Article Text

Original research
Antiplatelet effects of hydroxychloroquine in patients with systemic lupus erythematosus evaluated by the total thrombus-formation analysis system (T-TAS)
  1. Daisuke Hiraoka1,
  2. Jun Ishizaki1,
  3. Jun Yamanouchi1,2,
  4. Takatsugu Honda3,
  5. Toshiyuki Niiya3,
  6. Erika Horimoto1,
  7. Kenta Horie1,
  8. Hitoshi Yamasaki4,
  9. Takuya Matsumoto1,
  10. Koichiro Suemori1,
  11. Hitoshi Hasegawa5 and
  12. Katsuto Takenaka1
  1. 1Department of Hematology, Clinical Immunology and Infectious Diseases, Ehime University Graduate School of Medicine, Toon, Japan
  2. 2Division of Blood Transfusion and Cell Therapy, Ehime University Hospital, Toon, Japan
  3. 3Clinical Laboratory Department, Ehime University Hospital, Toon, Japan
  4. 4Internal medicine, Saiseikai Imabari Hospital, Imabari, Japan
  5. 5Rheumatic Center, Ozu Memorial Hospital, Ozu, Japan
  1. Correspondence to Dr Jun Ishizaki; ishizaki{at}m.ehime-u.ac.jp

Abstract

Objectives Hydroxychloroquine (HCQ) has been shown to reduce thrombotic events in patients with SLE. However, the antiplatelet effects of HCQ are only supported by the platelet aggregation assay, which is a non-physiological test. The total thrombus-formation analysis system (T-TAS) is a microchip-based flow chamber system that mimics physiological conditions and allows for the quantitative analysis of thrombogenicity. The present study investigated the antiplatelet effects of HCQ using T-TAS.

Methods This was a single-centre cross-sectional study on 57 patients with SLE. We measured the area under the pressure curve for 10 min (PL-AUC10) and the time to 10 kPa (T10) in patients with SLE using T-TAS and examined their relationships with the use of HCQ. PL-AUC10 and platelet aggregation were also measured at several HCQ concentrations using blood samples from healthy donors.

Results PL-AUC10 was significantly lower in the HCQ/real body weight (RBW) ≥5 mg/kg group than in the <5 mg/kg group, while T10 was similar, indicating that HCQ inhibited overall thrombus formation rather than the initiation of thrombus formation. The antiplatelet effects of HCQ were initially detected at HCQ/RBW of approximately 4 mg/kg and reached a plateau at around 5.5 mg/kg. The administration of HCQ/RBW >4.6 mg/kg clearly exerted antiplatelet effects. Additionally, HCQ inhibited thrombus formation in T-TAS and the platelet aggregation response to epinephrine in a dose-dependent manner.

Conclusions We demonstrated the antiplatelet effects of HCQ under conditions simulating the physiological environment by using T-TAS and identified the range of doses at which HCQ exerted antiplatelet effects.

  • Lupus Erythematosus, Systemic
  • Cardiovascular Diseases
  • Myocardial infarction

Data availability statement

Data are available upon reasonable request. Data that support the present results are available from the corresponding author on reasonable request.

http://creativecommons.org/licenses/by-nc/4.0/

This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/.

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WHAT IS ALREADY KNOWN ON THIS TOPIC

  • Hydroxychloroquine (HCQ) has been shown to reduce thrombotic events in patients with SLE.

  • The antiplatelet effects of HCQ are supported by the platelet aggregation assay, which is a non-physiological test.

  • The total thrombus-formation analysis system (T-TAS) is a microchip-based flow chamber system that mimics physiological conditions and allows for the quantitative analysis of thrombogenicity and evaluations of the therapeutic efficacy of antiplatelet drugs.

WHAT THIS STUDY ADDS

  • We demonstrated, for the first time, the antiplatelet effects of HCQ under conditions simulating the physiological environment by using T-TAS.

  • The results of T-TAS indicated that HCQ inhibited overall thrombus formation rather than the initiation of thrombus formation.

