The risk score for predicting clopidogrel response was built from a study population that has been described previously (Wu et al., 2020). The trial was registered (URL: www.chictr.org, number: ChiCTR-OCH-11001767). Two patients without data on fibrinogen were excluded; a total of 445 patients with ACS undergoing coronary stenting were included in this analysis. External validation of the HRPR risk score was obtained by using an independent study population (Clopidogrel‐associated genetic variants on inhibition of platelet activity and clinical outcome for acute coronary syndrome patients), which focused on the pharmacodynamics and clinical outcomes of clopidogrel. The validation dataset enrolled 196 patients who were diagnosed with ACS.

All subjects received DAPT with the combination of aspirin (300 mg loading dose, 100 mg once daily) and clopidogrel (300 mg loading dose, 75 mg once daily). The Medical Ethics Committee of Zhongshan Hospital approved this study. Identified data were anonymized, and privacy issues were kept confidential.

The main exclusion criteria included the following: 1) receiving other antiplatelet agents or oral anticoagulants; 2) history of cerebral hemorrhage and known relevant hematological deviations; 3) discontinuation of clopidogrel antithrombotic; 4) lack of follow-ups.

Baseline demographics and laboratory measurements were collected through electronic medical records. The fibrinogen concentration measurement was performed by the following steps: 1) premix blood samples with thrombin solution, 2) deposit the mixture as droplets on a glass surface, and 3) allow the droplet to clot and apply a paper strip on top. The distance that wicks down the strip of blood was precisely related to the fibrinogen concentration.

An approximate venous blood sample (2 ml) for genotyping was collected into a vacutainer containing anticoagulant Ethylene Diamine Tetra‐acetic Acid (EDTA) from patients upon recruitment. Another whole peripheral blood sample (4 ml) was collected to measure the on-treatment platelet reactivity with thrombelastography (TEG; Haemoscope Corp, Niles, IL, United States) after receiving a loading dose of clopidogrel.

The DNA sample of each patient was isolated using the QIAamp DNA Blood Kit (Qiagen, Hilden, Germany) according to standard procedure. The single nucleotide polymorphisms (SNPs) including the CYP2C19 phenotype, were conducted by polymerase chain reaction (PCR) and TaqMan genotyping assays (Light Cycler 480, Roche, CA, United States). LOF alleles of CYP2C19*2 and *3 gene variants were the point mutations of 681G > A (rs4244285) and 636 G > A (rs4986893), respectively.

Individuals were classified into 3 genotype groups according to previous literature as extensive metabolizers (*1/*1), intermediate metabolizers (*1/*2 or *1/*3), and poor metabolizers (*2/*2, *2/*3 or *3/*3), respectively. We recorded the number of CYP2C19 LOF alleles as 0, 1, and 2 in extensive, intermediate, and poor metabolizers, respectively.

We applied the Thrombelastograph Hemostasis Analyzer (Haemoscope Corp., Niles, Illinois, United States) with platelet mapping to measure platelet activity after medication with aspirin and clopidogrel. A blood sample (4 ml) was obtained for the TEG test, which is a point-of-care test to evaluate platelet and fibrin contributions to clot strength. The particular advantage of the TEG mapping system is that it evaluates antiplatelet effects after DAPT treatment simultaneously, including relative inhibition of platelet aggregation (IPA) and net residual (post-treatment) platelet activity. Adenosine diphosphate (ADP, 2 µmol/L 100 µL) induced maximal attitude (MA_ADP) was considered as the residual platelet reactivity after clopidogrel treatment.

The novel risk score is derived from the incorporation of 3 independent predictors on-clopidogrel HRPR, including 2 clinical and 1 genetic factor; it was named the GeneFA score based on the inclusion of CYP2C19 genotype, fibrinogen, and age. By analyzing the area under a combined receiver-operating characteristic (ROC) curve, age >60 years of age and fibrinogen value >310 mg/dl were identified as the optimal cut-offs. The total score was 4 and a cut-off score ≤2 represented high sensitivity and specificity to identify HRPR on clopidogrel in the proof-of-concept study. At present, the ABCD-GENE score was established to predict HRPR on clopidogrel among the European population based on 1 genetic and 4 clinical factors as follows: age >75 years (4 points), body mass index (BMI) > 30 kg/m², chronic kidney disease (evaluated glomerular filtration rate <60 ml/min, 4 points), diabetes mellitus, and CYP2C19 LOF alleles. A cut-off score ≥10 was regarded as the best specificity and sensitivity for the identification of HRPR for clopidogrel (Angiolillo et al., 2020), as shown in Table 1.

