Background

Two congenital bleeding diatheses have been identified in Thoroughbred horses: Glanzmann thrombasthenia (GT) and a second, novel diathesis associated with abnormal platelet function in response to collagen and thrombin stimulation.

Hypothesis/objectives

Platelet dysfunction in horses with this second thrombasthenia results from a secretory defect.

Animals

Two affected and 6 clinically normal horses.

Methods

Ex vivo study. Washed platelets were examined for (1) expression of the αIIb-β3 integrin; (2) fibrinogen binding capacity in response to ADP and thrombin; (3) secretion of dense and α-granules; (4) activation of the mammalian target of rapamycin (mTOR)-protein kinase B (AKT) signaling pathway; and (5) cellular distribution of phosphatidylinositol-4-phosphate-3-kinase, class 2B (PIK3C2B) and SH2 containing inositol-5'-phosphatase 1 (SHIP1).

Results

Platelets from affected horses expressed normal amounts of αIIb-β3 integrin and bound fibrinogen normally in response to ADP, but bound 80% less fibrinogen in response to thrombin. α-granules only released 50% as much Factor V as control platelets, but dense granules released their contents normally. Protein kinase B (AKT) phosphorylation was reduced after thrombin activation, but mTOR Complex 2 (mTORC2) and phosphoinositide-dependent kinase 1 (PDK1) signaling were normal. SH2-containing inositol-5'-phosphatase 1 (SHIP1) did not localize to the cytoskeleton of affected platelets and was decreased overall consistent with reduced AKT phosphorylation.

Conclusions and clinical significance

Defects in fibrinogen binding, granule secretion, and signal transduction are unique to this thrombasthenia, which we designate as atypical equine thrombasthenia.

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.