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•Filamin A associates with both inactive and active platelet integrin αIIbβ3 to differentially regulate bidirectional signaling.•Filamin A induces a structural transition in the αIIb CT to strongly link αIIbβ3 with actin to promote outside-in signaling.

•Kindlin-3 promotes EBI formation and regulates erythropoiesis.•By linking to F-actin and spectrin, kindlin-3 maintains the erythrocyte cytoskeleton to support normal erythrocyte shape and deformability.

Plasminogen/plasmin is a serine protease system primarily responsible for degrading fibrin within blood clots. Plasminogen mediates its functions by interacting with plasminogen receptors on the cell surface. H2B, one such plasminogen receptor, is found on the surface of several cell types including macrophages. Both basic and clinical studies support the role of plasminogen in the process of foam cell formation (FCF), a hallmark of atherosclerosis. Growing evidence also implicates serine protease‐activated receptors (PARs) in atherosclerosis. These receptors are also found on macrophages, and plasmin is capable of activating PAR1 and PAR4. The goal of this study was to determine the extent of H2B's contribution to plasminogen‐mediated FCF by macrophages and if PARs are involved in this process.

Plasminogen/plasmin is a serine protease system primarily responsible for degrading fibrin within blood clots. Plasminogen mediates its functions by interacting with plasminogen receptors on the cell surface. H2B, one such plasminogen receptor, is found on the surface of several cell types including macrophages. Both basic and clinical studies support the role of plasminogen in the process of foam cell formation (FCF), a hallmark of atherosclerosis. Growing evidence also implicates serine protease‐activated receptors (PARs) in atherosclerosis. These receptors are also found on macrophages, and plasmin is capable of activating PAR1 and PAR4. The goal of this study was to determine the extent of H2B's contribution to plasminogen‐mediated FCF by macrophages and if PARs are involved in this process. Treating macrophages with plasminogen increases their oxidized low‐density lipoprotein uptake and plasminogen‐mediated foam cell formation (Plg‐FCF) significantly. The magnitude of Plg‐FCF correlates with cell‐surface expression of the H2B level. H2B blockade or downregulation reduces Plg‐FCF, whereas its overexpression or high endogenous levels increases Plg‐FCF. Modulating PAR1 level in mouse macrophages affects Plg‐FCF. Activation/overexpression of PAR1 increases and its blockade/knockdown reduces this response. Confocal imaging indicates that both H2B and PAR1 colocalize with clathrin coated pits on the surface of macrophages, and reducing expression of clathrin or interfering with the clathrin‐coated pits integrity reduces Plg‐FCF. Our data indicate that the magnitude of Plg‐FCF by macrophages is proportional to the H2B levels and demonstrate for the first time that PAR1 is involved in this process and that the integrity of clathrin‐coated pits is required for the full effect of Plg‐induced FCF.

Current antithrombotic drugs, including widely used antiplatelet agents and anticoagulants, are associated with significant bleeding risk. Emerging experimental evidence suggests that the molecular and cellular mechanisms of hemostasis and thrombosis can be separated, thereby increasing the possibility of new antithrombotic therapeutic targets with reduced bleeding risk. We review new coagulation and platelet targets and highlight the interaction between integrin αMβ2 (Mac-1, CD11b/CD18) on leukocytes and GPIbα on platelets that seems to distinguish thrombosis from hemostasis.

Current antithrombotic drugs, including widely used antiplatelet agents and anticoagulants, are associated with significant bleeding risk. Emerging experimental evidence suggests that the molecular and cellular mechanisms of hemostasis and thrombosis can be separated, thereby increasing the possibility of new antithrombotic therapeutic targets with reduced bleeding risk. We review new coagulation and platelet targets and highlight the interaction between integrin αMβ2(Mac-1, CD11b/CD18) on leukocytes and GPIbα on platelets that seems to distinguish thrombosis from hemostasis.

Kindlins are essential for integrin-mediated cell adhesion. This study focuses on the evolutionary origin and subsequent functional specialization of kindlins and their role in evolutionary adaptation of cell adhesiveness in multicellular organisms.

The phagocytic function of macrophages plays a pivotal role in eliminating apoptotic cells and invading pathogens. Evidence implicating plasminogen (Plg), the zymogen of plasmin, in phagocytosis is extremely limited with the most recent in vitro study showing that plasmin acts on prey cells rather than on macrophages. Here, we use apoptotic thymocytes and immunoglobulin opsonized bodies to show that Plg exerts a profound effect on macrophage-mediated phagocytosis in vitro and in vivo. Plg enhanced the uptake of these prey by J774A.1 macrophage-like cells by 3.5- to fivefold Plg receptors and plasmin proteolytic activity were required for phagocytosis of both preys. Compared with Plg+/+ mice, Plg−/− mice exhibited a 60% delay in clearance of apoptotic thymocytes by spleen and an 85% reduction in uptake by peritoneal macrophages. Phagocytosis of antibody-mediated erythrocyte clearance by liver Kupffer cells was reduced by 90% in Plg−/− mice compared with Plg+/+ mice. A gene array of splenic and hepatic tissues from Plg−/− and Plg+/+ mice showed downregulation of numerous genes in Plg−/− mice involved in phagocytosis and regulation of phagocytic gene expression was confirmed in macrophage-like cells. Thus, Plg may play an important role in innate immunity by changing expression of genes that contribute to phagocytosis.

