Flow cytometric evaluation of disseminated intravascular coagulation in a canine endotoxemia model

Abstract

Sepsis is associated with substantial morbidity and mortality in dogs. Alterations in hemostasis by systemic inflammation play an important role in the pathophysiology of sepsis. To evaluate the functional hemostatic changes in sepsis, we evaluated coagulation profiles and flow cytometric measurement of P-selectin (CD62P) expression on platelets, as well as platelet-leukocyte aggregation from a lipopolysaccharide (LPS)-induced endotoxemia model in dogs (n = 7). A sublethal dose of LPS [1 mg/kg body weight (BW)] induced thrombocytopenia and increased activated partial thromboplastin time (aPTT), prothrombin time (PT), and D-dimer concentrations. Flow cytometry analysis showed a significant increase in P-selectin expression on platelets between 1 and 24 h of a total 48 h of the experiment. In addition, platelet-leukocyte aggregation was significantly increased in the early stage of endotoxemia (at 1 and < 6 h for platelet-monocyte aggregation and at 3 h for platelet-neutrophil aggregation). Our results suggest that CD62P expression on platelets and platelet-leukocyte aggregation, as measured by flow cytometry, can be useful biomarkers of disseminated intravascular coagulation (DIC) in canine sepsis. These functional changes contribute to our understanding of the pathophysiology of hemostasis in endotoxemia.

Figure 1

Measurement of platelet-leukocyte aggregation from PMA stimulated blood. Canine blood was incubated with phorbol myristate acetate (PMA, 10 μM) at 37°C for 20 min in vitro and platelet-leukocyte aggregation was measured using flow cytometry. CD14 antibody was used as a neutrophil/monocyte surface marker and CD61 was used as a platelet surface marker. Neutrophils/monocytes were logically gated based on forward/side scatter in flow cytometry (a) and discriminated by CD14 (b). Among the gated cell population shown in Figure 1(a) and (b), CD14 ^bright+/CD61⁺ double positive cells (upper right quadrant population) represented monocyte-platelet aggregation and CD14 ^dim+/CD61⁺-double-positive cells (upper left quadrant population) represented granulocyte-platelet aggregation (c).

Figure 2

Alterations in coagulation tests in control and in dogs given LPS. Dogs were slowly injected with lipopolysaccharide (LPS, 1 mg/kg BW from E. coli O111: B4, solid black line) or saline (control, grey line) and blood was periodically collected from the jugular vein. (a) Platelet counts; (b) Prothrombin time; (c) Activated partial thromboplastin time; and (d) D-dimer concentrations (all values from the controls were below detection limits). * P < 0.05, ** P < 0.01 versus control at each time point.

Figure 3

Alterations in CD62P (P-selectin) expression on platelets in control and in dogs given LPS. Dogs were injected with lipopolysaccharide (LPS, 1 mg/kg BW from E. coli O111: B4, solid black line) or saline (control, dashed line). Platelets were selected by sorting out CD61-PE-positive cells by flow cytometry. The percentage of CD62P-FITC positive cells compared to isotype control was calculated by histogram. The same letters designate a significant difference in the positive of CD62P-positive cells within or between groups.

Figure 4

Percentage of platelet-leukocyte aggregation by flow cytometry in control and LPS dogs. Platelet-neutrophil aggregation increased 3 h after lipopolysaccharide (LPS) injection (a), whereas platelet-leukocyte aggregation increased in monocytes from 3 to 6 h after LPS injection (b). The same letters designate a significant difference in platelet-leukocyte aggregation between groups.