Deirdre Nolfi-Donegan’s research while affiliated with University of Pittsburgh and other places
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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.
Background:
Hemophilia A is a hereditary bleeding disorder characterized by low factor VIII (FVIII) activity. Patients with severe bleeding phenotypes are at risk for significant bleeding-related morbidity and mortality including the development of hemophilic arthropathy. In recent years, prophylaxis with emicizumab, a bispecific monoclonal antibody, has supplanted prophylactic FVIII infusions due to its ease of self-administration, long half-life, and low annual bleeding rate (ABR), independent of tolerization status to alloantibodies against FVIII (inhibitors). However, it is unclear if emicizumab FVIII-mimic activity is sufficient to achieve hemostasis in moderate-severe bleeds, and therefore, FVIII replacement may still be necessary.
Despite its well-established benefits, the use of emicizumab in previously untreated patients (PUPs), and patients with under 20 exposures to FVIII is still not well understood due to a lack of long-term experience and understanding of its impact on the pathophysiology of FVIII inhibitors. Further investigation is also needed regarding the risk of inhibitor recurrence in patients with previous inhibitors and the concomitant use of emicizumab.
At our institution, patients on emicizumab with a history of inhibitors do not continue post-immune tolerance induction prophylactic FVIII infusions. We also initiate emicizumab before 20 exposures to FVIII, and sometimes before immune tolerance induction (ITI). We explored our patient population to understand inhibitor incidence and recurrence amidst emicizumab prophylaxis.
Methods: We performed a single center retrospective analysis of patients served by the Hemophilia Center of Western Pennsylvania. We identified patients with Hemophilia A on emicizumab prophylaxis. Relevant data was collected including demographics, inhibitor status at time of transition to emicizumab and in the available follow-up period, bleeding events after transition, and number of FVIII exposures before and after transition. Inhibitor recurrence was defined as laboratory evidence of inhibitor using chromogenic Bethesda assay and/or unexplained breakthrough bleeding events despite on-demand FVIII infusions.
Results:
Data was reviewed for 36 patients on emicizumab prophylaxis. Three patients were lost to follow-up. Median age was 15.3 years (range 1.2 - 82). Median follow-up time on emicizumab was 3.5 years (range 0.5 - 5). Two patients had < 20 FVIII exposures prior to emicizumab, and one of those had an inhibitor before emicizumab. In total, 15 patients (45%) were diagnosed with an inhibitor prior to emicizumab. Of these, 8 patients (53%) were tolerized prior to emicizumab, and 7 patients (47%) were not tolerized prior to emicizumab. Median peak inhibitor titer was 4.2 Bethesda units (range 0.7 - 281 Bethesda units). After transition to emicizumab, no tolerized patients for which inhibitor status was tested during the follow-up period had inhibitor recurrence, and 1 non-tolerized patient's inhibitor became undetectable after 4 years of emicizumab therapy (the same patient who had developed an inhibitor with < 20 FVIII exposures prior to emicizumab). Mean ABR was 0.33 events/year for tolerized patients (median 0, range 0 - 3); 0.17 events/year for non-tolerized patients (median 0, range 0 - 1); and 0.60 events/year for patients with no history of inhibitor (median 0, range 0 - 4). Mean number of FVIII exposures per year was 0.09 for tolerized patients (median 0, range 0 - 1); 0.07 for non-tolerized patients (median 0, range 0 - 1); and 1.2 for patients with no history of inhibitor (median 0, range 0 - 20).
Conclusion:
In our small cohort, no patient who was tolerized prior to emicizumab had recurrence of clinical or laboratory evidence of inhibitor while on emicizumab therapy, despite infrequent exposure to FVIII infusion. We did not have enough power to determine whether patients with < 20 FVIII exposures before emicizumab developed inhibitors, though one such patient had inhibitor resolution on emicizumab without ITI. Consistent with other works, overall ABRs in all patients on emicizumab therapy were low. These findings add to other studies on the safety of emicizumab and its impact on the natural history of FVIII inhibitors. Our study also suggests that continuing post-ITI prophylactic FVIII infusions in tolerized patients on emicizumab may not be necessary. Larger-scale studies are required to substantiate our findings.
Background:The growing epidemic of obesity is strongly linked to cardiovascular disease (CVD). Obesity leads to endothelial dysfunction, an instigating event that drives vascular injury and subsequent CVD, but the mechanisms by which obesity causes endothelial dysfunction remain unclear. Platelets circulate proximal to the endothelium and are traditionally thought to contribute to obesity-associated vasculopathy through thrombotic activation. However, platelets are metabolically active and release vaso-modulatory molecules into their environment and the role of these functions have not been examined in the context of obesity-induced vasculopathy. We previously showed that alterations in platelet mitochondrial function regulate platelet degranulation. Further, it has been reported that thrombospondin 1 (TSP1), a platelet factor that propagates endothelial dysfunction, is elevated in the plasma of obese humans. We hypothesized that obesity/weight gain modulates platelet mitochondrial function, which stimulates the release of TSP1 from platelets to propagate vascular dysfunction.
