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One mechanism for disrupting the MLL gene in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) is through partial tandem duplication (MLL-PTD); however, the mechanism by which MLL-PTD contributes to MDS and AML development and maintenance is currently unknown. Herein, we investigated hematopoietic stem/progenitor cell (HSPC) phenotypes of Mll-PTD knock-in mice. Although HSPCs (Lin−Sca1+Kit+ (LSK)/SLAM+ and LSK) in MllPTD/WT mice are reduced in absolute number in steady state because of increased apoptosis, they have a proliferative advantage in colony replating assays, CFU-spleen assays, and competitive transplantation assays over wild-type HSPCs. The MllPTD/WT-derived phenotypic short-term (ST)–HSCs/multipotent progenitors and granulocyte/macrophage progenitors have self-renewal capability, rescuing hematopoiesis by giving rise to long-term repopulating cells in recipient mice with an unexpected myeloid differentiation blockade and lymphoid-lineage bias. However, MllPTD/WT HSPCs never develop leukemia in primary or recipient mice, suggesting that additional genetic and/or epigenetic defects are necessary for full leukemogenic transformation. Thus, the Mll-PTD aberrantly alters HSPCs, enhances self-renewal, causes lineage bias, and blocks myeloid differentiation. These findings provide a framework by which we can ascertain the underlying pathogenic role of MLL-PTD in the clonal evolution of human leukemia, which should facilitate improved therapies and patient outcomes.

Definitive hematopoietic stem and progenitor cells (HSCs/Ps) originating from the yolk sac and/or para-aorta-splanchno-pleura/aorta-gonad-mesonephros are hypothesized to colonize the fetal liver, but mechanisms involved are poorly defined. The Rac subfamily of Rho GTPases has been shown to play essential roles in HSC/P localization to the bone marrow following transplantation. Here, we study the role of Rac1 in HSC/P migration during ontogeny and seeding of fetal liver. Using a triple-transgenic approach, we have deleted Rac1 in HSCs/Ps during very early embryonic development. Without Rac1, there was a decrease in circulating HSCs/Ps in the blood of embryonic day (E) 10.5 embryos, while yolk sac definitive hematopoiesis was quantitatively normal. Intraembryonic hematopoiesis was significantly impaired in Rac1-deficient embryos, culminating with absence of intra-aortic clusters and fetal liver hematopoiesis. At E10.5, Rac1-deficient HSCs/Ps displayed decreased transwell migration and impaired inter-action with the microenvironment in migration-dependent assays. These data suggest that Rac1 plays an important role in HSC/P migration during embryonic development and is essential for the emergence of intraembryonic hematopoiesis.

Myelodysplastic Syndromes (MDS) are heterogeneous clonal hematopoietic disorders, which are characterized by ineffective hematopoiesis and uni- or multi-lineage dysplasia. Despite the fact that a variety of genetic, epigenetic, and metabolic aberrations have been identified, the clinical features for MDS remain similar, indicating that there is a common underlying mechanism for MDS pathogenesis. Accumulating clinical and research evidence has shown the involvement of systemic inflammation and activated immune signaling in MDS pathogenesis. Hypoxia inducible factor-1α (HIF-1α) is a critical transcription factor for the hypoxic response, angiogenesis, and cancer development. Importantly, HIF-1α is also a key regulator for immune cell activation. To determine whether MDS patients have an activated HIF-1α signature, we analyzed a public gene expression array data set of CD34+ BM cells from a large cohort of MDS patients (n = 183). Although HIF-1α mRNA remains unchanged, some HIF-1α target genes relating with survival/cell growth, glucose metabolism, and invasion/metastasis were significantly activated in the broad spectrum of MDS. We further determined HIF-1α expression in the BM biopsies from MDS patients (n = 39) using immunohistochemistry staining. We found a high frequency of HIF-1α expressed cells in both low- and high-risk IPSS groups. Consistent with this human data, deficiency of frequently mutated genes in MDS resulted in an activated HIF-1α signature in mice. We found that the levels of Hif-1α protein in the c-Kit+ BM cells from Dnmt3a-KO, Runx1-KO, and Mll-partial tandem duplication (PTD) knock-in (MllPTD/WT) mice were significantly upregulated compared with those from WT controls. Although the expression level of Hif-1α