Limiting prothrombin activation to meizothrombin is compatible with survival but significantly alters hemostasis in mice

The activation of prothrombin (fII) is the penultimate step of hemostasis. Thrombin (fIIa) cleaves fibrinogen and directly activates platelets, via protease-activated receptors (PARs).¹  However, fIIa also controls its own production, via activation of factors V (fV), VIII (fVIII), XI (fXI), and protein C.²  Through these targets and others (eg, factor XIII [fXIII], thrombin-activatable fibrinolysis inhibitor), fIIa not only plays a pivotal role in hemostasis, but in other physiologic and pathologic processes (eg, development, inflammation, cancer biology).^3-7  Mouse studies have underlined the seminal importance of fII, because the genetic elimination of fII is not compatible with life.^3,4,8 

Prothrombin activation proceeds through 2 parallel activation pathways, depending on the site of first cleavage by the prothrombinase complex. One pathway features an intermediate that is not an active enzyme, prethrombin, whereas the second pathway is via an active enzyme precursor, meizothrombin (fIIa^MZ).^9-11  FIIa^MZ, as an active enzyme, is capable of participating in hemostasis and thrombosis, as well as other physiologic processes. FIIa^MZ, like fIIa, activates fV,¹²  fVIII,¹³  and fXI.¹⁴  Thus, fII^MZ may contribute to positive feedback of physiologic hemostasis. Both human and murine rfIIa^MZ demonstrate reduced fibrinogen cleavage capacity.^15,16  Further, rfIIa^MZ from both species demonstrates an increased activation potential for protein C, in the presence of thrombomodulin. Thus, fIIa^MZ has potentially distinct effects on hemostasis regulation from that of the mature fIIa enzyme. At least 2 described human mutations result in a partial limitation of the activation of prothrombin to meizothrombin. Both prothrombin-Dharhan (R271H)¹⁷  and prothrombin-Barcelona/Madrid (R271C)^18-21  limit fII activation to meizothrombin-like enzymes by removing a cleavage site. For each of the described pedigrees, individuals that were heterozygotes did not have a bleeding diathesis, whereas homozygotes had a mild to moderate bleeding tendency. Thus, although limitation of fII activation has a clear hemostatic effect, the broader biological impact of meizothrombin in vivo has not been defined. The fact that the spontaneous human mutations were viable in a homozygous state supports the feasibility for examining meizothrombin in a homozygous state in mice.

To better understand the biology of fII overall, and fIIa^MZ, specifically, we generated a mouse with knock-in targeted mutations in the endogenous F2 allele, resulting in 3 amino acid substitutions (R157A, R258A, and K281A) that lock fII after activation by factor Xa (fXa) to meizothrombin. These mutations in murine fII have previously been identified to limit activation to meizothrombin.¹⁶  The working hypothesis was that mice expressing a form of fII that is limited to activation to fIIa^MZ, would (1) be viable without spontaneous hemorrhage, (2) allow examination of the role of fIIa^MZ in hemostasis in vivo, and (3) provide a novel tool for study of the biology of prothrombin in physiologic and pathologic conditions.

For details of the generation of fII^MZ gene–targeted mice, please see the supplemental Methods, available on the Blood Web site. Mice that are heterozygous for the fII null allele (fII^+/−) and compound heterozygotes for the conditional fII allele and fII null (fII^lox/–) were previously described.⁸  In vivo and ex vivo (ie, prothrombin time [PT], activated partial thromboplastin time [aPTT], and quantitative polymerase chain reaction [qPCR]) experiments used outbred hybrid (129/Ola:C57/Bl6) littermate controls, except as noted using at least 6 generations–inbred C57/Bl6 animals. All experiments were approved by the Cincinnati Children’s Hospital Research Foundation Animal Care and Use Committee and complied with National Institutes of Health guidelines.

For details of the hematologic analysis, please see the supplemental Methods.

