Classification of venous thromboembolism (VTE). The clot is hot: inflammation, myeloid leukocytes, and venous thromboembolism.
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Animals↗
Biomedical subjects
A S Weyrich
Publications and source records attributed to A S Weyrich.
The evolving role of platelets in inflammation.
Platelets are small in size and simple in structure. Nevertheless, these anucleate cytoplasts utilize complex molecular systems to regulate a variety of biological functions. Here we review evolutionary paths, traditional roles, and previously unrecognized biological capacities of platelets that interface thrombosis with inflammation and potentially identify new roles in inflammatory diseases.
Blood Platelets↗
Differential regulation of matrix metalloproteinase-9 by monocytes adherent to collagen and platelets.
Circulating monocytes adhere to platelets and matrix proteins at sites of vascular injury, where engagement of specific surface tethering molecules mediates outside-in signaling and synthesis of gene products by the leukocytes. Here we demonstrate that interaction of isolated human monocytes with collagen induces matrix metalloproteinase-9 (MMP-9; gelatinase B) synthesis by monocytes, a process that is greatly enhanced in the presence of platelets. MMP-9 is a potent matrix degrading enzyme implicated in atherosclerotic plaque rupture, aneurysm formation, and other vascular syndromes. Synthesis of MMP-9 by monocytes is tightly regulated and synergistically increased following adhesion to collagen and platelets. Adhesion to control matrix proteins alone did not result in MMP-9 protein production and, similarly, adhesion of monocytes to platelets activated with thrombin in suspension was not sufficient to induce MMP-9 synthesis in the absence of monocyte adhesion to collagen. Interruption of intercellular contact between platelets and monocytes dramatically inhibited MMP-9 synthesis. These observations demonstrate that discrete adhesion-dependent signaling pathways govern MMP-9 synthesis by monocytes. The synthesis of MMP-9 by monocytes may be critical in vascular syndromes and other pathological processes that are dependent on dysregulated cell-cell and cell-matrix interactions.
Blood Platelets↗
Cell adhesion regulates gene expression at translational checkpoints in human myeloid leukocytes.
Engagement of adhesion molecules on monocytes and other myeloid leukocytes, which are effector cells of the innate immune system, not only tethers the leukocytes in place but also transmits outside-in signals that induce functional changes and alter gene expression. We found that a subset of mRNAs that are induced or amplified by adhesion of human monocytes to P-selectin via its surface ligand, P-selectin glycoprotein 1, have characteristics that suggest specialized translational control. One of these codes for urokinase plasminogen activator receptor (UPAR), a critical surface protease receptor and regulator of cell adhesion and migration. Although UPAR transcripts are induced by adhesion, rapid synthesis of the protein uses constitutive mRNA without a requirement for new transcription and is regulated by mammalian target of rapamycin, demonstrating new biologic roles for the signal-dependent translation pathway controlled by this intracellular kinase. The synthesis of UPAR in monocytic cells is also regulated by eukaryotic translation initiation factor 4E, a second key translational checkpoint, and phosphorylation of eukaryotic translation initiation factor 4E is induced by adhesion of monocytes to P-selectin. Translationally controlled display of UPAR by monocytes confers recognition of the matrix protein, vitronectin. Adhesion-dependent signaling from the plasma membrane to translational checkpoints represents a previously unrecognized mechanism for regulating surface phenotype that may be particularly important for myeloid leukocytes and other cells that are specialized for rapid inflammatory and vascular responses.
Cell Adhesion↗
Activated platelets mediate inflammatory signaling by regulated interleukin 1beta synthesis.
