The foundation for investigating factor XI as a target for inhibition in human cardiovascular disease.

Anticoagulant therapy is a mainstay in the management of patients with cardiovascular disease and related conditions characterized by a heightened risk for thrombosis. Acute coronary syndrome, chronic coronary syndrome, ischemic stroke, and atrial fibrillation are the most common. In addition to their proclivity for thrombosis, each of these four conditions is also characterized by local and systemic inflammation, endothelial/endocardial injury and dysfunction, oxidative stress, impaired tissue-level reparative capabilities, and immune dysregulation that plays a critical role in linking molecular events, environmental triggers, and phenotypic expressions. Knowing that cardiovascular disease and thrombosis are complex and dynamic, can the scientific community identify a common pathway or specific point of interface susceptible to pharmacological inhibition or alteration that is likely to be safe and effective? The contact factors of coagulation may represent the proverbial "sweet spot" and are worthy of investigation. The following review provides a summary of the fundamental biochemistry of factor XI, its biological activity in thrombosis, inflammation, and angiogenesis, new targeting drugs, and a pragmatic approach to managing hemostatic requirements in clinical trials and possibly day-to-day patient care in the future.

Factor XI: structure, function and therapeutic inhibition.

Arterial and venous thromboembolism is a major medical concern that requires therapeutic anticoagulation in various medical fields to prevent its drastic consequences. Despite significant advances in anticoagulant therapy, thrombosis remains a leading cause of morbidity and mortality worldwide. Traditional anticoagulants like heparin and vitamin K antagonists (VKAs) have shown efficacy in preventing and treating thrombosis but come with an inherent risk of bleeding due to their non-specific inhibition of multiple coagulation factors. Subsequent direct oral anticoagulants (DOACs), targeting specific factors such as Xa or thrombin, demonstrated improved safety profiles compared to VKAs, yet bleeding remains a concern. Accordingly, research is focused on developing anticoagulants with improved safety profiles. A safer class of anticoagulants would have broad appeal. The intrinsic pathway of coagulation, involving factor XI (FXI), has attracted attention as a potential target for safer anticoagulants. Preclinical studies and epidemiological data indicate that FXI deficiency or inhibition protects against thrombosis with minimal bleeding. Current research involves evaluating various FXI-directed strategies, and phase 2 studies have shown promising results in orthopedic surgery, atrial fibrillation, end-stage renal disease (ESRD), myocardial infarction, and ischemic stroke. Several agents, such as antisense oligonucleotides, monoclonal antibodies, small synthetic molecules, natural peptides, and aptamers, have been developed to inhibit FXI at different stages, offering potentially safer alternatives to traditional anticoagulants. However, the optimal balance between preventing thrombosis and the risk of bleeding associated with FXI inhibitors requires validation through extensive phase 3 clinical trials using definite clinical endpoints. Several of such trials are currently underway or planned to define the role of FXI inhibitors in clinical practice and determine the most suitable FXI inhibitor for each specific indication. The current review highlights the rationale behind developing FXI inhibitors, presenting the most advanced agents in development, summarizing completed clinical trials, and discussing ongoing research efforts.

Temporal biomarker concentration patterns during the early course of acute coronary syndrome.

OBJECTIVES: Biomarker concentrations and their changes during acute coronary syndrome (ACS) provide clinically useful information on pathophysiological processes, e.g. myocardial necrosis, hemodynamic stress and inflammation. However, current evidence on temporal biomarker patterns early during ACS is limited, and studies investigating multiple biomarkers are lacking. METHODS: We measured concentrations of high-sensitivity cardiac troponin T (hs-cTnT) and I (hs-cTnI), NT-terminal pro-B-type natriuretic peptide, C-reactive protein, and growth-differentiation factor-15 (GDF-15) in plasma samples obtained at randomization in ACS patients from the PLATelet inhibition and patient Outcomes (PLATO) trial. Linear regressions with interaction analyses were used to investigate the associations of biomarker concentrations with the time from symptom onset and to model temporal biomarker concentration patterns. RESULTS: The study population consisted of 16,944 patients (median age 62 years; 71.3 % males) with 6,853 (40.3 %) having ST-elevation myocardial infarction (STEMI) and 10,141 (59.7 %) having non-ST-elevation ACS (NSTE-ACS). Concentrations of all biomarkers were associated with time from symptom onset (pinteraction<0.001), apart for GDF-15 (pinteraction=0.092). Concentration increases were more pronounced in STEMI compared to NSTE-ACS. Temporal biomarker patterns for hs-cTnT and hs-cTnI were different depending on sex whereas biomarker patterns for the other biomarkers were similar in cohorts defined by age and sex. CONCLUSIONS: Temporal concentration patterns differ for various biomarkers early during ACS, reflecting the variability in the activation and duration of different pathophysiological processes, and the amount of injured myocardium. Our data emphasize that the time elapsed from symptom onset should be considered for the interpretation of biomarker results in ACS.

