Generation of transgenic mice expressing very low levels of PC.
The strategy designed to generate PC-insufficient mice employed a mouse PC (mPC) transgene that would allow a low level of PC expression in a PC–/–
background. For this, we used a cosmid-based approach, with an FVII-FX
chromosomal fragment that was minimally altered to contain an inactivated FVII
gene, followed sequentially by the complete 5′ proximal FVII-FX intergenic region (19
), and an inactivated FX
gene. This chromosomal segment provided the entire upstream promoter region of the FX
). In turn, this should allow appropriate temporal and spatial expression patterns, as well as proper γ-carboxylation, of the downstream PC cDNA, which has characteristics similar to those of FX. Since much less is currently known about the characteristics of the promoter elements of the murine PC
gene, the use of the known proximal promoter of the FX
gene was the preferred approach. In addition, the utilization of this large chromosomal fragment was anticipated to minimize the potential influence of neighboring genes on PC
transgene expression. Finally, the PC cDNA sequence was inserted downstream of this FX
5′ promoter, since use of the PC cDNA, rather than the PC
gene, would be expected to result in significantly reduced levels of PC expression, and this was the desired goal of the design. The final construct is diagrammed in Figure A.
Figure 1 Generation of low-PC transgenic mice. (A) Schematic diagram showing relevant features of the low PC DNA construct. A 12.5-kb fragment of an inactivated (by partial promoter deletion FVII gene and an 18-kb fragment of an inactive (by exon 1 deletion) FX (more ...)
The cosmid containing the PC cDNA was microinjected into C57BL/6 zygotes to generate PC transgenic founders. We screened a total of 84 potential founders for the presence of the PC transgene using a PCR strategy that differentiated between the endogenous PC gene and the mPC cDNA derived from the inserted transgene. Positive founders were identified by the presence of a 284-bp PCR fragment representing the PC cDNA (Figure B). The 497-bp PCR amplicon corresponds to a fragment of the endogenous PC gene (Figure B). However, this PCR approach does not distinguish PC+/– from PC+/+ mice. Thus, a second PCR analysis was employed for differentiating between the WT and null alleles in 2 groups of mice. Once identified, the PC+/– mice were mated with PC transgenic founders [PC+/+(PCTg)] to generate PC+/–(PCTg) offspring. Further breeding of these offspring with PC+/– mice generated progeny with total inactivation of both WT PC alleles but containing the PC transgene [PC–/–(PCTg)].
The pattern of PC transgene integration was analyzed by Southern blot hybridization of genomic DNA digested with KpnI. Hybridization using a 5′ probe for exon 1 of the FVII
gene (Figure , A and C) resulted in a 2.7-kb band representing 2 copies of the endogenous FVII
gene, which thus served as an internal control. Each transgenic mouse line contained at least 2 integration sites, indicated by the presence of different-sized PC transgene fragments. In addition, each represents a unique line distinguishable by variations in integration patterns. The 3.7-kb fragment (Figure C) was consistent with the PC transgene integrated in tandem repeats, and this signal was found in all transgenic lines. The fragment intensity indicated multiple copies of the PC
transgene and demonstrated that several different founder lines with potentially different levels of PC expression were obtained. PC–/–
progenies followed the predicted Mendelian inheritance distribution. Consistent with a previous report (16
embryos lacking the PC
transgene died soon after birth. In contrast, PC–/–(PCTg)
mice survived the neonatal lethality. This approach led to the generation of 4 novel, low PC–expressing transgenic mouse lines.
Spontaneous thrombosis in low-PC transgenic mice.
Plasma PC was determined in all of the transgenic lines using plasmid (pIRESNeo-mPC) antibodies specific for mPC, developed in our laboratory, in an ELISA format. The PC antigen level was not measurable in PC–/–(PCTg535) mice, which suggests a very low (<1%) level of WT PC in this transgenic line. This finding was consistent with the early onset and severity of thrombotic phenotypes associated with these mice. The detectable levels of plasma PC in other transgenic lines represent 1%, 3%, and 18% of WT levels for PC–/–(PCTg4), PC–/–(PCTg785), and PC–/–(PCTg527) mice, respectively (Figure A). In these mice, plasma PC levels strongly correlated with survival profiles (Figure B), with the poorest neonatal survival shown by PC–/–(PCTg535) mice when compared with PC–/–(PCTg4), PC–/–(PCTg785), or PC–/–(PCTg527) littermates. The strain-specific plasma PC levels also reflect the considerable differences in spontaneous phenotypes. Mice from the PC–/–(PCTg527) transgenic line expressed 18% of WT PC levels, compared to less than 4% found in PC–/–(PCTg4) or PC–/–(PCTg785) mice. This correlates with the lack of gross prothrombotic phenotypes in the transgenic mouse line expressing the highest levels of PC.
