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Differential roles of factors IX and XI in murine placenta and hemostasis under conditions of low tissue factor

Differential roles of factors IX and XI in murine placenta and hemostasis under conditions of low... Downloaded from http://ashpublications.org/bloodadvances/article-pdf/4/1/207/1552610/advancesadv2019000921.pdf by guest on 12 October 2021 REGULAR ARTICLE Differential roles of factors IX and XI in murine placenta and hemostasis under conditions of low tissue factor 1 1 1 1 1,2 1,2 3 Steven P. Grover, Clare M. Schmedes, Alyson C. Auriemma, Emily Butler, Molly L. Parrish, Adam Miszta, Audrey C. Cleuren, 4 4 5,6 5,6 1,2 1,2 1 Mayken Visser, Stefan Heitmeier, Jens J. Posma, Henri M. Spronk, Silvio Antoniak, Alisa S. Wolberg, Rafal Pawlinski, 7 1 David Gailani, and Nigel Mackman 1 2 UNC Blood Research Center, Division of Hematology and Oncology, Department of Medicine, and UNC Blood Research Center, Department of Pathology and Laboratory 3 4 Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC; Life Sciences Institute, University of Michigan, Ann Arbor, MI; Bayer AG, Wuppertal, Germany; 5 6 Laboratory for Clinical Thrombosis and Hemostasis, Department of Internal Medicine, and Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University Medical Center, Maastricht, The Netherlands; and Department of Pathology, Microbiology and Immunology, Vanderbilt University Medical Center, Nashville, TN The intrinsic tenase complex (FIXa-FVIIIa) of the intrinsic coagulation pathway and, to Key Points a lesser extent, thrombin-mediated activation of FXI, are necessary to amplify tissue factor � Factor IX, but not factor (TF)-FVIIa–initiated thrombin generation. In this study, we determined the contribution of XI, is essential for the murine FIX and FXI to TF-dependent thrombin generation in vitro. We further investigated postnatal survival of TF-dependent FIX activation in mice and the contribution of this pathway to hemostasis. mice under conditions Thrombin generation was decreased in FIX- but not in FXI-deficient mouse plasma. of low TF. Furthermore, injection of TF increased levels of FIXa-antithrombin complexes in both wild- 2/2 � Embryonic factor XI, type and FXI mice. Genetic studies were used to determine the effect of complete but not factor IX, pre- deficiencies of either FIX or FXI on the survival of mice expressing low levels of TF. Low-TF; vents enlargement of 2/y FIX male mice were born at the expected frequency, but none survived to wean. In blood pools in low-TF 2/2 contrast, low-TF;FXI mice were generated at the expected frequency at wean and had placentas. a 6-month survival equivalent to that of low-TF mice. Surprisingly, a deficiency of FXI, but not FIX, exacerbated the size of blood pools in low-TF placentas and led to acute hemorrhage and death of some pregnant dams. Our data indicate that FIX, but not FXI, is essential for survival of low-TF mice after birth. This finding suggests that TF-FVIIa–mediated activation of FIX plays a critical role in murine hemostasis. In contrast, FXI deficiency, but not FIX deficiency, exacerbated blood pooling in low-TF placentas, indicating a tissue-specific requirement for FXI in the murine placenta under conditions of low TF. Introduction The coagulation cascade can be divided into the extrinsic (tissue factor [TF]–factor [F] VIIa complex), intrinsic(FXIIa, FXIa,FIXa, andFVIIIa),and common (FXa,FVa,and thrombin)pathways. The extrinsic 2-4 pathway is the physiologic trigger of blood coagulation and activates both FIX and FX. TF-FVIIa–mediated activation of FIX was initially described by Francois Josso and colleagues and is commonly referred to as the “Josso loop.” The intrinsic pathway also contains a feedback loop in which thrombin activates FXI 5,6 leading to further generation of FIXa. These pathways are thought to play an importnant role in thrombin generation, because the TF-FVIIa complex is rapidly inhibited by TF pathway inhibitor (TFPI). Humans deficient in FVIII (hemophilia A) or FIX (hemophilia B) have hemostatic defects. Sim- 2/2 2/2 ilarly, FVIII and FIX mice exhibit increased bleeding after a hemostatic challenge, such as tail Submitted 3 September 2019; accepted 28 October 2019; published online 14 © 2020 by The American Society of Hematology January 2020. DOI 10.1182/bloodadvances.2019000921. The full-text version of this article contains a data supplement. 14 JANUARY 2020 x VOLUME 4, NUMBER 1 207 Downloaded from http://ashpublications.org/bloodadvances/article-pdf/4/1/207/1552610/advancesadv2019000921.pdf by guest on 12 October 2021 9,10 transection. FXI-deficient patients rarely have spontaneous with control plasma when thrombin generation was initiated with bleeding events, but increased bleeding occurs in some patients a low concentration of TF (Figure 1A; data not shown). on provocation, particularly in tissues with high fibrinolytic activity, Next, the contribution of FXI to thrombin generation was determined 11,12 such as the mouth, nose, and genitourinary tract. FXI is thought in mouse plasma in which activation of coagulation was initiated by 13,14 2/2 to stabilize the fibrin clot and protect it from fibrinolysis. FXI either silica or TF. As expected, silica-initiated thrombin generation mice have normal bleeding times in the tail vein transection model. 2/2 1/2 was significantly impaired in FXI PPP compared with FXI or 2/2 One study reported a significant increase in bleeding in FXI mice 1/1 FXI PPP (Figure 1B). In contrast, there was no significant in the saphenous vein injury model, but this was not confirmed in difference in thrombin generation (lag time, time to peak, peak, or an independent study. In vitro studies have shown that FXI 2/2 1/1 1/2 ETP) in FXI PPP compared with FXI or FXI PPP, at contributes to thrombin generation in human plasma initiated by a low concentration of TF (Figure 1C; data not shown). To exclude 18-20 low, but not high, concentrations of TF. the potential effects of contact pathway activation during blood collection, additional samples were collected in the presence of In contrast to studies with mice lacking components of the intrinsic corn trypsin inhibitor (CTI). However, even in the presence of CTI, pathway, a complete deficiency of components of the extrinsic or despite observing a robust reduction in silica-induced thrombin common pathways in mice leads to death during embryonic 21-28 generation (Figure 1D; data not shown), there was no difference in development or shortly after birth. For instance, 80% to 90% 1/1 1/2 2/2 2/2 21-23 2/2 TF-initiated thrombin generation between FXI , FXI , or FXI of TF embryos die at embryonic day (E) 9.5-E10.5. TF mice can be rescued with a transgene that expresses human TF PPP (Figure 1E; data not shown). (hTF) regulated by the human TF promoter. These “low-TF” mice Platelets provide an important surface for the activation of FXI 2/2 2/2 1/1 (murine TF [mTF ];hTF ) express ;1% levels of TF. Low- and may be important in FXI-mediated amplification of thrombin TF mice exhibit spontaneous hemostatic defects in various organs 35-37 generation. To account for the potential effects of platelets and have significantlyincreased bleeding in the tail vein transection we assessed thrombin generation in platelet-rich plasma (PRP). 1,30-32 model. Mice expressing ;1% levels of FVII have phenotypes When coagulation was initiated with silica, thrombin generation 33,34 similar to those of low-TF mice. It is notable that maternal blood 2/2 was significantly reduced in PRP from FXI mice compared pooling occurs in placentas of low-TF embryos but does not cause 1/2 1/1 to FXI or FXI controls (Figure 1F). In contrast, when any loss of embryos or fatal hemorrhages in the dams. The coagulation was initiated with TF, there was no difference in placenta is derived from embryonic tissue, indicating that blood 2/2 1/2 thrombin generation in PRP from FXI mice compared to FXI pooling is related to low levels of TF in the embryo. 1/1 or FXI controls (Figure 1G; data not shown). Collectively, these data in PPP and PRP show that, although FXI deficiency was In the current study, we investigated the contribution of FIX and FXI detectable in mouse plasma in contact silica-initiated assays, FXI to thrombin generation in vitro and in vivo under conditions of low did not contribute to thrombin generation in mouse plasma when TF. Plasma-based thrombin generation assays were used to assess initiated by TF. the relative contribution of FIX and FXI to TF-initiated thrombin generation. The ability of the TF-FVIIa complex to activate FIX in vivo TF activates FIX in vivo in the absence of FXI 2/2 was also determined in wild-type and FXI mice. Finally, a genetic To determine whether TF can activate FIX in vivo, mice were injected approach was employed to determine the effect of a complete with TF and plasma levels of FIXa-antithrombin (AT) complexes deficiency of either FIX or FXI on the survival of mice expressing low measured. The thrombin inhibitor dabigatran was used to prevent levels of TF. death associated with the formation of intravascular thrombi after Methods injection of TF and to block thrombin-mediated activation of FXI. Injection of TF into wild-type mice significantly increased the plasma Mice levels of FIXa-AT complexes compared with controls (Figure 2A). 