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  • 1
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    Online Resource
    Suppression of fibrin(ogen)-driven pathologies in disease models through controlled knockdown by lipid nanoparticle delivery of siRNA
    American Society of Hematology ; 2022
    In:  Blood Vol. 139, No. 9 ( 2022-03-03), p. 1302-1311
    Details
    In: Blood, American Society of Hematology, Vol. 139, No. 9 ( 2022-03-03), p. 1302-1311
    Abstract: Fibrinogen plays a pathologic role in multiple diseases. It contributes to thrombosis and modifies inflammatory and immune responses, supported by studies in mice expressing fibrinogen variants with altered function or with a germline fibrinogen deficiency. However, therapeutic strategies to safely and effectively tailor plasma fibrinogen concentration are lacking. Here, we developed a strategy to tune fibrinogen expression by administering lipid nanoparticle (LNP)-encapsulated small interfering RNA (siRNA) targeting the fibrinogen α chain (siFga). Three distinct LNP-siFga reagents reduced both hepatic Fga messenger RNA and fibrinogen levels in platelets and plasma, with plasma levels decreased to 42%, 16%, and 4% of normal within 1 week of administration. Using the most potent siFga, circulating fibrinogen was controllably decreased to 32%, 14%, and 5% of baseline with 0.5, 1.0, and 2.0 mg/kg doses, respectively. Whole blood from mice treated with siFga formed clots with significantly decreased clot strength ex vivo, but siFga treatment did not compromise hemostasis following saphenous vein puncture or tail transection. In an endotoxemia model, siFga suppressed the acute phase response and decreased plasma fibrinogen, D-dimer, and proinflammatory cytokine levels. In a sterile peritonitis model, siFga restored normal macrophage migration in plasminogen-deficient mice. Finally, treatment of mice with siFga decreased the metastatic potential of tumor cells in a manner comparable to that observed in fibrinogen-deficient mice. The results indicate that siFga causes robust and controllable depletion of fibrinogen and provides the proof-of-concept that this strategy can modulate the pleiotropic effects of fibrinogen in relevant disease models.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.2021014559
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2022
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
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  • 2
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    Hypofibrinogenemia with preserved hemostasis and protection from thrombosis in mice with an Fga  truncation mutation
    American Society of Hematology ; 2022
    In:  Blood Vol. 139, No. 9 ( 2022-03-03), p. 1374-1388
    Details
    In: Blood, American Society of Hematology, Vol. 139, No. 9 ( 2022-03-03), p. 1374-1388
    Abstract: Genetic variants within the fibrinogen Aα chain encoding the αC-region commonly result in hypodysfibrinogenemia in patients. However, the (patho)physiological consequences and underlying mechanisms of such mutations remain undefined. Here, we generated Fga270 mice carrying a premature termination codon within the Fga gene at residue 271. The Fga270 mutation was compatible with Mendelian inheritance for offspring of heterozygous crosses. Adult Fga270/270 mice were hypofibrinogenemic with ∼10% plasma fibrinogen levels relative to FgaWT/WT mice, linked to 90% reduction in hepatic Fga messenger RNA (mRNA) because of nonsense-mediated decay of the mutant mRNA. Fga270/270 mice had preserved hemostatic potential in vitro and in vivo in models of tail bleeding and laser-induced saphenous vein injury, whereas Fga−/− mice had continuous bleeding. Platelets from FgaWT/WT and Fga270/270 mice displayed comparable initial aggregation following adenosine 5′-diphosphate stimulation, but Fga270/270 platelets quickly disaggregated. Despite ∼10% plasma fibrinogen, the fibrinogen level in Fga270/270 platelets was ∼30% of FgaWT/WT platelets with a compensatory increase in fibronectin. Notably, Fga270/270 mice showed complete protection from thrombosis in the inferior vena cava stasis model. In a model of Staphylococcus aureus peritonitis, Fga270/270 mice supported local, fibrinogen-mediated bacterial clearance and host survival comparable to FgaWT/WT, unlike Fga−/− mice. Decreasing the normal fibrinogen levels to ∼10% with small interfering RNA in mice also provided significant protection from venous thrombosis without compromising hemostatic potential and antimicrobial function. These findings both reveal novel molecular mechanisms underpinning fibrinogen αC-region truncation mutations and highlight the concept that selective fibrinogen reduction may be efficacious for limiting thrombosis while preserving hemostatic and immune protective functions.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.2021012537
