Mutational landscape, inhibitor development, and health-care burden in non-severe haemophilia A: A single-centre Australian experience.

AIM: To characterise non-severe haemophilia A (HA) patients enrolled on the Australian Bleeding Disorders Registry (ABDR) treated through a state-wide Haemophilia Treatment Centre (HTC) with respect to their mutational profile, inhibitor risk and health-care burden. METHOD: We conducted a single-centre observational study of all non-severe HA patients treated at the Alfred Health HTC registered on the ABDR as of the 26th July 2023. Data were extracted from the ABDR and electronic medical record (EMR) regarding demographics, severity, genetic testing, treatment, inhibitors, bleeding events and procedures. Inhibitor risk was calculated as a function of exposure days (EDs) of FVIII replacement. RESULTS: There were 289 non-severe HA patients treated at the Alfred HTC registered on the ABDR as of July 2023, all of whom were adult patients aged > 18 years old. Genotyping had been performed in 228/289 (78.9%). Of the inhibitor analysis population, 14/193 (7.3%) had an inhibitor. The cumulative incidence of inhibitor development at 75 EDs was 31% (95% CI 13%-46%). The median cost of bypassing agents per inhibitor patient was $57,087.50/year. CONCLUSION: These results demonstrate a relatively high inhibitor prevalence and incidence risk in non-severe HA compared to previously published work, although this may partly reflect a smaller population size. High rates of genotyping have allowed representative mutational characterisation. The burden of care imposed by non-severe HA in terms of bleeding events, procedures and bypassing agent cost is larger than expected, particularly within the inhibitor population.

Exploring the effects of Factor Xa inhibitors on thrombin generation in people with haemophilia.

Optimal management of cardiovascular disease (CVD) in people with haemophilia (PWH) is a growing issue, given the continuing improvement in life expectancy among PWH. The evolving treatment paradigms targeting higher trough levels and the advent of non-factor replacement therapies (NFRT) means much of the 'protection' PWH were thought to have against CVD may be lost. There is a paucity of evidence regarding the safety of using anticoagulants in PWH. We designed a study assessing the thrombin generation (TG) of PWH of different severities and treatments, compared to non-haemophilia patients receiving a Factor Xa (FXa) inhibitor (apixaban or rivaroxaban), healthy controls, and assessing TG parameters of adding FXa inhibitor to the plasma of PWH receiving emicizumab prophylaxis. In total, 40 patients were included. TG was initiated with 5pM tissue factor (TF) using the calibrated automated thrombinoscope. Compared to those with mild haemophilia, patients receiving a FXa inhibitor had higher endogenous thrombin potential (ETP) (1278.42 vs 1831.36) and velocity index (40.71 vs 112.56), but both had a similar peak height (154.0 vs 262.63) and time to peak (both 5.83). People with severe haemophilia receiving emicizumab had significantly improved TG parameters compared to those not receiving emicizumab - ETP 1678.11 vs 809.96 and peak height 233.8 vs 92.05; however, when FXa inhibitor was added their TG parameters deteriorated to the severe haemophilia range (ETP 1179.60 and peak height 103.05). TG may provide additional useful information regarding the use of anticoagulants in PWH.

Differential effects of physiological agonists on the proteome of platelet-derived extracellular vesicles.

Arterial thrombosis manifesting as heart attack and stroke is the leading cause of death worldwide. Platelets are central mediators of thrombosis that can be activated through multiple activation pathways. Platelet-derived extracellular vesicles (pEVs), also known as platelet-derived microparticles, are granular mixtures of membrane structures produced by platelets in response to various activating stimuli. Initial studies have attracted interest on how platelet agonists influence the composition of the pEV proteome. In the current study, we used physiological platelet agonists of varying potencies which reflect the microenvironments that platelets experience during thrombus formation: adenosine diphosphate, collagen, thrombin as well as a combination of thrombin/collagen to induce platelet activation and pEV generation. Proteomic profiling revealed that pEVs have an agonist-dependent altered proteome in comparison to their cells of origin, activated platelets. Furthermore, we found that various protein classes including those related to coagulation and complement (prothrombin, antithrombin, and plasminogen) and platelet activation (fibrinogen) are attributed to platelet EVs following agonist stimulation. This agonist-dependent altered proteome suggests that protein packaging is an active process that appears to occur without de novo protein synthesis. This study provides new information on the influence of physiological agonist stimuli on the biogenesis and proteome landscape of pEVs.

