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![Heme-induced TLR4 signaling activates AKT to inhibit complex V activity. (A) Representative western blot and quantification of pAKT/total AKT in platelets treated with heme (2.5 μM) in the presence or absence of BX795 (TBK1 inh; 2 μM), N-[1-[2-(4-Morpholinyl) ethyl]-1H-benzimidazol-2-yl]-3-nitrobenzamide (IRAK1/4 inh; 100 nM) or (5Z)-7-Oxozeaenol (TAK1 inh; 5 nM). (B) Platelet mitochondrial complex V activity (C) the ratio of phosphorylated to total complex V beta subunit, (D) MitoSOX fluorescence and (E) thrombospondin-1 release in heme treated platelets pre-treated with or without ARQ092 (10 μM). Data are represented as Mean ± SEM. ****p < 0.0001, ***p < 0.001, **p < 0.01, ns -not significant. n = 4.](publication/356792915/figure/fig4/AS:1104651801899009@1640380921101/Heme-induced-TLR4-signaling-activates-AKT-to-inhibit-complex-V-activity-A.png)
Heme-induced TLR4 signaling activates AKT to inhibit complex V activity. (A) Representative western blot and quantification of pAKT/total AKT in platelets treated with heme (2.5 μM) in the presence or absence of BX795 (TBK1 inh; 2 μM), N-[1-[2-(4-Morpholinyl) ethyl]-1H-benzimidazol-2-yl]-3-nitrobenzamide (IRAK1/4 inh; 100 nM) or (5Z)-7-Oxozeaenol (TAK1 inh; 5 nM). (B) Platelet mitochondrial complex V activity (C) the ratio of phosphorylated to total complex V beta subunit, (D) MitoSOX fluorescence and (E) thrombospondin-1 release in heme treated platelets pre-treated with or without ARQ092 (10 μM). Data are represented as Mean ± SEM. ****p < 0.0001, ***p < 0.001, **p < 0.01, ns -not significant. n = 4.
Source publication
Hemolysis, a pathological component of many diseases, is associated with thrombosis and vascular dysfunction. Hemolytic products, including cell-free hemoglobin and free heme directly activate platelets. However, the effect of hemolysis on platelet degranulation, a central process in not only thrombosis, but also inflammatory and mitogenic signalin...
Contexts in source publication
Context 1
... the phosphorylation status of Akt in heme treated platelets by blocking TLR4 downstream signaling with TBK1, IRAK1/4 or TAK1 inhibitors. Heme treated platelets showed a significant increase in the level of Akt phosphorylation at serine 473 (pAkt -S473), which was decreased in heme-treated platelets pre-treated with blockers of TLR4 signaling (Fig. 4A). These data demonstrate that heme-mediated activation of TLR4 signaling stimulates downstream Akt ...
Context 2
... activation of Akt regulates complex V activity, we treated platelets with heme in the presence and absence of ARQ092, a small molecule that prevents phosphorylation of Akt at S473 [71], and measured complex V activity. While heme treatment inhibited complex V activity, this effect was significantly attenuated when Akt phosphorylation was blocked (Fig. 4B). Immunoprecipitation of the beta subunit of complex V from these platelets showed that heme induced significant phosphorylation of complex V, and this phosphorylation was attenuated when Akt activation was blocked with ARQ092 (Fig. 4C). Collectively, these data demonstrate that heme-induced TLR4 activation stimulates the downstream ...
Context 3
... inhibited complex V activity, this effect was significantly attenuated when Akt phosphorylation was blocked (Fig. 4B). Immunoprecipitation of the beta subunit of complex V from these platelets showed that heme induced significant phosphorylation of complex V, and this phosphorylation was attenuated when Akt activation was blocked with ARQ092 (Fig. 4C). Collectively, these data demonstrate that heme-induced TLR4 activation stimulates the downstream activation of Akt, which phosphorylates complex V and inhibits its activity. Further, pre-treatment of platelets with ARQ092 significantly attenuated heme-induced platelet mtROS production and TSP-1 release ( Fig. 4D and ...
Context 4
... was blocked with ARQ092 (Fig. 4C). Collectively, these data demonstrate that heme-induced TLR4 activation stimulates the downstream activation of Akt, which phosphorylates complex V and inhibits its activity. Further, pre-treatment of platelets with ARQ092 significantly attenuated heme-induced platelet mtROS production and TSP-1 release ( Fig. 4D and ...
Citations
... 4 Hemin, a degradation product of cell-free hemoglobin, has been shown to be a more potent agonist than hemoglobin for directly activating platelets. 5 It is a signaling molecule that mediates various biochemical processes, such as inflammation, transcription, and signal transduction, via transient binding to various proteins. 6,7 Annarapu GK. et al. showed that heminstimulated human platelet mitochondrial oxidant production induces targeted granule secretion. ...