  • The association analysis between the area under a flow pressure curve for 10 min (PL-AUC10) and the dose of HCQ suggested that the antiplatelet effects of HCQ were initially detected at HCQ/real body weight (RBW) of 4 mg/kg and reached a plateau around 5.5 mg/kg, while the administration of HCQ/RBW >4.6 mg/kg clearly exerted antiplatelet effects.

  • HCQ inhibited thrombus formation in T-TAS and the platelet aggregation response to epinephrine in a dose-dependent manner.

HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY

  • The identification of an ideal PL-AUC10 for the prevention of cardiovascular events may lead to future therapeutic strategies that prevent cardiovascular disease based on the optimal use of HCQ and antiplatelet agents.

Introduction

SLE is an autoimmune disease that is characterised by systemic and organ-targeted clinical manifestations due to extensive immune system dysregulation.1 Despite advances in SLE therapeutics, the mortality rate among patients with SLE remains twofold to threefold higher than that of the general population.2 3 Cardiovascular disease (CVD) is a leading cause of death in patients with SLE.3 Therefore, thromboprophylaxis is crucial for the management of patients with SLE.

The antimalarial medication, hydroxychloroquine (HCQ), is the mainstay treatment for SLE because it reduces SLE flares by 50%4 and prolongs the long-term survival of patients with SLE.5–7 HCQ reduces disease activity8–11 and glucocorticoid doses,5 10 12 both of which are SLE-related CVD risk factors.13 Therefore, HCQ is recommended for all patients unless contraindicated.14 Previous studies reported the protective effects of HCQ against thrombovascular events in patients with SLE with or without antiphospholipid antibodies.15–17 The antiplatelet effects of HCQ are supported by its inhibition of platelet aggregation in the platelet aggregation assay.18–21 However, the platelet aggregation assay is regarded as a non-physiological test due to its evaluation of aggregate formation induced by platelet aggregating agents without shear stress.22

The total thrombus-formation analysis system (T-TAS; Fujimori Kogyo, Yokohama, Japan) allows thrombogenicity in whole blood to be evaluated under flow conditions by applying whole blood to a microchip and recording flow pressure curves.23 T-TAS analyses thrombus formation using two microchips with different thrombogenic surfaces; a platelet (PL) chip for the evaluation of primary haemostatic ability and an atheroma (AR) chip for the assessment of fibrin-rich platelet thrombus formation.23 The PL chip contains collagen-coated capillaries, and its utility has been demonstrated not only in the assessment of platelet function24 25 but also in evaluations of the therapeutic efficacy of antiplatelet drugs.26 27 The principal advantage of T-TAS is its capability to evaluate thrombogenicity under conditions that closely resemble the physiological environment, which is unattainable with the platelet aggregation assay.

The antiplatelet effects of HCQ have not yet been examined using an ex vivo analysis mimicking physiological conditions, such as T-TAS. Therefore, the present study investigated the antiplatelet effects of HCQ in patients with SLE using T-TAS.

Methods

Study design and subjects

This was a single-centre, cross-sectional study. We collected whole blood samples from 57 patients with SLE and 10 healthy donors between December 2022 and January 2024. Written informed consent was obtained from all patients and healthy donors prior to participating in the study. The trial was conducted in accordance with the Declaration of Helsinki.

All patients fulfilled the revised American College of Rheumatology classification criteria or the Systemic Lupus International Collaborating Clinics classification criteria.28 29 We verbally confirmed that all patients adhered to the prescribed HCQ dose every day for at least 1 month before the T-TAS assay. Additionally, we assessed medication adherence using the Morisky-Green-Levine Scale (MGLS), and all patients demonstrated high adherence. Patients who were treated with antiplatelet agents were excluded. Patients with active infectious diseases, malignancies, pregnancy and menstruating at the time of blood collection were also excluded. Moreover, patients with a platelet count ≤130×109/L or a haematocrit (Ht) <25% were excluded because the analytical conditions of T-TAS are not suitable for samples with low platelet counts or low Ht levels.30 31 Information such as weight, height and medication usage was recorded at the time of blood collection.