Between a rock and a hard place: resumption of oral anticoagulant therapy after intracranial hemorrhage.

Intracranial hemorrhage (ICH) is the most feared and lethal complication of oral anticoagulant (OAC) therapy. Resumption of OAC after ICH has long posed a challenge for clinicians, complicated by the expanding range of anticoagulant agents available in modern clinical practice, including direct OACs and, more recently, factor XI and XII inhibitors. A review of the current literature found support for resuming OAC in the majority of patients after ICH based on pooled retrospective data showing that resumption is associated with a lower risk of mortality and thromboembolism without a significantly increased risk of recurrent hemorrhage. The optimal time to resume OAC is less clear; however, the available evidence suggests that the composite risk of both recurrent hemorrhage and thromboembolism is likely minimized, somewhere between 4 and 6 weeks, after ICH in most patients. Specific considerations to guide the optimal resumption time in the individual patient include ICH location, mechanism, and anticoagulant class. Patients with mechanical heart valves and intracerebral malignancy represent high-risk groups who require more nuanced decision making. Here, we appraise the literature with the aim of providing a practical guide for clinicians while also discussing priorities for future investigation.

Plasma from patients with vaccine-induced immune thrombotic thrombocytopenia displays increased fibrinolytic potential and enhances tissue-type plasminogen activator but not urokinase-mediated plasminogen activation.

BACKGROUND: Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare complication of adenovirus vector-based COVID-19 vaccines. VITT is associated with markedly raised levels of D-dimer; yet, how VITT modulates the fibrinolytic system is unknown. OBJECTIVES: We aimed to compare changes in fibrinolytic activity in plasma from patients with VITT, patients diagnosed with venous thromboembolism (VTE) after vaccination but without VITT (VTE-no VITT), and healthy vaccinated controls. METHODS: Plasma levels of plasmin-antiplasmin (PAP) complexes, plasminogen, and alpha-2-antiplasmin (α2AP) from 10 patients with VITT, 10 patients with VTE-no VITT, and 14 healthy vaccinated controls were evaluated by enzyme-linked immunosorbent assay and/or Western blotting. Fibrinolytic capacity was evaluated by quantitating PAP levels at baseline and after ex vivo plasma stimulation with 50-nM tissue-type plasminogen activator (tPA) or urokinase for 5 minutes. RESULTS: Baseline PAP complex levels in control and VTE-no VITT individuals were similar but were ∼7-fold higher in plasma from patients with VITT (P < .0001). VITT samples also revealed consumption of α2AP and fibrinogenolysis consistent with a hyperfibrinolytic state. Of interest, VITT plasma produced significantly higher PAP levels after ex vivo treatment with tPA, but not urokinase, compared to the other groups, indicative of increased fibrinolytic potential. This was not due to D-dimer as addition of D-dimer to VTE-no VITT plasma failed to potentiate tPA-induced PAP levels. CONCLUSION: A marked hyperfibrinolytic state occurs in patients with VITT, evidenced by marked elevations in PAP, α2AP consumption, and fibrinogenolysis. An unidentified plasma cofactor that selectively potentiates tPA-mediated plasminogen activation also appears to exist in the plasma of patients with VITT.

Decoding the role of platelets in tumour metastasis: enigmatic accomplices and intricate targets for anticancer treatments.

The canonical role of platelets as central players in cardiovascular disease by way of their fundamental role in mediating thrombosis and haemostasis is well appreciated. However, there is now a large body of experimental evidence demonstrating that platelets are also pivotal in various physiological and pathophysiological processes other than maintaining haemostasis. Foremost amongst these is the emerging data highlighting the key role of platelets in driving cancer growth, metastasis and modulating the tumour microenvironment. As such, there is significant interest in targeting platelets therapeutically for the treatment of cancer. Therefore, the purpose of this review is to provide an overview of how platelets contribute to the cancer landscape and why platelets present as valuable targets for the development of novel cancer diagnosis tools and therapeutics.