Platelets release preformed mediators and generate eicosanoids that regulate acute hemostasis and inflammation, but these anucleate cytoplasts are not thought to synthesize proteins or cytokines, or to influence inflammatory responses over time. Interrogation of an arrayed cDNA library demonstrated that quiescent platelets contain many messenger RNAs, one of which codes for interleukin 1beta precursor (pro-IL-1beta). Unexpectedly, the mRNA for IL-1beta and many other transcripts are constitutively present in polysomes, providing a mechanism for rapid synthesis. Platelet activation induces rapid and sustained synthesis of pro-IL-1beta protein, a response that is abolished by translational inhibitors. A portion of the IL-1beta is shed in its mature form in membrane microvesicles, and induces adhesiveness of human endothelial cells for neutrophils. Signal-dependent synthesis of an active cytokine over several hours indicates that platelets may have previously unrecognized roles in inflammation and vascular injury. Inhibition of beta3 integrin engagement markedly attenuated the synthesis of IL-1beta, identifying a new link between the coagulation and inflammatory cascades, and suggesting that antithrombotic therapies may also have novel antiinflammatory effects.
Antigens, CD↗
Integrins regulate the intracellular distribution of eukaryotic initiation factor 4E in platelets. A checkpoint for translational control.
Recent evidence from our laboratory demonstrates that platelets synthesize numerous proteins in a signal-dependent fashion (Pabla, R., Weyrich, A. S., Dixon, D. A., Bray, P. F., McIntyre, T. M., Prescott, S. M., and Zimmerman, G. A. (1999) J. Cell Biol. 144, 175-184; Weyrich, A. S., Dixon, D. A., Pabla, R., Elstad, M. R., McIntyre, T. M., Prescott, S. M., and Zimmerman, G. A. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 5556-5561). Protein synthesis in platelets is controlled at the translational level; however, the mechanisms of regulation are not known. Here we demonstrate that translation initiation factors are redistributed to mRNA-rich areas in aggregated platelets, an event that induces protein synthesis. Interrogation of cDNA arrays revealed that platelet-derived mRNAs are primarily associated with the cytoskeletal core. In contrast, eukaryotic initiation factor 4E (eIF4E), the essential mRNA cap-binding protein that controls global translation rates, is localized in the membrane skeleton and soluble fraction of platelets, physically separated from most mRNAs. Platelet activation redistributes eIF4E to the cytoskeleton and increases interactions of eIF4E with mRNA cap structures. Redistribution of eIF4E to the mRNA-rich cytoskeleton coincides with a marked increase in protein synthesis, a process that is blocked when intracellular actin is disrupted. Additional studies demonstrated that beta(3) integrins are the primary membrane receptor that distributes eIF4E within the cell. These results imply that integrins link receptor-mediated pathways with mRNA-rich cytoskeletal domains and thereby modulate the organization of intracellular translational complexes. They also indicate that the functional status of eIF4E is regulated by its intracellular distribution.
Arachidonic Acid↗
Fluid flow activates a regulator of translation, p70/p85 S6 kinase, in human endothelial cells.
Cellular phenotype is determined not only by genetic transcription but also by subsequent translation of mRNA into protein. Extracellular signals trigger intracellular pathways that distinctly activate translation. The 70/85-kDa S6 kinase (pp70(S6k)) is a central enzyme in the signal-dependent control of translation, but its regulation in endothelial cells is largely unknown. Here we show that fluid flow (in the absence of an exogenous mitogen) as well as humoral agonists activate endothelial pp70(S6k). Rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR), and wortmannin, a phosphatidylinositol 3-kinase inhibitor, blocked flow-induced pp70(S6k) activation; FK-506, a rapamycin analog with minimal mTOR inhibitory activity, and PD-98059, an inhibitor of the flow-sensitive mitogen-activated protein kinase pathway, had no effect. Synthesis of Bcl-3, a protein whose translation is controlled by an mTOR-dependent pathway, was induced by flow and inhibited by rapamycin and wortmannin. Transcriptional blockade did not abolish the flow-induced upregulation of Bcl-3. Fluid forces may therefore modify endothelial phenotype by specifically regulating translation of certain mRNA transcripts into protein.
3T3 Cells↗
Integrin-dependent control of translation: engagement of integrin alphaIIbbeta3 regulates synthesis of proteins in activated human platelets.
