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. 2009 Oct;119(10):2942-53.
doi: 10.1172/JCI39325.

Lack of Protein S in Mice Causes Embryonic Lethal Coagulopathy and Vascular Dysgenesis

Free PMC article

Lack of Protein S in Mice Causes Embryonic Lethal Coagulopathy and Vascular Dysgenesis

Tal Burstyn-Cohen et al. J Clin Invest. .
Free PMC article


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.


Figure 1
Figure 1. Mouse Pros1 gene targeting.
(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.)
Figure 2
Figure 2. Lethal embryonic coagulopathy in ProS-deficient mice.
Wild-type (A, C, and E) and Pros1–/– littermates (B, D, and F) at E15.5. (A and B) Principal superficial blood vessels that are readily visible in WT (A, arrowheads; n = 8) are not visible in Pros1–/– littermates (B, arrowheads; n = 10). Pros1–/– embryos present with macroscopic thrombi (B, asterisks). (C and D) Coronal brain sections (300 μm) of perfused WT and Pros1–/– embryos, respectively. Perfusion of WT brain yields clear tissue (C), but intravascular thrombi render vessels perfusion resistant in Pros1–/– brain tissue, and hemorrhages are prominent (D). (E and F) Perfusion-drained WT (E) and occluded, perfusion-resistant Pros1–/– (F) capillaries in 1-μm sections of perfused embryonic brains; white and red blood cells are trapped in a fibrin mesh. Red dashed lines delineate the boundaries of the microvessels. Scale bar in F applies to all panels: 500 μm in AD; 20 μm in E and F.
Figure 3
Figure 3. Severe hemorrhages and thrombi in Pros1–/– embryos.
(A and B) Carstairs staining of 10-μm coronal cryosections from nonperfused E13.5 embryo heads. Clotted blood cells stain magenta-purple; collagen, blue; and tissue, purple-blue. (A) Intact WT tissue with normal ventricles, no hemorrhages, and no clots. (B) Pros1–/– heads are hemorrhagic, with blood clots, enlarged brain ventricles, and prominent penetration of blood into the brain parenchyma. (CE) H&E staining of paraffin sections (4 μm) from nonperfused WT (C) and Pros1–/– embryos (D and E). Vessel walls are thick and well formed in WT (C) but are thin and discontinuous in Pros1–/– embryos (D), enabling leakage of blood cells into the surrounding tissue (D, arrowheads), which presents with pyknotic nuclei typical of ischemic damage (D, arrows). The vessel in D contains a thrombus (center). (E) Subectodermal, superficial hemorrhages are indicative of severe blood loss in Pros1–/– mice. Scale bar in E applies to all panels: 1,000 μm (A and B); 50 μm (C and D); and 100 μm (E).
Figure 4
Figure 4. Pros1–/– embryos present with a massive disseminated hypercoagulopathy and associated hemorrhages.
Carstairs differential stain of perfused E15.5 WT (A, C, E, and G) and Pros1–/– (B, D, F, and H) littermates stains loose blood cells yellow-orange; fibrin clots, magenta-purple; and collagen, blue. All WT vessels were cleared by perfusion in every tissue examined. In contrast, every Pros1–/– tissue displayed perfusion-resistant blood-bearing vessels with yellow-stained blood cells and magenta fibrin-positive clots (arrowheads in B, D, F, and H). (D) Note the massive hemorrhages present in the spinal cord lumen (central canal) and the right dorsal quadrant (yellow-orange stain). Scale bar in H applies to all panels: 400 μm in C and D; 100 μm in A, B, and EH.
Figure 5
Figure 5. ProS activity in mouse plasmas.
(A) aPC cofactor activity in mouse plasmas. Mean prolongation of clot time by aPC in Pros1+/+ plasmas (49.1 seconds) was taken as 100% aPC cofactor activity of ProS, and prolongations of individual plasmas were converted to % aPC cofactor activity. Points represent individual mouse plasmas. (B) Base clot times in the same assay as in A, but without aPC added. (C) ProS-direct anticoagulant activity in mouse plasmas. Thrombin generation profiles for 2 representative Pros1+/+ and 2 representative Pros1+/– mouse plasmas, after a stimulus of prothrombinase complex. (D) Lag times from profiles similar to those displayed in C, for 4 Pros1+/+ mice and 5 Pros1+/– mice. Statistical difference between cohorts of Pros1+/+ mice and Pros1+/– is shown in A, B, and D.
Figure 6
Figure 6. Immunohistochemical analysis of embryonic vasculature.
