Denuja Karunakaran

Controlled shedding of platelet glycoprotein (GP)VI and GPIb-IX-V by ADAM family metalloproteinases.

Background: Platelet glycoprotein (GP)VI that binds collagen, and GPIb–IX–V that binds von Willebrand factor, initiate thrombus formation.Objectives: In this study, we investigated the mechanisms of metalloproteinase‐mediated ectodomain shedding that regulate the surface expression of GPVI, GPIbα (the major ligand‐binding subunit) and GPV (that regulates thrombin‐dependent activation via GPIbα).Methods and results: Immunoblotting human platelet lysates using affinity‐purified antibodies against cytoplasmic domains of GPVI, GPIbα or GPV allowed simultaneous analysis of intact and cleaved receptor, and revealed (i) that a significant fraction of GPIbα, but not GPVI, exists in a cleaved state on platelets, even when isolated in the presence of metalloproteinase inhibitor (GM6001) or EDTA; (ii) the same‐sized membrane‐associated fragments of GPVI or GPIbα are generated by phorbol‐ester (PMA), the mitochondrial‐targeting reagent CCCP, the calmodulin inhibitor W7, or the thiol‐modifying reagent, N‐ethylmaleimide, that directly activates ADAM10/ADAM17; and (iii) GPV is shed by both metalloproteinase‐ and thrombin‐dependent mechanisms, depending on the concentration of thrombin. Based on the predicted cleavage area defined by these studies, ADAM10, but not ADAM17, cleaved a GPVI‐based synthetic peptide within the extracellular membrane‐proximal sequence (PAR^Q243YY) as analyzed by MALDI‐TOF‐MS. In contrast, ADAM17, but not ADAM10, cleaved within the GPIbα‐based peptide (LRG^V465LQ). Both ADAM10 and ADAM17 cleaved within a GPV‐based peptide (AQP^V494TT). Metalloproteinase‐mediated shedding of GPIbα from GPIb‐IX‐transfected or GPVI‐transfected cells induced by W7 or N‐ethylmaleimide was inhibited by mutagenesis of sequences identified from peptide analysis.Conclusions: These findings suggest surface levels of GPVI, GPIbα and GPV may be controlled by distinct mechanisms involving ADAM10 and/or ADAM17.

Platelet Receptor Proteolysis: A Mechanism for Downregulating Platelet Reactivity

The platelet plasma membrane is literally at the cutting-edge of recent research into proteolytic regulation of the function and surface expression of platelet receptors, revealing new mechanisms for how the thrombotic propensity of platelets is controlled in health and disease. Extracellular proteolysis of receptors irreversibly inactivates receptor-mediated adhesion and signaling, as well as releasing soluble fragments into the plasma where they act as potential markers or modulators. Platelet-surface sheddases, particularly of the metalloproteinase-disintegrin (ADAM) family, can be regulated by many of the same mechanisms that control receptor function, such as calmodulin association or activation of signaling pathways. This provides layers of regulation (proteinase and receptor), and a higher order of control of cellular function. Activation of pathways leading to extracellular shedding is concomitant with activation of intracellular proteinases such as calpain, which may also irreversibly deactivate receptors. In this review, platelet receptor shedding will be discussed in terms of (1) the identity of proteinases involved in receptor proteolysis, (2) key platelet receptors regulated by proteolytic pathways, and (3) how shedding might be regulated in normal physiology or future therapeutics. In particular, a focus on proteolytic regulation of the platelet collagen receptor, glycoprotein (GP)VI, illustrates many of the key biochemical, cellular, and clinical implications of current research in this area.

