New insights of glycoprotein Ib-IX-V complex organization and glycoprotein Ibα in platelet biogenesis.

PURPOSE OF REVIEW: Glycoprotein (GP) Ib-IX-V, a platelet surface receptor that plays a critical role in platelet adhesion and platelet-mediated immune responses, consists of GPIbα, GPIbß, GPIX, and GPV in a stoichiometry of 2 : 4 : 2 : 1. Forming a complex is essential for GPIb-IX-V to function. GPIb-IX-V also plays an important role in platelet biogenesis by regulating the number and size of platelets. Yet how GPIb-IX-V regulates platelet biogenesis remains elusive. This review will summarize recent findings in the complex organization of GPIb-IX-V and its role in platelet biogenesis. RECENT FINDINGS: Proteomics studies suggest that GPIbα, GPIbß, GPIX, and GPV form the complex in a ratio of 1 : 2 : 1 : 1, which is supported by analysis of molecular weight of GPIb-IX-V and GPIb-IX and the structure of entire GPIb-IX-V. To activate platelets, GPIbα requires binding of CLEC-2 to trigger signals. Furthermore, disrupting the GPIbα anchorage to filamin A causes defects in platelet budding away from proplatelets leading to giant platelets and a low platelet count. SUMMARY: New studies challenge the traditional model for the organization of GPIb-IX-V as a complex and indicate the role of GPIb-IX-V in platelet production. Those studies provide insights for GPIb-IX-V in the regulation of platelet activation and platelet biogenesis.

Deletion of Talin1 in Myeloid Cells Facilitates Atherosclerosis in Mice.

BACKGROUND: Integrin-regulated monocyte recruitment and cellular responses of monocyte-derived macrophages are critical for the pathogenesis of atherosclerosis. In the canonical model, talin1 controls ligand binding to integrins, a prerequisite for integrins to mediate leukocyte recruitment and induce immune responses. However, the role of talin1 in the development of atherosclerosis has not been studied. Our study investigated how talin1 in myeloid cells regulates the progression of atherosclerosis. METHODS: On an Apoe-/- background, myeloid talin1-deficient mice and the control mice were fed with a high-fat diet for 8 or 12 weeks to induce atherosclerosis. The atherosclerosis development in the aorta and monocyte recruitment into atherosclerotic lesions were analyzed. RESULTS: Myeloid talin1 deletion facilitated the formation of atherosclerotic lesions and macrophage deposition in lesions. Talin1 deletion abolished integrin ß2-mediated adhesion of monocytes but did not impair integrin α4ß1-dependent cell adhesion in a flow adhesion assay. Strikingly, talin1 deletion did not prevent Mn2+- or chemokine-induced activation of integrin α4ß1 to the high-affinity state for ligands. In an in vivo competitive homing assay, monocyte infiltration into inflamed tissues was prohibited by antibodies to integrin α4ß1 but was not affected by talin1 deletion or antibodies to integrin ß2. Furthermore, quantitative polymerase chain reaction and ELISA (enzyme-linked immunosorbent assay) analysis showed that macrophages produced cytokines to promote inflammation and the proliferation of smooth muscle cells. Ligand binding to integrin ß3 inhibited cytokine generation in macrophages, although talin1 deletion abolished the negative effects of integrin ß3. CONCLUSIONS: Integrin α4ß1 controls monocyte recruitment during atherosclerosis. Talin1 is dispensable for integrin α4ß1 activation to the high-affinity state and integrin α4ß1-mediated monocyte recruitment. Yet, talin1 is required for integrin ß3 to inhibit the production of inflammatory cytokines in macrophages. Thus, intact monocyte recruitment and elevated inflammatory responses cause enhanced atherosclerosis in talin1-deficient mice. Our study provides novel insights into the roles of myeloid talin1 and integrins in the progression of atherosclerosis.

Clearance of VWF by hepatic macrophages is critical for the protective effect of ADAMTS13 in sickle cell anemia mice.

