EMILIN2 regulates platelet activation, thrombus formation, and clot retraction.

Thrombosis, like other cardiovascular diseases, has a strong genetic component, with largely unknown determinants. EMILIN2, Elastin Microfibril Interface Located Protein2, was identified as a candidate gene for thrombosis in mouse and human quantitative trait loci studies. EMILIN2 is expressed during cardiovascular development, on cardiac stem cells, and in heart tissue in animal models of heart disease. In humans, the EMILIN2 gene is located on the short arm of Chromosome 18, and patients with partial and complete deletion of this chromosome region have cardiac malformations. To understand the basis for the thrombotic risk associated with EMILIN2, EMILIN2 deficient mice were generated. The findings of this study indicate that EMILIN2 influences platelet aggregation induced by adenosine diphosphate, collagen, and thrombin with both EMILIN2-deficient platelets and EMILIN2-deficient plasma contributing to the impaired aggregation response. Purified EMILIN2 added to platelets accelerated platelet aggregation and reduced clotting time when added to EMILIN2-deficient mouse and human plasma. Carotid occlusion time was 2-fold longer in mice with platelet-specific EMILIN2 deficiency, but stability of the clot was reduced in mice with both global EMILIN2 deficiency and with platelet-specific EMILIN2 deficiency. In vitro clot retraction was markedly decreased in EMILIN2 deficient mice, indicating that platelet outside-in signaling was dependent on EMILIN2. EMILIN1 deficient mice and EMILIN2:EMILIN1 double deficient mice had suppressed platelet aggregation and delayed clot retraction similar to EMILIN2 mice, but EMILIN2 and EMILIN1 had opposing affects on clot retraction, suggesting that EMILIN1 may attenuate the effects of EMILIN2 on platelet aggregation and thrombosis. In conclusion, these studies identify multiple influences of EMILIN2 in pathophysiology and suggest that its role as a prothrombotic risk factor may arise from its effects on platelet aggregation and platelet mediated clot retraction.

Plasminogen regulates cardiac repair after myocardial infarction through its noncanonical function in stem cell homing to the infarcted heart.

OBJECTIVES: The purpose of this study was to investigate the role of plasminogen (Plg) in stem cell-mediated cardiac repair and regeneration after myocardial infarction (MI). BACKGROUND: An MI induces irreversible tissue damage, eventually leading to heart failure. Bone marrow (BM)-derived stem cells promote tissue repair and regeneration after MI. Thrombolytic treatment with Plg activators significantly improves the clinical outcome in MI by restoring cardiac perfusion. However, the role of Plg in stem cell-mediated cardiac repair remains unclear. METHODS: An MI was induced in Plg-deficient (Plg(-/-)) and wild-type (Plg(+/+)) mice by ligation of the left anterior descending coronary artery. Stem cells were visualized by in vivo tracking of green fluorescent protein (GFP)-expressing BM cells after BM transplantation. Cardiac function, stem cell homing, and signaling pathways downstream of Plg were examined. RESULTS: Granulocyte colony-stimulating factor, a stem cell mobilizer, significantly promoted BM-derived stem cell (GFP(+)c-kit(+) cell) recruitment into the infarcted heart and stem cell-mediated cardiac repair in Plg(+/+) mice. However, Plg deficiency markedly inhibited stem cell homing and cardiac repair, suggesting that Plg is critical for stem cell-mediated cardiac repair. Moreover, Plg regulated C-X-C chemokine receptor type 4 (CXCR4) expression in stem cells in vivo and in vitro through matrix metalloproteinase-9. Lentiviral reconstitution of CXCR4 expression in BM cells successfully rescued stem cell homing to the infarcted heart in Plg-deficient mice, indicating that CXCR4 has a critical role in Plg-mediated stem cell homing after MI. CONCLUSIONS: These findings have identified a novel role for Plg in stem cell-mediated cardiac repair after MI. Thus, targeting Plg may offer a new therapeutic strategy for stem cell-mediated cardiac repair after MI.

Lp(a)/apo(a) modulate MMP-9 activation and neutrophil cytokines in vivo in inflammation to regulate leukocyte recruitment.