2/2 2/2 2/2 FIX and FXI mice were backcrossed 10 generations to the FXI mice were used to eliminate the thrombin-FXIa feedback 10,15 2/2 1/1 C57BL/6J background. Low-TF mice (mTF ;hTF ) were loop. Similar to the results in wild-type mice, TF injection significantly backcrossed 6 generations to the C57BL/6J background. increased the plasma levels of FIXa-AT complexes in thrombin- 2/2 Studies complied with National Institutes of Health Guide for the inhibited FXI mice (Figure 2B). These results indicate that the Care and Use of Laboratory Animals and were performed with the TF-FVIIa complex activates FIX in mice. approval of the Animal Care and Use Committee of the University of Effect of FIX deficiency on the survival of North Carolina at Chapel Hill. low-TF offspring All additional details of experimental procedures are given in the The FIX gene resides on the X chromosome. Therefore, to study the supplemental Methods. 2/2 1/1 1/y effect of FIX deficiency on low-TF mice, male mTF ;hTF ;FIX 1/2 1/2 1/2 Results and female mTF ;hTF ;FIX were crossed and the allele frequencies of offspring determined at various timepoints. No male Effect of either FIX or FXI deficiency on TF-initiated 2/y low-TF;FIX offspring were observed at postnatal day (P) 21, thrombin generation in murine plasma a significant reduction compared with the expected frequency The effect of FIX deficiency on TF-initiated thrombin generation in (Table 1). Next, offspring were analyzed at E14.5 to determine if 2/y mouse platelet-poor plasma (PPP) was determined. Endogenous low-TF;FIX offspring had survived development beyond E9.5 thrombin potential (ETP), time to peak, peak, and lag time were and E10.5 given that embryos lacking various coagulation factors 2/y measured. As expected, a significant reduction in peak thrombin dieatthistimepoint.Viablelow-TF;FIX offspring were observed generation and ETP was observed in FIX-deficient plasma compared at E14.5 (Table 1) and appeared grossly normal by histological 208 GROVER et al 14 JANUARY 2020 x VOLUME 4, NUMBER 1 Downloaded from http://ashpublications.org/bloodadvances/article-pdf/4/1/207/1552610/advancesadv2019000921.pdf by guest on 12 October 2021 Figure 1. Effect of deficiency of FIX or FXI on thrombin generation in murine plasma. (A) Thrombin generation in murine FIX-deficient PPP was initiated with low and high concentrations of +/+ hTF. Thrombin generation in mFXI-deficient PPP collected in the ab- FIX -/- sence of CTI was initiated with silica (B) or low and high doses of 600 FIX hTF (C). Thrombin generation in mFXI-deficient PPP collected in the presence of CTI was initiated with silica (D) or low and high doses +/+ FXI of hTF (E). Thrombin generation in mFXI-deficient PRP collected in +/- FXI the presence of CTI was initiated with silica (F) or low and high -/- FXI doses of hTF (G). Data are presented as individual values with 200 means 6 95% confidence intervals. *P , .05, 2-way analysis of var- 1/1 2/2 iance with post hoc Bonferroni test (FIX vs FIX ); **P , .001, 1/1 1-way analysis of variance with post hoc Bonferroni test (FXI 1/2 2/2 0.05 0.5 TF (pM) and FXI vs FXI ). BC ** 0 0 Silica 0.05 0.5 TF (pM) DE ** 0 0 Silica 0.05 0.5 TF (pM) FG ** 0 0 Silica 0.05 0.5 TF (pM) 14 JANUARY 2020 x VOLUME 4, NUMBER 1 FIX AND FXI IN MURINE HEMOSTASIS 209 ETP (nM-min) ETP (nM-min) ETP (nM-min) ETP (nM-min) ETP (nM-min) ETP (nM-min) ETP (nM-min) Downloaded from http://ashpublications.org/bloodadvances/article-pdf/4/1/207/1552610/advancesadv2019000921.pdf by guest on 12 October 2021 was not significantly different from the expected frequency of A B 4.69% (Table 2). Four additional crosses with sires and dams of 700 700 differing genotypes were conducted to yield larger numbers of low- ** 2/2 TF;FXI offspring. Similar to the results observed when crossing 2/2 F1 intermediates low-TF;FXI mice were generated at a frequency 500 500 that was not significantly different from the expected frequency 400 400 in these 4 additional breedings (supplemental Table 1). These observations indicate that, even when overall procoagulant potential is reduced under conditions of low TF, FXI is not necessary for 200 200 survival in mice. 100 100 Next, the effect of FXI deficiency on the survival of low-TF mice 0 0 between weaning and 6 months of age was determined. As 38,39 Ctrl TF Ctrl TF expected from previous studies, 27% (3 of 11) of low-TF; 1/1 -/- FXI mice died during the 6-month survival study, with evidence WT FXI of pulmonary hemorrhage in all mice. Importantly, survival did not 2/2 1/2 2/2 differ significantly between low-TF;FXI , low TF;FXI offspring, Figure 2. TF activates FIX in thrombin-inhibited wild-type and FXI mice. 1/1 and low-TF;FXI controls (Figure 3). Plasma levels of FIXa-AT complexes were quantified in dabigatran-pretreated 2/2 wild-type (WT) (A) or FXI (B) mice, administered either vehicle control or TF 2/2 Death of pregnant dams bearing low-TF;FXI (n 5 6-10 per group). A significant increase in plasma FIXa-AT complexes was ob- 2/2 served on administration of TF in both WT and FXI mice. Data are presented as embryos individual values with means 6 95% confidence intervals. *P , .0001; **P , .05; 2/2 While generating low-TF;FXI offspring, the death of pregnant unpaired Student t test. 1/2 1/(1/2) 1/2 1/2 1/(1/2) 2/2 mTF ;hTF ;FXI (n 5 6) and mTF ;hTF ;FXI 2/2 (n 5 5) dams bearing low-TF;FXI embryos occurred during late 2/y analysis (data not shown). Finally, the number of low-TF;FIX at gestation (E13.5-15.5) in all 5 breedings (Table 3). In breeding 1, death 2/y 1/2 1/(1/2) 1/2 P1 was analyzed. Male low-TF;FIX offspring were observed at occurred in 9% (2 of 33) of pregnancies in mTF ;hTF ;FXI 2/y 1/2 1/(1/2) 1/2 P1 (Table 1).However,2ofthe 5(40%)maleP1low-TF;FIX dams. Death of 20% to 40% of pregnant mTF ;hTF ;FXI 2/y offspring were dead. One low-TF;FIX P1 pup was pale and had dams in breedings 2 and 3 and of 30% to 33% of the pregnant 1/2 1/(1/2) 2/2 a dark abdomen, which was consistent with abdominal hemor- mTF ;hTF ;FXI dams in breedings 4 and 5 was rhage (supplemental Figure 1). One of the 6 (17%) male low-TF; observed. These results suggest that the death of the pregnant 1/y 2/2 FIX P1 pups was dead with no apparent phenotype. These dams was associated with the relative burden of low-TF;FXI results indicate that FIX is essential for postnatal survival of low- embryos and was largely independent of the maternal FXI TF mice. genotype. There was a trend toward increased death of the pregnant dams FXI deficiency does not affect the generation or 2/2 as the burden of low-TF;FXI embryos increased (Table 3). To survival of low-TF offspring further investigate whether there was an association between the 2/2 The effect of FXI deficiency on the generation and survival of frequency of low-TF;FXI offspring and maternal death, the 2/2 mice expressing low levels of TF was determined. Male and female burden of low-TF;FXI offspring was determined for pregnancies 1/2 1/2 1/2 F1 intermediates (mTF ;hTF ;FXI ) were crossed, and in which the dam survived vs those in which the dam died. Interestingly, 2/2 the allele frequencies of 117 offspring were determined. Six of dams that died carried a higher proportion of low-TF;FXI embryos 2/2 117 (5.13%) of the offspring at wean were low TF;FXI , which compared to the dams that survived (Table 3). 2/2 Table 1. Distribution of genotypes at weaning from a breeding to generate low-TF;FIX offspring Sex mTF hTF FIX Expected, % E14.5, n E14.5 observed, % E14.5 O/E, % P1, n P1 observed, % P1 O/E, % P21, n P21 observed, % P21 O/E, % F 1/21/(1/2) 1/1 12.5 7 18.4 147 3 6.6 52 15 18.5 148 F 1/21/(1/2) 1/2 12.5 7 18.4 147 7 15.5 147 13 16.0 128 M 1/21/(1/2) 1/y 12.5 1 2.6 21 10 22.2 178 15 18.5 148 M 1/21/(1/2) 2/y 12.5 4 10.5 84 8 17.7 142 9 11.1 89 F 2/21/(1/2) 1/1 12.5 5 13.2 106 4 8.8 70 8 9.9 79 F 2/21/(1/2) 1/2 12.5 5 13.2 106 2 4.4 35 12 14.8 118 M 2/21/(1/2) 1/y 12.5 5 13.2 106 6* 13.3 106 9 11.1 89 M 2/21/(1/2) 2/y 12.5 4 10.5 84 5† 11.1 89 0 0 0‡ Total 100.0 38 100.0 — 45 100.0 — 81 100.0 — 1/(1/2) 1/2 1/1 F, female; hTF , hTF or hTF ; M, male; O/E, observed/expected. *One of 6 dead. †Two of 5 dead. ‡P , .05 observed vs expected, 2-tailed Fisher’s exact test. 210 GROVER et al 14 JANUARY 2020 x VOLUME 4, NUMBER 1 FIXa-AT (pM) FIXa-AT (pM) Downloaded from http://ashpublications.org/bloodadvances/article-pdf/4/1/207/1552610/advancesadv2019000921.pdf by guest on 12 October 2021 Table 2. Distribution of genotypes at weaning from a breeding to 2/2 generate low-TF;FXI offspring mTF hTF FXI Expected, % Observed, n Observed, % O/E, % 1/1 — 1/1 1.56 4 3.42 219 1/1 — 1/2 3.13 4 3.42 109 1/1 — 2/2 1.56 4 3.42 219 1/11/(1/2) 1/1 4.69 6 5.13 109 1/11/(1/2) 1/2 9.38 10 8.55 91 +/+ LowTF; FXI (n=11) 1/11/(1/2) 2/2 4.69 2 1.71 36 +/- LowTF; FXI (n=21) 1/2 — 1/1 3.13 2 1.71 55 20 -/- LowTF; FXI (n=12) 1/2 — 1/2 6.25 8 6.84 109 1/2 — 2/2 3.13 3 2.56 82 0 30 60 90 120 150 180 1/21/(1/2) 1/1 9.38 16 13.68 146 Days 1/21/(1/2) 1/2 18.75 25 21.37 114 1/21/(1/2) 2/2 9.38 11 9.40 100 1/1 Figure 3. Survival of low-TF mice with different levels of FXI. Low-TF;FXI , 2/2 — 1/1 1.56 0 0.00 0* 1/2 2/2 low-TF;FXI , and low-TF;FXI mice were assessed in a 6-month survival study. 2/2 — 1/2 3.13 0 0.00 0* 1/1 As expected, 27% (3 of 11) of low-TF;FXI mice died by 6 months with evidence 1/2 2/2 — 2/2 1.56 0 0.00 0* of pulmonary hemorrhage. By comparison 10% (2 of 21) of low-TF;FXI and 25% 2/2 (3 of 12) of low-TF;FXI mice died by 6 months with evidence of pulmonary 2/21/(1/2) 1/1 4.69 8 6.84 146 hemorrhage. No significant difference in survival was observed between groups. 