For the prothrombin activation assay, citrated platelet-poor plasma was diluted 1:10 with sterile Tris-buffered saline. The peptide Gly-Pro-Arg-Pro (EMD Millipore) was added to a final concentration of 4 mM to prevent fibrin polymerization. Thromboplastin (Diapharma), reconstituted in sterile water, was added to the plasma. Aliquots were taken from this solution and directly added to sodium dodecyl sulfate sample buffer to stop the reaction, at time points as indicated in Results. Immunoblots of these samples were performed under reducing conditions, using an antibody specific for the N-terminus of prothrombin (antigen was a peptide within the first 50 amino acids of prothrombin, sc-23340, Santa Cruz Biotechnology). Detection would yield, in decreasing size, intact prothrombin, F1.2.A, F1.2, or F1.

Please see the supplemental Methods.

Cohorts of fII^WT and fII^MZ mice were infused with genotype-controlled, fluorescently-labeled platelets. Anesthetized animals then had mesenteric arterioles isolated, and 10% topical ferric chloride solution was applied for 5 minutes. An observer blinded to genotype monitored vessels for the time to the first thrombus formation and the time until occlusion or for 30 minutes.

Please see the supplemental Methods.

Tissues were fixed in 10% buffered formalin (Sigma) and embedded in paraffin. Sections were cut and subsequently stained with hematoxylin and eosin (Sigma), Masson’s trichrome, or Prussian blue (Electron Microscopy Services). Photomicrographs were captured using an Axioplan 2 microscope (Zeiss) equipped with an AxiocamHR camera and software (Axiovision 5.6.3; Zeiss).

As described,²²  a single-cell suspension of 4 × 10⁵ GFP-expressing Lewis lung carcinoma cells (LLC^GFP) was injected IV in cohorts of C57/Bl6 fII^WT and fII^MZ mice in parallel. Fluorescent pulmonary metastatic foci were evaluated 14 days after injection. For B16 melanoma experimental metastases, a single-cell suspension was injected (8 × 10⁴ cells) IV into cohorts of C57/Bl6 fII^WT, fII^MZ, and fII^+/− mice in parallel, as previously described.²³  Nineteen days after injection, lungs were removed, stained overnight in picric acid solution (Sigma), and the metastatic foci counted.

All statistical analyses were generated using the Mann-Whitney U test, except as follows: the χ² test was used to analyze breeding data and Fisher’s exact test was used to analyze the tail bleeding times and percent occlusion in intravital microscopy experiments. Statistical analyses were performed on GraphPad Prism version 5.04 (GraphPad Software).

To determine the in vivo consequences of expressing a form of prothrombin incapable of activation beyond the intermediate, meizothrombin, we introduced 3 amino acid substitutions into the endogenous allele, to remove both a fXa cleavage site and potential fIIa auto-cleavage sites: R157A, R268A, and K281A (Figure 1A).²⁴  The R157A and R268A cleavage site mutations have been previously identified to limit murine prothrombin activation to meizothrombin.¹⁶  In addition to these 3 amino acid changes, we introduced a novel BamHI endonuclease site in Exon 7 and a novel EcoRI site in Exon 8, without further modification of the amino acid coding sequence. Founder mice transmitted the mutant allele through the germline to yield mice heterozygous for the mutation (fII^MZ/WT). Analyses of intercrosses of fII^MZ/WT mice resulting in >800 progeny revealed an ∼50% decrease in the numbers of homozygous mutant animals (hereafter referred to as fII^MZ) relative to what would be expected based on a 1:2:1 Mendelian ratio (Table 1). Further studies were conducted to confirm the timing of failure of the homozygous animals. Analyses of embryos harvested at embryonic day 18.5 (E18.5; ie, the day before birth) from fII^MZ/WT breeding pairs did not reveal a significant difference in relative numbers of fII^WT and fII^MZ embryos (Table 2). This suggests the loss of homozygous fII^MZ animals occurs in the early postnatal timeframe. Consistent with this conclusion, evidence of abdominal hemorrhage in fII^MZ pups was observed (supplemental Figure 1). Homozygous fII^MZ mice identified at weaning survived well into adulthood without excess mortality. Of the >200 adult fII^MZ mice generated to date, none were found to suffer overt hemorrhage. Furthermore, homozygous fII^MZ females were capable of carrying litters to term, with 23 C57Bl/6 homozygous fII^MZ females successfully carrying 52 litters to term.