Lipoprotein(a) [Lp(a)] is an independent risk factor for cardiovascular diseases, but the mechanism is unclear. The pathogenic risk of Lp(a) is associated with elevated plasma concentration, small isoforms of apolipoprotein [apo(a)], the unique apolipoprotein of Lp(a), and a mimic of plasminogen. Inflammation is associated with both the initiation and recovery of cardiovascular diseases, and plasminogen plays an important role in leukocyte recruitment. Because Lp(a)/apo(a) is expressed only in primates, transgenic mice were generated, apo(a)tg and Lp(a)tg mice, to determine whether Lp(a)/apo(a) modifies plasminogen-dependent leukocyte recruitment or whether apo(a) has an independent role in vivo. Plasminogen activation was markedly reduced in apo(a)tg and Lp(a)tg mice in both peritonitis and vascular injury inflammatory models, and was sufficient to reduce matrix metalloproteinase-9 activation and macrophage recruitment. Furthermore, neutrophil recruitment and the neutrophil cytokines, CXCL1/CXCL2, were suppressed in apo(a)tg mice in the abdominal aortic aneurysm model. Reconstitution of CXCL1 or CXCL2 restored neutrophil recruitment in apo(a)tg mice. Apo(a) in the plasminogen-deficient background and Lp(a)tg mice were resistant to inhibition of macrophage recruitment that was associated with an increased accumulation of apo(a) in the intimal layer of the vessel wall. These data indicate that, in inflammation, Lp(a)/apo(a) suppresses neutrophil recruitment by plasminogen-independent cytokine inhibition, and Lp(a)/apo(a) inhibits plasminogen activation and regulates matrix metalloproteinase-9 activation and macrophage recruitment.

Genetic dissection of quantitative trait Loci for hemostasis and thrombosis on mouse chromosomes 11 and 5 using congenic and subcongenic strains.

Susceptibility to thrombosis varies in human populations as well as many inbred mouse strains. Only a small portion of this variation has been identified, suggesting that there are unknown modifier genes. The objective of this study was to narrow the quantitative trait locus (QTL) intervals previously identified for hemostasis and thrombosis on mouse distal chromosome 11 (Hmtb6) and on chromosome 5 (Hmtb4 and Hmtb5). In a tail bleeding/rebleeding assay, a reporter assay for hemostasis and thrombosis, subcongenic strain (6A-2) had longer clot stability time than did C57BL/6J (B6) mice but a similar time to the B6-Chr11(A/J) consomic mice, confirming the Hmtb6 phenotype. Six congenic and subcongenic strains were constructed for chromosome 5, and the congenic strain, 2A-1, containing the shortest A/J interval (16.6 cM, 26.6 Mbp) in the Hmtb4 region, had prolonged clot stability time compared to B6 mice. In the 3A-2 and CSS-5 mice bleeding time was shorter than for B6, mice confirming the Hmtb5 QTL. An increase in bleeding time was identified in another congenic strain (3A-1) with A/J interval (24.8 cM, 32.9 Mbp) in the proximal region of chromosome 5, confirming a QTL for bleeding previously mapped to that region and designated as Hmtb10. The subcongenic strain 4A-2 with the A/J fragment in the proximal region had a long occlusion time of the carotid artery after ferric chloride injury and reduced dilation after injury to the abdominal aorta compared to B6 mice, suggesting an additional locus in the proximal region, which was designated Hmtb11 (5 cM, 21.4 Mbp). CSS-17 mice crossed with congenic strains, 3A-1 and 3A-2, modified tail bleeding. Using congenic and subcongenic analysis, candidate genes previously identified and novel genes were identified as modifiers of hemostasis and thrombosis in each of the loci Hmtb6, Hmtb4, Hmtb10, and Hmtb11.

Lipoprotein(a) metabolism: potential sites for therapeutic targets.

Lipoprotein(a) [Lp(a)] resembles low-density lipoprotein (LDL), with an LDL lipid core and apolipoprotein B (apoB), but contains a unique apolipoprotein, apo(a). Elevated Lp(a) is an independent risk factor for coronary and peripheral vascular diseases. The size and concentration of plasma Lp(a) are related to the synthetic rate, not the catabolic rate, and are highly variable with small isoforms associated with high concentrations and pathogenic risk. Apo(a) is synthesized in the liver, although assembly of apo(a) and LDL may occur in the hepatocytes or plasma. While the uptake and clearance site of Lp(a) is poorly delineated, the kidney is the site of apo(a) fragment excretion. The structure of apo(a) has high homology to plasminogen, the zymogen for plasmin and the primary clot lysis enzyme. Apo(a) interferes with plasminogen binding to C-terminal lysines of cell surface and extracellular matrix proteins. Lp(a) and apo(a) inhibit fibrinolysis and accumulate in the vascular wall in atherosclerotic lesions. The pathogenic role of Lp(a) is not known. Small isoforms and high concentrations of Lp(a) are found in healthy octogenarians that suggest Lp(a) may also have a physiological role. Studies of Lp(a) function have been limited since it is not found in commonly studied small mammals. An important aspect of Lp(a) metabolism is the modification of circulating Lp(a), which has the potential to alter the functions of Lp(a). There are no therapeutic drugs that selectively target elevated Lp(a), but a number of possible agents are being considered. Recently, new modifiers of apo(a) synthesis have been identified. This review reports the regulation of Lp(a) metabolism and potential sites for therapeutic targets.

