Packaging of supplemented urokinase into naked alpha-granules of in vitro -grown megakaryocytes for targeted therapeutic delivery.

Our prior finding that uPA endogenously expressed and stored in the platelets of transgenic mice prevented thrombus formation without causing bleeding, prompted us to develop a potentially clinically relevant means of generating anti-thrombotic human platelets in vitro from CD34 + hematopoietic cell-derived megakaryocytes. CD34 + -megakaryocytes internalize and store in α-granules single-chain uPA (scuPA) and a uPA variant modified to be plasmin-resistant, but thrombin-activatable, (uPAT). Both uPAs co-localized with internalized factor V (FV), fibrinogen and plasminogen, low-density lipoprotein receptor-related protein 1 (LRP1), and interferon-induced transmembrane protein 3 (IFITM3), but not with endogenous von Willebrand factor (VWF). Endocytosis of uPA by CD34 + -\megakaryocytes was mediated in part via LRP1 and αIIbß3. scuPA-containing megakaryocytes degraded endocytosed intragranular FV, but not endogenous VWF, in the presence of internalized plasminogen, whereas uPAT-megakaryocytes did not significantly degrade either protein. We used a carotid-artery injury model in NOD-scid IL2rγnull (NSG) mice homozygous for VWF R1326H (a mutation switching binding VWF specificity from mouse to human glycoprotein IbmlIX) to test whether platelets derived from scuPA-MKs or uPAT-Mks would prevent thrombus formation. NSG/VWF R1326H mice exhibited a lower thrombotic burden after carotid artery injury compared to NSG mice unless infused with human platelets or MKs, whereas intravenous injection of either uPA-containing megakaryocytes into NSG/VWF R1326H generated sufficient uPA-containing human platelets to lyse nascent thrombi. These studies suggest the potential to deliver uPA or potentially other ectopic proteins within platelet α-granules from in vitro- generated megakaryocytes. Key points: Unlike platelets, in vitro-grown megakaryocytes can store exogenous uPA in its α-granules.uPA uptake involves LRP1 and αIIbß3 receptors and is functionally available from activated platelets.

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

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.

PAI-1 Deficiency Drives Pulmonary Vascular Smooth Muscle Remodeling and Pulmonary Hypertension.

Pulmonary arterial hypertension (PAH) is a progressive and potentially a rapidly fatal disease characterized by vasoconstriction and remodeling of small pulmonary arteries (PA) leading to increased pulmonary vascular resistance and right heart failure. Central to the remodeling process is a switch of the smooth muscle cells in small PAs (PASMC) to a proliferative, apoptosis-resistant phenotype. There is reason to suspect that the plasminogen activator system may play an important role in the remodeling program in PAH based on its roles in vascular post-injury restenosis, fibrosis, angiogenesis and tumorigenesis. Plasminogen activator inhibitor-1 (PAI-1) is the primary physiological inhibitor of the plasminogen activators - urokinase-type and tissue-type (uPA and tPA, respectively). Immunohisto- chemical and immunoblot analyses revealed that PAI-1 was deficient in smooth muscle areas of small remodeled PAs and early-passage PASMC from subjects with PAH compared to non-PAH controls. PAI1-/- male and female mice developed spontaneous pulmonary vascular remodeling and pulmonary hypertension (PH) as evidenced by significant increase in PA medial thickness, systolic right ventricular pressure, and right ventricular hypertrophy. Lastly, the uPA inhibitors upamostat (WX-671) and amiloride analog BB2-30F down-regulated mTORC1 and SMAD3, restored PAI-1 levels, reduced proliferation, and induced apoptosis in human PAH PASMC. We examined the effect of inhibition of uPA catalytic activity by BB2-30F on the development of SU5416/Hypoxia (SuHx)-induced PH in mice. Vehicletreated SuHx-exposed mice had up-regulated mTORC1 in small PAs, developed pulmonary vascular remodeling and PH, as evidenced by significant increase of PA MT, sRVP, RV hypertrophy, and a significant decrease in the pulmonary artery acceleration time/pulmonary ejection time (PAAT/PET) ratio compared to age- and sex-matched normoxia controls, whereas BB2-30F-treated group was protected from all these pathological changes. Taken together, our data strongly suggest that PAI-1 down- regulation in PASMC from human PAH lungs promotes PASMC hyper-proliferation, remodeling, and spontaneous PH due to unopposed uPA activation. Further studies are needed to determine the potential benefits of targeting the PAI-1/uPA imbalance to attenuate the progression and/or reverse pulmonary vascular remodeling and PH.