protein in the c-Kit+ BM cells from Tet2-KO mice was not changed, they had an activated HIF-1α signature. IDH1/2 mutations are also known to elevate Hif-1α proteins. These results suggest that the HIF-1α pathway is widely activated in MDS patients by MDS-genic mutations through different mechanisms. To determine whether HIF-1α is sufficient for developing MDS phenotypes, we generated blood specific inducible HIF-1α transgenic mice. Using Vav1-Cre/Rosa26-loxP-Stop-loxP (LSL) rtTA driver, stable HIF-1α can be induced in a doxycycline dependent manner. HIF-1α-induced mice developed thrombocytopenia, leukocytopenia, and macrocytic anemia. We found micromegakaryocyte and multi-segmented megakaryocyte formations which are a major diagnostic criteria for MDS. In the BM of anemic mice, we found significant reduction of basophilic and polychromatic erythroblasts. The central macrophages are known to form erythroblastic islands that play a critical role in the late stage of erythropoiesis. Both the frequency and absolute number of F4/80+ Ter119+ BM erythroblastic islands were significantly decreased in HIF-1α-induced mice compared to the control mice. We also found activation of both innate and adaptive immunity in HIF-1α-induced mice. Lineage specific HIF-1α induction revealed the cell-intrinsic effect of HIF-1α on aberrant megakaryocytopoiesis and cell-extrinsic effect of HIF-1α on macrocytic anemia development. We have previously shown that MllPTD/WT mice develop several MDS-associated features, such as increased self-renewal and apoptosis in HSPCs, expansion of myeloid progenitors, and skewed myeloid differentiation. Synergistic effects between MLL-PTD and RUNX1 mutant (S291fs) or Runx1 deletion, further accumulated the Hif-1α protein and caused a variety of MDS features that phenocopy the human MDS. To understand the role of HIF-1α in the context of MDS pathogenesis, we took genetic and pharmacologic approaches. Hif-1α deletion significantly abrogated disease development and rescued macrocytic anemia and thrombocytopenia. This is not due to the elimination of donor derived cells. After the deletion of Hif-1α, a majority of donor derived cells in PB were still CD11b+ cells. HIF-1α inhibition significantly reduced colony formation of MllPTD/WT/RUNX1-291fs cells and MllPTD/WT/Runx1D/D cells in vitro, and prolonged survival of MDS mice in vivo. In conclusion, we identified the sufficient and essential role of HIF-1α in developing MDS. These findings implicate that HIF-1α pathway may be the common underlying mechanism of MDS pathophysiology and could be an effective therapeutic target for a broad spectrum of MDS patients. Disclosures Shih: Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding.

A human megakaryoblastic cell line, designated CHRF-288–11, has been established in vitro through the use of adherent stromal cells in long- term human bone marrow culture. Long-term bone marrow cultures were required for the initial adaptation of the megakaryoblastic cells to culture conditions; however, once adapted, the cells were weaned from the stromal layer until they proliferated in the complete absence of any feeder layers. The seed cells for the establishment of this line were derived from a solid tumor; the cloned cell line derived from this tumor exhibits markers characteristic of megakaryocytes and platelets. Specifically, the cells express platelet peroxidase, platelet factor 4, and platelet Ca+(+)-adenosine triphosphatase (ATPase), glycoprotein IIb- IIIa (CDw41), factor VIII antigen, and the MY7 (CD13) and MY9 (CD33) antigens. The cells do not express the erythroid markers glycophorin A and hemoglobin, the myeloid marker myeloperoxidase, nor markers specific for T and/or B cells. The established cell line produces both basic fibroblast growth factor and transforming growth factor-beta, properties demonstrated previously for the solid tumor. The clonal cell population exhibited a unique, singular karyotype, indicating cellular homogeneity. The cells display a doubling time of approximately 33 hours in either 25% horse or calf serum. Treatment of the cells with 1 X 10(-8) mol/L phorbol 12-myristate 13-acetate (PMA) leads to the induction of multi-nucleation and hyperploidy in the cells, with approximately 35% of the cells exhibiting two or more nuclei per cell, and greater than 80% of the cells enlarging in size. The establishment of this unique cell line under defined culture conditions will be beneficial for the future study of megakaryocytic properties expressed by this cell line.