The plasminogen system in regulating stem cell mobilization.

The treatment of patients with hematopoietic progenitor and stem cells (HPSCs) to reconstitute hematopoiesis after myeloablative therapy or to repair ischemia after myocardial infarction has significantly improved clinical outcomes. Successful blood or bone marrow transplants require a sufficient number of HPSCs capable of homing to the injured site to regenerate tissue. Granulocyte-colony stimulating factor (G-CSF) is widely used clinically for stem cell mobilization. However, in some patients the response is poor, thus a better understanding of the mechanisms underlying G-CSF-regulated stem cell mobilization is needed. The pasminogen (Plg) system is the primary fibrinolytic pathway responsible for clot dissolution after thrombosis. Recent evidence suggests that Plg plays a pivotal role in stem cell mobilization from the bone marrow to the peripheral circulation, particularly in HPSC mobilization in response to G-CSF. This paper will discuss the potential mechanisms by which the Plg system regulates stem cell mobilization, focusing on stepwise proteolysis and signal transduction during HPSC egress from their bone marrow niche. Clear elucidation of the underlying mechanisms may lead to the development of new Plg-based therapeutic strategies to improve stem cell mobilization in treating hematological and cardiovascular diseases.

Challenges for heart disease stem cell therapy.

Cardiovascular diseases (CVDs) are the leading cause of death worldwide. The use of stem cells to improve recovery of the injured heart after myocardial infarction (MI) is an important emerging therapeutic strategy. However, recent reviews of clinical trials of stem cell therapy for MI and ischemic heart disease recovery report that less than half of the trials found only small improvements in cardiac function. In clinical trials, bone marrow, peripheral blood, or umbilical cord blood cells were used as the source of stem cells delivered by intracoronary infusion. Some trials administered only a stem cell mobilizing agent that recruits endogenous sources of stem cells. Important challenges to improve the effectiveness of stem cell therapy for CVD include: (1) improved identification, recruitment, and expansion of autologous stem cells; (2) identification of mobilizing and homing agents that increase recruitment; and (3) development of strategies to improve stem cell survival and engraftment of both endogenous and exogenous sources of stem cells. This review is an overview of stem cell therapy for CVD and discusses the challenges these three areas present for maximum optimization of the efficacy of stem cell therapy for heart disease, and new strategies in progress.

Plasminogen regulates stromal cell-derived factor-1/CXCR4-mediated hematopoietic stem cell mobilization by activation of matrix metalloproteinase-9.

OBJECTIVE: Granulocyte colony-stimulating factor (G-CSF) is a widespread therapeutic agent for stimulation of hematopoietic progenitor and stem cell (HPSC) mobilization from bone marrow (BM). Plasminogen (Plg) has been shown to be critical for HPSC mobilization. Here, we investigated the role of Plg in G-CSF-induced HPSC mobilization and the underlying mechanism. METHODS AND RESULTS: By using gene-targeted mice, our data show that Plg is required for G-CSF-induced HPSC egress to sinusoidal capillaries in BM and subsequent mobilization to peripheral circulation. G-CSF induced Plg-dependent activation of matrix metalloproteinase-9 (MMP-9) in BM, and MMP-9 neutralization or deficiency suppressed HPSC migration and mobilization. Reconstitution of MMP-9 activity by BM transplantation after lentiviral overexpression rescued HPSC mobilization in Plg-deficient mice, indicating that MMP-9 activation is required for Plg-mediated HPSC mobilization. Interestingly, after G-CSF simulation, Plg downregulated stromal cell-derived factor-1 in BM and spatiotemporally regulated the expression of C-X-C chemokine receptor type 4 (CXCR4) on mobilized HPSCs, and reconstitution of MMP-9 activity in Plg-deficient mice reversed CXCR4 expression on HPSCs in plasma and BM, suggesting that CXCR4 serves as a new downstream signal of Plg/MMP-9 in HPSC mobilization. CONCLUSIONS: Our data elucidated a novel mechanism that Plg regulates MMP-9-dependent CXCR4 expression to facilitate HPSC mobilization in response to G-CSF.