Lipoprotein(a), platelet function and cardiovascular disease.

Lipoprotein(a) (Lp(a)) is associated with atherothrombosis through several mechanisms, including putative antifibrinolytic properties. However, genetic association studies have not demonstrated an association between high plasma levels of Lp(a) and the risk of venous thromboembolism, and studies in patients with highly elevated Lp(a) levels have shown that Lp(a) lowering does not modify the clotting properties of plasma ex vivo. Lp(a) can interact with several platelet receptors, providing biological plausibility for a pro-aggregatory effect. Observational clinical studies suggest that elevated plasma Lp(a) concentrations are associated with worse long-term outcomes in patients undergoing revascularization. Furthermore, in these patients, those with elevated plasma Lp(a) levels derive more benefit from prolonged dual antiplatelet therapy than those with normal Lp(a) levels. The ASPREE trial in healthy older individuals treated with aspirin showed a reduction in ischaemic events in those who had a single-nucleotide polymorphism in LPA that is associated with elevated Lp(a) levels in plasma, without an increase in bleeding events. In this Review, we re-examine the role of Lp(a) in the regulation of platelet function and suggest areas of research to define further the clinical relevance to cardiovascular disease of the observed associations between Lp(a) and platelet function.

Can a best practice advisory improve anticoagulation prescribing to reduce stroke risk in patients with atrial fibrillation?

BACKGROUND: Atrial fibrillation (AF) is the most common cardiac rhythm disorder and a risk factor for stroke. Randomized trials have demonstrated that anticoagulation can reduce strokes in AF patients. Yet, widespread underutilization of this therapy continues. To address this practice gap, we designed a study to implement and evaluate the effectiveness of a best practice advisory (BPA) for an Atrial Fibrillation Decision Support Tool (AFDST) embedded within our electronic health record. METHODS: Our intervention is provider-facing, focused on decision support. Clinical setting is ambulatory patients being seen by primary care physicians. We prospectively enrolled 608 patients in our health system who are currently receiving less than optimal anticoagulation therapy as determined by the AFDST and randomized them to one of two arms - 1) usual care, in which the AFDST is available for use; or 2) addition of a BPA to the AFDST notifying clinicians that their patient stands to gain significant benefit from a change in current therapy. Primary outcome was effectiveness of the BPA measured by change to "appropriate thromboprophylaxis" based on the AFDST recommendation at 3 months post-enrollment. Secondary endpoints included Reach and Adoption from the RE-AIM (Reach, Effectiveness, Adoption, Implementation, & Maintenance) framework for implementation studies. RESULTS: Among 562 patients with a minimum follow-up of 3 months, addition of a BPA to the AFDST resulted in significant improvement in anticoagulation therapy, 5 % (12/248) versus 11 % (33/314) p = 0.02, odds ratio 2.31 (95 % CI, 1.17-4.87). CONCLUSIONS: A BPA added to an AF decision support tool improved anticoagulation therapy among AF patients in a primary care academic health system setting.

Cardiopulmonary Phenotypes of Post Acute Sequelae of Severe Acute Respiratory Syndrome Coronavirus 2: A Narrative Review.

The acute effects of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are well known; however, the long-term cardiopulmonary effects are less well characterized. The phenotypic expression of acute infection is heterogeneous, ranging from a complete absence of symptoms to shock, multisystem organ failure, and death. Patients with severe or critical coronavirus disease (COVID-19) who survive their initial illness can require a prolonged period of recovery lasting weeks to months. This specific patient group is part of a larger and even more heterogeneous group of patients who initially experience mild-to-moderate symptoms that fail to resolve over time. Collectively, patients recovering from severe or critical COVID-19 and those who continue to experience symptoms following a lower acuity infection are considered to have Post Acute Sequalae of SARS-CoV-2 infection (PASC). Using prognostic factors like myocardial infarction, myocarditis, pulmonary embolism, acute respiratory distress syndrome, need for mechanical ventilation or extracorporeal membrane oxygenation, and advanced pharmaceutical therapies that primarily occur or are instituted in the acute phase of illness one can begin to develop a taxonomy or corpus of PASC in its varied forms.

