COVID-19 Thrombotic Complications and Therapeutic Strategies.

Shortly after the emergence of coronavirus disease 2019 (COVID-19) in late 2019, clinicians rapidly recognized an apparent association between the disease and both arterial and venous thrombotic complications, which was confirmed in epidemiologic studies. Based on these data, hospitals empirically developed and implemented protocols with different strategies for anticoagulation of hospitalized COVID-19 patients. Subsequent randomized controlled trials (RCTs) clarified the role of anticoagulation in patients hospitalized with COVID-19 and recently discharged from the hospital. In this review, we discuss the epidemiology and pathophysiology of thrombosis in patients with COVID-19, observational comparative effectiveness analyses that provided hints of a benefit from anticoagulation, and finally the RCTs that established which patients with COVID-19 benefit from treatment-dose anticoagulation. These RCTs have demonstrated that hospitalized, noncritically ill patients with COVID-19 benefit from treatment-dose anticoagulation, but patients who are hospitalized and critically ill, discharged from the hospital, or not hospitalized do not benefit.

Ingestion of the Non-Nutritive Sweetener Erythritol, but Not Glucose, Enhances Platelet Reactivity and Thrombosis Potential in Healthy Volunteers-Brief Report.

BACKGROUND: Although artificial and non-nutritive sweeteners are widely used and generally recognized as safe by the US and European Union regulatory agencies, there have been no clinical trials to assess either long-term cardiovascular disease risks or short-term cardiovascular disease-relevant phenotypes. Recent studies report that fasting plasma levels of erythritol, a commonly used sweetener, are clinically associated with heightened incident cardiovascular disease risks and enhance thrombosis potential in vitro and in animal models. Effects of dietary erythritol on thrombosis phenotypes in humans have not been examined. METHODS: Using a prospective interventional study design, we tested the impact of erythritol or glucose consumption on multiple indices of stimulus-dependent platelet responsiveness in healthy volunteers (n=10 per group). Erythritol plasma levels were quantified with liquid chromatography tandem mass spectrometry. Platelet function at baseline and following erythritol or glucose ingestion was assessed via both aggregometry and analysis of granule markers released. RESULTS: Dietary erythritol (30 g), but not glucose (30 g), lead to a >1000-fold increase in erythritol plasma concentration (6480 [5930-7300] versus 3.75 [3.35-3.87] µmol/L; P<0.0001) and exhibited acute enhancement of stimulus-dependent aggregation responses in all subjects, agonists, and doses examined. Erythritol ingestion also enhanced stimulus-dependent release of the platelet dense granule marker serotonin (P<0.0001 for TRAP6 [thrombin activator peptide 6] and P=0.004 for ADP) and the platelet α-granule marker CXCL4 (C-X-C motif ligand-4; P<0.0001 for TRAP6 and P=0.06 for ADP). In contrast, glucose ingestion triggered no significant increases in stimulus-dependent release of either serotonin or CXCL4. CONCLUSIONS: Ingestion of a typical quantity of the non-nutritive sweetener erythritol, but not glucose, enhances platelet reactivity in healthy volunteers, raising concerns that erythritol consumption may enhance thrombosis potential. Combined with recent large-scale clinical observational studies and mechanistic cell-based and animal model studies, the present findings suggest that discussion of whether erythritol should be reevaluated as a food additive with the Generally Recognized as Safe designation is warranted. REGISTRATION: URL: https://www.clinicaltrials.gov; Unique identifier: NCT04731363.

Platelet RNA Biomarker of Ticagrelor-Responsive Genes Is Associated With Platelet Function and Cardiovascular Events.

