Platelet activation in cats with hypertrophic cardiomyopathy

Abstract

Background: Cats with hypertrophic cardiomyopathy (HCM) are at risk for development of systemic thromboembolic disease. However, the relationship between platelet activation state and cardiovascular parameters associated with HCM is not well described.

Objectives: To characterize platelet activation by flow cytometric evaluation of platelet P-selectin and semiquantitative Western blot analysis of soluble platelet-endothelial cell adhesion molecule-1 (sPECAM-1).

Animals: Eight normal healthy cats (controls) owned by staff and students of the School of Veterinary Medicine and 36 cats from the UC Davis Feline HCM Research Laboratory were studied.

Methods: Platelet-rich plasma (PRP) was used for all flow cytometry studies. Platelet surface CD41 and P-selectin expression were evaluated before and after ADP stimulation. sPECAM-1 expression was evaluated by Western blot analysis of platelet-poor plasma that had been stabilized with aprotinin. Standard echocardiographic studies were performed.

Results: Resting platelets from cats with severe HCM had increased P-selectin expression compared to controls, and expressed higher surface density of P-selectin reflected by their increased mean fluorescence intensities (MFI). Stimulation with ADP also resulted in significantly increased P-selectin MFI of platelets from cats with severe HCM. Increased P-selectin expression and MFI correlated with the presence of a heart murmur and end-systolic cavity obliteration (ESCO). sPECAM-1 expression from cats with moderate and severe HCM was significantly increased above those of control cats.

Conclusions and clinical importance: P-selectin and sPECAM expression may be useful biomarkers indicating increased platelet activation in cats with HCM.

Keywords: Hypertrophic cardiomyopathy; P-selectin; Platelet activation; Platelets; sPECAM-1.

Figure 1

(A, B) Representative forward scatter and side scatter images of resting control platelets (A) and resting platelets from a cat with severe hypertrophic cardiomyopathy (HCM) (B). Black oval (→) delineates the microvesicle population as defined by 0.5 μm‐standardized beads, while the larger black oval to the right (>) represents the platelet population. (C) Representative histograms of resting platelets from a control cat (light gray) and a cat with severe HCM (black) demonstrating the significant increase in platelet P‐selectin expression in cats with severe HCM. (D) Flow cytometric analysis of P‐selectin‐positive platelets demonstrates a significant difference between P‐selectin‐positive platelets in control cats and cats with severe HCM (P = .0275). (E) Platelets from cats with severe HCM have a higher mean fluorescence intensities of P‐selectin on their surface than platelets from control cats (P = .0028).

Figure 2

(A) When platelets are stimulated with the physiologic agonist adenosine diphosphate, there were no statistically significant differences in the percentage of P‐selectin‐positive platelets between any of the groups. (B) There were statistically significant differences in mean fluorescence intensities (MFI), and platelets from cats with severe hypertrophic cardiomyopathy had higher P‐selectin MFI on their surface than did control cats (P = .0055).

Figure 3

(A) When platelets were stimulated with adenosine diphosphate (ADP), cats with severe hypertrophic cardiomyopathy (HCM) expressed greater numbers of CD41‐positive microvesicles than did control cats (P = .0228). (B) Representative histograms of ADP‐activated platelets from a control cat (light gray) and a cat with severe HCM (black) demonstrating the significant increase in CD41‐positive microvesicles in cats with severe HCM.

Figure 4

(A) There was a significant increase in P‐selectin‐positive platelets in cats with murmurs (P = .011). (B) Platelets from cats with heart murmurs had significantly increased P‐selectin mean fluorescence intensities on their surface (P = .0017).

Figure 5

(A) Cats that had end‐systolic cavity obliteration (ESCO) on ultrasound examination expressed more P‐selectin‐positive cells (P = .02). (B) Cats that had ESCO on ultrasound examination also had a higher mean fluorescence intensities of P‐selectin on the cell surface (P = .002).

Figure 6

Mean relative densities of soluble platelet‐endothelial cell adhesion molecule‐1 from cats with moderate and severe hypertrophic cardiomyopathy were significantly higher than those of controls (P ≤ .001 and P ≤ .05, respectively).