Prothrombin expression levels were determined to confirm that homozygous fII^MZ neonates had normal levels of circulating fII protein. Total RNA, harvested from fII^WT and fII^MZ livers, revealed no significant difference in fII mRNA in either fII^WT or fII^MZ mice, whereas the expected decreases in mRNA levels for both fII^+/− and fII^lox/− control samples were observed (Figure 1B). Immunoblot analyses of plasma obtained from fII^WT and fII^MZ homozygous animals revealed no discernible differences in fII protein levels between genotypes (Figure 1C). Citrated plasma was also assayed for chromogenic fII activity, because it has been previously determined that both α-thrombin and meizothrombin have similar affinities for S-2238 chromogenic substrate.¹⁶  After activation of the fII in the plasma samples by ecarin, a similar level of chromogenic activity was observed between fII^WT and fII^MZ plasma samples (Figure 1D).

To confirm that the prothrombin generated from the fII^MZ animals was indeed limited to activation to meizothrombin, diluted plasma (1:10 with Tris-buffered saline) from fII^WT and fII^MZ animals was incubated with thromboplastin. Fibrin polymerization was blocked with the peptide Gly-Pro-Arg-Pro. Immunoblots (under reducing conditions) were performed using an antibody against an epitope at the N-terminus of the prothrombin molecule. Figure 2A details the expected fragments from fII activation in both fII^WT and fII^MZ plasma. Samples from fII^WT animals revealed a fragment consistent with activation to meizothrombin (fragment 1.2.A), which then migrated to a size consistent with fragment 1 over time (Figure 2B). However, in fII^MZ animals, no conversion beyond fIIa^MZ was detected (only fragment 1.2.A was present), even after incubation for 30 minutes at 37°C (Figure 2C).

Complete blood count analyses on blood from fII^WT and fII^MZ mice revealed no significant difference in the white blood cell (WBC) count, hemoglobin level, or platelet count (Table 3) between genotypes. In addition, analysis of the WBC differential revealed no significant difference between fII^WT and fII^MZ animals (data not shown) in terms of leukocyte subsets. Standard coagulation function analyses revealed a significant prolongation of both the PT and aPTT for fII^MZ animals compared with fII^WT animals (Table 3). This prolongation was expected given the known diminution of fIIa^MZ activity for fibrinogen. Predictably, there was no difference in the thrombin times between fII^WT and fII^MZ mice (Table 3), consistent with fII^MZ having no impact on plasma fibrinogen levels.

Comparative thrombin generation assays were performed on plasma from both fII^MZ and fII^WT mice (representative curves, Figure 3A-B). The fII^MZ animals had both a prolonged lag time compared with fII^WT (eg, a prolonged time to any [meizo]thrombin generation; Figure 3C) and a prolonged time to peak (meizo)thrombin production (Figure 3D). Furthermore, peak (meizo)thrombin production and area under the curve were found to be reduced in fII^MZ compared with fII^WT mice (Figure 3E-F, respectively). The Velocity Index, the rate of (meizo)thrombin generation, was also decreased in fII^MZ mice in contrast to fII^WT animals (data not shown).

To explore the contribution of fII^MZ toward activated protein C (aPC) generation, we challenged cohorts of fII^MZ and fII^WT mice with lipopolysaccharide (LPS), an inflammatory challenge known to activate the hemostatic cascade and result in aPC generation. Two hours after administration of LPS, fII^WT mice displayed the expected, and statistically significant, increase in aPC activity (supplemental Figure 2). The fII^MZ animals challenged with LPS did not display a significant difference in aPC activity compared with unchallenged fII^MZ animals. Interestingly, there was also not a statistically significant difference in aPC generation between the fII^MZ and fII^WT mice after LPS. In light of the data regarding reduced meizothrombin generation in fII^MZ animals, these data do not necessarily contradict the previously reported findings regarding activity of fIIa^MZ for protein C; this finding would also be expected with diminished overall fIIa^MZ activity secondary to diminished generation of fIIa^MZ from fII^MZ.