Integrins are widely expressed plasma membrane adhesion molecules that tether cells to matrix proteins and to one another in cell-cell interactions. Integrins also transmit outside-in signals that regulate functional responses of cells, and are known to influence gene expression by regulating transcription. In previous studies we found that platelets, which are naturally occurring anucleate cytoplasts, translate preformed mRNA transcripts when they are activated by outside-in signals. Using strategies that interrupt engagement of integrin alphaIIbbeta3 by fibrinogen and platelets deficient in this integrin, we found that alphaIIbbeta3 regulates the synthesis of B cell lymphoma 3 (Bcl-3) when platelet aggregation is induced by thrombin. We also found that synthesis of Bcl-3, which occurs via a specialized translation control pathway regulated by mammalian target of rapamycin (mTOR), is induced when platelets adhere to immobilized fibrinogen in the absence of thrombin and when integrin alphaIIbbeta3 is engaged by a conformation-altering antibody against integrin alphaIIbbeta3. Thus, outside-in signals delivered by integrin alphaIIbbeta3 are required for translation of Bcl-3 in thrombin-stimulated aggregated platelets and are sufficient to induce translation of this marker protein in the absence of thrombin. Engagement of integrin alpha2beta1 by collagen also triggered synthesis of Bcl-3. Thus, control of translation may be a general mechanism by which surface adhesion molecules regulate gene expression.
Antibodies, Monoclonal↗
Coengagement of ICAM-3 and Fc receptors induces chemokine secretion and spreading by myeloid leukocytes.
ICAM-3 is expressed at high levels on myeloid leukocytes, but its function on these cells is unknown. We tested the hypothesis that it transduces outside-in proinflammatory signals using immobilized mAbs to engage ICAM-3 on freshly isolated human monocytes and neutrophils. Two immobilized Abs that recognize epitopes in the extracellular domain 1 of ICAM-3, which is critical for recognition by the alphaL/beta2 integrin, potently induced secretion of MIP-1alpha, IL-8, and MCP-1 by monocytes and triggered IL-8 secretion by neutrophils. These chemokines are products of immediate-early genes that are induced when myeloid cells are activated. Chemokine secretion induced by "triggering" Abs was greater than that induced by isotype-matched immobilized Abs against ICAM-1, ICAM-2, PECAM-1, control Igs, or immobilized control proteins. Coengagement of ICAM-3 and Fc receptors (FcgammaRI or FcgammaRII) was required for maximal chemokine secretion by monocytes. Microscopy documented that there is also dramatic spreading of monocytes when surface ICAM-3 is engaged by immobilized Abs. Spreading was induced by Fab and F(ab')2 fragments of triggering anti-ICAM-3 mAb, demonstrating direct outside-in signaling, but was not required for chemokine secretion. These experiments indicate that ICAM-3 may transmit outside-in signals when it is engaged by beta2 integrins during myeloid cell-cell interactions in inflammatory lesions. Binding of Fc receptors by Ig in the local environment can amplify the responses.
Adult↗
Signal-dependent translation of a regulatory protein, Bcl-3, in activated human platelets.
Circulating human platelets lack nuclei, cannot synthesize mRNA, and are considered incapable of regulated protein synthesis. We found that thrombin-activated, but not resting, platelets synthesize Bcl-3, a member of the IkappaB-alpha family of regulatory proteins. The time- and concentration-dependent generation of Bcl-3 in platelets signaled by thrombin was blocked by translational inhibitors, by rapamycin, and by inhibitors of phosphatidylinositol-3-kinase, indicating that it occurs via a specialized translational control pathway that involves phosphorylation of the inhibitory protein 4E-BP1. After its synthesis in activated platelets Bcl-3 binds to the SH3 domain of Fyn (p59(fyn)), a Src-related tyrosine kinase. This, along with its expression in anucleate cells, suggests that Bcl-3 has previously unrecognized functions aside from modulation of transcription. We also demonstrate that platelets synthesize and secrete numerous proteins besides Bcl-3 after they adhere to fibrinogen, which mediates adhesion and outside-in signaling of these cells by engagement of alphaIIb/beta3 integrin. Taken together, these data demonstrate that regulated synthesis of proteins is a signal-dependent activation response of human platelets.
Adaptor Proteins, Signal Transducing↗
Engagement of P-selectin glycoprotein ligand-1 enhances tyrosine phosphorylation and activates mitogen-activated protein kinases in human neutrophils.
