldrich) by density gradient centrifugation. After preincubated with 5 μL Fc Block (BD Biosciences) for 10 minutes at room temperature (approximately 24-26 °C), peripheral blood mononuclear cells were gently mixed with monoclonal antibodies: APC-Cy7-labeled CD3 (BD Biosciences, clone: SK7), APC-labeled CD8 (BD Biosciences, clone: HIT8α), and BB700-labeled CD4 (BD Biosciences, clone: SK3), followed by incubation for 20 minutes at room temperature (approximately 24-26 °C). The samples were washed and analyzed via FACSCanto II flow cytometry to assess the proportions of CD3+, CD4+, and CD8+ T cells.

2.6. IL-10 and TGF-β1 concentration in PTR

The serum levels of IL-10 and TGF-β1 were analyzed using the Human IL-10 Instant ELISA kit (Invitrogen) and the Human TGF-β1 ELISA kit (Invitrogen), according to the manufacturer’s instructions.

2.7. Statistical analysis

Data were presented as mean ± standard deviation (SD). Statistical analyses were conducted using GraphPad Prism 9 (version 9.0.0; GraphPad Inc), with statistical significance set at P < .05. A t-test or Mann–Whitney U-test was used for comparisons between 2 groups, whereas a one-way analysis of variance was used for comparisons among 3 or more groups. Spearman’s rank correlation test was used for the correlation analysis.

3. Results

3.1. Characteristics of platelet alloantibody in PTR

Platelet alloantibody detection in the sera of 101 patients with PTR revealed that 46.53% of patients (47/101) lacked detectable antibodies (Figure 1). Anti-HLA antibodies were detected in 48.51% of patients (49/101). Notably, anti-CD36 antibodies, which target glycoprotein IV (GPIV, also known as CD36), were detected in 0.99% of patients (1/101). Additionally, antiglycoprotein (GP) antibodies were identified in 3.96% of patients (4/101). Among these, 2 patients had anti-GPIIb/IIIa antibodies, one had anti-GPIb/IX antibodies, and one had both anti-GPIIb/IIIa and anti-GPIa/IIa antibodies.

Figure 1.

Figure 1

Open in a new tab

Percentage of platelet alloantibodies in 101 patients with PTR.

3.2. Desialylation contributed to platelet destruction in PTR

Platelet desialylation and Neu1 expression were significantly higher in patients with PTR than in healthy donors (Figure 2A, B).

Figure 2.

Figure 2

Open in a new tab

Desialylation and neuraminidase expression in patients with PTR. (A) RCA-I binding (7.64 ± 4.03 vs 19.54 ± 11.21) and (B) Neu-1 expression (1.23 ± 1.24 vs 7.85 ± 6.68) on platelets in healthy donors (n = 101) and patients with PTR (n = 101). (C) RCA-I binding (19.12 ± 10.96 vs 20.11 ± 11.33) and (D) Neu-1 expression (7.08 ± 6.25 vs 8.41 ± 7.06) on platelets in patients with PTR with no detectable antibodies (n = 47) versus those with detectable antibodies (n = 54). (E) Platelet counts (8.64 ± 8.27 vs 17.62 ± 22.51) in desialylated PTR (n = 51) versus nondesialylated PTR (n = 44). Data are presented as mean ± standard deviation (SD). ∗P < .01, ∗∗P < .0001.

The 101 patients with PTR were divided into 2 groups according to the presence of platelet alloantibody in their sera: no detectable antibody group and detectable antibody group. The results showed no difference in the expression of RCA-I and Neu1 between the 2 groups (Figure 2C, D).

Based on the mean and SD of RCA-I in 101 healthy donors, an RCA-I binding in patients with PTR greater than the x¯+2SD (7.64+2 × 4.03) indicated increased desialylation. The 101 patients with PTR were also divided into desialylation PTR and nondesialylation PTR groups. Patients with PTR exhibiting high desialylation had lower platelet counts compared with those without desialylation (Figure 2E). Table 1 presents detailed characteristics of the 2 groups. No significant differences in sex, ethnicity, blood type, or platelet alloantibody levels were observed (P > .05). However, increased desialylation was more prevalent in patients with PTR who had concomitant hematologic disorders, such as leukemia, myelodysplastic syndrome, and aplastic anemia, likely due to frequent platelet transfusions in this population (P < .05).

Table 1.

Demographic characteristics of patients with PTR.