Figure 2 PC levels correlate with poor survival in unchallenged low-PC mice. (A) Plasma PC levels were measured in mice derived from WT and the various low-PC transgenic lines using ELISA methodology. In PC–/–(PCTg535), PC–/–(PC (more ...)
The majority of PC–/–(PCTg535) survivors developed early-onset hemorrhage that typically affected their legs and tails, leading to spontaneous amputation of the individual extremities or the entire legs and tails (Figure , A and B). Only 2% of these neonates survived to 6 months of age. Similarly, survivors from the PC–/–(PCTg4) and PC–/–(PCTg785) transgenic lines were also susceptible to thrombosis and hemorrhage, most frequently in the tails and feet (Figure , C–E). However, the disease onset of these latter mice was markedly different from that of PC–/–(PCTg535) mice, as offspring of PC–/–(PCTg4) or PC–/–(PCTg785) mice developed thrombotic phenotypes only after 3 months of age. Necrosis of the paws/legs was often accompanied by severe edematous swelling. Other macroscopically observable phenotypes included necrosis of the ear and the face (Figure , F and G). Despite their overall longer survival tendencies, 10% of progenies of PC–/–(PCTg4) and PC–/–(PCTg785) transgenic lines died spontaneously or were euthanized due to advanced disease. Macroscopic evidence of soft tissue hemorrhage was typically noted at postmortem examination. Evidence of focal hemorrhage was present in lungs, leg muscles, tails, paws, and in severe cases, associated localized necrosis in the lobes of the liver (Figure , H–J).
Figure 3 Gross phenotypes of the low-PC transgenic mice. (A and B) Early onset of severe hemorrhage in the tails and legs of PC–/–(PCTg535) littermates. (C–G) Late onset of thromboembolic phenotypes in PC–/–(PCTg785) and (more ...)
We determined tail bleeding times for PC–/–(PCTg785) mice at 4, 8, and 12 weeks of age to further discern whether their thrombotic phenotypes precede a hemorrhagic condition. Significant differences in bleeding times in PC–/–(PCTg785) mice at various ages were observed. At 4 weeks, there were no statistical differences in bleeding times between PC–/–(PCTg785) and WT mice. Similarly, bleeding times for WT mice at 8 and 12 weeks of age were comparable to those of the 4-week-old mice. In contrast, low-PC mice at 8 weeks of age showed a prothrombotic phenotype, as evidenced by a significantly shorter bleeding time. At 12 weeks, these mice became more hemorrhagic, as bleeding times were significantly prolonged (Figure K). These findings suggest that hemorrhage was a secondary event to thrombosis, i.e, consumptive coagulopathy was the underlying cause of their hemorrhagic phenotype. This finding was further strengthened by evidence of disseminated intravascular coagulation in 12-week-old mice, as indicated by a significant change in plasma D-dimer levels (data not shown).
Histological examination of the H&E-stained sections revealed large thrombus deposits in various vessels and organs in PC–/–(PCTg4) and PC–/–(PCTg785) transgenic mice at 4 months of age compared with WT littermates. Fibrinogen/fibrin deposits were frequently observed in the lung, heart, and liver (Figure , A–F). Enhanced leukocytic infiltration into lung alveoli was observed in PC–/–(PCTg4) and PC–/–(PCTg785) transgenic mice compared with WT mice (Figure , G and H). To examine whether enhanced leukocytic infiltration in the lung was a reaction to thrombus formation or a consequence of PC insufficiency, we assessed infiltration of leukocytes in mice at various ages. Total and differential cell counts were determined in bronchoalveolar lavage fluid (BALF) collected from mice at 4, 8, 12, and 16 weeks of age. Significant differences in lymphocyte counts were observed in low-PC mice compared with WT mice at 8 weeks of age. Furthermore, both lymphocyte and neutrophil counts increased with age in low-PC mice, while no differences in lymphocytes or neutrophils were observed in WT mice at various ages (Figure I). Given that 8-week-old low-PC mice showed signs of prothrombotic phenotypes, it is not fully clear whether leukocyte infiltration in mice of this age was promoted by enhanced thrombosis or by a severe PC deficiency. However, the finding that enhanced leukocytic infiltration advanced with age implies that lung inflammation is secondary to thrombosis in the older mice.
Figure 4 Histological analyses of various tissues from PC–/–(PCTg785) mice. (A–D) Enhanced fibrin deposition in the PC–/–(PCTg785) mice compared with WT littermates was found in the lung and the heart (arrowheads). (E and (more ...)