FIIa is a potent activator of platelet aggregation through activation of PARs.¹  To determine the potential of fIIa^MZ to activate platelets, we initiated fIIa or fIIa^MZ generation in platelet-rich plasma (PRP) derived from fII^WT and fII^MZ animals. Qualitative platelet aggregation was comparable between genotypes, with similar total aggregation (Figure 4A-B; fII^WT n = 7, fII^MZ n = 7). However, there was a prolonged time to both shape change and positive deflection in fII^MZ animals (Figure 4C-D). We postulate that the prolonged time to initiation of platelet aggregation in fII^MZ plasma was a function of the extended time to (meizo)thrombin generation. More notably, these data suggest that murine fIIa^MZ is capable of activating PAR-4.

Clot retraction is an important physiologic step in both hemostasis and wound repair that is dependent on fibrin polymer formation, platelet activation, and fXIII activity.^25-27  To directly determine whether fII^MZ supports clot retraction, we initiated coagulation with thromboplastin in both whole blood and PRP (platelet counts adjusted to 2.5 × 10⁵ platelets/µL) from fII^WT and fII^MZ animals. Although time to initial clot retraction was modestly delayed in fII^MZ blood and plasma samples, both fII^WT and fII^MZ displayed evidence of similar maximal clot retraction in both whole blood and PRP (supplemental Figure 3A-B,E-F). There was no qualitative difference in the size of clots by the end of the 90-minute observation period. Further, red blood cell (RBC) inclusion in thrombi was not significantly different between samples from fII^WT and fII^MZ mice (supplemental Figure 3C-D).

To determine the effect of limiting fII activation to meizothrombin on hemostasis in vivo, we employed a standard tail tip amputation assay. Cohorts of fII^MZ and fII^WT animals were challenged by amputation of 3 mm of the distal tail. All fII^WT mice had cessation of bleeding, with a mean time to cessation of 72 ± 6 seconds. However, all fII^MZ animals had bleeding for the entire 10-minute observation period (Figure 5A). Qualitative narrowing of the caliber of the bloodstream from the amputation site was sometimes observed in fII^MZ animals, but bleeding universally persisted throughout the observation period.

To define thrombotic potential in fII^MZ mice, we challenged fII^MZ and fII^WT mice in parallel with a ferric chloride mesenteric artery injury. Here, fluorescein-labeled platelets from donor mice of the same genotype as the challenged animals were infused before the procedure to allow real-time tracking of thrombus size and time to occlusion by intravital fluorescent microscopy. FII^WT mice demonstrated rapid formation of platelet aggregates that progressed steadily to full occlusion (Figure 5B). Although fII^MZ animals had clear evidence of platelet aggregation (Figure 5C), fII^MZ mice demonstrated a significantly increased time to first thrombus formation and prolonged time to occlusion relative to fII^WT mice, with a significant fraction of fII^MZ mice failing to form a stable occlusive thrombus during the 30-minute observation period (Figures 5D-E). In evaluation of the formation of thrombosis in the fII^MZ animals, the thrombi formed after ferric chloride injury was unstable with frequent emboli. In mice with no occlusive thrombi, a combination of delayed thrombus formation and embolization contributed to this phenotype (supplemental Video 1 [fII^WT animals] and supplemental Video 2 [fII^MZ animals]). Taken together with the platelet aggregation data, these data suggest the hypothesis that diminished fibrin polymer formation in fIIa^MZ mice results in a failure to stabilize the growing platelet aggregate and a corresponding protection from arterial occlusion.

Mice with genetic deficiencies of tissue factor, fII, fVII, fX, and fXIII^8,28-31  develop cardiac hemorrhage with iron deposition and subsequent fibrosis that progresses with age. To investigate whether fII^MZ predisposes adult animals to spontaneous hemorrhage, we examined organs from cohorts (10-12 weeks of age) of C57/Bl6 fII^WT and fII^MZ mice. No gross or microscopic evidence of hemorrhage was observed in the pulmonary, gastrointestinal, genitourinary, or central nervous systems of fII^WT or fII^MZ animals. As expected, fII^WT mice did not show any histologic evidence of cardiac pathologies (Figure 6A-C). In contrast, cardiac fibrosis was readily identified in both male and female fII^MZ mice (Figure 6D-E) and was most notable in the subepicardial zones. Prussian blue staining of the heart tissue revealed colocalization of iron deposits with areas of fibrosis (Figure 6F), suggesting chronic hemorrhage leading to the etiology of fibrosis in this setting.

