Platelet Priming and Activation in Naturally Occurring Thermal Burn Injuries and Wildfire Smoke Exposure Is Associated With Intracardiac Thrombosis and Spontaneous Echocardiographic Contrast in Feline Survivors

Abstract

Wildfires pose a major health risk for humans, wildlife, and domestic animals. We previously discovered pathophysiologic parallels between domestic cats with naturally occurring smoke inhalation and thermal burn injuries and human beings with similar injuries; these were characterized by transient myocardial thickening, cardiac troponin I elevation and formation of intracardiac thrombosis. While the underlying mechanisms remain unclear, results from murine models suggest that platelet priming and activation may contribute to a global hypercoagulable state and thrombosis. Herein, we evaluated and compared the degree of platelet activation, platelet response to physiologic agonists and levels of platelet-derived microvesicles (PDMV) in 29 cats with naturally occurring wildfire thermal injuries (WF), 21 clinically healthy cats with subclinical hypertrophic cardiomyopathy (HCM) and 11 healthy cats without HCM (CC). We also quantified and compared circulating PDMVs in WF cats to CC cats. In addition, we examined the association between thrombotic events, severity of burn injuries, myocardial changes, and the degree of platelet activation in cats exposed to wildfires. Flow cytometric detection of platelet surface P-selectin expression showed that WF cats had increased platelet response to adenosine diphosphate (ADP) and thrombin compared to the two control groups indicating the presence of primed platelets in circulation. In addition, cats in the WF group had increased circulating levels of PDMV, characterized by increased phosphatidylserine on the external leaflet. Cats in the WF group with documented intracardiac thrombosis had elevated platelet activation and platelet priming in the presence of ADP. While high dose arachidonic acid (AA) mostly resulted in platelet inhibition, persistent response to AA was noted among cats in the WF group with intracardiac thrombosis. Univariate and multiple logistic regression analyses demonstrated that increased platelet response to AA was independently associated with thrombotic events. This is the first study reporting the significant association between platelet priming and intracardiac thrombosis in domestic cats with naturally occurring wildfire-related injuries and smoke inhalation. Further studies are required to delineate additional mechanisms between inflammation and thrombosis, especially regarding platelet primers and the cyclooxygenase pathway.

One sentence summary: Platelet activation and shedding of platelet-derived microvesicles due to platelet priming is present following naturally occurring wildfire smoke exposure and thermal burn injuries in a population of domestic cats.

Keywords: hypercoagulability; hypertrophic cardiomyopathy (HCM); particulate matter <2.5 μm (PM) 2.5; primary hemostasis; thromboembolism.

Figure 1

Representative scatter plot diagrams and histograms of flow cytometric analysis of platelets in a cat with wildfire (WF) related injuries and 1 healthy control cat. (A,B) Platelets were identified by forward (FS) and side scatter (SS) properties. Note the shift in scatter properties (arrows) in platelets from a cat in WF group compared to those in the healthy control indicating shape change and degranulation. Arachidonic acid (AA)-treated (1 μM) platelets in a WF group cat resulted in a shift in scatter properties indicative of intact granules and size. (B) Representative histograms illustrating the number of platelets expressing P-selectin in the absence (resting) or presence of 20 μM ADP in a WF cat and healthy control. Note the upregulation in P-selectin in either resting or ADP-activated platelets in the WF cat compared to healthy control.

Figure 2

Increased platelet priming led to augmented response to adenosine diphosphate in cats with naturally occurring wildfire injuries and smoke exposure. Platelet activation, measured as P-selectin median fluorescence intensity (MFI) (A) and percentage of P-selectin positive events (B), was evaluated by flow cytometry in 29 cats exposed to wildfires (WF), 21 cats with subclinical hypertrophic cardiomyopathy (HCM) and 11 control cats without HCM (CC). Platelet rich plasma was activated with 20 μM ADP and compared to unstimulated platelets (resting) within each group. ADP resulted in significant elevation in P-selectin expression in all 3 groups. WF cats had increased magnitude in P-selectin density (MFI) compared to cats in the HCM and CC groups in response to ADP. *p < 0.05.

