Journal of Thrombosis and Haemostasis

Genetic predisposition to venous thrombosis may impact COVID-19 infection and its sequelae. Participants in the ongoing prospective cohort study, Million Veteran Program (MVP), who were tested for COVID-19, with European ancestry, were evaluated for associations with polygenic venous thromboembolic risk, Factor V Leiden mutation (FVL) (rs6025) and prothrombin gene 3 -UTR mutation (F2 G20210A)(rs1799963), and their interactions. Logistic regression models assessed genetic associations with VTE diagnosis, COVID-19 (positive) testing rates and outcome severity (modified WHO criteria), and post-test conditions, adjusting for outpatient anticoagulation medication usage, age, sex, and genetic principal components. 108,437 out of 464,961 European American MVP participants were tested for COVID-19 with 9786 (9%) positive. PRS(VTE), FVL, F2 G20210A were not significantly associated with the propensity of being tested for COVID-19. PRS(VTE) was significantly associated with a positive COVID-19 test in F5 wild type (WT) individuals (OR 1.05; 95% CI [1.02-1.07]), but not in FVL carriers (0.97, [0.91-1.94]). There was no association with severe outcome for FVL, F2 G20210A or PRS(VTE). Outpatient anticoagulation usage in the two years prior to testing was associated with worse clinical outcomes. PRS(VTE) was associated with prevalent VTE diagnosis among both FVL carriers or F5 wild type individuals as well as incident VTE in the two years prior to testing. Increased genetic propensity for VTE in the MVP was associated with increased COVID-19 positive testing rates, suggesting a role of coagulation in the initial steps of COVID-19 infection. Key PointsO_LIIncreased genetic predisposition to venous thrombosis is associated with increased COVID-19 positive testing rates. C_LIO_LIPRS for VTE further risk stratifies factor V Leiden carriers regarding their VTE risk. C_LI

BackgroundVascular thrombosis is common in patients with coronavirus disease 2019 (COVID-19). Etiologies underlying this complication are unclear. PurposeTo determine the prevalence of antiphospholipid (aPL), including lupus anticoagulant, anti-cardiolipin and anti-{beta}2-glycoprotein-1 antibodies, and its possible association with thrombotic manifestations of COVID-19. Data SourcesWe searched MEDLINE indexed journals on September 24, 2020 using the tool LitCovid and the pre-print server medRxIV. Study SelectionOriginal investigations (cross-sectional studies, cohort studies, case series, and research letters) on COVID-19 and thrombosis were included. Data ExtractionData were independently extracted, and compiled into spreadsheets based on the PRISMA principles. Data SynthesisHospitalized patients with COVID-19 showed a higher prevalence of lupus anticoagulant compared to non-COVID-19 patients. Temporally, lupus anticoagulant was generally positive early in the course of illness, whereas anti-cardiolipin and anti-{beta}2-glycoprotein-1 antibodies appeared to emerge later in the disease. Some patients who were aPL-negative at an early time-point after disease onset became aPL-positive at a later time-point. Lupus anticoagulant was independently associated with thrombosis in 60 COVID-19 patients in New York had who had 32 thrombotic events (8 arterial and 24 venous). In 88 patients in Wuhan, who had more than 20 each of arterial and venous thrombotic events, medium/high positivity for multiple aPL was significantly associated with arterial thrombosis. However, the association of aPL with thrombosis was not evident in reports that had an overall lower number of or predominantly venous thrombotic events. Analysis of pooled patients revealed that aPL were significantly more frequent in COVID-19 patients with stroke than stroke patients in the general population. Furthermore, injection of IgG aPL fractions from COVID-19 patients into mice accelerated venous thrombosis. LimitationLimited data and paucity of prospective studies. ConclusionThe aPL are prevalent in patients with COVID-19 and their presence is associated with thrombosis. Importantly, these antibodies may be a key mechanism of thrombosis in COVID-19. Follow-up studies are required to understand the relationship between aPL and the spectrum of vascular thrombosis during and after infection with SARS-CoV-2. Primary Funding SourceNone.