Plain Language Summary
What is the context? Hemolysis is a primary hematological disease. Hemolysis is a pathological complication of several diseases.
Hemin, a degradation product of cell-free hemoglobin, has been proven to be a more potent agonist than hemoglobin for directly activating platelets.
Platelet membrane glycoproteins (GP), including GPIb-IX and GPIIb/IIIa complexes, play crucial roles in platelet hemostasis.
Desialylation (loss of sialic acid residues) of GPIbα, is believed to regulate physiological platelet clearance through liver macrophages and hepatocytes.
What is new? In this study, we evaluated the effects of hemolysis on platelet clearance. We first analyzed the influence of hemin at 0-50 μM on platelets in vitro before exploring the mechanism underlying hemin-induced platelet activation and its role in platelet clearance in vitro and in vivo.
Our analyses suggest that: Hemin bound to GPIbα on the platelet surface with high affinity.
Platelet clearance occurred slowly in the liver and spleen after hemin treatment.
Platelets exhibited significant significantly reduced GPIbα surface expression and desialylation after hemin treatment.
Platelets exhibited significant significantly reduced GPIbα surface expression and desialylation after hemin treatment.
What is the impact? This study provides new insights into the role of hemin in the mechanisms of GPIbα-mediated platelets activation and clearance in diseases associated with hemolysis.
... Hemolysis is associated with coagulopathy and thrombosis through its effects on platelets, the endothelium, and macrophages. Human platelets exposed to heme or hemoglobin show a dosedependent increase in surface P-selectin and activated glycoprotein IIb/IIIa (49). This effect depends, at least in part, on hemebinding through TLR4, which leads to inhibition of platelet mitochondrial complex V, increased production of mitochondrial ROS, and release of a subset of platelet granules (49). ...
... Human platelets exposed to heme or hemoglobin show a dosedependent increase in surface P-selectin and activated glycoprotein IIb/IIIa (49). This effect depends, at least in part, on hemebinding through TLR4, which leads to inhibition of platelet mitochondrial complex V, increased production of mitochondrial ROS, and release of a subset of platelet granules (49). Heme-activated platelets release thrombospondin-1, which potentiates platelet activation and aggregation, enhances leukocyte migration, and inhibits endothelial nitric oxide signaling (49,50). ...
... This effect depends, at least in part, on hemebinding through TLR4, which leads to inhibition of platelet mitochondrial complex V, increased production of mitochondrial ROS, and release of a subset of platelet granules (49). Heme-activated platelets release thrombospondin-1, which potentiates platelet activation and aggregation, enhances leukocyte migration, and inhibits endothelial nitric oxide signaling (49,50). Heme exposure also boosts tissue factor expression in the endothelium and in macrophages (51,52). ...
OBJECTIVES
Cell-free hemoglobin (CFH) is a potent mediator of endothelial dysfunction, organ injury, coagulopathy, and immunomodulation in hemolysis. These mechanisms have been demonstrated in patients with sepsis, hemoglobinopathies, and those receiving transfusions. However, less is known about the role of CFH in the pathophysiology of trauma, despite the release of equivalent levels of free hemoglobin.
DATA SOURCES
Ovid MEDLINE, Embase, Web of Science Core Collection, and BIOSIS Previews were searched up to January 21, 2023, using key terms related to free hemoglobin and trauma.
DATA EXTRACTION
Two independent reviewers selected studies focused on hemolysis in trauma patients, hemoglobin breakdown products, hemoglobin-mediated injury in trauma, transfusion, sepsis, or therapeutics.
DATA SYNTHESIS
Data from the selected studies and their references were synthesized into a narrative review.
CONCLUSIONS
Free hemoglobin likely plays a role in endothelial dysfunction, organ injury, coagulopathy, and immune dysfunction in polytrauma. This is a compelling area of investigation as multiple existing therapeutics effectively block these pathways.
... In 2007, heme was demonstrated to mediate proinflammatory signalling in mouse macrophages via the CD14-TLR4-MyD88 axis, which suggested heme as a TLR4 agonist [37]. Up to date, the involvement of several cofactors (CD14, MD2), adaptor, and effector proteins in heme-triggered TLR4 signalling in macrophages, platelets, HEK293, peripheral blood mononuclear (PBMCs), microglial, dendritic, and endothelial cells has been described [14,16,17,20,[37][38][39][40][41][42][43][44], which was recently contextualized in the knowledge graph 'HemeKG' [19]. Corroborating the heme-TLR4 association, stimulating cells with heme induces signalling via NF-κB and AP1 resulting in the expression of proinflammatory mediators, such as TNF and the functional IL-8 homologue CXCL1 [37,39,45]. ...