BACKGROUND: Identifying patients with the optimal risk:benefit for ticagrelor is challenging. The aim was to identify ticagrelor-responsive platelet transcripts as biomarkers of platelet function and cardiovascular risk. METHODS: Healthy volunteers (n=58, discovery; n=49, validation) were exposed to 4 weeks of ticagrelor with platelet RNA data, platelet function, and self-reported bleeding measured pre-/post-ticagrelor. RNA sequencing was used to discover platelet genes affected by ticagrelor, and a subset of the most informative was summarized into a composite score and tested for validation. This score was further analyzed (1) in CD34+ megakaryocytes exposed to an P2Y12 inhibitor in vitro, (2) with baseline platelet function in healthy controls, (3) in peripheral artery disease patients (n=139) versus patient controls (n=30) without atherosclerosis, and (4) in patients with peripheral artery disease for correlation with atherosclerosis severity and risk of incident major adverse cardiovascular and limb events. RESULTS: Ticagrelor exposure differentially expressed 3409 platelet transcripts. Of these, 111 were prioritized to calculate a Ticagrelor Exposure Signature score, which ticagrelor reproducibly increased in discovery and validation cohorts. Ticagrelor's effects on platelets transcripts positively correlated with effects of P2Y12 inhibition in primary megakaryocytes. In healthy controls, higher baseline scores correlated with lower baseline platelet function and with minor bleeding while receiving ticagrelor. In patients, lower scores independently associated with both the presence and extent of atherosclerosis and incident ischemic events. CONCLUSIONS: Ticagrelor-responsive platelet transcripts are a biomarker for platelet function and cardiovascular risk and may have clinical utility for selecting patients with optimal risk:benefit for ticagrelor use.

N-Acetyl Cysteine Prevents Arterial Thrombosis in a Dose-Dependent Manner In Vitro and in Mice.

BACKGROUND: Platelet-rich thrombi occlude arteries causing fatal infarcts like heart attacks and strokes. Prevention of thrombi by current antiplatelet agents can cause major bleeding. Instead, we propose using N-acetyl cysteine (NAC) to act against the protein VWF (von Willebrand factor), and not platelets, to prevent arterial thrombi from forming. METHODS: NAC was assessed for its ability to prevent arterial thrombosis by measuring platelet accumulation rate and occlusion time using a microfluidic model of arterial thrombosis with human blood. Acute clot formation, clot stability, and tail bleeding were measured in vivo with the murine modified Folts model. The effect of NAC in the murine model after 6 hours was also measured to determine any persistent effects of NAC after it has been cleared from the blood. RESULTS: We demonstrate reduction of thrombi formation following treatment with NAC in vitro and in vivo. Human whole blood treated with 3 or 5 mmol/L NAC showed delayed thrombus formation 2.0× and 3.7× longer than control, respectively (P<0.001). Blood treated with 10 mmol/L NAC did not form an occlusive clot, and no macroscopic platelet aggregation was visible (P<0.001). In vivo, a 400-mg/kg dose of NAC prevented occlusive clots from forming in mice without significantly affecting tail bleeding times. A lower dose of NAC significantly reduced clot stability. Mice given multiple injections showed that NAC has a lasting and cumulative effect on clot stability, even after being cleared from the blood (P<0.001). CONCLUSIONS: Both preclinical models demonstrate that NAC prevents thrombus formation in a dose-dependent manner without significantly affecting bleeding time. This work highlights a new pathway for preventing arterial thrombosis, different from antiplatelet agents, using an amino acid derivative as an antithrombotic therapeutic.

PAR4 Antagonism in Patients With Coronary Artery Disease Receiving Antiplatelet Therapies.

BACKGROUND: BMS-986141 is a novel potent highly selective antagonist of PAR (protease-activated receptor) type 4. PAR4 antagonism has been demonstrated to reduce thrombus formation in isolation and in combination with factor Xa inhibition in high shear conditions in healthy people. We sought to determine whether PAR4 antagonism had additive antithrombotic effects in patients with coronary artery disease who were receiving antiplatelet therapy. METHODS: Forty-five patients with stable coronary heart disease and 10 healthy volunteers completed a phase 2a open-label 4-arm single-center study. Patients were allocated to 1 of 3 treatment arms for 7 days: (1) ticagrelor (90 mg BID), (2) aspirin (75 mg QD), or (3) the combination of ticagrelor and aspirin. Agonist-induced platelet aggregation, platelet activation, and ex vivo thrombus formation were measured before and 2 and 24 hours after a single oral 4-mg dose of BMS-986141 on the first study visit day in all participants. RESULTS: BMS-986141 demonstrated highly selective inhibition of PAR4-AP (agonist peptide)-induced platelet aggregation, P-selectin expression, and platelet-monocyte aggregate expression (P≤0.001 for all), which were unaffected by concomitant antiplatelet therapies. PAR4 antagonism reduced ex vivo thrombus area in high shear conditions in healthy volunteers (-21%; P=0.001) and in patients receiving ticagrelor alone (-28%; P=0.001), aspirin alone (-23%; P=0.018), or both in combination (-24%; P≤0.001). Plasma concentration of BMS-986141 correlated with PAR4-AP-induced platelet responses (P≤0.001 for all) and total thrombus area under high shear stress conditions (P≤0.01 for all). CONCLUSIONS: PAR4 antagonism has additive antithrombotic effects when used in addition to ticagrelor, aspirin, or their combination, in patients with stable coronary heart disease. REGISTRATION: URL: https://www.clinicaltrials.gov; Unique identifier: NCT05093790.

