About

Amrita Sarkar, Ph.D., is a Research Assistant Professor of Pediatrics (Hematology) and a Senior Principal Scientist in the Division of Hematology at Children’s Hospital of Philadelphia. Her research focuses on immune-mediated thrombotic disorders, including Antiphospholipid Syndrome (APS), Thrombotic Thrombocytopenic Purpura (TTP), and Adenovirus-Induced Immune Thrombotic Thrombocytopenia (AITT). She integrates in vivo and in vitro models to study thrombo-inflammation and develop novel, non-anticoagulant therapies. Dr. Sarkar has established various animal models for conditions such as thrombosis, sepsis, arthritis, and diabetes, investigating key disease drivers like complement activation, NET formation, and PF4:β2GPI complex-mediated thrombosis. She also utilizes human and rodent primary cell systems and microfluidic assays to model thrombosis using patient-derived plasma or plasma-derived IgGs. Her current work centers on dissecting the role of PF4:NET:β2GPI interactions in APS and TTP, with a focus on their unexplored connection to complement activation. Additionally, she applies nanotechnology and antibody engineering to explore targeted therapeutic strategies. Through this integrated approach, Dr. Sarkar aims to define the molecular links between thrombosis, immunity, and inflammation to advance precision therapies for autoimmune thrombotic diseases.

Research topics

• Internal medicine
• Medicine
• Immunology

Selected publications

• Understanding how a highly prevalent <i>GRK5</i> polymorphism affects platelets and enhances thrombotic risk

  Blood · 2026-01-20 · 4 citations

  articleOpen access

  ABSTRACT: Inherited genetic variants that modulate platelet function contribute significantly to thrombotic disorders, yet their mechanisms and clinical implications remain underexplored. Two genome-wide association studies identified an A→G variant (rs10886430) in the first intron of G protein-coupled receptor kinase 5 (GRK5), found in homozygosity in ∼5 million Americans. The homozygous GRK5 GG genotype is associated with an increased risk of stroke and venous thromboembolism, but the mechanistic link between this variant and thrombotic risk has remained unclear. To investigate this, we identified 3 GG individuals. GRK5 protein levels in GG platelets were 90% lower than in AA controls. The significant reduction in GRK5 levels in GG platelets led to elevated platelet responsiveness to thrombin and a protease-activated receptor 1 (PAR1) agonist but not a PAR4 agonist. These findings were corroborated in GRK5-/- induced pluripotent stem cell-derived megakaryocytes, transgenic Grk5-deficient murine platelets, and AA platelets exposed to a GRK5 inhibitor. We demonstrated that PAR1 internalization was reduced in GG platelets, leading to enhanced PAR1 signaling. Under venous shear in an endothelialized microfluidic system, GG platelets exhibited increased accumulation, which was reversed by PAR1 inhibition with vorapaxar. In an arterial murine thrombosis model following human platelet infusion, GG platelets also showed enhanced thrombus formation in vivo. This study provides, to our knowledge, the first experimental evidence directly linking a highly prevalent human GRK5 variant to defective PAR1 regulation and increased thrombotic risk. Together, these findings establish that the GRK5 GG genotype confers increased thrombotic potential through impaired PAR1 desensitization, providing mechanistic insight that connects human genetics, thrombin receptor signaling, and thrombotic disease.