... In sickle cell mice, heme triggers acute lung injury, nephrotoxicity, liver damage and vasoocclusion in a TLR4-dependent way, manifesting the classical complications in sickle cell disease [13,14,16,43,47]. The prothrombotic consequences of heme are thereby initiated through different processes such as the mobilization of adhesion proteins, elevated tissue factor expression in endothelial cells and platelet granule release [14,16,44,47]. In contrast to this detailed functional characterization of the heme-triggered TLR4 signalling mostly in mouse models, direct heme binding to mTLR4 [48] and mouse and human MD2 [48][49][50] was just recently demonstrated but lacks important details of the interaction on the molecular level. ...
... Numerous studies have shown a major role of TLR4 signalling in heme-driven proinflammatory, complementactivating and prothrombotic effects under haemolytic conditions [14,26,32,37,40,41,44,45]. In the present study, the heme-binding behaviour and capacity of TLR4, MD2 and the TLR4-MD2 complex was investigated on the molecular level. ...
Haemolytic disorders, such as sickle cell disease, are accompanied by the release of high amounts of labile heme into the intravascular compartment resulting in the induction of proinflammatory and prothrombotic complications in affected patients. In addition to the relevance of heme-regulated proteins from the complement and blood coagulation systems, activation of the TLR4 signalling pathway by heme was ascribed a crucial role in the progression of these pathological processes. Heme binding to the TLR4-MD2 complex has been proposed recently, however, essential mechanistic information of the processes at the molecular level, such as heme-binding kinetics, the heme-binding capacity and the respective heme-binding sites (HBMs) is still missing. We report the interaction of TLR4, MD2 and the TLR4-MD2 complex with heme and the consequences thereof by employing biochemical, spectroscopic, bioinformatic and physiologically relevant approaches. Heme binding occurs transiently through interaction with up to four HBMs in TLR4, two HBMs in MD2 and at least four HBMs in their complex. Functional studies highlight that mutations of individual HBMs in TLR4 preserve full receptor activation by heme, suggesting that heme interacts with TLR4 through different binding sites independently of MD2. Furthermore, we confirm and extend the major role of TLR4 for heme-mediated cytokine responses in human immune cells.
... Subsequently, Kho et al. [15] demonstrated that platelets were involved in the killing of erythrocytic stages of all four major human Plasmodium species. In 2021, Annarapu et al. [130] demonstrated that extracellular hemoglobin leads to platelet activation. This results in the production of mitochondrial reactive oxygen species and platelet degranulation. ...
... This results in the production of mitochondrial reactive oxygen species and platelet degranulation. Platelet factor-4 (PF-4, also known as CXCL4) accumulates in infected erythrocytes and is the chemokine involved in platelet activation, inducing production of mitochondrial reactive oxygen species [130]. These results indicate that platelets may be involved in oxidative damage of RBCs in canine babesiosis. ...
... Together with ICAM-1 and P-selectin, activates neutrophils to form NETs. [92,95,96,285,286] PF-4 (CXCL4) Platelet activation and induction of production of mitochondrial reactive oxygen species. [130] IP-10 (CXCL10) Contribution in the recruitment of granulocytes and macrophages, and phagocytosis. ...
Canine babesiosis is a tick-borne protozoan disease caused by intraerythrocytic parasites of the genus Babesia. The infection may lead to anemia in infected dogs. However, anemia is not directly caused by the pathogen. The parasite’s developmental stages only have a marginal role in contributing to a decreased red blood cell (RBC) count. The main cause of anemia in affected dogs is the immune response to the infection. This response includes antibody production, erythrophagocytosis, oxidative damage of RBCs, complement activation, and antibody-dependent cellular cytotoxicity. Moreover, both infected and uninfected erythrocytes are retained in the spleen and sequestered in micro-vessels. All these actions are driven by pro-inflammatory cytokines and chemokines, especially IFN-γ, TNF-α, IL-6, and IL-8. Additionally, imbalance between the actions of pro- and anti-inflammatory cytokines plays a role in patho-mechanisms leading to anemia in canine babesiosis. This article is a review of the studies on the pathogenesis of anemia in canine babesiosis and related diseases, such as bovine or murine babesiosis and human or murine malaria, and the role of pro-inflammatory cytokines and chemokines in the mechanisms leading to anemia in infected dogs.
... Activated PLCγ2 usually catalyzes phosphatidylinositol-4,5-bisphosphate (PIP 2 ) hydrolysis to inositol trisphosphate (IP 3 ) and diacylglycerol (DAG), leading to calcium mobilization and protein kinase C (PKC) activation [51]. A heme-induced increase in IP3 and DAG levels has not yet been described; however, elevated intracellular calcium levels and mobilization in platelets as well as PKC activation in neutrophils has already been monitored (in the presence of up to 20 µM heme) [46,57,58]. In general, calcium mobilization enables contractile activity through myosin, which leads to the characteristic shape change of activated platelets [50]. ...