The growth of Lewis lung carcinoma (LLC) was sustained in plasminogen-deficient mice when transplanted into the dorsal skin but was dramatically suppressed in another anatomic location, the footpad. This unanticipated negative effect of plasminogen deficiency on footpad tumor growth was entirely relieved by superimposing a deficit in fibrinogen. This finding was not simply an unusual feature of LLC tumors—T241 fibrosarcoma growth in the footpad was also restricted by plasminogen deficiency in a fibrinogen-dependent manner. The probable mechanistic basis for suppression of tumor growth was revealed through transmission electron microscopy studies of tumor tissues. Occlusive microvascular thrombi were commonplace within footpad tumors from plasminogen-deficient mice, whereas no such lesions were observed within either dorsal skin tumors from plasminogen-deficient mice or footpad tumors from mice that also lacked fibrinogen. The data infer that tumor growth in the footpad of plasminogen-deficient mice is compromised as a function of the formation and persistence of vaso-occlusive thrombi that limit tumor blood supply. These studies indicate that plasminogen and fibrinogen can serve as critical determinants of tumor growth, but their relative importance is dependent on the tumor microenvironment. Furthermore, these studies suggest that one target of plasmin(ogen) relevant to tumor progression in vivo is intravascular fibrin.

Abstract 2801 A relatively common mechanism through which Mixed-Lineage Leukemia (MLL) gene is disrupted in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) is a partial tandem duplication of its coding sequence (MLL-PTD), which duplicates the MLL N-terminal region and creates a larger in-frame oncogenic protein. We have previously shown that MLL interacts with and stabilizes the core binding factor (CBF) complex, a heterodimeric transcription factor complex composed of a DNA-binding subunit RUNX1 (CBFa) and a non-DNA-binding subunit CBFβ. MLL is required for CBF dependent regulation of the downstream target gene PU.11. In MDS and AML, RUNX1 is frequently disrupted by mutations, and the presence of MLL-PTD and/or RUNX1 mutations may negatively impact on clinical outcome. However, little is known how mutated MLL and RUNX1 contribute to MDS-associated ineffective hematopoiesis, clonal expansion and transformation into AML. Interestingly, MDS patients with either mutant RUNX1 or MLL-PTD tend to develop secondary AML harboring both RUNX1 and MLL mutations, suggesting a leukemogenic cooperation between the two genes through converging mechanisms. We hypothesized that the MLL-CBF nuclear signaling pathway maintains stem cell fitness, and the MDS-associated mutations co-opt this pathway leading to ineffective hematopoiesis, clonal expansion and transformation into AML. To address these questions, MLL-PTD+ and MLL-PTD(−) AML cell lines were compared. RUNX1 and CBFβ proteins were expressed at low levels in all 4 MLL-PTD+ AML cell lines (OCI-AML2, EOL, KOMP88, MUTZ11) compared with MLL-PTD(−) AML cell lines (OCI-AML5, Kasumi1, HL60, KG1a). In contrast to wild type MLL, which stabilized the CBF complex, MLL-PTD downregulated RUNX1 and CBFβ proteins in co-expression assays in 293T cells. This finding was further confirmed using the MllPTD/WT knock-in mouse model. In MllPTD/WT mice, the abnormal hematopoietic stem and progenitor cells (HSPCs) showed decreased expression levels of Runx1 (50%) and Cbfβ (30%) proteins and in turn PU.1 (30%) and its target gene, Mcl-1 (20%) compared with WT HSPCs. Consistent with this finding, HSPCs from MllPTD/WT mice had increased spontaneous apoptosis compared with WT HSPCs [(Lin−/cKit+/Sca-1+ (LSK), Annexin V positivity: 44% ±7.5% vs 25%±4.5%, p Disclosures: No relevant conflicts of interest to declare.

