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.

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.

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.

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.

Venous anomalies and thromboembolism.

Patients with venous anomalies are at increased risk of developing venous thromboembolism (VTE) and subsequent complications, but they are often under-recognised. While unprovoked VTE may trigger testing for inherited thrombophilias and malignancy screening, anatomic variants are considered less often. Venous anomalies increase the risk due to venous flow disturbance, resulting in hypertension, reduced flow velocity and turbulence. Recognition is important as endovascular or surgical intervention may be appropriate, these patients have a high rate of VTE recurrence if anticoagulation is ceased, and the anomalies can predispose to extensive VTE and severe post-thrombotic syndrome (PTS). In this case series, we present representative cases and radiological images of May-Thurner syndrome (MTS), inferior vena cava (IVC) variants and venous aneurysms, and review the available literature regarding optimal diagnosis and management in each condition.

IVIg-exposure and thromboembolic event risk: findings from the UK Biobank.

BACKGROUND: Arterial and venous thromboembolic events (TEEs) have been associated with intravenous Ig use, but the risk has been poorly quantified. We aimed to calculate the risk of TEEs associated with exposure to intravenous Ig. METHODS: We included participants from UK Biobank recruited over 3 years, data extracted September 2020.The study endpoints were incidence of myocardial infarction, other acute ischaemic heart disease, stroke, pulmonary embolism and other venous embolism and thrombosis.Predictors included known TEE risk factors: age, sex, hypertension, smoking status, type 2 diabetes mellitus, hypercholesterolaemia, cancer and past history of TEE. Intravenous Ig and six other predictors were added in the sensitivity analysis.Information from participants was collected prospectively, while data from linked resources, including death, cancer, hospital admissions and primary care records were collected retrospectively and prospectively.  FINDINGS: 14 794 of 502 492 individuals had an incident TEE during the study period. The rate of incident events was threefold higher in those with prior history of TEE (8 .7%) than those without previous history of TEE (3.0%).In the prior TEE category, intravenous Ig exposure was independently associated with increased risk of incident TEE (OR=3.69 (95% CI 1.15 to 11.92), p=0.03) on multivariate analysis. The number needed to harm by exposure to intravenous Ig in those with a history of TEE was 5.8 (95% CI 2.3 to 88.3).Intravenous Ig exposure did not increase risk of TEE in those with no previous history of TEE. INTERPRETATION: Intravenous Ig is associated with increased risk of further TEE in individuals with prior history of an event with one further TEE for every six people exposed. In practice, this will influence how clinicians consent for and manage overall TEE risk on intravenous Ig exposure.

The Emerging Threat of (Micro)Thrombosis in COVID-19 and Its Therapeutic Implications.

The recent emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the ensuing global pandemic has presented a health emergency of unprecedented magnitude. Recent clinical data has highlighted that coronavirus disease 2019 (COVID-19) is associated with a significant risk of thrombotic complications ranging from microvascular thrombosis, venous thromboembolic disease, and stroke. Importantly, thrombotic complications are markers of severe COVID-19 and are associated with multiorgan failure and increased mortality. The evidence to date supports the concept that the thrombotic manifestations of severe COVID-19 are due to the ability of SARS-CoV-2 to invade endothelial cells via ACE-2 (angiotensin-converting enzyme 2), which is expressed on the endothelial cell surface. However, in patients with COVID-19 the subsequent endothelial inflammation, complement activation, thrombin generation, platelet, and leukocyte recruitment, and the initiation of innate and adaptive immune responses culminate in immunothrombosis, ultimately causing (micro)thrombotic complications, such as deep vein thrombosis, pulmonary embolism, and stroke. Accordingly, the activation of coagulation (eg, as measured with plasma D-dimer) and thrombocytopenia have emerged as prognostic markers in COVID-19. Given thrombotic complications are central determinants of the high mortality rate in COVID-19, strategies to prevent thrombosis are of critical importance. Several antithrombotic drugs have been proposed as potential therapies to prevent COVID-19-associated thrombosis, including heparin, FXII inhibitors, fibrinolytic drugs, nafamostat, and dipyridamole, many of which also possess pleiotropic anti-inflammatory or antiviral effects. The growing awareness and mechanistic understanding of the prothrombotic state of COVID-19 patients are driving efforts to more stringent diagnostic screening for thrombotic complications and to the early institution of antithrombotic drugs, for both the prevention and therapy of thrombotic complications. The shifting paradigm of diagnostic and treatment strategies holds significant promise to reduce the burden of thrombotic complications and ultimately improve the prognosis for patients with COVID-19.

Understanding vaccine-induced thrombotic thrombocytopenia (VITT).

