Assessment of protein C anticoagulant pathway by thrombin generation assay in the presence of endothelial cells

Coll, Enriqueta; Robles‐Carrillo, Liza; Reyes, Eduardo; Francis, John L.; Amirkhosravi, Ali

doi:10.1111/jth.12353

Cited by 6 publications

(3 citation statements)

References 12 publications

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“…The kinetics of thrombin and subsequent APC formation are comparable to the results obtained by continuous thrombin generation measurement in plasma in the presence of purified TM, with the exception that peak levels were significantly lower. 27 Both thrombin and APC formation declined after 60 minutes, indicating that thrombin and APC are cleared in the plasma matrix by endogenous inhibitors and that thrombin-TM complexes are internalized by ECs. 28 Although these data confirm the ability of the ECFCplasma approach to study the reactivity of the PC pathway, it is unclear whether the model can accurately reflect differences in EC-dependent APC formation.…”

Section: Discussionmentioning

confidence: 97%

Ex Vivo Modeling of the PC (Protein C) Pathway Using Endothelial Cells and Plasma: A Personalized Approach

Schwarz

Müller

Yadegari

et al. 2023

ATVB

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Background: The endothelial cell–dependent PC (protein C) pathway is critically involved in the regulation of coagulation, anti-inflammatory, and cytoprotective signaling. Its reactivity shows high interindividual variability, and it contributes to prothrombotic disorders, such as the FVL (factor V Leiden) mutation. Methods: Endothelial colony–forming cells (ECFCs) were isolated from heparinized peripheral blood from healthy individuals and FVL carriers. Confluent monolayers of ECFCs were overlaid with plasma, and thrombin formation was initiated by addition of tissue factor (1 pmol/L). Subsequently, thrombin and APC formation rates were measured over time using oligonucleotide-based enzyme capture assays. To induce downregulation of thrombomodulin expression, ECFCs were stimulated with IL-1β (interleukin 1β). In vivo APC response rates were monitored in study participants after infusion of low-dose rFVIIa (recombinant activated factor VII). Results: The median peak APC concentration was 1.12 nmol/L in experiments with IL-1β stimulated ECFCs and 3.66 nmol/L without IL-1β. Although thrombin formation rates were comparable, APC formation rates were significantly higher in FVL carriers (n=6) compared to noncarriers (n=5) as evidenced by a higher ratio between the area under the curve of APC generation to the area under the curve of thrombin generation (median 0.090 versus 0.031, P =0.017). These ex vivo results were correlated with an increased APC response to rFVIIa-induced thrombin formation in FVL carriers in vivo. Conclusions: Patient-specific ex vivo modeling of the PC pathway was achieved using blood-derived ECFCs. The correlation between in and ex vivo APC response rates confirms that the autologous PC model accurately depicts the in vivo situation.

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Section: Discussionmentioning

confidence: 97%

Ex Vivo Modeling of the PC (Protein C) Pathway Using Endothelial Cells and Plasma: A Personalized Approach

Schwarz

Müller

Yadegari

et al. 2023

ATVB

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“…These experiments revealed that LPS upregulates parallel inducers of the coagulation cascade, TF and PtdSer, a finding that affirms and extends previous studies of the endothelium in vivo and in vitro (17,49) and that Angpt-1 exerts actions to blunt this response. While our in vitro studies using HUVECs have limitations inherent to studies of coagulation on cultured cells, it is a well-established model (51)(52)(53). Finally, in endotoxemic mice, Angpt-1 gene transfer improved both the early prothrombotic response of systemic inflammation and the late fibrin accumulation, suggesting an action most consistent with a normalization effect.…”

Section: Methodsmentioning

confidence: 92%

Tie2 protects the vasculature against thrombus formation in systemic inflammation

Higgins¹,

Ceunynck

Kellum

et al. 2018

Journal of Clinical Investigation

109

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“…Like APC inhibition, partial depletion of plasma PC produced fibrin deposition similar to control WB (P 5 .34), with a slight decrease in time to peak fibrin deposition. 43 PC deficiency dramatically altered the activity of the fusion protein, eliminating its inhibitory effect on overall fibrin deposition (P 5 .34; Figure 8B-D) and the decrease in peak fibrin generation rate seen in control WB (supplemental Table 10). In contrast, hTM/R6.5 produced a delay in time to peak fibrin generation in both PC-deficient and control WB, consistent with direct thrombin inhibition by the fusion protein (supplemental Table 10).…”

Section: Htm/r65 Is At Least Partially Pc Dependentmentioning

confidence: 99%

ICAM-1–targeted thrombomodulin mitigates tissue factor–driven inflammatory thrombosis in a human endothelialized microfluidic model

Greineder¹,

Johnston

Villa

et al. 2017

Blood Advances

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Assessment of protein C anticoagulant pathway by thrombin generation assay in the presence of endothelial cells

Cited by 6 publications

References 12 publications

Ex Vivo Modeling of the PC (Protein C) Pathway Using Endothelial Cells and Plasma: A Personalized Approach

Ex Vivo Modeling of the PC (Protein C) Pathway Using Endothelial Cells and Plasma: A Personalized Approach

Tie2 protects the vasculature against thrombus formation in systemic inflammation

ICAM-1–targeted thrombomodulin mitigates tissue factor–driven inflammatory thrombosis in a human endothelialized microfluidic model

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