Figure 3

Representative scatter dot plot diagrams, gating strategy and P-selectin expression in thrombin-activated platelets. (A) Platelets were first identified by forward (FS) and side scatter (SS) properties (*Platelet gate). With thrombin activation (0.01 U/ml), events were further gated based on scatter properties to either Quiescent (arrowhead) or Activated (arrow). Note the shift of cells from the Quiescent to Activated gates following thrombin treatment in a control cat while the number of events was similar in either gate in a cat exposed to wildfires. Platelet activation, measured as percentage of P-selectin positive events, or median fluorescence intensity (MFI) was compared before and after thrombin stimulation in 29 cats exposed to wildfires (WF), 21 cats with subclinical hypertrophic cardiomyopathy (HCM) and 11 control cats without HCM (CC). (B) In the Quiescent gate, thrombin resulted in elevated P-selectin in HCM and WF groups but no difference was noted among the three groups. (C) Only cats in the WF group had elevated levels of P-selectin (MFI) in the Activated gate following thrombin stimulation. *p > 0.05.

Figure 4

Cats with naturally occurring wildfire related (WF) injuries had increased activated platelets and platelet priming. Platelet activation in 29 WF cats with or without spontaneous echocardiographic contrast with or without intracardiac thrombi (SEC ± T) was measured as either percentage of P-selectin-positive platelets or P-selectin median florescence intensity (MFI) at rest, or in the presence of 20 μM ADP (A) or 0.01 U/ml thrombin (B). (A) Cats with SEC ± T had increased amount of P-selectin positive platelets in unstimulated (resting) platelets. (A,B) There was also evidence of increased platelet priming in cats with SEC ± T given that higher percentages of P-selectin positive platelets were found in cats with SEC ± T compared to those without following treatment of platelets with ADP or thrombin. *p < 0.05.

Figure 5

Ex vivo treatment of platelets with arachidonic acid results in platelet inhibition but not in those with spontaneous echocardiographic contrast with or without intracardiac thrombi. Platelet activation was assessed in 29 cats with wildfire (WF) injuries, measured as P-selectin median fluorescence intensity (MFI) (A) or percentage (%) of P-selectin positive platelets (B) at rest and in the presence of 1 μM arachidonic acid (AA) by flow cytometry. While AA caused a significant decrease in percentage of P-selectin positive platelets in cats with or without SEC ± T (A), those with SEC ± T sustained P-selectin density (MFI) following AA treatment compared to those without SEC ± T, which had significantly lower MFI (B). (C) Platelets from five healthy cats were unstimulated (rest) or treated with either 1 μM AA or 10 μM PGE₁. Phosphorylation of intracellular vasodilator-stimulated phosphoprotein (P-VASP) was measured by flow cytometry. Elevation in P-VASP MFI in the presence of AA indicates significant platelet inhibition. *p < 0.05.

Figure 6

Cats with naturally occurring wildfire injuries (WF) had elevated circulating platelet-derived microvesicles (PDMV). PDMV was measured in platelet poor plasma in 29 WF cats and 8 healthy control cats by flow cytometry. (A–C) Representative scatter dot plot diagrams of flow cytometric analysis of PDMV identified based on size, side scatter properties and co-expression of integrin β3 (CD61) and phosphatidylserine (PS) – P2. (A) Representative scatter plot generated from a cat with wildfire-related injuries, (B) from a control cat without hypertrophic cardiomyopathy and (C) positive control generated by activating platelets with 2 mM calcium and the calcium ionophore, A23187 (2.5 μM), which showed a significant increase in PDMV compared to resting sample. (D,E) WF cats not only had increased number of PDMV but a greater percentage of them had externalized PS compared to healthy controls. *p < 0.05.

Figure 7

(A) Myocardial thickness, measured based on maximum wall thickness of the left ventricle (LV) and left atrial function, assessed by left auricular flow velocity (LAuV) did not differ among cats exposed to wildfires with or without spontaneous echocardiographic contrast ± intracardiac thrombi (SEC ± T). (B) Platelet activation in 29 cats, measured as P-selectin median florescence intensity (MFI) at rest or in the presence of 20 μM ADP, thrombin or 1 μM arachidonic acid (AA). Platelet activation did not differ among cats with evidence of myocardial thickness. (C) Similarly, there was no evidence of increased platelet priming in cats with normal or low left auricular flow velocity (LAuV).