Tissue Factor Deficiency Causes Cardiac Fibrosis and Left Ventricular Dysfunction

Tissue factor deficiency causes cardiac fibrosis and left ventricular dysfunction

R. Pawlinski*, A. Fernandes*, B. Kehrle*, B. Pedersen*, G. Parry*, J. Erlich†, R. Pyo‡, D. Gutstein‡, J. Zhang‡, F. Castellinoʈ, E. Melis§, P. Carmeliet§, G. Baretton¶, T. Luther¶, M. Taubman‡, E. Rosenʈ, and N. Mackman*,**

*Department of Immunology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037; †Department of Nephrology, University of New South Wales, Prince of Wales Hospital, High Street, Sydney 2052, Australia; ‡Department of Medicine and Molecular Biology, Mount Sinai Medical Center, One Gustave L. Levy Place, Box 1269, New York, NY 10029; §Center for Transgene Technology and Gene Therapy, Flanders Interuniversity Institute for Biotechnology (VIB), KU Leuven, B-3000 Leuven, Belgium; ¶Institut fu¨r Pathologie, Universita¨tsklinikum Carl Gustav Carus der Technischen Universita¨t, 01307 Dresden, Germany; and ʈDepartment of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556

Edited by Philip W. Majerus, Washington University School of Medicine, St. Louis, MO, and approved October 4, 2002 (received for review August 20, 2002) Exposure of blood to tissue factor (TF) activates the extrinsic Materials and Methods (TF:FVIIa) and intrinsic (FVIIIa:FIXa) pathways of coagulation. In this Mice. Low-TF mice (line 47) were analyzed on either a mixed Ϸ study, we found that mice expressing low levels of human TF ( 1% genetic background (62.5% C57BL͞6J, 25% 129Sv, and 12.5% ؊/؊ of wild-type levels) in an mTF background had significantly BALB͞c) or a C57BL͞6J background (Ն97%). mTF⌬cyt/⌬cyt shorter lifespans than wild-type mice, in part, because of sponta- mice express normal levels of murine TF lacking the cytoplasmic neous fatal hemorrhages. All low-TF mice exhibited a selective domain (17). Low murine FVII mice (FVIItTA-FVII/tTA-FVII) were heart defect that consisted of hemosiderin deposition and fibrosis. generated by replacing the entire FVII gene with a transgene Direct intracardiac measurement demonstrated a 30% reduction expressing FVII under the control of a tTA responsive promoter (P < 0.001) in left ventricular function in 8-month-old low-TF (E.R., Z. Liang, A. Martin, and F.C., unpublished data). FbgϪ/Ϫ mice compared with age-matched wild-type mice. Mice expressing Ϫ Ϫ and FIX / mice have been described (11, 18). All studies were low levels of murine FVII (Ϸ1% of wild-type levels) exhibited a approved by The Scripps Research Institute Animal Care and similar pattern of hemosiderin deposition and fibrosis in their Use Committee and comply with National Institutes of Health hearts. In contrast, FIX؊/؊ mice, a model of hemophilia B, had guidelines. normal hearts. Cardiac fibrosis in low-TF and low-FVII mice appears to be caused by hemorrhage from cardiac vessels due to impaired Measurement of Cell Counts and Clotting Activity. White cell, red hemostasis. We propose that TF expression by cardiac myocytes cell and platelet counts, and hemoglobin and hematocrit levels provides a secondary hemostatic barrier to protect the heart from were determined by LabCorp (San Diego), using blood collected hemorrhage. from the inferior vena cava. Activated partial thromboplastin times (APTTs) and prothrombin times (PTs) were performed xpression of tissue factor (TF) by adventitial fibroblasts and using Automated APTT Reagent and Thromboplastin Reagent BIOCHEMISTRY Evascular smooth muscle cells surrounding blood vessels (Organon Teknika), respectively, and clotting times determined provides a hemostatic