The phagocytic function of macrophages plays a pivotal role in eliminating apoptotic cells and invading pathogens. Evidence implicating plasminogen (Plg), the zymogen of plasmin, in phagocytosis is extremely limited with the most recent in vitro study showing that plasmin acts on prey cells rather than on macrophages. Here, we use apoptotic thymocytes and immunoglobulin opsonized bodies to show that Plg exerts a profound effect on macrophage-mediated phagocytosis in vitro and in vivo. Plg enhanced the uptake of these prey by J774A.1 macrophage-like cells by 3.5- to fivefold Plg receptors and plasmin proteolytic activity were required for phagocytosis of both preys. Compared with Plg+/+mice, Plg−/−mice exhibited a 60% delay in clearance of apoptotic thymocytes by spleen and an 85% reduction in uptake by peritoneal macrophages. Phagocytosis of antibody-mediated erythrocyte clearance by liver Kupffer cells was reduced by 90% in Plg−/−mice compared with Plg+/+mice. A gene array of splenic and hepatic tissues from Plg−/−and Plg+/+mice showed downregulation of numerous genes in Plg−/−mice involved in phagocytosis and regulation of phagocytic gene expression was confirmed in macrophage-like cells. Thus, Plg may play an important role in innate immunity by changing expression of genes that contribute to phagocytosis.

This review considers recent developments concerning the role of integrins in vascular biology with a specific emphasis on integrin activation, and the crosstalk between integrins and growth factor receptors.

Kindlin-2, a widely distributed cytoskeletal protein, has been implicated in integrin activation, and its absence is embryonically lethal in mice. In the present study, we tested whether hemostasis might be perturbed in kindlin-2+/− mice. Bleeding time and carotid artery occlusion time were significantly prolonged in kindlin-2+/− mice. Whereas plasma concentrations/activities of key coagulation/fibrinolytic proteins and platelet counts and aggregation were similar in wild-type and kindlin-2+/− mice, kindlin-2+/− endothelial cells (ECs) showed enhanced inhibition of platelet aggregation induced by adenosine 5′-diphosphate (ADP) or low concentrations of other agonists. Cell-surface expression of 2 enzymes involved in ADP/adenosine 5′-monophosphate (AMP) degradation, adenosine triphosphate (ATP) diphosphohydrolase (CD39) and ecto-5′-nucleotidase (CD73) were increased twofold to threefold on kindlin-2+/− ECs, leading to enhanced ATP/ADP catabolism and production of adenosine, an inhibitor of platelet aggregation. Trafficking of CD39 and CD73 at the EC surface was altered in kindlin-2+/− mice. Mechanistically, this was attributed to direct interaction of kindlin-2 with clathrin heavy chain, thereby controlling endocytosis and recycling of CD39 and CD73. The interaction of kindlin-2 with clathrin was independent of its integrin binding site but still dependent on a site within its F3 subdomain. Thus, kindlin-2 regulates trafficking of EC surface enzymes that control platelet responses and hemostasis.

Kindlin-2, a widely distributed cytoskeletal protein, has been implicated in integrin activation, and its absence is embryonically lethal in mice. In the present study, we tested whether hemostasis might be perturbed in kindlin-2+/−mice. Bleeding time and carotid artery occlusion time were significantly prolonged in kindlin-2+/−mice. Whereas plasma concentrations/activities of key coagulation/fibrinolytic proteins and platelet counts and aggregation were similar in wild-type and kindlin-2+/−mice, kindlin-2+/−endothelial cells (ECs) showed enhanced inhibition of platelet aggregation induced by adenosine 5′-diphosphate (ADP) or low concentrations of other agonists. Cell-surface expression of 2 enzymes involved in ADP/adenosine 5′-monophosphate (AMP) degradation, adenosine triphosphate (ATP) diphosphohydrolase (CD39) and ecto-5′-nucleotidase (CD73) were increased twofold to threefold on kindlin-2+/−ECs, leading to enhanced ATP/ADP catabolism and production of adenosine, an inhibitor of platelet aggregation. Trafficking of CD39 and CD73 at the EC surface was altered in kindlin-2+/−mice. Mechanistically, this was attributed to direct interaction of kindlin-2 with clathrin heavy chain, thereby controlling endocytosis and recycling of CD39 and CD73. The interaction of kindlin-2 with clathrin was independent of its integrin binding site but still dependent on a site within its F3 subdomain. Thus, kindlin-2 regulates trafficking of EC surface enzymes that control platelet responses and hemostasis.

