Sex-Specific Platelet Activation Through Protease-Activated Receptors Reverses in Myocardial Infarction

Abstract

Objective: The platelet phenotype in certain patients and clinical contexts may differ from healthy conditions. We evaluated platelet activation through specific receptors in healthy men and women, comparing this to patients presenting with ST-segment-elevation myocardial infarction and non-ST-segment-elevation myocardial infarction. Approach and Results: We identified independent predictors of platelet activation through certain receptors and a murine MI model further explored these findings. Platelets from healthy women and female mice are more reactive through PARs (protease-activated receptors) compared with platelets from men and male mice. Multivariate regression analyses revealed male sex and non-ST-segment-elevation myocardial infarction as independent predictors of enhanced PAR1 activation in human platelets. Platelet PAR1 signaling decreased in women and increased in men during MI which was the opposite of what was observed during healthy conditions. Similarly, in mice, thrombin-mediated platelet activation was greater in healthy females compared with males, and lesser in females compared with males at the time of MI.

Conclusions: Sex-specific signaling in platelets seems to be a cross-species phenomenon. The divergent platelet phenotype in males and females at the time of MI suggests a sex-specific antiplatelet drug regimen should be prospectively evaluated.

Keywords: female; myocardial infarction; platelet activation; regression analysis; thrombin.

Figure 1.. Platelet activation and signaling in healthy subjects:

Platelets from healthy women and men were isolated and stimulated ex vivo with agonists for (A) PAR1 (TRAP-6), (B) the thromboxane receptor (U46619), and (C) the P2Y₁₂ receptor (ADP) at the indicated concentrations. Platelet activation was assessed by FACS for alpha granule secretion (surface p-selectin) as mean fold increase from baseline ± SEM. * P <0.05 between groups at the indicated time point by the Kruskal-Wallis test followed by Dunn's post-test correction. Data are representative of n=11 men and n=17 women. (D) Male and female platelets were assessed for adhesion to fibrinogen after 30 mins at 37 °C by confocal microscopy. Data are represented as mean platelet surface area ± SEM. (n=3 in each group, 4-5 random fields per subject, 10-20 platelets per field). *P=0.007 between groups, t-test. Phalloidin-PE stains actin cytoskeleton red. Yellow scale bar= 5μM. (E) Platelets were isolated from healthy male and female subjects, and incubated for 3 hours with either IBMX to inhibit phosphodiesterase or forskolin to stimulate adenylyl cyclase. Platelet lysates were flash frozen, and an ELISA was conducted to determine GαS activity by cAMP production. Data are presented as mean ± SEM, n=8 men and n=8 women. * P=0.0099 **P=0.009 and ***P=0.0002 between male and female groups, by Mann-Whitney U test. IBMX=3-isobutyl-1-methylxanthine (100 μM, 30 minutes) inhibits platelet phosphodiesterase. Forskolin (1 μM, 30 minutes) activates platelet adenylyl cyclase.

Figure 1.. Platelet activation and signaling in healthy subjects:

Platelets from healthy women and men were isolated and stimulated ex vivo with agonists for (A) PAR1 (TRAP-6), (B) the thromboxane receptor (U46619), and (C) the P2Y₁₂ receptor (ADP) at the indicated concentrations. Platelet activation was assessed by FACS for alpha granule secretion (surface p-selectin) as mean fold increase from baseline ± SEM. * P <0.05 between groups at the indicated time point by the Kruskal-Wallis test followed by Dunn's post-test correction. Data are representative of n=11 men and n=17 women. (D) Male and female platelets were assessed for adhesion to fibrinogen after 30 mins at 37 °C by confocal microscopy. Data are represented as mean platelet surface area ± SEM. (n=3 in each group, 4-5 random fields per subject, 10-20 platelets per field). *P=0.007 between groups, t-test. Phalloidin-PE stains actin cytoskeleton red. Yellow scale bar= 5μM. (E) Platelets were isolated from healthy male and female subjects, and incubated for 3 hours with either IBMX to inhibit phosphodiesterase or forskolin to stimulate adenylyl cyclase. Platelet lysates were flash frozen, and an ELISA was conducted to determine GαS activity by cAMP production. Data are presented as mean ± SEM, n=8 men and n=8 women. * P=0.0099 **P=0.009 and ***P=0.0002 between male and female groups, by Mann-Whitney U test. IBMX=3-isobutyl-1-methylxanthine (100 μM, 30 minutes) inhibits platelet phosphodiesterase. Forskolin (1 μM, 30 minutes) activates platelet adenylyl cyclase.