Abstract 334 A growing body of evidence points to a crucial role for fibrinogen in both tumor dissemination and the regulation of inflammation. Given this dual importance, we hypothesized that fibrinogen plays an important role in the progression of inflammation-driven cancers. To test this hypothesis, we induced colitis-associated cancer (CAC) in fibrinogen-deficient mice and controls using a combination of azoxymethane (AOM) and dextran sodium sulfate (DSS). Fibrinogen deficiency resulted in a dramatic diminution in the number of adenomas formed after AOM/DSS challenge in spite of the fact that overt gastrointestinal bleeding appeared more severe in Fib- animals relative to controls. These results suggest that while fibrin deposition is crucial for the maintenance of vascular integrity and control of blood loss in colitis, fibrin(ogen) appears to drive colitis-associated adenoma development/outgrowth. Fibrin(ogen) could promote CAC through multiple mechanisms. One intriguing possibility is that fibrin(ogen) interactions with leukocytes through the integrin αMβ2 are an important determinant of inflammation-induced tumor progression. To test this hypothesis, we explored CAC development in control mice and animals expressing a mutant form of fibrinogen (Fibγ390-396A) that maintains full clotting function and supports thrombus formation normally in vivo, but does not support binding to αMβ2. Consistent with our hypothesis, Fibγ390-396A mice developed significantly fewer adenomas after AOM/DSS challenge. In fact, the majority of Fibγ390-396A mice had no discernable adenomas while the phenotypic penetrance of adenoma development was 100% in control animals. Detailed analyses revealed that one mechanism coupling fibrin(ogen)-mediated αMβ2 engagement to adenoma formation/outgrowth is by supporting inflammatory events during the antecedent colitis. Fibγ390-396A mice manifested a dramatic diminution in inflammatory cell infiltrates, colonic edema, crypt loss and epithelial ulceration relative to control mice after chronic DSS exposure. Analyses of short-term DSS exposure revealed an ∼10-fold diminution in Fibγ390-396A mice relative to controls in the elaboration of key cytokines known to promote CAC progression (i.e., IL-6, TNF-α, IL-1β, IFN-γ). Previous studies suggest that these cytokines promote CAC, at least in part, through changes in epithelial cell function. Consistent with this view, the number of colonic epithelial cells staining positive for established activation markers (i.e., phosphorylated cJun and RelA/p65) were significantly diminished in Fibγ390-396A animals relative to control animals after short-term DSS exposure. Taken together, these data strongly suggest that one mechanism coupling fibrin(ogen) to adenoma progression is through αMβ2-mediated proinflammatory events early in the disease process which lead to epithelial damage/turnover important in adenoma formation. However, the role of fibrin(ogen) in adenoma progression does not appear to be limited to these early inflammatory events. The adenomas harvested from Fibγ390-396A mice were significantly smaller than those from control mice and less proliferative based on mitotic indices, suggesting an additional role for fibrin(ogen) in the growth of established adenomas. These studies demonstrate, for the first time, a unique link between fibrin(ogen) and the development of inflammation-driven malignancy. Given the importance of antecedent inflammation in the progression of numerous cancers, these studies suggest that therapies targeting fibrin(ogen)-αMβ2 interactions may be useful in preventing and/or treating this important subset of malignancies. Disclosures: No relevant conflicts of interest to declare.