Venous thromboembolism (VTE), comprising both deep vein thrombosis (DVT) and pulmonary embolism (PE) is a common, multi-causal disease with potentially serious short- and long-term complications. In clinical practice, there is a need for improved plasma biomarker-based tools for VTE diagnosis and risk prediction. We used multiplex proteomics profiling to screen plasma from patients with suspected acute VTE, and a case-control study of patients followed up after ending anticoagulant treatment for a first VTE. With replication in 5 independent studies, together totalling 1137 patients and 1272 controls, we identify Complement Factor H Related Protein (CFHR5), a regulator of the alternative pathway of complement activation, as a novel VTE associated plasma biomarker. Using GWAS analysis of 2967 individuals we identified a genome-wide significant pQTL signal on chr1q31.3 associated with CFHR5 levels. We showed that higher CFHR5 levels are associated with increased thrombin generation in patient plasma and that recombinant CFHR5 enhances platelet activation in vitro. Thrombotic complications are a frequent feature of COVID-19; in hospitalised patients we found CFHR5 levels at baseline were associated with short-time prognosis of disease severity, defined as maximum level of respiratory support needed during hospital stay. Our results indicate a clinically important role for regulation of the alternative pathway of complement activation in the pathogenesis of VTE and pulmonary complications in acute COVID-19. Thus, CFHR5 is a potential diagnostic and/or risk predictive plasma biomarker reflecting underlying pathology in VTE and acute COVID-19.

Ischemic stroke due to large vessel occlusion results from the blockage of a major cerebral artery by a clot; however, the origins and molecular composition of these clots remain poorly understood. Mechanical thrombectomy has become a standard treatment to remove obstructive clots, providing a unique opportunity to analyze their properties. We previously demonstrated that blood can clot into an amyloid-like form, generating fibrinaloid microclots (2-200 m) that are highly resistant to fibrinolysis. In this study, archived clots from eight ischemic stroke patients with large vessel occlusion were examined, using samples stored in the Walton Centre Clot Bank in Liverpool, UK. All clots exhibited strong, heterogeneous amyloid staining, revealing a pervasive amyloid component. These findings represent a previously unreported characteristic of stroke clots, highlighting the potential for amyloid-targeted therapies to overcome their fibrinolytic resistance and providing a foundational new insight into ischaemic stroke pathophysiology and treatment.

Thrombotic and thromboembolic complications have been shown to play a critical role in the clinical outcome of COVID-19. Emerging evidence has shown that exosomal miRNAs are functionally involved in a number of physiologic and pathologic processes. However, neither exosomes nor miRNAs have been hitherto investigated in COVID-19. To test the hypothesis that exosomal miRNAs are a key determinant of thrombosis in COVID-19, we enrolled patients positive for COVID-19. Circulating exosomes were isolated from equal amounts of serum and levels of exosomal miRNAs were quantified. We divided our population in two groups based on the serum level of D-dimer on admission. Strikingly, we found that exosomal miR-424 was significantly upregulated whereas exosomal miR-103a, miR-145, and miR-885 were significantly downregulated in patients in the high D-dimer group compared to patients in the low D-Dimer group (p<0.0001).

BackgroundFibrinogen is a critical coagulation factor that plays an essential role in thrombosis and is elevated in individuals with chronic inflammation. Here, we used fibrinogen as a representative quantitative measure of pro-coagulant risk and evaluated metabolites associated with fibrinogen levels through non-targeted plasma metabolomic profiling (Broad and Metabolon platforms). MethodsOur analysis included 10,533 individuals across six U.S. based cohorts representing diverse population groups. The cross-sectional relationship between each of 789 tested metabolites and plasma fibrinogen concentration was assessed with adjustment for relevant covariates such as age, sex, body mass index, and circulating lipoprotein levels. ResultsMeta-analysis of per-cohort results revealed 270 metabolites significantly associated with fibrinogen level (FDR adjusted p-value < 0.05). Lipid species such as glycerophospholipids, sphingolipids, and fatty acyls were prevalent among significantly associated metabolites; some of these may capture effects of inflammation, as supported by sensitivity analyses adjusted for C-reactive protein. Significant associations between fibrinogen levels and serotonin, thyroxine, and sex-hormone derivatives may capture endogenous influences on fibrinogen levels. Exogenous compounds and microbial co-metabolites significantly associated with fibrinogen also implicate lifestyle and microbiome risk-factors. Only a portion of fibrinogen-associated metabolites (30%) have been associated with a cardiovascular disease outcome in a prior study, suggesting the associations discovered here may provide insights on vascular biology which case-control studies may not yet be powered to detect. ConclusionsThese findings contribute to a growing list of metabolite biomarkers that may influence coagulation and inflammation pathways and may thereby contribute to vascular risk.