The main objective of this study was to identify the diagnostic ability of the GeneFA score for HRPR on clopidogrel in the Chinese ACS population following PCI as compared to the ABCD-GENE score.

The clinical outcome of the deviation population was major adverse cardiovascular events (MACE) during 12 months of follow-up through outpatient follow-ups and electronic medical record (EMR). The MACE was the composite of cardiovascular death, nonfatal myocardial infarction (MI), or stroke. The primary clinical endpoint of the validation dataset was MACE, including cardiovascular death, myocardial infarction, and revascularization for the targeted vascular lesion.

Cardiovascular death was regarded as any death with a demonstrable cardiovascular cause or any death not clearly attributable to a noncardiovascular cause. The diagnosis of MI is based on a new rise in troponin T ≥ 0.03 ng/ml associated with typical symptoms and/or typical electrocardiogram changes. The diagnosis of ischemic stroke requires clinical presentation and confirmation by computed tomography or magnetic resonance imaging of the head.

The descriptive statistics of continuous variables were expressed as means ± standard deviations (SD), and those of discrete variables were expressed as counts or percentages. Currently, the primary interest of this study was to test the specificity and sensitivity of the GeneFA score for prediction of HRPR on clopidogrel, which was defined as MA_ADP > 47.0 mm according to recent consensus statement (Tantry et al., 2013). In the developmental data set, a potential interaction of the risk score with MACE during follow-ups was evaluated, and the diagnostic ability of the GeneFA risk scoring system for MACE was tested in the validation data set.

One-way ANOVA was used to compare the differences of the continuous variables associated with HRPR, and a chi-squared test was performed to compare the distribution of categorical variables. We applied receiver-operating characteristic (ROC) curves to assess the discrimination performance of HRPR and ischemic events compared with GeneFA and ABCD-GENE scores. From the ROC curves, areas under the curve (AUCs) or c-statistics greater than 0.5 are considered to be of clinical value. Then, the GeneFA risk scoring system was performed to evaluate the discriminatory power of HRPR and ischemic events with the calculation of the area under the ROC in the validation data set.

Statistical analysis was performed using SPSS (IBM SPSS Statistics 22.0) and Prism 5 (GrandPad Software). A p value of 0.05 was considered as the threshold for statistical significance.

Patients enrolled in the developmental datasets (N = 445) were representative of the previous study. Throughout 1 year of follow-up, a total of 38 patients sustained ischemic events, including 32 with nonfatal acute myocardial infarction, 3 experiencing nonfatal ischemic stroke, and 3 died in the developmental group. Meanwhile, a total of 102 (22.9%) participants were demonstrated to have HRPR (MA_ADP > 47.0 mm) based on results of platelet activity measurements. Variables including demographic information, comorbidity, laboratory measurements, concomitant medication, procedural characteristics, and LOF carriers of enrolled participants were collected as clinical and genotype factors.

As demonstrated in Table 2, the percentage of patients with an age >60 was 77.5% among subjects with HRPR and 57.7% among those without HRPR (p = 0.004). Similarly, 61.8% of patients with HRPR displayed high fibrinogen, whereas only 26.8% of patients without HRPR displayed high fibrinogen (p < 0.001). Moreover, the carriage of CYP2C19 LOF was more frequently observed in subjects with HRPR. After adjustment for confounding factors, these three parameters were independently and significantly associated with HRPR.

On the basis of points assigned, patients were divided into five subgroups with a GeneFA score of 0–4 points. The different quintiles in absolute MA_ADP values are in the lower quintile (0–3 points): 17.8 (8.0–72.2) mm and in the upper quintile (3 and 4 points): 34.8 (12.6–83.2) mm.