barrier that activates coagulation when using a START4 Coagulation Analyzer (Diagnostica Stago, vascular integrity is disrupted (1). TF is also expressed by cardiac Parsippany, NJ). Levels of TAT in the plasma were determined muscle but not by skeletal muscle (1). TF functions as the ͞ using a commercial ELISA (Enzygnost, Dade Behring, Marburg, high-affinity cellular receptor for FVII VIIa (2). The coagula- Germany). The procoagulant activity of heart tissue extracts tion protease cascades are comprised of the extrinsic (TF:FVIIa) added to mouse plasma was determined using a one-stage and intrinsic (FVIIIa:FIXa) pathways, which together maintain clotting assay as described (13) and converted to activity units by hemostasis (3). comparison to a standard curve generated using mouse brain Many murine models of coagulation have been generated that thromboplastin. Factor VII assays were performed using a provide new insights into the role of the various procoagulant modification of the Coaset FVII assay (Chromogenix, Milan). A and anticoagulant proteins in hemostasis (4). For instance, standard curve was generated by combining wild-type mouse FVLeiden/Leiden mice, which express an FV variant that is resistant plasma with human FX-deficient plasma at varying ratios. to inactivation by activated protein C, and TMPro/Pro mice, which express a mutated version of thrombomodulin (TM) with re- Histology. Tissue sections were stained with hematoxylin͞eosin duced thrombin binding, both exhibit prothrombotic phenotypes (H&E), Prussian Blue, or Masson’s Trichrome. Macrophages with increased fibrin deposition in select tissues (5–7). Mice with were identified with a monoclonal antibody MOMA-2 (1:1,000; prohemorrhage phenotypes include models of hemophilia A (FVIIIϪ/Ϫ) and B (FIXϪ/Ϫ), as well as fibrinogen-deficient mice Serotec; ref. 20). (FbgϪ/Ϫ) and thrombocytopenic mice (NF-E2Ϫ/Ϫ) (8–12). Mice Measurement of Left Ventricular (LV) Function of the Hearts of Low-TF with complete deficiencies in TF, FVII, FX, FV, and prothrom- Mice. bin die in utero or shortly after birth (4). We and others have LV function of hearts of low-TF mice (8 months of age) and Ͻ age-matched C57BL͞6J mice was measured as described (20). generated mice expressing low levels ( 0.1–1% of wild-type ͞ levels) of human TF, murine FVII, and murine FV (13–15). We LV function (dp dt) and LV systolic pressures were obtained at have shown that low-TF mice have impaired uterine hemostasis a constant heart rate range of 480–510. The heart rate was (16). A similar phenotype is observed with low-FVII mice. decreased by increasing the level of isoflurane anesthesia. In this study, we performed a detailed characterization of

low-TF mice. These mice exhibited shorter lifespans than wild- This paper was submitted directly (Track II) to the PNAS ofﬁce. type mice. Histological analysis of various tissues of low-TF mice Abbreviations: Fbg, ﬁbrinogen; H&E, hematoxylin͞eosin; LV, left ventricular; TF, tissue revealed hemosiderin deposition and fibrosis selectively in their factor. hearts. Our data suggest that cardiac fibrosis in low-TF mice is **To whom correspondence should be addressed at: Departments of Immunology and Cell caused by hemorrhage from cardiac vessels due to impaired Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, C-204, La Jolla, hemostasis. CA 92037. E-mail: [email protected].

www.pnas.org͞cgi͞doi͞10.1073͞pnas.242501899 PNAS ͉ November 26, 2002 ͉ vol. 99 ͉ no. 24 ͉ 15333–15338 Downloaded by guest on September 30, 2021 Table 1. Clotting activity in low-TF mice Genotype mTFϩ/ϩ mTFϩ/Ϫ͞hTFϩ mTFϪ/Ϫ͞hTFϩ