In addition, cohorts of inbred C57/Bl6 fII^WT and fII^MZ mice were aged for a year before their organs were harvested. Notably, no loss of fII^MZ animals occurred during this time. Grossly, evidence of one ovarian hemorrhagic cyst and one intestinal hemorrhage were noted in the fII^MZ (n = 8) animals. There was no evidence of gross or microscopic hemorrhage in the fII^WT (n = 7) mice (supplemental Figure 4A-C). Abundant cardiac fibrosis was noted in both male and female fII^MZ animals (supplemental Figure 4D-E), whereas minimal, if any, cardiac fibrosis was noted in the fII^WT mice. Again, sites of fibrosis colocalized with sites of iron deposition (supplemental Figure 4F), likely as a result of local hemorrhage. There were no hemorrhagic findings in other organ systems.

To investigate the biological consequences of fII^MZ activity in a disease context distinct from a classical hemostasis or thrombosis challenge, we examined the contribution of fII^MZ to experimental tumor metastasis. FIIa can support metastasis through tumor cell–instrinsic mechanisms involving activation of PARs,³²  as well as through fibrinogen cleavage and platelet activation.^23,33,34  Our hypothesis was that fII^MZ animals would demonstrate reduced numbers of metastases. Cohorts of inbred C57Bl/6-derived fII^MZ and fII^WT mice were IV injected in parallel with LLC^GFP cells and the lungs harvested 14 days later. Although abundant metastases were uniformly present among the fII^WT animals, few, if any, fII^MZ animals had any evidence of metastatic foci (Figure 7A-C). This was similar to previous findings of LLC experimental lung metastases in animals with lowered levels of prothrombin.³⁵  To expand on these findings and further compare fII^MZ with diminished levels of prothrombin, we compared experimental metastases of B16 melanoma in fII^WT, fII^MZ, and fII^+/− animals. Again, fII^MZ animals demonstrated a significant protection from the development of lung metastases (Figure 7D,F-G). Evaluation of tumor metastases in mice with 50% prothrombin (fII^+/−) revealed an essentially identical pattern to fII^MZ animals (Figure 7E). This suggests that even modest diminutions in thrombin generation potential, either through diminution of circulating prothrombin levels or alteration of activation, have profound effects on metastatic potential of tumor cells.

Activation of thrombin follows 1 of 2 pathways, which produce functionally different intermediate species. The intermediate of the alternative prothrombin activation pathway prethrombin, lacks detectable protease activity. In contrast, meizothrombin, a short-lived activation intermediate of prothrombin,^9,11  has readily detectable protease activity with notably distinct substrate specificities from α-thrombin. The pathway of thrombin activation is dictated in part by the microenvironment or cellular surface on which the prothrombinase complex is assembled. Recent data have suggested that thrombin generation on platelets primarily proceeds via prethrombin,³⁶  whereas thrombin generation on the surface of RBCs tends to occur through meizothrombin.³⁷  Although it has been hypothesized that the pathway of fII activation is biologically relevant, given the short half-life of fIIa^MZ, the specific contribution of fIIa^MZ-mediated proteolysis to hemostasis and other physiologic or pathologic processes has been difficult to assess in vivo. Here, we demonstrate for the first time the consequences of limiting the activation of endogenous fII to an activation intermediate in vivo. Mice that express fII^MZ are viable, survive well into adulthood, and have reproductive success. FII^MZ mice have partial perinatal lethality, unlike fII^lox/− animals (mice with 10% of wild-type prothrombin levels). Similar to fII^lox/− animals, fII^MZ animals that survive to weaning have an essentially normal lifespan. FII^MZ animals have significantly delayed and decreased (meizo)thrombin generation, compared with fII^WT mice. FII^MZ animals also have delayed time to occlusion after a ferric chloride arterial injury and prolonged bleeding times.