Methods: Platelets were isolated from lean and obese humans and mitochondrial structure and function measured. To induce diet-induced obesity wildtype (WT) mice were fed with a high fat diet (HFD, 60 kcal% fat) for 10 weeks, and low fat diet (LFD, 10 kcal% fat)- fed WT mice were used as controls. Platelets and plasma from the mice were screened for mitochondrial proteins and plasma TSP-1 was measured by ELISA. A murine model with platelet-specific deletion of mitofusin-1 (MFN1; pltMFN1KO mice), a mitochondrial GTP-ase that increases mitochondrial function was generated and fed either HFD or LFD for 4-10 weeks. Further, endothelial-dependent- and independent- vascular relaxation was measured in both groups by stimulating aortic rings with acetylcholine (Ach) or sodium nitroprusside (SNP) using wire myography. Additionally, platelet-specific TSP1 knockout (plt-TSP1KO) mice were generated and fed with HFD for 10 weeks and measured plasma TSP1 levels.
Results: Platelets from obese subjects showed increased levels of MFN1 compared to lean subjects. WT mice fed with HFD had increased platelet MFN1 and plasma TSP1 levels compared to LFD-fed WT mice. Platelet-specific deletion of MFN1 in mice resulted in reduced plasma TSP1 levels compared to control PF4-cre mice after HFD challenge. We found that HFD caused impaired Ach-stimulated vasorelaxation (indicative of endothelial dysfunction) in control PF4-cre mice, and this effect was attenuated in pltMFN1KO mice. Further, we found that HFD-induced increases in plasma TSP1 levels were attenuated in plt-TSP1KO mice, indicating platelets are the major source of TSP1 in HFD induced obesity model.
Conclusion:These data demonstrate that obesity/weight gain induces platelet MFN1 expression, which drives TSP1 release from platelets to propagate vascular dysfunction. Our findings elucidate a novel platelet-centric mechanism underlying obesity-associated vasculopathy and have implications for the targeting of non-thrombotic platelet function as a therapeutic strategy for obesity-induced vasculopathy.
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 signaling, remains less clear. Our group showed that hemoglobin-induced platelet activation involved the production of mitochondrial reactive oxygen species (mtROS). However, the molecular mechanism by which extracellular hemolysis induces platelet mtROS production, and whether these mtROS regulate platelet degranulation remains unknown. Here, we demonstrate using isolated human platelets that cell free heme is a more potent agonist for platelet activation than hemoglobin, and stimulates the release of a specific set of molecules, including the glycoprotein thrombospondin-1 (TSP-1), from the α-granule of platelets. We uncover the mechanism of heme-mediated platelet mtROS production which is dependent on the activation of platelet toll-like receptor 4 (TLR4) signaling and leads to the downstream phosphorylation and inhibition of complex-V by the serine kinase Akt. Notably, inhibition of platelet TLR4 or Akt, or scavenging of mtROS prevents heme-induced granule release in vitro. Further, heme-dependent granule release is significantly attenuated in vivo in mice lacking TLR4 or those treated with the mtROS scavenger MitoTEMPO. These data elucidate a novel mechanism of TLR4-mediated mitochondrial regulation, establish the mechanistic link between hemolysis and platelet degranulation, and begin to define the heme and mtROS-dependent platelet secretome. These data have implications for hemolysis-induced thrombo-inflammatory signaling and for the consideration of platelet mitochondria as a therapeutic target in hemolytic disorders.
Background: Patients with Sickle Cell Disease (SCD) are at greater risk for thrombosis and the development of chronic vasculopathy, both of which are major contributors to morbidity and mortality in these patients. While thrombosis and vasculopathy are associated with hemolysis in SCD, the molecular mechanisms by which hemolysis propagates these conditions remains unclear. At the cellular level, we and others have shown that hemolytic components, including hemoglobin and its degradation product, free heme, directly activate platelets. Notably, activated platelets are not only central to thrombosis, but are also implicated in vasculopathy through their degranulation, which results in the release of vasoactive molecules. Though heme and hemoglobin-induced platelet activation has been widely studied, the effect of hemolytic products on platelet degranulation and the identity of the resulting platelet secretome remains less clear. We hypothesize that free heme activates a platelet signaling cascade resulting in platelet degranulation and the release of specific "secretome" molecules.
