Intrauterine lethality in Tfpi gene disrupted mice is differentially suppressed during mid‐ and late‐gestation by platelet TFPIα overexpression

Siebert, Amy E; Maroney, Susan A.; Martinez, Nicholas D.; Mast, Alan E.

doi:10.1111/jth.15299

Cited by 4 publications

(3 citation statements)

References 51 publications

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“…This finding is consistent with mice expressing transgenic TFPIα under control of the GP1bα promoter, which have a four-to fivefold increase in platelet TFPIα, but normal plasma TFPI levels. 54 Notably, the Tfpi Δα/Δα mice did not have increased plasma TAT complex levels indicating that the absence of TFPIα did not produce a systemic increase in inappropriate thrombin production.…”

Section: Discussionmentioning

confidence: 95%

The contribution of TFPIα to the hemostatic response to injury in mice

Marar

Martinez

Maroney

et al. 2021

Journal of Thrombosis and Haemostasis

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Background: Tissue factor pathway inhibitor (TFPI) is an essential regulator of coagulation, limiting thrombin generation and preventing thrombosis. In humans and mice, TFPIα is the sole isoform present in platelets.Objective: Here, we asked whether TFPIα, because of its release from platelets at sites of injury, has a unique role in limiting the hemostatic response.Methods: TFPIα-mutant (Tfpi Δα/Δα ) mice were generated by introducing a stop codon in the C-terminus. Platelet accumulation, platelet activation, and fibrin accumulation were measured following penetrating injuries in the jugular vein and cremaster muscle arterioles, and imaged by fluorescence and scanning electron microscopy. Time to bleeding cessation was recorded in the jugular vein studies.Results: Tfpi Δα/Δα mice were viable and fertile. Plasma TFPI levels were normal in the Tfpi Δα/Δα mice, no TFPI protein or activity was present in their platelets and thrombinantithrombin complex levels were indistinguishable from Tfpi +/+ littermates. There was a small, but statistically significant reduction in the time to bleeding cessation following jugular vein puncture injury in the Tfpi Δα/Δα mice, but no measurable changes in platelet or fibrin accumulation or in hemostatic plug architecture following injury of the micro-or macrovasculature. Conclusion:Loss of TFPIα expression does not produce a global prothrombotic state in mice. Platelet TFPIα is expected to be released or displayed in a focal manner at the site of injury, potentially accumulating to high concentrations in the narrow gaps between platelets. If so, the data from the vascular injury models studied here indicate this is not essential for a normal hemostatic response in mice.

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Section: Discussionmentioning

confidence: 95%

The contribution of TFPIα to the hemostatic response to injury in mice

Marar

Martinez

Maroney

et al. 2021

Journal of Thrombosis and Haemostasis

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“…TFPI deletion causes vascular abnormalities specifically in the central nervous system and defects in the cerebrovascular development are prevented by genetic deletion of FV, 100 suggesting that TFPI is an important regulator of thrombin‐dependent signaling events that modulate cerebrovascular development. These vascular development defects cannot be rescued by genetic overexpression of platelet TFPIα, 101 which is efficient in preventing death during early embryonic development. In addition, genetic reduction of TFPI expression leads to thrombotic perinatal lethality in mice carrying the prothrombotic FV Leiden mutation, 102 further emphasizing the crucial role of TFPI as a regulator of the common coagulation pathway during pre‐ and postnatal development.…”

Section: Implications Of Tfpi Loss On Signaling Pathways and Patholog...mentioning

confidence: 99%

Regulation of coagulation by tissue factor pathway inhibitor: Implications for hemophilia therapy

Mast

Ruf

2022

Journal of Thrombosis and Haemostasis

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Tissue factor pathway inhibitor (TFPI) is an alternatively spliced anticoagulant protein that primarily dampens the initiation phase of coagulation before thrombin is generated. As such, TFPI’s actions are localized to cells expressing TF and to sites of injury, where it is an important regulator of bleeding in hemophilia. The major splice isoforms TFPIα and TFPIβ localize to different sites within and surrounding the vasculature. Both forms directly inhibit factor Xa (FXa) via their Kunitz 2 domain and inhibit TF‐FVIIa via their Kunitz 1 domain in a tight complex primarily localized to cells. By forming complexes localized to distinct cellular microenvironments and engaging additional cell surface receptors, TFPI alters cellular trafficking and signaling pathways driven by coagulation proteases of the TF pathway. TFPIα, which circulates in complex with FV and protein S, also serves an inhibitor of FXa independent of the TF initiation complex and prevents the formation of an active prothrombinase. This regulation of thrombin generation in the context of vessel injury is effectively blocked by antibodies to Kunitz 2 domain of TFPI and exploited as a therapy to restore efficient hemostasis in hemophilia.

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“…Many genes are critically important for different stages of embryonic development and therefore animals with their constitutive knockout die during embryonic development [1][2][3]. Conditional knockout organisms are required to study the functions of the affected genes on later stages of development and throughout the adult lifespan.…”

Section: Introductionmentioning

confidence: 99%

Limitations of Tamoxifen Application for In Vivo Genome Editing Using Cre/ERT2 System

Ilchuk

Stavskaya

Varlamova

et al. 2022

IJMS

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Inducible Cre-dependent systems are frequently used to produce both conditional knockouts and transgenic mice with regulated expression of the gene of interest. Induction can be achieved by doxycycline-dependent transcription of the wild type gene or OH-tamoxifen-dependent nuclear translocation of the chimeric Cre/ERT2 protein. However, both of these activation strategies have some limitations. We analyzed the efficiency of knockout in different tissues and found out that it correlates with the concentration of the hydroxytamoxifen and endoxifen—the active metabolites of tamoxifen—measured by LC-MS in these tissues. We also describe two cases of Cdk8floxed/floxed/Rosa-Cre-ERT2 mice tamoxifen-induced knockout limitations. In the first case, the standard scheme of tamoxifen administration does not lead to complete knockout formation in the brain or in the uterus. Tamoxifen metabolite measurements in multiple tissues were performed and it has been shown that low recombinase activity in the brain is due to the low levels of tamoxifen active metabolites. Increase of tamoxifen dosage (1.5 fold) and duration of activation (from 5 to 7 days) allowed us to significantly improve the knockout rate in the brain, but not in the uterus. In the second case, knockout induction during embryonic development was impossible due to the negative effect of tamoxifen on gestation. Although DNA editing in the embryos was achieved in some cases, the treatment led to different complications of the pregnancy in wild-type female mice. We propose to use doxycycline-induced Cre systems in such models.

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Intrauterine lethality in Tfpi gene disrupted mice is differentially suppressed during mid‐ and late‐gestation by platelet TFPIα overexpression

Cited by 4 publications

References 51 publications

The contribution of TFPIα to the hemostatic response to injury in mice

The contribution of TFPIα to the hemostatic response to injury in mice

Regulation of coagulation by tissue factor pathway inhibitor: Implications for hemophilia therapy

Limitations of Tamoxifen Application for In Vivo Genome Editing Using Cre/ERT2 System

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