  PublisherOA PDFDOI

• Destabilization of PF4-antigenic complexes in heparin-induced thrombocytopenia

  Blood · 2025-03-26 · 9 citations

  article

  ABSTRACT: Heparin-induced thrombocytopenia (HIT) is initiated by antibodies that recognize large antigenic complexes composed of multiple molecules of cationic platelet factor 4 (PF4) and polyanions such as unfractionated heparin (UFH) that bind to each other primarily through electrostatic interactions. We asked whether the formation and stability of these HIT antigenic or ultralarge immune complexes (ULICs) would be inhibited by biocompatible synthetic polycationic molecules shown previously to dissociate UFH from antithrombin III and to inhibit polyphosphates. Members of this family of molecules, designated universal heparin reversal agents (UHRAs), inhibited formation and dissociated preformed ultralarge PF4-UFH (antigenic) complexes (ULCs), dissociated ULICs composed of the HIT-like monoclonal antibody KKO and ULCs, blocked binding of human HIT immunoglobulin G antibodies to PF4/heparin, binding of KKO to platelets, KKO-induced adhesion of platelets to activated human endothelium under flow, and microvascular thrombosis induced by KKO in a mouse model of HIT. These data suggest that UHRAs might provide a rationale intervention that acts at an early step in the pathogenesis of HIT to enhance the benefits and lessen the risks of nonheparin anticoagulants. Destabilization of immune complexes using polycationic inhibitors might also find a role in management of other polyanion PF4-antibody-mediated conditions, including vaccine-induced thrombocytopenia/thrombosis, postviral, and autoimmune HIT.

  PublisherDOI

• G6b-B antibody-based cis-acting platelet receptor inhibitors (CAPRIs) as a new family of anti-thrombotic therapeutics

  bioRxiv (Cold Spring Harbor Laboratory) · 2024-05-14 · 4 citations

  preprintOpen access

  Platelets are highly reactive fragments of megakaryocytes that play a fundamental role in thrombosis and hemostasis. Predictably, all conventional anti-platelet therapies elicit bleeding, raising the question whether the thrombotic activity of platelets can be targeted separately. In this study, we describe a novel approach of inhibiting platelet activation through the use of bispecific single-chain variable fragments (bi-scFvs), termed cis-acting platelet receptor inhibitors (CAPRIs) that harness the immunoreceptor tyrosine-based inhibition motif (ITIM)-containing co-inhibitory receptor G6b-B (G6B) to suppress immunoreceptor tyrosine-based (ITAM)-containing receptor-mediated platelet activation. CAPRI-mediated hetero-clustering of G6B with either the ITAM-containing GPVI-FcR γ-chain complex or FcγRIIA (CD32A) inhibited collagen- or immune complex-induced platelet aggregation. G6B-GPVI CAPRIs strongly and specifically inhibited thrombus formation on collagen under arterial shear, whereas G6B-CD32A CAPRI strongly and specifically inhibited thrombus formation to heparin-induced thrombocytopenia, vaccine-induced thrombotic thrombocytopenia and antiphospholipid syndrome complexes on Von Willebrand Factor-coated surfaces and photochemical-injured endothelial cells under arterial shear. Our findings provide proof-of-concept that CAPRIs are highly effective at inhibiting ITAM receptor-mediated platelet activation, laying the foundation for a novel family of anti-thrombotic therapeutics with potentially improved efficacy and fewer bleeding outcomes compared with current anti-platelet therapies. .

  PublisherOA PDFDOI

• Platelet Factor 4 Enhances Endothelial Cell Antimicrobial Activity <i>in Vitro</i> and Promotes Bacterial Clearance <i>In Vivo</i>, Improving Sepsis Outcomes