... Apart from PLC activation (see above), PI3K can catalyze Akt phosphorylation, explaining its activation in platelets upon incubation with heme (2.5 µM) [57]. Heme-triggered platelet activation along with Akt phosphorylation was demonstrated to be dependent on TLR4 [57]. ...
... Apart from PLC activation (see above), PI3K can catalyze Akt phosphorylation, explaining its activation in platelets upon incubation with heme (2.5 µM) [57]. Heme-triggered platelet activation along with Akt phosphorylation was demonstrated to be dependent on TLR4 [57]. Although the TLR4 signaling pathway is highly pronounced in the HemeThrom-bKG network, it did not emerge during crosstalk analysis, since it is not yet recognized for its contribution to procoagulant processes in the databases. ...
Excess labile heme, occurring under hemolytic conditions, displays a versatile modulator in the blood coagulation system. As such, heme provokes prothrombotic states, either by binding to plasma proteins or through interaction with participating cell types. However, despite several independent reports on these effects, apparently contradictory observations and significant knowledge gaps characterize this relationship, which hampers a complete understanding of heme-driven coagulopathies and the development of suitable and specific treatment options. Thus, the computational exploration of the complex network of heme-triggered effects in the blood coagulation system is presented herein. Combining hemostasis- and heme-specific terminology, the knowledge available thus far was curated and modeled in a mechanistic interactome. Further, these data were incorporated in the earlier established heme knowledge graph, "HemeKG", to better comprehend the knowledge surrounding heme biology. Finally, a pathway enrichment analysis of these data provided deep insights into so far unknown links and novel experimental targets within the blood coagulation cascade and platelet activation pathways for further investigation of the prothrombotic nature of heme. In summary, this study allows, for the first time, a detailed network analysis of the effects of heme in the blood coagulation system.
Background:
Cell-free hemoglobin (CFH) and free heme are potent mediators of endotheliopathy and organ injury in sepsis, but their roles in other hemolytic pathologies are not well-defined. A prime example is trauma where early hemolysis may initiate damage and predict outcome. Here, we investigated the presence of plasma CFH, heme, and their major scavengers after traumatic injury.
Methods:
Adult patients who presented as highest-level activations were prospectively enrolled at a level 1 trauma center between 2021 and 2023. Venous blood was collected upon arrival (pretransfusion) and 6, 12, and 24 hours after admittance for quantification of CFH, haptoglobin, heme, and hemopexin.
Results:
We studied 119 mostly male subjects (101:18) with a median age of 48 years (interquartile range [IQR], 31-64 years) and an Injury Severity Score of 22 (IQR, 11-29); the majority had suffered blunt force trauma. The 28-day mortality rate was 11%. Cell-free hemoglobin was high upon emergency department arrival (10.9 μM; IQR, 6.8-17.6) and then declined but remained elevated compared with normative levels during the monitoring period (>5 vs. ~0.2 μM). The initial drop in CFH was attributed to haptoglobin binding and clearance. Notably, there was a subgroup of patients with two- to threefold higher levels of CFH on emergency department arrival (median, 25 μM). Patients with these highest CFH levels had longer hospital stays and more frequent complications.
Conclusion:
Cell-free hemoglobin is elevated in trauma patients very early after injury and may impact outcome. While further work is needed, early correction of hemolysis could provide benefit.
Level of evidence:
Prognostic Study; Level III.
Thrombosis and inflammation are intimately linked and synergistically contribute to the pathogenesis of numerous thromboinflammatory diseases, including sickle cell disease (SCD). While platelets are central to thrombogenesis and inflammation, the molecular mechanisms of crosstalk between the 2 remain elusive. High-mobility group box 1 (HMGB1) regulates inflammation and stimulates platelet activation through Toll-like receptor 4. However, it remains unclear whether HMGB1 modulates other thrombotic agonists to regulate platelet activation. Herein, using human platelets, we demonstrate that HMGB1 significantly enhanced ADP-mediated platelet activation. Furthermore, inhibition of the purinergic receptor P2Y12 attenuated HMGB1-dependent platelet activation. Mechanistically, we show that HMGB1 stimulated ADP secretion, while concomitantly increasing P2Y12 levels at the platelet membrane. We show that in SCD patients, increased plasma HMGB1 levels were associated with heightened platelet activation and surface P2Y12 expression. Treatment of healthy platelets with plasma from SCD patients enhanced platelet activation and surface P2Y12, and increased sensitivity to ADP-mediated activation, and these effects were linked to plasma HMGB1. We conclude that HMGB1-mediated platelet activation involves ADP-dependent P2Y12 signaling, and HMGB1 primes platelets for ADP signaling. This complementary agonism between ADP and HMGB1 furthers the understanding of thromboinflammatory signaling in conditions such as SCD, and provides insight for therapeutic P2Y12 inhibition.
