TY - ABST

T1 - The Canine Platelet Secretome (CAPS): Proteomic Analysis of Thrombin-stimulated Release

AU - Cremer, Signe Emilie

AU - Catalfamo, J.L.

AU - Kristensen, Annemarie Thuri

AU - Goggs, R.A.N.

AU - Brooks, M. B:

PY - 2018

Y1 - 2018

N2 - Background: Domestic dogs share the same environment as their owners and represent a valuable animal model to study naturally‐occurring human disease. Proteomics holds promise for discovery of cancer biomarkers, however comparative platelet proteomics are lacking.Aims: To establish a protocol for shotgun proteomic identification and quantification of proteins released from agonist‐activated canine platelets.Methods: Washed platelets were isolated from ACD‐A anticoagulated blood from a hound mixed‐breed dog by serial centrifugation. Stirred, washed platelets (1.0×109/mL) were stimulated with saline or 50 nM gamma thrombin at 37°C for 6 minutes. Protease inhibitors were added and followed by centrifugation to remove cells and debris. The supernatant was spun at 50,000× g to yield soluble and pellet (microparticle) fractions. The former was concentrated. Protein concentration was measured in both fractions. SDS‐PAGE was used to separate proteins present in soluble and pellet fractions (Figure 1). For shotgun proteomic analysis, the gel was divided into sections and in‐gel trypsin digested. Tryptic peptides were separated by nano‐LC (liquid chromatography) followed by tandem mass‐spectrometry (MS/MS). Proteins were identified with Sequest software searching a canine database. Identified proteins had minimally 1 unique peptide and were sorted based on +/‐ ≥2 peptides. A ratio of ≥2 for MS1 abundance (activated/control) was used to identify proteins released.

AB - Background: Domestic dogs share the same environment as their owners and represent a valuable animal model to study naturally‐occurring human disease. Proteomics holds promise for discovery of cancer biomarkers, however comparative platelet proteomics are lacking.Aims: To establish a protocol for shotgun proteomic identification and quantification of proteins released from agonist‐activated canine platelets.Methods: Washed platelets were isolated from ACD‐A anticoagulated blood from a hound mixed‐breed dog by serial centrifugation. Stirred, washed platelets (1.0×109/mL) were stimulated with saline or 50 nM gamma thrombin at 37°C for 6 minutes. Protease inhibitors were added and followed by centrifugation to remove cells and debris. The supernatant was spun at 50,000× g to yield soluble and pellet (microparticle) fractions. The former was concentrated. Protein concentration was measured in both fractions. SDS‐PAGE was used to separate proteins present in soluble and pellet fractions (Figure 1). For shotgun proteomic analysis, the gel was divided into sections and in‐gel trypsin digested. Tryptic peptides were separated by nano‐LC (liquid chromatography) followed by tandem mass‐spectrometry (MS/MS). Proteins were identified with Sequest software searching a canine database. Identified proteins had minimally 1 unique peptide and were sorted based on +/‐ ≥2 peptides. A ratio of ≥2 for MS1 abundance (activated/control) was used to identify proteins released.

U2 - 10.1002/rth2.12125

DO - 10.1002/rth2.12125

M3 - Conference abstract in journal

VL - 2

JO - Research and Practice in Thrombosis and Haemostasis

JF - Research and Practice in Thrombosis and Haemostasis

SN - 2475-0379

IS - S1

M1 - PB055

Y2 - 18 July 2018 through 21 July 2018

ER -