Mycobacterium tuberculosis induces the miR-33 locus to reprogram autophagy and host lipid metabolism

Mycobacterium tuberculosis (Mtb) survives in macrophages by evading delivery to the lysosome and promoting the accumulation of lipid bodies, which serve as a bacterial source of nutrients. We found that by inducing the microRNA (miRNA) miR-33 and its passenger strand miR-33*, Mtb inhibited integrated pathways involved in autophagy, lysosomal function and fatty acid oxidation to support bacterial replication. Silencing of miR-33 and miR-33* by genetic or pharmacological means promoted autophagy flux through derepression of key autophagy effectors (such as ATG5, ATG12, LC3B and LAMP1) and AMPK-dependent activation of the transcription factors FOXO3 and TFEB, which enhanced lipid catabolism and Mtb xenophagy. These data define a mammalian miRNA circuit used by Mtb to coordinately inhibit autophagy and reprogram host lipid metabolism to enable intracellular survival and persistence in the host.

Macrophage Mitochondrial Energy Status Regulates Cholesterol Efflux and Is Enhanced by Anti-miR33 in Atherosclerosis

RATIONALE Therapeutically targeting macrophage reverse cholesterol transport is a promising approach to treat atherosclerosis. Macrophage energy metabolism can significantly influence macrophage phenotype, but how this is controlled in foam cells is not known. Bioinformatic pathway analysis predicts that miR-33 represses a cluster of genes controlling cellular energy metabolism that may be important in macrophage cholesterol efflux. OBJECTIVE We hypothesized that cellular energy status can influence cholesterol efflux from macrophages, and that miR-33 reduces cholesterol efflux via repression of mitochondrial energy metabolism pathways. METHODS AND RESULTS In this study, we demonstrated that macrophage cholesterol efflux is regulated by mitochondrial ATP production, and that miR-33 controls a network of genes that synchronize mitochondrial function. Inhibition of mitochondrial ATP synthase markedly reduces macrophage cholesterol efflux capacity, and anti-miR33 required fully functional mitochondria to enhance ABCA1-mediated cholesterol efflux. Specifically, anti-miR33 derepressed the novel target genes PGC-1α, PDK4, and SLC25A25 and boosted mitochondrial respiration and production of ATP. Treatment of atherosclerotic Apoe(-/-) mice with anti-miR33 oligonucleotides reduced aortic sinus lesion area compared with controls, despite no changes in high-density lipoprotein cholesterol or other circulating lipids. Expression of miR-33a/b was markedly increased in human carotid atherosclerotic plaques compared with normal arteries, and there was a concomitant decrease in mitochondrial regulatory genes PGC-1α, SLC25A25, NRF1, and TFAM, suggesting these genes are associated with advanced atherosclerosis in humans. CONCLUSIONS This study demonstrates that anti-miR33 therapy derepresses genes that enhance mitochondrial respiration and ATP production, which in conjunction with increased ABCA1 expression, works to promote macrophage cholesterol efflux and reduce atherosclerosis.

Compromised ITAM‐based platelet receptor function in a patient with immune thrombocytopenic purpura

Summary.  Background: Receptors on platelets that contain immunoreceptor tyrosine‐based activation motifs (ITAMs) include collagen receptor glycoprotein (GP) VI, and FcγRIIa, a low affinity receptor for immunoglobulin (Ig) G. Objectives: We examined the function of GPVI and FcγRIIa in a patient diagnosed with immune thrombocytopenic purpura (ITP) who had unexplained pathological bruising despite normalization of the platelet count with treatment. Methods and Results: Patient platelets aggregated normally in response to ADP, arachadonic acid and epinephrine, but not to GPVI agonists, collagen or collagen‐related peptide, or to FcγRII‐activating monoclonal antibody (mAb) 8.26, suggesting ITAM receptor dysfunction. Plasma contained an anti‐GPVI antibody by MAIPA and aggregated normal platelets. Aggregating activity was partially (∼60%) blocked by FcγRIIa‐blocking antibody, IV.3, and completely blocked by soluble GPVI ectodomain. Full‐length GPVI on the patient platelet surface was reduced to ∼10% of normal levels, and a ∼10‐kDa GPVI cytoplasmic tail remnant and cleaved FcγRIIa were detectable by western blot, indicating platelet receptor proteolysis. Plasma from the patient contained ∼150 ng mL−1 soluble GPVI by ELISA (normal plasma, ∼15 ng mL−1) and IgG purified from patient plasma caused FcγRIIa‐mediated, EDTA‐sensitive cleavage of both GPVI and FcγRIIa on normal platelets. Conclusions: In ITP patients, platelet autoantibodies can curtail platelet receptor function. Platelet ITAM receptor dysfunction may contribute to the increased bleeding phenotype observed in some patients with ITP.