ABSTRACT: Although it is caused by a single-nucleotide mutation in the ß-globin gene, sickle cell anemia (SCA) is a systemic disease with complex, incompletely elucidated pathologies. The mononuclear phagocyte system plays critical roles in SCA pathophysiology. However, how heterogeneous populations of hepatic macrophages contribute to SCA remains unclear. Using a combination of single-cell RNA sequencing and spatial transcriptomics via multiplexed error-robust fluorescence in situ hybridization, we identified distinct macrophage populations with diversified origins and biological functions in SCA mouse liver. We previously found that administering the von Willebrand factor (VWF)-cleaving protease ADAMTS13 alleviated vaso-occlusive episode in mice with SCA. Here, we discovered that the ADAMTS13-cleaved VWF was cleared from the circulation by a Clec4f+Marcohigh macrophage subset in a desialylation-dependent manner in the liver. In addition, sickle erythrocytes were phagocytized predominantly by Clec4f+Marcohigh macrophages. Depletion of macrophages not only abolished the protective effect of ADAMTS13 but exacerbated vaso-occlusive episode in mice with SCA. Furthermore, promoting macrophage-mediated VWF clearance reduced vaso-occlusion in SCA mice. Our study demonstrates that hepatic macrophages are important in the pathogenesis of SCA, and efficient clearance of VWF by hepatic macrophages is critical for the protective effect of ADAMTS13 in SCA mice.

LFA-1 Activation in T-Cell Migration and Immunological Synapse Formation.

Integrin LFA-1 plays a critical role in T-cell migration and in the formation of immunological synapses. LFA-1 functions through interacting with its ligands with differing affinities: low, intermediate, and high. Most prior research has studied how LFA-1 in the high-affinity state regulates the trafficking and functions of T cells. LFA-1 is also presented in the intermediate-affinity state on T cells, however, the signaling to activate LFA-1 to the intermediate-affinity state and the role of LFA-1 in this affinity state both remain largely elusive. This review briefly summarizes the activation and roles of LFA-1 with varied ligand-binding affinities in the regulation of T-cell migration and immunological synapse formation.

Endothelial VWF is critical for the pathogenesis of vaso-occlusive episode in a mouse model of sickle cell disease.

Vaso-occlusive episode (VOE) is a common and critical complication of sickle cell disease (SCD). Its pathogenesis is incompletely understood. von Willebrand factor (VWF), a multimeric plasma hemostatic protein synthesized and secreted by endothelial cells and platelets, is increased during a VOE. However, whether and how VWF contributes to the pathogenesis of VOE is not fully understood. In this study, we found increased VWF levels during tumor necrosis factor (TNF)-induced VOE in a humanized mouse model of SCD. Deletion of endothelial VWF decreased hemolysis, vascular occlusion, and organ damage caused by TNF-induced VOE in SCD mice. Moreover, administering ADAMTS13, the VWF-cleaving plasma protease, reduced plasma VWF levels, decreased inflammation and vaso-occlusion, and alleviated organ damage during VOE. These data suggest that promoting VWF cleavage via ADAMTS13 may be an effective treatment for reducing hemolysis, inflammation, and vaso-occlusion during VOE.

Deletion of platelet CLEC-2 decreases GPIbα-mediated integrin αIIbß3 activation and decreases thrombosis in TTP.

Microvascular thrombosis in patients with thrombotic thrombocytopenic purpura (TTP) is initiated by GPIbα-mediated platelet binding to von Willebrand factor (VWF). Binding of VWF to GPIbα causes activation of the platelet surface integrin αIIbß3. However, the mechanism of GPIbα-initiated activation of αIIbß3 and its clinical importance for microvascular thrombosis remain elusive. Deletion of platelet C-type lectin-like receptor 2 (CLEC-2) did not prevent VWF binding to platelets but specifically inhibited platelet aggregation induced by VWF binding in mice. Deletion of platelet CLEC-2 also inhibited αIIbß3 activation induced by the binding of VWF to GPIbα. Using a mouse model of TTP, which was created by infusion of anti-mouse ADAMTS13 monoclonal antibodies followed by infusion of VWF, we found that deletion of platelet CLEC-2 decreased pulmonary arterial thrombosis and the severity of thrombocytopenia. Importantly, prophylactic oral administration of aspirin, an inhibitor of platelet activation, and therapeutic treatment of the TTP mice with eptifibatide, an integrin αIIbß3 antagonist, reduced pulmonary arterial thrombosis in the TTP mouse model. Our observations demonstrate that GPIbα-mediated activation of integrin αIIbß3 plays an important role in the formation of thrombosis in TTP. These observations suggest that prevention of platelet activation with aspirin may reduce the risk for thrombosis in patients with TTP.