Aspirin Versus Clopidogrel for Long-Term Maintenance Monotherapy After Percutaneous Coronary Intervention: The HOST-EXAM Extended Study.

BACKGROUND: Long-term outcomes of antiplatelet monotherapy in patients who receive percutaneous coronary intervention are unknown. The HOST-EXAM (Harmonizing Optimal Strategy for Treatment of Coronary Artery Stenosis-Extended Antiplatelet Monotherapy) Extended study reports the posttrial follow-up results of the original HOST-EXAM trial. METHODS: From March 2014 through May 2018, 5438 patients who maintained dual antiplatelet therapy without clinical events for 12±6 months after percutaneous coronary intervention with drug-eluting stents were randomly assigned in a 1:1 ratio to receive clopidogrel (75 mg once daily) or aspirin (100 mg once daily). The primary end point (a composite of all-cause death, nonfatal myocardial infarction, stroke, readmission attributable to acute coronary syndrome, and Bleeding Academic Research Consortium type 3 or greater bleeding), secondary thrombotic end point (cardiac death, nonfatal myocardial infarction, ischemic stroke, readmission attributable to acute coronary syndrome, and definite or probable stent thrombosis), and bleeding end point (Bleeding Academic Research Consortium type 2 or greater bleeding) were analyzed during the extended follow-up period. Analysis was performed on the per-protocol population (2431 patients in the clopidogrel group and 2286 patients in the aspirin group). RESULTS: During a median follow-up of 5.8 years (interquartile range, 4.8-6.2 years), the primary end point occurred in 12.8% and 16.9% in the clopidogrel and aspirin groups, respectively (hazard ratio, 0.74 [95% CI, 0.63-0.86]; P<0.001). The clopidogrel group had a lower risk for the secondary thrombotic end point (7.9% versus 11.9%; hazard ratio, 0.66 [95% CI, 0.55-0.79]; P<0.001) and secondary bleeding end point (4.5% versus 6.1%; hazard ratio, 0.74 [95% CI, 0.57-0.94]; P=0.016). There was no significant difference in the incidence of all-cause death between the 2 groups (6.2% versus 6.0%; hazard ratio, 1.04 [95% CI, 0.82-1.31]; P=0.742). Landmark analysis at 2 years showed that the beneficial effect of clopidogrel was consistent throughout the follow-up period. CONCLUSIONS: During an extended follow-up of >5 years after randomization, clopidogrel monotherapy compared with aspirin monotherapy was associated with lower rates of the composite net clinical outcome in patients without clinical events for 12±6 months after percutaneous coronary intervention with drug-eluting stents. REGISTRATION: URL: https://www. CLINICALTRIALS: gov; Unique identifier: NCT02044250.

Dual Antiplatelet Therapy After Embolic Stroke of Undetermined Source: A Subgroup Analysis of the CHANCE-2 Trial.