BackgroundThe American Society of Hematology (ASH) recommends assessing venous thromboembolism (VTE) and major bleeding risk to optimize pharmacological VTE prophylaxis for medical inpatients. However, the clinical utility of model-guided approaches remains unknown. MethodsOur objective was to estimate differences in VTE and major bleeding event rates and efficiency with prophylaxis guided by risk models versus prophylaxis based on physician judgment. Patients were adults admitted to one of 10 Cleveland Clinic hospitals between December 2017 and January 2020. We compared physician practice with hypothetical prophylaxis recommended by model- based prophylaxis strategies, including ASH-recommended risk scores (Padua and IMPROVE) and locally derived Cleveland Clinic risk prediction models. For each strategy we quantified the prophylaxis rate, VTE and major bleeding rates, and the incremental number-needed-to-treat (NNT) to prevent one event (VTE or bleeding). ResultsPhysicians prescribed prophylaxis to 62% of patients whereas model-based strategies recommended prophylaxis for 17-87%. Model-guided prophylaxis produced more VTEs and fewer major bleeds than physicians, but total events varied among strategies. Overall, per 1,000 patients, model- based strategies produced 14.0-16.1 events compared with 14.3 for physicians. The Padua/IMPROVE models recommended prophylaxis for the fewest patients but caused the most total events. The most efficient model-based strategy recommended prophylaxis to 28% of patients with an incremental NNT (relative to no prophylaxis) of 80. Compared to physicians, it reduced prophylaxis by 55% and total events by 0.14%. ConclusionsPhysicians often prescribed inappropriate prophylaxis, highlighting the need for decision support. A model-based strategy maximized efficiency, reducing both events and prophylaxis relative to physicians.

BackgroundVenous thromboembolism (VTE) is a preventable complication of hospitalization. Risk-stratification is the cornerstone of prevention. The Caprini and Padua are the most commonly used risk-assessment models to quantify VTE risk. Both models perform well in select, high-risk cohorts. While VTE risk-stratification is recommended for all hospital admissions, few studies have evaluated the models in a large, unselected cohort of patients. MethodsWe analyzed consecutive first hospital admissions of 1,252,460 unique surgical and non-surgical patients to 1,298 VA facilities nationwide between January 2016 and December 2021. Caprini and Padua scores were generated using the VAs national data repository. We first assessed the ability of the two RAMs to predict VTE within 90 days of admission. In secondary analyses, we evaluated prediction at 30 and 60 days, in surgical versus non-surgical patients, after excluding patients with upper extremity DVT, in patients hospitalized [&ge;]72 hours, after including all-cause mortality in the composite outcome, and after accounting for prophylaxis in the predictive model. We used area under the receiver-operating characteristic curves (AUC) as the metric of prediction. ResultsA total of 330,388 (26.4%) surgical and 922,072 (73.6%) non-surgical consecutively hospitalized patients (total n=1,252,460) were analyzed. Caprini scores ranged from 0-28 (median, interquartile range: 4, 3-6); Padua scores ranged from 0-13 (1, 1-3). The RAMs showed good calibration and higher scores were associated with higher VTE rates. VTE developed in 35,557 patients (2.8%) within 90 days of admission. The ability of both models to predict 90-day VTE was low (AUCs: Caprini 0.56 [95% CI 0.56-0.56], Padua 0.59 [0.58-0.59]). Prediction remained low for surgical (Caprini 0.54 [0.53-0.54], Padua 0.56 [0.56-0.57]) and non-surgical patients (Caprini 0.59 [0.58-0.59], Padua 0.59 [0.59-0.60]). There was no clinically meaningful change in predictive performance in patients admitted for [&ge;]72 hours, after excluding upper extremity DVT from the outcome, after including all-cause mortality in the outcome, or after accounting for ongoing VTE prophylaxis. ConclusionsCaprini and Padua risk-assessment model scores have low ability to predict VTE events in a cohort of unselected consecutive hospitalizations. Improved VTE risk-assessment models must be developed before they can be applied to a general hospital population.