EMILIN2 (Elastin microfibril interface located protein), potential modifier of thrombosis.

BACKGROUND: Elastin microfibril interface located protein 2 (EMILIN2) is an extracellular glycoprotein associated with cardiovascular development. While other EMILIN proteins are reported to play a role in elastogenesis and coagulation, little is known about EMILIN2 function in the cardiovascular system. The objective of this study was to determine whether EMILIN2 could play a role in thrombosis. RESULTS: EMILIN2 mRNA was expressed in 8 wk old C57BL/6J mice in lung, heart, aorta and bone marrow, with the highest expression in bone marrow. In mouse cells, EMILIN2 mRNA expression in macrophages was higher than expression in endothelial cells and fibroblasts. EMILIN2 was identified with cells and extracellular matrix by immunohistochemistry in the carotid and aorta. After carotid ferric chloride injury, EMILIN2 was abundantly expressed in the thrombus and inhibition of EMILIN2 increased platelet de-aggregation after ADP-stimulated platelet aggregation. CONCLUSIONS: These results suggest EMILIN2 could play a role in thrombosis as a constituent of the vessel wall and/or a component of the thrombus.

Mouse chromosome 17 candidate modifier genes for thrombosis.

Two overlapping quantitative trait loci (QTLs) for clot stability, Hmtb8 and Hmtb9, were identified on mouse chromosome 17 in an F2 intercross derived from C57BL/6J (B6) and B6-Chr17(A/J) (B6-Chr17) mouse strains. The intervals were in synteny with a QTL for thrombotic susceptibility on chromosome 18 in a human study, and there were 23 homologs between mouse and human. The objective of this study was to determine whether any of these genes in the syntenic region are likely candidates as modifiers for clot stability. Seven genes, Twsg1, Zfp161, Dlgap1, Ralbp1, Myom1, Rab31, and Emilin2, of the 23 genes with single nucleotide polymorphisms (SNPs) in the mRNA-UTR had differential expression in B6 and A/J mice. Dlgap1, Ralbp1, Myom1, and Emilin2 also had nonsynonymous SNPs. In addition, two other genes had nonsynonymous SNPs, Lama1 and Ndc80. Of these nine candidate genes, Emilin2 was selected for further analysis since other EMILIN (Elastin Microfibril Interface Located Protein) proteins have known functions in vascular structure and coagulation. Differences were found between B6 and A/J mice in vessel wall architecture and EMILIN2 protein in plasma, carotid vessel wall, and thrombi formed after ferric chloride injury. In B6-Chr17(A/J) mice both clot stability and Emilin2 mRNA expression were higher compared to those in B6 and A/J mice, suggesting the exposure of epistatic interactions. Although other homologous genes in the QTL region cannot be ruled out as causative genes, further investigation of Emilin2 as a candidate gene for thrombosis susceptibility is warranted.

Does plasmin have anticoagulant activity?

The coagulation and fibrinolytic pathways regulate hemostasis and thrombosis, and an imbalance in these pathways may result in pathologic hemophilia or thrombosis. The plasminogen system is the primary proteolytic pathway for fibrinolysis, but also has important proteolytic functions in cell migration, extracellular matrix degradation, metalloproteinase activation, and hormone processing. Several studies have demonstrated plasmin cleavage and inactivation of several coagulation factors, suggesting plasmin may be not only be the primary fibrinolytic enzyme, but may have anticoagulant properties as well. The objective of this review is to examine both in vitro and in vivo evidence for plasmin inactivation of coagulation, and to consider whether plasmin may act as a physiological regulator of coagulation. While several studies have demonstrated strong evidence for plasmin cleavage and inactivation of coagulation factors FV, FVIII, FIX, and FX in vitro, in vivo evidence is lacking for a physiologic role for plasmin as an anticoagulant. However, inactivation of coagulation factors by plasmin may be useful as a localized anticoagulant therapy or as a combined thrombolytic and anticoagulant therapy.

A physiological function for apolipoprotein(a): a natural regulator of the inflammatory response.