2/21/(1/2) 1/2 9.38 8 6.84 73 P . .05, log-rank test. 2/21/(1/2) 2/2 4.69 6 5.13 109 Total 100.00 117 100.00 — proteolytically cleave pro-hepatocyte growth factor (pro-HGF) *Expected embryonic lethality. to active HGF. Therefore, levels of TFPI and pro-HGF were 2/2 1/1 analyzed in placentas from FXI and FXI embryos. If FXIa were a major proteolytic inactivator of TFPI, increased expression 2/2 of full-length TFPI protein would be expected in FXI placentas. FXI deficiency is associated with increased size of 2/2 However, TFPI protein expression in FXI placentas was signifi- blood pools in low-TF placentas 1/1 cantly lower than that in FXI controls (Figure 4D-E). In contrast, 2/2 Necropsies were conducted on 9 of the 11 dams carrying low-TF; levels of pro-HGF protein were significantly higher in FXI 2/2 1/1 FXI embryos that had either died (n 5 8) or had been euthanized placentas than in FXI controls (Figure 4D, F), suggesting that due to uterine hemorrhage (n 5 1) during late gestation. Free blood FXIa regulates activation of pro-HGF in the placenta. was observed in the uterus of 3 of 9 (33%) of the females that died 2/2 with evidence of ruptured low-TF;FXI placentas, suggesting that Discussion 2/2 some of the pregnant females carrying low-TF;FXI embryos died The FIXa;FVIIIa complex has been proposed to sustain activation of of acute uterine or placental hemorrhage. coagulation and thrombin generation after inhibition of the TF-FVIIa 2/2 The size of the maternal blood pools in low-TF;FXI placentas complex by TFPI. Importantly, the TF-FVIIa complex is known to 1/2 1/1 1-3,42 was compared to those in low-TF;FXI placentas and low-TF;FXI activate both FIX and FX in vitro. Indeed, an in vitro study placentas removed from dead or euthanized dams (Figure 4A-B). comparing activation of FIX and FX by the TF-FVIIa complex The average size of the maternal blood pools was significantly concluded that FIX is the major substrate. In addition, individuals 2/2 increased in low-TF;FXI placentas compared with low-TF; with a point mutation in FIX that reduces interaction with TF-FVIIa 1/1 2/2 FXI placentas (Figure 4B). In addition, 5 of 17 low-TF;FXI but not FXI have a mild form of hemophilia B. Furthermore, basal placentas appeared to have ruptured (Figure 4B). Of note, FIX levels of FIXa activation are reduced in FVII-deficient (,7% levels) deficiency did not affect the size of the blood pools (Figure 4B). but not in FXI-deficient (,8% levels) individuals. Taken together, 2/2 Importantly, no abnormalities were observed in FXI placentas these in vitro and in vivo studies indicate that the TF-FVIIa complex is with 50% or 100% levels of mouse TF (Figure 4A). a major activator of FIX. Given the observation that FXI deficiency exacerbated placental Consistent with the important contribution of TF-FVIIa–mediated blood pooling under conditions of low TF, F9, F11, and F12 gene activation of FIX to thrombin generation, a deficiency of FIX markedly expression was assessed in wild-type mouse placentas. A high level 45-47 blunted TF-initiated thrombin generation in human plasma. A of F11 mRNA, but not of F9 or F12 mRNA, was observed in the similar approach has been taken to investigating the contribution placenta (Figure 4C), suggesting that local FXI expression plays of thrombin-mediated FXI activation to thrombin generation. In a role in the placenta. FXI-deficient human plasma, thrombin generation is impaired at 20,48,49 The contribution of FIX-independent functions of FXI in the exacerba- a low, but not a high, initiating concentration of TF. Taken tion of blood pooling in low-TF placentas was investigated. FXIa has together, these data suggest that, in humans, TF-FVIIa–mediated been shown to proteolytically inactivate TFPI. Conversely, FXIa can activation of FIX and, to a lesser extent, thrombin-mediated 14 JANUARY 2020 x VOLUME 4, NUMBER 1 FIX AND FXI IN MURINE HEMOSTASIS 211 Survival (%) Downloaded from http://ashpublications.org/bloodadvances/article-pdf/4/1/207/1552610/advancesadv2019000921.pdf by guest on 12 October 2021 2/2 Table 3. Observed low-TF;FXI offspring at weaning from additional breedings and death of females Survived Died Breeding type Expected, % Dam FXI genotype Death/pregnancy, n/n (%) n/N at P21 (litters) Observed, % n/N at E13-15.5 (litters) Observed, % 1 4.69 1/2 2/23 (9) — ——— 2 10.26 1/2 2/5 (40) 1/25 (3) 4.0 4/14 (2)* 29.0 3 10.26 1/2 2/10 (20) 3/40 (6) 7.5 7/19 (2)* 37.0 4 10.26 2/2 2/6 (33) 2/19 (3) 10.5 2/7 (1) 29.0 5 20.52 2/2 3/10 (30) 3/22 (7) 13.6 6/17 (3) 35.3 2/2 1/(1/2) 2/2 *P , .05, difference in observed frequency of mTF ;hTF ;FXI in surviving mothers vs dead mothers assessed by 2-tailed Fisher’s exact test. activation of FXI both contribute to thrombin generation. However, TF was combined with a complete deficiency of FIX. Surprisingly, 2/y it is unclear whether these amplificatory pathways also contribute the expected number of male low-TF;FIX embryos was observed to thrombin generation and hemostasis in mice. at E14.5, indicating that these embryos survived the E9.5 to 10.5 2/2 21-23 period associated with the death of mTF embryos. Birth In the present study, a deficiency in FIX resulted in a modest, but presents a strong hemostatic challenge as pups are squeezed significant, impairment in thrombin generation in mouse PPP at through the birth canal. Mice with a complete absence of different a low, but not a high, initiating dose of TF. In contrast, no defect in components of the extrinsic (FVII) and common (FV, FX, and TF-initiated thrombin generation was observed in FXI-deficient prothrombin) pathways exhibit a high rate of death at birth and in the mouse PPP at this same low initiating dose of TF. Inhibition of 24-27 immediate postnatal period. Some of these pups lacking key exogenous contact activation by the FXIIa inhibitor CTI at the time of coagulation factors appear to die of acute abdominal hemorrhage blood collection failed to reveal a contribution of FXI to TF-initiated after birth and appear pale rather than pink, with dark abdomens thrombin generation in PPP. Platelets have been identified as an 24-27 due to blood pooling. Consistent with this phenotype, 2 of 5 35-37 important physiologic surface for the activation of FXI by thrombin. 2/y (40%) of the low-TF;FIX male mice died after birth, and 1 pup TF-initiated thrombin generation, however, was unchanged in FXI- was pale with a dark abdomen, consistent with death due to an deficient PRP. These data support the involvement of FIX, but not acute abdominal hemorrhage. In addition, 1 of 6 (17%) of the low-TF FXI, in TF-initiated thrombin generation in mouse plasma. It is male mice also died at birth, which was likely caused by a hemostatic interesting tonotethatinmurinePPP thecontributionofFIX to TF- defect. initiated thrombin generation was observed only at 0.05 pM of TF, whereas in human PPP, impaired thrombin generation was Low-TF mice backcrossed 7 generations to a C57BL/BJ back- observed in FIX-deficient plasma at doses as high as 1 pM. This ground demonstrate a spontaneous hemostatic defect that results result suggests that thrombin generation in humans may have in the death of 40% to 60% of mice by 6 months of age, primarily as 38,39 a greater dependence on FIX than in mice. a result of spontaneous hemorrhage in the lung. In contrast, 2/2 FIX mice do not exhibit a spontaneous hemostatic defect, but Given the similarity in the components and general mechanism of have increased bleeding upon challenge, such as a tail transection, the hemostatic systems in humans and mice, it is somewhat despite the presence of normal levels of TF. The postnatal lethality surprising that FXI does not seem to serve a similar role in the 2/y associated with low TF;FIX mice, therefore, represents a marked amplification of coagulation in these 2 mammals. Interestingly, it exacerbation of the phenotype observed in either low-TF mice or was recently demonstrated that, unlike humans, most of the FXI FIX-deficient mice. Importantly, the data suggest that FIX is in mice is localized to the vascular surface through binding to essential for the survival of low-TF mice during the trauma of birth glycosaminoglycans, owing to an additional cluster of basic residues 50 and in the immediate postnatal period. on the Apple 4 domain of mouse FXI. This process leads to three- to four-fold lower levels of circulating FXI in mice compared to To evaluate the contribution of thrombin-mediated activation of FXI, humans. attempts were made to generate low-TF mice lacking FXI. Low-TF; 2/2 FXI mice were generated at the expected frequency at wean To confirm the ability of the TF-FVIIa complex to activate murine FIX 1/2 2/2 from multiple breedings involving both FXI and FXI dams, in in vivo, dabigatran-anticoagulated mice were injected with exoge- 2/y contrast to the attempts to generate low-TF;FIX mice. Moreover, nous TF, and FIXa generation was determined by measurement of 2/2 the survival of low-TF;FXI mice was not different from that of low- plasma levels of FIXa-AT complexes. Injection of TF into wild-type 1/2 1/1 TF;FXI or low-TF;FXI mice between wean and 6 months of mice resulted in generation of significant levels of FIXa. This finding 2/2 2/2 age. This result differs from the reduced survival of low-TF;PAR4 was also replicated in FXI mice, which eliminated the notion of mice during the same period. The results from this genetic any contribution of the thrombin-FXI pathway to the observed approach suggest that FIX, but not FXI, contributes to thrombin generation of FIXa. This approach provides the first direct evidence generation and hemostasis under conditions of reduced TF in mice. of a functional Josso loop in mice. 2/2 Having demonstrated the ability of the TF-FVIIa complex to activate During the generation of low-TF;FXI mice, several pregnant FIX in vivo in mice, we used a complementary genetic approach to dams died during late gestation. Unexpectedly, in pregnant dams 2/2 investigate the contribution of TF-FVIIa–mediated activation