Venous Thromboembolism and Estrogen-Containing Gender-Affirming Hormone Therapy.

Gender-affirming therapy involves the use of hormones to develop the physical characteristics of the identified gender and suppressing endogenous sex hormone production. Venous thromboembolism (VTE) is a known risk of exogenous estrogen therapy, and while evidence of VTE risk among transgender women using modern gender-affirming hormone therapy (GAHT) is still emerging, it is thought to affect up to 5% of transgender women. Historically, GAHT was associated with a high risk of VTE; however, modern preparations are less thrombogenic mainly due to significantly lower doses used as well as different preparations. This review presents the available literature regarding the following four topics: (1) risk of VTE among transgender women receiving estradiol GAHT, (2) how the route of administration of estradiol affects the VTE risk, (3) perioperative management of GAHT, (4) VTE risk among adolescents on GAHT. There is a need for large, longitudinal studies of transgender women using GAHT to further characterize VTE risk and how this is affected by factors such as patient age, duration of GAHT use, tobacco use, body mass index, and comorbidities. Future studies in these areas could inform the development of clinical guidelines to improve the care of transgender people.

Congenital fibrinogen disorders: Strengthening genotype-phenotype correlations through novel genetic diagnostic tools.

Congenital fibrinogen disorders or CFDs are heterogenous, both in clinical manifestation and array of culprit molecular lesions. Correlations between phenotype and genotype remain poorly defined. This review examines the genetic landscape discovered to date for this rare condition. The question of a possible oligogenic model of inheritance influencing phenotypic heterogeneity is raised, with discussion of the benefits and challenges of sequencing technology used to enhance discovery in this space. Considerable work lies ahead in order to achieve diagnostic and prognostic precision and subsequently provide targeted management to this complex cohort of patients.

Mortality data from omission of early thromboprophylaxis in critically ill patients highlights the importance of an individualised diagnosis-related approach.

BACKGROUND: Venous thromboembolism (VTE) prophylaxis is effective in reducing VTE events, however, its impact on mortality is unclear. We examined the association between omission of VTE prophylaxis within the first 24 h after intensive care unit (ICU) admission and hospital mortality. METHODS: Retrospective analysis of prospectively collected data from the Australian New Zealand Intensive Care Society Adult Patient Database. Data were obtained for adult admissions between 2009 and 2020. Mixed effects logistic regression models were used to evaluate the association between omission of early VTE prophylaxis and hospital mortality. RESULTS: Of the 1,465,020 ICU admissions, 107,486 (7.3%) did not receive any form of VTE prophylaxis within the first 24 h after ICU admission without documented contraindication. Omission of early VTE prophylaxis was independently associated with 35% increased odds of in-hospital mortality (odds ratios (OR): 1.35; 95% CI: 1.31-1.41). The associations between omission of early VTE prophylaxis and mortality varied by admission diagnosis. In patients diagnosed with stroke (OR: 1.26, 95% CI: 1.05-1.52), cardiac arrest (OR: 1.85, 95% CI: 1.65-2.07) or intracerebral haemorrhage (OR: 1.48, 95% CI: 1.19-1.84), omission of VTE prophylaxis was associated with increased risk of mortality, but not in patients diagnosed with subarachnoid haemorrhage or head injury. CONCLUSIONS: Omission of VTE prophylaxis within the first 24 h after ICU admission was independently associated with increased risk of mortality that varied by admission diagnosis. Consideration of early thromboprophylaxis may be required for patients with stroke, cardiac arrest and intracerebral haemorrhage but not in those with subarachnoid haemorrhage or head injury. The findings highlight the importance of individualised diagnosis-related thromboprophylaxis benefit-harm assessments.

Activated Coagulation FXII: A Unique Target for In Vivo Molecular Imaging.