Spontaneous enhanced thrombosis was evident in PC–/–(PCTg4) and PC–/–(PCTg785) mice, as indicated by increased plasma D-dimer levels, which suggests enhanced fibrinolysis (Figure A). Accordingly, the thrombin-antithrombin (TAT) levels in these PC-insufficient mice were also significantly elevated, which suggests increased thrombin generation (Figure B), presumably due to loss of the ability to regulate FVa and FVIIIa by aPC. In addition, these increased plasma thrombin levels may account for the platelet activation observed in low-PC mice, as shown by flow cytometric analysis of P-selectin (P-Sel) expression on the surface of activated platelets (Figure C).
Figure 5 Detection of various prothrombotic and proinflammatory markers in plasma of female low-PC mice. Levels of plasma D-dimer (A), TAT (B), and P-Sel (expressed as mean fluorescence intensity [MFI]) (C), aPTT (D), platelet counts (E), granulocyte (more ...)
Prolonged activated partial thromboplastin times (aPTTs) (Figure D), but not prothrombin times, were noted in low-PC mice. Consumptive coagulopathy may account for the discernible reduction in the aPTTs, given the prothrombotic phenotypes observed. In addition, platelet levels were higher in PC–/–(PCTg785) mice than in WT mice (Figure E). Consistent with increased platelet production, spleens from these low-PC mice were consistently larger than those from WT mice of the same age and gender (low PC, 410 ± 65.6 mg; WT, 110 ± 36.1 mg; P < 0.05). H&E stains confirmed the presence of increased numbers of megakaryocytes in the spleen. Megakaryocyte counts were also determined in bone marrows of these mice. Consistent with the increased spleen megakaryocyte population, the data showed that bone marrow megakaryocyte counts were higher in PC–/–(PCTg785) than in WT mice (low-PC, 6.68%; WT, 2.44%; P < 0.05).
Low-PC transgenic mice develop proinflammatory phenotypes.
Beyond its pivotal role in maintaining hemostasis, PC also exhibits a crucial function in directly or indirectly combating inflammatory challenges imposed on the host immune system (21
). PC exerts its antiinflammatory function both by inhibiting thrombin generation and thus overriding downstream effects of the thrombin-induced proinflammatory response (22
) and by signaling through EPCR in a PAR-1–dependent manner (23
). PC also prevents inflammation by downregulating inflammatory signaling mediators, such as IL-6 (18
). Accordingly, various markers were measured to evaluate the resting inflammatory potential in these low-PC mice. Higher total wbc counts were observed in low-PC mice compared with WT mice, and granulocytes accounted for more than 40% of the total wbcs (Figure F). Moreover, an elevated unchallenged plasma level of IL-6 (Figure G), but not TNF-α (data not shown), was seen in these PC–/–(PCTg785)
We addressed the question of whether this basal hyperinflammatory state was due to an inflammatory effect of the PC deficiency or to the corresponding high thrombin levels by assessing the inflammatory state in PC–/–(PCTg785) mice after continual administration of hirudin to 12-week-old mice for 3 days. Mice treated with hirudin have a significant reduction in plasma thrombin activity as indicated by a 4-fold increase in aPTT compared with that of controls, at day 3 after pump implantation. No side effects, e.g., bleeding, were associated with hirudin treatment over this time. Despite the substantial decrease in active thrombin levels, the inflammatory state of these mice remained unchanged; specifically, wbc counts and IL-6 (Figure , H–I) levels did not significantly decrease over the 3-day treatment. These unchanged inflammatory conditions implied that inflammation in low-PC mice was likely a thrombin-independent event. Moreover, direct determination of plasma leukotriene B4 in 4- to 8-week-old mice further suggested that low-PC mice have inflammation at ages 4 and 8 weeks (Figure J). These data suggest that PC deficiency promotes inflammation in the absence of obvious thrombosis.
Spontaneous abortion in very low–PC transgenic mothers.
This study further shows that although low levels of PC had no impact on male or female fertility, a severe PC insufficiency inhibited the ability of females to sustain pregnancy to full term. To examine whether the potential cause of pregnancy failure was of maternal influence, or whether the PC–/– offspring determine the fatal course of pregnancy, breedings were established by mating PC–/–(PCTg785) female mice with either PC–/–(PCTg785) or PC+/+(PCTg785) male mice to generate progenies of 100% PC–/–(PCTg785) or 100% PC+/–(PCTg785) mice, respectively. Neither of the matings led to full-term pregnancies. Spontaneous abortions affected all fetuses, regardless of the fetal PC genotypes. These findings strongly suggest that the underlying pathology of the pregnancy failure was strictly a maternal factor. Control breedings with PC+/+(PCTg785) females excluded the possibility that disruption of pregnancy-related gene(s) at the site of PC transgene integration played a role in the spontaneous abortions observed in these transgenic mice.