Pathogenesis of refractory ITP: Overview.

A subset of individuals with 'primary' or 'idiopathic' immune thrombocytopenia (ITP) who fail to respond to conventional first- and second-line agents or who lose responsiveness are considered to have 'refractory' disease (rITP), placing them at increased risk of bleeding and complications of intensive treatment. However, the criteria used to define the refractory state vary among studies, which complicates research and clinical investigation. Moreover, it is unclear whether rITP is simply 'more severe' ITP, or if there are specific pathogenic pathways that are more likely to result in refractory disease, and whether the presence or development of rITP can be established or anticipated based on these differences. This paper reviews potential biological features that may be associated with rITP, including genetic and epigenetic risk factors, dysregulation of T cells and cytokine networks, antibody affinity and specificity, activation of complement, impaired platelet production and alterations in platelet viability and clearance. These findings indicate the need for longitudinal studies using novel clinically available methodologies to identify and monitor pathogenic T cells, platelet antibodies and other clues to the development of refractory disease.

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

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.

Vaccine-induced immune thrombotic thrombocytopenia.

Within the first months of the COVID-19 vaccination campaign, previously healthy recipients who developed severe thrombosis (often cerebral and/or splanchnic vasculature) and thrombocytopenia typically after adenoviral vector-based vaccination were identified. Similarities between this syndrome, vaccine-induced immune thrombotic thrombocytopenia (VITT), and heparin-induced thrombocytopenia prompted recognition of the role of antiplatelet factor 4 (PF4) antibodies and management strategies based on IV immunoglobulin and nonheparin anticoagulants, which improved outcome. We update current understanding of VITT and potential involvement of anti-PF4 antibodies in thrombotic disorders.

Neutrophil extracellular trap stabilization by platelet factor 4 reduces thrombogenicity and endothelial cell injury.

Neutrophil extracellular traps (NETs) are abundant in sepsis, and proposed NET-directed therapies in sepsis prevent their formation or accelerate degradation. Yet NETs are important for microbial entrapment, as NET digestion liberates pathogens and NET degradation products (NDPs) that deleteriously promote thrombosis and endothelial cell injury. We proposed an alternative strategy of NET-stabilization with the chemokine, platelet factor 4 (PF4, CXCL4), which we have shown enhances NET-mediated microbial entrapment. We now show that NET compaction by PF4 reduces their thrombogenicity. In vitro, we quantified plasma thrombin and fibrin generation by intact or degraded NETs and cell-free (cf) DNA fragments, and found that digested NETs and short DNA fragments were more thrombogenic than intact NETs and high molecular weight genomic DNA, respectively. PF4 reduced the thrombogenicity of digested NETs and DNA by interfering, in part, with contact pathway activation. In endothelial cell culture studies, short DNA fragments promoted von Willebrand factor release and tissue factor expression via a toll-like receptor 9-dependent mechanism. PF4 blocked these effects. Cxcl4-/- mice infused with cfDNA exhibited higher plasma thrombin anti-thrombin (TAT) levels compared to wild-type controls. Following challenge with bacterial lipopolysaccharide, Cxcl4-/- mice had similar elevations in plasma TAT and cfDNA, effects prevented by PF4 infusion. Thus, NET-stabilization by PF4 prevents the release of short fragments of cfDNA, limiting the activation of the contact coagulation pathway and reducing endothelial injury. These results support our hypothesis that NET-stabilization reduces pathologic sequelae in sepsis, an observation of potential clinical benefit.

Modulation of ultralarge immune complexes in heparin-induced thrombocytopenia.