Healthy donors were recruited in Ehime University Hospital. Among healthy donors, there were no individuals with a history of bleeding disorders and none were pregnant, menstruating or taking antiplatelet agents.

Assessment of platelet thrombus formation in patients with SLE using T-TAS

The PL chip was used to assess the effects of HCQ on primary haemostasis. Blood was collected by venous puncture into 3 mL benzylsulfonyl-d-Arg-Pro-4-amidinobenzylamide (BAPA) tubes (Fujimori Kogyo, Tokyo, Japan). BAPA functions as an anticoagulant that inhibits thrombin and factor Xa, blocking the coagulation cascade and allowing PL assays to specifically measure the platelet thrombus formation process only (primary haemostasis).32 According to the T-TAS user’s manual, measurements are conducted after 1 hour for platelet resting and within 3 hours after the collection of whole blood samples. A whole blood sample is pumped into the PL chip at a shear rate of 1500 s-1, which corresponds to the arterial wall shear rate.27 Platelets then adhere to and aggregate on the collagen surface of the capillaries in the PL chip, causing capillary occlusion. The area under a flow pressure curve for 10 min (PL-AUC10) and the time to 10 kPa (T10) were assessed. PL-AUC10 and T10 indicate total thrombogenicity and the onset of platelet thrombus formation, respectively.31

Measurement of PL-AUC10 and platelet aggregation at different HCQ concentrations using blood samples from healthy donors

Whole blood samples were collected by venous puncture into 3 mL BAPA tubes from 10 healthy donors. HCQ (FUJIFILM Wako Chemical Corporation, Tokyo, Japan) was dissolved in phosphate-buffered saline (PBS) and added to whole blood samples to achieve HCQ concentrations of 1, 2 and 10 µg/mL. An equal amount of PBS was added to samples to prepare HCQ 0 µg/mL. Since measurements need to be conducted within 3 hours of blood collection, the incubation time of samples and HCQ was set at 2 hours at room temperature. PL-AUC10 were assessed as described above.

Whole blood samples were also collected by venous puncture into 6 mL tubes containing 3.2% citrate from seven healthy donors. HCQ dissolved in PBS was added to whole blood samples to achieve HCQ concentrations of 1 and 10 µg/mL. An equal amount of PBS was added to samples to prepare HCQ 0 µg/mL. Since measurements need to be conducted within 4 hours after blood collection,33 the incubation time of samples and HCQ was set at 1 hour at room temperature. Samples were then centrifuged at 200×g at room temperature for 10 min to prepare platelet-rich plasma (PRP). Samples were further centrifuged at 1500×g for 15 min to prepare platelet-poor plasma (PPP). The platelet aggregation assay was performed on CN-6500 (SYSMEX CORPORATION, Hyogo, Japan) using light transmission aggregometry, with 0% light transmittance set with PRP and 100% transmittance set with PPP. Platelet-aggregating agents and their final concentrations were as follows: epinephrine 10 µmol/L, ADP 2 and 10 µmol/L and collagen 2 µg/mL. All agents were purchased from SYSMEX CORPORATION and their final concentrations were set according to the protocol provided by SYSMEX CORPORATION. Results were expressed as maximum aggregation intensity (%).

Statistical analysis

Values are expressed as medians and IQRs or as numbers and percentages. To investigate the relationship between T-TAS measurements and the dose of HCQ, patients with SLE administered HCQ (HCQ users) were divided into two groups using a cut-off value for HCQ/real body weight (RBW) of 5 mg/kg, which is the target dose of HCQ in the 2023 EULAR (European Alliance of Associations for Rheumatology) recommendations.14 Continuous non-parametric values were compared using the Mann-Whitney U test between two groups and the Kruskal-Wallis test and Dunn’s multiple comparison test among the three groups. Categorical values were compared using Fisher’s direct probability test between the groups. Correlational analyses were performed using Spearman’s rank correlation. We used restricted cubic splines (RCS) to examine the non-linear relationship between HCQ/RBW and PL-AUC10. We defined the lowest tertile of PL-AUC10 among HCQ users as an obvious antiplatelet effect of HCQ. The validity of this definition was evaluated with the receiver operating characteristic (ROC) curve to discriminate between HCQ users with the obvious antiplatelet effect and HCQ non-users, and T-TAS microscopic images of thrombus formation. The ROC curve was constructed using a logistic regression to evaluate the validity of the definition of the obvious antiplatelet effect and to identify the optical cut-off point discerning this effect among HCQ users. The cut-off point was defined as the maximum sum of sensitivity and specificity. Differences within paired groups were examined using the Wilcoxon signed-rank test. P values <0.05 were considered to be significant. All analyses were performed using GraphPad PRISM V.9.4.1 (GraphPad Software, San Diego, California, USA).