  Blood · 2024-11-05 · 1 citations

  articleOpen access

  Introduction: In this study, we investigated mechanism(s) by which the platelet-specific chemokine, platelet factor 4 (PF4), interacts with bacteria to enhance host-defense function and improve outcome in sepsis. Sepsis, a life-threatening dysregulated response to infection, is the leading cause of mortality worldwide for which there are no targeted treatments. Upon sensing pathogens, platelets release high concentrations of PF4, the most abundant protein in platelet α granules. Due to its strong positive charge, following release, PF4 is rapidly sequestered from the circulation by binding to polyanionic glycosaminoglycans on the surface of leukocytes and endothelial glycocalyx. PF4 also interacts specifically with CXCR3B, a chemokine receptor that is constitutively expressed by ECs, and forms non-specific electrostatic interactions with anionic polymers in the bacterial cell wall. We previously found that PF4 improves outcomes in the murine lipopolysaccharide endotoxemia model and enhances in vitro bacterial capture by neutrophil extracellular traps. We now show that PF4-coating of bacteria reduces systemic coagulopathy, limits bacterial dissemination, and improves survival in a murine model of polymicrobial sepsis, in part through the novel mechanism of enhancing the antimicrobial function of endothelial cells (ECs). Methods: Effects of PF4 on EC killing of live bacteria: HUVECs were exposed to live Escherichia (E) coli pre-treated with PF4 (0-25µg/mL) for 5hr. Every hour, ECs were washed and treated with 1% penicillin-streptomycin to remove excess unbound bacteria, and cell lysates were plated on agar plates overnight to count colony forming units (CFUs). To assess the role of receptor CXCR3 in facilitating PF4-mediated bacteria uptake, experiments were repeated in HUVECs pre-treated with small molecule inhibitor (10mM AMG-487; 1hr) prior to E coli ± PF4 (0-25µg/mL) exposure. Visualizing PF4-mediated bacterial clearance in vivo: Wildtype (WT) and PF4-/- mice were intravenously (IV) infused with fluorescent E coli (8x1011 CFU) and subjected to lung confocal intravital imaging. Liver and spleen residual CFUs were quantified at 15min and 2hr post E coli infusion. Effects of PF4 on polymicrobial sepsis outcomes: Contents from the cecum (cecal slurry, CS) of WT C57BL6 donor mice (100mg/mL) were pre-treated with vehicle or PF4 (100µg; 20min) and injected IV into WT or PF4-/- recipient mice to induce immediate bacteremia. Animals were evaluated for sepsis severity for up to 96hr, and survival analysis was performed using the Mantel-Cox test. A separate cohort of WT and PF4-/- mice was given IV CS (400mg/mL) pre-incubated with vehicle or PF4 (8µg; 20min). Blood was drawn at 24hr post-CS infusion and subjected to complete blood count to assess thrombocytopenia. Thrombin anti-thrombin (TAT) and VWF levels were measured by ELISAs in plasma as indicators of intravascular coagulopathy and endothelial activation. Plasma was also subjected to multiplex Olink analyses for inflammatory markers. To assess bacterial load, blood and liver, spleen, and lung homogenates were plated on agar plates to count for CFUs. Results: PF4 increased E coli uptake by HUVECs at 2hr but reduced intracellular E coli proliferation at 3-5hr. Such enhanced bacterial uptake effects by PF4 was prevented by blocking CXCR3. Lung intravital microscopy demonstrate that following E coli infusion, PF4-/- mice had accelerated platelet accumulation in the pulmonary microvasculature and impaired bacterial clearance as compared to WT mice. In the CS model of polymicrobial sepsis, PF4 reduced sepsis severity and mortality in both WT and PF4 -/- mice, and decreased bacterial load in blood, lung, and liver 24hr post-CS inoculation. WT mice also exhibited reduced leukopenia, thrombocytopenia, TAT, VWF, and inflammatory cytokine levels 24hr post-CS challenge. Conclusion: In vitro: PF4 crosslinks E coli, initially enhancing bacterial uptake by ECs via CXCR3, but prevent subsequent intracellular proliferation. In vivo: PF4 promotes bacterial clearance while dampening coagulopathy and inflammation, leading to improved outcomes in polymicrobial sepsis. These studies suggest that PF4 binding to bacteria may be an important host defense mechanism. The ability of PF4-based therapeutics to enhance clearance of microbes merits further translational studies in sepsis.

  PublisherOA PDFDOI

• Anti-Phospholipid Syndrome (APS) Antibody (Ab)-Induced Thrombosis Can be Blocked By Platelet Factor 4 (PF4)-Directed Abs: A Novel Therapeutic Approach for APS?