Inflammatory bowel disease has been recognized as an important risk factor in the development of colon cancer for decades. However, the factors that support the inflammatory response in this setting are not fully defined. Given that activation of the hemostatic system is a consistent feature of colitis, and previous studies have shown that hemostatic factors are key regulators of inflammation in other settings, we hypothesized that the platelet/fibrinogen axis plays an important role in inflammatory colitis. In order to explore whether fibrinogen interactions with leukocytes or platelets contribute to colitis, we challenged gene-targeted mice expressing mutant forms of fibrinogen lacking the binding motifs recognized by the leukocyte integrin αMβ2 (Fibγ390–396A mice) or the platelet integrin αIIbβ3 (FibγΔ5 mice) with dextran sodium sulfate (DSS). The severity of colonic injury and inflammation in Fibγ390–396A mice were dramatically diminished relative to control animals based on multiple histopathological criteria, including edema, erosion/ulceration, crypt loss, and inflammatory cell infiltration. DSS-challenged Fibγ390–396A mice also exhibited significantly diminished circulating levels of the inflammatory cytokine IL-6. These studies support the general conclusion that fibrinogen-αMβ2 interactions are an important determinant of inflammatory processes within the colon. Complementary studies of FibγΔ5 mice suggest that fibrinogen interaction with the platelet integrin αIIbβ3 is also a determinant of colitis-associated pathologies. Here, initial studies of FibγΔ5 mice revealed that the loss of platelet-fibrinogen interactions results in a significant, but counterintuitive, diminution in colitis-associated GI bleeding and weight loss. Fibrinogen-supported platelet deposition and activation are likely to contribute to inflammatory disease processes though multiple mechanisms, but a contribution of the many proinflammatory cytokines and chemokines present within the platelet secretome may be a significant factor. In addition to supporting colitis, hemostatic factors contribute to the more complex process of colitis-associated cancer (CAC) progression. In this regard, it is notable that Fibγ390–396A mice developed significantly fewer colonic adenomas relative to control mice when challenged with a combination of azoxymethane, a DNA alkylator, and DSS. Indeed, ~30% of Fibγ390–396A mice had no discernable adenomas, while penetrance was 100% in control mice. Furthermore, the tumors harvested from Fibγ390–396A mice were significantly smaller than those observed in wild-type mice, resulting in a profound diminution in total tumor burden. These results support the conclusion that the platelet/fibrinogen axis is a major determinant of inflammatory colitis and colitis-associated cancer progression, and therapies designed to disrupt inflammatory pathways at the level of hemostatic factors could be useful in the treatment or prevention of colitis or colitis-associated colon cancer.

While genetic delivery of recombinant anti-sickling β-globin genes have been shown to correct murine sickle cell anemia (SCA), correction of SCA by delivery of a natural hemoglobin, fetal hemoglobin (HbF), the proportion of genetically modified hematopoietic stem cells (HSC), or amount of HbF necessary to correct the disease is unknown. We designed a lentivirus vector carrying γ-globin exons with β-globin regulatory elements and non-coding sequences, GbG. First, GbG or mock transduced Berkeley sickle HSC were transplanted using a myeloablative (lethal irradiation) transplant model, to acheive full donor chimerism. GbG mice showed high HbF expression (HbF 41 ± 5% measured by HPLC) that was sustained in primary (6 mo) and secondary (7.5 mo) transplant recipients, and resulted in effective correction of hematological and functional RBC parameters, and reduction of inflammation that results from sickle cell disease. We found significantly reduced irreversibly sickled cells (2.3 ± 0.7% in GbG versus 10.2 ± 0.3% in mock mice; p30% HbF/F-cell and >60% F-cells consistently corrected SCA. The mean HSC transduction (assessed by secondary HbF+ CFU-S at 6 months post transplant) was 50% and 30% in the myeloablative and reduced intensity transplant models, respectively, with 1–3 GbG copies/ cell. Furthermore, three GbG mice showed correction of SCA with 20% HSC transduction, a clinically achievable goal. Taken together, this study is the first demonstration of correction of SCA with gene therapy using γ-globin, and defines critical determinants for effective gene therapy of this disease. Mouse Model Hb (g/dl) RBC 106/ul) Reticulocyte (%) WBC (K/ul) *p