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2022
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
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  • 3
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    Factor XIII cross-links fibrin(ogen) independent of fibrin polymerization in experimental acute liver injury
    American Society of Hematology ; 2021
    In:  Blood Vol. 137, No. 18 ( 2021-05-6), p. 2520-2531
    Details
    In: Blood, American Society of Hematology, Vol. 137, No. 18 ( 2021-05-6), p. 2520-2531
    Abstract: Intravascular fibrin clot formation follows a well-ordered series of reactions catalyzed by thrombin cleavage of fibrinogen leading to fibrin polymerization and cross-linking by factor XIIIa (FXIIIa). Extravascular fibrin(ogen) deposits are observed in injured tissues; however, the mechanisms regulating fibrin(ogen) polymerization and cross-linking in this setting are unclear. The objective of this study was to determine the mechanisms of fibrin polymerization and cross-linking in acute liver injury induced by acetaminophen (APAP) overdose. Hepatic fibrin(ogen) deposition and cross-linking were measured following APAP overdose in wild-type mice, mice lacking the catalytic subunit of FXIII (FXIII−/−), and in FibAEK mice, which express mutant fibrinogen insensitive to thrombin-mediated fibrin polymer formation. Hepatic fibrin(ogen) deposition was similar in APAP-challenged wild-type and FXIII−/− mice, yet cross-linking of hepatic fibrin(ogen) was dramatically reduced ( & gt;90%) by FXIII deficiency. Surprisingly, hepatic fibrin(ogen) deposition and cross-linking were only modestly reduced in APAP-challenged FibAEK mice, suggesting that in the APAP-injured liver fibrin polymerization is not strictly required for the extravascular deposition of cross-linked fibrin(ogen). We hypothesized that the oxidative environment in the injured liver, containing high levels of reactive mediators (eg, peroxynitrite), modifies fibrin(ogen) such that fibrin polymerization is impaired without impacting FXIII-mediated cross-linking. Notably, fibrin(ogen) modified with 3-nitrotyrosine adducts was identified in the APAP-injured liver. In biochemical assays, peroxynitrite inhibited thrombin-mediated fibrin polymerization in a concentration-dependent manner without affecting fibrin(ogen) cross-linking over time. These studies depict a unique pathology wherein thrombin-catalyzed fibrin polymerization is circumvented to allow tissue deposition and FXIII-dependent fibrin(ogen) cross-linking.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.2020007415
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2021
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
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  • 4
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    Plasmin-mediated cleavage of high-molecular-weight kininogen contributes to acetaminophen-induced acute liver failure
    American Society of Hematology ; 2021
    In:  Blood Vol. 138, No. 3 ( 2021-07-22), p. 259-272
    Details
    In: Blood, American Society of Hematology, Vol. 138, No. 3 ( 2021-07-22), p. 259-272
    Abstract: Acetaminophen (APAP)-induced liver injury is associated with activation of coagulation and fibrinolysis. In mice, both tissue factor–dependent thrombin generation and plasmin activity have been shown to promote liver injury after APAP overdose. However, the contribution of the contact and intrinsic coagulation pathways has not been investigated in this model. Mice deficient in individual factors of the contact (factor XII [FXII] and prekallikrein) or intrinsic coagulation (FXI) pathway were administered a hepatotoxic dose of 400 mg/kg of APAP. Neither FXII, FXI, nor prekallikrein deficiency mitigated coagulation activation or hepatocellular injury. Interestingly, despite the lack of significant changes to APAP-induced coagulation activation, markers of liver injury and inflammation were significantly reduced in APAP-challenged high-molecular-weight kininogen-deficient (HK−/−) mice. Protective effects of HK deficiency were not reproduced by inhibition of bradykinin-mediated signaling, whereas reconstitution of circulating levels of HK in HK−/− mice restored