Demographics Patients with PTR (n = 101) Nondesialylation PTR group (n = 46) Desialylation PTR group (n = 55) P value
Sex
 Female 68 34 33 .14
 Male 33 12 22
Ethnicity
 Han 54 25 29 .57
 Zhuang 37 18 19
 Miao 1 0 1
 Yao 8 3 5
 Dong 1 0 1
Blood type (2 patients were not provided)
 A 22 14 8 .06
 B 30 13 17
 O 41 13 28
 AB 6 4 2
Platelet alloantibody
 No detectable antibody 47 23 24 .52
 Detectable antibody 54 23 31
Primary disease
 Nonhematologic system diseases 26 17 9 .02
 Hematologic system diseases 75 29 46
Open in a new tab

PTR, platelet transfusion refractoriness.

3.3. Sera of patients with PTR have differential effects on platelet desialylation and apoptosis

When selecting sera for platelet desialylation and apoptosis, we prioritized samples with sufficient residual volume after clinical testing to ensure feasible and reliable experimental outcomes. Due to limitations in clinical samples, only sera with adequate volumes were included in this study, resulting in a total of 60 samples: 15 from patients with PTR with anti-HLA antibodies, 15 from patients with PTR with no detectable antibodies, 15 from healthy donors, 1 from a patient with PTR with anti-CD36 antibodies, and 14 previously stored sera at –80 °C from patients with PTR with anti-CD36 antibodies. All sera from patients with PTR induced an increase in RCA-I binding (P > .05), which was significantly higher than that observed in the healthy donor group (P < .05; Figure 3A). The anti-CD36 antibody group showed more platelet apoptosis, as assessed by Annexin V, than the other groups (P < .05). However, no significant differences were observed among the groups with anti-HLA antibodies and no detectable antibodies, and the donor group (P > .05; Figure 3B). Additionally, Spearman’s rank correlation analysis revealed no significant correlation between the ability of sera from patients with PTR to trigger desialylation and apoptosis (P > .05; Figure 3C). This suggests that these 2 processes may be relatively independent of each other.

Figure 3.

Figure 3

Open in a new tab

Effects of sera and corresponding IgG from patients with PTR on platelet desialylation and apoptosis. (A) RCA-I binding (5.01 ± 1.35 vs 9.55 ± 3.53 vs 9.72 ± 5.22 vs 9.67 ± 4.55) and (B) Annexin V exposure (5.75 ± 1.58 vs 6.12 ± 4.87 vs 7.03 ± 6.03 vs 18.95 ± 20.64)induced by sera from healthy donors (n = 15), patients with PTR with no detectable antibodies (n = 15), with anti-HLA antibodies (n = 15), and with anti-CD36 antibodies (n = 15). (C) Correlation analysis of platelet apoptosis and desialylation induced by sera from patients with PTR (n = 45). (D) RCA-I binding (1.20 ± 0.21 vs 1.16 ± 0.28) and (E) Annexin V exposure (1.18 ± 0.98 vs 1.12 ± 0.51) induced by PTR sera and their corresponding purified IgG (n = 22). Data are presented as mean ± standard deviation (SD). ∗P < .05, ∗∗P < .01. FI: fold increase, defined as the ratio of RCA-I binding and Annexin V exposure on platelets incubated with PTR sera (or IgG) relative to those incubated with control sera (or IgG).

To examine whether the IgG fraction caused platelet desialylation and apoptosis, 22 serum samples, including detectable and no detectable antibodies from 101 patients with PTR, were used to obtain the purified IgG fraction. The results showed no significant difference between the sera of patients with PTR and their corresponding IgG fractions in their capacity to trigger platelet desialylation and apoptosis (P > .05; Figure 3D, E). These findings indicate that IgG antibodies are the primary effectors in inducing these effects, rather than other factors such as cytokines.

3.4. Anti-CD36 antibodies induced platelet apoptosis via FcγR

After assessing desialylation and apoptosis in 15 serum samples containing anti-CD36 antibodies, only 8 samples had sufficient residual volume for further experiments. Sera were co-incubated in vitro with platelets pretreated either with or without the FcγRIIA inhibitor (IV.3). The results showed no significant change in RCA-I binding in the presence of the FcγRIIA inhibitor (P > .05; Figure 4A). In the presence of the FcγRIIA inhibitor, Annexin V exposure on platelets was significantly reduced (P < .05; Figure 4B).

Figure 4.

Figure 4

Open in a new tab

Effects of FcγRIIA inhibitor IV.3 on platelet desialylation and apoptosis induced by sera from patients with PTR with anti-CD36 antibodies (n = 8). (A) RCA-I binding and (B) Annexin V exposure. ∗P < .01.

3.5. T-lymphocyte-mediated immune dysregulation was involved in platelet clearance in PTR

Due to limited blood sample volumes, T-lymphocyte subpopulations were analyzed in 31 patients with PTR and 31 healthy donors. In healthy donors, the proportion of CD3+ T and CD4+ T cells was higher than that in the PTR group (P < .05; Figure 5A, B), whereas the proportion of CD8+ T cells was lower than that observed in the PTR group (P < .05; Figure 5C). The concentration of TGF-β in sera was lower in patients with PTR, whereas the IL-10 concentration was higher, compared with that in healthy donors (P < .05; Table 2).