Evaluating chest pain in patients with post COVID conditions permission to think outside of the box.

Chest pain is among the most common symptoms of post-COVID-19 Conditions (PCC) that prompts medical attention. Because the SARS-CoV-2 virus has proclivity for many organs and organ systems in the chest, ranging from the heart, lungs, great vessels, lymphatics, and peripheral nerves, clinicians evaluating patients with chest pain must consider a broad differential diagnosis and take a comprehensive approach to management.

Vascular endothelial growth factor-D plasma levels and VEGFD genetic variants are independently associated with outcomes in patients with cardiovascular disease.

AIMS: The vascular endothelial growth factor (VEGF) family is involved in pathophysiological mechanisms underlying cardiovascular (CV) diseases. The aim of this study was to investigate the associations between circulating VEGF ligands and/or soluble receptors and CV outcome in patients with acute coronary syndrome (ACS) and chronic coronary syndrome (CCS). METHODS AND RESULTS: Levels of VEGF biomarkers, including bFGF, Flt-1, KDR (VEGFR2), PlGF, Tie-2, VEGF-A, VEGF-C, and VEGF-D, were measured in the PLATO ACS cohort (n = 2091, discovery cohort). Subsequently, VEGF-D was also measured in the STABILITY CCS cohort (n = 4015, confirmation cohort) to verify associations with CV outcomes. Associations between plasma VEGF-D and outcomes were analysed by multiple Cox regression models with hazard ratios (HR [95% CI]) comparing the upper vs. the lower quartile of VEGF-D. Genome-wide association study (GWAS) of VEGF-D in PLATO identified SNPs that were used as genetic instruments in Mendelian randomization (MR) meta-analyses vs. clinical endpoints. GWAS and MR were performed in patients with ACS from PLATO (n = 10 013) and FRISC-II (n = 2952), and with CCS from the STABILITY trial (n = 10 786). VEGF-D, KDR, Flt-1, and PlGF showed significant association with CV outcomes. VEGF-D was most strongly associated with CV death (P = 3.73e-05, HR 1.892 [1.419, 2.522]). Genome-wide significant associations with VEGF-D levels were identified at the VEGFD locus on chromosome Xp22. MR analyses of the combined top ranked SNPs (GWAS P-values; rs192812042, P = 5.82e-20; rs234500, P = 1.97e-14) demonstrated a significant effect on CV mortality [P = 0.0257, HR 1.81 (1.07, 3.04) per increase of one unit in log VEGF-D]. CONCLUSION: This is the first large-scale cohort study to demonstrate that both VEGF-D plasma levels and VEGFD genetic variants are independently associated with CV outcomes in patients with ACS and CCS. Measurements of VEGF-D levels and/or VEGFD genetic variants may provide incremental prognostic information in patients with ACS and CCS.

Genetic ablation of diabetes-associated gene Ccdc92 reduces obesity and insulin resistance in mice.

Multiple genome-wide association studies (GWAS) have identified specific genetic variants in the coiled-coil domain containing 92 (CCDC92) locus that is associated with obesity and type 2 diabetes in humans. However, the biological function of CCDC92 in obesity and insulin resistance remains to be explored. Utilizing wild-type (WT) and Ccdc92 whole-body knockout (KO) mice, we found that Ccdc92 KO reduced obesity and increased insulin sensitivity under high-fat diet (HFD) conditions. Ccdc92 KO inhibited macrophage infiltration and fibrosis in white adipose tissue (WAT), suggesting Ccdc92 ablation protects against adipose tissue dysfunction. Ccdc92 deletion also increased energy expenditure and further attenuated hepatic steatosis in mice on an HFD. Ccdc92 KO significantly inhibited the inflammatory response and suppressed the NLR Family Pyrin Domain Containing 3 (NLRP3) inflammasome in WAT. Altogether, we demonstrated the critical role of CCDC92 in metabolism, constituting a potential target for treating obesity and insulin resistance.

