Cerebral malaria (CM) from Plasmodium falciparum is a major cause of death in African children. Since bradykinin (BK) is a mediator of vasogenic edema, we hypothesized that it contributes to the pathogenesis of CM in Kenyan children and Plasmodium berghei ANKA-infected (PbA-infected) C57BL/6J mice in experimental CM (ECM). Cleaved plasma high-molecular-weight kininogen (cHK) is a marker for BK release. 40% of children with central nervous system malaria had plasma cHK versus 18% of children with uncomplicated malaria. Wild-type PbA-infected mice with ECM had circulating cHK, elevated BK levels, and reduced HK and prekallikrein activity/antigen levels. HK-null (Kng1-/-), combined BK B1- and B2 receptor-null (Bdkrb1-/-Bdkrb2-/-), BK B2 receptor-null (Bdkrb2-/-), or BK B1 receptor-null (Bdkrb1-/-) mice were protected significantly from neurologic deterioration and brain edema compared with wild-type mice. F12-/- mice were not protected from neurological deterioration. Prekallikrein-null (Klkb1-/-), prolylcarboxypeptidase hypomorph (Prcpgt/gt), and brain endothelial cell conditional KO of PRCP (Prcpfl/fl Cre) mice with ECM had reduced neurologic deterioration and brain edema. Adjuvant plasma kallikrein inhibition combined with artesunate treatment in PbA-infected mice reversed neurologic deterioration and brain edema and significantly prolonged survival over artesunate alone. BK-induced vasogenic edema contributes to human and murine CM.

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.

Hereditary hemorrhagic telangiectasia (HHT) results in recurrent severe epistaxis, chronic gastrointestinal bleeding, and other morbid manifestations. Pomalidomide was recently demonstrated to be efficacious for bleeding in HHT in the landmark PATH-HHT trial. Given that the vascular remodeling that results in bleeding improvement may require several months, characteristics associated with treatment response would be invaluable to plan treatment. Using data from PATH-HHT (NCT03910244), we identified characteristics associated with change from baseline in Epistaxis Severity Score (ESS, a well-validated 10-point HHT bleeding scale and the trial’s primary endpoint) using multivariable-adjusted repeated measures models. Characteristics associated with a better response to pomalidomide at weeks 16-24 of the trial included a higher (more severe) baseline ESS (change from baseline, -0.76 [95% CI -0.99,-0.54] points per 1 point higher baseline score, P<0.001) and concomitant antifibrinolytic therapy (-0.73 [95% CI -1.48, 0.01] vs. no therapy, p=0.05). Characteristics associated with a less robust response were older age (0.29 [95% CI 0.05, 0.53] per 10 years older, P=0.02) and underlying ACVRL1 germline mutation (0.62 [95% CI -0.04,1.27] vs. ENG, SMAD4 or mutation not known, p=0.06]. Characteristics notably associated with placebo response (placebo effect) were patient-reported outcome measures, such that those experiencing more severe disease at baseline had less placebo effect. In conclusion, several baseline patient and disease characteristics were associated with pomalidomide response, findings that may help guide patient selection for the use of pomalidomide in HHT. Clinicaltrials.gov identifier: NCT03910244

Venous thromboembolism (VTE) is an important cause of morbidity and mortality in individuals with active cancer. Most cancer-associated VTE occurs in the ambulatory setting in individuals receiving systemic therapy. Multiple anticoagulants are currently available for the treatment of cancer-associated VTE; a major drawback is the risk of bleeding. No drugs are currently approved for primary prevention of VTE in cancer in the outpatient setting. Adherence/compliance issues, drug-drug interactions, and concern about renal and hepatic metabolism are other ongoing concerns with these agents. Thus, there are major unmet needs in the quest to optimize the treatment and prevention of VTE in cancer. Factor XI (FXI) has only a small impact on hemostasis but contributes significantly to thrombosis. It is therefore a potential therapeutic target with less concern for enhancing bleeding. Several early-phase studies suggest that FXI inhibitors have reduced the risk of bleeding at therapeutic doses in the prevention of venous and arterial events in cardiology and postoperative settings. Multiple studies are now addressing the utility of this new class of drugs in cancer-associated VTE, including completed, ongoing, and planned trials of primary prevention and treatment. This narrative review addresses the role of FXI in thrombosis, the rationale for FXI inhibitors in primary and secondary prevention settings in malignancy and provides an overview of preliminary results and future directions.