Although we did not directly measure factor XIII (fXIII) activation by fII^MZ, indirect evidence points to at least adequate activation. Clot retraction in fII^MZ plasma was normal, both in PRP and in whole blood. FXIII activity is required in the process of clot retraction and RBC retention within the thrombus.^25-27  As additional evidence that fII^MZ activates fXIII, RBC retention in whole-blood thrombi after clot retraction was not significantly different between fII^WT and fII^MZ animals. Similarly, PAR-1 activation by fII^MZ has not been directly measured. However, the homozygous fII^MZ animals do not have the midgestation loss seen in PAR-1⁻³⁸  and fII⁻ animals,^3,4  which would suggest that activation of PAR-1 by fIIa^MZ is adequate to support successful embryonic development.

Both human and murine rfIIa^MZ have decreased polymerization potential of fibrin.^15,16  Our findings with in vivo fII^MZ are compatible with these results. Prolongation of the PT and aPTT are both suggestive of a decreased polymerization of fibrin (the end point of both assays). Although delayed, fIIa^MZ also induces platelet aggregation qualitatively similar to α-thrombin. However, fII^MZ animals were unable to achieve hemostasis after a standard tail amputation bleeding time. This is in contrast to animals expressing a form of fibrinogen that cannot be cleaved by thrombin (Fib^AEK). Fib^AEK mice demonstrated partial cessation of bleeding in a standard tail amputation bleeding time.³⁹  This suggests that, although inadequate fibrin polymerization contributes to abnormal hemostasis in fII^MZ mice, positive feedback for self-activation also likely participates in the failure to achieve hemostasis.

Intravital microscopy, after mesenteric arteriole ferric chloride injury, also reveals insight into the failure to form thrombi. Although fII^MZ animals formed platelet aggregates at the site of vessel injury, these aggregates were not stable. The aggregates formed after ferric chloride injury in the fII^WT controls quickly evolved into occlusive thrombi. In stark contrast, fully occlusive thrombi never formed in fII^MZ mice, rather smaller aggregates embolized from the initial thrombus in fII^MZ animals precluding occlusion. Taken together, this suggests that, in the setting of animals expressing only fII^MZ, platelet aggregation is adequate, but insufficient positive feedback to the hemostatic cascade and diminished fibrinogen cleavage lead to unstable platelet aggregates. Thus, a lack of both physiologic hemostasis and diminished thrombosis is observed in fII^MZ mice.

Previous studies have reported the effects of recombinant human and murine meizothrombin (rhfIIa^MZ and rmfIIa^MZ, respectively), both in vitro and in vivo.^16,40-42  Similar to our current work, rmfIIa^MZ demonstrated significantly diminished fibrinogen cleavage capabilities in vitro. However, in the same in vitro studies of rmfIIa^MZ, increased protein C activation in the presence of thrombomodulin was observed.¹⁶  Our data suggest that the activation of protein C is not significantly different in the fII^MZ and fII^WT animals after an LPS challenge. Direct comparison between the in vitro findings and the in vivo analysis of aPC generation reported here is complicated by the overall reduced activation potential of meizothrombin in the homozygous mutant mice. Thus, we cannot further address the activity of fIIa^MZ for protein C. The other significant difference in the published account of rmfIIa^MZ and our current findings using knock-in animals is that rmfIIa^MZ was evaluated not as a zymogen, but as a fully active enzyme. Here, we were able to explore the consequences of limiting all activation of fII to meizothrombin. Although in vitro data suggested that rhfIIa^MZ would be capable of positive feedback of the hemostatic cascade, our data in murine meizothrombin suggest otherwise.^12,13  Further studies will be needed to assess the in vivo activation of factors V, VIII, and XI by meizothrombin. Because (meizo)thrombin generation is significantly altered in the fII^MZ compared with fII^WT animals, this suggests meizothrombin is less effective at supporting activation of at least one of these factors. Our data challenges the hypothesis that fIIa^MZ contributes to physiologic hemostasis.

Another modification of fII leading to a specificity difference, fII^WE, has diminished fibrinogen cleavage while preserving activation of protein C.^43,44  Exploiting this difference in specificities, fIIa^WE has been administered pharmaceutically as an anticoagulant^43,45  and antiinflammatory⁴⁶  agent. In addition, animals have been generated that express only the fII^WE zymogen.⁵  However, unlike the fII^MZ animals, mice expressing only fII^WE are not viable. The difference in viability between fII^WE and fII^MZ animals illustrates that fII^MZ does not affect physiologic hemostasis to the same degree. Further, fII^MZ animals may have an advantage in studying fII specificity alterations in physiologic or disease states.