BACKGROUND: The atherosclerotic sources of embolism are a significant contributor to embolic stroke of undetermined source (ESUS). However, there is limited evidence for the efficacy of intensive dual antiplatelet therapy for ESUS. We conducted an investigation to determine whether gene-directed dual antiplatelet therapy could reduce the risk of recurrent stroke in patients with ESUS. METHODS: CHANCE-2 (Clopidogrel in High-Risk Patients with Acute Nondisabling Cerebrovascular Events-II) was an investigator-initiated, multicenter, randomized, double-blind, placebo-controlled trial that objectively compared ticagrelor plus aspirin and clopidogrel plus aspirin in patients with minor stroke or transient ischemic attack who carried CYP2C19 loss-of-function alleles in China. All study participants were classified into ESUS and non-ESUS groups for the prespecified exploratory analysis. Cox proportional hazards models were used to assess the interaction of the state of ESUS with the effects of dual antiplatelet therapy with ticagrelor-aspirin versus clopidogrel-aspirin, adjusting for sociodemographic and clinical factors. RESULTS: The subgroup analysis comprised 5796 participants (90.4% of the total 6412 participants) in the CHANCE-2 trial, with a median age of 64.9 years (range, 57.0-71.4 years), of whom 1964 (33.9%) were female. These participants underwent diffusion-weighted imaging as part of the study protocol. After systematic evaluation, 15.2% of patients (881/5796) were deemed to have ESUS. The incidence of stroke recurrence in patients with ESUS was found to be 5.6% in the ticagrelor-aspirin group and 9.2% in the clopidogrel-aspirin group (hazard ratio, 0.57 [95% CI, 0.33-0.99]; P=0.04). In patients without ESUS, the respective incidence rates were 5.6% and 7.5% (hazard ratio, 0.72 [95% CI, 0.58-0.90]; P<0.01). The P value was 0.56 for the treatment × ESUS status interaction effect. CONCLUSIONS: In this prespecified exploratory analysis, ticagrelor with aspirin was superior to clopidogrel with aspirin for preventing stroke at 90 days in patients with acute ischemic stroke or transient ischemic attack who carried CYP2C19 loss-of-function alleles and were classified as ESUS. REGISTRATION: URL: https://www.clinicaltrials.gov; Unique identifier: NCT04078737.

Baseline Stroke Risk and Efficacy of Dual-Antiplatelet Therapy: A Post Hoc Analysis of the POINT Trial.

BACKGROUND: High-risk transient ischemic attacks and minor ischemic strokes are followed by a variable risk of ischemic stroke. We aimed to determine how baseline stroke risk modified the efficacy of clopidogrel-aspirin (referred to here as dual-antiplatelet therapy [DAPT]) for transient ischemic attack and minor ischemic stroke. METHODS: We performed an unplanned secondary analysis of the POINT trial (Platelet-Oriented Inhibition in New Transient Ischemic Attack and Minor Ischemic Stroke). We first evaluated the associations of the CHA2DS2-VASc and stroke prognosis instrument II (SPI-II) scores with the risk of incident ischemic stroke and major hemorrhage (intracranial hemorrhage or major systemic hemorrhage). We then tested for heterogeneity of the relative and absolute treatment effect of DAPT relative to aspirin across low- and high-risk patient subgroups. RESULTS: A total of 4841 trial participants were included in this analysis, with 2400 participants assigned to treatment with short-term DAPT and 2430 participants to treatment with aspirin and placebo. The dichotomized SPI-II score, but not the CHA2DS2-VASc score (P=0.18), was associated with the risk of incident ischemic stroke. A high-risk SPI-II score (>3) was associated with greater risk of incident ischemic stroke (hazard ratio of incident ischemic stroke relative to low-risk SPI-II score of 1.84 [95% CI, 1.44-2.35]; P<0.001) and numerically greater risk of major hemorrhage though not meeting statistical significance (hazard ratio, 1.80 [95% CI, 0.90-3.57]; P=0.10). The relative risk reduction with DAPT was similar across SPI-II strata (Pinteraction=0.31). The absolute risk reduction for ischemic stroke with DAPT compared with aspirin was nearly 4-fold higher (2.80% versus 0.76%; number needed to treat, 31 versus 131) in the high-risk SPI-II stratum relative to the low-risk stratum. The absolute risk increase for major hemorrhage with DAPT compared with aspirin was 3-fold higher (0.84% versus 0.30%; number needed to harm, 119 versus 331) in the high-risk SPI-II stratum relative to the low-risk stratum. CONCLUSIONS: Stratification by baseline stroke risk identifies a patient subgroup that derives greater absolute benefit from treatment with DAPT. REGISTRATION: URL: https://www.clinicaltrials.gov; Unique identifier: NCT00991029.

Convolutional neuronal network for identifying single-cell-platelet-platelet-aggregates in human whole blood using imaging flow cytometry.