Kindlin-2, a widely distributed cytoskeletal protein, has been implicated in integrin activation, and its absence is embryonically lethal in mice and causes severe developmental defects in zebrafish. Knockdown of kindlin-2 levels in endothelial cells resulted in defective adhesive and migratory responses, suggesting that angiogenesis might be aberrant even with partial reduction of kindlin-2. This hypothesis has now been tested in the kindlin-2+/− mice. RM1 prostate tumors grown in kindlin-2+/− mice had fewer blood vessels, which were thinner and shorter and supported less tumor growth compared with wild-type littermates. The vessels that did form in the kindlin-2+/− mice lacked smooth muscle cells and pericytes and had thinner basement membranes, indicative of immature vessels. VEGF-induced angiogenesis in matrigel implants was also abnormal in the kindlin-2+/− mice. Vessels in the kindlin-2+/− mice were leaky, and BM transplantation from kindlin-2+/− to WT mice did not correct this defect. Endothelial cells derived from kindlin-2+/− mice had integrin expression levels similar to WT mice but reduced αVβ3-dependent signaling, migration, adhesion, spreading, and tube formation. Developmental angiogenesis was markedly impaired by kindlin-2 morpholinos in zebrafish. Taken together, kindlin-2 plays an important role in pathologic and developmental angiogenesis, which arises from defective activation of integrin αVβ3.

Kindlin-2, a widely distributed cytoskeletal protein, has been implicated in integrin activation, and its absence is embryonically lethal in mice and causes severe developmental defects in zebrafish. Knockdown of kindlin-2 levels in endothelial cells resulted in defective adhesive and migratory responses, suggesting that angiogenesis might be aberrant even with partial reduction of kindlin-2. This hypothesis has now been tested in the kindlin-2+/−mice. RM1 prostate tumors grown in kindlin-2+/−mice had fewer blood vessels, which were thinner and shorter and supported less tumor growth compared with wild-type littermates. The vessels that did form in the kindlin-2+/−mice lacked smooth muscle cells and pericytes and had thinner basement membranes, indicative of immature vessels. VEGF-induced angiogenesis in matrigel implants was also abnormal in the kindlin-2+/−mice. Vessels in the kindlin-2+/−mice were leaky, and BM transplantation from kindlin-2+/−to WT mice did not correct this defect. Endothelial cells derived from kindlin-2+/−mice had integrin expression levels similar to WT mice but reduced αVβ3-dependent signaling, migration, adhesion, spreading, and tube formation. Developmental angiogenesis was markedly impaired by kindlin-2 morpholinos in zebrafish. Taken together, kindlin-2 plays an important role in pathologic and developmental angiogenesis, which arises from defective activation of integrin αVβ3.

The Kindlin family of intracellular proteins has recently emerged as key regulators of cellular functions and cell-matrix interactions. The 3 members of this family, Kindlin-1, -2, and -3, perform an essential role in activation of integrin adhesion receptors, and expression of at least 1 Kindlin paralog is required to enable integrin activation in physiologically relevant settings. In humans, deficiencies in Kindlin-3 lead to a number of abnormalities affecting hemostasis, the immune system, and bone function, whereas the lack of Kindlin-1 causes profound skin defects. The importance of Kindlins is underscored by the results of animal knockout studies, which clearly show the indispensable and nonredundant functions of all 3 Kindlins in development and normal physiology. This review discusses recent progress in the studies of Kindlin protein family, emphasizing newly identified functions and potential mechanisms underlying differential activities of the family members.

The Kindlin family of intracellular proteins has recently emerged as key regulators of cellular functions and cell-matrix interactions. The 3 members of this family, Kindlin-1, -2, and -3, perform an essential role in activation of integrin adhesion receptors, and expression of at least 1 Kindlin paralog is required to enable integrin activation in physiologically relevant settings. In humans, deficiencies in Kindlin-3 lead to a number of abnormalities affecting hemostasis, the immune system, and bone function, whereas the lack of Kindlin-1 causes profound skin defects. The importance of Kindlins is underscored by the results of animal knockout studies, which clearly show the indispensable and nonredundant functions of all 3 Kindlins in development and normal physiology. This review discusses recent progress in the studies of Kindlin protein family, emphasizing newly identified functions and potential mechanisms underlying differential activities of the family members.

There are three kindlin family members in vertebrates, which have high-sequence homology and a common organization signature with a C-terminal 4.1, ezrin, radixin, moesin (FERM) domain bisected by a pleckstrin-homology domain. Although the cell and tissue distributions of the three kindlins differ, there is a consistent and close interrelationship between kindlins and integrins, and alterations of kindlin expression affect integrin-dependent functions. However, in-vivo data on the functions of the kindlins and their mechanisms of action have been lacking.