Structured AbstractO_ST_ABSImportanceC_ST_ABSTicagrelor inhibits platelet function, prevents myocardial infarction, and causes bleeding. A comprehensive analysis of the on- and off-target platelet effects of ticagrelor that underlie its clinical effects is lacking. ObjectiveTo test the hypothesis that platelet transcripts that change in response to ticagrelor exposure are associated with platelet function or bleeding. DesignA discovery cohort of healthy volunteers were sequentially exposed to aspirin, aspirin washout, and ticagrelor. Messenger RNA sequencing (mRNAseq) of purified platelets was performed pre/post each exposure. We defined the ticagrelor exposure signature (TES) as the ratio of mean expression of up-vs. down-regulated genes by ticagrelor that were prioritized based on lasso regression, weighted gene co-expression networks, and isoform level analyses. A separate healthy cohort was recruited to validate ticagrelors effects on TES genes measured using Nanostring. Platelet function was measured at baseline and in response to ticagrelor exposure in all participants. Self-reported bleeding was systematically queried during periods of ticagrelor exposure. SettingAn early phase, academic, clinical research unit. ParticipantsSelf-reported, healthy volunteers age > 30 and < 75, non-smoking, taking no daily prescribed medications. ExposuresTicagrelor (90 mg twice daily) and aspirin (81 mg/day and 325 mg/day) each for 4 weeks. Main outcomes and measuresExpression levels of platelet messenger RNA, platelet count, mean platelet volume, and 9 different measures of ex vivo platelet function (aggregated into a previously described platelet function score), and self-reported bleeding at baseline and after each exposure. ResultsIn the discovery cohort (n = 58, mean age 43, 39 female) platelet mRNAseq identified (FDR < 5%) 1820 up- and 1589 down-regulated genes associated with ticagrelor exposure. We prioritized 84 of these transcripts to calculate a TES score, which was increased by ticagrelor and unaffected by either dose of aspirin. In an independent cohort (n = 49, mean age 44, 24 female) we validated that ticagrelor exposure (beta = 0.48, SE = 0.08, p < 0.0001) increases TES scores. In combined analyses of discovery and validation cohorts, when TES levels were calculated using baseline platelet RNA, higher TES levels were associated with lower levels of baseline platelet function (meta-analysis beta = -0.60, standard error [SE] 0.29, P = 0.04) and self-reported bleeding during ticagrelor exposure (meta-analysis beta = 0.28, standard error [SE] = 0.14, P = 0.04). In contrast, we found no associations between bleeding with baseline platelet count, platelet volume, or platelet function. Conclusions and RelevanceTicagrelor exposure reproducibly and specifically changes a set of platelet transcripts, the baseline levels of which are a biomarker for platelet function and bleeding tendency on ticagrelor. Key PointsO_ST_ABSQuestionC_ST_ABSWhat are the global effects of ticagrelor exposure on platelets beyond platelet inhibition? FindingsIn an experimental human study of different antiplatelet therapies, we comprehensively characterized the effects of ticagrelor on platelet messenger RNA (mRNA). We found that 4 weeks of 90mg twice daily ticagrelor therapy specifically and reproducibly changes the levels of selected platelet mRNA. At baseline, volunteers with levels of platelet gene expression that mimic ticagrelor exposure had lower levels of platelet function and when exposed to ticagrelor a greater tendency for minor bleeding. MeaningBy using ticagrelor exposure as a molecular probe, we identified a platelet RNA biomarker that may identify patients at higher risk for ticagrelor-associated bleeding.

BackgroundPCSK9 chaperones the hepatic low-density lipoprotein receptor (LDLR) for lysosomal degradation, elevating serum LDL cholesterol and increasing the risk of atherosclerotic heart disease. Though the major effect on the hepatic LDLR comes from secreted PCSK9, the details of PCSK9 reuptake into the hepatocyte remain unclear. In both tissue culture and animal models, heparan sulfate proteoglycans (HSPGs) on hepatocytes act as co-receptors to promote PCSK9 reuptake. We hypothesized that if this PCSK9:HSPG interaction is important in humans, disrupting the interaction with unfractionated heparin (UFH) would acutely displace PCSK9 from the liver and increase plasma PCSK9. MethodsWe obtained remnant plasma samples from 160 subjects undergoing cardiac catheterization before and after administration of intravenous UFH. PCSK9 levels were determined using a commercial ELISA. ResultsMedian plasma PCSK9 concentrations were 113 ng/ml prior to UFH and 119 ng/ml afterwards. This difference was not significantly different (p = 0.83, 95% CI of difference: -6.23 to 6.31 ng/ml). Tests for equivalence provided 95% confidence that UFH administration would not raise PCSK9 levels by more than 4.7% of the baseline value. No predefined subgroups exhibited an effect of UFH on circulating PCSK9. ConclusionAdministration of UFH does not result in a clinically meaningful effect on circulating PCSK9 in humans. These results suggest that disruption of the PCSK9:HSPG interaction does not affect PCSK9 reuptake into the liver. Further, they cast doubt on the clinical utility of disrupting the PCSK9:HSPG interaction as a therapeutic strategy for PCSK9 inhibition. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=102 SRC="FIGDIR/small/22279796v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@f04518org.highwire.dtl.DTLVardef@48638eorg.highwire.dtl.DTLVardef@803104org.highwire.dtl.DTLVardef@12167c_HPS_FORMAT_FIGEXP M_FIG C_FIG Clinical PerspectiveO_ST_ABSWhat is new?C_ST_ABSO_LIPrior tissue culture and animal data suggest that PCSK9 interacts with hepatic heparan sulfate proteoglycans to enter the liver and raise cholesterol levels C_LIO_LIWe found no evidence that heparin, a competitive inhibitor of heparan sulfate proteoglycans, acutely affects PCSK9 in humans C_LI What are the clinical implications?O_LIHeparin is unlikely to be successfully repurposed as a PCSK9 inhibitor C_LI