Imaging flow cytometry is an attractive method to investigate individual cells by optical properties. However, imaging flow cytometry applications with clinical relevance are scarce so far. Platelet aggregation naturally occurs during blood coagulation to form a clot. However, aberrant platelet aggregation is associated with cardiovascular disease under steady-state conditions in the blood. Several types of so-called antiplatelet drugs are frequently described to reduce the risk of stroke or cardiovascular diseases. However, an efficient monitoring method is missing to identify the presence and frequency of platelet-platelet aggregates in whole blood on a single cell level. In this work, we employed imaging flow cytometry to identify fluorescently labeled platelets in whole blood with a conditional gating strategy. Images were post-processed and aligned. A convolutional neural network was designed to identify platelet-platelet aggregates of two, three, and more than three platelets, and results were validated against various data set properties. In addition, the neural network excluded erythrocyte-platelet aggregates from the results. Based on the results, a parameter for detecting platelet-platelet aggregates, the weighted platelet aggregation, was developed. If employed on a broad scale with proband and patient samples, our method could aid in building a future diagnostic marker for cardiovascular disease and monitoring parameters to optimize drug prescriptions in such patient groups.

Platelet functions in lymphatic filariasis patients.

Filariasis is a chronic disease where parasitic worms survive in human hosts even for decades and lead to complications like lymphedema and elephantiasis. Despite the persistent existence of filarial parasites in human hosts, fatal and thrombotic complications are not known, unlike other parasitic diseases like malaria. This suggests that filarial parasites might be affecting the host's platelet functions. This study was conducted to examine platelet functions in confirmed filariasis patients and healthy controls. Results showed that filariasis patients had larger platelets, inhibited aggregation, and slower speed of aggregation, compared to controls. However, in vivo markers of platelet activation and degranulation (beta thromboglobulin and soluble P-selectin) were not affected. Observations suggested that there is increased platelet turnover, cellular apoptosis and inhibited platelet functions in filariasis patients compared to controls. Platelet function inhibition was not associated with the duration of disease, lymphedema-affected organs, or gender of patients. This study confirms that filarial parasites modulate platelet functions in human hosts.

Effects of Body Mass Index and Body Weight on Plasma Concentration of Ticagrelor and Platelet Aggregation Rate in Patients with Unstable Angina in a Chinese Han Population.

Background: The aim of this study was to investigate the impact of body mass index (BMI) and body weight on the concentrations of ticagrelor and the ticagrelor metabolite, AR-C124910XX, as well as the platelet aggregation rate (PAR) in a Chinese Han population with unstable angina (UA). Specifically, it focused on these parameters following the administration of dual antiplatelet therapy (DAPT) comprising aspirin and ticagrelor. Methods: A total of 105 patients with UA were included in the study. Measurement of the platelet aggregation rate induced by adenosine diphosphate (PAR-ADP) was performed before, as well as 3 and 30 days after DAPT treatment. The plasma concentrations of ticagrelor and AR-C124910XX were detected at 3 and 30 days after DAPT treatment. We conducted correlation analyses to assess the effects of BMI and body weight on the concentrations of ticagrelor and AR-C124910XX, on PAR-ADP, and on the inhibition of platelet aggregation induced by adenosine diphosphate (IPA-ADP) at both 3 and 30 days after DAPT treatment. Results: The BMI and body weight were positively correlated with baseline PAR-ADP (r = 0.205, p = 0.007; r = 0.122, p = 0.022). The PAR-ADP at 3 and 30 days after DAPT treatment were significantly lower than at baseline (61.56% ± 10.62%, 8.02% ± 7.52%, 12.90% ± 7.42%, p < 0.001). There was a negative correlation between body weight and the concentrations of ticagrelor and AR-C124910XX at 3 days following DAPT treatment (r = -0.276, p < 0.001; r = -0.337, p < 0.001). Additionally, BMI showed a similar negative correlation with the concentrations of ticagrelor and AR-C124910XX (r = -0.173, p = 0.009; r = -0.207, p = 0.002). At 30 days after treatment, both body weight and BMI were negatively correlated with ticagrelor (r = -0.256, p < 0.001; r = -0.162, p = 0.015) and its metabolite (r = -0.352, p < 0.001; r = -0.202, p = 0.002). Body weight was positively correlated with PAR-ADP (r = 0.171, p = 0.010) and negatively correlated with IPA-ADP (r = -0.163, p = 0.015) at 30 days after treatment. Similarly, BMI was positively correlated with PAR-ADP (r = 0.217, p = 0.001) and negatively correlated with IPA-ADP (r = -0.211, p = 0.001) at the same time point. Conclusions: BMI and body weight are key factors influencing the pharmacokinetics and pharmacodynamics of ticagrelor in Chinese Han patients with UA following DAPT treatment that includes ticagrelor. Both BMI and body weight were positively correlated with PAR-ADP at baseline and 30 days after DAPT treatment. Clinical Trial Registration: ChiCTR2100044938, https://www.chictr.org.cn/.