Figure 5.

Figure 5

Open in a new tab

Percentages of (A) CD3+ (54.47 ± 10.61 vs 13.12 ± 15.81), (B) CD4+ (51.05 ± 7.18 vs 43.95 ± 15.90), and (C) CD8+ (36.58 ± 5.70 vs 47.58 ± 16.87) T cells in healthy donors (n = 31) and patients with PTR (n = 31). Data are presented as mean ± standard deviation (SD). ∗P < .05, ∗∗P < .01, ∗∗∗P < .0001.

Table 2.

Comparison in detection levels of serum cytokines between healthy donors and patients with PTR.

Group Cases (n) IL-10 (pg/mL)a TGF-β1 (pg/mL)b
Donor 40 1.27 ± 1.50 198.29 ± 107.47
PTR 19 2.94 ± 2.41 55.74 ± 44.72
Open in a new tab

IL, interleukin; PTR, platelet transfusion refractoriness; TGF-β1, transforming growth factor-beta 1.

a

P < .05.

b

P < .0001.

4. Discussion

This study revealed previously unrecognized pathways involved in platelet clearance in patients with PTR, including desialylation, apoptosis, and T-lymphocyte-mediated immune dysregulation.

Several studies have demonstrated that PTR caused by anti-CD36 antibodies is frequently observed in Asian and African populations, including in China. Two types of CD36 deficiency have been identified: type I, in which CD36 is not expressed on platelets and monocytes, and type II, in which CD36 is deficient only on platelets. Individuals with type I deficiency are at risk of developing anti-CD36 isoantibodies. Our previous study showed that the frequency of CD36 deficiency was 4.13% in the Guangxi population, with type I at 1.69% and type II at 2.44%. Notably, CD36 deficiency prevalence among the Zhuang ethnic minority in Guangxi reached 5.76% [26]. Conversely, in the Kunming population, CD36 deficiency was 1.68%, comprising 0.24% and 1.44% type I and II, respectively [27]. Similarly, in the Guangzhou population, the deficiency was 1.8%, with 0.5% type I and 1.3% type II [28]. Despite these relatively low prevalence, anti-CD36 antibodies remain a clinically significant PTR risk factor, particularly in the Guangxi Zhuang Autonomous Region. Our laboratory has well documented platelet alloimmunity caused by anti-CD36 antibodies [[29], [30], [31], [32], [33], [34]]. Thus, CD36 has emerged as an important risk factor for immune-mediated thrombocytopenia in the Guangxi population [35].

PTR remains a major clinical complication, particularly in patients who require long-term transfusion support. Platelet desialylation plays an important role in this condition [9,14,36,37]. Our findings demonstrated that RCA-I binding in platelets from patients with PTR was higher than that in healthy donors, suggesting that platelet desialylation may be involved in the occurrence of PTR. Additionally, the presence of Neu1 in patients with PTR was enhanced, which may indicate that the translocation of Neu1 within platelets in patients with PTR leads to platelet desialylation.

In PTR, among 101 patients, classification based on antibody detectability revealed that desialylation occurred regardless of whether alloantibodies were detectable. As shown in Figure 2C and D, RCA-I binding and Neu 1 expression in patients with PTR were similar between the groups with and without detectable antibodies, suggesting that platelet desialylation may occur in a broader subset of patients with PTR than previously recognized. Furthermore, our study demonstrated that desialylation contributed to platelet destruction in PTR, as evidenced by lower platelet counts in the high desialylation group than in the nondesialylation group. Notably, increased desialylation was not associated with variables such as sex, ethnicity, blood type, or the presence of platelet alloantibody. This finding corroborates recent studies indicating that the reduction of sialic acid is a prevalent pathological phenomenon in patients with PTR [[38], [39], [40]].