BACKGROUNDBoth clinical and genetic factors drive the risk of venous thromboembolism. However, whether clinically recorded risk factors and genetic variants can be combined into a clinically applicable predictive score remains unknown. METHODSUsing Cox proportional-hazard models, we analyzed the association of risk factors with the likelihood of venous thromboembolism in U.K. Biobank, a large prospective cohort. We created a novel ten point clinical score using seven established clinical risk factors for venous thromboembolism. We also generated a polygenic risk score of 21 single nucleotide polymorphisms to quantify genetic risk. The genetic score was categorized into high risk (top two deciles of scores), intermediate risk (deciles three to eight), and low risk (lowest two deciles). The discrete clinical score led to the following approximate decile categorizations: high risk (5 to 10 points), intermediate risk (3 to 4 points), and low risk (0 to 2 points). RESULTSAmongst the 502,536 participants in the U.K. Biobank, there were 4,843 events of venous thromboembolism. Analyses of established clinical risk factors and the most commonly used medications revealed that participants were at decreased risk of venous thromboembolism if they had ever used oral contraceptive pills (hazard ratio, 0.88; 95% confidence interval [CI], 0.79 to 0.99) or if they currently used bendroflumethiazide (hazard ratio, 0.84; 95% CI, 0.74 to 0.95), cod liver oil capsules (hazard ratio, 0.87; 95% CI, 0.77 to 0.99), or atenolol (hazard ratio, 0.79; 95% CI, 0.68 to 0.91). Participants were at significantly increased risk of venous thromboembolism if they were at high clinical risk (hazard ratio, 5.98; 95% CI, 5.43 to 6.59) or high genetic risk (hazard ratio, 2.28; 95% CI, 2.07 to 2.51) relative to participants at low clinical or genetic risk, respectively. Combining clinical risk factors with genetic risk factors produced a model that better predicted risk of venous thromboembolism than either model alone (P<0.001). Participants at high clinical and genetic risk in the combined score had over an eightfold increased risk of venous thromboembolism relative to participants at low risk (hazard ratio, 8.27; 95% CI 7.59 to 9.00). CONCLUSIONSBy assessing venous thromboembolic events in over 500,000 participants, we identified several known and novel associations between risk factors and venous thromboembolism. Participants in the high risk group of a combined score, consisting of clinical and genetic factors, were over eight times more likely to experience venous thromboembolism than participants in the low risk group.

Background and AimsInfective endocarditis (IE) is a life-threatening condition known to cause stroke. Swift diagnosis and antibiotic treatment are crucial for preventing cerebral and systemic embolism, therefore reducing mortality and morbidity. However, diagnosing IE can be challenging. In this study, we aimed to assess whether analyzing cerebral thrombi retrieved by endovascular thrombectomy from stroke patients with IE could aid in the diagnosis and shed light on the composition signature of endocarditic thrombi. MethodsWe compared cerebral thrombi from three groups of ischemic stroke patients: those with definite infective endocarditis (IE) (n=10), those with cardioembolic stroke and concomitant infections other than IE (n=10 CE-I+), and those with cardioembolic stroke without infections (n=30 CE-I-). Our multiparameter analysis encompassed histological examinations, molecular biology and microbiological tests to detect microorganisms within the thrombi and to comprehensively assess their structural composition and immune signatures. ResultsWe directly detected invading pathogens through histology or PCR in all cerebral thrombi from IE patients, while none of the control thrombi exhibited such pathogens. Thrombi from IE patients displayed a distinct composition, characterized by a significant lower content of red blood cells, reduced CD14+ monocytes, increased von Willebrand Factor density, and a cell-dominant pattern of Neutrophil Extracellular Traps (NETs) deposition. ConclusionsComprehensive analysis of cerebral thrombi from stroke patients with suspected IE sustains early, definitive endocarditis diagnosis by detecting pathogens and immunothrombotic changes.