L-SIGN is a receptor on liver sinusoidal endothelial cells for SARS-CoV-2 virus.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), remains a pandemic. Severe disease is associated with dysfunction of multiple organs, but some infected cells do not express ACE2, the canonical entry receptor for SARS-CoV-2. Here, we report that the C-type lectin receptor L-SIGN interacted in a Ca2+-dependent manner with high-mannose-type N-glycans on the SARS-CoV-2 spike protein. We found that L-SIGN was highly expressed on human liver sinusoidal endothelial cells (LSECs) and lymph node lymphatic endothelial cells but not on blood endothelial cells. Using high-resolution confocal microscopy imaging, we detected SARS-CoV-2 viral proteins within the LSECs from liver autopsy samples from patients with COVID-19. We found that both pseudo-typed virus enveloped with SARS-CoV-2 spike protein and authentic SARS-CoV-2 virus infected L-SIGN-expressing cells relative to control cells. Moreover, blocking L-SIGN function reduced CoV-2-type infection. These results indicate that L-SIGN is a receptor for SARS-CoV-2 infection. LSECs are major sources of the clotting factors vWF and factor VIII (FVIII). LSECs from liver autopsy samples from patients with COVID-19 expressed substantially higher levels of vWF and FVIII than LSECs from uninfected liver samples. Our data demonstrate that L-SIGN is an endothelial cell receptor for SARS-CoV-2 that may contribute to COVID-19-associated coagulopathy.

Kupffer cell receptor CLEC4F is important for the destruction of desialylated platelets in mice.

The liver has recently been identified as a major organ for destruction of desialylated platelets. However, the underlying mechanism remains unclear. Kupffer cells, which are professional phagocytic cells in the liver, comprise the largest population of resident tissue macrophages in the body. Kupffer cells express a C-type lectin receptor, CLEC4F, that recognizes desialylated glycans with an unclear in vivo role in mediating platelet destruction. In this study, we generated a CLEC4F-deficient mouse model (Clec4f-/-) and found that CLEC4F was specifically expressed by Kupffer cells. Using the Clec4f-/- mice and a newly generated platelet-specific reporter mouse line, we revealed a critical role for CLEC4F on Kupffer cells in mediating destruction of desialylated platelets in the liver in vivo. Platelet clearance experiments and ultrastructural analysis revealed that desialylated platelets were phagocytized predominantly by Kupffer cells in a CLEC4F-dependent manner in mice. Collectively, these findings identify CLEC4F as a Kupffer cell receptor important for the destruction of desialylated platelets induced by bacteria-derived neuraminidases, which provide new insights into the pathogenesis of thrombocytopenia in disease conditions such as sepsis.

Heightened activation of embryonic megakaryocytes causes aneurysms in the developing brain of mice lacking podoplanin.

During early embryonic development in mammals, including humans and mice, megakaryocytes (Mks) first originate from primitive hematopoiesis in the yolk sac. These embryonic Mks (eMks) circulate in the vasculature with unclear function. Herein, we report that podoplanin (PDPN), the ligand of C-type lectin-like receptor (CLEC-2) on Mks/platelets, is temporarily expressed in neural tissue during midgestation in mice. Loss of PDPN or CLEC-2 resulted in aneurysms and spontaneous hemorrhage, specifically in the lower diencephalon during midgestation. Surprisingly, more eMks/platelets had enhanced granule release and localized to the lower diencephalon in mutant mouse embryos than in wild-type littermates before hemorrhage. We found that PDPN counteracted the collagen-1-induced secretion of angiopoietin-1 from fetal Mks, which coincided with enhanced TIE-2 activation in aneurysm-like sprouts of PDPN-deficient embryos. Blocking platelet activation prevented the PDPN-deficient embryo from developing vascular defects. Our data reveal a new role for PDPN in regulating eMk function during midgestation.