hepatotoxicity. Fibrinolysis activation was observed in mice after APAP administration. Western blotting, enzyme-linked immunosorbent assay, and mass spectrometry analysis showed that plasmin efficiently cleaves HK into multiple fragments in buffer or plasma. Importantly, plasminogen deficiency attenuated APAP-induced liver injury and prevented HK cleavage in the injured liver. Finally, enhanced plasmin generation and HK cleavage, in the absence of contact pathway activation, were observed in plasma of patients with acute liver failure due to APAP overdose. In summary, extrinsic but not intrinsic pathway activation drives the thromboinflammatory pathology associated with APAP-induced liver injury in mice. Furthermore, plasmin-mediated cleavage of HK contributes to hepatotoxicity in APAP-challenged mice independently of thrombin generation or bradykinin signaling.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.2020006198
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2021
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
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  • 5
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    Targeting neutrophil PKM2 for stroke treatment
    American Society of Hematology ; 2022
    In:  Blood Vol. 139, No. 8 ( 2022-02-24), p. 1131-1132
    Details
    In: Blood, American Society of Hematology, Vol. 139, No. 8 ( 2022-02-24), p. 1131-1132
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.2021014199
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2022
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
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  • 6
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    Mechanism of ICH with tPA thrombolysis
    American Society of Hematology ; 2021
    In:  Blood Vol. 138, No. 1 ( 2021-07-08), p. 8-9
    Details
    In: Blood, American Society of Hematology, Vol. 138, No. 1 ( 2021-07-08), p. 8-9
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.2021011268
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2021
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
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  • 7
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    Protective Role of Plasminogen Deficiency in Non-Alcoholic Fatty Liver Disease and Glucose Dysmetabolism
    American Society of Hematology ; 2021
    In:  Blood Vol. 138, No. Supplement 1 ( 2021-11-05), p. 1026-1026
    Details
    In: Blood, American Society of Hematology, Vol. 138, No. Supplement 1 ( 2021-11-05), p. 1026-1026
    Abstract: Obesity is global health problem with 40% of the world population being classified as overweight (BMI & gt; 25) and 13% as obese (BMI & gt; 30). Obesity drives chronic metabolic inflammation leading to metabolic syndrome, cardiovascular disease, fatty liver disease, Type II diabetes, and certain cancers. A documented clinical manifestation of obesity is perturbed and dysregulated hemostatic system leading to a procoagulant and anti-fibrinolytic state that ultimately results in an increased risk of thrombosis. Previous work suggested that clotting system components thrombin and fibrin engage in reciprocal mechanisms and contribute to the development of obesity. Specifically, we have shown that fibrin accumulates within obese adipose tissue and liver of obesity patients and mice challenged with a high fat diet (HFD) and colocalizes with macrophages, a key driver of inflammation in obesity. Despite the fact that obesity is known to be linked to an impaired fibrinolytic system, a potential functional contribution of fibrinolytic proteases to the development of obesity and associated downstream diseases has been understudied. Here, we tested the hypothesis that elimination of the fibrinolytic protease plasminogen would increase HFD-driven fibrin deposition and exacerbate macrophage accumulation and subsequent weight gain and obesity-associated pathologies. Contrary to our hypothesis, plasminogen-deficient (Plg-) mice gained as much weight as the Plg+ mice after 20 weeks on HFD. However, whereas the liver mass of HFD-challenged Plg+ mice was significantly higher than that of low fat diet (LFD)-fed mice, the livers of HFD-fed Plg- mice had a mass comparable to LFD-fed mice. HFD-fed Plg- mice had reduced hepatocellular damage, measured by plasma ALT activity, as well as reduced hepatosteatosis, measured by hepatic triglyceride content and liver histology, compared to HFD-fed Plg+ mice. Circulating cholesterol levels in HFD-fed Plg -/- mice were comparable to LFD-fed Plg- mice, while it was significantly elevated in HFD-fed Plg+ mice. While the epididymal white adipose tissue mass was higher in HFD-fed Plg- mice compared to HFD-fed Plg+ mice, the brown adipose tissue mass was comparable. However, there was an upregulation of uncoupling protein-1 (UCP-1) expression BAT of HFD-fed Plg- mice. Glucose clearance was more efficient in HFD-fed Plg- mice compared to HFD-fed Plg+ mice in a glucose tolerance test. Collectively, our data suggest that plasmin(ogen) contributes to HFD-induced fatty liver disease and glucose dysmetabolism. Disclosures No relevant conflicts of interest to declare.