Results

Patient characteristics

Patient characteristics are shown in table 1. The present study included 57 Japanese patients with SLE. The median age of patients was 44 years (IQR 36–56), with 49 females and 8 males, and the median disease duration was 10.2 years (IQR 3.3–18.8). Lupus disease activity at the time of blood collection was assessed by the Systemic Lupus Erythematosus Diseases Activity Index 2000,34 with a median score of 1 (IQR 0–2).

Table 1

Patient characteristics

No significant differences were observed in characteristics between patients with SLE treated with (HCQ users, n=39) and without (HCQ non-users, n=18) HCQ (table 1). Furthermore, no significant differences were noted in characteristics between the HCQ/RBW <5 mg/kg group (n=25) and ≥5 mg/kg group (n=14), except for the administration of belimumab (BLM) (table 1). When HCQ users were divided using the cut-off for HCQ/ideal body weight (IBW) of 5 mg/kg, no significant differences were noted in their characteristics (online supplemental table 1).

Supplemental material

Relationship between T-TAS measurements and the administered dose of HCQ in patients with SLE

To investigate the relationship between T-TAS measurements and the administered dose of HCQ, PL-AUC10 and T10 were compared among three groups of patients with SLE: HCQ non-users (n=18) and the HCQ/RBW <5 mg/kg (n=25) and ≥5 mg/kg (n=14) groups. As shown in figure 1A, the Kruskal-Wallis test revealed a significant difference in PL-AUC10 among the three groups (p=0.0009). No significant difference in PL-AUC10 was noted between HCQ non-users and the HCQ/RBW <5 mg/kg group (median PL-AUC10, 377.5 vs 380.4, p=1.0). PL-AUC10 was significantly lower in the HCQ/RBW ≥5 mg/kg group than in the <5 mg/kg group (median PL-AUC10, 283.3 vs 380.4, p=0.001). The Kruskal-Wallis test showed no significant differences in T10 among the three groups (p=0.19) (figure 1B). Based on these results, we focused on PL-AUC10 in subsequent analyses.

Figure 1

Comparison of T-TAS measurements by HCQ dosages in patients with SLE. (A) Bar graph of PL-AUC10 among HCQ non-users and the HCQ/RBW <5 and ≥5 mg/kg groups. (B) Bar graph of T10 among HCQ non-users and the HCQ/RBW <5 and ≥5 mg/kg groups. AUC, area under the curve; HCQ, hydroxychloroquine; PL, platelet; RBW, real body weight; T10, time to 10 kPa; T-TAS, total thrombus-formation analysis system. Data represent the median±IQR. Comparisons were performed using the Kruskal-Wallis test and Dunn’s multiple comparison.

We examined the relationship between PL-AUC10 and HCQ/RBW. A correlation was detected between HCQ/RBW and PL-AUC10 (rs=−0.47, p=0.002). When RCS were fit between HCQ/RBW and PL-AUC10, a smooth curve was obtained (figure 2). This curve indicated that PL-AUC10 started to decrease at HCQ/RBW of approximately 4 mg/kg and reached a plateau at around 5.5 mg/kg. PL-AUC10 was significantly lower in HCQ users with the lowest tertile of PL-AUC10, which was defined as an obvious antiplatelet effect in the present study, than in HCQ non-users (median PL-AUC10 (IQR), 241.3 (194.6–289.6) vs 377.5 (332.4–412.0), p<0.0001). This definition allowed us to discern between these patients with AUC 0.93. Furthermore, in visual assessments (figure 3 and online supplemental video), thrombus formation was suppressed more in HCQ users with the lowest tertile of PL-AUC10 than in HCQ non-users. Therefore, this definition was considered to be reasonable as the obvious antiplatelet effect of HCQ in the present study. The cut-off point for the obvious antiplatelet effect obtained from the ROC curve was HCQ/RBW of 4.6 mg/kg, with a sensitivity of 84.6%, specificity of 80.8% and AUC of 0.83.