Effective targeting of cancer stem cells (CSCs) requires neutralization of self-renewal and chemoresistance, but these phenotypes are often regulated by distinct molecular mechanisms. Here we report the ability to target both of these phenotypes via CD55, an intrinsic cell surface complement inhibitor, which was identified in a comparative analysis between CSCs and nonCSCs in endometrioid cancer models. In this context, CD55 functions in a complement-independent manner and required lipid raft localization for CSC maintenance and cisplatin resistance. CD55 regulated self-renewal and core pluripotency genes via ROR2/JNK signaling and in parallel cisplatin resistance via lymphocyte-specific protein tyrosine kinase (LCK) signaling, which induced DNA repair genes. Targeting LCK signaling via saracatinib, an inhibitor currently undergoing clinical evaluation, sensitized chemoresistant cells to cisplatin. Collectively, our findings identify CD55 as a unique signaling node that drives self-renewal and therapeutic resistance through a bifurcating signaling axis and provides an opportunity to target both signaling pathways in endometrioid tumors.

BACKGROUND:Despite antiplatelet therapy, some patients remain at high ischemic risk because of drug nonresponsiveness or high residual platelet reactivity. We aimed to target an orphan platelet GPCR (G protein-coupled receptor) from the OR (olfactory receptor) family as a novel antithrombotic strategy. METHODS:Using an engineered reporter cell line expressing human OR2L13, an orphan GPCR implicated in limiting platelet reactivity, we conducted a high-throughput screen of 8000 nonodorant bioactive compounds with counterscreen validation. Subsequent studies assessed platelet function in healthy subjects and patients with coronary artery and peripheral artery disease. Phospho-proteomics revealed key signaling pathways, whereas ex vivo and in vivo studies evaluated the impact of a lead compound on platelet signaling, biomechanics, and thrombosis in both arterial and venous vasculature. RESULTS:We identified 6 OR2L13 (olfactory receptor family 2 subfamily L member 13)-specific agonists that suppressed platelet aggregation and α-granule exocytosis through multiple receptors, suggesting a shared downstream mediator. The lead agonist (CCF0054500) phosphorylated platelet HSP27 (heat shock protein 27), disrupting the actin cytoskeleton and reducing clot retraction (clot area, 70.6 versus 5.2; P<0.0001), an effect reversed by HSP27 inhibition. In a murine arterial injury model, CCF0054500 decreased platelet accumulation by 88.9% (P<0.0003) without affecting fibrin generation or hemostasis. In a myocardial infarction model with high residual platelet reactivity, CCF0054500 lowered platelet reactivity (P<0.0001) and improved left ventricular function (P=0.007). CONCLUSIONS:We describe and characterize the first nonolfactory probe for the purpose of inhibiting platelet activation and thrombosis through downstream HSP27 in a comprehensive investigation using a first-of-its-kind platelet inhibitor targeting an orphan platelet GPCR.

Cerebral malaria (CM) due to Plasmodium falciparum ( Pf ) infection is a major cause of death in African children. Bradykinin (BK) is a mediator of vasogenic edema. It could contribute to the pathogenesis of central nervous system malaria in Kenyan children and P. berghei ANKA ( PbA ) infected C57BL/6J mice with experimental cerebral malaria. Cleaved plasma high molecular weight kininogen (cHK) is a marker for prior BK release. 40% of children with central nervous system malaria had plasma cHK versus 18% of children with uncomplicated malaria. Wild-type PbA -infected mice had circulating plasma cHK, elevated BK levels, and reduced HK and prekallikrein levels. HK null ( Kng1 -/- ), combined BK B1 and B2 receptor null ( Bdkrb1 -/- / Bdkrb2 -/- ), BK B2 ( Bdkrb2 -/- ) or BK B1 ( Bdkrb1 -/- ) receptor null mice were protected from neurologic deterioration and brain edema compared to wild-type mice. F12 -/- mice were not protected from neurological deterioration. Prekallikrein null ( Klkb1 -/- ), prolylcarboxypeptidase hypomorphs ( Prcp gt/gt ), and brain endothelial cell conditional knockout of PRCP ( Prcp fl/fl Cre) mice had reduced neurologic deterioration and brain edema. Adjuvant plasma kallikrein inhibition combined with artesunate treatment of PbA -infected mice reversed neurologic deterioration and brain edema and prolonged survival relative to artesunate alone. BK-induced vasogenic edema contributes to human and murine CM.