Methods: Washed platelets were prepared from whole blood collected from healthy human volunteers in 0.32% sodium citrate (n=6). Platelets were treated with heme (2.5µM) in the presence or absence of MitoTEMPO (10 µM) - a mitochondrial oxidant (mtROS) scavenger, or ARQ092 (10 µM), a small molecule that prevents phosphorylation of the serine/threonine kinase Akt at S473. Platelet mtROS was measured by fluorescence spectroscopy using MitoSOX Red. Thrombospondin-1 (TSP1), CXCL7, Fibroblast Growth Factor (FGF) basic, TGFβ, IL-1β, PDGF-B, angiostatin, kininogen, CD40L and PAI-1 were quantified in heme treated platelet releasates in the presence and absence of MitoTEMPO by dot blot. The enzymatic activity of mitochondrial electron transport complex V was measured spectrophotometrically by kinetic assay.
Results:
We found that heme stimulated the release of a specific set of molecules from the α-granule of platelets, including TSP1, CXCL7, FGF basic, TGFβ, IL-1β, PDGF-B, angiostatin, and kininogen; but did not stimulate the release of CD40L or PAI-1. Mechanistic studies demonstrate that the release of several of these molecules was dependent on heme-induced activation of platelet Akt which inhibits mitochondrial complex V, resulting in mtROS production. Consistent with this mechanism, the heme-stimulated release of TSP1, CXCL7, FGF basic, IL-1β, PDGF-B, and angiostatin were significantly attenuated by preventing Akt phosphorylation at Ser473 with ARQ092, which also prevented complex V inhibition and mtROS production. Direct scavenging of mtROS with MitoTEMPO also attenuated heme-induced release of these molecules.
Conclusion: These data, for the first time, begin to characterize the platelet secretome released in response to free heme. Further, they demonstrate a novel molecular pathway in which extracellular heme induces the activation of platelet Akt to inhibit mitochondrial complex V, ultimately inducing mtROS. Notably, this study suggests that release of a proportion of the heme-induced secretome is regulated by mtROS production and can be suppressed by mtROS scavengers. Ongoing studies are further characterizing the hemolysis-induced platelet secretome, the downstream effects of secretome products on vascular function, and the extent of regulation of the secretome by mtROS. These studies provide a mechanistic link between hemolysis and platelet degranulation, by which the release of mitogens can lead to the pathogenesis of vascular wall dysfunction. These studies also suggest that platelet mtROS may represent a novel therapeutic target to attenuate vascular dysfunction in hemolytic disorders including SCD.
Note: The finding discussed in the above abstract are available as preprint in bioRxiv; doi: https://doi.org/10.1101/2021.08.02.454816
Disclosures
No relevant conflicts of interest to declare.
Hemolysis is a pathological component of many diseases and 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 signaling, remains less clear. Our group showed that hemoglobin-induced platelet activation involved the production of mitochondrial reactive oxygen species (mtROS). However, the molecular mechanism by which extracellular hemolysis induces platelet mtROS production, and whether the mtROS regulate platelet degranulation remains unknown. Here, we demonstrate using isolated human platelets that cell free heme is a more potent agonist for platelet activation than hemoglobin, and stimulates the release of a specific set of molecules from the α-granule of platelets, including the glycoprotein thrombospondin-1 (TSP-1). We uncover the mechanism of heme-mediated platelet mtROS production which is dependent on the activation of platelet TLR4 signaling and leads to the downstream phosphorylation of complex-V by the serine kinase Akt. Notably, inhibition of platelet TLR4 or Akt, or scavenging mtROS prevents heme-induced granule release in vitro. Further, heme-dependent granule release is significantly attenuated in vivo in mice lacking TLR4 or those treated with the mtROS scavenger MitoTEMPO. These data elucidate a novel mechanism of TLR4-mediated mitochondrial regulation, establish the mechanistic link between hemolysis and platelet degranulation, and begin to define the heme and mtROS-dependent platelet secretome. These data have implications for hemolysis-induced thrombo-inflammatory signaling and for the consideration of platelet mitochondria as a therapeutic target in hemolytic disorders.
Key points
Heme induces platelet mtROS production by inhibiting complex-V activity via TLR4 signaling.
Heme stimulated platelet granule secretion is regulated by mtROS.
