False-positive activated protein C (APC) sensitivity ratio caused by freezing and by contamination of plasma with platelets

Shizuka, Reiko; Kanda, Tsugiyasu; Amagai, H; Kobayashi, Isao

doi:10.1016/0049-3848(95)00046-1

Cited by 18 publications

(10 citation statements)

References 3 publications

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“…Sample handling has previously been shown to be a critical factor in the phenotypic analysis of APC resistance. Lower sensitivity ratios have been observed in freeze-thawed samples (Girolami et al, 1993;Trossaert et al, 1995), apparently in relation to platelet contamination (Shizuka, 1995). We found that false-low APCsr occur in samples left for more than 24 h post venepuncture and falsepositive results are common with freeze-thawed samples.…”

Section: Discussionmentioning

confidence: 58%

Effect of platelet phospholipid exposure on activated protein C resistance: implications for thrombophilia screening

1996

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The effect of platelet contamination and freeze-thawing on the activated protein C sensitivity ratio (APCsr) was determined. With increasing platelet count there was a progressive reduction in the ratio. Filtration of samples through a 0.2 microm filter before or after freeze-thawing abolished the development of resistance to the addition of activated protein C indicating that the phenomenon is due to the presence of a particulate factor. Contamination of normal plasma with platelets from a patient with homozygous factor V (FV) deficiency was also associated with the same development of resistance to activated protein C, indicating that the phenomenon was not due to exposure of platelet-derived factor V that might be inaccessible to APC. 82% (96/117) of FVQ506 and 32% (138/430) of FVR506 individuals had APC resistance on analysis of unfiltered plasma. However, 85% (42/50) of FVQ506 individuals had APC resistance on analysis of filtered plasma, whilst only 1/50 FV R506 individuals had APC resistance after filtration. For the purpose of identifying individuals at increased risk of venous thromboembolism due to the presence of the FVQ506 and associated APC resistance a PCR-based genotypic analysis is recommended.

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Section: Discussionmentioning

confidence: 58%

Effect of platelet phospholipid exposure on activated protein C resistance: implications for thrombophilia screening

1996

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“…This can potentially account for the poor anticoagulant activity of APC on activated platelets. In addition, it has been noted previously that APC resistance assays using frozen, but not fresh plasma, containing platelets are associated with frequent false-positive APC sensitivity ratios (41,42). It has been hypothesized that components released from platelets during freezing and thawing may serve as substrates and/or inhibitors of APC interfering with this assay (41).…”

Section: Vitronectin As a Cofactor In Pai-1 Inhibition Of Apc 15569mentioning

confidence: 99%

Vitronectin Functions as a Cofactor for Rapid Inhibition of Activated Protein C by Plasminogen Activator Inhibitor-1

Rezaie

2001

Journal of Biological Chemistry

123

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Activated protein C (APC) is a natural anticoagulant in plasma that down-regulates the coagulation cascade by degrading factors Va and VIIIa. In addition to its anticoagulant function, APC is also known to possess a profibrinolytic property. This property of APC has been attributed to its ability to neutralize PAI-1, thereby increasing the concentration of tissue plasminogen activator in plasma leading to up-regulation of the fibrinolytic cascade. This hypothesis, however, has not been well established, since the concentration of PAI-1 in plasma is low, and its reactivity with APC is very slow in a purified system. Here we demonstrate that vitronectin enhances the reactivity of PAI-1 with APC ϳ300-fold making PAI-1 the most efficient inhibitor of APC thus far reported (k 2 ‫؍‬ 1.8 ؋ 10 5 M ؊1 s ؊1 ). We further show that PAI-1 inhibition of the Glu 192 3 Gln mutant of APC is enhanced ϳ40-fold, independent of vitronectin, suggesting that vitronectin partially overcomes the inhibitory interaction of PAI-1 with Glu 192 . Additionally, we show that PAI-1 inhibition of the Lys 37 -Lys 38 -Lys 39 3 Pro-Gln-Glu mutant of APC is severely impaired, suggesting that, similar to tissue plasminogen activator, the basic 39-loop of APC plays a critical role in the reaction. Together, these results suggest that vitronectin functions as a cofactor to promote the profibrinolytic activity of APC.

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“…Whatever the reason, this contamination is a problem, as many clotting tests have been reported to be influenced by residual platelets. A typical example of such an influence is the APTT test when it is used to detect lupus anticoagulants (Sletnes et al , 1992; Chantarangkul et al , 2002), to measure heparin (van den Besselaar et al , 1990) or to test for APC resistance (Shizuka et al , 1995; Sidelmann et al , 1995; Luddington et al , 1996). This effect is particularly relevant after the freezing and thawing of plasma, as this process disrupts platelets.…”

Section: Discussionmentioning

confidence: 99%

“…Both test formulations are commercially available. Soon after the unmodified APTT‐based test became available, it was shown to be affected by the storage of plasma (Trossaert et al , 1995) in conjunction with platelet contamination (Shizuka et al , 1995; Sidelmann et al , 1995; Luddington et al , 1996). However, these observations were made on relatively small plasma sample numbers and only with the first generation of the commercial, unmodified, APTT‐based method.…”

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confidence: 99%

Standardization of activated protein C resistance testing: effect of residual platelets in frozen plasmas assessed by commercial and home‐made methods

et al. 2003

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Summary. We investigated 42 plasmas prepared at different centrifugation speeds with three activated protein C (APC) resistance methods. The APC ratio for fresh plateletpoor plasma declined significantly after freezing and thawing. This effect was more evident with the original method (average reduction 11AE3%) than with either the home-made (3AE8%) or the modified method (3AE2%). No significant decrease in the APC ratio was observed after freezing and thawing of platelet-free plasmas from the same patients.When frozen platelet-poor plasma was centrifuged at high speed after thawing and before testing, there was no significant decrease in the APC ratio, in comparison with fresh platelet-poor plasma using the home-made and modified methods.

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False-positive activated protein C (APC) sensitivity ratio caused by freezing and by contamination of plasma with platelets

Cited by 18 publications

References 3 publications

Effect of platelet phospholipid exposure on activated protein C resistance: implications for thrombophilia screening

Effect of platelet phospholipid exposure on activated protein C resistance: implications for thrombophilia screening

Vitronectin Functions as a Cofactor for Rapid Inhibition of Activated Protein C by Plasminogen Activator Inhibitor-1

Standardization of activated protein C resistance testing: effect of residual platelets in frozen plasmas assessed by commercial and home‐made methods

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