BackgroundProthrombotic antiphospholipid antibodies (aPL) found in patients with antiphospholipid syndrome (APS) are a recognised risk factor for ischemic stroke. However, it is unclear if aPL cause injury post thrombolysis leading to worse outcomes. We investigated whether aPL exacerbate reperfusion injury and sought to translate our findings in endothelial colony forming cells (ECFC) isolated from patients with APS. MethodsTransient ischemic stroke was induced in adult rats injected with serum-derived IgG from patients with APS (APS-IgG, containing aPL) or healthy controls (HC-IgG). Infarct size and intracellular signalling processes involved in ischemia-reperfusion injury were determined post reperfusion. In vitro, human umbilical vein endothelial cells (HUVEC) treated with IgG, as well as APS and HC ECFC, were exposed to hypoxia (0.1% O2). Cell death and relevant signalling mechanisms were assessed following reperfusion and compared to matched normoxic cultures. ResultsIn vivo, APS-IgG induced >2-fold larger infarcts and lower levels of active phosphorylated Akt, a key pro-survival kinase, compared to HC-IgG. In vitro, aPL-mediated cell death and suppression of Akt phosphorylation was confirmed in HUVEC exposed to IgG and hypoxia-reperfusion. Consistent with these findings, higher rates of cell death and reduced Akt phosphorylation following reperfusion were observed in ex vivo APS ECFC compared to HC ECFC. Treatment with the immunomodulating agent hydroxychloroquine ameliorated ECFC death and this effect was more pronounced in APS-derived cells. ConclusionPatient-derived IgG aPL exacerbate cell death following reperfusion in a novel in vivo stroke model for APS, as well as in vitro HUVEC cultures. These observations are mimicked in ex vivo APS ECFC. Our findings describe a novel pathogenic role for aPL in mediating tissue injury in addition to their known thrombogenic properties and indicate potential for pharmacological intervention.

We measured plasma levels of fibrinogen, plasminogen, tissue plasminogen activator (t-PA) and plasminogen activation inhibitor 1 (PAI-1) in blood from 37 patients with severe coronavirus disease-19 (COVID-19) and 23 controls. PAI-1, t-PA and fibrinogen levels were significantly higher in the COVID-19 group. Increased levels of PAI-1 likely result in lower plasmin activity and hence decreased fibrinolysis. These observations provide a partial explanation for the fibrin- mediated increase in blood viscosity and hypercoagulability that has previously been observed in COVID-19. Our data suggest that t-PA administration may be problematic, but that other interventions designed to enhance fibrinolysis might prove useful in the treatment of the coagulopathy that is often associated with severe COVID-19.

BackgroundAcute coronary syndrome (ACS) is caused by arterial thrombosis and is associated with sustained activation of coagulation. Clotting requires interactions of coagulation factors with aminophospholipids (aPL): phosphatidylserine (PS) and phosphatidylethanolamine (PE) on membrane surfaces. The aPL composition of circulating membranes in coronary disease has not been characterized. Furthermore, the contribution of external-facing aPL to elevated thrombotic risk in ACS is unknown. Methods and resultsThrombin generation was measured on platelet, leukocyte and extracellular vesicles (EV) from patients with ACS (n = 24), stable coronary artery disease (CAD, n = 18), risk factor positive (RF, n = 23) and healthy controls (HC, n = 24). The aPL composition on the surface of EV, platelets and leukocytes was determined using lipidomics. Leukocytes, platelets and EV externalized PE- and PS-containing fatty acids ranging from C16:0-20:4. These included both diacyl and plasmalogen forms, with significant increases stimulated by agonist activation. Thrombin generation on the surface of EV and leukocytes was higher in ACS than HC. Also, thrombin generation was higher for EV from CAD and RF, than HC. EV counts were higher in CAD and ACS compared with HC. Thrombin generation correlated positively with plasma EV counts and membrane surface area. ConclusionThe aPL membrane of EV and leukocytes may contribute to the activation of coagulation in CAD and ACS. Targeting EV formation/clearance and the aPL surface of EV and leukocyte membranes represents a novel anti-thrombotic target in CAD and ACS. Condensed abstractAcute coronary syndrome (ACS) is associated with sustained activation of coagulation, requiring procoagulant aminophospholipids (aPL). However, the aPL composition of circulating membranes and their contribution to thrombotic risk in ACS is undetermined. Lipidomics demonstrated that leukocytes, platelets and extracellular vesicles (EV) externalized aPL-containing fatty acids ranging from C16:0-20:4. Thrombin generation on the surface of EV and leukocytes was higher in ACS patients than healthy controls (HC). EV counts were higher in ACS compared with HC and correlated positively with thrombin generation. In summary, aPL in the outer membranes of EV and leukocytes may contribute to elevated thrombotic risk in ACS. Highlights What is new?O_LIThe aPL profile of platelets, leukocytes and EV in patients with ACS, CAD, RF and HC is defined for the first-time using lipidomics. C_LIO_LIThrombin generation on the surface of unstimulated leukocytes, is elevated in patients with ACS compared with HC. C_LIO_LIThrombin generation on the surface of EV is elevated in patients with ACS, CAD and RF compared with HC. C_LIO_LIEV counts in patients with ACS/CAD/RF were elevated compared with HC and correlate positively with thrombin generation. C_LI Clinical Perspective What are the clinical implications?O_LIThe membranes of EV and leukocytes may contribute to the activation of coagulation in ACS. C_LIO_LIThe aPL in EV and leukocyte membranes represent a novel target for reducing thrombotic risk in ACS. C_LIO_LITargeting EV formation/clearance could reduce thrombotic risk in CAD and ACS. C_LI

