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Evaluation of procoagulant tissue factor expression in canine hemangiosarcoma cell lines

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  • 1 Department of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14850.
  • | 2 Department of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14850.
  • | 3 Department of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14850.
  • | 4 Department of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14850.

Abstract

OBJECTIVE To evaluate expression of procoagulant tissue factor (TF) by canine hemangiosarcoma cells in vitro.

SAMPLES 4 canine hemangiosarcoma cell lines (SB-HSA [mouse-passaged cutaneous tumor], Emma [primary metastatic brain tumor], and Frog and Dal-1 [primary splenic tumors]) and 1 nonneoplastic canine endothelial cell line (CnAoEC).

PROCEDURES TF mRNA and TF antigen expression were evaluated by quantitative real-time PCR assay and flow cytometry, respectively. Thrombin generation was measured in canine plasma and in coagulation factor–replete or specific coagulation factor–deficient human plasma by calibrated automated thrombography. Corn trypsin inhibitor and annexin V were used to examine contributions of contact activation and membrane-bound phosphatidylserine, respectively, to thrombin generation.

RESULTS All cell lines expressed TF mRNA and antigen, with significantly greater expression of both products in SB-HSA and Emma cells than in CnAoEC. A greater percentage of SB-HSA cells expressed TF antigen, compared with other hemangiosarcoma cell lines. All hemangiosarcoma cell lines generated significantly more thrombin than did CnAoEC in canine or factor-replete human plasma. Thrombin generation induced by SB-HSA cells was significantly lower in factor VII–deficient plasma than in factor-replete plasma and was abolished in factor X–deficient plasma; residual thrombin generation in factor VII–deficient plasma was abolished by incubation of cells with annexin V. Thrombin generation by SB-HSA cells was unaffected by the addition of corn trypsin inhibitor.

CONCLUSIONS AND CLINICAL RELEVANCE Hemangiosarcoma cell lines expressed procoagulant TF in vitro. Further research is needed to determine whether TF can be used as a biomarker for hemostatic dysfunction in dogs with hemangiosarcoma.

Abstract

OBJECTIVE To evaluate expression of procoagulant tissue factor (TF) by canine hemangiosarcoma cells in vitro.

SAMPLES 4 canine hemangiosarcoma cell lines (SB-HSA [mouse-passaged cutaneous tumor], Emma [primary metastatic brain tumor], and Frog and Dal-1 [primary splenic tumors]) and 1 nonneoplastic canine endothelial cell line (CnAoEC).

PROCEDURES TF mRNA and TF antigen expression were evaluated by quantitative real-time PCR assay and flow cytometry, respectively. Thrombin generation was measured in canine plasma and in coagulation factor–replete or specific coagulation factor–deficient human plasma by calibrated automated thrombography. Corn trypsin inhibitor and annexin V were used to examine contributions of contact activation and membrane-bound phosphatidylserine, respectively, to thrombin generation.

RESULTS All cell lines expressed TF mRNA and antigen, with significantly greater expression of both products in SB-HSA and Emma cells than in CnAoEC. A greater percentage of SB-HSA cells expressed TF antigen, compared with other hemangiosarcoma cell lines. All hemangiosarcoma cell lines generated significantly more thrombin than did CnAoEC in canine or factor-replete human plasma. Thrombin generation induced by SB-HSA cells was significantly lower in factor VII–deficient plasma than in factor-replete plasma and was abolished in factor X–deficient plasma; residual thrombin generation in factor VII–deficient plasma was abolished by incubation of cells with annexin V. Thrombin generation by SB-HSA cells was unaffected by the addition of corn trypsin inhibitor.

CONCLUSIONS AND CLINICAL RELEVANCE Hemangiosarcoma cell lines expressed procoagulant TF in vitro. Further research is needed to determine whether TF can be used as a biomarker for hemostatic dysfunction in dogs with hemangiosarcoma.

Supplementary Materials

    • Supplementary Figure 1 (PDF 299 kb)

Contributor Notes

Address correspondence to Ms. Witter (lew226@cornell.edu).

Mr. Lean's present address is School of Veterinary Science, Faculty of Science, The University of Queensland, 4343 Gatton, Queensland, Australia.