The podoplanin-CLEC-2 interaction promotes platelet-mediated melanoma pulmonary metastasis.

BACKGROUND: Podoplanin (PDPN) expressed on tumour cells interacts with platelet C-type lectin-like receptor 2 (CLEC-2). This study aimed to investigate the role of the PDPN-platelet CLEC-2 interaction in melanoma pulmonary metastasis. METHODS: Murine melanoma B16-F0 cells, which have two populations that express podoplanin, were sorted by FACS with anti-podoplanin staining to obtain purified PDPN + and PDPN- B16-F0 cells. C57BL/6J mice transplanted with CLEC-2-deficient bone marrow cells were used for in vivo experiments. RESULTS: The in vivo data showed that the number of metastatic lung nodules in WT mice injected with PDPN + cells was significantly higher than that in WT mice injected with PDPN- cells and in WT or CLEC-2 KO mice injected with PDPN- cells. In addition, our results revealed that the platelet Syk-dependent signalling pathway contributed to platelet aggregation and melanoma metastasis. CONCLUSIONS: Our study indicates that the PDPN-CLEC-2 interaction promotes experimental pulmonary metastasis in a mouse melanoma model. Tumour cell-induced platelet aggregation mediated by the interaction between PDPN and CLEC-2 is a key factor in melanoma pulmonary metastasis.

A rare case of glanzmann thrombasthenia with concomitant factor VII deficiency.

Glanzmann thrombasthenia and clotting factor VII deficiency are rare autosomal recessive bleeding disorders. But the occurrence of both in the same person is an extremely rare phenomenon. Here, we present the case of a young female from Sindh, Pakistan that got diagnosed with Glanzmann thrombasthenia and concomitant moderate factor VII deficiency, a combination not previously reported in the country. The patient exhibited typical clinical manifestations including menorrhagia, nasal bleeds, and prolonged bleeding after minor injuries, compounded by a positive family history and consanguinity. Laboratory investigations revealed marked anemia, prolonged bleeding time, and abnormal platelet aggregation studies consistent with Glanzmann thrombasthenia. The identification of this rare combination relied on comprehensive clinical evaluation, emphasizing the importance of family history in suspected cases. Management involved platelet transfusions, tranexamic acid, and Factor VII replacement, resulting in clinical improvement.

Characterization of a novel mouse platelet transfusion model.

BACKGROUND AND OBJECTIVES: Platelet transfusions are increasing with medical advances. Based on FDA criteria, platelet units are assessed by in vitro measures; however, it is not known how platelet processing and storage duration affect function in vivo. Our study's aim was to develop a novel platelet transfusion model stored in mouse plasma that meets FDA criteria adapted to mice, and transfused fresh and stored platelets are detectable in clots in vivo. STUDY DESIGN AND METHODS: Platelet units stored in mouse plasma were prepared using a modified platelet-rich plasma (PRP) collection protocol. Characteristics of fresh and stored units, including pH, cell count, in vitro measures of activity, including activation and aggregation, and post-transfusion recovery (PTR), were determined. Lastly, a tail transection assay was conducted using mice transfused with fresh or stored units, and transfused platelets were identified by confocal imaging. RESULTS: Platelet units had acceptable platelet and white cell counts and were negative for bacterial contamination. Fresh and 1-day stored units had acceptable pH; the platelets were activatable by thrombin and adenosine diphosphate, agreeable with thrombin, had acceptable PTR, and were present in vivo in clots of recipients after tail transection. In contrast, 2-day stored units had clinically unacceptable quality. CONCLUSION: We developed mouse platelets for transfusion analogous to human platelet units using a modified PRP collection protocol with maximum storage of 1 day for an 'old' unit. This provides a powerful tool to test how process modifications and storage conditions affect transfused platelet function in vivo.