During inflammation, P-selectin on activated platelets and endothelial cells initiates adhesion of leukocytes through interactions with P-selectin glycoprotein ligand-1 (PSGL-1). We investigated whether ligation of PSGL-1 also transmits signals into leukocytes. Neutrophils incubated with anti-PSGL-1 monoclonal antibodies, but not with Fab fragments of these antibodies, rapidly increased tyrosine phosphorylation of proteins with relative molecular masses of 105-120, 70-84, and 42-44 kDa. PSGL-1-dependent adhesion of neutrophils to P-selectin increased tyrosine phosphorylation of similarly sized proteins. Cytochalasin B did not prevent the tyrosine phosphorylation induced by ligation of PSGL-1, suggesting that an intact cytoskeleton is not required for signaling. Engagement of PSGL-1 activated the GTPase Ras through a mechanism that did not require tyrosine phosphorylation of PSGL-1 or association of the Shc.Grb2.Sos1 complex with PSGL-1. Engagement of PSGL-1 activated the 42-44-kDa extracellular signal-regulated kinase family of mitogen-activated protein (MAP) kinases through a pathway that required activation of the MAP kinase kinase. Ligation of PSGL-1 also stimulated secretion of interleukin-8. The tyrosine kinase inhibitor, genistein, blocked tyrosine phosphorylation and secretion of interleukin-8, whereas the MAP kinase kinase inhibitor PD98059 partially inhibited secretion of interleukin-8. Tyrosine phosphorylation stimulated through PSGL-1 on selectin-tethered leukocytes may propagate a signaling cascade that is integrated with signals generated by other mediators.
Antibodies, Monoclonal↗
Oncostatin M is a proinflammatory mediator. In vivo effects correlate with endothelial cell expression of inflammatory cytokines and adhesion molecules.
Oncostatin M is a member of the IL-6 family of cytokines that is primarily known for its effects on cell growth. Endothelial cells have an abundance of receptors for oncostatin M, and may be its primary target. We determined if oncostatin M induces a key endothelial cell function, initiation of the inflammatory response. We found that subcutaneous injection of oncostatin M in mice caused an acute inflammatory reaction. Oncostatin M in vitro stimulated: (a) polymorphonuclear leukocyte (PMN) transmigration through confluent monolayers of primary human endothelial cells; (b) biphasic PMN adhesion through rapid P-selectin expression, and delayed adhesion mediated by E-selectin synthesis; (c) intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 accumulation; and (d) the expression of PMN activators IL-6, epithelial neutrophil activating peptide-78, growth-related cytokine alpha and growth-related cytokine beta without concomitant IL-8 synthesis. The nature of the response to oncostatin M varied with concentration, suggesting high and low affinity oncostatin M receptors independently stimulated specific responses. Immunohistochemistry showed that macrophage-like cells infiltrating human aortic aneurysms expressed oncostatin M, so it is present during a chronic inflammatory reaction. Therefore, oncostatin M, but not other IL-6 family members, fulfills Koch's postulates as an inflammatory mediator. Since its effects on endothelial cells differ significantly from established mediators like TNFalpha, it may uniquely contribute to the inflammatory cycle.
Animals↗
Vitamin C blocks inflammatory platelet-activating factor mimetics created by cigarette smoking.
Cigarette smoking within minutes induces leukocyte adhesion to the vascular wall and formation of intravascular leukocyte-platelet aggregates. We find this is inhibited by platelet-activating factor (PAF) receptor antagonists, and correlates with the accumulation of PAF-like mediators in the blood of cigarette smoke-exposed hamsters. These mediators were PAF-like lipids, formed by nonenzymatic oxidative modification of existing phospholipids, that were distinct from biosynthetic PAF. These PAF-like lipids induced isolated human monocytes and platelets to aggregate, which greatly increased their secretion of IL-8 and macrophage inflammatory protein-1alpha. Both events were blocked by a PAF receptor antagonist. Similarly, blocking the PAF receptor in vivo blocked smoke-induced leukocyte aggregation and pavementing along the vascular wall. Dietary supplementation with the antioxidant vitamin C prevented the accumulation of PAF-like lipids, and it prevented cigarette smoke-induced leukocyte adhesion to the vascular wall and formation of leukocyte-platelet aggregates. This is the first in vivo demonstration of inflammatory phospholipid oxidation products and it suggests a molecular mechanism coupling cigarette smoke with rapid inflammatory changes. Inhibition of PAF-like lipid formation and their intravascular sequela by vitamin C suggests a simple dietary means to reduce smoking-related cardiovascular disease.
