Reduced platelet glycoprotein Ibα shedding accelerates thrombopoiesis and COX-1 recovery: implications for aspirin dosing regimen

Abstract

Cardiovascular (CV) disease prevention with low-dose aspirin can be less effective in patients with a faster recovery of platelet (PLT) cyclooxygenase (COX)-1 activity during the 24-hour dosing interval. We previously showed that incomplete suppression of TXA2 over 24 hours can be rescued by a twice daily aspirin regimen. Here we show that reduced PLT glycoprotein (GP)Ibα shedding characterizes patients with accelerated COX-1 recovery and may contribute to higher thrombopoietin (TPO) production and higher rates of newly formed PLT, escaping aspirin inhibition over 24 hours. Two hundred aspirin-treated patients with high CV risk (100 with type 2 diabetes mellitus) were stratified according to the kinetics of PLT COX-1 activity recovery during the 10- to 24-hour dosing interval. Whole proteome analysis showed that PLT from patients with accelerated COX-1 recovery were enriched in proteins involved in cell survival, inhibition of apoptosis and cellular protrusion formation. In agreement, we documented increased plasma TPO, megakaryocyte maturation and proplatelet formation, and conversely increased PLT galactose and reduced caspase 3, phosphatidylserine exposure and ADAM17 activation, translating into diminished GPIbα cleavage and glycocalicin (GC) release. Treatment of HepG2 cells with recombinant GC led to a dose-dependent reduction of TPO mRNA in the liver, suggesting that reduced GPIbα ectodomain shedding may unleash thrombopoiesis. A cluster of clinical markers, including younger age, non-alcoholic fatty liver disease, visceral obesity and higher TPO/GC ratio, predicted with significant accuracy the likelihood of faster COX-1 recovery and suboptimal aspirin response. Circulating TPO/GC ratio, reflecting a dysregulation of PLT lifespan and production, may provide a simple tool to identify patients amenable to more frequent aspirin daily dosing.

Figure 1.

Subjects with faster kinetics of recovery of serum thromboxane B₂ during the 24-hour aspirin dosing interval, display increased platelet COX-1 expression and urinary 11-dehydro-thromboxane B₂ excretion as compared to those with normal serum thromboxane B₂ recovery. (A) Study design. Linear fitting of serum thromboxane B₂ (sTXB₂) measured 10 and 24 hours post-aspirin intake in patients without (B) (n=100), and with type 2 diabetes mellitus (T2DM) (C) (n=100) stratified in tertiles according to sTXB₂ (ex vivo index of COX-1-dependent TXA₂ production) slope (n=33/tertile). Patients in the third sTXB₂ slope tertile display significantly faster recovery of sTXB₂ vs. first tertile (P<0.001), during the 24 hours between 2 witnessed aspirin administrations. sTXB₂ values are as median and interquartile range. Comparison of subjects in the third vs. first sTXB₂ slope tertile for platelet transcript levels of COX-1 mRNA (D) (n=20 vs. n=24) and urinary 11-dehydro-TXB₂ (E) (n=66 vs. n=66). Significance was calculated by Mann-Whitney U test.

Figure 2.

Proteomic analysis shows activation of pathways “formation of cellular protrusions”, “formation of proplatelets”, “cell survival”, “production of reactive oxygen species”, and inhibition of “apoptosis” in platelets from patients with faster COX-1 recovery. Proteomic analysis using ingenuity pathway analysis (IPA) revealed activation of “formation of cellular protrusions”, “formation of proplatelets”, “cell survival”, “production of reactive oxygen species”, and inhibition of “apoptosis” pathways in platelets of third vs. first serum thromboxane B₂ (sTXB₂) tertile in patients without (A, E), and with type 2 diabetes mellitus (T2DM) (B, E). Further details are reported in the Online Supplementary Figures S1, S2 and S3. Validation of proteomic data by western blot, assessing 78 kDa glucose-regulated protein (GRP78) (C) (n=4/tertile), COX-1 (D) (n=4/tertile) and Rab27B (F) (n=4/tertile) in patients from third tertile vs. first tertile and healthy subjects (HS) (n=4), using b-Actin as loading control. Significance was calculated by Student’s t-test.

Figure 3.

Enhanced megakaryocyte maturation and proplatelet formation in patients with faster COX-1 recovery. Representative immunofluorescence of megakaryocytes (Mk) (A) and proplatelet formation (PPF) (B). The proportion of mature Mk was measured by flow cytometry, as the percentage and mean fluorescence intensity (MFI) of CD41-positive cells (healthy subjects n=5; first tertile: all n=8, type 2 diabetes mellitus (T2DM) n=4, no T2DM n=4; third tertile: all n=10, T2DM n=4, no T2DM n=6) (C, D); and as the percentage and MFI of GPIbα (CD42b)-positive cells (healthy subjects n=5; first tertile: all n=8, T2DM n=4, no T2DM n=4; third tertile: all n=11, T2DM n=5, no T2DM n=6) (C, D). PPF was quantified as the proportion of Mk displaying at least one proplatelet with respect to the total number of adhered Mk (healthy subjects n=5; first tertile: all n=7, T2DM n=4, no T2DM n=3; third tertile: all n=8, T2DM n=4, no T2DM n=4) (E).