Cilostazol versus Aspirin or Clopidogrel for Reducing Post-Stroke Aspiration Pneumonia: A Nationwide Retrospective Cohort Study.

INTRODUCTION: The association between the use of cilostazol as a post-stroke antiplatelet medication and a reduction in post-stroke pneumonia has been suggested. However, whether cilostazol has a greater preventive effect against post-stroke aspiration pneumonia (AP) than other antiplatelet medications remains unclear. Thus, this study aimed to evaluate whether cilostazol has a greater preventive effect against post-stroke AP than aspirin or clopidogrel. METHODS: Through the Japanese Diagnosis Procedure Combination database, we identified patients who were hospitalized for ischemic stroke between April 2012 and September 2019. We performed 1:1 propensity score matching between patients who received cilostazol alone at discharge and those who received aspirin or clopidogrel alone at discharge. The primary outcome was the 90-day readmission for post-stroke AP. The occurrence of recurrent ischemic stroke within 90 days was also evaluated. RESULTS: Among the 305,543 eligible patients with ischemic stroke, 65,141 (21%), 104,157 (34%), and 136,245 (45%) received cilostazol, aspirin, and clopidogrel, respectively. Propensity score matching generated 65,125 pairs. The cilostazol group had a higher proportion of 90-day post-stroke readmissions with AP than the aspirin or clopidogrel groups (1.5% vs. 1.2%, p < 0.001). The proportion of patients with recurrent ischemic stroke within 90 days was also higher in the cilostazol group (2.4% vs. 2.2%, p = 0.017). CONCLUSION: The present study suggests that cilostazol may not have a greater effect on preventing post-stroke AP within 90 days than other antiplatelet medications. Nevertheless, further randomized controlled trials with longer follow-up periods are warranted.

Histopathological Features of Cancer-Associated Venous Thromboembolism: Presence of Intrathrombus Cancer Cells and Prothrombotic Factors.

BACKGROUND: Cancer-associated venous thromboembolism (VTE) is a critical complication in patients with cancer. However, the pathological findings of VTE are limited. Here, we investigated the histopathological features of cancer-associated VTE in human autopsy cases. METHODS: We clinically examined the autopsy cases of VTE with (n=114) and without cancer (n=66) and immunohistochemically analyzed the expression of prothrombotic factors in intrathrombus cancer cells, the thrombus contents of erythrocytes, fibrin, platelets, citrullinated histone H3, and degree of organization. RESULTS: Vascular wall invasion or small cell clusters of cancer cells was observed in thrombi in 27.5% of deep vein thrombosis and 25.9% of pulmonary embolism cases. The majority of the cancer cells in deep vein thrombi appeared to be invading the vessel wall, whereas the majority of pulmonary thrombi had cancer cell clusters, consistent with embolization via blood flow. These cancer cells were immunohistochemically positive for TF (tissue factors) or podoplanin in up to 88% of VTE cases. The frequency of TF-positive monocyte/macrophages in thrombi was higher in cancer-associated VTE than that in VTE without cancer. Citrullinated histone H3 was predominantly observed in the early stages of organizing thrombi. There was no significant difference in thrombus components between VTE with cancer and without cancer groups. CONCLUSIONS: Vascular wall invasion or cancer cell clusters in thrombi might influence thrombogenesis of cancer-associated VTE. TF and podoplanin in cancer cells and in monocyte/macrophages may induce coagulation reactions and platelet aggregation. Neutrophil extracellular traps may play a role in the early stages of VTE, regardless of cancer status.

Aging Is Associated With Organ-Specific Alterations in the Level and Expression Pattern of von Willebrand Factor.