ABSTRACT:Hereditary hemorrhagic telangiectasia (HHT), an autosomal dominant vasculopathy affecting 1 in 5000 individuals, is the second most common inherited bleeding disorder worldwide. Despite this prevalence, comprehensive data on disease manifestations and complications remain limited. To address this gap, the US Congress allocated funding leading to the Comprehensive HHT Outcomes Registry of the United States (CHORUS), a prospective, 15-center longitudinal registry enrolling unselected patients with confirmed HHT. In this initial report, we describe findings from the first 600 participants, with a median age of 53 (range, 0-88) years and 60% female. Despite most participants developing typical HHT manifestations by age 13 years, the majority (63%) were not diagnosed until mid-to-late adulthood. Recurrent spontaneous epistaxis occurred in 95% of participants, chronic gastrointestinal bleeding in 30%, and heavy menstrual bleeding in 35% of postmenarche females, together resulting in moderate-to-severe mucosal bleeding in 76%. Iron deficiency and/or anemia were diagnosed in 68%, with 41% requiring IV iron and 25% requiring red cell transfusions. Serious complications of solid-organ arteriovenous malformations were frequent, including intracranial hemorrhage (3%), pulmonary hemorrhage (2%), venous thromboembolism (7%), arterial thromboembolism (11%), heart failure (7%), and pulmonary hypertension (7%). These data from CHORUS, the first national US registry of its kind, provide reliable, real-world estimates of the incidence, prevalence, and severity of numerous HHT manifestations and complications. HHT has a high burden of moderate-to-severe bleeding, anemia, thrombosis, and major neurologic and cardiopulmonary complications. There is a mean interval between first symptoms and diagnosis of >2 decades, during which substantial, serious, and preventable HHT morbidity, including early intracranial hemorrhage, may occur. This trial was registered at clinicaltrials.gov as NCT06259292.

ABSTRACT:Hereditary hemorrhagic telangiectasia (HHT) causes severe recurrent epistaxis, chronic gastrointestinal bleeding, solid organ arteriovenous malformations, and significant anemia. Although it is the second most common inherited bleeding disorder worldwide, no approved therapies exist. The multicenter, US randomized, controlled PATH-HHT trial demonstrated efficacy of short-term pomalidomide treatment of epistaxis in HHT. However, questions regarding long-term safety, effectiveness, and utility for HHT-associated gastrointestinal bleeding remain. The PATH-HHT ATLAS (after trial longitudinal assessment study) was a multicenter US longitudinal observational study evaluating patients enrolled in PATH-HHT who continued pomalidomide after PATH-HHT through a poststudy drug access program, with ongoing close monitoring per PATH-HHT protocol and as mandated by the US Food and Drug Administration. Sixty-two patients treated with pomalidomide for HHT for up to 4.4 years were included. Significant, durable improvements in mean epistaxis severity score (5.55 points [baseline] to 2.80 points [month 12]; P< .0001) and mean hematologic support score (9.11 red cell unit equivalents [RUEs] during 6 months pretreatment to 5.73 RUEs [months 7-12]; P = .0056) were observed. Pomalidomide was less effective for gastrointestinal bleeding than for epistaxis, particularly in patients with high red blood cell transfusion requirements. Thirty-one patients (50%) underwent dose reduction from 4 mg daily, primarily due to neutropenia, but most maintained effectiveness at 2 or 3 mg daily. Over 105.2 patient-years of pomalidomide treatment, 0 patients developed peripheral neuropathy, 1 developed venous thromboembolism, and 5 developed serious infections. In conclusion, pomalidomide was effective for HHT-associated epistaxis over extended durations, albeit with nontrivial potential toxicities, and may be considered for certain patients with HHT. This trial was registered at www.clinicaltrials.gov as NCT07018401.

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.

INTRODUCTION:Therapeutic development in hereditary hemorrhagic telangiectasia (HHT), the second-most-common inherited bleeding disorder, is rapidly accelerating. Therefore, modern clinical trials in HHT must be equipped with rigorous, fit-for-purpose hematologic endpoints. AREAS COVERED:Due to the inadequacy of solitary hematologic measurements (hemoglobin, red cell transfusions and quantity of iron infused used as separate measurements), the recent International Consensus Report on Standardization in HHT advised widespread implementation of two composite hematologic measurements, the Hematologic Support Score and Hematologic Impact Score. The Hematologic Support Score is a composite measurement of total hematologic support (red cell transfusions and quantity of iron infused) administered over a time period, and the Hematologic Impact Score provides the total hematologic impact of an intervention, in red-cell unit equivalents, by integrating change in hemoglobin, red cell transfusions, and quantity of iron infused. EXPERT OPINION:This article, authored by 16 hematologist-experts in HHT, hemostasis, and iron deficiency, explains these measures in depth and the importance of their implementation in clinical studies of major disorders of chronic bleeding, such as HHT, gastrointestinal angiodysplasia, and heavy menstrual bleeding. We urge clinicians, investigators, study sponsors, and regulatory bodies to adopt and promote the use of these common-sense measurements in clinical studies of chronic bleeding interventions.