  Blood · 2024-11-05

  articleOpen access1st authorCorresponding

  Background: APS is an autoimmune thromboinflammatory disease characterized by thrombocytopenia, thrombosis, and/or complications in pregnancy. APS occurs in association with one or more antiphospholipid (aPL) Abs/IgGs whose major target is b2-glycoprotein I (b2GPI), a protein previously shown to bind to human (h) PF4, a platelet-specific chemokine with a high affinity for polyanions including DNA in neutrophil extracellular traps (NETs) and von Willebrand factor (vWF). No further studies have been conducted to characterize the role of hPF4:β2GPI complexes in APS-related thrombosis. We hypothesized that b2GPI binds to hhPF4 adhered to NETs or vWF to form potent, prothrombotic antigenic complexes. Aim: We now aim to determine whether PF4:b2GPI:NETs or vWF are central to APS-mediated thrombosis and evaluate whether hPF4-blocking antibodies can prevent these events. Methods: The following anti-hPF4 monoclonal (mo) Abs were studied: (1) the murine heparin-induced thrombocytopenia (HIT)-like anti-hPF4 KKO, (2) IgG4 humanized KKO (G4KKO), (3) humanized vaccine-induced immune thrombotic thrombocytopenia (VITT)-like anti-hPF4 Ab 1E12, (4) deglycosylated 1E12 (DG1E12), and (5) a murine PF4-tetramer disaggregating moAb RTO. These Abs were infused through Bioflux microfluidic channels coated with NETs, released from healthy donor neutrophils stimulated with phorbol 12-myristate 13-acetate and then treated with β2GPI (20 µg/ml), hPF4 (6.5 µg/ml) and APS IgGs (100 µg/ml). A polyclonal anti-β2GPI Ab (a gift from Dr. McCrae) was used to visualize β2GPI binding to NETs. In a separate set of studies, human umbilical vein endothelial cells (HUVECs) were grown to confluence in Bioflux channels and then subjected to a hematoporphyrin (HP)-induced photochemical injury that stimulates vWF release. Whole blood from healthy donors was supplemented with additional β2GPI (20 µg/ml), PF4 (25 µg/ml) and purified IgGs (100 µg/ml) from four distinct patients with “triple-positive” APS with associated thrombi and was then flowed through the HUVEC-lined channels with or without the various anti-hPF4 moAbs. Some studies included DNase1 (100 U/mL, Sigma-Aldrich) or ADAMTS13 (0.7 µg/ml, R&amp;D Systems). Results: In studies with NET-lined microfluidic channels, we did not detect binding of b2GPI to unmodified NETs, but found b2GPI adhered well to PF4-coated NETs. β2GPI binding to PF4-coated NETs was completely inhibited by VITT-like moAb 1E12, but unaffected by HIT-like Ab KKO, which binds to a different antigenic site on PF4. In the experiments in which whole blood was infused through HUVEC-lined channels following HP injury, platelet thrombosis and complement deposition were significantly higher when all three proteins - PF4, β2GPI and APS IgG - were present. Inclusion of either DNaseI or ADAMTS13 partially blocked thrombosis. Inclusion of the HIT-like G4KKO, the VITT-like DG1E12 and RTO showed marked inhibition of platelet- and complement-rich thrombi in the presence of APS IgG. Conclusions: Our studies support the importance of PF4:b2GPI:NET and vWF complexes in APS in in vitro model systems. b2GPI requires the presence of PF4 to be anchored to NETs. This interaction can be blocked by inclusion of the VITT-site-binding moAb 12E12, but not to the HIT-site-binding moAb KKO, suggesting that the hhPF4:b2GPI:NET complex involves the VITT site. The fact that both ADAMTS13 as well as DNase1 can reduce APS Ab-induced thrombosis in our HUVEC-lined microfluidic system, suggests that PF4:b2GPI forms complex on both NETs and VWF. Anti-hPF4 Abs that block either the HIT or VITT sites on hPF4 or disrupt PF4 tetramerization can effectively prevent thrombi on both NETs and vWF. These studies provide new mechanistic insights into a central role for PF4 thrombosis in the presence of anti-b2GPI+ APS Abs and identify potential new non-anticoagulant therapeutics that may be useful in this challenging prothrombotic setting.