BACKGROUND: Current clinical imaging of thromboembolic diseases often relies on indirect detection of thrombi, which may delay diagnosis and ultimately the institution of beneficial, potentially lifesaving treatment. Therefore, the development of targeting tools that facilitate the rapid, specific, and direct imaging of thrombi using molecular imaging is highly sought after. One potential molecular target is FXIIa (factor XIIa), which initiates the intrinsic coagulation pathway but also activates the kallikrein-kinin system, thereby initiating coagulation and inflammatory/immune responses. As FXII (factor XII) is dispensable for normal hemostasis, its activated form (FXIIa) represents an ideal molecular target for diagnostic and therapeutic approaches, the latter combining diagnosis/identification of thrombi and effective antithrombotic therapy. METHODS: We conjugated an FXIIa-specific antibody, 3F7, to a near-infrared (NIR) fluorophore and demonstrated binding to FeCl3-induced carotid thrombosis with 3-dimensional fluorescence emission computed tomography/computed tomography and 2-dimensional fluorescence imaging. We further demonstrated ex vivo imaging of thromboplastin-induced pulmonary embolism and detection of FXIIa in human thrombi produced in vitro. RESULTS: We demonstrated imaging of carotid thrombosis by fluorescence emission computed tomography/computed tomography and measured a significant fold increase in signal between healthy and control vessels from mice injected with 3F7-NIR compared with mice injected with nontargeted probe (P=0.002) ex vivo. In a model of pulmonary embolism, we measured increased NIR signal in lungs from mice injected with 3F7-NIR compared with mice injected with nontargeted probe (P=0.0008) and healthy lungs from mice injected with 3F7-NIR (P=0.021). CONCLUSIONS: Overall, we demonstrate that FXIIa targeting is highly suitable for the specific detection of venous and arterial thrombi. This approach will allow direct, specific, and early imaging of thrombosis in preclinical imaging modalities and may facilitate monitoring of antithrombotic treatment in vivo.

A systems immunology study comparing innate and adaptive immune responses in adults to COVID-19 mRNA and adenovirus vectored vaccines.

Identifying the molecular mechanisms that promote optimal immune responses to coronavirus disease 2019 (COVID-19) vaccination is critical for future rational vaccine design. Here, we longitudinally profile innate and adaptive immune responses in 102 adults after the first, second, and third doses of mRNA or adenovirus-vectored COVID-19 vaccines. Using a multi-omics approach, we identify key differences in the immune responses induced by ChAdOx1-S and BNT162b2 that correlate with antigen-specific antibody and T cell responses or vaccine reactogenicity. Unexpectedly, we observe that vaccination with ChAdOx1-S, but not BNT162b2, induces an adenoviral vector-specific memory response after the first dose, which correlates with the expression of proteins with roles in thrombosis with potential implications for thrombosis with thrombocytopenia syndrome (TTS), a rare but serious adverse event linked to adenovirus-vectored vaccines. The COVID-19 Vaccine Immune Responses Study thus represents a major resource that can be used to understand the immunogenicity and reactogenicity of these COVID-19 vaccines.

Improving the rate of inferior vena cava filter retrieval through multidisciplinary engagement.

Background: The placement of retrievable inferior vena cava (IVC) filters occurs commonly, but retrieval rates remain low. Consequently, there is an unmet clinical need to ensure appropriate follow-up and retrieval of these devices. Objectives: To determine the association between an IVC filter surveillance team with filter retrievals or a documented filter plan, time to retrieval, and incidence of filter complications or recurrent venous thromboembolism. Methods: Ambidirectional cohort study evaluating consecutive IVC filter insertions before and after the implementation of a multidisciplinary surveillance team (MDST). We report an odds ratio (OR) with 95% CIs, adjusted by age, sex, weight, and malignancy status. Results: Overall, 453 patients were included, with 272 individuals in the pre-MDST cohort and 181 individuals in the post-MDST cohort. The MDST was associated with a higher composite primary outcome of IVC filter retrieval or a documented filter plan from 79.4% in the pre-MDST cohort to 96.1% in the post-MDST cohort (OR, 6.44; 95% CI, 3.06-15.84). Compared with the pre-MDST cohort, IVC filter retrieval rates were higher in the post-MDST cohort (52.6%-73.5%, respectively; (OR, 2.50; 95% CI, 1.67-3.78). The MDST was associated with a shorter median time-to-filter retrieval (187-150 days, hazard ratio, 1.78; 95% CI, 1.39-2.29), but there was no significant difference when comparing symptomatic or clinically significant IVC filter complications, recurrent venous thromboembolism, or mortality. Conclusion: Our study demonstrates the importance of a structured program to ensure timely IVC filter retrieval and ultimately improve patient care.