Vaccine-induced thrombotic thrombocytopenia (VITT) is a rare, but serious, syndrome characterised by thrombocytopenia, thrombosis, a markedly raised D-dimer and the presence of anti-platelet factor-4 (PF4) antibodies following COVID-19 adenovirus vector vaccination. VITT occurs at a rate of approximately 2 per 100 000 first-dose vaccinations and appears exceedingly rare following second doses. Our current understanding of VITT pathogenesis is based on the observations that patients with VITT have antibodies that bind to PF4 and have the ability to form immune complexes that induce potent platelet activation. However, the precise mechanisms that lead to pathogenic VITT antibody development remain a source of active investigation. Thrombosis in VITT can manifest in any vascular bed and affect multiple sites simultaneously. While there is a predilection for splanchnic and cerebral venous sinus thrombosis, VITT also commonly presents with deep vein thrombosis and pulmonary embolism. Pillars of management include anticoagulation with a non-heparin anticoagulant, intravenous immunoglobulin and 'rescue' therapies, such as plasma exchange for severe cases. VITT can be associated with a high mortality rate and significant morbidity, but awareness and optimal therapy have significantly improved outcomes in Australia. A number of questions remain unanswered, including why VITT is so rare, reasons for the predilection for thrombosis in unusual sites, how long pathological antibodies persist, and the optimal duration of anticoagulation. This review will provide an overview of the presentation, diagnostic workup and management strategies for patients with VITT.

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.

Advances in the Management of Acute Venous Thromboembolism and New Therapeutic Agents.

Important advances in the understanding and management of venous thromboembolism (VTE) have enhanced our ability to diagnose, prevent, and treat VTE. In this narrative review, we discuss how recent advances in the understanding and management of VTE are changing practice, highlight ongoing unmet needs in VTE management, and outline how novel therapeutic targets with little or no influence on hemostasis may help address these unmet needs.

C-Reactive Protein and Its Structural Isoforms: An Evolutionary Conserved Marker and Central Player in Inflammatory Diseases and Beyond.

C-reactive protein (CRP) is an evolutionary highly conserved member of the pentraxin superfamily of proteins. CRP is widely used as a marker of inflammation, infection and for risk stratification of cardiovascular events. However, there is now a large body of evidence, that continues to evolve, detailing that CRP directly mediates inflammatory reactions and the innate immune response in the context of localised tissue injury. These data support the concept that the pentameric conformation of CRP dissociates into pro-inflammatory CRP isoforms termed pCRP* and monomeric CRP. These pro-inflammatory CRP isoforms undergo conformational changes that facilitate complement binding and immune cell activation and therefore demonstrate the ability to trigger complement activation, activate platelets, monocytes and endothelial cells. The dissociation of pCRP occurs on the surface of necrotic, apoptotic, and ischaemic cells, regular ß-sheet structures such as ß-amyloid, the membranes of activated cells (e.g., platelets, monocytes, and endothelial cells), and/or the surface of microparticles, the latter by binding to phosphocholine. Therefore, the deposition and localisation of these pro-inflammatory isoforms of CRP have been demonstrated to amplify inflammation and tissue damage in a broad range of clinical conditions including ischaemia/reperfusion injury, Alzheimer's disease, age-related macular degeneration and immune thrombocytopaenia. Given the potentially broad relevance of CRP to disease pathology, the development of inhibitors of CRP remains an area of active investigation, which may pave the way for novel therapeutics for a diverse range of inflammatory diseases.

Platelets as Central Actors in Thrombosis-Reprising an Old Role and Defining a New Character.

Platelets have long been considered simple anucleate cells that rapidly adhere and aggregate at sites of vascular injury. However, recent in vivo experimental data have shed new light on the platelet response to vascular injury. These data have unexpectedly revealed that platelet thrombus formation is a highly dynamic process and yields a platelet thrombus with a distinct hierarchical structure composed of a "core" of highly activated platelets and a "shell" of platelets in a low activation state. This has given rise to the concept that therapeutic targeting of the propagating thrombus shell may hold promise as a means to target thrombosis while sparing hemostasis. While platelets have been historically considered central to arterial thrombosis, they have been traditionally viewed as minor contributors to the formation of venous thrombosis. However, this concept has recently been challenged with the emergence of a large body of evidence highlighting the important proinflammatory function of platelets. The proinflammatory function of platelets is afforded by their ability to induce neutrophil extracellular trap formation, enhance leucocyte recruitment, and secrete granular contents such as high mobility group protein B1 and polyphosphate. These proinflammatory processes trigger coagulation, via the intrinsic pathway, and are central to the formation of venous thrombosis, a condition now appreciated to be a form of sterile inflammation. These data now place platelets at the center stage in orchestrating the thromboinflammatory response underpinning venous thrombosis and have provided new hope that novel platelet-targeted therapeutics may represent a safe and effective approach to prevent venous thrombosis.