Leukocyte-derived microparticles (MPs) are markers of cardiovascular diseases and contribute to pathogenesis by their interaction with various cell types. The presence and activation state of a multifunctional leukocyte receptor, integrin αMβ2 (CD11b/18), on MPs derived from human neutrophils (PMNs) were examined. αMβ2 expression was significantly enhanced on MPs derived from stimulated compared with resting PMNs. Furthermore, αMβ2 on MPs from stimulated but not resting PMNs was in an activated conformation because it was capable of binding activation-specific monoclonal antibodies (CBRM1/5 and mAb24) and soluble fibrinogen. MPs expressing active αMβ2 interacted with and were potent activators of resting platelets as assessed by induction of P-selectin expression and activation of αIIbβ3. With the use of function-blocking antibodies and MPs obtained from αM−/−-deficient mice, we found that engagement of GPIbα on platelets by αMβ2 on MPs plays a pivotal role in MP binding. Platelet activation by MPs occurs by a pathway dependent on Akt phosphorylation. PSGL-1/P-selectin interaction also is involved in the conjugation of MPs to platelets, and the combination of blocking reagents to both αMβ2/GPIbα and to PSGL-1/P-selectin completely abrogates MP-induced platelet activation. Thus, cooperation of these 2 receptor/counterreceptor systems regulates the prothrombotic properties of PMN-derived MPs.

Leukocyte-derived microparticles (MPs) are markers of cardiovascular diseases and contribute to pathogenesis by their interaction with various cell types. The presence and activation state of a multifunctional leukocyte receptor, integrin αMβ2(CD11b/18), on MPs derived from human neutrophils (PMNs) were examined. αMβ2expression was significantly enhanced on MPs derived from stimulated compared with resting PMNs. Furthermore, αMβ2on MPs from stimulated but not resting PMNs was in an activated conformation because it was capable of binding activation-specific monoclonal antibodies (CBRM1/5 and mAb24) and soluble fibrinogen. MPs expressing active αMβ2interacted with and were potent activators of resting platelets as assessed by induction of P-selectin expression and activation of αIIbβ3. With the use of function-blocking antibodies and MPs obtained from αM−/−-deficient mice, we found that engagement of GPIbα on platelets by αMβ2on MPs plays a pivotal role in MP binding. Platelet activation by MPs occurs by a pathway dependent on Akt phosphorylation. PSGL-1/P-selectin interaction also is involved in the conjugation of MPs to platelets, and the combination of blocking reagents to both αMβ2/GPIbα and to PSGL-1/P-selectin completely abrogates MP-induced platelet activation. Thus, cooperation of these 2 receptor/counterreceptor systems regulates the prothrombotic properties of PMN-derived MPs.

Plasminogen (Plg) facilitates inflammatory cell recruitment, a function that depends upon its binding to Plg receptors (Plg-Rs). However, the Plg-Rs that are critical for cell migration are not well defined. Three previously characterized Plg-Rs (α-enolase, annexin 2, and p11) and a recently identified Plg-R (histone H2B [H2B]) were assessed for their contribution to Plg binding and function on macrophages. Two murine macrophage cell lines (RAW 264.7 and J774A.1) and mouse peritoneal macrophages induced by thioglycollate were analyzed. All 4 Plg-Rs were present on the surface of these cells and showed enhanced expression on the thioglycollate-induced macrophages compared with peripheral blood monocytes. Using blocking Fab fragments to each Plg-R, H2B supported approximately 50% of the Plg binding capacity, whereas the other Plg-Rs contributed less than 25%. Anti-H2B Fab also demonstrated a major role of this Plg-R in plasmin generation and matrix invasion. When mice were treated intravenously with anti-H2B Fab, peritoneal macrophage recruitment in response to thioglycollate was reduced by approximately 45% at 24, 48, and 72 hours, with no effect on blood monocyte levels. Taken together, these data suggest that multiple Plg-Rs do contribute to Plg binding to macrophages, and among these, H2B plays a very prominent and functionally important role.

Plasminogen (Plg) facilitates inflammatory cell recruitment, a function that depends upon its binding to Plg receptors (Plg-Rs). However, the Plg-Rs that are critical for cell migration are not well defined. Three previously characterized Plg-Rs (α-enolase, annexin 2, and p11) and a recently identified Plg-R (histone H2B [H2B]) were assessed for their contribution to Plg binding and function on macrophages. Two murine macrophage cell lines (RAW 264.7 and J774A.1) and mouse peritoneal macrophages induced by thioglycollate were analyzed. All 4 Plg-Rs were present on the surface of these cells and showed enhanced expression on the thioglycollate-induced macrophages compared with peripheral blood monocytes. Using blocking Fab fragments to each Plg-R, H2B supported approximately 50% of the Plg binding capacity, whereas the other Plg-Rs contributed less than 25%. Anti-H2B Fab also demonstrated a major role of this Plg-R in plasmin generation and matrix invasion. When mice were treated intravenously with anti-H2B Fab, peritoneal macrophage recruitment in response to thioglycollate was reduced by approximately 45% at 24, 48, and 72 hours, with no effect on blood monocyte levels. Taken together, these data suggest that multiple Plg-Rs do contribute to Plg binding to macrophages, and among these, H2B plays a very prominent and functionally important role.