A novel phosphocholine-mimetic inhibits a pro-inflammatory conformational change in C-reactive protein.

C-reactive protein (CRP) is an early-stage acute phase protein and highly upregulated in response to inflammatory reactions. We recently identified a novel mechanism that leads to a conformational change from the native, functionally relatively inert, pentameric CRP (pCRP) structure to a pentameric CRP intermediate (pCRP*) and ultimately to the monomeric CRP (mCRP) form, both exhibiting highly pro-inflammatory effects. This transition in the inflammatory profile of CRP is mediated by binding of pCRP to activated/damaged cell membranes via exposed phosphocholine lipid head groups. We designed a tool compound as a low molecular weight CRP inhibitor using the structure of phosphocholine as a template. X-ray crystallography revealed specific binding to the phosphocholine binding pockets of pCRP. We provide in vitro and in vivo proof-of-concept data demonstrating that the low molecular weight tool compound inhibits CRP-driven exacerbation of local inflammatory responses, while potentially preserving pathogen-defense functions of CRP. The inhibition of the conformational change generating pro-inflammatory CRP isoforms via phosphocholine-mimicking compounds represents a promising, potentially broadly applicable anti-inflammatory therapy.

A Randomized Trial of Nafamostat for Covid-19.

A Randomized Trial of Nafamostat for Covid-19Nafamostat mesylate is a potent in vitro antiviral that inhibits the host transmembrane protease serine 2 enzyme used by SARS-CoV-2 for cell entry. Morpeth et al report the results of an open-label randomized clinical trial of nafamostat for noncritically ill patients with Covid-19.

C-reactive protein, immunothrombosis and venous thromboembolism.

C-reactive protein (CRP) is a member of the highly conserved pentraxin superfamily of proteins and is often used in clinical practice as a marker of infection and inflammation. There is now increasing evidence that CRP is not only a marker of inflammation, but also that destabilized isoforms of CRP possess pro-inflammatory and pro-thrombotic properties. CRP circulates as a functionally inert pentameric form (pCRP), which relaxes its conformation to pCRP* after binding to phosphocholine-enriched membranes and then dissociates to monomeric CRP (mCRP). with the latter two being destabilized isoforms possessing highly pro-inflammatory features. pCRP* and mCRP have significant biological effects in regulating many of the aspects central to pathogenesis of atherothrombosis and venous thromboembolism (VTE), by directly activating platelets and triggering the classical complement pathway. Importantly, it is now well appreciated that VTE is a consequence of thromboinflammation. Accordingly, acute VTE is known to be associated with classical inflammatory responses and elevations of CRP, and indeed VTE risk is elevated in conditions associated with inflammation, such as inflammatory bowel disease, COVID-19 and sepsis. Although the clinical data regarding the utility of CRP as a biomarker in predicting VTE remains modest, and in some cases conflicting, the clinical utility of CRP appears to be improved in subsets of the population such as in predicting VTE recurrence, in cancer-associated thrombosis and in those with COVID-19. Therefore, given the known biological function of CRP in amplifying inflammation and tissue damage, this raises the prospect that CRP may play a role in promoting VTE formation in the context of concurrent inflammation. However, further investigation is required to unravel whether CRP plays a direct role in the pathogenesis of VTE, the utility of which will be in developing novel prophylactic or therapeutic strategies to target thromboinflammation.

Improving compliance with venous thromboembolism prophylaxis guidelines in obese inpatients.

Obesity is a well-recognised risk factor for venous thromboembolism, and increased dosing of pharmacological prophylaxis is recommended in obese inpatients. We performed a clinical decision support intervention by adjusting order sentences of prophylactic enoxaparin within our electronic medical records, which significantly improved appropriate dosing for thromboprophylaxis in obese inpatients at our institution.