  PublisherOA PDFDOI

• Antiphospholipid Syndrome (APS) Is a Platelet Factor 4 (PF4)-Centric Immunothrombotic Disorder

  Blood · 2024-11-05

  articleOpen access

  Background: APS is a devastating immune-mediated disorder characterized by thrombosis and pregnancy complications in association with autoantibodies (aPL Abs) that bind β2-glycoprotein 1 (β2GP1), among other antigens. The physiological role of β2GP1 is uncertain, and the mechanism by which anti-β2GP1 aPL Abs lead to thrombosis is not fully elucidated. In 2010, it was shown that PF4, a platelet-specific chemokine released in large amounts following platelet activation, forms tetramers that bind to β2GP1 dimers. This PF4:β2GP1 interaction enhances recognition of β2GP1 by aPL Abs. Furthermore, aPL Abs induce the release of neutrophil extracellular traps (NETs), webs of decondensed chromatin, that also bind PF4. PF4:NET complexes are key antigenic targets involved in the pathogenesis of heparin-induced thrombocytopenia (HIT) and in vaccine-induced thrombocytopenia (VITT). Aim: We propose that PF4 bridges β2GP1 to NETs, leading to the formation of PF4:β2GP1:NET complexes that act as an important antigenic target in APS. To address this hypothesis, we studied the formation of PF4:β2GP1:NET complexes and asked whether they contribute to the prothrombotic state in in vitro assays and in a murine model of APS. Methods: We studied plasma and isolated IgGs from four patients with “triple-positive” APS and thrombosis. Two distinct in vitro-binding studies were performed: i. Dynamic light-scattering (DLS) studies were done using β2GP1, PF4 and calf thymus (ct) DNA plus aPL IgGs in a Malvern Zetasizer Nano-ZS. In some experiments RTO, a monoclonal Ab that disrupts the PF4 tetramers, was added. ii. Isolated human neutrophils were adhered to fibronectin-coated Bioflux microfluidic channels, and then stimulated with phorbol myristyl acetate to induce NET release. NET-lined channels were then infused with β2GP1 and/or PF4. Functional studies to examine the importance of PF4:β2GP1:NETs in APS thrombosis were conducted in two systems: i. Microfluidic channels coated with fibronectin were lined with human umbilical endothelial cells (HUVECs) and then photochemically-injured with hematoporphyrin and blue-light exposure prior to adding whole blood from healthy donors to which β2GP1 and/or PF4 and/or aPL IgGs had been added. ii. A passive immunization model of isolated aPL IgGs were infused into double-transgenic human PF4/FcgRIIA-expressing mice or wildtype (WT) mice or mice lacking PF4 (mPF4-/- mice). Rolling and adherence of neutrophils on cremaster venules were quantified before and after infusion of aPL IgGs. Platelet and neutrophil accumulation in laser injury-induced thrombi were measured. Results: DLS studies showed that β2GP1 forms large complexes with PF4 plus ctDNA that binds aPL IgGs. Infused RTO decreased the formation of these large immune complexes in a dose-dependent fashion. In the NET microfluidic system, we observed that β2GP1 bound to exposed NETs only when PF4 was present. Additionally, just as in HIT and VITT, the ability of APS IgGs to lead to platelet-rich thrombus formation on the injured HUVECs was dependent on the inclusion of PF4. In a passive immunization murine model, we show that the induction of APS led to slowing of neutrophil rolling and neutrophil arrest on cremaster venules of mice in a PF4-dependent fashion, similar to that seen in HIT and VITT murine models. Moreover, infusion of aPL IgGs caused either hPF4/FcgRIIA or WT mice to develop occlusive platelet-rich thrombi at sites of laser injury in both arterioles and venules. Enhanced neutrophil accumulation developed in venules post-laser injury. Importantly, mice lacking PF4 were protected from enhanced arteriolar and venular thrombosis in the presence of APS IgGs. These findings were similar to those seen in prior studies of HIT- and VITT-induced prothrombotic mice models. Conclusion: Our studies indicate that thrombosis in APS involves generation of PF4:NET complexes, as in HIT and VITT. In APS, β2GP1 is incorporated into these complexes forming PF4:β2GP1:NETs that are bound by anti- β2GP1 aPL IgG. In a passive murine immunization model of APS, these aPL IgGs increase neutrophil adhesiveness as well as platelet and neutrophil incorporation into laser-injury-induced thrombi in a PF4-dependent manner. Additional studies will be needed to relate these finding to clinical presentation and to ask whether strategies that block the formation of PF4:b2GP1:NET complexes would be an effective adjunctive therapy for APS.

