Ex vivo effects of low dose delayed release rapamycin on agonist-induced platelet aggregation, activation and procoagulant platelet phenotypes in domestic cats

Figures

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  Fig. 1. A summary of workflow used in this study. Eight cats were treated with low dose (0.3 mg/kg) delayed release rapamycin (LDDRR) orally every 7 days for 4 consecutive weeks. Blood was collected at 3, 24 and 48 h following the final dose of rapamycin. Platelet-rich plasma (PRP) was isolated and part of the isolated PRP was used for stepwise assessment of mitochondrial membrane potential and PS exposure and the remaining part was used to assess platelet activation detected by P-selectin.

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  Fig. 2. Platelet aggregation, measured as (A) area under the curve (AUC), (B) aggregation unit (AU) and (C) velocity (AU/min), was evaluated by whole blood electrical impedance aggregometry in 8 cats. Diluted whole blood was treated with 6.25µM adenosine diphosphate (ADP) before or at 24 and 48 h after-LDDRR treatment for 4 weeks. No difference in platelet aggregation was found at 24 and 48 h when compared to baseline measurements. Dotted lines represent third quartile, median and first quartile. Width of the plots demonstrate frequency at which values occur.

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  Fig. 3. Ex vivo measurement of mitochondrial membrane potential in live platelets. Platelets isolated from 8 cats were loaded with tetramethylrhodamine methyl ester (TMRM) before stimulation with thrombin, thrombin/collagen (COL) and thrombin/convulxin (CVX). The extent of mitochondrial membrane potential loss was determined by measuring the percent change (%) in TMRM-positive platelets and fold change (FC) (log10) in TMRM median fluorescence intensity (MFI). (A) First, platelets were identified on flow cytometry using forward (FS) and side scatter (SS) properties and (B) gating for TMRM-positive platelets were established in fluorescent-minus-one (FMO) controls in thrombin + COL stimulated platelets. (C) Histograms demonstrating the loss of TMRM following stimulation with thrombin + CVX (red) compared to the resting (unstimulated) platelets (blue) at baseline. At 24 and 48 h after rapamycin treatment, a graduate increase in TMRM positive platelets was shown demonstrating mitochondrial protective effect despite stimulation with thrombin and CVX. (D) Violin plots demonstrating that rapamycin at 3, 24 and 48 h prevented the loss of mitochondrial membrane potential upon stimulation by all…

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  Fig. 4. Ex vivo effect of rapamycin on platelet phosphatidylserine externalization in 3-, 24-, and 48-hours post-treatment. Magnitude of phosphatidylserine (PS) flip was measured as percent change (%) based on baseline measurements. PS on platelets was detected by annexin V (AV) by flow cytometry following activation by thrombin, thrombin/ collagen (COL) and thrombin/ convulxin (CVX). (A) Resting or unstimulated platelets had minimal externalized PS. (B) Fluorescent minus one (FMO) control in thrombin + COL treated platelets was used to establish gating to identify AV-positive platelets. (C) Histograms showing an increase in PS depicted by a shift to the right after stimulation with thrombin + COL (dark purple) sample compared to unstimulated platelets (blue) at baseline and 24, 48 h post rapamycin dosing. (D) Violin plots showing that PS flip was prevented at 24 h post rapamycin treatment in thrombin (p = 0.006) and thrombin + COL activated (p = 0.02) platelets. *p < 0.05. ** p < 0.01. Dotted lines represent third quartile, median and first quartile. Width of the plots demonstrate frequency at which values occur.

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  Fig. 5. The magnitude of platelet alpha granule secretion, measured by P-selectin, after stimulation of platelets with ADP, thrombin, thrombin + COL or thrombin + CVX at 3 time points post rapamycin treatment in 8 cats. Platelets from 8 cats were activated with adenosine diphosphate (ADP) or thrombin in the presence or absence of collagen (COL) and convulxin (CVX). The magnitude of alpha granule secretion was measured as fold change (log 10) in P-selectin median fluorescence intensity (MFI) based on unstimulated (resting) platelets. (A) Resting platelets showing minimal P-selectin expression based on (B) fluorescence-minus-one control (FMO) to establish P-selectin gate. (C) Histograms showing platelets before (blue) and after ADP stimulation (pink) at baseline along with 24, 48 h post rapamycin. Modulation in P-selectin upregulation was most profound at 24 h. (D) Violin plots showing that rapamycin significantly modulated platelet activation in response to ADP at 24 and 48 h and thrombin + COL at 3 h post rapamycin. *p < 0.05. **p < 0.005. Dotted lines represent third quartile, median and first quartile. Width of the plots demonstrate frequency at which values occur.

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  Fig. 6. Heat maps showing the platelet procoagulant tendency scores in response to thrombin, thrombin/collagen (COL) and thrombin/convulxin (CVX) in 8 cats at 3, 24 and 48 h after rapamycin treatment. A procoagulant tendency score out of 3 was assigned to each cat at different time points based on the agonist groups using procoagulant platelet markers, loss of mitochondrial membrane potential (ΔΨm), phosphatidylserine (PS) externalization and P-selectin expression. A score of 3 represents the highest procoagulant tendency in an individual while a score of 0 represents resting or apoptotic platelets. Rapamycin reduced procoagulant tendency at 24 and 48 h after rapamycin when platelets were stimulated with thrombin in the presence or absence of COL. To a lesser degree, procoagulant propensity scores were reduced at 24 h after rapamycin treatment when platelets were activated.