Biomarker Concentrations and Their Temporal Changes in Patients With Myocardial Infarction and Nonobstructive Compared With Obstructive Coronary Arteries: Results From the PLATO Trial.

Background The pathobiology of myocardial infarction (MI) with nonobstructive coronary arteries (MINOCA) is often uncertain. Investigating biomarker concentrations and their changes may offer novel pathophysiological insights. Methods and Results In this post hoc study of the PLATO (Platelet Inhibition and Patient Outcomes) trial, concentrations of hs-cTnT (high-sensitivity cardiac troponin T), NT-proBNP (N-terminal pro-B-type natriuretic peptide), hs-CRP (high-sensitivity C-reactive protein), and GDF-15 (growth differentiation factor 15) were measured in patients with MINOCA at baseline (n=554) and at 1-month follow-up (n=107). For comparisons, biomarkers were also measured in patients with MI with obstructive (stenosis ≥50%) coronary artery disease (baseline: n=11 106; follow-up: n=2755]). Adjusted linear regression models were used to compare concentrations and their short- and long-term changes. The adjusted geometric mean ratios (GMRs) in patients with MINOCA (median age, 61 years; 50.4% women) indicated lower hs-cTnT (GMR, 0.77 [95% CI, 0.68-0.88]) but higher hs-CRP (GMR, 1.21 [95% CI, 1.08-1.37]) and GDF-15 concentrations (GMR, 1.06 [95% CI, 1.02-1.11]) at baseline compared with patients with MI with obstructive coronary artery disease, whereas NT-proBNP concentrations were similar. Temporal decreases in hs-cTnT, NT-proBNP, and hs-CRP concentrations until 1-month follow-up were more pronounced in patients with MINOCA. At follow-up, patients with MINOCA had lower concentrations of hs-cTnT (GMR, 0.71 [95% CI, 0.60-0.84]), NT-proBNP (GMR, 0.45 [95% CI, 0.36-0.56]), and hs-CRP (GMR, 0.68 [95% CI, 0.53-0.86]). One-month GDF-15 concentrations were similar between both groups with MI. Conclusions Biomarker concentrations suggest greater initial inflammatory activity, similar degree of myocardial dysfunction, and less pronounced myocardial injury during the acute phase of MINOCA compared with MI with obstructive coronary artery disease but also faster myocardial recovery. Registration URL: http://www.clinicaltrials.gov; Unique identifier: NCT00391872.

Apixaban or Warfarin in Patients with an On-X Mechanical Aortic Valve.

Apixaban, Warfarin, and On-X Mechanical Aortic ValvesAlthough vitamin K antagonists are the only oral anticoagulants approved with mechanical heart valves, this trial examined whether apixaban could be safely used in patients with an On-X mechanical aortic valve. A total of 863 such patients were assigned apixaban 5 mg twice daily or warfarin (target international normalized ratio 2.0 to 3.0). A total of 20 thrombotic events occurred in the apixaban group (4.2%/patient-year) and 6 events in the warfarin group (1.3%/patient-year). Major bleeding rates were 3.6%/patient-year with apixaban and 4.5%/patient-year with warfarin.

Biomarker-Based Prediction of Recurrent Ischemic Events in Patients With Acute Coronary Syndromes.

