Abstract

TFPI and thrombomodulin are the two primary endothelium associated anticoagulant proteins. Studies using an in vitro model system of purified coagulation proteins have shown that TFPI and thrombomodulin act synergistically to quench tissue factor mediated thrombin generation via neutralization of prothrombinase activity. However, it is unclear how these two proteins cooperate within different vascular beds in vivo, particularly in the brain that has large amounts of tissue factor procoagulant activity. Mice with decreased thrombomodulin function do not have fibrin deposition within the brain suggesting that other anticoagulants, such as TFPI, may be important for prevention of cerebral thrombosis. Consistent with this hypothesis, we have demonstrated that partial TFPI deficiency induces intravascular fibrin deposition in the brain of the mice with decreased thrombomodulin function suggesting that TFPI may be the primary anticoagulant within the brain vasculature. Yet, studies of mouse and human tissues have shown that TFPI is expressed at much lower levels in brain than in other tissues. Therefore, we undertook studies to characterize the expression of two alternatively spliced isoforms of TFPI, TFPIα and TFPIβ, in mouse brain. In situ hybridization studies detected abundant expression of TFPIα and TFPIβ on brain endothelium as well as expression by individual astrocytes within the parenchyma of the cerebral cortex. Using RPL-19 as a housekeeper gene, quantitative real time PCR analysis of TFPIα and TFPIβ mRNA in mouse brain demonstrated that TFPIα message is 9-fold more abundant that TFPIβ message. This analysis also demonstrated that brain expresses far less TFPIα and TFPIβ mRNA than any other tissue (TFPIα: 18-fold less than lung, 19-fold less than heart, 372-fold less than placenta; TFPIβ: 15-fold less than lung, 41-fold less than heart, 68-fold less than placenta). Since TFPI mRNA was readily detected within mouse brain endothelium in the in situ hybridization studies, we hypothesized that the low TFPIα and TFPIβ expression in the brain is due to low relative numbers of endothelial cells in brain when compared to other tissues rather than low expression of TFPI by brain endothelium. Quantitative PCR analysis was repeated with TFPI expression levels normalized to CD-31 and VE-cadherin, two relatively specific markers for endothelial cells. In this analysis, placenta has by far the highest level of TFPIα and TFPIβ expression most likely because both the endothelial cells and trophoblasts of the placenta produce TFPI while trophoblasts produce smaller amounts of CD-31 or VE-cadherin. The relative amount of TFPIα mRNA produced by brain endothelium is 7-fold greater than that of lung endothelial cells and 60% that of heart endothelial cells. The amount of TFPIβ mRNA produced by brain endothelium is 20-fold greater than that of lung endothelial cells and 10% that of heart endothelial cells. Therefore, in contrast to previous reports, these data demonstrate that brain endothelium produces abundant amounts of TFPIα and TFPIβ mRNA. The variable amount of TFPI produced by these vascular beds likely contributes to the tissue specific thrombosis observed in mouse models of tissue factor mediated disease. Finally, western blot analysis demonstrated that TFPIβ is the primary TFPI isoform produced by mouse brain indicating that regulation of TFPI isoform production occurs at least in part at the level of protein translation. Taken together, the data demonstrate that TFPI is an abundant anticoagulant protein on brain endothelium where it acts to prevent fibrin deposition within the cerebral vasculature. In mice, this anticoagulant activity is produced primarily by TFPIβ.

Disclosures: No relevant conflicts of interest to declare.

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