Background Hereditary hemorrhagic telangiectasia (HHT) is the second most common inherited bleeding disorder worldwide and results in substantial medical and psychosocial morbidity due to recurrent, often severe epistaxis, chronic gastrointestinal bleeding, hematologic support-dependent iron deficiency anemia, and serious bleeding and nonbleeding complications of visceral arteriovenous malformations. With disease-modifying therapeutics entering clinical trials, disease-specific health-related quality of life (HRQoL) instruments capable of longitudinal assessment of HHT-specific manifestations are essential. Objectives To externally validate and define the features relevant to clinical application of the HHT-specific Quality of Life scale. Methods The HHT-specific Quality of Life (HHT-QoL) scale was developed as an easily administered, 4-item instrument assessing physical, social, personal, and work impacts due to HHT longitudinally. Utilizing comprehensive, longitudinal data from pomalidomide for the treatment of HHT, the largest HHT randomized clinical trial to date, we evaluated internal consistency and external validity of the HHT-QoL and defined essential characteristics for clinical application. Results The HHT-QoL demonstrated good internal consistency and external validity, including directionally and clinically consistent correlations with the Epistaxis Severity Score, bleeding duration as assessed by epistaxis diary, and multiple well-validated generic HRQoL instruments measuring emotional distress, social roles and activities, and fatigue. Development of an HHT HRQoL severity classification utilizing HHT-QoL total scores was successful (0 as no limitation, 1-3 as mild, 4-7 as moderate, and 8-16 points as severe limitation), and the minimal clinically important difference was calculated to be 1.1 points (best rounded to 1 point as the scores are integers) utilizing anchor-based and distribution-based methods. Conclusion This study successfully externally validates the HHT-QoL. The HHT-QoL is a validated disease-specific HRQoL instrument with defined severity classification and minimal clinically important difference, making it ideal for use in clinical trials of bleeding in HHT.

BACKGROUND:Over the past few years, new information has emerged in the management of both immune thrombotic thrombocytopenic purpura (iTTP) and congenital (or hereditary) thrombotic thrombocytopenic purpura (cTTP). METHODS:In March 2024, the International Society on Thrombosis and Haemostasis (ISTH) formed a multidisciplinary panel comprising hematologists, intensivists, nephrologists, pathologists, patient representatives, and a methodology team. The panel discussed all treatment questions related to thrombotic thrombocytopenic purpura (TTP) using the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) method to appraise evidence and formulate recommendations. RESULTS:For patients with cTTP in remission, a new strong recommendation was issued for the use of recombinant ADAMTS-13 over fresh frozen plasma in the context of moderate certainty evidence. The panel also revised a previous recommendation and suggested using fresh frozen plasma over a watch-and-wait approach for patients with cTTP in remission based on very low certainty evidence should recombinant. The panel reviewed and referenced new publications supporting therapeutic efficacy, potential survival benefit, and cost considerations of adding caplacizumab to therapeutic plasma exchange, corticosteroids, and rituximab, but concluded that no change was warranted to the previous recommendations in the management of iTTP. Good practice statements on the concomitant use of antithrombotic agents were marginally modified. CONCLUSIONS:For patients with iTTP, no change to 2020's recommendations. For patients with cTTP, the panel supports ADAMTS-13 replacement. Where accessible, recombinant ADAMTS-13 provides the most favorable balance of benefits and risks. Otherwise, fresh frozen plasma may still be effective. Shared decision-making should include the benefits, the potential harms, and the burden of care.