  PublisherOA PDFDOI

• The PF4-Centric Immunothrombotic Disorders, HIT, Vitt and APS, Lead to Complement Activation Responsive to Complement-Blocking Therapeutics

  Blood · 2024-11-05

  articleOpen access1st authorCorresponding

  Background: Heparin-induced thrombocytopenia (HIT) and vaccine-induced immune thrombocytopenia with thrombosis (VITT)are highly prothrombotic disorders mediated by platelet -activating anti-PF4 antibodies (Abs). Studies from our lab and others have shown that PF4 bound to von Willebrand factor (VWF) strings and neutrophil extracellular traps (NETs) creates a dense, antigenic target for pathogenic HIT and VITT Abs conducive for both FcgRIIA-mediated cell activation and complement activation. We hypothesize that in antiphospholipid syndrome (APS), b2 glycoprotein I (b2GPI) - known to bind to PF4 - also forms a similar antigenic target for APS Abs and that inhibition of the terminal complement pathway will be effective in ameliorating the thrombosis in all three PF4-mediated immunothrombotic disorders. Aim: Toinvestigate the in vitro and in vivo effects of C5 inhibitor in microfluidic and animal models of thrombosis in HIT, VITT and APS. Methods: Confluent-lined HUVECs were injured with hematoporphyrin to release VWF strings. Human whole blood from healthy donors was pre-incubated with eculizumab, a humanized monoclonal antibody that binds to C5 and prevents activation of complement terminal complex (10 µg/ml and 15 µg/ml) for 15 minutes. HIT, VITT or APS IgGs (100 µg/ml), each isolated from three distinct patients, along with PF4 (25 µg/ml) were added and incubated for another 15-20 minutes. Samples were then flowed through microfluidics channels and in vitro thrombus formation measured by epifluorescence microscopy of aggregated platelets every 5 minutes for 15 minutes. The channels were washed and stained for complement component C5b9 and imaged by confocal microscopy. In in vivo studies, FcgRIIA+/hPF4+/mPF4-/- (HIT) mice were injected with anti-mouseC5 Ab BB5.1 24h before injection of HIT-like moAb KKO to induce thrombocytopenia and thrombosis. Neutrophil rolling and platelet-fibrin clot were analyzed in the cremaster muscle injury model. Results: In vitro data showed a dose-dependent inhibitory effect of eculizumab on thrombus formation and C5b9 deposition in HIT-, VITT- and APS-mediated microfluidic thrombosis models. Inhibition of the terminal complement complex by BB5.1 had a moderate effect on in vivo thrombocytopenia, but significantly inhibited neutrophil adhesion to the endothelium, as well as both venous and arterial thrombus formation in the HIT murine model. Conclusions: These studies suggest that C5 inhibitors, eculizumab and BB5.1, can successfully inhibit complement and platelet activation in HIT-, VITT- and APS-mediated prothrombotic settings. Our data support the important role of complement activation in the progression of thrombosis in these related, antibody-mediated disorders. This concept provides a rationale for therapeutic interventions that act upstream of thrombin generation and might thereby amplify the benefits and reduce the risks of total reliance on anticoagulants for management of these diseases.