of FIX that died, several of the placentas from low-TF;FXI embryos to murine embryonic development and hemostasis. Mice were were found to have large maternal blood pools. Previous studies crossed to generate offspring in which expression of a low level of have demonstrated that placentas from low-TF offspring contain 212 GROVER et al 14 JANUARY 2020 x VOLUME 4, NUMBER 1 Downloaded from http://ashpublications.org/bloodadvances/article-pdf/4/1/207/1552610/advancesadv2019000921.pdf by guest on 12 October 2021 +/- -/- +/- -/- FXI FXI LTF;FXI LTF;FXI 2mm BD 100 +/+ -/- FXI FXI TFPI 44kDa 100kDa proHGF 38kDa E Actin +/- -/y +/- -/- LTF LTF;FIX LTF;FIX LTF;FXI LTF;FXI (n=11) (n=5) (n=4) (n=10) (n=17) CE F Liver Placenta **** *** ** ** 0.1 100 0.01 50 200 0.001 0 0 +/+ -/- +/+ -/- F9 F11 F12 FXI FXI FXI FXI Figure 4. Analysis of the contribution of FIX and FXI to placental blood pooling under conditions of low TF. (A) Representative images of placentas from 2 dams 2/2 1/2 1/2 1/(1/2); from breeding 3 (top row) and 4 (bottom row), each showing a large blood pool in a low-TF;FXI placenta. Genotypes have been abbreviated to FXI (mTF ;hTF 1/2 2/2 1/1 1/(1/2); 2/2 1/2 2/2 1/(1/2); 1/2 2/2 2/2 1/(1/2); 2/2 FXI ), FXI (mTF ;hTF FXI ), low TF;FXI (mTF ;hTF FXI ), and low TF;FXI (mTF ;hTF FXI ). Blood pools are indicated by arrow- heads or asterisks in placentas of E14.5 sections stained with hematoxylin and eosin. (B) The percentage of placental area occupied by maternal blood pools in placentas of different genotypes was quantified at E13.5-E15.5 (n 5 4-17 per group). Filled circles indicate ruptured placentas. A significant increase in the percentage area occupied by 2/2 1/1 1/y 1/1 blood pools was observed in low-TF;FXI placentas compared with low-TF;FXI placentas. *P , .01, Kruskal-Wallis with post hoc Dunn’s tests; low TF;FIX ;FXI 2/2 (LTF) vs low TF;FXI . Data represented as individual values with median. (C) F9, F11, and F12 gene expression in the placenta at E14.5 was assessed by quantitative polymerase chain reaction and expressed relative to expression of these genes in the liver. Data are the mean 6 SD. **P , .0001 unpaired Student t test (liver vs placenta). 1/1 2/2 (D) Representative western blots of TFPI, pro-HGF, and b-actin in tissue lysates from FXI and FXI placentas. Densitometric analysis of TFPI (E) and pro-HGF (F) in 2/2 1/1 FXI placentas and FXI controls (n 5 8 per group) normalized against b actin expression. Data are presented as individual values with the median. ***P , .01; ****P , .05, Mann-Whitney U test. small maternal blood pools in the labyrinth layer. It is important to The death of the pregnant dams appeared to be related to the 2/2 note that, over a 20-year period, our group has not observed rupture rupture of low-TF;FXI placentas and subsequent uterine hemor- 1/2 1/2 2/2 2/2 of placentas from low-TF embryos or death of pregnant mTF rhage. Both FXI and FXI dams carrying low-TF;FXI 2/2 dams. No defect was observed in placentas from FXI offspring, embryos died during mid- to late gestation, suggesting that in which TF levels were normal, suggesting that a role of FXI in the embryonic, but not maternal, FXI limits the size of the placental placenta is revealed only under conditions of low TF. blood pools in low-TF placentas. A trend toward an increased rate 14 JANUARY 2020 x VOLUME 4, NUMBER 1 FIX AND FXI IN MURINE HEMOSTASIS 213 Relative expression (AU) Relative blood pool area (%) Litter 2 Litter 1 TFPI densitometry (AU) ProHGF densitometry (AU) Downloaded from http://ashpublications.org/bloodadvances/article-pdf/4/1/207/1552610/advancesadv2019000921.pdf by guest on 12 October 2021 of death of dams was observed as the expected frequency of low-TF; placentas had significantly reduced numbers of labyrinth tropho- 2/2 53 FXI embryos increased in different breedings. In addition, death blast cells with poorly developed maternal lacunae. Importantly, 2/2 of the pregnant dams in each of 5 breedings was associated with significantly higher levels of pro-HGF were observed in FXI 2/2 the percentage of low-TF;FXI embryos carried by the dam. Indeed, placentas, suggesting that FXIa could function as an important pregnant dams that died had a significantly higher percentage of activator of HGF during placental development. A reduction in HGF 2/2 low-TF;FXI embryos than did dams that survived. Interestingly, the caused by an absence of FXI may explain the observed increase in murine placenta was found to express high levels of F11 mRNA, but blood pooling in low-TF placentas. not of F9 or F12 mRNA, which suggests a specific role for FXI in the In summary, in this study, the ability of the TF-FVIIa complex to murine placenta. In contrast, FXI is not expressed in human placenta. activate FIX in vivo was confirmed. Furthermore, under conditions These contrasting findings provide evidence of another potential of low TF, a complete deficiency in FIX was found to result in species-specific difference in FXI biology between humans and postnatal death of mice. This finding is consistent with a critical mice. It is possible that FXI expression in the mouse placenta serves function for TF-FVIIa–mediated activation of FIX in thrombin generation. as an adaptive response to the reduced levels of circulating FXI. In contrast, under conditions of low TF, a complete deficiency in FXI We have previously shown that placentas of low-TF embryos contain wascompatiblewithmurinesurvival. However, under conditions of low blood pools. Maternal lacunae are separated by a barrier formed of TF, FXI, but not FIX, appeared to be essential for preventing expansion labyrinth trophoblast cells. Ultrastructural analysis of low-TF placen- of placental blood pools, supporting a tissue-specific requirement for tas revealed a reduction in the number of contacts between FXI under conditions of low TF in mice. trophoblasts and a thinning of the barrier spanning the blood space Acknowledgments between adjacent trabeculae. This mechanism appears to be the one underlying the coalescence of maternal lacunae and formation The authors thank Ying Zhang for expert technical assistance and of blood pools. It was unclear whether the primary defect in low-TF Bentley Midkiff in the Translational Pathology Laboratory for expert placentas was hemostatic or structural. It is notable that there is also technical assistance. a reduction in contacts between mesoderm and endoderm in the This work was supported by a John C. Parker Professorship 2/2 21 yolk sac of TF embryos at E9.5. Interestingly, blood pools in (N.M.). S.P.G. is supported by an American Heart Association low-TF placentas were reduced by concomitant deletion of TFPI, postdoctoral fellowship (19POST34370026). M.L.P. was supported presumably because of increased local thrombin generation. by a grant from the National Science Foundation (1559922). The UNC Translational Pathology Laboratory is supported in part by grants It is possible that FXI contributes to thrombin generation in the from the National Institutes of Health, National Cancer Institute placenta. It has recently been shown that human FXI can bypass FIX 2/2 17 (5P30CA016080-42 and U54-CA156733), National Institute and restore hemostasis in FIX mice. Indeed, 2 studies have 17,52 of Environmental Health Sciences (3P30 EOS010126-17), the shown that FXIa can activate FV, FVIII, and FX. High levels of University Cancer Research Fund, and the North Carolina Bio- FXI in the placenta may drive a similar pathway that enhances local technology Center (2015-IDG-1007). thrombin generation. A deficiency of FXI would further reduce thrombin levels under low-TF conditions and could contribute to the Authorship observed increase in maternal blood pools in placentas from low- 2/2 TF;FXI embryos. In addition, it is possible that levels of TF are Contribution: S.P.G., N.M., A.M., A.S.W., A.C.C., M.V., and S.H. lower in the placenta than in other tissues, making this tissue more designed the experiments; S.P.G., C.M.S., A.C.A., E.B., A.M., M.L.P., dependent on thrombin-mediated activation of FXI. Interestingly, A.C.C., and M.V. conducted the experiments and analyzed the data; FXIa has also been shown to proteolytically inactivate TFPI. It is S.P.G., N.M., A.M., A.S.W., A.C.C., M.V., S.H., J.J.P., H.M.S., S.A., D.G., and R.P. interpreted the data; S.P.G. and N.M. drafted the possible that loss of FXI in the placenta leads to increased levels of TFPI that further decrease FXa and thrombin generation in low-TF manuscript; S.P.G., N.M., A.S.W., A.C.C., S.H., H.M.S., S.A., R.P., 1/1 2/2 placentas. However, analysis of placentas from FXI and FXI and D.G. edited the manuscript; and all authors read and approved embryos revealed that loss of FXI resulted in a paradoxical decrease the manuscript before submission. in TFPI expression that would not explain the observed phenotype. Conflict-of-interest disclosure: N.M. and H.M.S. are consultants An alternative explanation for the increase in blood pooling in low- for Bayer. S.H. is an employee of Bayer. The remaining authors TF placentas lacking FXI is that FXIa regulates factors required for declare no competing financial interests. placenta development. For instance, FXIa can also serve as ORCID profiles: S.P.G., 0000-0001-8709-8394; A.S.W., 0000- a noncanonical activator of pro-HGF to active HGF. HGF has 0002-2845-2303. been found to serve as an important regulator of placental development. Deletion of HGF severely impaired placental devel- Correspondence: Nigel Mackman, Department of Medicine, opment between E13.5 and E15.5 resulting in the death of the University of North Carolina at Chapel Hill, 8004B Mary Ellen Jones 53,54 embryos. This is the same period during which blood pooling Building, 116 Manning Dr, Chapel Hill, NC 27599; e-mail: nmackman@ 2/2 2/2 was observed in low-TF and low-TF;FXI placentas. HGF med.unc.edu. References 1. Grover SP, Mackman N. Tissue factor: an essential mediator of hemostasis and trigger of thrombosis. Arterioscler Thromb Vasc Biol. 2018;38(4):709-725. 2. Josso F, Prou-Wartelle O. Interaction of tissue factor and factor VII at the earliest phase of coagulation. 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Differential roles of factors IX and XI in murine placenta and hemostasis under conditions of low tissue factor