Animals↗
Activated platelets signal chemokine synthesis by human monocytes.
Human blood monocytes adhere rapidly and for prolonged periods to activated platelets that display P-selectin, an adhesion protein that recognizes a specific ligand on leukocytes, P-selectin glycoprotein-1. We previously demonstrated that P-selectin regulates expression and secretion of cytokines by stimulated monocytes when it is presented in a purified, immobilized form or by transfected cells. Here we show that thrombin-activated platelets induce the expression and secretion of monocyte chemotactic protein-1 and IL-8 by monocytes. Enhanced monokine synthesis requires engagement of P-selectin glycoprotein-1 on the leukocyte by P-selectin on the platelet. Secretion of the chemokines is not, however, directly signaled by P-selectin; instead, tethering of the monocytes by P-selectin is required for their activation by RANTES (regulated upon activation normal T cell expressed presumed secreted), a platelet chemokine not previously known to induce immediate-early gene products in monocytes. Adhesion of monocytes to activated platelets results in nuclear translocation of p65 (RelA), a component of the NF-kappaB family of transcription factors that binds kappaB sequences in the regulatory regions of monocyte chemotactic protein-1, IL-8, and other immediate-early genes. However, expression of tissue factor, a coagulation protein that also has a kappaB sequence in the 5' regulatory region of its gene, is not induced in monocytes adherent to activated platelets. Thus, contact of monocytes with activated platelets differentially affects the expression of monocyte products. These experiments suggest that activated platelets regulate chemokine secretion by monocytes in inflammatory lesions in vivo and provide a model for the study of gene regulation in cell-cell interactions.
Blood Platelets↗
Platelet-activating factor (PAF): signalling and adhesion in cell-cell interactions.
Signalling by PAF is closely linked to adhesive interactions between cells of the inflammatory and vascular systems. It acts as a juxtacrine signal that alters the activity of beta 2 integrins on myeloid leukocytes (Figure 1), and works in concert with P-selectin at the surfaces of endothelial cells (Figure 2 and text). Observations in models of flow and in vivo support the original experiments using cultured endothelium under static conditions that indicated that PAF acts at this vascular interface. P-selectin modifies and integrates signals delivered through the PAF receptor on monocytes (Figure 4). Adhesion via P-selectin and engagement of beta 2 integrins modify signals leading to PAF synthesis (text and Figure 5). The intimate relationship between adhesive events and signalling by PAF may be a critical determinant in its roles in physiologic and pathologic responses.
Animals↗
Time course of coronary vascular endothelial adhesion molecule expression during reperfusion of the ischemic feline myocardium.
The time course of endothelial P-selectin, ICAM-1, and E-selectin expression was studied in a feline model of myocardial ischemia and reperfusion. Cats were subjected to 90 min of myocardial ischemia followed by 0, 10, 20, 60, 150, or 270 min of reperfusion. At the end of reperfusion, the coronary vasculature was examined immunohistochemically to localize monoclonal antibodies (mAbs) PB1.3, RR1/1, and Cy1787 directed against P-selectin, ICAM-1, and E-selectin, respectively. Immunohistochemical localization for P-selectin, recognized by mAb PB1.3, was maximally expressed 20 min after reperfusion in 60 +/- 6% of coronary venules (P < 0.05 compared to non-reperfused controls), and covered 59 +/- 3% of the endothelial cell perimeter of immunostained coronary venules. Immunolocalization of mAb PB1.3 gradually declined at 60, 150, and 270 min of reperfusion. Immunohistochemical localization of mAb RR1/1 (anti-ICAM-1) in endothelial cells of coronary venules was observed to a modest extent in non-ischemic myocardium and at 10, 20, and 60 min of reperfusion, but was significantly increased following 150 and 270 min of reperfusion (P < 0.05 compared non-reperfused controls). At 270 min post-reperfusion, mAb RR1/1 was seen in 50 +/- 4% of coronary venules. Endothelial immunolocalization of mAb Cy1787 (anti-E-selectin) was only observed in 13 +/- 1 and 14 +/- 3% of coronary venules after 150 and 270 min of reperfusion, respectively, suggesting that pronounced expression of E-selectin does not occur within 270 min after reperfusion. These results demonstrate sequential expression of three major endothelial cell adherence molecules in situ following myocardial ischemia and reperfusion. The timing of endothelial cell expressed P-selectin and ICAM-1 could coordinate neutrophil trafficking during the early stages of reperfusion.