Thrombin-mediated proteolysis is central to hemostasis, directly controlling both platelet activation and fibrin deposition as well as positively- and negatively-regulating further thrombin generation through the activation of factors XI, VIII, V and protein C. Thrombin also appears to be important in multiple processes distinct from traditional hemostasis, but detailed genetics-based studies have been impeded by the uniform embryonic and perinatal failure of mice with constitutive prothrombin (fII) deficiency. To develop an experimental setting to explore the importance of fII in vascular biology, tissue repair, the inflammatory response and disease processes in adult animals, we have generated mice carrying a conditional prothrombin knockout allele (fIIlox). In the absence of Cre-mediated recombination, homozygous fIIlox/lox mice or compound heterozygous mice carrying one fIIlox allele and one constitutive-null allele were found to carry ~20% and ~10% the normal level of circulating prothrombin, respectively. These fIIlox/lox and fIIlox/− mice exhibited normal developmental and reproductive success, survived well into adulthood and young adults exhibited no appreciable spontaneous bleeding events or other pathologies. However, one-year-old fIIlox/− mice developed modest focal hemosiderin deposits and fibrosis within the heart, consistent with chronic low grade hemorrhage in this tissue. No gross or microscopic pathologies were observed in any other tissue examined in mice carrying an intact conditional knockout allele, regardless of age or gender. The induction of Cre recombinase in adult fIIlox mice using the poly I:C-inducible Mx1-Cre system resulted in the rapid and near-complete recombination of the fIIlox allele within the liver, loss of hepatic fII mRNA, elimination of detectable circulating prothrombin, and profound derangements in coagulation function. The life-expectancy in adults genetically-depleted of prothrombin was found to be very short (generally 5–7 days), and the loss of viability was associated with the development of severe hemorrhagic events within multiple tissues, most prominently in the heart (100% phenotypic penetrance) and brain (~50% phenotypic penetrance). Microscopic analysis of hearts from mice following deletion of the fII allele revealed widespread hemorrhage within the myocardium, particularly in the subepicardial region, focal ischemia and necrosis, neutrophil infiltrates and early granulation tissue. Gross evidence of blood was observed within the pleural cavity in nearly half of Cre-induced fIIlox/− mice at autopsy, and the lack of appreciable hemorrhage within lung tissue favored the heart as the source of this free blood. Less common hemorrhagic events were observed several other tissues, including skeletal muscle, intestines and testes. Examination of brains from mice lacking fII revealed widespread bleeding in the central nervous system, including dural-based hemorrhage and bleeding into both the brain parenchyma and ventricles. These results imply that a robust hemostatic system is essential to limit spontaneous bleeding events in tissues under repetitive mechanical or pulsatile stress. These findings also demonstrate that low levels of fII are compatible with long-term survival in adult mice, affording the ability to examine fII in a diverse spectrum of disease and physiologic processes. As evidence of this concept, mice lacking fII were challenged with S. aureus peritonitis prior to the onset of hemorrhage. Similar to mice lacking fibrinogen, these animals were found to have a profound deficit in their ability to control this infection, pointing to the need for polymerized fibrin for the effective clearance of this bacteria from the peritoneal cavity.