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Downloaded from http://ashpublications.org/bloodadvances/article-pdf/4/1/207/1552610/advancesadv2019000921.pdf by guest on 12 October 2021 REGULAR ARTICLE Differential roles of factors IX and XI in murine placenta and hemostasis under conditions of low tissue factor 1 1 1 1 1,2 1,2 3 Steven P. Grover, Clare M. Schmedes, Alyson C. Auriemma, Emily Butler, Molly L. Parrish, Adam Miszta, Audrey C. Cleuren, 4 4 5,6 5,6 1,2 1,2 1 Mayken Visser, Stefan Heitmeier, Jens J. Posma, Henri M. Spronk, Silvio Antoniak, Alisa S. Wolberg, Rafal Pawlinski, 7 1 David Gailani, and Nigel Mackman 1 2 UNC Blood Research Center, Division of Hematology and Oncology, Department of Medicine, and UNC Blood Research Center, Department of Pathology and Laboratory 3 4 Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC; Life Sciences Institute, University of Michigan, Ann Arbor, MI; Bayer AG, Wuppertal, Germany; 5 6 Laboratory for Clinical Thrombosis and Hemostasis, Department of Internal Medicine, and Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University Medical Center, Maastricht, The Netherlands; and Department of Pathology, Microbiology and Immunology, Vanderbilt University Medical Center, Nashville, TN The intrinsic tenase complex (FIXa-FVIIIa) of the intrinsic coagulation pathway and, to Key Points a lesser extent, thrombin-mediated activation of FXI, are necessary to amplify tissue factor � Factor IX, but not factor (TF)-FVIIa–initiated thrombin generation. In this study, we determined the contribution of XI, is essential for the murine FIX and FXI to TF-dependent thrombin generation in vitro. We further investigated postnatal survival of TF-dependent FIX activation in mice and the contribution of this pathway to hemostasis. mice under conditions Thrombin generation was decreased in FIX- but not in FXI-deficient mouse plasma. of low TF. Furthermore, injection of TF increased levels of FIXa-antithrombin complexes in both wild- 2/2 � Embryonic factor XI, type and FXI mice. Genetic studies were used to determine the effect of complete but not factor IX, pre- deficiencies of either FIX or FXI on the survival of mice expressing low levels of TF. Low-TF; vents enlargement of 2/y FIX male mice were born at the expected frequency, but none survived to wean. In blood pools in low-TF 2/2 contrast, low-TF;FXI mice were generated at the expected frequency at wean and had placentas. a 6-month survival equivalent to that of low-TF mice. Surprisingly, a deficiency of FXI, but not FIX, exacerbated the size of blood pools in low-TF placentas and led to acute hemorrhage and death of some pregnant dams. Our data indicate that FIX, but not FXI, is essential for survival of low-TF mice after birth. This finding suggests that TF-FVIIa–mediated activation of FIX plays a critical role in murine hemostasis. In contrast, FXI deficiency, but not FIX deficiency, exacerbated blood pooling in low-TF placentas, indicating a tissue-specific requirement for FXI in the murine placenta under conditions of low TF. Introduction The coagulation cascade can be divided into the extrinsic (tissue factor [TF]–factor [F] VIIa complex), intrinsic(FXIIa, FXIa,FIXa, andFVIIIa),and common (FXa,FVa,and thrombin)pathways. The extrinsic 2-4 pathway is the physiologic trigger of blood coagulation and activates both FIX and FX. TF-FVIIa–mediated activation of FIX was initially described by Francois Josso and colleagues and is commonly referred to as the “Josso loop.” The intrinsic pathway also contains a feedback loop in which thrombin activates FXI 5,6 leading to further generation of FIXa. These pathways are thought to play an importnant role in thrombin generation, because the TF-FVIIa complex is rapidly inhibited by TF pathway inhibitor (TFPI). Humans deficient in FVIII (hemophilia A) or FIX (hemophilia B) have hemostatic defects. Sim- 2/2 2/2 ilarly, FVIII and FIX mice exhibit increased bleeding after a hemostatic challenge, such as tail Submitted 3 September 2019; accepted 28 October 2019; published online 14 © 2020 by The American Society of Hematology January 2020. DOI 10.1182/bloodadvances.2019000921. The full-text version of this article contains a data supplement. 14 JANUARY 2020 x VOLUME 4, NUMBER 1 207 Downloaded from http://ashpublications.org/bloodadvances/article-pdf/4/1/207/1552610/advancesadv2019000921.pdf by guest on 12 October 2021 9,10 transection. FXI-deficient patients rarely have spontaneous with control plasma when thrombin generation was initiated with bleeding events, but increased bleeding occurs in some patients a low concentration of TF (Figure 1A; data not shown). on provocation, particularly in tissues with high fibrinolytic activity, Next, the contribution of FXI to thrombin generation was determined 11,12 such as the mouth, nose, and genitourinary tract. FXI is thought in mouse plasma in which activation of coagulation was initiated by 13,14 2/2 to stabilize the fibrin clot and protect it from fibrinolysis. FXI either silica or TF. As expected, silica-initiated thrombin generation mice have normal bleeding times in the tail vein transection model. 2/2 1/2 was significantly impaired in FXI PPP compared with FXI or 2/2 One study reported a significant increase in bleeding in FXI mice 1/1 FXI PPP (Figure 1B). In contrast, there was no significant in the saphenous vein injury model, but this was not confirmed in difference in thrombin generation (lag time, time to peak, peak, or an independent study. In vitro studies have shown that FXI 2/2 1/1 1/2 ETP) in FXI PPP compared with FXI or FXI PPP, at contributes to thrombin generation in human plasma initiated by a low concentration of TF (Figure 1C; data not shown). To exclude 18-20 low, but not high, concentrations of TF. the potential effects of contact pathway activation during blood collection, additional samples were collected in the presence of In contrast to studies with mice lacking components of the intrinsic corn trypsin inhibitor (CTI). However, even in the presence of CTI, pathway, a complete deficiency of components of the extrinsic or despite observing a robust reduction in silica-induced thrombin common pathways in mice leads to death during embryonic 21-28 generation (Figure 1D; data not shown), there was no difference in development or shortly after birth. For instance, 80% to 90% 1/1 1/2 2/2 2/2 21-23 2/2 TF-initiated thrombin generation between FXI , FXI , or FXI of TF embryos die at embryonic day (E) 9.5-E10.5. TF mice can be rescued with a transgene that expresses human TF PPP (Figure 1E; data not shown). (hTF) regulated by the human TF promoter. These “low-TF” mice Platelets provide an important surface for the activation of FXI 2/2 2/2 1/1 (murine TF [mTF ];hTF ) express ;1% levels of TF. Low- and may be important in FXI-mediated amplification of thrombin TF mice exhibit spontaneous hemostatic defects in various organs 35-37 generation. To account for the potential effects of platelets and have significantlyincreased bleeding in the tail vein transection we assessed thrombin generation in platelet-rich plasma (PRP). 1,30-32 model. Mice expressing ;1% levels of FVII have phenotypes When coagulation was initiated with silica, thrombin generation 33,34 similar to those of low-TF mice. It is notable that maternal blood 2/2 was significantly reduced in PRP from FXI mice compared pooling occurs in placentas of low-TF embryos but does not cause 1/2 1/1 to FXI or FXI controls (Figure 1F). In contrast, when any loss of embryos or fatal hemorrhages in the dams. The coagulation was initiated with TF, there was no difference in placenta is derived from embryonic tissue, indicating that blood 2/2 1/2 thrombin generation in PRP from FXI mice compared to FXI pooling is related to low levels of TF in the embryo. 1/1 or FXI controls (Figure 1G; data not shown). Collectively, these data in PPP and PRP show that, although FXI deficiency was In the current study, we investigated the contribution of FIX and FXI detectable in mouse plasma in contact silica-initiated assays, FXI to thrombin generation in vitro and in vivo under conditions of low did not contribute to thrombin generation in mouse plasma when TF. Plasma-based thrombin generation assays were used to assess initiated by TF. the relative contribution of FIX and FXI to TF-initiated thrombin generation. The ability of the TF-FVIIa complex to activate FIX in vivo TF activates FIX in vivo in the absence of FXI 2/2 was also determined in wild-type and FXI mice. Finally, a genetic To determine whether TF can activate FIX in vivo, mice were injected approach was employed to determine the effect of a complete with TF and plasma levels of FIXa-antithrombin (AT) complexes deficiency of either FIX or FXI on the survival of mice expressing low measured. The thrombin inhibitor dabigatran was used to prevent levels of TF. death associated with the formation of intravascular thrombi after Methods injection of TF and to block thrombin-mediated activation of FXI. Injection of TF into wild-type mice significantly increased the plasma Mice levels of FIXa-AT complexes compared with controls (Figure 2A). 