Studies in murine models of ITP have shown that monoclonal anti-GPIb antibodies can lead to increased desialylation [10]. In humans, the mechanism of ITP appears to be more complex as anti-GPIIb/IIIa antibodies may induce platelet activation in some human platelets through an FcγRIIa-dependent mechanism [10]. Zheng et al. [36] reported that sera containing anti-GPIIb /IIIa antibodies showed a higher capacity for desialylation through neuraminidase translocation, although another study found no significant difference in desialylation between anti-GPIb and anti-GPIIb/IIIa antibodies [12]. These findings collectively highlight the diversity of immune mechanisms involved in ITP, including distinct antibody specificities that may lead to desialylation and subsequent platelet clearance. This diversity underscores the complexity of immune-mediated thrombocytopenic disorders. In the context of PTR, the immune profile of patients, such as the presence of anti-CD36 antibodies, anti-HLA antibodies, or absence of detectable antibodies, also affected the degree of platelet desialylation and apoptosis. When pooled platelets from healthy donors were incubated with PTR sera, increased desialylation was observed across all groups, including the subgroup without detectable antibodies. This suggests that PTR sera themselves, rather than any specific alloantibody, trigger platelet desialylation in vitro. Nonetheless, sera containing anti-CD36 antibodies significantly promoted platelet apoptosis, suggesting their potential clinical relevance in predicting platelet apoptosis in patients with PTR. This may be particularly significant in regions such as Guangxi, where anti-CD36 antibodies are more prevalent, and greater attention should be paid. There was no notable difference between patients’ sera and their respective IgG fractions in triggering sialic acid cleavage and phosphatidylserine exposure. This result is consistent with findings from Marini et al. [41] and Zheng et al. [36], who reported similar IgG-mediated effects in patients with thrombocytopenia.

Earlier research has shown that in ITP, the platelet desialylation ability of anti-GPIbα antibody is independent of FcγR [7,10,42]. Given the distinctive regional immunologic profile of the Guangxi population, in which anti-CD36 antibodies rank as the second most frequent cause of PTR, we investigated whether FcγR engagement modulates desialylation and apoptosis in patients with PTR induced by anti-CD36 antibodies. The induction of platelet apoptosis by these antibodies was significantly inhibited by IV.3, an FcγRIIA blocker, suggesting that anti-CD36 antibodies may trigger platelet apoptosis through FcγR signaling. However, platelet desialylation remained unaffected by IV.3, which is consistent with findings reported by Li et al. [10] and Zhang et al. [43], suggesting that platelet desialylation may involve mechanisms distinct from the FcγR pathway.

Many studies have reported that the initial trigger of ITP development may be abnormal immune regulation mediated by T-lymphocytes [17,18,22,44,45]. However, similar data in PTR are limited [6]. Our results demonstrated that CD4+ T cells in patients with PTR were reduced, whereas CD8+ T cells were increased despite their limited population. As reported by Qiu et al. [11], CD8+ T cells mediate platelet desialylation, contributing to the pathogenesis of ITP. It is plausible that the elevated CD8+ T-cell population in PTR may similarly induce desialylation. T-lymphocytes are crucial for maintaining immune balance. In patients with PTR, this balance may be disrupted, potentially triggering excessive immune reactions against platelets and resulting in their elimination. IL-10 and TGF-β1, cytokines with bidirectional immunomodulatory effects, are the main cytokines secreted by T cells and are key mediators of their immunosuppressive and anti-inflammatory effects [46,47]. An increased serum concentration of IL-10 was observed in PTR, likely exerting suppressive effects on excessive immune responses and helping prevent hyperinflammation and tissue damage [48]. Similar findings have been reported in patients with systemic lupus erythematosus [49,50] but these differ from the trends observed in ITP [51,52]. Similar to IL-10, TGF-β1 also contributes to maintaining immune homeostasis. In contrast to elevated IL-10 levels, patients with PTR exhibited decreased concentrations of TGF-β1, which is consistent with findings reported by Wang et al. [52]. The coexistence of elevated IL-10 levels and reduced TGF-β1 concentrations in patients with PTR may reflect complex immunomodulatory mechanisms or the underlying primary disease. Although these variations are intriguing, they do not necessarily allow definitive conclusions. Instead, they suggest a complex immune modulation process in these individuals.

Despite these insights, this study had some limitations. First, the PakPlus ELISA kit used in this study had limited ability to detect low-affinity antibodies and antibodies against antigens not included in its panel, such as HPA-15. Consequently, the absence of detectable antibodies in some patients with PTR could not exclude the presence of other clinically significant antibodies. Second, serum selection for experiments was based on the availability of sufficient residual volumes following clinical testing. This limited availability precluded randomized selection and prevented the replication of experiments using platelets from multiple donors. Consequently, some experiments were performed using pooled platelets from 10 fresh O-type apheresis donors. Thus, although our findings may indicate potential trends, future studies with larger, prospectively designed cohorts are needed for detailed exploration. Furthermore, the limited sample prevented the investigation of potential therapeutic interventions. For example, the impact of the neuraminidase inhibitor, 2,3-dehydro-2-deoxy-N-acetylneuraminic acid, on desialylation could not be assessed, which would have provided more direct evidence for the proposed mechanism. [53,54]. Finally, although cytokine alterations and T-cell imbalances were observed, the causal relationship between these immune parameters and desialylation or apoptosis could not be thoroughly investigated. Further mechanistic studies are required to elucidate the pathways linking immune dysfunction to platelet clearance and to identify new therapeutic targets for PTR management.