alpha1-Adrenergic receptor (alpha1-ARs) subtypes (alpha1A, alpha1B, and alpha1D) regulate multiple signal pathways, such as phospholipase C, protein kinase C (PKC), and mitogen-activated protein kinases. We employed oligonucleotide microarray technology to explore the effects of both short- (1 h) and long-term (18 h) activation of the alpha1A-AR to enable RNA changes to occur downstream of earlier well characterized signaling pathways, promoting novel couplings. Polymerase chain reaction (PCR) studies confirmed that PKC was a critical regulator of alpha1A-AR-mediated gene expression, and secreted interleukin (IL)-6 also contributed to gene expression alterations. We next focused on two novel signaling pathways that might be mediated through alpha1A-AR stimulation because of the clustering of gene expression changes for cell adhesion/motility (syndecan-4 and tenascin-C) and hyaluronan (HA) signaling. We confirmed that alpha1-ARs induced adhesion in three cell types to vitronectin, an interaction that was also integrin-, FGF7-, and PKC-dependent. alpha1-AR activation also inhibited cell migration, which was integrin- and PKC-independent but still required secretion of FGF7. alpha1-AR activation also increased the expression and deposition of HA, a glycosaminoglycan, which displayed two distinct structures: pericellular coats and long cable structures, as well as increasing expression of the HA receptor, CD44. Long cable structures of HA can bind leukocytes, which this suggests that alpha1-ARs may be involved in proinflammatory responses. Our results indicate alpha1-ARs induce the secretion of factors that interact with the extracellular matrix to regulate cell adhesion, motility and proinflammatory responses through novel signaling pathways.

High-throughput genomic technology identified an association between a single nucleotide polymorphism (SNP), a proline (P387) rather than the predominant alanine (A387) at position 387 in thrombospondin-4 (TSP-4) and premature myocardial infarction. The inflammatory hypothesis of atherosclerosis invokes a prominent role of leukocytes and cytokines in pathogenesis. As the expression of TSP-4 by vascular cells permits its exposure to circulating leukocytes, the interactions of human neutrophils (polymorphonuclear leukocytes [PMNs]) with both TSP-4 variants were investigated. Phorbol 12-myristate 13-acetate (PMA)–stimulated PMNs adhered and migrated well and equally on the TSP-4 variants. Integrin αMβ2was identified as the TSP-4 receptor mediating these responses, and the 3 epidermal growth factor (EGF)–like domains of TSP-4 harboring the SNPs interacted with the αMI-domain. Despite the similarity in these responses, the P387 variant induced more robust tyrosine phosphorylation of the stress-related mitogen-activated protein kinases (MAPKs): p38MAPK and c-Jun NH2-terminal kinase (JNK), as well as signal transducer and activator of transcription-1 (STAT1) and heat shock protein 27 (HSP27) than the A387 variant. Additionally, cells adherent to P387 TSP-4 variant released 4-fold more H2O2and secreted 2-fold more interleukin 8 (IL-8) as compared with the A387. H2O2release and p38MAPK activation were totally inhibited by blockade of αMβ2. Thus, αMβ2plays a central role in proinflammatory activities of TSP-4 (P387) and may contribute to the prothrombotic phenotype associated with this variant.

High-throughput genomic technology identified an association between a single nucleotide polymorphism (SNP), a proline (P387) rather than the predominant alanine (A387) at position 387 in thrombospondin-4 (TSP-4) and premature myocardial infarction. The inflammatory hypothesis of atherosclerosis invokes a prominent role of leukocytes and cytokines in pathogenesis. As the expression of TSP-4 by vascular cells permits its exposure to circulating leukocytes, the interactions of human neutrophils (polymorphonuclear leukocytes [PMNs]) with both TSP-4 variants were investigated. Phorbol 12-myristate 13-acetate (PMA)–stimulated PMNs adhered and migrated well and equally on the TSP-4 variants. Integrin αMβ2 was identified as the TSP-4 receptor mediating these responses, and the 3 epidermal growth factor (EGF)–like domains of TSP-4 harboring the SNPs interacted with the αMI-domain. Despite the similarity in these responses, the P387 variant induced more robust tyrosine phosphorylation of the stress-related mitogen-activated protein kinases (MAPKs): p38MAPK and c-Jun NH2-terminal kinase (JNK), as well as signal transducer and activator of transcription-1 (STAT1) and heat shock protein 27 (HSP27) than the A387 variant. Additionally, cells adherent to P387 TSP-4 variant released 4-fold more H2O2 and secreted 2-fold more interleukin 8 (IL-8) as compared with the A387. H2O2 release and p38MAPK activation were totally inhibited by blockade of αMβ2. Thus, αMβ2 plays a central role in proinflammatory activities of TSP-4 (P387) and may contribute to the prothrombotic phenotype associated with this variant.