2/2 2/2 2/2 FIX and FXI mice were backcrossed 10 generations to the FXI mice were used to eliminate the thrombin-FXIa feedback 10,15 2/2 1/1 C57BL/6J background. Low-TF mice (mTF ;hTF ) were loop. Similar to the results in wild-type mice, TF injection significantly backcrossed 6 generations to the C57BL/6J background. increased the plasma levels of FIXa-AT complexes in thrombin- 2/2 Studies complied with National Institutes of Health Guide for the inhibited FXI mice (Figure 2B). These results indicate that the Care and Use of Laboratory Animals and were performed with the TF-FVIIa complex activates FIX in mice. approval of the Animal Care and Use Committee of the University of Effect of FIX deficiency on the survival of North Carolina at Chapel Hill. low-TF offspring All additional details of experimental procedures are given in the The FIX gene resides on the X chromosome. Therefore, to study the supplemental Methods. 2/2 1/1 1/y effect of FIX deficiency on low-TF mice, male mTF ;hTF ;FIX 1/2 1/2 1/2 Results and female mTF ;hTF ;FIX were crossed and the allele frequencies of offspring determined at various timepoints. No male Effect of either FIX or FXI deficiency on TF-initiated 2/y low-TF;FIX offspring were observed at postnatal day (P) 21, thrombin generation in murine plasma a significant reduction compared with the expected frequency The effect of FIX deficiency on TF-initiated thrombin generation in (Table 1). Next, offspring were analyzed at E14.5 to determine if 2/y mouse platelet-poor plasma (PPP) was determined. Endogenous low-TF;FIX offspring had survived development beyond E9.5 thrombin potential (ETP), time to peak, peak, and lag time were and E10.5 given that embryos lacking various coagulation factors 2/y measured. As expected, a significant reduction in peak thrombin dieatthistimepoint.Viablelow-TF;FIX offspring were observed generation and ETP was observed in FIX-deficient plasma compared at E14.5 (Table 1) and appeared grossly normal by histological 208 GROVER et al 14 JANUARY 2020 x VOLUME 4, NUMBER 1 Downloaded from http://ashpublications.org/bloodadvances/article-pdf/4/1/207/1552610/advancesadv2019000921.pdf by guest on 12 October 2021 Figure 1. Effect of deficiency of FIX or FXI on thrombin generation in murine plasma. (A) Thrombin generation in murine FIX-deficient PPP was initiated with low and high concentrations of +/+ hTF. Thrombin generation in mFXI-deficient PPP collected in the ab- FIX -/- sence of CTI was initiated with silica (B) or low and high doses of 600 FIX hTF (C). Thrombin generation in mFXI-deficient PPP collected in the presence of CTI was initiated with silica (D) or low and high doses +/+ FXI of hTF (E). Thrombin generation in mFXI-deficient PRP collected in +/- FXI the presence of CTI was initiated with silica (F) or low and high -/- FXI doses of hTF (G). Data are presented as individual values with 200 means 6 95% confidence intervals. *P , .05, 2-way analysis of var- 1/1 2/2 iance with post hoc Bonferroni test (FIX vs FIX ); **P , .001, 1/1 1-way analysis of variance with post hoc Bonferroni test (FXI 1/2 2/2 0.05 0.5 TF (pM) and FXI vs FXI ). BC ** 0 0 Silica 0.05 0.5 TF (pM) DE ** 0 0 Silica 0.05 0.5 TF (pM) FG ** 0 0 Silica 0.05 0.5 TF (pM) 14 JANUARY 2020 x VOLUME 4, NUMBER 1 FIX AND FXI IN MURINE HEMOSTASIS 209 ETP (nM-min) ETP (nM-min) ETP (nM-min) ETP (nM-min) ETP (nM-min) ETP (nM-min) ETP (nM-min) Downloaded from http://ashpublications.org/bloodadvances/article-pdf/4/1/207/1552610/advancesadv2019000921.pdf by guest on 12 October 2021 was not significantly different from the expected frequency of A B 4.69% (Table 2). Four additional crosses with sires and dams of 700 700 differing genotypes were conducted to yield larger numbers of low- ** 2/2 TF;FXI offspring. Similar to the results observed when crossing 2/2 F1 intermediates low-TF;FXI mice were generated at a frequency 500 500 that was not significantly different from the expected frequency 400 400 in these 4 additional breedings (supplemental Table 1). These observations indicate that, even when overall procoagulant potential is reduced under conditions of low TF, FXI is not necessary for 200 200 survival in mice. 100 100 Next, the effect of FXI deficiency on the survival of low-TF mice 0 0 between weaning and 6 months of age was determined. As 38,39 Ctrl TF Ctrl TF expected from previous studies, 27% (3 of 11) of low-TF; 1/1 -/- FXI mice died during the 6-month survival study, with evidence WT FXI of pulmonary hemorrhage in all mice. Importantly, survival did not 2/2 1/2 2/2 differ significantly between low-TF;FXI , low TF;FXI offspring, Figure 2. TF activates FIX in thrombin-inhibited wild-type and FXI mice. 1/1 and low-TF;FXI controls (Figure 3). Plasma levels of FIXa-AT complexes were quantified in dabigatran-pretreated 2/2 wild-type (WT) (A) or FXI (B) mice, administered either vehicle control or TF 2/2 Death of pregnant dams bearing low-TF;FXI (n 5 6-10 per group). A significant increase in plasma FIXa-AT complexes was ob- 2/2 served on administration of TF in both WT and FXI mice. Data are presented as embryos individual values with means 6 95% confidence intervals. *P , .0001; **P , .05; 2/2 While generating low-TF;FXI offspring, the death of pregnant unpaired Student t test. 1/2 1/(1/2) 1/2 1/2 1/(1/2) 2/2 mTF ;hTF ;FXI (n 5 6) and mTF ;hTF ;FXI 2/2 (n 5 5) dams bearing low-TF;FXI embryos occurred during late 2/y analysis (data not shown). Finally, the number of low-TF;FIX at gestation (E13.5-15.5) in all 5 breedings (Table 3). In breeding 1, death 2/y 1/2 1/(1/2) 1/2 P1 was analyzed. Male low-TF;FIX offspring were observed at occurred in 9% (2 of 33) of pregnancies in mTF ;hTF ;FXI 2/y 1/2 1/(1/2) 1/2 P1 (Table 1).However,2ofthe 5(40%)maleP1low-TF;FIX dams. Death of 20% to 40% of pregnant mTF ;hTF ;FXI 2/y offspring were dead. One low-TF;FIX P1 pup was pale and had dams in breedings 2 and 3 and of 30% to 33% of the pregnant 1/2 1/(1/2) 2/2 a dark abdomen, which was consistent with abdominal hemor- mTF ;hTF ;FXI dams in breedings 4 and 5 was rhage (supplemental Figure 1). One of the 6 (17%) male low-TF; observed. These results suggest that the death of the pregnant 1/y 2/2 FIX P1 pups was dead with no apparent phenotype. These dams was associated with the relative burden of low-TF;FXI results indicate that FIX is essential for postnatal survival of low- embryos and was largely independent of the maternal FXI TF mice. genotype. There was a trend toward increased death of the pregnant dams FXI deficiency does not affect the generation or 2/2 as the burden of low-TF;FXI embryos increased (Table 3). To survival of low-TF offspring further investigate whether there was an association between the 2/2 The effect of FXI deficiency on the generation and survival of frequency of low-TF;FXI offspring and maternal death, the 2/2 mice expressing low levels of TF was determined. Male and female burden of low-TF;FXI offspring was determined for pregnancies 1/2 1/2 1/2 F1 intermediates (mTF ;hTF ;FXI ) were crossed, and in which the dam survived vs those in which the dam died. Interestingly, 2/2 the allele frequencies of 117 offspring were determined. Six of dams that died carried a higher proportion of low-TF;FXI embryos 2/2 117 (5.13%) of the offspring at wean were low TF;FXI , which compared to the dams that survived (Table 3). 2/2 Table 1. Distribution of genotypes at weaning from a breeding to generate low-TF;FIX offspring Sex mTF hTF FIX Expected, % E14.5, n E14.5 observed, % E14.5 O/E, % P1, n P1 observed, % P1 O/E, % P21, n P21 observed, % P21 O/E, % F 1/21/(1/2) 1/1 12.5 7 18.4 147 3 6.6 52 15 18.5 148 F 1/21/(1/2) 1/2 12.5 7 18.4 147 7 15.5 147 13 16.0 128 M 1/21/(1/2) 1/y 12.5 1 2.6 21 10 22.2 178 15 18.5 148 M 1/21/(1/2) 2/y 12.5 4 10.5 84 8 17.7 142 9 11.1 89 F 2/21/(1/2) 1/1 12.5 5 13.2 106 4 8.8 70 8 9.9 79 F 2/21/(1/2) 1/2 12.5 5 13.2 106 2 4.4 35 12 14.8 118 M 2/21/(1/2) 1/y 12.5 5 13.2 106 6* 13.3 106 9 11.1 89 M 2/21/(1/2) 2/y 12.5 4 10.5 84 5† 11.1 89 0 0 0‡ Total 100.0 38 100.0 — 45 100.0 — 81 100.0 — 1/(1/2) 1/2 1/1 F, female; hTF , hTF or hTF ; M, male; O/E, observed/expected. *One of 6 dead. †Two of 5 dead. ‡P , .05 observed vs expected, 2-tailed Fisher’s exact test. 210 GROVER et al 14 JANUARY 2020 x VOLUME 4, NUMBER 1 FIXa-AT (pM) FIXa-AT (pM) Downloaded from http://ashpublications.org/bloodadvances/article-pdf/4/1/207/1552610/advancesadv2019000921.pdf by guest on 12 October 2021 Table 2. Distribution of genotypes at weaning from a breeding to 2/2 generate low-TF;FXI offspring mTF hTF FXI Expected, % Observed, n Observed, % O/E, % 1/1 — 1/1 1.56 4 3.42 219 1/1 — 1/2 3.13 4 3.42 109 1/1 — 2/2 1.56 4 3.42 219 1/11/(1/2) 1/1 4.69 6 5.13 109 1/11/(1/2) 1/2 9.38 10 8.55 91 +/+ LowTF; FXI (n=11) 1/11/(1/2) 2/2 4.69 2 1.71 36 +/- LowTF; FXI (n=21) 1/2 — 1/1 3.13 2 1.71 55 20 -/- LowTF; FXI (n=12) 1/2 — 1/2 6.25 8 6.84 109 1/2 — 2/2 3.13 3 2.56 82 0 30 60 90 120 150 180 1/21/(1/2) 1/1 9.38 16 13.68 146 Days 1/21/(1/2) 1/2 18.75 25 21.37 114 1/21/(1/2) 2/2 9.38 11 9.40 100 1/1 Figure 3. Survival of low-TF mice with different levels of FXI. Low-TF;FXI , 2/2 — 1/1 1.56 0 0.00 0* 1/2 2/2 low-TF;FXI , and low-TF;FXI mice were assessed in a 6-month survival study. 2/2 — 1/2 3.13 0 0.00 0* 1/1 As expected, 27% (3 of 11) of low-TF;FXI mice died by 6 months with evidence 1/2 2/2 — 2/2 1.56 0 0.00 0* of pulmonary hemorrhage. By comparison 10% (2 of 21) of low-TF;FXI and 25% 2/2 (3 of 12) of low-TF;FXI mice died by 6 months with evidence of pulmonary 2/21/(1/2) 1/1 4.69 8 6.84 146 hemorrhage. No significant difference in survival was observed between groups. 