Background: Acetylsalicylic acid (ASA) has been shown to reduce recurrent deep venous thrombosis (DVT) rates. However, limited mechanistic data is available regarding the efficacy of ASA on DVT resolution and measures of post-thrombotic syndrome (PTS), particularly in female subjects. Furthermore, real-world sex-specific data on ASA prescription and withdrawal rates in secondary DVT prevention patients are sparse. Methods: First the effects of ASA pretreatment on thrombogenesis were assessed in both female and male C57BL/6 mice (n=149) utilizing serial intravital microscopy in an extended murine DVT model. Next the effects of ASA on DVT resolution and vein wall inflammation were evaluated in male mice with IVC stasis DVT using histopathological and scanning electron microscopy analyses. Next, in a single-center clinical study, secondary DVT prevention patients with a history of unprovoked lower extremity DVT were assessed for ASA and anticoagulant prescription and withdrawal rates over 24 months. Results: In murine stasis DVT, utilizing serial IVM for 24 hours (n=82), ASA pretreatment (3 mg/kg/day) significantly reduced thrombogenesis and accelerated DVT resolution in males (Fig 1A p<0.05), with a higher ASA dose (15 mg/kg/day) required in females for efficacy (Fig 1B p>0.05 all, Fig 1C p<0.05 all). In complete-stasis DVT (n=67), ASA pretreatment reduced day 8 thrombus (Fig 1D-E, p<0.001), fibrin-rich area and fibrin fibril density, and increased plasminogen expression. ASA further reduced vein wall fibrosis and fibroblasts (Fig 1F-H, p<0.05), as well as vein wall MMP activity. Next, in a cohort of 131 patients with unprovoked DVT followed over 24 months, 13.3% of patients discontinued anticoagulation therapy (Fig 1I-J, p trend <0.001). Discontinuation occurred more often in females (19.5% vs. 9.7% in males, p interaction =0.047). ASA was infrequently prescribed in patients who stopped anticoagulation (3/7 males, 0/8 females). Conclusions: Preventative ASA treatment reduced murine thrombogenesis and improved DVT resolution, with females requiring a higher ASA dose to achieve efficacy. ASA further reduced vein wall injury and inflammation. Clinically, patients with unprovoked DVT showed increasing rates of anticoagulant withdrawal over 24 months, especially in female patients. Such patients further infrequently receive ASA. These results may motivate reconsideration of preventative ASA to reduce recurrent DVT and PTS in patients ineligible for anticoagulant therapy.

Platelets circulating in the bloodstream are relatively quiescent but become "activated" upon encountering stimulants or "agonists" at the site of blood vessel injury. Proaggregatory and procoagulant platelets represent two distinct populations of activated platelets. While proaggregatory platelets facilitate the cessation of bleeding, or "hemostasis," by forming a plug of platelets clumped together through fibrinogen bridges, procoagulant platelets dramatically accelerate the coagulation cascade, culminating in fibrin clot formation. An interesting aspect of procoagulant platelets is that their morphology exhibits certain features of "necrosis" and "apoptosis." They may thus represent a form of cell death in platelets, albeit with an important role in thrombosis and hemostasis. This article introduces the concept of procoagulant platelets, their relevance to health and disease, and a comparison of existing methods for their analysis. It then provides comprehensive protocols for analyzing procoagulant platelets, investigating the mechanisms of their formation, and assessing their prothrombotic role in facilitating coagulation. The article concludes with a discussion of key steps, limitations, and troubleshooting principles for the described methods.

Hereditary hemorrhagic telangiectasia (HHT, Osler-Weber-Rendu disease) is the second most common inherited bleeding disorder worldwide, affecting approximately 1 in 5000 people. Development of disease-modifying and efficacious hemostatic agents to treat HHT has finally begun after decades without such medical therapies. However, the lack of consensus on standardized severity definitions, outcome criteria, and terminology remains a major obstacle to clinical investigation and therapeutic development in HHT. Additionally, with the ongoing repurposing of antiangiogenic agents and emerging development of novel HHT-specific therapies, comparative clinical trials are expected in the future. Therefore, to end the problematic heterogeneity hindering these efforts, the Global Research and Medical Advisory Board (GRMAB) of the Cure HHT Foundation, an international group of recognized HHT experts, convened a conference of expert HHT clinician-investigators from within GRMAB as well as invited external experts in HHT and bleeding disorders generally, patients with HHT, and patient advocates to define standard terminology and definitions for outcomes and severity classification for bleeding and anemia in HHT. These criteria and definitions should be adopted by regulators, investigators, and the pharmaceutical industry in the development and performance of interventional clinical trials and cohort studies to allow improved comparability between clinical trials, facilitate communication between clinicians and investigators, improve therapeutic guideline development, and provide a standardized framework for regulatory agencies. SEARCH STRATEGY AND SELECTION CRITERIA: Evidence for this report was systematically identified and evaluated utilizing two search strategies in Ovid MEDLINE, described in full detail in the Appendix, pp.4-13. The searches were conducted on January 7, 2025. The titles and abstracts of each record were reviewed, and the inclusion criteria were applied to all search results to identify full text articles to be retrieved for further review. All retrieved full texts were then reviewed to reach a final determination if a study met the inclusion criteria. Included references were then compiled into evidence tables, which were then utilized by the International Consensus Report Working Group throughout the development of report recommendations.