Supplemental material

Figure 2

The smooth curve generated by restricted cubic splines with four knots between HCQ/RBW (mg/kg) and PL-AUC10. AUC, area under the curve; HCQ, hydroxychloroquine; PL, platelet; RBW, real body weight.

Figure 3

Microscopic images of thrombus formation in T-TAS. These are representative cases from each group: (A) a case of a HCQ non-user (PL-AUC10 381.9), (B) a case of HCQ/RBW <5 mg/kg (PL-AUC10 380.4) and (C) a case of HCQ/RBW ≥5 mg/kg (PL-AUC10 267.6). The presented images were captured 4 min after the initiation of the T-TAS assay. HCQ, hydroxychloroquine; RBW, real body weight; T-TAS, total thrombus-formation analysis system.

We then compared PL-AUC10 and T10 among the three groups of patients with SLE: HCQ non-users (n=18) and the HCQ/IBW <5 mg/kg (n=22) and ≥5 mg/kg (n=17) groups. The Kruskal-Wallis test revealed a significant difference in PL-AUC10 among the three groups (p=0.002) (online supplemental figure 1A). No significant difference was noted in PL-AUC10 between HCQ non-users and the HCQ/IBW <5 mg/kg group (median PL-AUC10, 377.5 vs 385.6, p=1.0). PL-AUC10 was significantly lower in the HCQ/IBW ≥5 mg/kg group than in the <5 mg/kg group (median PL-AUC10, 286.1 vs 385.6, p=0.004). Furthermore, the Kruskal-Wallis test revealed that T10 did not significantly differ among the three groups (p=0.22) (online supplemental figure 1). HCQ/IBW correlated with PL-AUC10 (rs=−0.43, p=0.006). The data analysed by IBW were similar to those by RBW.

Supplemental material

We also conducted a visual assessment of thrombus formation using T-TAS. Many thrombi formed across a wide area in HCQ non-users and the HCQ/RBW <5 mg/kg group (figure 3A,B). However, in the HCQ/RBW ≥5 mg/kg group, the area of thrombus formation was localised and fewer thrombi formed (figure 3C). More detailed processes of thrombus formation through videos are available in online supplemental video.

Relationships between PL-AUC10 and organ complications, comorbidities, other medications and autoantibodies in patients with SLE

We examined the effects of organ complications, comorbidities, other medications and autoantibodies on PL-AUC10 levels. As shown in table 2, PL-AUC10 were not associated with the presence of lupus nephritis and anti-phospholipid antibody syndrome. In addition, PL-AUC10 was also not associated with hypertension, diabetes mellitus or dyslipidaemia.

Table 2

Relationships between PL-AUC10 and organ complications, comorbidities, other medications and autoantibodies

PL-AUC10 was not associated with glucocorticoids or other immunosuppressive agents, except BLM. PL-AUC10 was significantly lower in BLM non-users (n=48) than in BLM users (n=9) (357.8 vs 399.5, p=0.020) (table 2). However, patients with SLE with HCQ/RBW ≥5 mg/kg were slightly more prevalent among BLM non-users than among BLM users (n=14 (29%) versus n=0 (0%), p=0.095). In addition, among BLM non-users, PL-AUC10 was significantly lower in the HCQ/RBW ≥5 mg/kg group than in HCQ non-users (median 288.3 vs 377.5, p=0.001) and the <5 mg/kg group (median 288.3 vs 367.9, p=0.004). These results indicate that PL-AUC10 was reduced in the HCQ/RBW ≥5 mg/kg group of BLM non-users, suggesting that BLM did not affect PL-AUC10. PL-AUC10 was not associated with the positivity of anti-double-stranded DNA, anti-Smith, anti-β2 glycoprotein I or anti-cardiolipin antibodies or lupus anticoagulant. These results indicate that PL-AUC10 in patients with SLE was not affected by organ complications, comorbidities, autoantibodies, glucocorticoids or immunosuppressive agents.