BackgroundCancer is associated with an increased risk of acute ischemic stroke (AIS) including large vessel occlusions (LVO). Whether cancer status affects outcomes in patients with LVO that undergo endovascular thrombectomy (EVT) remains unknown. Methods and ResultsAll consecutive patients undergoing EVT for LVO were recruited into a prospective ongoing multi-center database and the data was retrospectively analyzed. Patients with active cancer were compared to patients with cancer in remission. Association of cancer status with 90-day functional outcome and mortality were calculated in multivariable analyses. We identified 154 patients with cancer and LVO that underwent EVT (mean age 74{+/-}11, 43% men, median NIHSS 15). Of the included patients, 70 (46%) had a remote history of cancer or cancer in remission and 84 (54%) had active disease. Outcome data at 90 days post-stroke was available for 138 patients (90%) and was classified as favorable in 53 (38%). Patients with active cancer were younger and more often smoked but did not significantly differ from those without malignancy in other risk factors, stroke severity, stroke subtype or procedural variables. Favorable outcome rates among patients with active cancer did not significantly differ compared to those seen in patients without active cancer but mortality rates were significantly higher among patients with active cancer on univariate and multivariable analyses. ConclusionsOur study suggests that EVT is safe and efficacious in patients with history of malignancy as well as in those with active cancer at the time of stroke onset although mortality rates are higher among patients with active cancer. Clinical PerspectivesO_ST_ABSWhat is new?C_ST_ABSO_LIThe results of the current study suggest that EVT is safe and efficacious in patients with history of malignancy as well as in those with active cancer at the time of stroke onset although mortality rates are significantly higher and favorable outcome rates tend to be lower among patients with active cancer. C_LI What are the clinical implications?O_LIThe results imply that all cancer patients with LVO stroke should be considered for EVT regardless of cancer status. C_LI

BackgroundHigher rates of venous and arterial thromboembolism have been noted in coronavirus disease-2019 (COVID-19). There has been limited research on the impact of anticoagulant and antiplatelet choice in COVID-19. MethodsThis was a single-centre retrospective cohort study of 933 patients with COVID-19 infection presenting between 01/02/2020 and 31/05/2020. Survival time at 90 days post-diagnosis and thromboembolism development were the measured outcomes. ResultsOf 933 total patients, mean age was 68 years and 54.4% were male. 297 (31.8%) did not survive at 90 days. A Cox proportional hazards model analysis found no statistically significant relationship between anticoagulant or antiplatelet choice and survival (p<0.05). 57 (6.3%) developed thromboembolism. Antiplatelet choice was not shown to have a statistically significant relationship with thromboembolism development. Warfarin and direct oral anticoagulant (DOAC) use did not have a statistically significant impact on thromboembolism development (p<0.05). Therapeutic low-molecular-weight heparin (LMWH) use was associated with increased thromboembolism risk (Odds ratio = 14.327, 95% CI 1.904 - 107.811, p = 0.010). ConclusionsAntiplatelet choice was shown to have no impact on survival or thromboembolism development in COVID-19. Anticoagulant choice did not impact survival or thromboembolism development, aside from LMWH. Therapeutic LMWH use was associated with increased risk of thromboembolism. However, it should be noted that the sample size for patients using therapeutic LMWH was small (n=4), and there may be confounding variables affecting both LMWH use and thromboembolism development. These findings should be repeated with a larger sample of patients using therapeutic LMWH with additional adjustment for cofounding variables.