Proximal colon-derived O-glycosylated mucus encapsulates and modulates the microbiota.

Colon mucus segregates the intestinal microbiota from host tissues, but how it organizes to function throughout the colon is unclear. In mice, we found that colon mucus consists of two distinct O-glycosylated entities of Muc2: a major form produced by the proximal colon, which encapsulates the fecal material including the microbiota, and a minor form derived from the distal colon, which adheres to the major form. The microbiota directs its own encapsulation by inducing Muc2 production from proximal colon goblet cells. In turn, O-glycans on proximal colon-derived Muc2 modulate the structure and function of the microbiota as well as transcription in the colon mucosa. Our work shows how proximal colon control of mucin production is an important element in the regulation of host-microbiota symbiosis.

Neutrophils lacking ERM proteins polarize and crawl directionally but have decreased adhesion strength.

Ezrin/radixin/moesin (ERM) proteins are adaptors that link the actin cytoskeleton to the cytoplasmic domains of membrane proteins. Leukocytes express mostly moesin with lower levels of ezrin but no radixin. When leukocytes are activated, ERMs are postulated to redistribute membrane proteins from microvilli into uropods during polarization and to transduce signals that influence adhesion and other responses. However, these functions have not been tested in leukocytes lacking all ERMs. We used knockout (KO) mice with neutrophils lacking ezrin, moesin, or both proteins (double knockout [DKO]) to probe how ERMs modulate cell shape, adhesion, and signaling in vitro and in vivo. Surprisingly, chemokine-stimulated DKO neutrophils still polarized and redistributed ERM-binding proteins such as PSGL-1 and CD44 to the uropods. Selectin binding to PSGL-1 on moesin KO or DKO neutrophils activated kinases that enable integrin-dependent slow rolling but not those that generate neutrophil extracellular traps. Flowing neutrophils of all genotypes rolled normally on selectins and, upon chemokine stimulation, arrested on integrin ligands. However, moesin KO and DKO neutrophils exhibited defective integrin outside-in signaling and reduced adhesion strength. In vivo, DKO neutrophils displayed normal directional crawling toward a chemotactic gradient, but premature detachment markedly reduced migration from venules into inflamed tissues. Our results demonstrate that stimulated neutrophils do not require ERMs to polarize or to move membrane proteins into uropods. They also reveal an unexpected contribution of moesin to integrin outside-in signaling and adhesion strengthening.

Monocyte upregulation of podoplanin during early sepsis induces complement inhibitor release to protect liver function.

Multiple organ failure in sepsis is a progressive failure of several interdependent organ systems. Liver dysfunction occurs early during sepsis and is directly associated with patient death; however, the underlying mechanism of liver dysfunction is unclear. Platelet transfusion benefits patients with sepsis, and inhibition of complement activation protects liver function in septic animals. Herein, we explored the potential link between platelets, complement activation, and liver dysfunction in sepsis. We found that deletion of platelet C-type lectin-like receptor 2 (CLEC-2) exacerbated liver dysfunction in early sepsis. Platelet CLEC-2-deficient mice exhibited higher complement activation, more severe complement attack in the liver, and lower plasma levels of complement inhibitors at early time points after E. coli infection. Circulating monocytes expressed the CLEC-2 ligand podoplanin in early sepsis, and podoplanin binding induced release of complement inhibitors from platelets. Injection of complement inhibitors released from platelets reduced complement attack and attenuated liver dysfunction in septic mice. These findings indicate a new function of platelets in the regulation of complement activation during sepsis.

Th1 Cells Rolling on Selectins Trigger DAP12-Dependent Signals That Activate Integrin αLß2.