P-selectin glycoprotein ligand 1 (PSGL-1, CD162) and integrin αMβ2 (Mac-1, CD11bCD18) are leukocyte adhesion molecules essential for innate immunity and inflammation. The interaction of PSGL-1 with P-selectin (CD62P) mediates tethering, rolling, and weak adhesion of leukocytes, during which they become sufficiently activated in situ by locally released or displayed cytokines and chemoattractants for integrin-mediated firm adhesion. However, communication between P-selectin and the integrin, whether P-selectin can trigger β2-integrin activation, remains controversial. We found that P-selectin immunoglobulin chimera and PSGL-1 monoclonal antibodies (mAbs) increased adhesion of human neutrophils to immobilized, but not soluble, fibrinogen. This intermediate state of neutrophil adhesion was defined by moderate clustering of integrin αMβ2, no increase in CBRM1/5 (a mAb specific for the activation epitope on the αM subunit) recognition, and no increase in surface expression of αMβ2, whereas phorbol myristate acetate (PMA) induced extensive changes in these 3 parameters. Furthermore, platelet-activating factor or interleukin 8 acted in concert with P-selectin for further enhancing the activation of αMβ2. We thus propose a model in which P-selectin induces an intermediate state of integrin activation and then cooperates with other extracellular stimuli to support maximal adhesion of human neutrophils.

P-selectin glycoprotein ligand 1 (PSGL-1, CD162) and integrin αMβ2 (Mac-1, CD11bCD18) are leukocyte adhesion molecules essential for innate immunity and inflammation. The interaction of PSGL-1 with P-selectin (CD62P) mediates tethering, rolling, and weak adhesion of leukocytes, during which they become sufficiently activated in situ by locally released or displayed cytokines and chemoattractants for integrin-mediated firm adhesion. However, communication between P-selectin and the integrin, whether P-selectin can trigger β2-integrin activation, remains controversial. We found that P-selectin immunoglobulin chimera and PSGL-1 monoclonal antibodies (mAbs) increased adhesion of human neutrophils to immobilized, but not soluble, fibrinogen. This intermediate state of neutrophil adhesion was defined by moderate clustering of integrin αMβ2, no increase in CBRM1/5 (a mAb specific for the activation epitope on the αM subunit) recognition, and no increase in surface expression of αMβ2, whereas phorbol myristate acetate (PMA) induced extensive changes in these 3 parameters. Furthermore, platelet-activating factor or interleukin 8 acted in concert with P-selectin for further enhancing the activation of αMβ2. We thus propose a model in which P-selectin induces an intermediate state of integrin activation and then cooperates with other extracellular stimuli to support maximal adhesion of human neutrophils.

Previous studies demonstrated that integrin αMβ2 (CD11b/18, Mac-1) forms a physical complex with the urokinase-type plasminogen activator receptor (uPAR/CD87) on leukocytes. In this study, we used human peripheral blood neutrophils and transfected cells expressing αMβ2, uPAR, or both receptors to show that the integrin can directly interact with urokinase (uPA). We demonstrate that αMβ2 supported adhesion and migration of these cells to uPA, and, in each case, blockade of αMβ2 suppressed the response. Within uPA, both the kringle and proteolytic domains are recognized by αMβ2, which are distinct from the growth factor domain that binds to uPAR. Within the αM subunit of the integrin, the I domain interacts with uPA, which is distinct from the region that interacts with uPAR. On cells expressing uPAR and αMβ2, both receptors mediated adhesion and migration. This cooperation was particularly apparent in the responses of neutrophils to uPA, where blockade of αMβ2 reduced uPAR-mediated responses and engagement of uPAR enhanced recognition of uPA by αMβ2. Thus, recognition of uPA by αMβ2 allows for formation of a multicontact trimolecular complex, in which a single uPA ligand may bind simultaneously to both uPAR and αMβ2. This complex may play an important role in the control of inflammatory cell migration and vascular homeostasis.

Previous studies demonstrated that integrin αMβ2(CD11b/18, Mac-1) forms a physical complex with the urokinase-type plasminogen activator receptor (uPAR/CD87) on leukocytes. In this study, we used human peripheral blood neutrophils and transfected cells expressing αMβ2, uPAR, or both receptors to show that the integrin can directly interact with urokinase (uPA). We demonstrate that αMβ2supported adhesion and migration of these cells to uPA, and, in each case, blockade of αMβ2suppressed the response. Within uPA, both the kringle and proteolytic domains are recognized by αMβ2, which are distinct from the growth factor domain that binds to uPAR. Within the αMsubunit of the integrin, the I domain interacts with uPA, which is distinct from the region that interacts with uPAR. On cells expressing uPAR and αMβ2, both receptors mediated adhesion and migration. This cooperation was particularly apparent in the responses of neutrophils to uPA, where blockade of αMβ2reduced uPAR-mediated responses and engagement of uPAR enhanced recognition of uPA by αMβ2. Thus, recognition of uPA by αMβ2allows for formation of a multicontact trimolecular complex, in which a single uPA ligand may bind simultaneously to both uPAR and αMβ2. This complex may play an important role in the control of inflammatory cell migration and vascular homeostasis.