We established timed matings of PC–/–(PCTg785) females with PC–/–(PCTg785) or PC+/+(PCTg785) males to determine the stages of embryonic lethality in low-PC mothers. Embryos retrieved from PC–/–(PCTg785) mothers at 6.5 days postcoitum (dpc) appear to have normal developmental morphology. However, enhanced bleeding and fibrin deposition were noticeable at the ectoplacental cone region in low-PC mothers but were absent in WT mice (Figure , A and B). At this stage, embryonic development in low-PC and WT mothers was morphologically indistinguishable, and no evidence of trophoblast giant cell death was associated with embryos derived from PC–/–(PCTg785) mothers. Staining for proliferating cell nuclear antigen (PCNA) indicated ongoing active proliferation (data not shown). However, at 7.5 dpc, multiple hemorrhage and extensive fibrin deposition were enhanced in the low-PC mothers (Figure , C and D). Embryonic growth was restricted, and TUNEL assays revealed extensive trophoblast giant cell death in all embryos derived from PC–/–(PCTg785) mothers (Figure , E and F). By 8.5 dpc, most embryos were either growth retarded or in advanced stages of resorption (Figure , G and H).
Figure 6 Spontaneous abortion in PC–/–(PCTg785) mothers. (A and B) H&E stains of embryos in utero showing enhanced bleeding and fibrin deposition at the ectoplacental cone region and area surrounding a 6.5-dpc embryo. (C and D) Intense (more ...)
To gain additional insight into the cause of these failed pregnancies, we explored whether enhanced thrombosis was partly responsible for spontaneous abortion, employing females from the 4 low-PC transgenic lines generated in this study. It was found that only the PC–/–(PCTg527) transgenic females could sustain pregnancy and deliver healthy pups. The fact that fetal development was normal through 5.5 dpc in all lines implied that blastocyst development prior to implantation is not PC dependent. This observation supports the concept that maternal plasma PC is needed to sustain embryonic development, likely by regulating the extent of bleeding associated with endometrial remodeling during trophoblast implantation.
We examined whether not only enhanced thrombosis but also preexisting maternal inflammation contributed to pregnancy loss in PC–/–(PCTg785) mothers. Consistent with their proinflammatory phenotypes, recruitment of leukocytes to the ectoplacental cone region at 7.5 dpc was markedly enhanced in low-PC mice, suggestive of an inflammatory response at the implantation site (Figure , I and J).
The finding that resting low-PC mice displayed a baseline proinflammatory condition raised the question of whether enhanced inflammation contributes to the spontaneous abortion in the low-PC mothers. To examine whether normal pregnancies in PC–/–(PCTg527) mice were the result of reduced maternal inflammation, we compared the thrombotic and inflammatory responses of PC–/–(PCTg785) and PC–/–(PCTg527) mice. Consistent with their lack of spontaneous thrombosis, PC–/–(PCTg527) mice did not show evidence of an inflammatory response. Granulocyte counts were comparable to those of WT mice (Figure F). Additionally, plasma D-dimer and TAT levels in PC–/–(PCTg527) mice were indistinguishable from those same values in WT mice (Figure , A and B) but significantly lower than the levels found in mice derived from the PC–/–(PCTg785) transgenic line. Similarly, unlike in PC–/–(PCTg785) transgenic mice, IL-6 was undetectable in PC–/–(PCTg527) mice (data not shown). These findings together indicate a lack of spontaneous inflammation in PC–/–(PCTg527) mice, the mouse strain expressing the highest levels of PC.
Rescue of embryonic lethality in PC-insufficient mothers.
While it seems very clear that maternal PC is necessary to maintain pregnancies, we rigorously examined this concept using an ovarian transplant strategy. Ovaries from PC–/–(PCTg785) females were transplanted into WT mothers, and the recipient females were mated with PC–/–(PCTg785) males. From 7 recipients, 35 pups were born, all of which were genetically PC–/–(PCTg785). As described above, no births occurred when PC–/–(PCTg785) biological mothers were mated with males of any PC genotype or when WT mouse ovaries were transplanted into genetic PC–/–(PCTg785) females. Since a very small portion of WT ovary tissue was allowed to remain in the transplanted female to assist revascularization with transplanted tissue, it is possible that pups could occasionally have been born with a WT allele, although this would be statistically rare. This did not occur in the breedings that are reported here, and all pups were born from PC–/–(PCTg785) ova, which would have been expected to heavily predominate. These experiments show that a restoration of maternal PC rescues embryonic development that is otherwise lethal beyond 7.5 dpc in mothers with severe deficiency in PC. Thus, it is further confirmed that maternal PC is required to sustained pregnancy beyond 7.5 dpc.