ObjectiveCatastrophic antiphospholipid syndrome (CAPS), characterized by widespread thrombosis and multi-organ failure, is associated with high morbidity and mortality. We previously established complement activation as a pathogenic driver of CAPS and identified rare germline variants in complement-regulatory genes including Complement Receptor 1 (CR1) in 50% of CAPS. MethodsWe quantified CR1 expression by flow cytometry across hematopoietic cell types. CRISPR/Cas9 genome editing of TF-1 (erythroleukemia) 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 CR1 expression in patients with reduced CR1 expression. Functional impact of low CR1 expression was assessed by complement-mediated cell killing using modified Ham (mHam) assay, cell-bound complement degradation products through flow cytometry and circulatory immune complexes (CIC) in serum samples through ELISA. ResultsCR1 expression in erythrocytes was markedly reduced on CAPS erythrocytes (n=9, 21.80%) compared to healthy controls (HC; n=32, 82.40%), with promoter hypermethylation emerging as a plausible epigenetic mechanism for CR1 downregulation. A 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 CIC, which are bound and cleared by CR1 on erythrocytes, were higher in acute CAPS (n=3, 25.55 {micro}g Eq/ml) than healthy controls (n=3, 7.445 {micro}g Eq/ml). Five patients were treated with C5 inhibition which mitigated thrombosis. ConclusionGenetic or epigenetic-mediated CR1 deficiency is a potential hallmark of CAPS and predicts response to C5 inhibition.

BackgroundIt was recently reported that highly elevated plasma activity of the ADP-scavenging enzyme creatine kinase (CK), to >10 times the upper reference limit (URL), is independently associated with fatal or non-fatal bleeding during treatment for ST-segment elevation myocardial infarction (OR 2.6 [95% CI, 1.8 to 2.7]/log CK increase). Evidence indicates that CK attenuates ADP-dependent platelet aggregation. This study investigates whether moderately elevated CK in non-ST-segment elevation acute coronary syndromes (NSTE-ACS) is associated with major bleeding. MethodsThe Thrombolysis In Myocardial Ischemia (TIMI) 3B trial compared rt-PA (35 to 80 mg) with placebo, and early catheterization with conservative management in patients with NSTE-ACS. Main outcomes of the current study are the independent association of peak plasma CK (CKmax) with adjudicated fatal or non-fatal major bleeding (primary), and with combined major bleeding, stroke, and all-cause mortality (secondary) in multivariable binomial logistic regression analysis, with co-variables including age, sex, BMI, SBP, creatinine, and treatment assignment. Discrimination was assessed with C-statistics. ResultsThe study included 1473 patients (66% men, 80% white, mean age 59 y, SE 0.3). CKmax ranged between 15 and 19045 IU/L (mean (SE), 450(24) IU/L; i.e. 2 times URL). Major bleeding occurred in 2.0% (mean age 65(1.3) y; mean CKmax 1015(318) IU/L; 6 times URL), and the combined outcome in 4.3% of the patients, adjusted OR per log CK increase respectively 3.1 [1.6 to 5.8] for major bleeding, and 3.9 [2.5 to 6.1] for the combined outcome; C-index 0.8 for both outcomes. DiscussionThe presented data add to the existing evidence that proportionate to its plasma activity, the ADP-binding enzyme CK is strongly and independently associated with non-fatal and fatal major bleeding during ACS treatment. CK might increase the accuracy of prediction models for major bleeding in patients treated with antithrombotic or thrombolytic drugs for ACS. ClinicalTrials.gov identifierNCT00000472

ObjectiveAn unexpectedly large number of people infected with Covid-19 had experienced a thrombotic event. This study aims to assess the associations between Covid-19 infection and thromboembolism including myocardial infarction (MI), ischaemic stroke, deep-vein thrombosis (DVT), and pulmonary embolism (PE). Patients and MethodsA self-controlled case-series study was conducted covering the whole of Scotlands general population. The study population comprised individuals with confirmed (positive test) Covid-19 and at least one thromboembolic event between March 2018 and October 2020. Their incidence rates during the risk interval (5 days before to 56 days after the positive test) and the control interval (the remaining periods) were compared intra-personally. ResultsAcross Scotland, 1,449 individuals tested positive for Covid-19 and experienced a thromboembolic event. The risk of thromboembolism was significantly elevated over the whole risk period but highest in the 7 days following the positive test (IRR 12.01, 95% CI 9.91-14.56) in all included individuals. The association was also present in individuals not originally hospitalised for Covid-19 (IRR 4.07, 95% CI 2.83-5.85). Risk of MI, stroke, PE and DVT were all significantly higher in the week following a positive test. The risk of PE and DVT was particularly high and remained significantly elevated even 56 days following the test. ConclusionConfirmed Covid-19 infection was associated with early elevations in risk with MI, ischaemic stroke, and substantially stronger and prolonged elevations with DVT and PE both in hospital and community settings. Clinicians should consider thromboembolism, especially PE, among people with Covid-19 in the community.