Lack of Protein S in mice causes embryonic lethal coagulopathy and vascular dysgenesis
Tal Burstyn-Cohen, … , Mary Jo Heeb, Greg Lemke
Tal Burstyn-Cohen, … , Mary Jo Heeb, Greg Lemke
Published September 1, 2009
Citation Information: J Clin Invest. 2009;119(10):2942-2953. https://doi.org/10.1172/JCI39325.
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Lack of Protein S in mice causes embryonic lethal coagulopathy and vascular dysgenesis

Abstract

Protein S (ProS) is a blood anticoagulant encoded by the Pros1 gene, and ProS deficiencies are associated with venous thrombosis, stroke, and autoimmunity. These associations notwithstanding, the relative risk that reduced ProS expression confers in different disease settings has been difficult to assess without an animal model. We have now described a mouse model of ProS deficiency and shown that all Pros1–/– mice die in utero, from a fulminant coagulopathy and associated hemorrhages. Although ProS is known to act as a cofactor for activated Protein C (aPC), plasma from Pros1+/– heterozygous mice exhibited accelerated thrombin generation independent of aPC, and Pros1 mutants displayed defects in vessel development and function not seen in mice lacking protein C. Similar vascular defects appeared in mice in which Pros1 was conditionally deleted in vascular smooth muscle cells. Mutants in which Pros1 was deleted specifically in hepatocytes, which are thought to be the major source of ProS in the blood, were viable as adults and displayed less-severe coagulopathy without vascular dysgenesis. Finally, analysis of mutants in which Pros1 was deleted in endothelial cells indicated that these cells make a substantial contribution to circulating ProS. These results demonstrate that ProS is a pleiotropic anticoagulant with aPC-independent activities and highlight new roles for ProS in vascular development and homeostasis.

Authors

Tal Burstyn-Cohen, Mary Jo Heeb, Greg Lemke

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Figure 1

Mouse Pros1 gene targeting.

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Mouse Pros1 gene targeting. (A) ProS structure: gamma carboxyglutami...
(A) ProS structure: gamma carboxyglutamic acid (Gla) domain, thrombin-sensitive region (TSR), EGF-like repeats, and SHBG domain containing 2 laminin G repeats. (B) Mouse Pros1 locus with exon 1 encoding the ProS signal peptide (sig pep). (C) Targeting vector: Exons 11–15 were flanked by loxP sites. PstI (blue), BglI (black), and HpaI (green) sites used to characterize targeting. (D) Southern blot of DNA from an ES cell clone, using Pstl and BglI digests and the 5′ and 3′ external probes indicated in C (left 2 lanes). PstI diagnostic digest of genomic DNA from WT (+/+), heterozygous (fl/+), and homozygous (fl/fl) floxed mice (middle 3 lanes). Crossing floxed mice to the EIIA-Cre general deleter generated a KO Pros1 allele (far right lane). HpaI-digested genomic DNA from WT and KO/+ mice blotted and hybridized with the 5′ probe (right 2 lanes). (E) qPCR of Pros mRNA from E17.5 WT (n = 8) and KO (n = 6) embryos; mean ± 1 SD. (F) Western blot of protein from WT (+/+) and heterozygous Pros1+/– (KO/+) mice, with an anti-ProS antibody generated against the amino terminus of the protein (upper blot) and anti–β-actin (lower blot). ProS appears as a full-length protein (upper band) and as a thrombin-cleaved form (lower band). Both lanes were run on the same gel but were noncontiguous. Transcripts from nontargeted aminoterminal exons 4–5 are present in Pros1+/– heterozygotes (E) but apparently do not code for a stable protein. (See also Figure 9A for a related immunoblot.)