  PublisherOA PDFDOI

• Platelet Factor 4 Enhances Antimicrobial Function of the Endothelium and Improves Outcome in a Murine Model of Sepsis

  Blood · 2023-11-02

  article

  Introduction: In this study, we investigated mechanism(s) by which the platelet-specific chemokine platelet factor 4 (PF4) interacts with bacteria to enhance host-defense function and improve outcome in sepsis. Sepsis, a life-threatening dysregulated response to infection, is the leading cause of mortality worldwide for which there are no targeted treatments. Upon sensing pathogens, platelets release high concentrations of PF4, a positively charged chemokine with high affinity for polyanions including polymers in the bacterial cell wall, the endothelial glycocalyx and cell-free DNA. We previously found that PF4 improves outcomes in the murine lipopolysaccharide endotoxemia and enhances in vitro bacterial capture by neutrophil extracellular traps. We now show that PF4-coating of bacteria reduces coagulopathy, limits bacterial dissemination, and improves survival in a murine model of polymicrobial sepsis, in part through the novel mechanism of enhancing the antimicrobial function of endothelial cells (ECs). Methods: Effects of PF4 on bacterial internalization by ECs was assessed by incubating heat-inactivated Staphylococcus (S) aureus conjugated to pHrodo, a dye that only fluoresces following cellular internalization, with PF4 (0-100mg/mL) and then expose those complexes to human umbilical vein endothelial cells (HUVECs) ± tumor necrosis factor a (TNFa) to simulate inflammation. Internalized S aureus fluorescent signal was visualized 16hr post-exposure. HUVECs were stained for von Willebrand factor (VWF) release to indicate bacteria-induced endothelial activation. Effects of PF4 on EC killing of bacteria was studied by exposing HUVECs to live Escherichia (E) coli pre-treated with PF4 (0-25mg/mL) for 1hr. ECs were washed to remove excess unbound bacteria, and cell lysates were plated on agar plates for 24hr to count colony forming units (CFUs). To investigate the effects of PF4-bacteria interactions on sepsis survival outcomes, contents from the cecum (cecal slurry, CS) of C57BL6 donor mice (100mg/mL) were pre-treated with PF4 (100mg) for 20min and injected intravenously (IV) into wildtype (WT) or PF4 -/-recipient mice to induce immediate bacteremia. Animals were evaluated for sepsis severity for up to 72hr, and survival analysis was performed using the Mantel-Cox test. A separate cohort of WT mice was given IV CS (400mg/mL) pre-incubated with PF4 (8mg). Blood was drawn at 3 to 24hr post-CS infusion and subjected to complete blood count. Thrombin anti-thrombin (TAT) and VWF levels were measured by ELISAs in plasma as indicators of intravascular coagulopathy and endothelial activation. To assess bacterial load, blood and liver and spleen homogenates were plated on agar plates to count for CFUs. Results: PF4 coating of bacteria enhanced bacterial uptake by HUVECs, peaking at 20-50 µg/mL of PF4. TNFa-stimulated HUVECs more readily internalized PF4-coated bacteria at 1-20 µg/mL of PF4. Although PF4 promoted S aureus uptake, it limited bacteria-induced VWF secretion by HUVECs. PF4 also enhanced EC killing of E coli upon bacterial internalization. In the CS model of murine polymicrobial sepsis, PF4 prevented CS-induced mortality in both WT and PF4 -/- mice ( Fig. 1). PF4-pretreatment of CS also reduced leukopenia and thrombocytopenia 3-6hr post-CS challenge in WT mice. Mice given PF4-treated CS exhibited lower TAT and VWF levels as compared to untreated-CS animals. Lastly, PF4 reduced bacterial load in blood, liver, and spleen 24hr post-CS inoculation ( Fig. 2). Conclusion: PF4 enhances bacterial uptake and killing by the endothelium in vitro, promotes bacterial clearance while preventing bacteria-induced coagulopathy and endothelial dysfunction leading to improved survival in the murine CS model of polymicrobial sepsis. These studies suggest that PF4 binding to bacteria may be an important host defense mechanism. Further studies are underway to define the contribution of EC antimicrobial activity to the observed outcomes. The ability of PF4-based therapeutics to enhance clearance of microbes merits further translational studies in sepsis.