5. Conclusion

In summary, this study investigated several distinct and potentially contributory factors involved in PTR. Notably, we confirmed that platelet desialylation, a phenomenon previously associated with PTR, correlates strongly with decreased platelet counts and can occur independently of traditionally identifiable platelet alloantibodies. This study also highlighted the significant role of anti-CD36 antibodies, which are highly prevalent in the Guangxi population, in inducing marked platelet apoptosis and consequently, accelerating platelet destruction. Additionally, our findings reveal alterations in circulating T-lymphocyte populations and cytokine profiles in a proportion of patients with PTR, suggesting a potential quantitative alteration in T-cell immunity that warrants further functional investigation. Collectively, these data enhance the current understanding of the pathogenesis of PTR. Our results emphasize the multifactorial nature of this clinical condition and highlight potential therapeutic targets for addressing abnormal platelet clearance in patients with PTR.

Acknowledgments

We would like to thank Editage (www.editage.cn) for their English language editing assistance.

Funding

This work was supported by the Chinese Society of Blood Transfusion Weigao Research Foundation (grant number CSBT-WG-2023-06); and the Fourth-Cycle Medical Key Laboratory Construction Project of Nanning City.

Author Contributions

Y. Z.: writing-original draft, investigation, funding acquisition, data curation. Z.Z.: investigation, formal analysis. H.M.: methodology, resources. L.L.: supervision, conceptualization. C.S.: resources. Y.C.: data curation. F.L.: methodology. Y.H.: methodology. G.W.: writing-review and editing, funding acquisition.

Relationship Disclosure

There are no competing interests to disclose.