(A and B) Sections of dorsal superficial artery stained with the smooth muscle cell marker α-SMA. Vessels are delineated by dashed lines. VSMCs show intense α-SMA staining throughout the circumference of the WT vessel (A) but only weak noncontinuous staining in the Pros1–/– vessel (B). (C and D) Double staining of ECs with CD144/VE-cadherin (green) and VSMCs with α-SMA (red). (C) WT arterioles have a luminal EC layer surrounded by a VSMC layer. (D) Pros1–/– arteriole shows only residual α-SMA signal and disorganized, nonuniform endothelial and VSMC marker staining. (E and F) Double staining of spinal cord microvasculature for CD31/PECAM-1 (green) and fibrin (red). (E) WT small-diameter vessels (apposed arrowheads) without fibrin clots. (F) Pros1–/– spinal cord vasculature with fibrin-positive immunoreactivity within vessels and aneurysms (apposed arrowheads). (G and H) Vessel ECs revealed by CD144/VE-cadherin in brain vasculature. (G) WT tissue shows elongated and uniform vessels, with tight interendothelial junctions. (H) Pros1–/– mice fail to form tight vessels, with ECs dispersed in clusters. (I and J) Yolk sac vasculature. Blood-filled vessels are seen in WT (I) but not in Pros1–/– yolk sac (J). (K and L) PECAM-1/CD31 immunoreactivity in yolk sac. CD31 staining reveals an intricate vascular network in WT yolk sac (K), but Pros1–/– yolk sac vasculature shows reduced vascular density, smaller-caliber vessels, and blind ends that fail to anastomose (asterisks). Scale bar in L applies to all panels: 150 μm in A, B, E, F, K, and L; 40 μm in C, D, G, and H; 500 μm in I and J.
Figure 7
Figure 7. Defective vasculature in Pros1+/– heterozygotes and other mutants.
(A and B) Two vessels, one aberrant and one normal, from the same Pros1+/– embryo. Normal vessels (A) show discrete layers of luminal ECs and outer VSMCs, visualized by staining for CD144/VE-cadherin (green) and α-SMA (red). Defective vessels (B) show nonuniform α-SMA staining and intermixing of endothelial and smooth muscle layers, with break points (arrowheads). (C and D) Muzzles (C) and intestines (D) of wild-type (left) and Pros1+/– adult mice, 1 hour after injection with 0.1% EB. (EJ) FITC-conjugated tomato lectin (green; E and F) was injected into the tail vein of adult mice together with EB (red; G and H). Tomato lectin labels vascular ECs. EB binds serum albumin and is confined to the interior of intact vessels; in leaky vessels, the dye leaches into tissue parenchyma. (E and G) Pros1+/+ brain vasculature maintains EB (red) within vessels (green). (F and H) Leakage of EB (H, arrowheads) in Pros1+/– brain. (I and J) Green and red channels of Pros1+/+ and Pros1+/– vessels, respectively, merged. Scale bar in J applies to all immunofluorescence panels: 30 μm in A and B; 100 μm in EJ. (KO) Modified Miles assays of EB penetration into liver parenchyma (see Methods) of WT mice versus Pros1+/– heterozygotes (K), Gas6–/– mice (L), Tyro3–/–, Axl–/–, and Mer–/– mice (M), Tie2-Cre/Pros1fl/fl mutants (N), and Sm22-Cre/Pros1fl/fl mutants (O). EB (0.1%) was injected for the measurements in K, and 1% was injected for LO. The cohort of WT mice is the same for M and O. The number of mice analyzed per genetic group (n) is indicated. Data are mean ± 1 SEM.
Figure 8
Figure 8. Coagulopathy without hemorrhage in Alb-Cre/Pros1fl/fl and Tie2-Cre/Pros1fl/fl embryos.
CD31 visualization of ECs (green; left panels), fibrin visualization of clotted blood (red; middle panels), and merged CD31/fibrin images (right panels) in sections of E15.5 embryonic brain from WT (AC), Alb-Cre/Pros1fl/fl (DF), and Tie2-Cre/Pros1fl/fl mice (GI). Scale bar in I applies to all panels: 50 μm.
Figure 9
Figure 9. Elimination of ProS from hepatocytes in adult Alb-Cre/Pros1fl/fl mice and EC contribution of ProS to the circulation.
(A) Immunoblots of ProS (top) and β-actin (bottom) of protein extracts of purified hepatocytes prepared from WT, Alb-Cre/Pros1fl/+, and Alb-Cre/Pros1fl/fl adult mice. Alb-Cre/Pros1fl/+ and Alb-Cre/Pros1fl/fl lanes were run on the same gel but were noncontiguous. (B) ELISA measurements of serum ProS levels in individual adult (8 weeks or older) mice of the indicated genotypes. (See Methods for ELISA/antibody protocols.) Levels in individual mice are expressed as a percentage of the mean value of measurements from 17 different wild-type mice (Combined WT). Mean values and P values for comparisons between wild-type, EIIA-Cre/Pros1fl/+ heterozygous, and Alb-Cre/Pros1fl/fl and tie2-Cre/Pros1fl/fl homozygous sera are indicated. HC, hepatocytes. (C) Measurement of aPC cofactor activity (prolongation of clotting time in seconds) in plasmas from mice of the indicated genotypes, in assays performed as those in Figure 5A (see Methods), expressed as percentage of WT. Mean values and P values for comparisons between heterozygous Alb-Cre/Pros1fl/+, Tie2-Cre/Pros1fl/+, and WT plasmas are indicated. Mean prolongation of clot time by aPC in WT mice (54.8 seconds) was taken as 100% aPC cofactor activity of ProS.

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