During inflammation, both neutrophils and effector T cells use selectins to roll and integrins to arrest in postcapillary venules. In both cell types, chemokines can transduce signals that convert integrin αLß2 to a high-affinity conformation, which interacts with ICAM-1 to mediate arrest. In neutrophils, selectins also trigger an immunoreceptor-like signaling cascade that converts integrin αLß2 to an intermediate-affinity conformation, which interacts with ICAM-1 to slow rolling. It is not known whether selectins induce similar signaling events in T cells. Ag engagement causes phosphorylation of ITAMs on the TCR; these motifs recruit kinases and adaptors that lead to the activation of αLß2. We found that mouse Th1 cells rolling on P- or E-selectin triggered signals that promoted αLß2-dependent slow rolling on ICAM-1 in vitro and in vivo. The selectin signaling cascade resembled that used by the TCR, except that unexpectedly, Th1 cells employed the ITAM-bearing protein DAP12, which was not known to be expressed in these cells. Importantly, outside-in signaling through ligand-occupied αLß2 also required DAP12. Cooperative selectin and chemokine signaling in Th1 cells promoted αLß2-dependent slow rolling and arrest in vitro and in vivo and migration into Ag-challenged tissues in vivo. Our findings reveal an important function for DAP12 in Th1 cells and a new mechanism to recruit effector T cells to sites of inflammation.

Sialylation on O-glycans protects platelets from clearance by liver Kupffer cells.

Most platelet membrane proteins are modified by mucin-type core 1-derived glycans (O-glycans). However, the biological importance of O-glycans in platelet clearance is unclear. Here, we generated mice with a hematopoietic cell-specific loss of O-glycans (HC C1galt1-/- ). These mice lack O-glycans on platelets and exhibit reduced peripheral platelet numbers. Platelets from HC C1galt1-/- mice show reduced levels of α-2,3-linked sialic acids and increased accumulation in the liver relative to wild-type platelets. The preferential accumulation of HC C1galt1-/- platelets in the liver was reduced in mice lacking the hepatic asialoglycoprotein receptor [Ashwell-Morell receptor (AMR)]. However, we found that Kupffer cells are the primary cells phagocytosing HC C1galt1-/- platelets in the liver. Our results demonstrate that hepatic AMR promotes preferential adherence to and phagocytosis of desialylated and/or HC C1galt1-/- platelets by the Kupffer cell through its C-type lectin receptor CLEC4F. These findings provide insights into an essential role for core 1 O-glycosylation of platelets in their clearance in the liver.

Circulating soluble P-selectin must dimerize to promote inflammation and coagulation in mice.

Leukocyte adhesion to P-selectin on activated platelets and endothelial cells induces shedding of the P-selectin ectodomain into the circulation. Plasma soluble P-selectin (sP-selectin) is elevated threefold to fourfold in patients with cardiovascular disease. Circulating sP-selectin is thought to trigger signaling in leukocytes that directly contributes to inflammation and thrombosis. However, sP-selectin likely circulates as a monomer, and in vitro studies suggest that sP-selectin must dimerize to induce signaling in leukocytes. To address this discrepancy, we expressed the entire ectodomain of mouse P-selectin as a monomer (sP-selectin) or as a disulfide-linked dimer fused to the Fc portion of mouse immunoglobulin G (sP-selectin-Fc). Dimeric sP-selectin-Fc, but not monomeric sP-selectin, triggered integrin-dependent adhesion of mouse leukocytes in vitro. Antibody-induced oligomerization of sP-selectin or sP-selectin-Fc was required to trigger formation of neutrophil extracellular traps. Injecting sP-selectin-Fc, but not sP-selectin, into mice augmented integrin-dependent adhesion of neutrophils in venules, generated tissue factor-bearing microparticles, shortened plasma-clotting times, and increased thrombus frequency in the inferior vena cava. Furthermore, transgenic mice that overexpressed monomeric sP-selectin did not exhibit increased inflammation or thrombosis. We conclude that elevated plasma sP-selectin is a consequence rather than a cause of cardiovascular disease.

Replacing the Promoter of the Murine Gene Encoding P-selectin with the Human Promoter Confers Human-like Basal and Inducible Expression in Mice.