2/21/(1/2) 1/2 9.38 8 6.84 73 P . .05, log-rank test. 2/21/(1/2) 2/2 4.69 6 5.13 109 Total 100.00 117 100.00 — proteolytically cleave pro-hepatocyte growth factor (pro-HGF) *Expected embryonic lethality. to active HGF. Therefore, levels of TFPI and pro-HGF were 2/2 1/1 analyzed in placentas from FXI and FXI embryos. If FXIa were a major proteolytic inactivator of TFPI, increased expression 2/2 of full-length TFPI protein would be expected in FXI placentas. FXI deficiency is associated with increased size of 2/2 However, TFPI protein expression in FXI placentas was signifi- blood pools in low-TF placentas 1/1 cantly lower than that in FXI controls (Figure 4D-E). In contrast, 2/2 Necropsies were conducted on 9 of the 11 dams carrying low-TF; levels of pro-HGF protein were significantly higher in FXI 2/2 1/1 FXI embryos that had either died (n 5 8) or had been euthanized placentas than in FXI controls (Figure 4D, F), suggesting that due to uterine hemorrhage (n 5 1) during late gestation. Free blood FXIa regulates activation of pro-HGF in the placenta. was observed in the uterus of 3 of 9 (33%) of the females that died 2/2 with evidence of ruptured low-TF;FXI placentas, suggesting that Discussion 2/2 some of the pregnant females carrying low-TF;FXI embryos died The FIXa;FVIIIa complex has been proposed to sustain activation of of acute uterine or placental hemorrhage. coagulation and thrombin generation after inhibition of the TF-FVIIa 2/2 The size of the maternal blood pools in low-TF;FXI placentas complex by TFPI. Importantly, the TF-FVIIa complex is known to 1/2 1/1 1-3,42 was compared to those in low-TF;FXI placentas and low-TF;FXI activate both FIX and FX in vitro. Indeed, an in vitro study placentas removed from dead or euthanized dams (Figure 4A-B). comparing activation of FIX and FX by the TF-FVIIa complex The average size of the maternal blood pools was significantly concluded that FIX is the major substrate. In addition, individuals 2/2 increased in low-TF;FXI placentas compared with low-TF; with a point mutation in FIX that reduces interaction with TF-FVIIa 1/1 2/2 FXI placentas (Figure 4B). In addition, 5 of 17 low-TF;FXI but not FXI have a mild form of hemophilia B. Furthermore, basal placentas appeared to have ruptured (Figure 4B). Of note, FIX levels of FIXa activation are reduced in FVII-deficient (,7% levels) deficiency did not affect the size of the blood pools (Figure 4B). but not in FXI-deficient (,8% levels) individuals. Taken together, 2/2 Importantly, no abnormalities were observed in FXI placentas these in vitro and in vivo studies indicate that the TF-FVIIa complex is with 50% or 100% levels of mouse TF (Figure 4A). a major activator of FIX. Given the observation that FXI deficiency exacerbated placental Consistent with the important contribution of TF-FVIIa–mediated blood pooling under conditions of low TF, F9, F11, and F12 gene activation of FIX to thrombin generation, a deficiency of FIX markedly expression was assessed in wild-type mouse placentas. A high level 45-47 blunted TF-initiated thrombin generation in human plasma. A of F11 mRNA, but not of F9 or F12 mRNA, was observed in the similar approach has been taken to investigating the contribution placenta (Figure 4C), suggesting that local FXI expression plays of thrombin-mediated FXI activation to thrombin generation. In a role in the placenta. FXI-deficient human plasma, thrombin generation is impaired at 20,48,49 The contribution of FIX-independent functions of FXI in the exacerba- a low, but not a high, initiating concentration of TF. Taken tion of blood pooling in low-TF placentas was investigated. FXIa has together, these data suggest that, in humans, TF-FVIIa–mediated been shown to proteolytically inactivate TFPI. Conversely, FXIa can activation of FIX and, to a lesser extent, thrombin-mediated 14 JANUARY 2020 x VOLUME 4, NUMBER 1 FIX AND FXI IN MURINE HEMOSTASIS 211 Survival (%) Downloaded from http://ashpublications.org/bloodadvances/article-pdf/4/1/207/1552610/advancesadv2019000921.pdf by guest on 12 October 2021 2/2 Table 3. Observed low-TF;FXI offspring at weaning from additional breedings and death of females Survived Died Breeding type Expected, % Dam FXI genotype Death/pregnancy, n/n (%) n/N at P21 (litters) Observed, % n/N at E13-15.5 (litters) Observed, % 1 4.69 1/2 2/23 (9) — ——— 2 10.26 1/2 2/5 (40) 1/25 (3) 4.0 4/14 (2)* 29.0 3 10.26 1/2 2/10 (20) 3/40 (6) 7.5 7/19 (2)* 37.0 4 10.26 2/2 2/6 (33) 2/19 (3) 10.5 2/7 (1) 29.0 5 20.52 2/2 3/10 (30) 3/22 (7) 13.6 6/17 (3) 35.3 2/2 1/(1/2) 2/2 *P , .05, difference in observed frequency of mTF ;hTF ;FXI in surviving mothers vs dead mothers assessed by 2-tailed Fisher’s exact test. activation of FXI both contribute to thrombin generation. However, TF was combined with a complete deficiency of FIX. Surprisingly, 2/y it is unclear whether these amplificatory pathways also contribute the expected number of male low-TF;FIX embryos was observed to thrombin generation and hemostasis in mice. at E14.5, indicating that these embryos survived the E9.5 to 10.5 2/2 21-23 period associated with the death of mTF embryos. Birth In the present study, a deficiency in FIX resulted in a modest, but presents a strong hemostatic challenge as pups are squeezed significant, impairment in thrombin generation in mouse PPP at through the birth canal. Mice with a complete absence of different a low, but not a high, initiating dose of TF. In contrast, no defect in components of the extrinsic (FVII) and common (FV, FX, and TF-initiated thrombin generation was observed in FXI-deficient prothrombin) pathways exhibit a high rate of death at birth and in the mouse PPP at this same low initiating dose of TF. Inhibition of 24-27 immediate postnatal period. Some of these pups lacking key exogenous contact activation by the FXIIa inhibitor CTI at the time of coagulation factors appear to die of acute abdominal hemorrhage blood collection failed to reveal a contribution of FXI to TF-initiated after birth and appear pale rather than pink, with dark abdomens thrombin generation in PPP. Platelets have been identified as an 24-27 due to blood pooling. Consistent with this phenotype, 2 of 5 35-37 important physiologic surface for the activation of FXI by thrombin. 2/y (40%) of the low-TF;FIX male mice died after birth, and 1 pup TF-initiated thrombin generation, however, was unchanged in FXI- was pale with a dark abdomen, consistent with death due to an deficient PRP. These data support the involvement of FIX, but not acute abdominal hemorrhage. In addition, 1 of 6 (17%) of the low-TF FXI, in TF-initiated thrombin generation in mouse plasma. It is male mice also died at birth, which was likely caused by a hemostatic interesting tonotethatinmurinePPP thecontributionofFIX to TF- defect. initiated thrombin generation was observed only at 0.05 pM of TF, whereas in human PPP, impaired thrombin generation was Low-TF mice backcrossed 7 generations to a C57BL/BJ back- observed in FIX-deficient plasma at doses as high as 1 pM. This ground demonstrate a spontaneous hemostatic defect that results result suggests that thrombin generation in humans may have in the death of 40% to 60% of mice by 6 months of age, primarily as 38,39 a greater dependence on FIX than in mice. a result of spontaneous hemorrhage in the lung. In contrast, 2/2 FIX mice do not exhibit a spontaneous hemostatic defect, but Given the similarity in the components and general mechanism of have increased bleeding upon challenge, such as a tail transection, the hemostatic systems in humans and mice, it is somewhat despite the presence of normal levels of TF. The postnatal lethality surprising that FXI does not seem to serve a similar role in the 2/y associated with low TF;FIX mice, therefore, represents a marked amplification of coagulation in these 2 mammals. Interestingly, it exacerbation of the phenotype observed in either low-TF mice or was recently demonstrated that, unlike humans, most of the FXI FIX-deficient mice. Importantly, the data suggest that FIX is in mice is localized to the vascular surface through binding to essential for the survival of low-TF mice during the trauma of birth glycosaminoglycans, owing to an additional cluster of basic residues 50 and in the immediate postnatal period. on the Apple 4 domain of mouse FXI. This process leads to three- to four-fold lower levels of circulating FXI in mice compared to To evaluate the contribution of thrombin-mediated activation of FXI, humans. attempts were made to generate low-TF mice lacking FXI. Low-TF; 2/2 FXI mice were generated at the expected frequency at wean To confirm the ability of the TF-FVIIa complex to activate murine FIX 1/2 2/2 from multiple breedings involving both FXI and FXI dams, in in vivo, dabigatran-anticoagulated mice were injected with exoge- 2/y contrast to the attempts to generate low-TF;FIX mice. Moreover, nous TF, and FIXa generation was determined by measurement of 2/2 the survival of low-TF;FXI mice was not different from that of low- plasma levels of FIXa-AT complexes. Injection of TF into wild-type 1/2 1/1 TF;FXI or low-TF;FXI mice between wean and 6 months of mice resulted in generation of significant levels of FIXa. This finding 2/2 2/2 age. This result differs from the reduced survival of low-TF;PAR4 was also replicated in FXI mice, which eliminated the notion of mice during the same period. The results from this genetic any contribution of the thrombin-FXI pathway to the observed approach suggest that FIX, but not FXI, contributes to thrombin generation of FIXa. This approach provides the first direct