BACKGROUND: In patients with acute coronary syndrome (ACS), there is residual and variable risk of recurrent ischemic events. OBJECTIVES: This study aimed to develop biomarker-based prediction models for 1-year risk of cardiovascular (CV) death and myocardial infarction (MI) in patients with ACS undergoing percutaneous coronary intervention. METHODS: We included 10,713 patients from the PLATO (A Comparison of Ticagrelor [AZD6140] and Clopidogrel in Patients With Acute Coronary Syndrome) trial in the development cohort and externally validated in 3,508 patients from the TRACER (Thrombin Receptor Antagonist for Clinical Event Reduction in Acute Coronary Syndrome) trial. Variables contributing to risk of CV death/MI were assessed using Cox regression models, and a score was derived using subsets of variables approximating the full model. RESULTS: There were 632 and 190 episodes of CV death/MI in the development and validation cohorts. The most important predictors of CV death/MI were the biomarkers, growth differentiation factor 15, and N-terminal pro-B-type natriuretic peptide, which had greater prognostic value than all candidate variables. The final model included 8 items: age (A), biomarkers (B) (growth differentiation factor 15 and N-terminal pro-B-type natriuretic peptide), and clinical variables (C) (extent of coronary artery disease, previous vascular disease, Killip class, ACS type, P2Y12 inhibitor). The model, named ABC-ACS ischemia, was well calibrated and showed good discriminatory ability for 1-year risk of CV death/MI with C-indices of 0.71 and 0.72 in the development and validation cohorts, respectively. For CV death, the score performed better, with C-indices of 0.80 and 0.84 in the development and validation cohorts, respectively. CONCLUSIONS: An 8-item score for the prediction of CV death/MI was developed and validated for patients with ACS undergoing percutaneous coronary intervention. The ABC-ACS ischemia score showed good calibration and discrimination and might be useful for risk prediction and decision support in patients with ACS. (A Comparison of Ticagrelor [AZD6140] and Clopidogrel in Patients With Acute Coronary Syndrome [PLATO]; NCT00391872; Trial to Assess the Effects of Vorapaxar [SCH 530348; MK-5348] in Preventing Heart Attack and Stroke in Participants With Acute Coronary Syndrome [TRACER]; NCT00527943).

Association of Blood Pressure-Related Increase in Vascular Stiffness on Other Measures of Target Organ Damage in Youth.

BACKGROUND: Hypertension-related increased arterial stiffness predicts development of target organ damage (TOD) and cardiovascular disease. We hypothesized that blood pressure (BP)-related increased arterial stiffness is present in youth with elevated BP and is associated with TOD. METHODS: Participants were stratified by systolic BP into low- (systolic BP <75th percentile, n=155), mid- (systolic BP ≥80th and <90th percentile, n=88), and high-risk BP categories (≥90th percentile, n=139), based on age-, sex- and height-specific pediatric BP cut points. Clinic BP, 24-hour ambulatory BP monitoring, anthropometrics, and laboratory data were obtained. Arterial stiffness measures included carotid-femoral pulse wave velocity and aortic stiffness. Left ventricular mass index, left ventricular systolic and diastolic function, and urine albumin/creatinine were collected. ANOVA with Bonferroni correction was used to evaluate differences in cardiovascular risk factors, pulse wave velocity, and cardiac function across groups. General linear models were used to examine factors associated with arterial stiffness and to determine whether arterial stiffness is associated with TOD after accounting for BP. RESULTS: Pulse wave velocity increased across groups. Aortic distensibility, distensibility coefficient, and compliance were greater in low than in the mid or high group. Significant determinants of arterial stiffness were sex, age, adiposity, BP, and LDL (low-density lipoprotein) cholesterol. Pulse wave velocity and aortic compliance were significantly associated with TOD (systolic and diastolic cardiac function and urine albumin/creatinine ratio) after controlling for BP. CONCLUSIONS: Higher arterial stiffness is associated with elevated BP and TOD in youth emphasizing the need for primary prevention of cardiovascular disease.

Current and novel biomarkers of thrombotic risk in COVID-19: a Consensus Statement from the International COVID-19 Thrombosis Biomarkers Colloquium.

Coronavirus disease 2019 (COVID-19) predisposes patients to thrombotic and thromboembolic events, owing to excessive inflammation, endothelial cell activation and injury, platelet activation and hypercoagulability. Patients with COVID-19 have a prothrombotic or thrombophilic state, with elevations in the levels of several biomarkers of thrombosis, which are associated with disease severity and prognosis. Although some biomarkers of COVID-19-associated coagulopathy, including high levels of fibrinogen and D-dimer, were recognized early during the pandemic, many new biomarkers of thrombotic risk in COVID-19 have emerged. In this Consensus Statement, we delineate the thrombotic signature of COVID-19 and present the latest biomarkers and platforms to assess the risk of thrombosis in these patients, including markers of platelet activation, platelet aggregation, endothelial cell activation or injury, coagulation and fibrinolysis as well as biomarkers of the newly recognized post-vaccine thrombosis with thrombocytopenia syndrome. We then make consensus recommendations for the clinical use of these biomarkers to inform prognosis, assess disease acuity, and predict thrombotic risk and in-hospital mortality. A thorough understanding of these biomarkers might aid risk stratification and prognostication, guide interventions and provide a platform for future research.