Relationship between HCQ concentrations and PL-AUC10 or platelet aggregation in blood samples from healthy donors

To investigate the relationship between PL-AUC10 and HCQ concentrations, HCQ was added to whole blood samples from ten healthy donors to achieve HCQ concentrations of 0, 1, 2 and 10 µg/mL, and PL-AUC10 was measured. In comparison with HCQ 0 µg/mL, significant decreases in PL-AUC10 were observed at 1, 2 and 10 µg/mL (p=0.037, p=0.009 and p=0.014, respectively; figure 4A). With an increase in HCQ concentrations (0, 1, 2 and 10 µg/mL), decreases were observed in the median values of PL-AUC10 (388.8, 365.9, 322.2 and 313.0, respectively) (figure 4A).

Figure 4

PL-AUC10 and maximum platelet aggregation at various HCQ concentrations in healthy donors. (A) Changes in PL-AUC10 induced by increases in HCQ concentrations. (B–E) Changes in maximum platelet aggregation to the following agents induced by increases in HCQ concentrations: (B) epinephrine 10 µmol/L, (C) ADP 2 µmol/L, (D) ADP 10 µmol/L and (E) collagen 2 µg/mL. ADP, adenosine diphosphate; AUC, area under the curve; HCQ, hydroxychloroquine; n.s., not significant; PL, platelet. Data represent the median±IQR. Differences within paired groups were examined using the Wilcoxon signed-rank test. *p<0.05, **p<0.01

To examine the relationship between platelet aggregation and HCQ concentrations, HCQ was also added to whole blood samples obtained from seven healthy donors to achieve HCQ concentrations of 0, 1 and 10 µg/mL. Platelet aggregation in response to the following platelet aggregating agents was measured; epinephrine 10 µmol/L, ADP 2 µmol/L, ADP 10 µmol/L and collagen 2 µg/mL. Compared with HCQ 0 µg/mL, significant decreases in the maximum aggregation intensity to epinephrine 10 µmol/L were observed at 1 and 10 µg/mL (p=0.016 and p=0.016, respectively; figure 4B). As the HCQ concentration increased (0, 1 and 10 µg/mL), decreases were noted in the median values of the maximum aggregation intensity to epinephrine 10 µmol/L (74.0%, 55.0% and 40.8%, respectively) (figure 4B). On the other hand, in the aggregation assay to other platelet aggregating agents, HCQ dose-dependent decreases in the maximum aggregation intensity were not detected (figure 4C–E).

Discussion

Here, we demonstrated, for the first time, the antiplatelet effects of HCQ in patients with SLE under conditions closely simulating the physiological environment by using T-TAS. The main results obtained were as follows: (1) PL-AUC10 was significantly lower in the HCQ/RBW ≥5 mg/kg group than in the <5 mg/kg group, while T10 was similar, indicating that HCQ inhibited overall thrombus formation rather than the initiation of thrombus formation; (2) the results from the association analysis of PL-AUC10 and the dose of HCQ suggested that the antiplatelet effects of HCQ were initially detected at HCQ/RBW of approximately 4 mg/kg and reached a plateau at around 5.5 mg/kg, and the administration of HCQ/RBW >4.6 mg/kg induced an obvious antiplatelet effect; (3) PL-AUC10 in patients with SLE were not affected by organ complications, comorbidities, autoantibodies, glucocorticoids or immunosuppressive agents; (4) HCQ inhibited overall thrombus formation in T-TAS and the platelet aggregation response to epinephrine in a dose-dependent manner; and (5) the data analysed by IBW were similar to those by RBW.