Figure 1.. Platelet activation and signaling in healthy subjects:

Platelets from healthy women and men were isolated and stimulated ex vivo with agonists for (A) PAR1 (TRAP-6), (B) the thromboxane receptor (U46619), and (C) the P2Y₁₂ receptor (ADP) at the indicated concentrations. Platelet activation was assessed by FACS for alpha granule secretion (surface p-selectin) as mean fold increase from baseline ± SEM. * P <0.05 between groups at the indicated time point by the Kruskal-Wallis test followed by Dunn's post-test correction. Data are representative of n=11 men and n=17 women. (D) Male and female platelets were assessed for adhesion to fibrinogen after 30 mins at 37 °C by confocal microscopy. Data are represented as mean platelet surface area ± SEM. (n=3 in each group, 4-5 random fields per subject, 10-20 platelets per field). *P=0.007 between groups, t-test. Phalloidin-PE stains actin cytoskeleton red. Yellow scale bar= 5μM. (E) Platelets were isolated from healthy male and female subjects, and incubated for 3 hours with either IBMX to inhibit phosphodiesterase or forskolin to stimulate adenylyl cyclase. Platelet lysates were flash frozen, and an ELISA was conducted to determine GαS activity by cAMP production. Data are presented as mean ± SEM, n=8 men and n=8 women. * P=0.0099 **P=0.009 and ***P=0.0002 between male and female groups, by Mann-Whitney U test. IBMX=3-isobutyl-1-methylxanthine (100 μM, 30 minutes) inhibits platelet phosphodiesterase. Forskolin (1 μM, 30 minutes) activates platelet adenylyl cyclase.

Figure 2. (A). Predictors of Platelet Reactivity at the Time of MI:

(A) Demographic of the post-MI study population. When stratifying for sex and NSTEMI/STEMI, except for HDL, the study populations were similar. There were 60 NSTEMI and 57 STEMI patients, and males comprised a similar proportion of each of these cohorts (30-33%.) Data are shown as mean ± SD. Level of significance is noted. HDL=high density lipoprotein. LDL=low density lipoprotein. LVEF=left ventricular ejection fraction. BMI=Body Mass Index. All data presented reflect variables at the time of patient evaluation in the emergency department. (B) Blood was drawn from women and men at the time of STEMI or NSTEMI diagnosis, prior to coronary angiography and prior to loading with a P2Y₁₂ receptor antagonist. Isolated platelets were stimulated for 15 mins with an agonist for: PAR1 (TRAP-6, 10 μM), the thromboxane receptor (U46619, 10 μM), or the P2Y₁₂ receptor (ADP, 10 μM). Platelet activation was assessed by FACS for alpha granule secretion (surface p-selectin) as mean fold increase from baseline ± 95% C. I. Differences between women and men for each agonist was assessed by the Mann-Whitney U test. *P=0.0001. **P=0.0473. NS=Not significant. n=80 males, n=37 females. (C) Independent predictors of platelet reactivity as forest plots illustrating multivariate-adjusted mean parameter estimates ± 95% C.I. following regression analyses for platelet function in response to surface receptors agonists at 10 μM. Red=PAR1, Green=Thromboxane, Blue=P2Y₁₂. Level of significance is noted. Data are representative of 60 NSTEMI and 57 STEMI patients. CKD=chronic kidney disease. CAD=existing coronary artery disease. PAD=peripheral artery disease.