Animals↗
Monocyte tethering by P-selectin regulates monocyte chemotactic protein-1 and tumor necrosis factor-alpha secretion. Signal integration and NF-kappa B translocation.
Adhesion molecules that tether circulating leukocytes to endothelial cells may also transduce or modulate outside-in signals for cellular activation, providing an initial regulatory point in the inflammatory response. Adhesion of human monocytes to P-selectin, the most rapidly expressed endothelial tethering factor, increased the secretion of monocyte chemotactic protein-1 (MCP-1) and tumor necrosis factor-alpha (TNF-alpha) by the leukocytes when they were stimulated with platelet-activating factor. Increased cytokine secretion was specifically inhibited by G1, an anti-P-selectin mAb that prevents P-selectin from binding to its ligand (P-selectin glycoprotein ligand-1) on myeloid cells. Moreover, tethering by P-selectin specifically enhanced nuclear translocation of nuclear factor-kappa B (NF-kappa B), a transcription factor required for expression of MCP-1, TNF-alpha, and other immediate-early genes. These results demonstrate that P-selectin, through its ligands on monocytes, may locally regulate cytokine secretion in inflamed tissues.
Animals↗
Quantification of neutrophil migration following myocardial ischemia and reperfusion in cats and dogs.
Endothelial cell dysfunction and cardiac myocyte injury resulting from ischemia and reperfusion have been associated with accumulation of neutrophils in the myocardium. To determine whether the accumulation is related primarily to intravascular sequestration or extravascular infiltration of neutrophils during the early period of reperfusion, we morphometrically quantified the tissue distribution of neutrophils in cats and dogs. At the end of the reperfusion period, the base of the heart was cross-clamped to preserve neutrophil location at the moment of death. Point-counting methods were used to determine the distribution of neutrophils inside and outside coronary arterioles and venules (< or = 100 microns in diameter) as well as coronary capillaries 5-10 microns in diameter in 0.5-microns-thick, plastic-embedded sections. Ischemia-reperfusion resulted in a threefold increase in neutrophil number in the lumen of arterioles and venules at 60 min of reperfusion and up to a sevenfold increase at 270 min of reperfusion (P < .05) compared to time-matched control nonischemic hearts. The ratio of intravascular neutrophils in venules to arterioles was 2:1. Intracapillary neutrophils increased, but not significantly, at 60 min of reperfusion. At 270 min of reperfusion, intracapillary neutrophils increased 11-fold (P < .05). The percentage of total neutrophils that accumulated outside arterioles and venules in cat hearts was 8% at 60 min of reperfusion (not significant, NS) and 28% at 270 min of reperfusion (P < .05). In dog hearts, the percentages were 26% (NS) and 44% (P < .05), respectively. The percentage of total neutrophils that accumulated outside capillaries was < 6% in both cat and dog hearts (NS). The combination of rapid intravascular sequestration, delayed extravascular infiltration, and low incidence of neutrophil-cardiac myocyte contact in situ in these two species suggests that neutrophil-mediated cardiac myocyte injury during early reperfusion may initially depend on diffusion of inflammatory mediators and subsequently require direct contact between neutrophils and cardiac myocytes.
Animals↗