Activation of the hemostatic system is a common pathological feature of severe inflammatory processes. In the context of inflammatory joint disease, articular fibrin deposition is a prominent feature that has been hypothesized to contribute to the derangements in immune/inflammatory processes and tissue reorganization that lead to debilitating and irreversible damage within arthritic joints. We directly examined the role of fibrin(ogen) in the progression of inflammatory joint disease using the well-established experimental setting of collagen-induced arthritis (CIA). Mice with genetically-imposed fibrinogen deficiency (Fib−/−) were found to exhibit significantly reduced macroscopic arthritic disease incidence, progression, and severity in the paws relative to littermate controls throughout the evaluation period. Consistent with the macroscopic observations, qualitative and quantitative histological analysis of distal- and knee-joint sections revealed a significant decrease in all joint disease parameters (e.g., inflammatory cell infiltrates, synovial hyperplasia, edema, and cartilage/bone loss) in Fib−/− mice as compared to fibrinogen-sufficient controls. Fibrin(ogen) has been shown to promote pro-inflammatory activity through engagement of the leukocyte integrin receptor αMβ2 (Mac-1). To determine if fibrinogen/αMβ2 interactions promote the pathogenesis of arthritis, we compared CIA progression in mice expressing fibrinogen that lacks the leukocyte integrin receptor αMβ2 binding motif (i.e., Fibγ390–396A mice). We observed decreased gross and histological inflammatory joint disease in Fibγ390–396A mice relative to control animals. This diminution in arthritic disease severity was comparable to that observed in Fib−/− mice despite the maintenance of normal clotting function in Fibγ390–396A animals. In comparison, CIA studies in mice expressing a mutant form of fibrinogen lacking the platelet integrin receptor αIIbβ3 binding motif showed that this feature of fibrinogen was not an essential driver of arthritis. The reduction in macroscopic and histological arthritic disease observed in CIA-challenged Fibγ390–396A mice was accompanied by reduced local mRNA levels of the pro-inflammatory cytokines TNF α, IL-6, and IL-1β in paws relative to paws from control animals. This data suggests that fibrin(ogen) may function upstream of local production of these critical inflammatory mediators. Consistent with this hypothesis, fibrinogen deficiency did not diminish arthritic disease driven by TNFα-overexpression. Together, these data support the view that local fibrin deposition is an important determinant of inflammatory joint disease and that one mechanism by which fibrin promotes arthritic disease is through leukocyte activation events downstream of αMβ2 engagement.

The local activation of coagulation system proteases and secondary deposition of fibrin may constitute important “danger” or “damage” signals that promote leukocyte activation events at sites of infection or injury. Consistent with this general hypothesis, we previously showed that mice expressing a mutant form of fibrinogen lacking the leukocyte integrin receptor αMβ2-binding motif displayed compromised clearance of bacteria in a model of S. aureus-induced peritonitis. Here, we show that mice with a complete genetic deficiency in fibrinogen (Fib−/−) show an even more profound sensitivity to S. aureus peritonitis. Fib−/− mice were found to succumb to bacterial doses that were rarely fatal in wildtype mice following intraperitoneal injection of wildtype S. aureus. This profound difference in survival profile was also seen when mice were challenged with S. aureus lacking either the bacterial fibrinogen-receptor (ClfA) or the bacterial prothrombin activator, coagulase. These results indicate that efficient bacterial clearance in control animals is related to host leukocyte engagement of fibrin(ogen) as opposed to bacterial engagement of this hemostatic factor. Following intraperitoneal inoculation with one billion CFU of S. aureus, control mice successful cleared >99% of the bacteria within 1 hour, whereas the number of bacteria retrieved within peritoneal lavage fluid of Fib−/− mice remained similar to the original input CFU. Microscopy studies and bacterial fate studies revealed that the rapid loss of viable bacteria in control animals was not due to either fibrinogen-dependent dissemination to other tissues nor the simple association of live bacteria with peritoneal surfaces. Neutrophils and macrophages appear to participate in early bacterial clearance in animals of both genotypes, but these cells appear to be overwhelmed and engorged with microbes in Fib-/− mice. Leukocyte composition and trafficking into the peritoneal cavity was not appreciably altered in fibrin(ogen)-deficient mice. Furthermore, B cells, T cells, mast cells and complement factors C3 and C5 were not found to be major factors in the fibrinogen-dependent bacterial clearance. Taken together, the available data indicate that a failure to fully implement leukocyte-mediated antimicrobial mechanisms is the primary impediment to bacterial clearance in Fib−/− mice. The findings are consistent with the hypothesis that leukocyte engagement of provisional fibrin matrices within challenged tissues is a critical parameter in leukocyte “target recognition” in vivo.