Figure 2. (A). Predictors of Platelet Reactivity at the Time of MI:

(A) Demographic of the post-MI study population. When stratifying for sex and NSTEMI/STEMI, except for HDL, the study populations were similar. There were 60 NSTEMI and 57 STEMI patients, and males comprised a similar proportion of each of these cohorts (30-33%.) Data are shown as mean ± SD. Level of significance is noted. HDL=high density lipoprotein. LDL=low density lipoprotein. LVEF=left ventricular ejection fraction. BMI=Body Mass Index. All data presented reflect variables at the time of patient evaluation in the emergency department. (B) Blood was drawn from women and men at the time of STEMI or NSTEMI diagnosis, prior to coronary angiography and prior to loading with a P2Y₁₂ receptor antagonist. Isolated platelets were stimulated for 15 mins with an agonist for: PAR1 (TRAP-6, 10 μM), the thromboxane receptor (U46619, 10 μM), or the P2Y₁₂ receptor (ADP, 10 μM). Platelet activation was assessed by FACS for alpha granule secretion (surface p-selectin) as mean fold increase from baseline ± 95% C. I. Differences between women and men for each agonist was assessed by the Mann-Whitney U test. *P=0.0001. **P=0.0473. NS=Not significant. n=80 males, n=37 females. (C) Independent predictors of platelet reactivity as forest plots illustrating multivariate-adjusted mean parameter estimates ± 95% C.I. following regression analyses for platelet function in response to surface receptors agonists at 10 μM. Red=PAR1, Green=Thromboxane, Blue=P2Y₁₂. Level of significance is noted. Data are representative of 60 NSTEMI and 57 STEMI patients. CKD=chronic kidney disease. CAD=existing coronary artery disease. PAD=peripheral artery disease.

Figure 3.. PAR-mediated platelet activation in healthy individuals and following MI:

(A) Platelets were isolated from healthy women and men (n=10; n=5 in each group) then stimulated with human thrombin for 15 minutes (0=1.0 u/mL). * <0.05 between groups, assessed one-way ANOVA followed by Bonferroni's multiple comparisons test. (B) Platelet were isolated from women and men at the time of NSTEMI (n=10; n=5 in each group) then stimulated with human thrombin (0=1.0 u/mL) for 15 minutes. *P=0.028 and **P=0.054 assessed one-way ANOVA followed by Bonferroni's multiple comparisons test. Platelet activation was assessed by FACS for alpha granule secretion (surface p-selectin) as mean fold increase from baseline ± SEM. (C) Platelets were isolated from healthy women and men (n=10; n=5 in each group) then stimulated with the PAR4 peptide GYPGKF. (D) Platelets were isolated from women and men at the time of NSTEMI (n=10; n=5 in each group) then stimulated with the PAR4 peptide GYPGKF. Platelet activation was assessed by FACS for alpha granule secretion (surface p-selectin) as mean fold increase from baseline ± SEM. P=NS between heathy males and females and between NSTEMI males and females at each concentration assessed by one-way ANOVA followed by Bonferroni's multiple comparisons test.

Figure 3.. PAR-mediated platelet activation in healthy individuals and following MI:

(A) Platelets were isolated from healthy women and men (n=10; n=5 in each group) then stimulated with human thrombin for 15 minutes (0=1.0 u/mL). * <0.05 between groups, assessed one-way ANOVA followed by Bonferroni's multiple comparisons test. (B) Platelet were isolated from women and men at the time of NSTEMI (n=10; n=5 in each group) then stimulated with human thrombin (0=1.0 u/mL) for 15 minutes. *P=0.028 and **P=0.054 assessed one-way ANOVA followed by Bonferroni's multiple comparisons test. Platelet activation was assessed by FACS for alpha granule secretion (surface p-selectin) as mean fold increase from baseline ± SEM. (C) Platelets were isolated from healthy women and men (n=10; n=5 in each group) then stimulated with the PAR4 peptide GYPGKF. (D) Platelets were isolated from women and men at the time of NSTEMI (n=10; n=5 in each group) then stimulated with the PAR4 peptide GYPGKF. Platelet activation was assessed by FACS for alpha granule secretion (surface p-selectin) as mean fold increase from baseline ± SEM. P=NS between heathy males and females and between NSTEMI males and females at each concentration assessed by one-way ANOVA followed by Bonferroni's multiple comparisons test.