Heart PCA, arbitrary units 232 Ϯ 80 (3) 98 Ϯ 80 (3) 0.2 Ϯ 0.2 (3) PT, s 10.47 Ϯ 0.15 (3) 10.48 Ϯ 0.05 (4) 10.53 Ϯ 0.32 (4) APTT, s 26.37 Ϯ 0.90 (3) 29.85 Ϯ 0.76 (4) 31.53 Ϯ 0.90 (4) TAT, ng͞ml 1.78 Ϯ 0.44 (3) 0.74 Ϯ 0.28 (5) 0.04 Ϯ 0.02 (5)

PT, prothrombin time; APTT, activated partial thromboplastin time; PCA, procoagulant activity. Numbers in parentheses indicate the number of mice used in each analysis.

Data Analysis. Statistical analysis was performed using a two- and then became widespread throughout the myocardium tailed unpaired Student’s t test, and differences were determined (Fig. 2B). to be statistically significant at a P value of Ͻ0.05. Decreased LV Function in the Hearts of Low-TF Mice. Hemodynamic Results studies revealed that low-TF mice had a marked impairment of Characterization of Low-TF Mice. Low-TF mice (mTFϪ/Ϫ͞hTFϩ heart contractility manifested by a significant decrease in dp͞dt line 47) contain a minigene (hTF) that directs cell type-specific at every heart rate examined (Fig. 3). At normal mouse heart expression of human TF that is similar to the expression of rates (480–510 beats per min), the dp͞dt and the LV pressure murine TF (13). Immunohistochemical analysis indicated that were decreased by 30% (P Ͻ 0.001). These results indicate that human TF was expressed by adventitial cells surrounding blood the cardiac fibrosis in the hearts of low-TF mice significantly vessels (data not shown). However, quantitation of the proco- impairs LV function. agulant activity of tissue extracts from various tissues indicated that these mice expressed low levels of TF (Ϸ1% of wild-type Detailed Histological Analysis of the Hearts of Low-TF Mice. We levels; ref. 13). For example, the procoagulant activity of tissue examined hearts from Ͼ100 low-TF mice at different ages. We extracts from hearts of low-TF mice was very low compared with observed golden-brown granular deposits in the myocardium in control mice (Table 1), indicating a deficiency of TF in the heart. tissue sections stained with H&E (Fig. 4A). We suspected that We evaluated the clotting activity and response to hemostatic these deposits were hemosiderin, which is an insoluble protein challenge of low-TF mice (4–8 weeks of age) on a C57BL͞6J produced by phagocyte digestion of hematin. We used Prussian background. No significant differences were found in whole Blue staining to confirm the presence of iron in the deposits in blood samples collected from low-TF mice (n ϭ 5) and serial sections (Fig. 4B). Immunohistochemical studies showed ϩ Ϫ ϩ mTF / ͞hTF (n ϭ 5) littermate mice with regard to platelet, that the hemosiderin-laden cells stained with MOMA-2 (not red cell, and white cells counts, and hematocrit and hemoglobin shown), which specifically recognizes macrophages (19). Hemo- (not shown). In addition, low-TF mice had APTTs and PTs that siderin deposits were associated with areas of fibrosis (Fig. 4C). were similar to control mice (Table 1). These results indicate that No hemosiderin was seen in 1-month-old mice (not shown). In the low levels of TF in these mice maintain hemostasis under 3-month-old mice, hemosiderin was observed in the subepicar- normal conditions. In contrast, low-TF mice exhibited an ab- dium and within the myocardium (Fig. 5A; see also Fig. 6A). In normal response to hemostatic challenge. Low-TF mice showed older mice (8 months), hemosiderin was observed throughout a significantly prolonged occlusion time (99 Ϯ 40 min, mean Ϯ the myocardium selectively deposited around capillaries (Fig. SD, n ϭ 7; P Ͻ 0.001) compared with C57BL͞6J mice (44 Ϯ 18 5B) and larger vessels (not shown). No hemosiderin was ob- min, n ϭ 9) in a Rose Bengal model of carotid artery injury (S. served in the hearts of wild-type C57BL͞6J mice (not shown). Day, J. Reeve, B.P., N.M., and W. Fay, unpublished data). We Hemosiderin in the heart is most likely derived from eryth- also observed that low-TF mice have low levels of circulating rocytes that have hemorrhaged into the myocardium. Indeed, we ϩ Ϫ ϩ TAT complexes compared with mTF / ͞hTF littermate mice identified interstitial hemorrhages in the hearts of low-TF mice and wild-type C57BL͞6J mice (Table 1), indicating that low-TF (Fig. 5 C and D), and these hemorrhages were often associated mice generate lower levels of thrombin. Taken together, these results suggest that low-TF mice would be prone to excessive hemorrhage in the event of vessel injury.