Targeting macrophage necroptosis for therapeutic and diagnostic interventions in atherosclerosis

Necroptosis promotes necrotic core and vulnerable atherosclerosis in humans and mice and is a prospective therapeutic and diagnostic tool. Atherosclerosis results from maladaptive inflammation driven primarily by macrophages, whose recruitment and proliferation drive plaque progression. In advanced plaques, macrophage death contributes centrally to the formation of plaque necrosis, which underlies the instability that promotes plaque rupture and myocardial infarction. Hence, targeting macrophage cell death pathways may offer promise for the stabilization of vulnerable plaques. Necroptosis is a recently discovered pathway of programmed cell necrosis regulated by RIP3 and MLKL kinases that, in contrast to apoptosis, induces a proinflammatory state. We show herein that necroptotic cell death is activated in human advanced atherosclerotic plaques and can be targeted in experimental atherosclerosis for both therapeutic and diagnostic interventions. In humans with unstable carotid atherosclerosis, expression of RIP3 and MLKL is increased, and MLKL phosphorylation, a key step in the commitment to necroptosis, is detected in advanced atheromas. Investigation of the molecular mechanisms underlying necroptosis showed that atherogenic forms of low-density lipoprotein increase RIP3 and MLKL transcription and phosphorylation—two critical steps in the execution of necroptosis. Using a radiotracer developed with the necroptosis inhibitor necrostatin-1 (Nec-1), we show that 123I-Nec-1 localizes specifically to atherosclerotic plaques in Apoe−/− mice, and its uptake is tightly correlated to lesion areas by ex vivo nuclear imaging. Furthermore, treatment of Apoe−/− mice with established atherosclerosis with Nec-1 reduced lesion size and markers of plaque instability, including necrotic core formation. Collectively, our findings offer molecular insight into the mechanisms of macrophage cell death that drive necrotic core formation in atherosclerosis and suggest that this pathway can be used as both a diagnostic and therapeutic tool for the treatment of unstable atherosclerosis.

Programmed autologous cleavage of platelet receptors

Summary.  Platelet adhesion receptors play a critical role in vascular pathophysiology, and control platelet adhesion, activation and aggregation in hemostasis, thrombotic disease and atherogenesis. One of the key emerging mechanisms for regulating platelet function is the programmed autologous cleavage of platelet receptors. Induced by ligand binding or platelet activation, proteolysis at extracellular (ectodomain shedding) or intracellular (cytoplasmic domain deactivation) sites down‐regulates the adheso‐signaling function of receptors, thereby controlling not only platelet responsiveness, but in the case of ectodomain shedding, liberating soluble ectodomain fragments into plasma where they constitute potential modulators or markers. This review discusses the underlying mechanisms for dual proteolytic pathways of receptor regulation, and the impact of these pathways on thrombus formation and stability in vivo.

Therapeutic Inhibition of miR-33 Promotes Fatty Acid Oxidation but Does Not Ameliorate Metabolic Dysfunction in Diet-Induced Obesity