Animals expressing fII^MZ developed cardiac fibrosis, worsening with age, similar to mice with low levels of tissue factor, fII, fVII, fX, and fXIII.^8,28-31  Areas of fibrosis colocalized with areas of iron deposition, likely related to recurrent small hemorrhages. Unlike mice with low levels of fII (both fII^lox/− mice and transgenic mice expressing low levels of human prothrombin) that only develop modest cardiac fibrosis at over one year of age,⁸  fII^MZ mice developed substantial cardiac fibrosis by 8 to 10 weeks of age. Also unlike previous accounts of cardiac fibrosis hemostatic factor deficiencies, we did not observe a gender predisposition to the development of cardiac fibrosis. Thus, although (meizo)thrombin-induced platelet aggregation is preserved, this was not sufficient to prevent the cardiac parenchymal bleeding that leads to cardiac fibrosis. That this bleeding is limited to the heart, as opposed to bleeding patterns seen in the low tissue factor and low fVII animals, suggests tissue differences in hemostasis requirements to prevent hemorrhagic complications.⁴⁷ 

As proof of concept that fII^MZ animals will provide a novel tool to study the biology of fII in disease, we examined the effect of limitation of the activation of fII in the setting of tumor metastasis. We found that mice with fII^MZ have dramatically reduced experimental metastasis compared with fII^WT animals. This was not necessarily a predictable result, because fibrin(ogen) and platelets both contribute to early protection of metastatic foci via protection from natural killer cells.^23,33  FII^MZ retains, albeit reduced, fibrinogen cleavage potential, and essentially normal platelet activation capacity. That mice with fII^MZ had very few metastases is most likely related to the low positive feedback in generation of (meizo)thrombin combined with diminished fibrinogen cleavage and polymerization and a delay in platelet activation. However, other possible mechanisms cannot be excluded, such as diminished PAR activation in either tumor cells or nonmalignant stromal cells. Interestingly, fII^+/− animals displayed a similar phenotype in tumor metastasis. FII^+/− animals have ∼75% of peak thrombin generation seen in fII^+/+ mice.⁵  Protection from metastases in fII^MZ and fII^+/− underscores that tumor metastasis is quite sensitive to derangements in thrombin generation.

The current studies demonstrate that mice expressing only fII^MZ have adequate hemostasis for successful survival and reproduction and that these animals provide a unique tool to further assess the role of both prothrombin and meizothrombin in physiologic and pathologic processes. Because coagulation factors are more closely tied to nonhemostatic processes, the ability to study altered enzyme specificity in vivo will allow further elucidation of the mechanistic contribution of fII to disease. Tools, such as fII^MZ animals, will be crucial to determine how the activation and activity of fII modify disease severity.

The authors acknowledge Russell Ware for his insightful comments, and thank Gregory Adams, Whitney Miller, Malinda Frederick, Leah Rosenfeldt, Carolina Cruz, and Cheryl Rewerts for their excellent technical assistance.

This study was supported by National Institutes of Health, National Heart, Lung and Blood Institute grants K08HL105672 (E.S.M.) and R01HL102101 (D.D.W.).

Contribution: E.S.M., M.A.S., M.J.F., J.S.P., N.L.E., C.T.E., D.D.W., and J.L.D. designed research; M.A.S., K.W.K., K.E.M., D.R.S., M.C., A.B., and E.S.M. performed research; E.S.M., M.A.S., N.L.E., C.T.E., D.D.W., and J.L.D analyzed data; and E.S.M., M.A.S., M.J.F., and J.S.P. wrote the manuscript.

Conflict-of-interest disclosure: E.S.M. has served on an advisory board for US WorldMeds and received honoraria from Baxalta for matters unrelated to this research. J.S.P. has served on an advisory board for US WorldMeds on matters unrelated to this research. The remaining declare no competing financial interests.

Correspondence: Eric S. Mullins, Division of Hematology, Cancer and Blood Diseases Institute, Cincinnati Children’s Research Foundation, MLC 7015, 3333 Burnet Ave, Cincinnati, OH 45229-3039; e-mail: eric.mullins@cchmc.org.