Thrombosis and inflammation are intimately linked and synergistically contribute to the pathogenesis of a number of vascular diseases. On a cellular level, while the platelet is central to thrombus formation as well as an active mediator of inflammation, the molecular mechanisms of cross-talk between thrombosis and inflammation remain elusive. High-Mobility Group Box 1 protein (HMGB1) is an inflammatory regulator that also stimulates platelet activation through its interaction with toll-like receptor 4 (TLR4). However, it remains unclear whether cross-talk between HMGB1 and traditional thrombotic agonists exists to modulate platelet activation. Using isolated human platelets, we tested whether HMGB1 treatment affects platelet activation mediated by traditional agonists. We found that HMGB1 enhances ADP-mediated platelet activation, but not platelet activation stimulated by thrombin or collagen. Further, inhibition of the canonical ADP purinergic P2Y 12 receptor attenuates HMGB1-dependent platelet activation. Mechanistically, we discovered that HMGB1 activates platelet surface TLR4 to release ADP from the platelet and concomitantly increase the localization of P2Y 12 on the platelet membrane. These data demonstrate that ADP-dependent P2Y 12 activation contributes to HMGB1 mediated platelet activation, while HMGB1 primes platelets for an enhanced activation response to ADP. These novel findings further our understanding of thrombo-inflammatory signaling and provide new insight for therapeutic P2Y 12 inhibition.
Key Points
HMGB1 enhances ADP-mediated platelet activation but not platelet activation stimulated by collagen or thrombin.
HMGB1 stimulates platelet ADP release and increases platelet surface localization of P2y12 receptors via TLR4-dependent mechanism(s).
Visual Abstract
Caption: HMGB1 activates TLR4 to activate platelets, release platelet ADP, and upregulate P2Y 12 at the platelet surface.
Citations (9)
... 70,71 A recent study has also shown that high-mobility group box 1 can increase platelet surface P2Y 12 (a purinergic receptor) and ADP-mediated platelet activation in SCD patients. 72 This complementary agonism between ADP and highmobility group box 1 further enhances our understanding of the thrombo-inflammatory profile in SCD and provides insight for therapeutic P2Y 12 inhibition. 72 Persistent platelet activation is now increasingly recognized to play a role in the pathophysiology of thrombosis in SCD and is further exacerbated during acute VOCs. ...
... 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. ...
... Given KHE's rapid progression and local aggressiveness, especially in cases associated with KMP where bleeding is a possibility, biopsies should be approached with caution. KMP is a clinical hallmark of KHE, characterized by decreased platelet count, reduced fibrinogen levels, elevated D-dimer levels, and a hypocoagulable state potentially leading to intermittent bleeding [16]. The incidence of KMP correlates positively with the tumor's diameter. ...
... One of the possible causes of SCD is a single-point mutation in the Hb-β gene. This mutation leads to the replacement of glutamic acid by valine, which results in the accumulation of abnormal hemoglobin (sickle Hb) [11,12]. Under low oxygen conditions, sickle Hb (HbS) tends to polymerize (a phenomenon known as sickling) and cause intravascular hemolysis, repeated polymerization, vaso-occlusive crisis (VOC), and ischemia-reperfusion injury [13][14][15]. ...
... In addition to P-selectin, activated platelets release other platelet factors and adhesion proteins, such as TSP-1. 36,37 The expression of TSP-1 glycoprotein was detected in both platelets and PDCs ( Figure 2B). It is reported that TSP-1 could enhance the killing of cytotoxic complexes released from CTLs, 22,38,39 which may also contribute to the cytotoxicity of PDCs. ...
... Hematopoietic stem cells sustain immune injury mediated by CD8 + T cells and cytokines (interferon-γ, tumor necrosis factor-α), aplastic crisis just not severe and permanent as aplastic anemia. Most cases involve infection (Staphylococcus [9], human parvovirus B19 [10,11], hepatitis virus [12,13]) or exposure to non-cytotoxic drugs (isoniazid, thiamazole, interferon, amikacin) [1,2]. ...
... Mitochondrial function is dependent on the regulation of bioenergetics and the interplay between mitochondrial and nuclear genomes. Mitochondria generate energy in the form of ATP through the electron transport chain (ETC) and OXPHOS, involving five multisubunit complexes [8,9]. These complexes are encoded by both nuclear DNA (nDNA) and mitochondrial DNA (mtDNA). ...
... Hydroxyurea benefits the body in various ways, stimulates fetal Hb synthesis, and reduces inflammation and oxidative stress [151]. Several clinical trials have shown that hydroxyurea reduces the incidence of VOC and improves overall clinical outcomes in SCD patients [152]. ...
... Neither vaccines nor antibiotic prophylaxis guarantee full protection from IMD [10]. There have been some reports of eculizumab recipients developing IMD caused by vaccine serogroups as well as disease caused by non-groupable (NG) meningococcal strains [12,15,19]. Meningococcal vaccination is not part of the national immunization program for children in Türkiye, but MenACWY-TT, MenACWY-CRM, MenACWY-D, and 4CMenB are privately available. ...