Protein C Activity in Dogs: Adaptation of a Commercial Human Colorimetric Assay and Evaluation of Effects of Storage Time and Temperature

Fry, Michael M.; Snyder, Karl R.; Tobias, Karen M.; Williamson, Baye G.; Reed, Gregory A.

doi:10.4061/2011/751849

Cited by 3 publications

(3 citation statements)

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“…A chromogenic assay with the same analytic principles as those previously published in dogs was used to measure plasma protein C activity in this case. Protein C is stable in both human and canine samples for at least 2 weeks when stored frozen at −20°C . In this case, samples taken at 2 and 6 weeks after diagnosis showed a persistent and marked decrease in plasma protein C concentrations and in the absence of any conditions known to cause an acquired protein C deficiency, the findings are consistent with a congenital deficiency.…”

Section: Discussionsupporting

confidence: 55%

“…Chromogenic assays are recommended for initial evaluation of protein C deficiency in humans because interferences that falsely prolong or shorten clotting times in clot‐based assays cannot operate in chromogenic assays whose analytical principles are independent of coagulation pathways . A canine‐specific assay for measurement of protein C is not available but adaption of a human chromogenic assay has been validated for use in dogs . Published reference values for protein C activity in dogs, measured with functional chromogenic assays developed for use in humans, have been established at 75%‐135% .…”

Section: Discussionmentioning

confidence: 99%

“…A canine‐specific assay for measurement of protein C is not available but adaption of a human chromogenic assay has been validated for use in dogs . Published reference values for protein C activity in dogs, measured with functional chromogenic assays developed for use in humans, have been established at 75%‐135% . A chromogenic assay with the same analytic principles as those previously published in dogs was used to measure plasma protein C activity in this case.…”

Section: Discussionmentioning

confidence: 99%

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Congenital protein C deficiency and thrombosis in a dog

Kelly¹,

Juvet²,

Moore³

2020

Veterinary Internal Medicne

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Congenital protein C deficiency is an important cause of thrombosis in humans but is not described in dogs. A 4-year-old Hungarian Vizsla was presented for investigation of acute onset of ascites. Computed tomography of the chest and abdomen and echocardiography confirmed a large thrombus within the right ventricle. A cause for thrombosis was not initially identified. The clinical signs resolved rapidly and the dog was administered clopidogrel and discharged. Plasma protein C activity measured 2 and 6 weeks later was markedly lower than expected on both occasions. All known causes of acquired protein C deficiency were excluded, and the dog was diagnosed with a congenital protein C deficiency. After diagnosis, the administration of clopidogrel was stopped and administration of rivaroxaban was started. The dog remains well with no evidence of recurrent thrombosis with 6 months of follow-up. K E Y W O R D S antiphospholipid, clopidogrel, rivaroxaban, thromboembolism

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Section: Discussionsupporting

confidence: 55%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Congenital protein C deficiency and thrombosis in a dog

Kelly¹,

Juvet²,

Moore³

2020

Veterinary Internal Medicne

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Purification of protein C from canine plasma

et al. 2014

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BackgroundIn order to characterize the functional properties of canine protein C (CnPC), the zymogen needs to be purified from plasma. The goals of this study were (1) to purify protein C from fresh frozen canine plasma by barium chloride and ammonium sulphate precipitation, followed by immunoaffinity chromatography using a monoclonal mouse antibody against human protein C (HPC4) and (2) to characterize this protein’s structure.ResultsThe purified protein contained three glycosylated forms of a heavy chain (~49 kDa) and a glycosylated light chain (~25 kDa). Tandem mass spectra of the peptides obtained following trypsin digestion and liquid chromatography identified this protein to be protein C (vitamin K-dependent protein C precursor, gi|62078422) with 100% probability. Three glycosylation sites (Asn139, Asn202, and Asn350) were identified by detection of peptides containing an N-linked glycosylation consensus sequon with a 3-dalton increase in mass following incubation of the protein with PNGase F in 18O-labeled water. Following incubation with Protac (a specific activator of protein C), the heavy chain showed a slight decrease in molecular size and amidolytic activity measured by a synthetic chromogenic substrate containing an amide bond [H-D-(γ-carbobenzoxyl)-lysyl-prolyl-arginine-paranitroanilide diacetate salt]. The amidolytic activity was increased by ~303-fold in the final protein preparation compared to that in plasma. The purified protein showed concentration-dependent anti-factor V and anti-factor VIII activities in canine plasma in coagulometric factor assays.ConclusionsThese studies showed that CnPC could be purified from plasma using HPC4 and that this protein showed amidolytic and anti-coagulant properties upon activation with Protac.

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Comparing the Effect of DOAC-Stop® and DOAC-Remove® on Apixaban, Rivaroxaban and Dabigatran Prior to Thrombophilia and Lupus Testing

Malik,

Ward,

Erskine

2024

Br J Biomed Sci

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BackgroundDirect oral anticoagulants (DOACs) interfere with coagulation assays potentially leading to inaccurate results. This study determined the effectiveness of DOAC-stop® and DOAC-remove® in overcoming DOAC interference. It aimed to investigate the extent to which apixaban, rivaroxaban, and dabigatran had an effect on thrombophilia and lupus tests using normal plasma, as well as whether DOACs interfere with true-positive results by testing abnormal controls.MethodsApixaban (0.03 mg/mL), rivaroxaban (0.01 mg/mL), and dabigatran (0.019 mg/mL) stock solutions were made and added to the normal pool at three different concentrations (200, 400 and 600 ng/mL) and to the abnormal controls at a single concentration. These samples and untreated DOAC controls were tested before and after adding either DOAC-stop® or DOAC-remove®. The measured parameters included protein C, protein S, antithrombin III (ATIII), DRVVS, DRVVC, PTT-LA and DOAC concentration. The normal pool spiked with DOAC was repeated seven times for each DOAC at each concentration level and the abnormal controls spiked with DOAC were repeated four times at a single concentration level for each DOAC.ResultsIn the normal pool, dabigatran and rivaroxaban affected all lupus anticoagulant tests, whereas apixaban only affected DRVVS and DRVVC. While dabigatran led to false-positive protein S deficiency and falsely elevated ATIII. Both DOAC-stop® and DOAC-remove® brought the thrombophilia results and all falsely elevated lupus anticoagulant results back within the normal range for apixaban and rivaroxaban. For dabigatran all the affected lupus anticoagulant tests remained abnormal following DOAC-remove®, unlike DOAC-stop® treatment, where only DRVVS and DRVVC at 600 ng/mL remained abnormal. In abnormal controls, all DOACs falsely elevated the lupus anticoagulant tests, whereas dabigatran caused false negative ATIII results, that were corrected (remained abnormal) with DOAC-stop® and DOAC-remove®. DOAC-stop® showed a greater reduction in lupus anticoagulant results than DOAC-remove®, causing a false-negative DRVVT ratio for rivaroxaban.ConclusionDOAC-stop® is more effective than DOAC-remove® in removing all DOACs below the reference range, whereas DOAC-remove® failed to remove dabigatran.

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Protein C Activity in Dogs: Adaptation of a Commercial Human Colorimetric Assay and Evaluation of Effects of Storage Time and Temperature

Cited by 3 publications

References 20 publications

Congenital protein C deficiency and thrombosis in a dog

Congenital protein C deficiency and thrombosis in a dog

Purification of protein C from canine plasma

Comparing the Effect of DOAC-Stop® and DOAC-Remove® on Apixaban, Rivaroxaban and Dabigatran Prior to Thrombophilia and Lupus Testing

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