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood-2021-145352
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2021
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
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  • 8
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    Fibrin(ogen) Promotes Immune Cell Infiltration, Dysbiosis and ROS Production in Experimental Colitis
    American Society of Hematology ; 2021
    In:  Blood Vol. 138, No. Supplement 1 ( 2021-11-05), p. 443-443
    Details
    In: Blood, American Society of Hematology, Vol. 138, No. Supplement 1 ( 2021-11-05), p. 443-443
    Abstract: Elevated stool fibrinogen has recently been shown to predict disease course in ulcerative colitis (UC), suggesting that fibrin(ogen) in the colitis microenvironment promotes UC pathogenesis. This conclusion is consistent with previous studies from our laboratory showing that fibrin(ogen)/leukocyte interactions mediated by the integrin receptor α Mβ 2 promote experimental colitis and colitis-associated cancer, but the mechanisms are not well defined. To delineate the mechanism coupling fibrinogen to colitis pathogenesis, we induced colitis in Fib WT and mice carrying a mutant fibrinogen lacking the α Mβ 2 binding motif (Fibg 390-396A) with Dextran Sulfate Sodium (DSS). We performed flow cytometric, protein analyses of colons and fecal microbiome and metabolomics analyses after DSS exposure. Five days after DSS challenge, a timepoint prior to significant epithelial damage, we observed significantly diminished infiltration of natural killer cells, T cells, dendritic cells, macrophages and neutrophils in colons harvested from DSS-challenged Fibg 390-396A mice relative to Fib WT mice. We also observed significantly diminished proinflammatory cytokine production by NK cells, macrophages and dendritic cells isolated from DSS-challenged Fibg 390-396A colons. It is well-established that the microbiome composition is a major determinant of colitis in humans and mice. One mechanism by which microbiome contents alter the course of colitis is by the elaboration of certain fecal metabolites. NMR-based fecal metabolomics analyses demonstrated no significant changes in short-chain fatty acids (acetate, propionate, and butyrate) and various amino acids (valine, proline, alanine) however we found significantly less uracil in fecal from Fibg 390-296A relative to Fib WT mice following DSS exposure. Uracil is a key ligand for Duox2 (dual NADPH oxidase), a gut epithelial specific enzyme, that drives reactive oxygen species production by gut epithelial in response to dysbiosis. Notably, Duox2 is the highest induced gene in inflammatory bowel disease (IBD) and has been identified as a risk gene. Our molecular analyses showed no difference in colonic Duox2 expression between genotypes at baseline, but Duox2 expression significantly increased in Fib WT relative to Fibg 390-296A after just 5 days of DSS exposure. Further western blot analyses revealed that Duox2 expression only in the colonic epithelial cells and not in the lamina propria cells. To determine if fibrinogen cause dysbiosis in DSS-induced colitis model, we performed shotgun sequencing on fecal samples from Fib WT and Fibg 390-396A mice at baseline and after 7 days of DSS challenge. Interesting shotgun sequencing analyses revealed major fibrin genotype dependent significant differences in the microbiomes of Fib WT and Fibg 390-396A mice at baseline as well as following DSS challenge. Our studies are the first to show that fibrin(ogen) is a major determinant of the gut-microbiome in the context of experimental colitis. Altogether, these studies demonstrate that fibrin(ogen) in the colitis microenvironment promotes the infiltration and activation of multiple leukocyte subsets that drive colitis pathogenesis. These results also suggest that fibrin(ogen) promotes colitis-associated dysbiosis and Duox2 expression, leading to ROS production that contributes to colitis pathogenesis and possibley tumorigenesis. Overall, these studies suggest that fibrin(ogen)-α Mβ 2 interactions represent an attractive therapeutic target for IBD without incurring the potential bleeding risks associated with anticoagulants or other modalities targeting fibrin deposition. Disclosures No relevant conflicts of interest to declare.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood-2021-153148