Survival of Low-TF Mice. In initial studies, we observed that low-TF mice on a mixed genetic background had shorter lifespans (Fig. 1) compared with C57BL͞6J mice (21). Low-TF mice had even shorter lifespans on a C57BL͞6J background compared with a mixed background (Fig. 1). Autopsies revealed that 17% of the low-TF mice died of spontaneous hemorrhages in the brain, lung, and gastrointestinal tract (not shown). Although we cannot exclude the possibility that we have underestimated the death rate due to acute hemorrhage, it appears that the majority of low-TF mice are dying prematurely of another cause.

Fibrosis in the Hearts of Low-TF Mice. Histological examination of the major organs (brain, lung, heart, liver, kidney, and spleen) of low-TF mice revealed fibrosis in the heart but no defects in the other organs. Cardiac fibrosis increased with age, with the rate of fibrosis being faster in the hearts of low-TF mice on the Fig. 1. Survival of low-TF mice on mixed and C57BL͞6J backgrounds. Kaplan– C57BL͞6J background compared with the mixed background Meier plots showing survival proﬁles of 77 low-TF mice on a mixed background (Fig. 2A). Fibrosis was initially observed in the subepicardium (Mixed) and 61 low-TF mice on a C57BL͞6J background (C57).

15334 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.242501899 Pawlinski et al. Downloaded by guest on September 30, 2021 Fig. 2. Fibrosis in hearts of low-TF mice. (A) The degree of fibrosis was scored (0–5) in heart sections stained with Masson’s Trichrome. Fibrosis was scored in hearts from 35 mice on a mixed background and 31 hearts on a C57BL͞6J background. (B) Cross-sections of hearts stained with Masson’s Trichrome demonstrate subepicardial and myocardial fibrosis in the hearts of low-TF mice (8 and 23 months) and no fibrosis in control mice (8 months). Normal myocardium stains red-brown and fibrotic tissue stains blue. Original magnification of the panels from the cross-sections was ϫ100. Hearts shown are from low-TF mice on a mixed genetic background. LV, left ventricle. BIOCHEMISTRY with cardiac myocyte necrosis (Fig. 5D). In other hearts, we the mTFϪ/Ϫ͞hTFϩ(line 31) mice succumb to fatal hemorrhages observed infiltration of leukocytes into the myocardium (Fig. in the brain and lung. Fig. 6 A and B shows hemosiderin 5E). Fibrosis and hemosiderin were seen throughout the hearts deposition in hearts of mTFϪ/Ϫ͞hTFϩ(line 47) and mTFϪ/Ϫ͞ of older low-TF mice (Fig. 5F). These results suggest that hTFϩ(line 31) mice, respectively. These results indicate that an hemosiderin deposition is derived from erythrocytes hemorrhag- independent line of low-TF mice have hemosiderin deposition in ing into the myocardium. their hearts.