Background:Antibodies to β2-glycoprotein I (β2GPI) cause thrombosis in antiphospholipid syndrome, however the role of β2GPI itself in regulation of coagulation pathways in vivo is not well understood. Methods:We developed β2GPI-deficient mice (Apoh -/- ) by deleting exon 2 and 3 of Apoh using CRISPR/Cas9 and compared the propensity of wild-type (WT) and Apoh -/- mice to develop thrombosis using rose bengal and FeCl 3 -induced carotid thrombosis, laser-induced cremaster arteriolar injury, and inferior vena cava (IVC) stasis models. We also compared tail bleeding times and assessed platelet activation in WT and Apoh -/- mice in the absence and presence of exogenous β2GPI. Results:Compared to WT littermates, Apoh -/- mice demonstrated a prolonged time to occlusion of the carotid artery after exposure to rose bengal or FeCl 3 , and reduced platelet and fibrin accumulation in cremasteric arterioles after laser injury. Similarly, significantly smaller thrombi were retrieved from the IVC of Apoh -/- mice 48 hours after IVC occlusion. The activated partial thromboplastin time (aPTT) and prothrombin time, as well as aPTT reagent- and tissue factor-induced thrombin generation times using plasma from Apoh -/- and WT mice revealed no differences. However, we observed significant prolongation of tail bleeding in Apoh -/- mice, and reduced P-selectin expression and binding of fibrinogen to the activated α2bβ3 integrin on platelets from these mice after stimulation with low thrombin concentrations; these changes were reversed by exogenous β2GPI. An antibody to PAR3 blocked thrombin-induced activation of WT, but not Apoh -/- platelets, as well as the ability of β2GPI to restore the activation response of Apoh -/- platelets to thrombin. β2GPI deficiency did not affect platelet activation by a PAR4-activator peptide, or ADP. Conclusions:In mice, β2GPI may mediate procoagulant activity by enhancing the ability of PAR3 to present thrombin to PAR4, promoting platelet activation at low thrombin concentrations. Key Points:β2GPI deficient mice are protected from experimental arterial, venous, and microvascular thrombosis.β2GPI deficient mice display prolonged tail bleeding times and reduced PAR3-facilitated platelet activation by low concentrations of thrombin.

OBJECTIVES:Catastrophic antiphospholipid syndrome (CAPS) is a complement-driven thrombotic disorder, characterised by widespread thrombosis and multiorgan failure. We identified rare germline variants including complement receptor 1 (CR1) in 50% of patients with CAPS. Here, we define CR1 dysregulation mechanisms (genetic/epigenetic) underlying complement-mediated thrombosis in CAPS and support C5 inhibition as a potential therapy. METHODS:We quantified CR1 expression by flow cytometry across haematopoietic cell types. CRISPR/Cas9 genome editing of TF-1 (erythroleukaemia) cells was performed to generate CR1 'knock-out' and 'knock-in' lines with patient-specific CR1 variants. Multiomics analysis was performed to investigate the role of methylation in patients with reduced CR1 expression. Functional impact of low CR1 was assessed by complement-mediated cell killing using modified Ham assay, cell-bound complement degradation products through flow cytometry, and circulatory immune complexes in serum samples through ELISA. RESULTS:CR1 expression in erythrocytes was markedly reduced on CAPS erythrocytes (n = 9, 21.80%) compared to healthy controls (HCs; n = 35, 84.04%), with promoter hypermethylation emerging as a plausible epigenetic mechanism for CR1 downregulation. Novel germline variant (CR1-V2125L; rs202148801) mitigated CR1 expression and increased complement-mediated cell death of knock-in cell lines. Erythrocytes from the patient with the CR1-V2125L variant had low CR1 expression. Levels of circulating immune complexes, which are bound and cleared by CR1 on erythrocytes, were higher in acute CAPS (n = 3, 25.55 µg Eq/mL) than HCs (n = 3, 7.445 µg Eq/mL). Five patients were treated with C5 inhibition which mitigated thrombosis. CONCLUSIONS:Genetic or epigenetic-mediated CR1 deficiency is a potential hallmark of CAPS and predicts response to C5 inhibition.