To gain insight into the mechanism by which the αMI-domain of integrin αMβ2interacts with multiple and unrelated ligands, the identity of the neutrophil inhibitory factor (NIF) recognition site was sought. A systematic strategy in which individual amino acid residues within three previously implicated segments were changed to those in the αLI-domain, which is structurally very similar but does not bind NIF, was implemented. The capacity of the resulting mutants, expressed as glutathione S-transferase fusion proteins, to recognize NIF was assessed. These analyses ultimately identified Asp149, Arg151, Gly207, Tyr252, and Glu258as critical for NIF binding. Cation binding, a function of the metal ion-dependent adhesion site (MIDAS) motif, was assessed by terbium luminescence to evaluate conformational perturbations induced by the mutations. All five mutants bound terbium with unaltered affinities. When the five residues were inserted into the αLI-domain, the chimera bound NIF with high affinity. Another ligand of αMβ2, C3bi, which is known to use the same segments of the αMI-domain in engaging the receptor, failed to bind to the chimeric αLI-domain. Thus, the αMI-domain appears to present a mosaic of exposed amino acids within surface loops on its MIDAS face, and different ligands interact with different residues to attain high affinity binding.

The midsegment of the β3subunit has been implicated in the ligand and cation binding functions of the β3integrins. This region may contain a metal ion-dependent adhesion site (MIDAS) and fold into an I domain-like structure. Two recombinant fragments, β3-(95–373) and β3-(95–301), were expressed and found to bind fibrinogen. Whereas 0.1 mmCa2+supported ligand binding to both recombinant fragments, 1.0 mmCa2+suppressed binding to the longer but not the shorter fragment. These properties suggest that β3-(95–373) contains both the ligand-competent (LC) and inhibitory (I) cation binding sites, and β3-(95–301) lacks the I site. In equilibrium dialysis experiments, β3-(95–373) contained two divalent cation binding sites, one reactive with either Mg2+or Ca2+and one Ca2+-specific, whereas β3-(95–301) lacked the Ca2+-specific site. Mutant forms of β3-(95–373) suggested that the LC site is a MIDAS motif involving Asp119, Ser121, Ser123, Asp217, and/or Glu220as coordination sites, and the I site was dependent upon residues within β3-(301–323). In a molecular model of β3-(95–373), a second Ca2+could be docked onto a flexible loop in close proximity to the MIDAS. These results indicate that the ligand competent and Ca2+-specific inhibitory cation binding sites are distinct and reside in β3-(95–373).

Leukocyte migration is the hallmark of inflammation, and integrin αMβ2 and its ligand fibrinogen (Fg) are key participants in this cellular response. Cells expressing wild-type or mutant αMβ2 and Fg or its derivatives have been used to dissect the molecular requirements for this receptor–ligand pair to mediate cell migration. The major conclusions are that (a) Fg, its D fragment, and its P1 and P2 αMβ2 recognition peptides support a chemotactic response; (b) when the I domain of αL was replaced with the I domain of αM, the chimeric receptor supported cell migration to Fg; however, the αM subunit, containing the I domain but lacking the β2 subunit, supported migration poorly, thus, the αMI domain is necessary but not sufficient to support chemotaxis, and efficient migration requires the β2 subunit and αMI domain; and (c) in addition to supporting cell migration, P2 enhanced αMβ2-mediated chemotaxis to Fg and the P1 peptide. This activation was associated with exposure of the activation-dependent epitope recognized by monoclonal antibody 7E3 and was observed also with human neutrophils. Taken together, these data define specific molecular requirements for αMβ2 to mediate cell migration to Fg derivatives and assign a novel proinflammatory activity to the P2 peptide.

Plasminogen plays an integral role in the inflammatory response, and this participation is likely to depend on its interaction with cell surfaces. It has previously been reported that isolation of human neutrophils from blood leads to a spontaneous increase in their plasminogen-binding capacity, and the basis for this up-regulation has been explored as a model for mechanisms for modulation of plasminogen receptor expression. Freshly isolated human peripheral blood neutrophils exhibited relatively low plasminogen binding, but when cultured for 20 hours, they increased this capacity dramatically, up to 50-fold. This increase was abolished by soybean trypsin inhibitor and was susceptible to carboxypeptidase B treatment, implicating proteolysis and exposure of carboxy-terminal lysines in the enhanced interaction. In support of this hypothesis, treatment of neutrophils with elastase, cathepsin G, or plasmin increased their plasminogen binding, and specific inhibitors of elastase and cathepsin G suppressed the up-regulation that occurred during neutrophil culture. When neutrophils were stimulated with phorbol ester, their plasminogen binding increased rapidly, but this increase was insensitive to the protease inhibitors. These results indicate that plasminogen binding to neutrophils can be up-regulated by 2 distinct pathways. A major pathway with the propensity to markedly up-regulate plasminogen binding depends upon the proteolytic remodeling of the cell surface. In response to thioglycollate, neutrophils recruited into the peritoneum of mice were shown to bind more plasminogen than those in peripheral blood, suggesting that modulation of plasminogen binding by these or other pathways may also occur in vivo.