BACKGROUND: Heparin-induced thrombocytopenia (HIT) is a serious thrombotic disorder caused by ultralarge immune complexes (ULICs) containing platelet factor 4 (PF4) and heparin that form the HIT antigen, together with a subset of anti-PF4 antibodies. ULICs initiate prothrombotic responses by engaging Fcγ receptors on platelets, neutrophils, and monocytes. Contemporary anti-thrombotic therapy for HIT is neither entirely safe nor entirely successful and acts downstream of ULIC formation and Fcγ receptor-initiated generation of thrombin. OBJECTIVES: To determine whether HIT antigen and ULIC formation and stability could be modified favorably by inhibiting PF4-heparin interactions with fondaparinux, together with blocking formation of PF4 tetramers using a humanized monoclonal anti-PF4 antibody (hRTO). METHODS: Results: The combination of fondaparinux and hRTO inhibited HIT antigen formation, promoted antigen dissociation, inhibited ULIC formation, and promoted ULIC disassembly at concentrations below the effective concentration of either alone and blocked Fcγ receptor-dependent induction of factor Xa activity by monocytic THP1 cells and activation of human platelets in whole blood. Combined with hRTO, fondaparinux inhibited HIT antigen and immune complex formation and activation through Fcγ receptors at concentrations at or below those used clinically to inhibit FXa coagulant activity. CONCLUSIONS: HIT antigen and immune complexes are dynamic and amenable to modulation. Fondaparinux can be converted from an anticoagulant that acts at a downstream amplification step into a rationale, disease-specific intervention that blocks ULIC formation. Interventions that prevent ULIC formation and stability might increase the efficacy, permit use of lower doses, shorten the duration of antithrombotic therapy, and help prevent this serious thrombotic disorder.

Platelet factor 4 (CXCL4/PF4) upregulates matrix metalloproteinase-2 (MMP-2) in gingival fibroblasts.

Periodontitis is a chronic inflammatory disease characterized by the release of matrix metalloproteinases (MMPs) from resident connective tissue cells in tooth-supporting tissues (periodontium). Platelet activation, and the attendant release of pro-inflammatory chemokines such as platelet factor 4 (CXCL4/PF4), are associated with periodontitis although the associated biochemical pathways remain undefined. Here we report that recombinant PF4 is internalized by cultured human gingival fibroblasts (hGFs), resulting in significant (p < 0.05) upregulation in both the production and release of MMP-2 (gelatinase A). This finding was corroborated by elevated circulating levels of MMP-2 (p < 0.05) in PF4-overexpressing transgenic mice, relative to controls. We also determined that PF4 induces the phosphorylation of NF-κB; notably, the suppression of NF-κB signaling by the inhibitor BAY 11-7082 abrogated PF4-induced MMP-2 upregulation. Moreover, the inhibition of surface glycosaminoglycans (GAGs) blocked both PF4 binding and NF-κB phosphorylation. Partial blockade of PF4 binding to the cells was achieved by treatment with either chondroitinase ABC or heparinase III, suggesting that both chondroitin sulfate and heparan sulfate mediate PF4 signaling. These results identify a novel pathway in which PF4 upregulates MMP-2 release from fibroblasts in an NF-κB- and GAG-dependent manner, and further our comprehension of the role of platelet signaling in periodontal tissue homeostasis.

Human neutrophil peptides 1-3 protect the murine urinary tract from uropathogenic Escherichia coli challenge.

Antimicrobial peptides (AMPs) are critical to the protection of the urinary tract of humans and other animals from pathogenic microbial invasion. AMPs rapidly destroy pathogens by disrupting microbial membranes and/or augmenting or inhibiting the host immune system through a variety of signaling pathways. We have previously demonstrated that alpha-defensins 1-3 (DEFA1A3) are AMPs expressed in the epithelial cells of the human kidney collecting duct in response to uropathogens. We also demonstrated that DNA copy number variations in the DEFA1A3 locus are associated with UTI and pyelonephritis risk. Because DEFA1A3 is not expressed in mice, we utilized human DEFA1A3 gene transgenic mice (DEFA4/4) to further elucidate the biological relevance of this locus in the murine urinary tract. We demonstrate that the kidney transcriptional and translational expression pattern is similar in humans and the human gene transgenic mouse upon uropathogenic Escherichia coli (UPEC) stimulus in vitro and in vivo. We also demonstrate transgenic human DEFA4/4 gene mice are protected from UTI and pyelonephritis under various UPEC challenges. This study serves as the foundation to start the exploration of manipulating the DEFA1A3 locus and alpha-defensins 1-3 expression as a potential therapeutic target for UTIs and other infectious diseases.

Minimal role for the alternative pathway in complement activation by HIT immune complexes.

BACKGROUND: Anti-platelet factor 4 (PF4)/heparin immune complexes that cause heparin-induced thrombocytopenia (HIT) activate complement via the classical pathway. Previous studies have shown that the alternative pathway of complement substantially amplifies the classical pathway of complement activation through the C3b feedback cycle. OBJECTIVES: These studies sought to examine the contributions of the alternative pathway to complement activation by HIT antibodies. METHODS: Using IgG monoclonal (KKO) and/or patient-derived HIT antibodies, we compared the effects of classical pathway (BBK32 and C1-esterase inhibitor [C1-INH]), alternative pathway (anti-factor B [fB] or factor D [fD] inhibitor) or combined classical and alternative pathway inhibition (soluble complement receptor 1 [sCR1]) in whole blood or plasma. RESULTS: Classical pathway inhibitors BBK32 and C1-INH and the combined classical/alternative pathway inhibitor sCR1 prevented KKO/HIT immune complex-induced complement activation, including release of C3 and C5 activation products, binding of immune complexes to B cells, and neutrophil activation. The alternative pathway inhibitors fB and fD, however, did not affect complement activation by KKO/HIT immune complexes. Similarly, alternative pathway inhibition had no effect on complement activation by unrelated immune complexes consisting of anti-dinitrophenyl (DNP) antibody and the multivalent DNP--keyhole limpet hemocyanin antigen. CONCLUSIONS: Collectively, these findings suggest the alternative pathway contributes little in support of complement activation by HIT immune complexes. Additional in vitro and in vivo studies are required to examine if this property is shared by most IgG-containing immune complexes or if predominance of the classic pathway is limited to immune complexes composed of multivalent antigens.

SARS-CoV-2 vaccination and ITP in patients with de novo or preexisting ITP.

Cases of de novo immune thrombocytopenia (ITP), including a fatality, following SARS-CoV-2 vaccination in previously healthy recipients led to studying its impact in preexisting ITP. In this study, 4 data sources were analyzed: the Vaccine Adverse Events Reporting System (VAERS) for cases of de novo ITP; a 10-center retrospective study of adults with preexisting ITP receiving SARS-CoV-2 vaccination; and surveys distributed by the Platelet Disorder Support Association (PDSA) and the United Kingdom (UK) ITP Support Association. Seventy-seven de novo ITP cases were identified in VAERS, presenting with median platelet count of 3 [1-9] ×109/L approximately 1 week postvaccination. Of 28 patients with available data, 26 responded to treatment with corticosteroids and/or intravenous immunoglobulin (IVIG), and/or platelet transfusions. Among 117 patients with preexisting ITP who received a SARS-CoV-2 vaccine, 19 experienced an ITP exacerbation (any of: ≥50% decline in platelet count, nadir platelet count <30 × 109/L with >20% decrease from baseline, and/or use of rescue therapy) following the first dose and 14 of 70 after a second dose. Splenectomized persons and those who received 5 or more prior lines of therapy were at highest risk of ITP exacerbation. Fifteen patients received and responded to rescue treatment. In surveys of both 57 PDSA and 43 UK patients with ITP, prior splenectomy was associated with worsened thrombocytopenia. ITP may worsen in preexisting ITP or be identified de novo post-SARS-CoV2 vaccination; both situations responded well to treatment. Proactive monitoring of patients with known ITP, especially those postsplenectomy and with more refractory disease, is indicated.

Mesenchymal stromal cells enhance self-assembly of a HUVEC tubular network through uPA-uPAR/VEGFR2/integrin/NOTCH crosstalk.

Endothelial cells (ECs) degrade the extracellular matrix of vessel walls and contact surrounding cells to facilitate migration during angiogenesis, leading to formation of an EC-tubular network (ETN). Mesenchymal stromal cells (MSC) support ETN formation when co-cultured with ECs, but the mechanism is incompletely understood. We examined the role of the urokinase-type plasminogen activator (uPA) system, i.e. the serine protease uPA, its inhibitor PAI-1, receptor uPAR/CD87, clearance by the low-density lipoprotein receptor-related protein (LRP1) and their molecular partners, in the formation of ETNs supported by adipose tissue-derived MSC. Co-culture of human umbilical vein ECs (HUVEC) with MSC increased mRNA expression levels of uPAR, MMP14, VEGFR2, TGFß1, integrin ß3 and Notch pathway components (Notch1 receptor and ligands: Dll1, Dll4, Jag1) in HUVECs and uPA, uPAR, TGFß1, integrin ß3, Jag1, Notch3 receptor in MSC. Inhibition at several steps in the activation process indicates that uPA, uPAR and LRP1 cross-talk with αv-integrins, VEGFR2 and Notch receptors/ligands to mediate ETN formation in HUVEC-MSC co-culture. The urokinase system mediates ETN formation through the coordinated action of uPAR, uPA's catalytic activity, its binding to uPAR and its nuclear translocation. These studies identify potential targets to help control aberrant angiogenesis with minimal impact on healthy vasculature.

Divergent impacts of tocilizumab and colchicine in COVID-19-associated coagulopathy: the role of alpha-defensins.

Patients who are severely affected by coronavirus disease 2019 (COVID-19) may develop a delayed onset 'cytokine storm', which includes an increase in interleukin-6 (IL-6). This may be followed by a pro-thrombotic state and increased D-dimers. It was anticipated that tocilizumab (TCZ), an anti-IL-6 receptor monoclonal antibody, would mitigate inflammation and coagulation in patients with COVID-19. However, clinical trials with TCZ have recorded an increase in D-dimer levels. In contrast to TCZ, colchicine reduced D-dimer levels in patients with COVID-19. To understand how the two anti-inflammatory agents have diverse effects on D-dimer levels, we present data from two clinical trials that we performed. In the first trial, TCZ was administered (8 mg/kg) to patients who had a positive polymerase chain reaction test for COVID-19. In the second trial, colchicine was given (0·5 mg twice a day). We found that TCZ significantly increased IL-6, α-Defensin (α-Def), a pro-thrombotic peptide, and D-dimers. In contrast, treatment with colchicine reduced α-Def and Di-dimer levels. In vitro studies show that IL-6 stimulated the release of α-Def from human neutrophils but in contrast to colchicine, TCZ did not inhibit the stimulatory effect of IL-6; raising the possibility that the increase in IL-6 in patients with COVID-19 treated with TCZ triggers the release of α-Def, which promotes pro-thrombotic events reflected in an increase in D-dimer levels.

Erythrocytes identify complement activation in patients with COVID-19.

COVID-19, the disease caused by the SARS-CoV-2 virus, can progress to multisystem organ failure and viral sepsis characterized by respiratory failure, arrhythmias, thromboembolic complications, and shock with high mortality. Autopsy and preclinical evidence implicate aberrant complement activation in endothelial injury and organ failure. Erythrocytes express complement receptors and are capable of binding immune complexes; therefore, we investigated complement activation in patients with COVID-19 using erythrocytes as a tool to diagnose complement activation. We discovered enhanced C3b and C4d deposition on erythrocytes in COVID-19 sepsis patients and non-COVID sepsis patients compared with healthy controls, supporting the role of complement in sepsis-associated organ injury. Our data suggest that erythrocytes may contribute to a precision medicine approach to sepsis and have diagnostic value in monitoring complement dysregulation in COVID-19-sepsis and non-COVID sepsis and identifying patients who may benefit from complement targeted therapies.

Complement mediates binding and procoagulant effects of ultralarge HIT immune complexes.

Heparin-induced thrombocytopenia (HIT) is a prothrombotic disorder mediated by ultra-large immune complexes (ULICs) containing immunoglobulin G (IgG) antibodies to a multivalent antigen composed of platelet factor 4 and heparin. The limitations of current antithrombotic therapy in HIT supports the need to identify additional pathways that may be targets for therapy. Activation of FcγRIIA by HIT ULICs initiates diverse procoagulant cellular effector functions. HIT ULICs are also known to activate complement, but the contribution of this pathway to the pathogenesis of HIT has not been studied in detail. We observed that HIT ULICs physically interact with C1q in buffer and plasma, activate complement via the classical pathway, promote codeposition of IgG and C3 complement fragments (C3c) on neutrophil and monocyte cell surfaces. Complement activation by ULICs, in turn, facilitates FcγR-independent monocyte tissue factor expression, enhances IgG binding to the cell surface FcγRs, and promotes platelet adhesion to injured endothelium. Inhibition of the proximal, but not terminal, steps in the complement pathway abrogates monocyte tissue factor expression by HIT ULICs. Together, these studies suggest a major role for complement activation in regulating Fc-dependent effector functions of HIT ULICs, identify potential non-anticoagulant targets for therapy, and provide insights into the broader roles of complement in immune complex-mediated thrombotic disorders.