Footnotes

Handling Editor: Professor Michael Makris

References

  • 1.Blandin L., Dougé A., Fayard A., Bay J.O., Berlie G., Pereira B., et al. Platelet transfusion refractoriness and anti-HLA immunization. Transfusion. 2021;61:1700–1704. doi: 10.1111/trf.16358. [DOI] [PubMed] [Google Scholar]
  • 2.Saris A., Pavenski K. Human leukocyte antigen alloimmunization and alloimmune platelet refractoriness. Transfus Med Rev. 2020;34:250–257. doi: 10.1016/j.tmrv.2020.09.010. [DOI] [PubMed] [Google Scholar]
  • 3.Ma R.X., Ma Y.J., Cui Q.Y., Wu D.P., Tang X.W. Construction and validation of an assistant decision-making model for platelet transfusion refractoriness in patients with acute myeloid leukemia. Clin Appl Thromb-Hemost. 2024;30 doi: 10.1177/10760296241278345. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Liu Y.D., Liang B., Liu Y., Wei G.Q., Wu W.J., Yang L.X., et al. Cytokine release syndrome is an independent risk factor associated with platelet transfusion refractoriness after CAR-T therapy for relapsed/refractory acute lymphoblastic leukemia. Front Pharmacol. 2021;12 doi: 10.3389/fphar.2021.702152. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Song X.F., Qi J.Q., Fang K., Li X.Q., Han Y. A meta-analysis of risk factors associated with platelet transfusion refractoriness. Int J Hematol. 2023;117:863–875. doi: 10.1007/s12185-023-03557-3. [DOI] [PubMed] [Google Scholar]
  • 6.Chen X.Y., Zhao Y.H., Lv Y., Xie J. Immunological platelet transfusion refractoriness: current insights from mechanisms to therapeutics. Platelets. 2024;35 doi: 10.1080/09537104.2024.2306983. [DOI] [PubMed] [Google Scholar]
  • 7.Quach M.E., Dragovich M.A., Chen W.C., Syed A.K., Cao W.P., Liang X., et al. Fc-independent immune thrombocytopenia via mechanomolecular signaling in platelets. Blood. 2018;131:787–796. doi: 10.1182/blood-2017-05-784975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Li J., Sullivan J.A., Ni H. Pathophysiology of immune thrombocytopenia. Curr Opin Hematol. 2018;25:373–381. doi: 10.1097/MOH.0000000000000447. [DOI] [PubMed] [Google Scholar]
  • 9.Zhou Y., Liang L.Y., Su C.S., Mo H.H., Chen Y., Lu F., et al. Analysis of correlation between platelet desialylation,apoptosis and platelet alloantibody and CD8+ T cells in platelet transfusion refractoriness. J Exp Hematol. 2025;33:1138–1144. doi: 10.19746/j.cnki.issn.1009-2137.2025.04.031. [DOI] [PubMed] [Google Scholar]
  • 10.Li J., van der Wal D.E., Zhu G.H., Xu M., Yougbare I., Ma L., et al. Desialylation is a mechanism of Fc-independent platelet clearance and a therapeutic target in immune thrombocytopenia. Nat Commun. 2015;6:7737. doi: 10.1038/ncomms8737. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Qiu J.H., Liu X.N., Li X.Q., Zhang X., Han P.P., Zhou H., et al. CD8+ T cells induce platelet clearance in the liver via platelet desialylation in immune thrombocytopenia. Sci Rep. 2016;6 doi: 10.1038/srep27445. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Tao L.L., Zeng Q.S., Li J., Xu M., Wang J.J., Pan Y., et al. Platelet desialylation correlates with efficacy of first-line therapies for immune thrombocytopenia. J Hematol Oncol. 2017;10:46. doi: 10.1186/s13045-017-0413-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Quach M.E., Chen W.C., Li R.H. Mechanisms of platelet clearance and translation to improve platelet storage. Blood. 2018;131:1512–1521. doi: 10.1182/blood-2017-08-743229. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Zhang Q.H., Huang M., Thomas E.R., Wang L., Liu J., Li X., et al. The role of platelet desialylation as a biomarker in primary immune thrombocytopenia: mechanisms and therapeutic perspectives. Front Immunol. 2024;15 doi: 10.3389/fimmu.2024.1409461. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Jiang Y., Cheng Y.J., Ma S.L., Li T., Chen Z., Zuo X.X., et al. Systemic lupus erythematosus-complicating immune thrombocytopenia: from pathogenesis to treatment. J Autoimmun. 2022;132 doi: 10.1016/j.jaut.2022.102887. [DOI] [PubMed] [Google Scholar]
  • 16.Baroni Pietto M.C., Lev P.R., Glembotsky A.C., Marín Oyarzún C.P., Gomez G., Collado V., et al. Pathogenic mechanisms contributing to thrombocytopenia in patients with systemic lupus erythematosus. Platelets. 2022;33:743–754. doi: 10.1080/09537104.2021.1988547. [DOI] [PubMed] [Google Scholar]
  • 17.Semple J.W., Provan D. The immunopathogenesis of immune thrombocytopenia: T cells still take center-stage. Curr Opin Hematol. 2012;19:357–362. doi: 10.1097/MOH.0b013e3283567541. [DOI] [PubMed] [Google Scholar]
  • 18.Olsson B., Andersson P.O., Jernås M., Jacobsson S., Carlsson B., Carlsson L.M., et al. T-cell-mediated cytotoxicity toward platelets in chronic idiopathic thrombocytopenic purpura. Nat Med. 2003;9:1123–1124. doi: 10.1038/nm921. [DOI] [PubMed] [Google Scholar]