Hemosiderin Deposition in Hearts of an Independent Line of Low-TF Analysis of Mice Expressing TF Lacking the Cytoplasmic Domain. We Mice. Previously, we demonstrated that we could rescue mTFϪ/Ϫ have shown that TF is located within the specialized adhesion embryos with two independent transgenic lines that express low junctions (intercalated discs) between cardiac myocytes (22), levels (line 47) and very low levels (line 31) of human TF (13, 14). suggesting that TF may contribute to the structural integrity of Indeed, line 47 provided long-term survival of mTFϪ/Ϫ͞ the myocardium. In addition, the TF cytoplasmic domain has hTFϩ(line 47) mice (see Fig. 1), whereas all mTFϪ/Ϫ͞hTFϩ(line been shown to bind to the cytoskeleton via actin binding protein 31) mice die within 8 weeks of birth (14). The majority (77%) of

Fig. 3. LV function in the hearts of low-TF mice. LV function was performed Fig. 4. Hemosiderin deposits in the hearts of low-TF mice. Serial sections of on 8-month-old low-TF mice (n ϭ 6) on a C57BL͞6J background and age- a low-TF mouse on a mixed background (8 months of age) were stained with matched C57BL͞6J mice (n ϭ 5). (Left) LV function was measured at different H&E (A), Prussian Blue (B), and Masson’s Trichrome (C). Hemosiderin appears heart rates. (Center and Right) LV function and LV pressure at normal heart as a golden-brown deposit with H&E and stains blue with Prussian Blue. rates (480–510 beats per min). (Original magniﬁcation ϫ400.)

Pawlinski et al. PNAS ͉ November 26, 2002 ͉ vol. 99 ͉ no. 24 ͉ 15335 Downloaded by guest on September 30, 2021 Fig. 5. Histological analysis of the hearts of low-TF mice. Heart sections were stained with H&E. (Original magniﬁcations: A, ϫ100; B, ϫ1,000; C, ϫ250; D–F, ϫ400.) Hearts of low-TF mice (line 47) at 3 months (A), 5 months (C), 8 months (B and D), 11 months (F), and 14 months (E) of age are shown. Hemosiderin (brown) is observed subepicardially (A) and perivascularly (B). (C and D) Interstitial hemorrhages and necrosis of a cardiac myocyte (nuclear pyknosis and loss of cross striations; arrow; D). (E) Leukocyte inﬁltration and cardiac myocyte necrosis. (F) Fibrosis (light pink) and associated hemosiderin deposition with some remaining cardiac myocytes (dark pink). Hearts shown are from low-TF mice on a mixed background. Blood vessels (bv), epicar- dium (epi), and hemosiderin (arrowheads) are indicated.

280 (23). We tested the hypothesis that TF plays a structural role hemostasis. No hemosiderin or fibrosis was observed in in the heart via interaction of the TF cytoplasmic domain with 6-month-old mTF⌬cyt/⌬cyt mice (not shown), indicating that de- the cytoskeleton by examining the hearts of mTF⌬cyt/⌬cyt mice letion of the TF cytoplasmic domain does not result in hemo- that express normal levels (100%) of murine TF lacking the siderin deposition in the heart. cytoplasmic domain (17). Importantly, these mice have normal Hemosiderin Deposition in Hearts of Low-FVII Mice. Low-FVII mice were generated by replacing the FVII gene with a transgene that expresses FVII under the control of a tTA-responsive promoter. These mice express low levels of FVII (Ͻ1% of wild-type levels) and 50% of these mice die within 45 days of birth mostly because of brain hemorrhages (E.R., unpublished data). We observed hemosiderin deposition and fibrosis in the hearts of low-FVII mice (Fig. 6 C and D). Interstitial hemorrhages were also observed in the hearts of low-FVII mice (not shown). Thus, the cardiac phenotype of low-FVII mice was remarkably similar to that of low-TF mice.