Objective—miR-33 has emerged as an important regulator of lipid homeostasis. Inhibition of miR-33 has been demonstrated as protective against atherosclerosis; however, recent studies in mice suggest that miR-33 inhibition may have adverse effects on lipid and insulin metabolism. Given the therapeutic interest in miR-33 inhibitors for treating atherosclerosis, we sought to test whether pharmacologically inhibiting miR-33 at atheroprotective doses affected metabolic parameters in a mouse model of diet-induced obesity. Approach and Results—High-fat diet (HFD) feeding in conjunction with treatment of male mice with 10 mg/kg control anti-miR or anti-miR33 inhibitors for 20 weeks promoted equivalent weight gain in all groups. miR-33 inhibitors increased plasma total cholesterol and decreased serum triglycerides compared with control anti-miR, but not compared with PBS-treated mice. Metrics of insulin resistance were not altered in anti-miR33–treated mice compared with controls; however, respiratory exchange ratio was decreased in anti-miR33–treated mice. Hepatic expression of miR-33 targets Abca1 and Hadhb were derepressed on miR-33 inhibition. In contrast, protein levels of putative miR-33 target gene SREBP-1 or its downstream targets genes Fasn and Acc were not altered in anti-miR33–treated mice, and hepatic lipid accumulation did not differ between groups. In the adipose tissue, anti-miR33 treatment increased Ampk gene expression and markers of M2 macrophage polarization. Conclusions—We demonstrate in a mouse model of diet-induced obesity that therapeutic silencing of miR-33 may promote whole-body oxidative metabolism but does not affect metabolic dysregulation. This suggests that pharmacological inhibition of miR-33 at doses known to reduce atherosclerosis may be a safe future therapeutic.

GPIba-selective activation of platelets induces platelet signaling events comparable to GPVI activation events

Platelet glycoprotein (GP)Ib-IX-V, which binds von Willebrand factor (VWF), and GPVI, which binds collagen, form an adhesion-signaling complex on platelets and mediate platelet adhesion in flowing blood. Platelet activation following engagement of GPIb-IX-V/GPVI by VWF/collagen is critical for initiation and development of a protective thrombus across a site of damaged or exposed endothelium. We examined platelet aggregation and signaling following selective engagement of platelet GPIbα (the major ligand-binding subunit of GPIb-IX-V) by a multivalent surface-expressed GPIbα-binding VWF-A1 domain on COS-7 cells. COS-7 cells expressing the VWF-A1 domain containing an R543W mutation (a gain-of-function mutation found in Type 2B von Willebrands Disease) were used as a selective agonist for GPIb-IX-V. When incubated in a cell-to-platelet ratio of up to 1 : 1200, VWF-A1/R543W cells caused rapid, spontaneous aggregation of washed platelets that was GPIbα- and αIIbβ3-dependent (blocked by inhibitory anti-VWF-A1, anti-GPIbα and anti-αIIbβ3 antibodies). Platelet aggregation was also sensitive to inhibitors of Src, phosphoinositide 3-kinase (PI3-kinase) or Syk, confirming a role for these proteins in GPIbα-mediated signal transduction. Platelet tyrosine phosphorylation patterns and specific tyrosine phosphorylation of Syk after GPIbα engagement by VWF-A1/R543W was comparable to that induced by engagement of GPVI by collagen or collagen-related peptide (CRP). These data indicate signaling events triggered by specific ligation of GPIbα can lead to robust platelet activation and help define GPIb-IX-V as both an adhesion and signaling receptor on platelets.

Phagocytosis of IgG-coated polystyrene beads by macrophages induces and requires high membrane order.

The biochemical composition and biophysical properties of cell membranes are hypothesized to affect cellular processes such as phagocytosis. Here, we examined the plasma membranes of murine macrophage cell lines during the early stages of uptake of immunoglobulin G (IgG)‐coated polystyrene particles. We found that the plasma membrane undergoes rapid actin‐independent condensation to form highly ordered phagosomal membranes, the biophysical hallmark of lipid rafts. Surprisingly, these membranes are depleted of cholesterol and enriched in sphingomyelin and ceramide. Inhibition of sphingomyelinase activity impairs membrane condensation, F‐actin accumulation at phagocytic cups and particle uptake. Switching phagosomal membranes to a cholesterol‐rich environment had no effect on membrane condensation and the rate of phagocytosis. In contrast, preventing membrane condensation with the oxysterol 7‐ketocholesterol, even in the presence of ceramide, blocked F‐actin dissociation from nascent phagosomes and particle uptake. In conclusion, our results suggest that ordered membranes function to co‐ordinate F‐actin remodelling and that the biophysical properties of phagosomal membranes are essential for phagocytosis.