  • 19.Chow L., Aslam R., Speck E.R., Kim M., Cridland N., Webster M.L., et al. A murine model of severe immune thrombocytopenia is induced by antibody- and CD8+ T cell-mediated responses that are differentially sensitive to therapy. Blood. 2010;115:1247–1253. doi: 10.1182/blood-2009-09-244772. [DOI] [PubMed] [Google Scholar]
  • 20.Wang Z.S., Lang T., Li Y., Zhang X.Y., Abdur M., Mao M. Hypermethylation of the FOXP3 gene regulates Tregs immunodysregulation in chronic idiopathic thrombocytopenic purpura. Allergol Immunopath. 2024;52:30–37. doi: 10.15586/aei.v52i4.1091. [DOI] [PubMed] [Google Scholar]
  • 21.Jiang Y.Z., Li J.Z., Huang J., Zhang Z.C., Liu X.C., Wang N.N., et al. Targeted proteomics profiling reveals valuable biomarkers in the diagnosis of primary immune thrombocytopaenia. Br J Haematol. 2025;206:133–143. doi: 10.1111/bjh.19760. [DOI] [PubMed] [Google Scholar]
  • 22.Malik A., Sayed A.A., Han P.P., Tan M.M.H., Watt E., Constantinescu-Bercu A., et al. The role of CD8+ T-cell clones in immune thrombocytopenia. Blood. 2023;141:2417–2429. doi: 10.1182/blood.2022018380. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Ma L., Simpson E., Li J., Xuan M., Xu M., Baker L., et al. CD8+ T cells are predominantly protective and required for effective steroid therapy in murine models of immune thrombocytopenia. Blood. 2015;126:247–256. doi: 10.1182/blood-2015-03-635417. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Li J., Karakas D., Xue F., Chen Y.Y., Zhu G.H., Yucel Y.H., et al. Desialylated platelet clearance in the liver is a novel mechanism of systemic immunosuppression. Research (Wash D C) 2023;6:0236. doi: 10.34133/research.0236. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Song T.J., Zhang Y., Huang J., Liu Z.W. Transfusion-induced platelet antibodies and regulatory T cells in multiply transfused patients. J Clin Lab Anal. 2021;35 doi: 10.1002/jcla.23864. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Zhong Z.L., Shen W.D., Zhou Y., Liu J.L., Li H.Y., Lu F., et al. Characteristic and structure analysis of CD36 deficiency in Han,Zhuang and Yao population from Guangxi region in China. Chin J Blood Transfus. 2014;27:14–17. [Google Scholar]
  • 27.Lyu Q., Peng M., Chen Q., Ji X., Wang Z., Li Q., et al. Frequency and molecular basis of CD36 deficiency among platelet donors in Kunming, China. Platelets. 2023;34 doi: 10.1080/09537104.2023.2176168. [DOI] [PubMed] [Google Scholar]
  • 28.Xu X., Ye X., Xia W., Liu J., Ding H., Deng J., et al. Studies on CD36 deficiency in South China: Two cases demonstrating the clinical impact of anti-CD36 antibodies. Thromb Haemost. 2013;110:1199–1206. doi: 10.1160/TH13-05-0435. [DOI] [PubMed] [Google Scholar]
  • 29.Wu G.G., Zhou Y., Zhong Z.L., Li L.L., Liu J.L., Shen W.D. Experimental study on the platelet transfusion refractoriness mediated by the anti-CD36: four cases reports in China. Chin J Blood Transfus. 2014;27:18–21. [Google Scholar]
  • 30.Zhou Y., Li L.L., Zhong Z.L., Liu X.J., Liu J.L., Shen W.D., et al. Anti-CD36 mediated platelet transfusion refractoriness and related cases after stem cell transplantation. J Exp Hematol. 2018;26:541–546. doi: 10.7534/j.issn.1009-2137.2018.02.039. [DOI] [PubMed] [Google Scholar]
  • 31.Wu G.G., Zhou Y., Li L.L., Shen W.D., Zhong Z.L., Liu J.L. Experimental study on the four cases of fetal and neonatal alloimmune thrombocytopenia mediated with anti-CD36 antibodies. Transfus. 2014;54:21A(S11-010B) [Google Scholar]
  • 32.Zhou Y., Zhong Z.L., Lu F., Liu J.L., Li L.L., Wu G.G. Experimental study on incidence of anti-CD36 antibodies in puerperae and platelet apoptosis induced by anti-CD36antibodies. J Clin Hematol. 2021;34:531–535. doi: 10.13201/j.issn.1004-2806.2021.08.001. [DOI] [Google Scholar]
  • 33.Shen W.D., Zhou Y., Li L.L., Zhong Z.L., Wu G.G. Study on a case of neonatal alloimmune thrombocytopenia associated with anti-CD36 and CD36 deficiency frequency in Guangxi population in China. Transfus. 2015;55:120A(SP168) [Google Scholar]
  • 34.Wu G.G., Zhou Y., Li L.L., Zhong Z.L., Li H.C., Li H.Y., et al. Platelet immunology in China: research and clinical applications. Transfus Med Rev. 2017;31:118–125. doi: 10.1016/j.tmrv.2016.12.001. [DOI] [PubMed] [Google Scholar]
  • 35.Li L.L., Zhong Z.L., Zhou Y., Li H.C., Lu F., Jiang L.H., et al. Platelet donor database: a study on the specialist donor database for the patients with alloimmune thrombocytopenia in the Chinese population and the assessment of completeness. Chin Med J (Engl) 2021;135:234–236. doi: 10.1097/CM9.0000000000001561. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Zheng S.S., Ahmadi Z., Leung H.H.L., Wong R., Yan F., Perdomo J.S., et al. Antiplatelet antibody predicts platelet desialylation and apoptosis in immune thrombocytopenia. Haematologica. 2022;107:2195–2205. doi: 10.3324/haematol.2021.279751. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Kuter D.J. Novel therapies for immune thrombocytopenia. Br J Haematol. 2022;196:1311–1328. doi: 10.1111/bjh.17872. [DOI] [PubMed] [Google Scholar]