Analysis of Hearts of FIX؊/؊ and Fbg؊/؊ Mice. We analyzed the hearts of FIXϪ/Ϫ and FbgϪ/Ϫ mice to determine whether hemosiderin deposition in the heart was a common phenotype in prohemorrhagic mice. We did not observe hemosiderin in the hearts of either FIXϪ/Ϫ (13 weeks of age) or FbgϪ/Ϫ (6 months of age) mice (not shown). These results indicate that hemosiderin in the heart is selective for deficiencies in the extrinsic pathway of coagulation. Discussion Fig. 6. Analysis of hearts of mice with deﬁciencies in the extrinsic pathway of coagulation. Heart sections were stained with Prussian Blue (A–C)or We used low-TF mice to test the hypothesis that TF expression Masson’s Trichrome (D). Shown are heart sections from an mTFϪ/Ϫ͞hTFϩ(line by cardiac myocytes contributes to hemostasis in the heart. 47) mouse (12 weeks of age; A), an mTFϪ/Ϫ͞hTFϩ(line 31) mouse (8 weeks of Low-TF mice had shortened lifespans compared with wild-type age; B), and an FVIItTA-FVII/tTA-FVII mouse (9 weeks of age; C and D). mice, in part, because of spontaneous hemorrhages. However,

15336 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.242501899 Pawlinski et al. Downloaded by guest on September 30, 2021 fatal hemorrhages did not appear to account for the death of the majority of the mice. Analysis of the major organs of these low-TF mice revealed striking fibrosis in their hearts. LV function of 8-month-old low-TF mice was reduced by 30% (P Ͻ 0.001) compared with age-matched wild-type C57BL͞6J mice. This decrease was not sufficiently large to induce congestive heart failure, but the extensive fibrosis may cause fatal arrhyth- mias in the low-TF mice and contribute to their shortened lifespan. Low-TF mice had normal platelet counts and hemoglobin levels, but had reduced TAT levels and impaired responses to hemostatic challenge, suggesting that these mice may be prone to excessive hemorrhage in the event of blood vessel injury. Of note, low-TF mice had low levels of functional procoagulant activity in their hearts, indicating reduced TF expression by cardiac myocytes. Studies with low-TF mice on two different genetic backgrounds demonstrated a reduced survival on a C57BL͞6J background compared with a mixed background. Interestingly, FVLeiden/Leiden mice developed intravascular thrombosis in the perinatal period on a mixed (129Sv-C57BL͞ 6J) background, but not on a C57BL͞6J background (7). Sim- ilarly, TMPro/Pro mice had more fibrin deposition on a mixed Fig. 7. Model showing how TF contributes to primary and secondary hemo- (129Sv-C57BL͞6J) background than on a C57BL͞6J back- static barriers in cardiac and skeletal muscle. Skeletal muscle contains only a ͞ primary TF hemostatic barrier surrounding blood vessels (dark pink), whereas ground (24). These results suggest that the C57BL 6J back- cardiac muscle contains both primary and secondary (light pink) TF hemostatic ground is prohemorrhagic compared with the mixed background barriers. Endothelial cell layer is shown in green and blood is shown in red. and may explain why low-TF mice on a C57BL͞6J background have an increased rate of cardiac fibrosis and a reduced lifespan. The cause of the cardiac fibrosis was investigated by detailed low-TF and low-FVII mice, but not in hearts of FIXϪ/Ϫ mice. At histological analysis of the hearts of low-TF mice at different present, it has not been determined whether severely FVII- ages. Hemosiderin deposition in younger mice was predomi- deficient individuals surviving into adulthood develop cardiac nantly subepicardial. In older mice, hemosiderin was observed fibrosis. around capillaries and larger vessels throughout the myocar- In skeletal muscle, we propose that coagulation is initiated by dium. Hemosiderin may result from hemorrhage of erythrocytes the TF:FVIIa complex, but requires the FVIIIa:FIXa complex to into the myocardium. Indeed, we observed interstitial hemor- generate sufficient levels of FXa and thrombin to maintain rhages in the myocardium. Importantly, low-FVII mice exhibited hemostasis. Our model may explain why hemophiliacs bleed