Structural similarities between apolipoprotein(a) (apo(a)), the unique apoprotein of lipoprotein(a), and plasminogen, the zymogen of plasmin, can interfere with functions of plasmin (ogen) in vitro. The purpose of this study was to evaluate the role of apo(a) in inflammation in vivo using apo(a) transgenic mice and to determine if effects are plasminogen-dependent using backgrounds that are either plasminogen-replete or plasminogen-deficient. After administration of peritoneal inflammatory stimuli, thioglycollate, bioimplants or lipopolysaccharide, the number of responding peritoneal neutrophils and macrophages were quantified. Apo(a), in either wild-type or plasminogen deficient backgrounds, inhibited neutrophil recruitment but had no effect on plasminogen-dependent macrophage recruitment. Macrophage-inflammatory protein-2, a neutrophil chemokine, was reduced in apo(a) mice, and injection of this chemokine prior to thioglycollate restored neutrophil recruitment in apo(a) transgenic mice. In the lipopolysaccharide model, mice with apo(a), unlike mice without apo(a), did not increase neutrophil recruitment in response to the stimulus. In the bioimplant model, neutrophil recruitment and neutrophil cytokines were reduced in apo(a)tg mice but only in a plasminogen-deficient background. These results indicate for the first time that apo(a), independent of plasminogen interaction, inhibits neutrophil recruitment in vivo in diverse peritoneal inflammatory models. Hence, apo(a) may function as a cell specific suppressor of the inflammatory response.

Quantitative trait locus analysis for hemostasis and thrombosis.

Susceptibility to thrombosis varies in human populations as well as many in inbred mouse strains. The objective of this study was to characterize the genetic control of thrombotic risk on three chromosomes. Previously, utilizing a tail-bleeding/rebleeding assay as a surrogate of hemostasis and thrombosis function, three mouse chromosome substitution strains (CSS) (B6-Chr5(A/J), Chr11(A/J), Chr17(A/J)) were identified (Hmtb1, Hmtb2, Hmtb3). The tail-bleeding/rebleeding assay is widely used and distinguishes mice with genetic defects in blood clot formation or dissolution. In the present study, quantitative trait locus (QTL) analysis revealed a significant locus for rebleeding (clot stability) time (time between cessation of initial bleeding and start of the second bleeding) on chromosome 5, suggestive loci for bleeding time (time between start of bleeding and cessation of bleeding) also on chromosomes 5, and two suggestive loci for clot stability on chromosome 17 and one on chromosome 11. The three CSS and the parent A/J had elevated clot stability time. There was no interaction of genes on chromosome 11 with genes on chromosome 5 or chromosome 17. On chromosome 17, twenty-three candidate genes were identified in synteny with previously identified loci for thrombotic risk on human chromosome 18. Thus, we have identified new QTLs and candidate genes not previously known to influence thrombotic risk.

Inflammatory macrophage migration requires MMP-9 activation by plasminogen in mice.

Inflammation plays a critical role in the development of cardiovascular diseases. Infiltration of leukocytes to sites of injury requires their exit from the blood and migration across basement membrane; this process has been postulated to require remodeling of the ECM. Plasminogen (Plg) is a protease that binds to the ECM and, upon conversion to plasmin, degrades multiple ECM proteins. In addition, plasmin directly activates MMPs. Here, we used Plg(-/-) mice to investigate the role of Plg in inflammatory leukocyte migration. After induction of peritonitis by thioglycollate injection, we found that Plg(-/-) mice displayed diminished macrophage trans-ECM migration and decreased MMP-9 activation. Furthermore, injection of the active form of MMP-9 in Plg(-/-) mice rescued macrophage migration in this model. We used periaortic application of CaCl2 to induce abdominal aortic aneurysm (AAA) and found that Plg(-/-) mice displayed reduced macrophage infiltration and were protected from aneurysm formation. Administration of active MMP-9 to Plg(-/-) mice promoted macrophage infiltration and the development of AAA. These data suggest that Plg regulates macrophage migration in inflammation via activation of MMP-9, which, in turn, regulates the ability of the cells to migrate across ECM. Thus, targeting the Plg/MMP-9 pathway may be an attractive approach to regulate inflammatory responses and AAA development.

Genetic background determines response to hemostasis and thrombosis.