  • 38.Zheng S.S., Perdomo J.S. Desialylation and apoptosis in immune thrombocytopenia: implications for pathogenesis and treatment. Curr Issues Mol Biol. 2024;46:11942–11956. doi: 10.3390/cimb46110709. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.An O., Deppermann C. Platelet lifespan and mechanisms for clearance. Curr Opin Hematol. 2024;31:6–15. doi: 10.1097/MOH.0000000000000792. [DOI] [PubMed] [Google Scholar]
  • 40.Chen Y.W., Luo L.P., Zheng Y.Z., Zheng Q.Y., Zhang N., Gan D.H., et al. Association of platelet desialylation and circulating follicular helper T cells in patients with thrombocytopenia. Front Immunol. 2022;13 doi: 10.3389/fimmu.2022.810620. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Marini I., Zlamal J., Faul C., Holzer U., Hammer S., Pelzl L., et al. Autoantibody-mediated desialylation impairs human thrombopoiesis and platelet lifespan. Haematologica. 2021;106:196–207. doi: 10.3324/haematol.2019.236117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Zhou J., Xu Y.Y., Shu J.H., Jiang H.J., Huang L.L., Xu M., et al. GPIbα CAAR T cells function like a Trojan horse to eliminate autoreactive B cells to treat immune thrombocytopenia. Haematologica. 2024;109:2256–2270. doi: 10.3324/haematol.2023.283874. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Zhang S., Sun C.L., Huang Q.X., Du J.H., Xia Y., Zhou K.X., et al. The role of protein kinase C and the glycoprotein Ibα cytoplasmic tail in anti-glycoprotein Ibα antibody-induced platelet apoptosis and thrombocytopenia. Thromb Res. 2024;244 doi: 10.1016/j.thromres.2024.109210. [DOI] [PubMed] [Google Scholar]
  • 44.Han P.P., Hou Y., Zhao Y.J., Liu Y., Yu T.S., Sun Y.Q., et al. Low-dose decitabine modulates T-cell homeostasis and restores immune tolerance in immune thrombocytopenia. Blood. 2021;138:674–688. doi: 10.1182/blood.2020008477. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Vrbensky J.R., Nazy I., Clare R., Larché M., Arnold D.M. T cell-mediated autoimmunity in immune thrombocytopenia. Eur J Haematol. 2022;108:18–27. doi: 10.1111/ejh.13705. [DOI] [PubMed] [Google Scholar]
  • 46.Cañete P.F., Sweet R.A., Gonzalez-Figueroa P., Papa I., Ohkura N., Bolton H., et al. Regulatory roles of IL-10-producing human follicular T cells. J Exp Med. 2019;216:1843–1856. doi: 10.1084/jem.20190493. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Kapur R., Semple J.W. Platelet TGF-β triggers immunosuppression in ITP. Blood. 2024;144:7–8. doi: 10.1182/blood.2024024825. [DOI] [PubMed] [Google Scholar]
  • 48.Ouyang W.J., O’Garra A. IL-10 family cytokines IL-10 and IL-22: from basic science to clinical translation. Immunity. 2019;50:871–891. doi: 10.1016/j.immuni.2019.03.020. [DOI] [PubMed] [Google Scholar]
  • 49.Żak-Gołąb A., Cieślik P., Siekiera U., Kuśmierz D., Hrycek A., Holecki M. The impact of the IL-10 gene polymorphism on mRNA expression and IL-10 serum concentration in Polish lupus patients. Int J Mol Sci. 2024;25:5511. doi: 10.3390/ijms25105511. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50.Talaat R.M., Alrefaey S.A., Bassyouni I.H., Ashour M.E., Raouf A.A. Genetic polymorphisms of interleukin 6 and interleukin 10 in Egyptian patients with systemic lupus eythematosus. Lupus. 2016;25:255–264. doi: 10.1177/0961203315615219. [DOI] [PubMed] [Google Scholar]
  • 51.Liu Q.F., Liu Y. Role of IL-10 and IL-22 cytokines in patients with primary immune thrombocytopenia and their clinical significance. J Clin Lab Anal. 2022;36 doi: 10.1002/jcla.24573. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 52.Wang X.F., Li F., Li Y., Sun L.H., Meng Y.H., Fan X.H., et al. Decreased levels of immune-regulatory cytokines in patients with immune thrombocytopenia and long-lasting overexpression of these cytokines in the splenectomized patients. J Leukocyte Biol. 2021;110:335–341. doi: 10.1002/JLB.5AB0521-621RR. [DOI] [PubMed] [Google Scholar]
  • 53.Liu L.Y., Zhang B., Song C.D., Li P.F., Yang M., Ren X.Q., et al. Successful treatment with oseltamivir phosphate in children with ITP who failed first-line therapy: a case series report. Ann Hematol. 2024;103:405–408. doi: 10.1007/s00277-023-05581-z. [DOI] [PubMed] [Google Scholar]
  • 54.Revilla N., Corral J., Miñano A., Mingot-Castellano M.E., Campos R.M., Velasco F., et al. Multirefractory primary immune thrombocytopenia; targeting the decreased sialic acid content. Platelets. 2019;30:743–751. doi: 10.1080/09537104.2018.1513476. [DOI] [PubMed] [Google Scholar]

Articles from Research and Practice in Thrombosis and Haemostasis are provided here courtesy of Elsevier

RESOURCES