Proteomic profiling of the thrombin-activated canine platelet secretome (CAPS)

Abstract

Domestic dogs share the same environment as humans, and they represent a valuable animal model to study naturally-occurring human disease. Platelet proteomics holds promise for the discovery of biomarkers that capture the contribution of platelets to the pathophysiology of many disease states, however, canine platelet proteomic studies are lacking. Our study objectives were to establish a protocol for proteomic identification and quantification of the thrombin-activated canine platelet secretome (CAPS), and to compare the CAPS proteins to human and murine platelet proteomic data. Washed platelets were isolated from healthy dogs, and stimulated with saline (control) or gamma-thrombin (releasate). Proteins were separated by SDS-page, trypsin-digested and analyzed by liquid chromatography and tandem mass spectrometry (MS). CAPS proteins were defined as those with a MS1-abundance ratio of two or more for releasate vs. unstimulated saline control. A total of 1,918 proteins were identified, with 908 proteins common to all dogs and 693 characterized as CAPS proteins. CAPS proteins were similar to human and murine platelet secretomes and were highly represented in hemostatic pathways. Differences unique to CAPS included replacement of platelet factor 4 with other cleavage products of platelet basic protein (e.g. interleukin-8), novel proteins (e.g. C-C motif chemokine 14), and proteins in relatively high (e.g. protease nexin-1) or low (e.g. von Willebrand factor) abundance. This study establishes the first in-depth platelet releasate proteome from healthy dogs with a reference database of 693 CAPS proteins. Similarities between CAPS and the human secretome confirm the utility of dogs as translational models of human disease, but we also identify differences unique to canine platelets. Our findings provide a resource for further investigations into disease-related CAPS profiles, and for comparative pathway analyses of platelet activation among species.

The canine activated platelet secretome (CAPS): A translational model of thrombin‐evoked platelet activation response

“…Beta‐actin was the exclusive form in the dog. The majority of this beta‐actin is likely derived from canine platelet microparticles, 39,40 as seen in our previous CAPS study, in which the secreted proteins were categorized as soluble or particulate fractions 24 …”

“…Recently, we reported a shotgun proteomic characterization of the subset of activation-released secretion platelet proteins: the canine activated platelet secretome (CAPS). 24 Two thousand platelet-associated proteins were identified, with 693 designated as CAPS proteins; however, the sample size was limited. In the present study, in-solution tryptic digests replaced the in-gel digests used in our preliminary study.…”

Platelet Proteome Changes in Dogs with Chronic Heart Failure

“…In humans, proteomic studies of platelets have been performed in heart diseases (coronary artery disease, mitral valve disease and CHF), as well as other pathological conditions such as sepsis, Alzheimer's disease, diabetes mellitus, and uremia (13)(14)(15). In a pacing-induced pig heart failure model, platelet proteins were involved in cellular processes ranging from proliferation to apoptosis, as well as in ammation and cytoskeletal changes, providing that signal transduction pathways in platelets might be key mediators of platelet contributions to cardiac failure (16).…”

“…There are two studies reporting the platelet proteome in dogs, and both of them were performed in three healthy subjects (15,18). Cremer et al (15) reported a total of 693 platelet proteins that were involved in coagulation, hemostasis, proteolysis, and organonitrogen compound metabolic process, and Trichler et al (18) reported a total of 5.974 platelet proteins associated with various biological process such as response to stress, transport, cellular nitrogen compound metabolic process and signal transduction. However, with the exception of two studies in healthy dogs (15,18), no platelet proteomic study has been performed in sick dogs.…”

“…Cremer et al (15) reported a total of 693 platelet proteins that were involved in coagulation, hemostasis, proteolysis, and organonitrogen compound metabolic process, and Trichler et al (18) reported a total of 5.974 platelet proteins associated with various biological process such as response to stress, transport, cellular nitrogen compound metabolic process and signal transduction. However, with the exception of two studies in healthy dogs (15,18), no platelet proteomic study has been performed in sick dogs. Thus, this study aimed to investigate the changes in global platelet proteomes in dogs with CHF, to clarify the possible role of platelets in the physiopathology of this disease.…”