at a remarkably similar phenotype with hemosiderin and fibrosis in sites of low-TF expression, such as joints and skeletal muscle BIOCHEMISTRY their hearts. Mice expressing normal levels of murine TF lacking (26), where the secondary TF-dependent hemostatic barrier is the cytoplasmic domain had no hemosiderin in their hearts. low or absent. Low-TF and low-FVII mice do not bleed in Taken together, we propose that the most likely cause of cardiac skeletal muscle, suggesting that there are sufficient levels of the fibrosis in the hearts of low-TF and low-FVII mice is hemorrhage TF:VIIa complex formed at this site to maintain hemostasis. It from cardiac vessels resulting from impaired hemostasis. is notable that FbgϪ/Ϫ mice and patients classified as afibrino- Why do low-TF and low-FVII mice exhibit a hemostatic deficit genemic exhibit a relatively low frequency of spontaneous selectively in the heart? The most likely explanation is that the bleeding events, which may be because of sufficient platelet heart represents a special tissue in which there is repetitive minor activation and platelet plug formation to control the bleeds (8). mechanical injury to blood vessels, especially at the surface of Similarly, the absence of hemosiderin deposition or fibrosis in the heart, that produces hemorrhage in mice with severe TF or the hearts of FbgϪ/Ϫ mice may be due to normal platelet FVII deficiency. Additionally, the presence of the TF:FVII activation, which would compensate for the loss of fibrinogen complex at the interface between cardiac myocytes and endo- and prevent a hemostatic defect in the heart. Deficiencies in TF thelial cells may contribute to the stability of capillaries. Future or FVII would result in reduced levels of thrombin, which would studies of mice with a cardiac-specific knockout of TF will affect both fibrin deposition and platelet activation. In sum, our directly address the role of cardiac myocyte TF in the heart. data demonstrates that the intrinsic and extrinsic pathways of We propose a model of tissue-specific hemostasis (Fig. 7). In blood coagulation do not contribute equally to hemostasis in this model, blood vessels in all tissues, such as skeletal muscle, different tissues. have a primary hemostatic barrier due to TF expression by Clinically, administration of recombinant FVIIa (NovoSeven) pericytes, vascular smooth muscle cells, and adventitial fibro- at high doses restores hemostasis in individuals with deficiencies blasts (1, 25). In addition to this primary hemostatic barrier, the in FVII, FVIII, or FIX, and is an effective therapy for treating heart has a secondary hemostatic barrier due to TF expression hemorrhages in hemophiliacs with inhibitory antibodies to FVIII by cardiac myocytes (Fig. 7). Other tissues that have a secondary (27, 28). Conversely, targeting the TF:FVIIa complex with new TF-dependent hemostatic barrier include the brain and uterus. antithrombotic drugs holds promise for reducing life-threatening Our model may explain why mice with deficiencies in either the thrombosis (29–31). Our data from low-TF and low-FVII mice extrinsic (low TF and low FVII) or the intrinsic (FVIIIϪ/Ϫ and Ϫ/Ϫ suggest that prolonged use of these drugs could cause adverse FIX ) pathways exhibit different phenotypes. For instance, effects on cardiac hemostasis. low-TF and low-FVII mice have severely impaired uterine Ϫ/Ϫ Ϫ/Ϫ hemostasis, whereas FVIII and FIX mice have normal We thank D. Stafford for the FIXϪ/Ϫ mice, W. Ruf for the mTF⌬cyt/⌬cyt uterine hemostasis (10, 12, 16). The current study strongly mice, M. Szeto for breeding the mice, C. Johnson for preparing the suggests that hemostasis in the heart is primarily regulated by manuscript, and W. Aird, T. Edgington, and W. Boisvert for critical the TF:FVIIa-driven extrinsic pathway and is independent of reading of the manuscript. This work was supported by National Insti- FVIIIa:FIXa, because hemosiderin is observed in hearts of tutes of Health Grants P01 HL16411, R01 HL65226, and HL19982.

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