Figure 4.

Higher circulating levels of thrombopoietin and platelet count and lower glycocalicin and glycocalicin index at 24 hours afer witnessed aspirin intake in patients with faster COX-1 recovery. Comparison of thrombopoietin (TPO) (A), glycocalicin (GC) (B), platelet (PLT) count (C) and glycocalicin index (GCI) (D) between first vs. third serum thromboxane B₂ (sTXB₂) slope tertile in all patients (n=132). Comparison of TPO (A) and GC (B) between healthy subjects (HS) (n=5) vs. first and vs. third sTXB₂ slope tertile in all patients (n=132). Significance was calculated by Mann-Whitney U test. Correlation between GCI and TPO in all investigated patients (E). Spearman correlation coefficient and P value are reported. Significance was calculated by Mann-Whitney U test.

Figure 5.

Lower glycocalicin circulating levels in platelets from patients with faster COX-1 recovery depend on higher GPIbcx expression, lower phosphatidylserine expression and lower ADAM17 activation, and enhance thrombopoietin mRNA transcription in liver cells. GPIbα protein levels in platelets of healthy subjects (HS) (n=4) vs. first (n=4) vs. third (n=4) tertile in all patients (A). ADAM17 levels in platelets of HS (n=3) vs. first (n=4) vs. third serum thromboxane B₂ (sTXB₂) slope tertile (n=4) in all patients (B). Phosphatidylserine (PS)-positive platelets (%CD41a+/AnV+) in the first (n=34) vs. third tertile (n=20) in all patients (C). PS-positive platelets (%CD41a+/AnV+) (D) and active-ADAM17 cleaved form (E) in 4 healthy subjects treated with low-dose aspirin, at 10 and 24 hours post aspirin. Treatment of HepG2 cells (n=4) with an increasing concentrations of human recombinant glycocalicin (rGC, 0.5, 1 and 2 µg/mL) is associated, after 1 hour of incubation, with a significant dose-dependent reduction in thrombopoietin (TPO) mRNA (F). Significance was calculated by Mann-Whitney U test or by Student’s t test.

Figure 6.

Higher platelet desialylation rate in patients with faster COX-1 recovery.

Figure 7.

Multivariable logistic regression analyses, receiver operating characteristic curve for the prediction of poor aspirin response and proposed model depicting the mechanisms involving platelet lifespan that may limit the extent and duration of aspirin effect over 24 hours. Determinants of the accelerated recovery of COX-1 activity in patients with (A) and without type 2 diabetes mellitus (T2DM) (B) assessed by multivariable logistic regression analysis. Receiver operating characteristic (ROC) curve for the prediction of poor aspirin response (C, D). ROC and the relative area under the curve (AUC) showing the ability of the model in discriminating between the third vs. first serum thromboxane B₂ (sTXB₂) slope tertile. Among patients with T2DM, the combination of younger age (standard deviation [SD]=6.38 years), presence of non-alcoholic fatty liver disease (NAFLD), higher platelet (PLT) count (SD=55.66 mL) and higher thrombopoietin/glycocalicin (TPO/GC) ratio (1^st tertile: <60; 2^nd tertile: from 60 to 138; third tertile >138) yielded an AUC value of 0.883 (95% confidence interval [CI]: 0.799-0.966) in distinguishing patients in third sTXB₂ slope tertile from first tertile patients (C). In comparison with a model including only clinical/hemocromocytometric variables (age, NAFLD and PLT count) the addition of TPO/GC ratio yielded a significant increase in AUC (from 0.754 to 0.883; P for difference 0.015). Among patients without T2DM, higher waist-to-hip ratio (WHR) (SD=0.066), lower mean platelet volume (SD=0.93 mL) and higher TPO/GC ratio (first tertile: <76; second tertile: from 76 to 147; third tertile >147) yielded an AUC value of 0.880 (95% CI: 0.794-0.966) in distinguishing patients in third sTXB₂ slope tertile from first tertile patients (D). Aspirin-treated patients were stratified according to the kinetics of COX-1 recovery over the 10- to 24-hour dosing interval. In poor aspirin responders we showed: i) increased plasma thrombopoietin, megakaryocyte (Mk) maturation and proplatelet formation (PPF) reflecting enhanced PLT production; ii) increased PLT desialylation, lower phosphatidylserine exposure, lower PLT sheddase ADAM17 and plasma glycocalicin and increased glycoprotein (GP)Ibα expression, altogether reflecting defective PLT GPIbα shedding; iii) a proteomic signature characterized by activation of cell survival and inhibition of apoptosis. Younger age, NAFLD and visceral obesity, higher PLT count together with higher thrombopoietin-to-glycocalicin ratio, predict suboptimal aspirin response (E).