Based on the observed frequencies of HRPR in relation to GeneFA risk score incorporation with clinical and genotype factors, the HRPR ratio in the development cohort was further presented according to each point (Figure 1). A relative higher clinically HRPR was observed in upper quintile with representative score of 3 (52.90%) and 4 (59.10%), meanwhile it was found less frequently in the score 0 (6.70%), 1 (15.10%), and 2 (16.70%) group, respectively.

Based on the cut-off risk scores of GeneFA >3 and ABCD-GENE ≥10, 445 participants were further categorized into low and high-risk groups (Figure 2A). When participants were divided into high and low risk groups according to the GeneFA score, participants with a GeneFA score >2 had an increased risk of HRPR (54.3 vs. 14.7%, p < 0.001) on clopidogrel (Figure 2A). Similarly, subjects with ABCD-GEN ≥10 also had an increased risk of HRPR (36.0 vs. 18.4%, p < 0.001). Moreover, the GeneFA score exhibited a better prediction for the presence of HRPR as compared to the ABCD-GENE score (p < 0.001).

On the basis of the cut-off, the GeneFA score significantly separated patients at higher and lower risk for MACE. As illustrated in Figure 2B, the high GeneFA score group exhibited a significantly higher ischemic rate compared to the low score group (20.7 vs. 5.4%, p < 0.001). The high ABCD-GENE score group displayed a significantly higher ischemic rate as compared with the low score group (14.9 vs. 6.3%, p = 0.005), and a similar modest ability was demonstrated to predict post-PCI ischemic events between the GeneFA and ABCD-GENE scores.

The ROC curve showed that the GeneFA score had better discriminative power in predicting HRPR on clopidogrel in the development cohort as compared to ABCD-GENE score reflected by the AUC value (c-statistic: 0.709 for the GeneFA score and 0.638 for the ABCD-GENE score, respectively) (Figure 3A). Meanwhile, the sensitivity analysis illustrated that both the GeneFA and ABCD-GENE risk score seemed to have moderate predictive power in predicting MACE based on AUC value (c-statistic: 0.696 for GeneFA and 0.654 for the ABCD-GENE risk score, respectively) (Figure 3B).

Then, we externally validated the developed prediction model in previous a clinical trial including 196 patients with 42 (21.4%) developing HRPR and 28 (14.3%) recent ischemic events during 1-year follow-up. External validation of each score was performed by GeneFA and ABCD-GENE. During the follow-up period, the HRPR on clopidogrel was significantly higher in high-risk groups as compared to low-risk groups according to the GeneFA risk score (65.6 and 12.8%, p = 0.001). Patients with a high GeneFA risk score were associated with a significantly higher rate of incidence of thrombosis, including hospitalization for revascularization, than patients with a low risk score (41.9 vs. 9.1%, p = 0.025).

We also compared the present risk score for predictive accuracy of HRPR incidence with the ABCD-GENE score in the validation population. The present new risk score had a similarly high prognostic value for HRPR incidence (C-statistic: 0.855 for GeneFA and 0.843 for ABCD-GENE) and ischemic recurrence (C-statistic: 0.726 for GeneFA and 0.724 for ABCD-GENE) on clopidogrel as compared to ABCD-GENE (Figure 4).

The GeneFA risk score had a moderate predictive ability for HRPR on clopidogrel for CAD patients in Chinese populations. The predictive value was consistent with the ABCD-GENE score, suggesting it to be clinically useful in HRPR identification.

There are several limitations to this study. First, some risk predictors in other scoring systems did not display statistical significance in the present study, which could be attributed to the limited sample size and short follow-up timeline. Therefore, a future large-scale clinical study is needed to confirm our hypothesis. Second, the study has the limitations inherent to the prospective cohort study, resulting in possible bias from selective prescription, and incidence of clinically relevant bleeding might be underestimated limited by the medical records. Third, items in some clinical outcomes could not be obtained owing to short follow-up periods, which might influence the score distribution and discriminative power. Thus, the results of this study need to be interpreted with caution in future clinical setting. Finally, we applied TEG as a measurement of platelet aggregation, which does not represent the functional gold standard for platelet function analysis. The HRPR for aspirin was not presented in this study, which caused the bias of MACE occurrence due to aspirin medication.