Figure 4:. Platelet Reactivity in a mild murine model of MI.

(A) Male and female C57BL/6J mice underwent sham surgery LAD infarction by cryoablation, followed immediately by retro-orbital infusion of 2% methylene blue to indicate areas of LV perfusion. Perfused tissue is blue and ischemic tissue is pink. LV region at risk quantified as % LV area (mean % of LV area ± SEM for n=5 mice). Representative images are shown for male and female mice. LAD=left anterior descending coronary artery. LV=left ventricle. Differences between groups was assessed by t-test, and not found to be significant 3 days following MI. Platelets were stimulated ex vivo with (B) thrombin or (C) PAR4 specific agonist GYPGKF. Platelet activation was assessed by FACS (surface p-selectin). Data are represented as mean fold increase over baseline (0) ± SEM for n=5 mice, each performed in quadruplicate. * P <0.05 between groups, assessed by one-way ANOVA followed by Bonferroni's multiple comparisons test. (D) Platelet calcium mobilization in males and female mice at the time of MI. 5 minutes after LAD occlusion, platelet Ca²⁺ mobilization was assessed by FURA-2 as Abs_{340 nm} /Abs_380nm and the ratio expressed as fluorescence, F/baseline Fluorescence, F₀. Data are presented as mean ± SEM for n=5 mice in each group.

Figure 4:. Platelet Reactivity in a mild murine model of MI.

(A) Male and female C57BL/6J mice underwent sham surgery LAD infarction by cryoablation, followed immediately by retro-orbital infusion of 2% methylene blue to indicate areas of LV perfusion. Perfused tissue is blue and ischemic tissue is pink. LV region at risk quantified as % LV area (mean % of LV area ± SEM for n=5 mice). Representative images are shown for male and female mice. LAD=left anterior descending coronary artery. LV=left ventricle. Differences between groups was assessed by t-test, and not found to be significant 3 days following MI. Platelets were stimulated ex vivo with (B) thrombin or (C) PAR4 specific agonist GYPGKF. Platelet activation was assessed by FACS (surface p-selectin). Data are represented as mean fold increase over baseline (0) ± SEM for n=5 mice, each performed in quadruplicate. * P <0.05 between groups, assessed by one-way ANOVA followed by Bonferroni's multiple comparisons test. (D) Platelet calcium mobilization in males and female mice at the time of MI. 5 minutes after LAD occlusion, platelet Ca²⁺ mobilization was assessed by FURA-2 as Abs_{340 nm} /Abs_380nm and the ratio expressed as fluorescence, F/baseline Fluorescence, F₀. Data are presented as mean ± SEM for n=5 mice in each group.

Figure 4:. Platelet Reactivity in a mild murine model of MI.

(A) Male and female C57BL/6J mice underwent sham surgery LAD infarction by cryoablation, followed immediately by retro-orbital infusion of 2% methylene blue to indicate areas of LV perfusion. Perfused tissue is blue and ischemic tissue is pink. LV region at risk quantified as % LV area (mean % of LV area ± SEM for n=5 mice). Representative images are shown for male and female mice. LAD=left anterior descending coronary artery. LV=left ventricle. Differences between groups was assessed by t-test, and not found to be significant 3 days following MI. Platelets were stimulated ex vivo with (B) thrombin or (C) PAR4 specific agonist GYPGKF. Platelet activation was assessed by FACS (surface p-selectin). Data are represented as mean fold increase over baseline (0) ± SEM for n=5 mice, each performed in quadruplicate. * P <0.05 between groups, assessed by one-way ANOVA followed by Bonferroni's multiple comparisons test. (D) Platelet calcium mobilization in males and female mice at the time of MI. 5 minutes after LAD occlusion, platelet Ca²⁺ mobilization was assessed by FURA-2 as Abs_{340 nm} /Abs_380nm and the ratio expressed as fluorescence, F/baseline Fluorescence, F₀. Data are presented as mean ± SEM for n=5 mice in each group.