  PublisherDOI

• Platelet factor 4 limits neutrophil extracellular trap– and cell-free DNA–induced thrombogenicity and endothelial injury

  JCI Insight · 2023-11-21 · 35 citations

  articleOpen access

  Plasma cell-free DNA (cfDNA), a marker of disease severity in sepsis, is a recognized driver of thromboinflammation and a potential therapeutic target. In sepsis, plasma cfDNA is mostly derived from neutrophil extracellular trap (NET) degradation. Proposed NET-directed therapeutic strategies include preventing NET formation or accelerating NET degradation. However, NET digestion liberates pathogens and releases cfDNA that promote thrombosis and endothelial cell injury. We propose an alternative strategy of cfDNA and NET stabilization with chemokine platelet factor 4 (PF4, CXCL4). We previously showed that human PF4 (hPF4) enhances NET-mediated microbial entrapment. We now show that hPF4 interferes with thrombogenicity of cfDNA and NETs by preventing their cleavage to short-fragment and single-stranded cfDNA that more effectively activates the contact pathway of coagulation. In vitro, hPF4 also inhibits cfDNA-induced endothelial tissue factor surface expression and von Willebrand factor release. In vivo, hPF4 expression reduced plasma thrombin-antithrombin (TAT) levels in animals infused with exogenous cfDNA. Following lipopolysaccharide challenge, Cxcl4-/- mice had significant elevation in plasma TAT, cfDNA, and cystatin C levels, effects prevented by hPF4 infusion. These results show that hPF4 interacts with cfDNA and NETs to limit thrombosis and endothelial injury, an observation of potential clinical benefit in the treatment of sepsis.

  PublisherDOI

• Treatment of thrombocytopenia and thrombosis in HIT in mice using deglycosylated KKO: a novel therapeutic?

  Blood Advances · 2023-05-17 · 11 citations

  articleOpen access1st authorCorresponding

  Heparin-induced thrombocytopenia (HIT) is characterized by thrombocytopenia associated with a highly prothrombotic state due to the development of pathogenic antibodies that recognize human platelet factor 4 (hPF4) complexed with various polyanions. Although nonheparin anticoagulants are the mainstay of care in HIT, subsequent bleeding may develop, and the risk of developing new thromboembolic events remain. We previously described a mouse immunoglobulin G2bκ (IgG2bκ) antibody KKO that mimics the sentinel features of pathogenic HIT antibodies, including binding to the same neoepitope on hPF4-polyanion complexes. KKO, like HIT IgGs, activates platelets through FcγRIIA and induces complement activation. We then questioned whether Fc-modified KKO could be used as a novel therapeutic to prevent or treat HIT. Using the endoglycosidase EndoS, we created deglycosylated KKO (DGKKO). Although DGKKO retained binding to PF4-polyanion complexes, it inhibited FcγRIIA-dependent activation of PF4-treated platelets triggered by unmodified KKO, 5B9 (another HIT-like monoclonal antibody), and IgGs isolated from patients with HIT. DGKKO also decreased complement activation and deposition of C3c on platelets. Unlike the anticoagulant fondaparinux, injection of DGKKO into HIT mice lacking mouse PF4, but transgenic for hPF4 and FcγRIIA, prevented and reversed thrombocytopenia when injected before or after unmodified KKO, 5B9, or HIT IgG. DGKKO also reversed antibody-induced thrombus growth in HIT mice. In contrast, DGKKO was ineffective in preventing thrombosis induced by IgG from patients with the HIT-related anti-PF4 prothrombotic disorder, vaccine-induced immune thrombotic thrombocytopenia. Thus, DGKKO may represent a new class of therapeutics for targeted treatment of patients with HIT.

  PublisherOA PDFDOI

Frequent coauthors

• Mortimer Poncz

  Children's Hospital of Philadelphia

  46 shared

• Lubica Rauova

  Children's Hospital of Philadelphia

  40 shared

• Kandace Gollomp

  Children's Hospital of Philadelphia

  23 shared

• M. Anna Kowalska

  Polish Academy of Sciences

  17 shared

• Douglas B. Cines

  University of Pennsylvania

  15 shared

• Gowthami M. Arepally

  Duke Medical Center

  14 shared

• Jérôme Rollin

  Université de Tours

  12 shared

• Friederike Jönsson

  Institut Pasteur

  12 shared

Education

• PhD, Physiology

  University of Calcutta

  2013