evidence generation and hemostasis under conditions of reduced TF in mice. of a functional Josso loop in mice. 2/2 Having demonstrated the ability of the TF-FVIIa complex to activate During the generation of low-TF;FXI mice, several pregnant FIX in vivo in mice, we used a complementary genetic approach to dams died during late gestation. Unexpectedly, in pregnant dams 2/2 investigate the contribution of TF-FVIIa–mediated activation of FIX that died, several of the placentas from low-TF;FXI embryos to murine embryonic development and hemostasis. Mice were were found to have large maternal blood pools. Previous studies crossed to generate offspring in which expression of a low level of have demonstrated that placentas from low-TF offspring contain 212 GROVER et al 14 JANUARY 2020 x VOLUME 4, NUMBER 1 Downloaded from http://ashpublications.org/bloodadvances/article-pdf/4/1/207/1552610/advancesadv2019000921.pdf by guest on 12 October 2021 +/- -/- +/- -/- FXI FXI LTF;FXI LTF;FXI 2mm BD 100 +/+ -/- FXI FXI TFPI 44kDa 100kDa proHGF 38kDa E Actin +/- -/y +/- -/- LTF LTF;FIX LTF;FIX LTF;FXI LTF;FXI (n=11) (n=5) (n=4) (n=10) (n=17) CE F Liver Placenta **** *** ** ** 0.1 100 0.01 50 200 0.001 0 0 +/+ -/- +/+ -/- F9 F11 F12 FXI FXI FXI FXI Figure 4. Analysis of the contribution of FIX and FXI to placental blood pooling under conditions of low TF. (A) Representative images of placentas from 2 dams 2/2 1/2 1/2 1/(1/2); from breeding 3 (top row) and 4 (bottom row), each showing a large blood pool in a low-TF;FXI placenta. Genotypes have been abbreviated to FXI (mTF ;hTF 1/2 2/2 1/1 1/(1/2); 2/2 1/2 2/2 1/(1/2); 1/2 2/2 2/2 1/(1/2); 2/2 FXI ), FXI (mTF ;hTF FXI ), low TF;FXI (mTF ;hTF FXI ), and low TF;FXI (mTF ;hTF FXI ). Blood pools are indicated by arrow- heads or asterisks in placentas of E14.5 sections stained with hematoxylin and eosin. (B) The percentage of placental area occupied by maternal blood pools in placentas of different genotypes was quantified at E13.5-E15.5 (n 5 4-17 per group). Filled circles indicate ruptured placentas. A significant increase in the percentage area occupied by 2/2 1/1 1/y 1/1 blood pools was observed in low-TF;FXI placentas compared with low-TF;FXI placentas. *P , .01, Kruskal-Wallis with post hoc Dunn’s tests; low TF;FIX ;FXI 2/2 (LTF) vs low TF;FXI . Data represented as individual values with median. (C) F9, F11, and F12 gene expression in the placenta at E14.5 was assessed by quantitative polymerase chain reaction and expressed relative to expression of these genes in the liver. Data are the mean 6 SD. **P , .0001 unpaired Student t test (liver vs placenta). 1/1 2/2 (D) Representative western blots of TFPI, pro-HGF, and b-actin in tissue lysates from FXI and FXI placentas. Densitometric analysis of TFPI (E) and pro-HGF (F) in 2/2 1/1 FXI placentas and FXI controls (n 5 8 per group) normalized against b actin expression. Data are presented as individual values with the median. ***P , .01; ****P , .05, Mann-Whitney U test. small maternal blood pools in the labyrinth layer. It is important to The death of the pregnant dams appeared to be related to the 2/2 note that, over a 20-year period, our group has not observed rupture rupture of low-TF;FXI placentas and subsequent uterine hemor- 1/2 1/2 2/2 2/2 of placentas from low-TF embryos or death of pregnant mTF rhage. Both FXI and FXI dams carrying low-TF;FXI 2/2 dams. No defect was observed in placentas from FXI offspring, embryos died during mid- to late gestation, suggesting that in which TF levels were normal, suggesting that a role of FXI in the embryonic, but not maternal, FXI limits the size of the placental placenta is revealed only under conditions of low TF. blood pools in low-TF placentas. A trend toward an increased rate 14 JANUARY 2020 x VOLUME 4, NUMBER 1 FIX AND FXI IN MURINE HEMOSTASIS 213 Relative expression (AU) Relative blood pool area (%) Litter 2 Litter 1 TFPI densitometry (AU) ProHGF densitometry (AU) Downloaded from http://ashpublications.org/bloodadvances/article-pdf/4/1/207/1552610/advancesadv2019000921.pdf by guest on 12 October 2021 of death of dams was observed as the expected frequency of low-TF; placentas had significantly reduced numbers of labyrinth tropho- 2/2 53 FXI embryos increased in different breedings. In addition, death blast cells with poorly developed maternal lacunae. Importantly, 2/2 of the pregnant dams in each of 5 breedings was associated with significantly higher levels of pro-HGF were observed in FXI 2/2 the percentage of low-TF;FXI embryos carried by the dam. Indeed, placentas, suggesting that FXIa could function as an important pregnant dams that died had a significantly higher percentage of activator of HGF during placental development. A reduction in HGF 2/2 low-TF;FXI embryos than did dams that survived. Interestingly, the caused by an absence of FXI may explain the observed increase in murine placenta was found to express high levels of F11 mRNA, but blood pooling in low-TF placentas. not of F9 or F12 mRNA, which suggests a specific role for FXI in the In summary, in this study, the ability of the TF-FVIIa complex to murine placenta. In contrast, FXI is not expressed in human placenta. activate FIX in vivo was confirmed. Furthermore, under conditions These contrasting findings provide evidence of another potential of low TF, a complete deficiency in FIX was found to result in species-specific difference in FXI biology between humans and postnatal death of mice. This finding is consistent with a critical mice. It is possible that FXI expression in the mouse placenta serves function for TF-FVIIa–mediated activation of FIX in thrombin generation. as an adaptive response to the reduced levels of circulating FXI. In contrast, under conditions of low TF, a complete deficiency in FXI We have previously shown that placentas of low-TF embryos contain wascompatiblewithmurinesurvival. However, under conditions of low blood pools. Maternal lacunae are separated by a barrier formed of TF, FXI, but not FIX, appeared to be essential for preventing expansion labyrinth trophoblast cells. Ultrastructural analysis of low-TF placen- of placental blood pools, supporting a tissue-specific requirement for tas revealed a reduction in the number of contacts between FXI under conditions of low TF in mice. trophoblasts and a thinning of the barrier spanning the blood space Acknowledgments between adjacent trabeculae. This mechanism appears to be the one underlying the coalescence of maternal lacunae and formation The authors thank Ying Zhang for expert technical assistance and of blood pools. It was unclear whether the primary defect in low-TF Bentley Midkiff in the Translational Pathology Laboratory for expert placentas was hemostatic or structural. It is notable that there is also technical assistance. a reduction in contacts between mesoderm and endoderm in the This work was supported by a John C. Parker Professorship 2/2 21 yolk sac of TF embryos at E9.5. Interestingly, blood pools in (N.M.). S.P.G. is supported by an American Heart Association low-TF placentas were reduced by concomitant deletion of TFPI, postdoctoral fellowship (19POST34370026). M.L.P. was supported presumably because of increased local thrombin generation. by a grant from the National Science Foundation (1559922). The UNC Translational Pathology Laboratory is supported in part by grants It is possible that FXI contributes to thrombin generation in the from the National Institutes of Health, National Cancer Institute placenta. It has recently been shown that human FXI can bypass FIX 2/2 17 (5P30CA016080-42 and U54-CA156733), National Institute and restore hemostasis in FIX mice. Indeed, 2 studies have 17,52 of Environmental Health Sciences (3P30 EOS010126-17), the shown that FXIa can activate FV, FVIII, and FX. High levels of University Cancer Research Fund, and the North Carolina Bio- FXI in the placenta may drive a similar pathway that enhances local technology Center (2015-IDG-1007). thrombin generation. A deficiency of FXI would further reduce thrombin levels under low-TF conditions and could contribute to the Authorship observed increase in maternal blood pools in placentas from low- 2/2 TF;FXI embryos. In addition, it is possible that levels of TF are Contribution: S.P.G., N.M., A.M., A.S.W., A.C.C., M.V., and S.H. lower in the placenta than in other tissues, making this tissue more designed the experiments; S.P.G., C.M.S., A.C.A., E.B., A.M., M.L.P., dependent on thrombin-mediated activation of FXI. Interestingly, A.C.C., and M.V. conducted the experiments and analyzed the data; FXIa has also been shown to proteolytically inactivate TFPI. It is S.P.G., N.M., A.M., A.S.W., A.C.C., M.V., S.H., J.J.P., H.M.S., S.A., D.G., and R.P. interpreted the data; S.P.G. and N.M. drafted the possible that loss of FXI in the placenta leads to increased levels of TFPI that further decrease FXa and thrombin generation in low-TF manuscript; S.P.G., N.M., A.S.W., A.C.C., S.H., H.M.S., S.A., R.P., 1/1 2/2 placentas. However, analysis of placentas from FXI and FXI and D.G. edited the manuscript; and all authors read and approved embryos revealed that loss of FXI resulted in a paradoxical decrease the manuscript before submission. in TFPI expression that would not explain the observed phenotype. Conflict-of-interest disclosure: N.M. and H.M.S. are consultants An alternative explanation for the increase in blood pooling in low- for Bayer. S.H. is an employee of Bayer. The remaining authors TF placentas lacking FXI is that FXIa regulates factors required for declare no competing financial interests. placenta development. 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