The platelet aggregation assay is considered the historical standard for a platelet function test.22 However, this assay evaluates the aggregates that form only after the addition of platelet aggregating agents under conditions where platelets are isolated from other whole blood components. Furthermore, these aggregates are not formed under high shear stress. Therefore, these conditions do not wholly replicate the process of platelet adhesion, activation and aggregation as they naturally occur in the physiological environment.22 In addition, due to the complexity of the procedures and conditions, adverse events may occur. Cornwell et al reported that HCQ reduced ADP-induced platelet aggregation.20 In contrast, Achuthan et al showed that HCQ significantly reduced platelet aggregation with arachidonic acid (AA), while HCQ did not show a significant reduction in platelet aggregation with ADP or collagen.19

T-TAS is an instrument that is capable of quantitatively analysing the formation of platelet thrombi at high shear stress, which corresponds to arterial wall shear stress, by applying whole blood to microchips coated with collagen.23 T-TAS comprehensively analyses platelet function, including platelet adhesion, activation and aggregation, under conditions that more closely resemble the physiological environment. In addition to the points described above, the advantages of T-TAS over the platelet aggregation assay are its ease of use and stability. In contrast, the disadvantage of this system is that it is unable to specifically evaluate the activity of a pathway that may be targeted by an antiplatelet agent; however, it has the ability to assess overall thrombogenicity. In addition, the analytical conditions of T-TAS are not suitable for samples with low platelet counts or low Ht levels.

Recent studies using T-TAS have been reported. T-TAS was shown to be useful for diagnosing von Willebrand disease and patients suspected of platelet function defects.24 25 T-TAS was also a useful index for evaluating the total effects of dual antiplatelet agents in patients with various CVD.26 27 In the present study, we detected the dose-dependent antiplatelet effects of HCQ in patients with SLE by using T-TAS. PL-AUC10 was significantly lower in the HCQ/RBW ≥5 mg/kg group than in the <5 mg/kg group, while T10 did not significantly differ between these groups. This result indicates that HCQ inhibits thrombus growth rather than the initiation of thrombus formation because PL-AUC10 and T10 reflect total thrombogenicity and the onset of platelet thrombus formation, respectively. PL-AUC10 in patients with SLE was not associated with organ complications, comorbidities, autoantibodies, glucocorticoids or immunosuppressive agents, except for BLM. BLM non-users had significantly lower PL-AUC10 than BLM users. However, this difference did not appear to be due to the absence of BLM, but rather to the higher number of patients receiving HCQ ≥5 mg/kg among BLM non-users, which may have led to the decrease in PL-AUC10. Therefore, BLM was considered to have no effect on PL-AUC10.

The platelet aggregation assay provides information primarily related to the pathways affected by antiplatelet agents because it assesses responses to various aggregating agents. In the present study, HCQ decreased the platelet aggregation response to epinephrine in a dose-dependent manner. Since epinephrine is a stimulant of the AA cascade,35 the antiplatelet effects of HCQ may be involved in this cascade. Although the mechanisms underlying the antiplatelet effects of HCQ have not yet been elucidated in detail, previous studies reported the involvement of HCQ in the AA cascade, such as the inhibition of phospholipase A2, AA release and thromboxane A2-induced platelet aggregation.19 36 37 Based on these findings and the present results, HCQ appears to inhibit thrombus growth through its effects on the AA cascade. Therefore, the combination of T-TAS and the platelet aggregation assay provides comprehensive information on primary haemostasis.