Differential effect of clopidogrel and ticagrelor on leukocyte count in relation to patient characteristics, biomarkers and genotype: a PLATO substudy.

Inflammation plays a key role in cardiovascular disease by contributing to atherothrombosis. The PLATelet inhibition and patient Outcomes (PLATO) study (NCT00391872) compared ticagrelor to clopidogrel in patients with acute coronary syndromes and demonstrated fewer cardiovascular events with ticagrelor but lower white blood cell counts (WBC) with clopidogrel. In this further analysis of the PLATO biomarker substudy, we assessed associations between WBC and clinical characteristics, biomarker levels, and CYP2C19 polymorphisms.On-treatment mean (SD) WBC in the clopidogrel group was mildly reduced at each stage of follow-up compared with either the ticagrelor group (1 month: 7.27 (2.1) and 7.67 (2.23) x109/L for clopidogrel and ticagrelor, respectively; p < .001) or following cessation of clopidogrel (7.23 (1.97) x109/L, at 6 months vs 7.56 (2.28) x109/L after treatment cessation; P < .001). This occurred independently of baseline biomarkers and CYP2C19 genotype (where known). Adjusting for clinical characteristics and other biomarkers, no significant interaction was detected between clinical risk factors and the observed effect of clopidogrel on WBC.Clopidogrel weakly suppresses WBC, independent of clinical characteristics, baseline inflammatory biomarker levels, and CYP2C19 genotype. Further work is required to determine the mechanism for this effect and whether it contributes to clopidogrel's efficacy as well as therapeutic interaction with anti-inflammatory drugs.

Efficacy and safety of next-generation tick transcriptome-derived direct thrombin inhibitors.

Despite their limitations, unfractionated heparin (UFH) and bivalirudin remain standard-of-care parenteral anticoagulants for percutaneous coronary intervention (PCI). We discovered novel direct thrombin inhibitors (DTIs) from tick salivary transcriptomes and optimised their pharmacologic activity. The most potent, ultravariegin, inhibits thrombin with a Ki of 4.0 pM, 445-fold better than bivalirudin. Unexpectedly, despite their greater antithrombotic effect, variegin/ultravariegin demonstrated less bleeding, achieving a 3-to-7-fold wider therapeutic index in rodent thrombosis and bleeding models. When used in combination with aspirin and ticagrelor in a porcine model, variegin/ultravariegin reduced stent thrombosis compared with antiplatelet therapy alone but achieved a 5-to-7-fold lower bleeding time than UFH/bivalirudin. Moreover, two antibodies screened from a naïve human antibody library effectively reversed the anticoagulant activity of ultravariegin, demonstrating proof-of-principle for antidote reversal. Variegin and ultravariegin are promising translational candidates for next-generation DTIs that may reduce peri-PCI bleeding in the presence of antiplatelet therapy.

Vascular Smooth Muscle Cells in Aortic Aneurysm: From Genetics to Mechanisms.

Aortic aneurysm, including thoracic aortic aneurysm and abdominal aortic aneurysm, is the second most prevalent aortic disease following atherosclerosis, representing the ninth-leading cause of death globally. Open surgery and endovascular procedures are the major treatments for aortic aneurysm. Typically, thoracic aortic aneurysm has a more robust genetic background than abdominal aortic aneurysm. Abdominal aortic aneurysm shares many features with thoracic aortic aneurysm, including loss of vascular smooth muscle cells (VSMCs), extracellular matrix degradation and inflammation. Although there are limitations to perfectly recapitulating all features of human aortic aneurysm, experimental models provide valuable tools to understand the molecular mechanisms and test novel therapies before human clinical trials. Among the cell types involved in aortic aneurysm development, VSMC dysfunction correlates with loss of aortic wall structural integrity. Here, we discuss the role of VSMCs in aortic aneurysm development. The loss of VSMCs, VSMC phenotypic switching, secretion of inflammatory cytokines, increased matrix metalloproteinase activity, elevated reactive oxygen species, defective autophagy, and increased senescence contribute to aortic aneurysm development. Further studies on aortic aneurysm pathogenesis and elucidation of the underlying signaling pathways are necessary to identify more novel targets for treating this prevalent and clinical impactful disease.