In humans and mice, megakaryocytes/platelets and endothelial cells constitutively synthesize P-selectin and mobilize it to the plasma membrane to mediate leukocyte rolling during inflammation. TNF-α, interleukin 1ß, and LPS markedly increase P-selectin mRNA in mice but decrease P-selectin mRNA in humans. Transgenic mice bearing the entire human SELP gene recapitulate basal and inducible expression of human P-selectin and reveal human-specific differences in P-selectin function. Differences in the human SELP and murine Selp promoters account for divergent expression in vitro, but their significance in vivo is not known. Here we generated knockin mice that replace the 1.4-kb proximal Selp promoter with the corresponding SELP sequence (Selp(KI)). Selp(KI) (/) (KI) mice constitutively expressed more P-selectin on platelets and more P-selectin mRNA in tissues but only slightly increased P-selectin mRNA after injection of TNF-α or LPS. Consistent with higher basal expression, leukocytes rolled more slowly on P-selectin in trauma-stimulated venules of Selp(KI) (/) (KI) mice. However, TNF-α did not further reduce P-selectin-dependent rolling velocities. Blunted up-regulation of P-selectin mRNA during contact hypersensitivity reduced P-selectin-dependent inflammation in Selp(KI) (/-) mice. Higher basal P-selectin in Selp(KI) (/) (KI) mice compensated for this defect. Therefore, divergent sequences in a short promoter mediate most of the functionally significant differences in expression of human and murine P-selectin in vivo.

O-glycans direct selectin ligands to lipid rafts on leukocytes.

Palmitoylated cysteines typically target transmembrane proteins to domains enriched in cholesterol and sphingolipids (lipid rafts). P-selectin glycoprotein ligand-1 (PSGL-1), CD43, and CD44 are O-glycosylated proteins on leukocytes that associate with lipid rafts. During inflammation, they transduce signals by engaging selectins as leukocytes roll in venules, and they move to the raft-enriched uropods of polarized cells upon chemokine stimulation. It is not known how these glycoproteins associate with lipid rafts or whether this association is required for signaling or for translocation to uropods. Here, we found that loss of core 1-derived O-glycans in murine C1galt1(-/-) neutrophils blocked raft targeting of PSGL-1, CD43, and CD44, but not of other glycosylated proteins, as measured by resistance to solubilization in nonionic detergent and by copatching with a raft-resident sphingolipid on intact cells. Neuraminidase removal of sialic acids from wild-type neutrophils also blocked raft targeting. C1galt1(-/-) neutrophils or neuraminidase-treated neutrophils failed to activate tyrosine kinases when plated on immobilized anti-PSGL-1 or anti-CD44 F(ab')2. Furthermore, C1galt1(-/-) neutrophils incubated with anti-PSGL-1 F(ab')2 did not generate microparticles. In marked contrast, PSGL-1, CD43, and CD44 moved normally to the uropods of chemokine-stimulated C1galt1(-/-) neutrophils. These data define a role for core 1-derived O-glycans and terminal sialic acids in targeting glycoprotein ligands for selectins to lipid rafts of leukocytes. Preassociation of these glycoproteins with rafts is required for signaling but not for movement to uropods.

Blocking neutrophil integrin activation prevents ischemia-reperfusion injury.

Neutrophil recruitment, mediated by ß2 integrins, combats pyogenic infections but also plays a key role in ischemia-reperfusion injury and other inflammatory disorders. Talin induces allosteric rearrangements in integrins that increase affinity for ligands (activation). Talin also links integrins to actin and other proteins that enable formation of adhesions. Structural studies have identified a talin1 mutant (L325R) that perturbs activation without impairing talin's capacity to link integrins to actin and other proteins. Here, we found that mice engineered to express only talin1(L325R) in myeloid cells were protected from renal ischemia-reperfusion injury. Dissection of neutrophil function in vitro and in vivo revealed that talin1(L325R) neutrophils had markedly impaired chemokine-induced, ß2 integrin-mediated arrest, spreading, and migration. Surprisingly, talin1(L325R) neutrophils exhibited normal selectin-induced, ß2 integrin-mediated slow rolling, in sharp contrast to the defective slow rolling of neutrophils lacking talin1 or expressing a talin1 mutant (W359A) that blocks talin interaction with integrins. These studies reveal the importance of talin-mediated activation of integrins for renal ischemia-reperfusion injury. They further show that neutrophil arrest requires talin recruitment to and activation of integrins. However, although neutrophil slow rolling requires talin recruitment to integrins, talin-mediated integrin activation is dispensable.