BACKGROUND: Thrombosis is the fatal and disabling consequence of cardiovascular diseases, the leading cause of mortality and morbidity in Western countries. Two inbred mouse strains, C57BL/6J and A/J, have marked differences in susceptibility to obesity, atherosclerosis, and vessel remodeling. However, it is unclear how these diverse genetic backgrounds influence pathways known to regulate thrombosis and hemostasis. The objective of this study was to evaluate thrombosis and hemostasis in these two inbred strains and determine the phenotypic response of A/J chromosomes in the C57BL/6J background. METHODS: A/J and C57Bl/6J mice were evaluated for differences in thrombosis and hemostasis. A thrombus was induced in the carotid artery by application of the exposed carotid to ferric chloride and blood flow measured until the vessel occluded. Bleeding and rebleeding times, as surrogate markers for thrombosis and hemostasis, were determined after clipping the tail and placing in warm saline. Twenty-one chromosome substitution strains, A/J chromosomes in a C57BL/6J background, were screened for response to the tail bleeding assay. RESULTS: Thrombus occlusion time was markedly decreased in the A/J mice compared to C57BL/6J mice. Tail bleeding time was similar in the two strains, but rebleeding time was markedly increased in the A/J mice compared to C57BL/6J mice. Coagulation times and tail morphology were similar, but tail collagen content was higher in A/J than C57BL/6J mice. Three chromosome substitution strains, B6-Chr5A/J, B6-Chr11A/J, and B6-Chr17A/J, were identified with increased rebleeding time, a phenotype similar to A/J mice. Mice heterosomic for chromosomes 5 or 17 had rebleeding times similar to C57BL/6J mice, but when these two chromosome substitution strains, B6-Chr5A/J and B6-Chr17A/J, were crossed, the A/J phenotype was restored in these doubly heterosomic progeny. CONCLUSION: These results indicate that susceptibility to arterial thrombosis and haemostasis is remarkably different in C57BL/and A/J mice. Three A/J chromosome substitution strains were identified that expressed a phenotype similar to A/J for rebleeding, the C57Bl/6J background could modify the A/J phenotype, and the combination of two A/J QTL could restore the phenotype. The diverse genetic backgrounds and differences in response to vascular injury induced thrombosis and the tail bleeding assay, suggest the potential for identifying novel genetic determinants of thrombotic risk.

The functions of plasminogen in cardiovascular disease.

Plasminogen (Plg) and its derivative serine protease, plasmin, together with the activators, inhibitors, modulators, and substrates of the Plg network, are postulated to regulate a wide variety of biologic responses that could influence cardiovascular disease. The development of Plg-deficient mice has provided an incisive approach to test these proposed functions in vivo. Several different models of atherosclerosis, restenosis, aneurysm, and thrombosis have been analyzed in these mice and have demonstrated profound effects of Plg on these events as well as on the inflammatory response, which contributes to these cardiovascular diseases. Plasmin (ogen) may influence the progression of cardiovascular diseases through its degradation of matrix proteins, including fibrin; its activation of matrix metalloproteinases; its regulation of growth factor and chemokine pathways; or its influence on directed cell migration. Dissection of these mechanisms represents a future challenge toward understanding the roles of Plg in vivo.

In vivo plasminogen deficiency reduces fat accumulation.

Obesity and non-insulin dependent diabetes are associated with a decrease in fibrinolysis, which is mediated by the plasminogen system. The purpose of the current study was to investigate the role of the plasminogen system in the reduced body weight of the plasminogen deficient (Plg-/-) mice. In this study we have found that the reduced body weight in Plg-/- mice is due to a reduced rate of the adipose tissue (25% less) and whole body fat (30% less) accumulation during growth in Plg-/- compared to wild-type (WT) littermates. When the mice are fed a high fat-lipogenic diet, adipose tissue accumulation increases in the Plg-/- mice indicating that the capacity for lipid filling of cells was not blocked. In addition, glycerol phosphate dehydrogenase, a marker of late differentiation, was not different in the depots from WT and Plg-/- mice. The number of stromal cells (number x 10(5)/g adipose tissue), isolated from inguinal (Plg-/- 3.4 +/- 1.2. n = 6; WT 0.17 +/- 0.07, n = 7, p < 0.02) and gonadal (Plg-/- 11.0 +/- 0.4, n = 6; WT 3.1 +/- 0.7, n = 7, p < 0.05) fat depots. was markedly higher in the depots from the Plg-/- mice than WT mice. Differentiation of stromal cells in culture from the Plg-/- mice was reduced compared to cells from WT mice. These results suggest that differences in the stromal cell population are responsible for the reduced adipose tissue accumulation in the Plg-/- mice, and that the plasminogen system plays an important role in adipose tissue accumulation.