HCQ exerts immunomodulatory effects, such as the alkalinisation of lysosomes with interference in phagocytosis and the suppression of inflammatory cytokine production,38 which reduces SLE flares by 50%4 and prolongs the long-term survival of patients with SLE.5–7 On the other hand, retinopathy has been identified as a major side effect of HCQ.39 Melles et al reported that the risk of retinal toxicity in patients with a mean daily use of HCQ/RBW >5 mg/kg, 4.0–5.0 mg/kg and<4.0 mg/kg was approximately 10%, less than 2% and almost 0% within 10 years of treatment, respectively, and approximately 40, 20 and 6% after 20 years, respectively, suggesting that the risk of toxicity is very low for doses <5 mg/kg.39 On the other hand, Jorge et al demonstrated that HCQ/RBW ≤5 mg/kg was associated with an adjusted OR of 1.98 for any SLE flare relative to >5 mg/kg, indicating that the threshold for flares is approximately 5 mg/kg.40 Based on these findings, the recommended dose of HCQ has undergone a shift from ‘should not exceed 5 mg/kg (RBW)’ in the 2019 EULAR recommendations41 to ‘a target dose of 5 mg/kg (RBW)’ in the 2023 EULAR recommendations.14 In the present study, we found that the antiplatelet effects of HCQ were initially detected at HCQ/RBW of approximately 4 mg/kg and reached a plateau at around 5.5 mg/kg, and the administration of HCQ/RBW >4.6 mg/kg exerted an obvious antiplatelet effect. Collectively, previous findings and the present results indicate that a HCQ dose of 5 mg/kg (RBW) may not only contribute to the control of disease activity, but also exert antiplatelet effects.

The present study has several limitations that need to be addressed. We did not monitor HCQ concentrations in the whole blood of patients with SLE. Therefore, we were unable to evaluate adherence to HCQ based on blood concentration monitoring. Moreover, medication adherence was neither supervised nor secured by medication boxes; however, we confirmed medication adherence both verbally and with the MGLS. Consequently, in the present study, we classified HCQ users into two subgroups based on the prescribed HCQ dose per RBW. Furthermore, we did not examine the relationship between T-TAS measurements and clinical outcomes, such as cardiovascular events, due to the cross-sectional design of the study. In addition, since the extent to which PL-AUC10 indicates thrombogenicity has not yet been examined, it is impossible to assess the extent of the antiplatelet effects of HCQ in clinical practice. The identification of an ideal PL-AUC10 for the prevention of cardiovascular events may lead to future therapeutic strategies that prevent CVD based on the optimal use of HCQ and antiplatelet agents. Moreover, this was a single centre study and all subjects were Japanese (Asian background), and its sample size was relatively small because patients with low platelet counts, low Ht levels or antiplatelet agents were excluded. In the present study, most patients with SLE were in remission or had low disease activity. Therefore, we were unable to evaluate PL-AUC10 in patients with high disease activity, which is a known risk factor for cardiovascular events. To address these limitations, a multinational, multicentre prospective study that investigates the relationships among T-TAS measurements, HCQ concentrations, high disease activity and clinical outcomes is needed in the future.

In conclusion, we demonstrated the dose-dependent antiplatelet effects of HCQ under conditions simulating the physiological environment by using T-TAS. T-TAS has potential as a useful tool for evaluating thrombogenicity in various collagen diseases.

Data availability statement

Data are available upon reasonable request. Data that support the present results are available from the corresponding author on reasonable request.

Ethics statements

Patient consent for publication

Ethics approval

This study involves human participants and was approved by the Institutional Review Boards at Ehime University Hospital (Approval No. 2212003). Participants gave informed consent to participate in the study before taking part.

Acknowledgments

We thank the patients and healthy donors for their cooperation and for providing their consent to participate in this study. We also thank Suzuna Matsuhiro for her technical assistance.

References

Supplementary materials

Footnotes

  • Contributors DH and JI were involved in the study concept and design. DH, JI, EH, KH, HY, TM and KS contributed to data collection. DH and KH performed the T-TAS assay. TH and TN performed the platelet aggregation assay. DH, JI, JY, HH and KT performed the data analysis. DH, JI, JY, HH and KT interpreted the results and wrote the first version of the manuscript. All authors critically revised the manuscript and approved the final version. JI is responsible for the overall content as guarantor.

  • Funding The authors have not declared a specific grant for this research from any funding agency in the public, commercial or not-for-profit sectors.

  • Competing interests None declared.

  • Patient and public involvement Patients and/or the public were not involved in the design, or conduct, or reporting or dissemination plans of this research.

  • Provenance and peer review Not commissioned; externally peer reviewed.

  • Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise.