Background: Since publication of the American Society of Hematology 2019 Guidelines for Immune Thrombocytopenia (ITP), new clinical trials have been completed and new treatments have become available. Objective: These evidence-based guidelines from the American Society of Hematology (ASH) are a focused update of the 2019 ASH ITP guidelines, centering on treatment of adults with primary ITP. Methods: ASH formed a multidisciplinary guideline panel including two patient representatives that was balanced to minimize potential bias from conflicts of interest. The University of Oklahoma supported the guideline development process, including updating or performing systematic evidence reviews (up to July 19, 2025). The panel prioritized clinical questions and outcomes according to their importance for clinicians and patients and identification of areas with new data or where application of new methods would be of benefit. The panel used the Grading of Recommendations Assessment, Development and Evaluation (GRADE) approach, including GRADE Evidence-to-Decision frameworks and the GRADE framework for multiple comparisons, to assess evidence and make recommendations, which were subject to public comment. Results: The panel agreed on recommendations in adults with primary ITP regarding initial therapy and second-line treatment in patients who failed first-line corticosteroids. The panel also issued good practice statements on thrombopoietic agent switching and splenectomy. Conclusions: Novel recommendations of these guidelines include: (1) initial therapy with a combination of rituximab plus corticosteroids or a thrombopoietic agent plus corticosteroids rather than corticosteroids alone (conditional recommendation) and (2) a thrombopoietic agent (strong recommendation) or rituximab (conditional recommendation) for patients who failed first-line corticosteroids.

Antiphospholipid syndrome (APS) is an immunothrombotic disorder, frequently attributed to autoantibodies that bind β2-glycoprotein I (β2GPI). A study showed that the platelet-specific chemokine, platelet factor 4 (PF4), binds to β2GPI, enhancing recognition of β2GPI by APS antibodies. APS antibodies induce the release of neutrophil extracellular traps (NETs), webs of decondensed chromatin that bind both PF4 and b2GPI. We propose that PF4 bridges β2GPI to NETs (and other PF4-targeted polyanions), leading to the formation of prothrombotic PF4:b2GPI:NET immunotargets in APS. Dynamic light-scattering studies of isolated IgGs from four patients with triple-positive APS show formation of PF4:β2GPI:NET complexes that bind APS antibodies. NETs released in a microfluidic system bound b2GPI, but only in the presence of PF4, forming a multimolecular APS antigenic target. Whole blood infused through a photochemically-injured, endothelium-lined microfluidic channel formed platelet-, fibrin-, and complement- rich thrombi that bound APS antibody only in the presence of PF4. Thrombi were reduced in size if either ADAMTS13 or DNase1 was infused. In a murine APS model, wildtype and transgenic mice expressing platelet human PF4 ± FcgRIIA developed more intense neutrophil rolling along veins, and more extensive thrombus formation following laser injury to cremaster arterioles and venules, whereas mice lacking PF4 did not. Three antigenically distinct anti-hPF4 monoclonal antibodies blocked thrombosis in vitro, and neutrophil rolling and thrombosis in vivo. Our studies provide new insights into the basis of APS that has mechanistic parallels to other known PF4 immunothrombotic disorders and offer potential diagnostic and non-anticoagulant therapeutic strategies for clinical management.

BACKGROUND:Heparin-induced thrombocytopenia (HIT) is an antibody-mediated disorder associated with thrombosis developing in response to anticoagulation with heparin. Monocytes targeted by HIT antibodies contribute to the prothrombotic state, but structural and functional alterations of the activated monocytes have not been described. OBJECTIVES:To study morphologic and functional changes in monocytes caused by HIT antibodies interacting with membrane-associated platelet factor 4 (PF4) in vitro. METHODS:THP-1, isolated human, or FcγRIIA-positive and FcγRIIA-negative mouse monocytes were incubated with recombinant human PF4 and/or anti-PF4/heparin antibodies followed by scanning electron microscopy and confocal microscopy. RESULTS:Binding of PF4 to monocytes induced formation of "knobs" ∼150 nm in size that protruded from the cell surface. Addition of pathogenic HIT-like monoclonal antibodies (KKO) caused profound remodeling of the cell membrane and time-dependent formation and clustering of KKO/PF4/glycosaminoglycan complexes into large "blebs" ranging in size from 500 to 1200 nm. Dynamic confocal microscopy revealed formation of monocyte-derived microvesicles in response to PF4 and KKO. In contrast, RTO, a monoclonal antibody that blocks PF4 oligomerization and prevents thrombocytopenia/thrombosis in an animal HIT model, inhibited PF4-induced modification of monocyte surfaces. Comparing monocytes from transgenic mice expressing hFcγRIIA to wild-type mice lacking FcγRIIA indicated that bleb formation results from clustering of knobs caused by bivalent HIT antibodies through crosslinking of FcγRIIA. CONCLUSIONS:Binding of pathogenic HIT antibodies to PF4-containing antigenic complexes assembled on the monocyte surface promotes large-scale plasma membrane remodeling as part of cell activation through the FcγRIIA receptors, resulting in the release of procoagulant microvesicles, which together may contribute to thrombosis in HIT.

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.

The McMaster Immune Thrombocytopenia (ITP) Summit, held on October 27, 2023, was an educational seminar from leading experts in immune thrombocytopenia and related disorders geared toward hematologists, internists, immunologists, and clinical and translational scientists. The focus of the Summit was to review the mechanisms, diagnosis, and treatment of primary versus secondary ITP. Specific objectives were to describe the unique features of secondary ITP, and to review its mechanisms in the context of autoimmune disease and infection. The key messages in this Summit were: (1) ITP is a heterogeneous disease, and genetic and immunologic insights may help classify patient subtypes; (2) exploring the autoimmune mechanisms and their association with hypogammaglobulinemia in patients with secondary ITP could improve our understanding of ITP and its subtypes; (3) investigating the mechanisms of ITP in the context of infections caused by viruses such as CMV, HIV, dengue, and hepatitis C, or bacteria such as H. pylori , or vaccinations could provide insight into the causes of ITP. A better understanding of secondary ITP could help elucidate the pathogenesis of ITP.

The sole FDA-approved drug treatment for ischemic stroke is tissue-type plasminogen activator (tPA). However, upregulation of JNK mitogen-activated protein kinase (MAPK) and endothelin 1 (ET-1) by tPA after stroke contributes to impaired cerebrovascular autoregulation. Wild-type (wt) tPA can bind to the lipoprotein-related receptor (LRP), which mediates vasodilation, or NMDA receptors (NMDA-Rs), exacerbating vasoconstriction. Elevations in IL-6, a marker of inflammation that accompanies stroke, are reported to be an adverse prognostic factor. We hypothesized that IL-6 released into CSF after stroke by wt-tPA through activation of NMDA-Rs and upregulation of ET-1 and JNK contribute to impairment of cerebrovascular autoregulation and increased histopathology. Results show that IL-6 was increased post stroke in pigs, which was increased further by wt-tPA. Co-administration of the IL-6 antagonist LMT-28 with wt-tPA prevented impairment of cerebrovascular autoregulation and necrosis of hippocampal cells. wt-tPA co-administered with the JNK inhibitor SP 600125 and the ET-1 antagonist BQ 123 blocked stroke-induced elevation of IL-6. Co-administration of LMT-28 with wt-tPA blocked the augmentation of JNK and ET-1 post stroke. In conclusion, IL-6 released after stroke, which is enhanced by wt-tPA through activation of NMDA-Rs and upregulation of ET-1 and JNK, impairs cerebrovascular autoregulation and increases histopathology. Strategies that promote fibrinolysis while limiting activation of NMDA-Rs and upregulation of IL-6 may improve the benefit/risk ratio compared to wt-tPA in treatment of stroke.

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.

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&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.

Fibrinolytics delivered into the general circulation lack selectivity for nascent thrombi, reducing efficacy and increasing the risk of bleeding. Urokinase-type plasminogen activator (uPA) transgenically expressed within murine platelets provided targeted thromboprophylaxis without causing bleeding but is not clinically feasible. Recent advances in generating megakaryocytes prompted us to develop a potentially clinically relevant means to produce "antithrombotic" platelets from CD34+ hematopoietic stem cell-derived in vitro-grown megakaryocytes. CD34+ megakaryocytes internalize and store in alpha granules (alpha-granules) single-chain uPA (scuPA) and a plasmin-resistant thrombinactivatable variant (uPAT). Both uPAs colocalized with internalized factor V (FV), fibrinogen and plasminogen, low-density lipoprotein receptor-related protein 1 (LRP1), and interferoninduced transmembrane protein 3, but not with endogenous von Willebrand factor (VWF). Endocytosis of uPA by CD34+ megakaryocytes was mediated, in part, via LRP1 and alpha IIb beta 3. scuPA-containing megakaryocytes degraded endocytosed intragranular FV but not endogenous VWF in the presence of internalized plasminogen, whereas uPATmegakaryocytes did not significantly degrade either protein. We used a carotid artery injury model in nonobese diabetic-severe combined immunodeficiency IL2r gamma null (NSG) mice homozygous for VWFR1326H(a mutation switching binding VWF specificity from mouse to human glycoprotein Ib alpha) to test whether platelets derived from scuPA- or uPATmegakaryocytes would prevent thrombus formation. NSG/VWFR1326H mice exhibited a lower thrombotic burden after carotid artery injury compared with NSG mice unless infused with human platelets or megakaryocytes, whereas intravenous injection of uPAmegakaryocytes generated sufficient uPA-containing human platelets to lyse nascent thrombi. These studies describe the use of in vitro-generated megakaryocytes as a potential platform for delivering uPA or other ectopic proteins within platelet alpha-granules to sites of vascular injury.

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.

BACKGROUND:Immunoglobulin G antibodies (Abs) to platelet factor 4 (PF4) complexed to heparin (PF4/H) commonly occur after H exposure but cause life-threatening complications of H-induced thrombocytopenia (HIT) in only a few patients. Presently, only platelet activation assays reliably distinguish anti-PF4/H Abs that cause disease (HIT Abs) from those likely to be asymptomatic (AAbs). OBJECTIVES:Recent studies indicate that complement activation is an important serologic property of HIT Abs and is essential for IgG Fc receptor IIA-mediated cellular activation. As platelet activation by HIT Abs also relies on IgG Fc receptor IIA activation, we correlated the complement- and platelet-activating properties of anti-PF4/H Abs in a clinically annotated patient cohort. METHODS:Clinical and laboratory features of patients with HIT (n = 8) and AAbs+ (n = 14) were correlated with properties of complement, platelet, and monocyte/neutrophil activation. RESULTS:Expected clinical and laboratory differences were seen between HIT and AAb+ patients, with HIT patients having lower mean platelet counts, greater percentage drop in platelet counts, higher 4T and HIT expert probability scores, higher anti-PF4 polyclonal and immunoglobulin G Ab levels, and serotonin release assay positivity. Ex vivo assays revealed significant differences in complement activation by HIT vs AAb+ patients, with the extent of complement activation closely correlated with percent serotonin release by anti-PF4/H Abs and matrix metalloproteinase-9 and interleukin-8 release in whole blood. CONCLUSION:These findings suggest that complement activation strongly correlates with cellular activation endpoints, including platelet and monocyte/neutrophil activation, and if confirmed in a larger prospective study, may serve as a "functional" biomarker for pathogenic HIT Abs.

KKO is a HIT (heparin-induced thrombocytopenia)-like mouse monoclonal antibody (moAb) that binds to platelet factor 4 (PF4) when complexed to polyanions like heparin and neutrophil extracellular traps (NETs). Human (h) PF4 protected NETs from DNases and was further enhanced by KKO as it activates platelets and neutrophils via both FcgRIIA and complement, causing prothrombotic state in transgenic mice expressing FcgRIIA and hPF4. We posited through that if Fc activity of KKO were eliminated then it may stabilize NETs and potentially be therapeutic by stabilizing NETs without FcgRIIA and complement activation. We deglycosylated Fc region of KKO to make DGKKO, and tested if we can avoid detrimental effects without interfering with KKO protection of PF4:NET complexes. We also speculated that DGKKO may bind to hPF4:polyanions and prevent them from inducing HIT. Binding efficacy of DGKKO to PF4-heparin complexes was studied by ELISA and dynamic light scattering. Platelet and complement activation were also studied for DGKKO, and we found that DGKKO still bind to PF4-polyanion complexes, with no platelet activation and significantly lower complement activation. DGKKO also enhanced NET nuclease resistance to improve microbial capture by PF4-NETs, preventing thrombocytopenia and improving survival in murine sepsis models. Also, our humanized in vitro microfluidics studies as well as HIT mice studies showed that infusion of DGKKO prevented both thrombocytopenia and thrombosis in a murine model of HIT. These results showed that Fc-modified KKO has protective effects in at least two models of thromboinflammation. Further studies are underway to examine the role of NET stabilization by modified KKO in other thromboinflammatory disorders. R35 to Mortimer Poncz and CHOP Foerderer Grant to Amrita Sarkar

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 αIIbβ3. 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 αIIbβ3 receptors and is functionally available from activated platelets.

( N Engl J Med. 2023;389:540–548) Immune thrombocytopenia (ITP) is an autoimmune disorder characterized by immunoglobulin G antiplatelet antibodies that lead to reduced platelet production and increased platelet clearance. It affects 1 in 3 people per 10,000 in the United States. ITP is more common in women of childbearing age, and during pregnancy, platelet counts may decrease, requiring special medical attention. Immunoglobulin G antiplatelet antibodies can also affect the fetus. The clinical variability of ITP complicates diagnosis and treatment, and uncertainty remains for many aspects of management. This article focuses on recent research on the risk assessment and management of primary ITP.

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 G2b & kappa; (IgG2b & kappa;) 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 Fc & gamma;RIIA 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 Fc & gamma;RIIA-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 Fc & gamma;RIIA, 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.

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.

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 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.