Integrin α4β1on the surface of T lymphocytes interacts with vascular cell adhesion molecule-1 (VCAM-1) and fibronectin during migration of lymphocytes from the blood to sites of inflammation. Migrating lymphocytes actively modify their environment through a number of mechanisms including proteolysis of the extracellular matrix by matrix metalloproteinases (MMP) synthesized by the cells. In this study, expression of MMP upon α4β1-mediated adhesion of leukocytes to two major ligands, the IIICS-1 domain of fibronectin and VCAM-1, has been examined. Adhesion of T lymphoblastoid Jurkat cells to the CS-1 peptide induced expression of mRNA for two MMPs, gelatinase A (MMP-2) and gelatinase B (MMP-9). As evaluated by relative RT-PCR and Northern blot analyses, the level of mRNA was upregulated about 4- to 5-fold for both MMPs compared to control cells maintained in suspension. With time, both enzymes were detected in conditioned media and inside the cells, and their identities were verified by Western blotting and gelatin zymography. Adhesion of Jurkat cells to the second major α4β1ligand, VCAM-1, upregulated mRNA for MMP-2 (3.5-fold) and failed to induce expression of mRNA for MMP-9. Accordingly, only MMP-2 protein was detected in conditioned media of cells adherent to VCAM-1. Occupancy of α4β1on the surface of suspended cells with soluble CS-1 peptide or VCAM-1 did not upregulate synthesis and release of MMPs. A similar pattern of induction of MMPs after adhesion to CS-1 and VCAM-1 was observed in T lymphocytes isolated from human blood. These results demonstrate that adhesion of T lymphocytes through α4β1to different ligands, which bind to similar or overlapping sites in the integrin, induces intracellular events leading to distinct patterns of MMPs biosynthesis.

Background.Vascular remodeling following arterial injury is characterized by an initial inflammatory reaction. Prior experiments using peritoneal inflammatory models have shown that the plasminogen system plays a role in the intensity of the inflammatory response. This study was undertaken to test the hypothesis that an absence of plasminogen would lead to a decrease in vascular remodeling.

αVβ3, a broadly distributed member of the integrin family of adhesion receptors, has been implicated in a variety of physiological and pathophysiological events, including control of bone density, angiogenesis, apoptosis, tumor growth, and metastasis. Recently, it has been shown that activation of αVβ3, its transition from a low- to a high-affinity/avidity state, influences its recognition of certain ligands. Bone sialoprotein (BSP) is recognized as an important ligand for αVβ3in processes ranging from bone formation to the homing of metastatic tumor cells. Here, the influence of αVβ3activation on the adhesion and migration of relevant cells to BSP has been examined. Stimulation of lymphoblastoid, osteoblastoid, and human umbilical vein endothelial cells (HUVEC) with PMA or Mn2+markedly enhanced αVβ3-dependent adhesion to BSP. αVβ3-mediated migration of HUVEC or osteoblastic cells to BSP was substantially enhanced by stimulation, demonstrating that αVβ3activation enhances both adhesive and migratory responses. However, adhesion and/or migration of certain tumor cell lines, including M21 melanoma and MDA MB435 and SKBR3 breast carcinoma cell lines, to BSP was constitutively high and was not augmented by αVβ3-activating stimuli. Inhibitors of the intracellular signaling molecules, phosphatidylinositol 3-kinase with wortmannin, hsp90-dependent kinases with geldanamycin, and calpain with calpeptin, but not MAPKK with PD98059, reduced the high spontaneous adhesion and migration of the M21 cells to BSP, consistent with the constitutive activation of the receptor on these tumor cells. These results indicate that the activation state of αVβ3can regulate cell migration and adhesion to BSP and, by extension, to other ligands of this receptor. The constitutive activation of αVβ3on neoplastic cells may contribute to tumor growth and metastatic potential.

Susceptibility to doxorubicin-induced nephropathy (DIN), a toxic model for the induction of proteinuria in mice, is related to the single-nucleotide polymorphism (SNP) C6418T of the Prkdc gene encoding for the DNA-repair enzyme DNA-PKcs. In addition, plasminogen (Plg) has been reported to play a role in glomerular damage. Here, we investigated the interdependence of both factors for the development of DIN. Genotyping confirmed the SNP of the Prkdc gene in C57BL/6 (PrkdcC6418/C6418) and 129S1/SvImJ (PrkdcT6418/T6418) mice. Intercross of heterozygous 129SB6F1 mice led to 129SB6F2 hybrids with Mendelian inheritance of the SNP. After doxorubicin injection, only homozygous F2 mice with PrkdcT6418/T6418 developed proteinuria. Genetic deficiency of Plg (Plg−/−) in otherwise susceptible 129S1/SvImJ mice led to resistance to DIN. Immunohistochemistry revealed glomerular binding of Plg in Plg+/+ mice after doxorubicin injection involving histone H2B as Plg receptor. In doxorubicin-resistant C57BL/6 mice, Plg binding was absent. In conclusion, susceptibility to DIN in 129S1/SvImJ mice is determined by a hierarchical two-hit process requiring the C6418T SNP in the Prkdc gene and subsequent glomerular binding of Plg. This article has an associated First Person interview with the first author of the paper.