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2021
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
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  • 9
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    Abstract PO-127: A uPA/uPAR axis in both the tumor cell and stromal compartment drives PDAC disease progression
    American Association for Cancer Research (AACR) ; 2021
    In:  Cancer Research Vol. 81, No. 22_Supplement ( 2021-11-15), p. PO-127-PO-127
    Details
    In: Cancer Research, American Association for Cancer Research (AACR), Vol. 81, No. 22_Supplement ( 2021-11-15), p. PO-127-PO-127
    Abstract: Pancreatic ductal adenocarcinoma (PDAC) is a lethal solid tumor malignancy with a 5-year survival rate of 9%. In both patients and animal models of disease, PDAC is associated with robust coagulation system activity. Intriguingly, in addition to being a rich source of procoagulant factors, PDAC tumors highly express fibrinolytic system components. Supporting this concept, urokinase plasminogen activator (uPA) and uPA receptor (uPAR) expression positively correlates with reduced overall patient survival. Here, we tested the hypothesis that the expression and activity of plasminogen activation (PA) system components are functionally linked to PDAC tumor growth and disease progression. We generated C57Bl/6-derived KPC (i.e., KRasG12D, TRP53R172H) PDAC cell lines in which uPA and uPAR were knocked out using CRISPR-Cas9. We then analyzed orthotopic tumor growth and experimental metastasis in mice carrying null or functional mutations in uPA, uPAR, or plasminogen to evaluate the interplay of PA components derived from tumor cells and/or stromal cells in mediating PDAC progression. Although both KPC cell CRISPR variants retained procoagulant function, elimination of tumor cell uPA or uPAR yielded significantly smaller tumors when compared to Cas9 control tumor cells in wildtype mice. Similarly, the growth of WT KPC tumor cells in C57Bl/6 background uPA-KO or uPAR-KO mice also resulted in reduced tumor growth. To our surprise, the metastasis potential of WT KPC tumor cells in uPA-KO or uPAR-KO mice did not change when compared to wildtype mice. Regarding to the uPA/uPAR axis downstream effector plasminogen, the growth of WT KPC tumors in plasminogen-KO mice was also significantly reduced, but not to the same extent as when eliminating uPA or uPAR. In addition, eliminating plasminogen drastically reduced WT KPC tumor cells metastasis potential. In conclusion, our data suggest a mechanism whereby uPA functions through uPAR in both the tumor cell and stromal cell compartments to promote PDAC progression through plasminogen-dependent and -independent mechanisms. Citation Format: Yi Yang, Sara R. Abrahams, Aditi Kothari, Harshi Matada, Keely Davey, Alisa S. Wolberg, Matthew J. Flick. A uPA/uPAR axis in both the tumor cell and stromal compartment drives PDAC disease progression [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-127.
    Type of Medium: Online Resource
    ISSN: 0008-5472 , 1538-7445
    URL: Article
    DOI: 10.1158/1538-7445.PANCA21-PO-127
    RVK:
    XA 36000
    RVK:
    XA 10000
    Language: English
    Publisher: American Association for Cancer Research (AACR)
    Publication Date: 2021
    detail.hit.zdb_id: 2036785-5
    detail.hit.zdb_id: 1432-1
    detail.hit.zdb_id: 410466-3
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  • 10
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    Pancreatic Ductal Adenocarcinoma Causes Bleeding Phenotype in Mice Associated with Loss of Procoagulant Activity and Increased Thrombomodulin Sensitivity
    American Society of Hematology ; 2022
    In:  Blood Vol. 140, No. Supplement 1 ( 2022-11-15), p. 11278-11278
    Details
    In: Blood, American Society of Hematology, Vol. 140, No. Supplement 1 ( 2022-11-15), p. 11278-11278
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood-2022-169582
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2022
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
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