BACKGROUND: VWF (von Willebrand factor) is an endothelial-specific procoagulant protein with a major role in thrombosis. Aging is associated with increased circulating levels of VWF, which presents a risk factor for thrombus formation. METHODS: Circulating plasma, cellular protein, and mRNA levels of VWF were determined and compared in young and aged mice. Major organs were subjected to immunofluorescence analyses to determine the vascular pattern of VWF expression and the presence of platelet aggregates. An in vitro model of aging, using extended culture time of endothelial cells, was used to explore the mechanism of age-associated increased VWF levels. RESULTS: Increased circulating plasma levels of VWF with elevated levels of larger multimers, indicative of VWF functional activity, were observed in aged mice. VWF mRNA and cellular protein levels were significantly increased in the brains, lungs, and livers but not in the kidneys and hearts of aged mice. Higher proportion of small vessels in brains, lungs, and livers of aged mice exhibited VWF expression compared with young, and this was concomitant with increased platelet aggregate formation. Prolonged culture of endothelial cells resulted in increased cell senescence that correlated with increased VWF expression; VWF expression was specifically detected in senescent cultured endothelial cells and abolished in response to p53 knockdown. A significantly higher proportion of VWF expressing endothelial cells in vivo exhibited senescence markers SA-ß-Gal (senescence-associated ß-galactosidase) and p53 in aged mouse brains compared with that of the young. CONCLUSIONS: Aging elicits a heterogenic response in endothelial cells with regard to VWF expression, leading to organ-specific increase in VWF levels and alterations in vascular tree pattern of expression. This is concomitant with increased platelet aggregate formation. The age-associated increase in VWF expression may be modulated through the process of cell senescence, and p53 transcription factor contributes to its regulation.

A Hematoma in the Anterior Abdominal Muscle in a Woman Receiving Venlafaxine: A Literature Review of the Reports on Similar Cases.

INTRODUCTION: Venlafaxine (VEN) is a selective norepinephrine reuptake inhibitor (SNRI) that mainly helps treat major depressive disorder and anxiety and panic disorders. It works by inhibiting the reuptake of serotonin (5-hydroxytryptamine, 5-HT) and noradrenaline (NA) by presynaptic neurons. Additionally, VEN administration has been linked with a bleeding predisposition that may be due to the inhibition of NA and 5-HT uptake by platelets which have their own receptors on their surface and are implicated in platelet aggregation. CASE PRESENTATION: Herein, we report a case of a 54-year-old patient treated with VEN, who presented with a hematoma in the anterior abdominal muscle. We also present the observational studies and case reports highlighting the association of SNRIs use with various hemorrhagic complications ranging from gastrointestinal hemorrhage or vaginal bleeding to bleeding during or after surgery due to either thrombocytopenia or impaired platelet aggregation. CONCLUSION: Given the cases of either reductions in the platelet count or impairment of platelet activity accompanied by bleeding events, every clinician should be aware of these possible adverse effects when prescribing SNRIs.

Inhibition of mitochondrial calcium transporters alters adp-induced platelet responses.

INTRODUCTION: ADP-stimulated elevation of cytosolic Ca2+ is an important effector mechanism for platelet activation. The rapidly elevating cytosolic Ca2+ is also transported to mitochondrial matrix via Mitochondrial Ca2+ Uniporter (MCU) and extruded via Na+/Ca2+/Li+ Exchanger (NCLX). However, the exact contribution of MCU and NCLX in ADP-mediated platelet responses remains incompletely understood. METHODS AND RESULTS: The present study aimed to elucidate the role of mitochondrial Ca2+ transport in ADP-stimulated platelet responses by inhibition of MCU and NCLX with mitoxantrone (MTX) and CGP37157 (CGP), respectively. As these inhibitory strategies are reported to cause distinct effects on matrix Ca2+ concentration, we hypothesized to observe opposite impact of MTX and CGP on ADP-induced platelet responses. Platelet aggregation profiling was performed by microplate-based spectrophotometery while p-selectin externalization and integrin αIIbß3 activation were analyzed by fluorescent immunolabeling using flow cytometery. Our results confirmed the expression of both MCU and NCLX mRNAs with relatively low abundance of NCLX in human platelets. In line with our hypothesis, MTX caused a dose-dependent inhibition of ADP-induced platelet aggregation without displaying any cytotoxicity. Likewise, ADP-induced p-selectin externalization and integrin αIIbß3 activation was also significantly attenuated in MTX-treated platelets. Concordantly, inhibition of NCLX with CGP yielded an accelerated ADP-stimulated platelet aggregation which was associated with an elevation of p-selectin surface expression and αIIbß3 activation. CONCLUSION: Together, these findings uncover a vital and hitherto poorly characterized role of mitochondrial Ca2+ transporters in ADP-induced platelet activation.

Low on-clopidogrel ADP- and TRAP-6-induced platelet aggregation in patients with atrial fibrillation undergoing percutaneous coronary intervention: an observational pilot study.

High on-clopidogrel platelet reactivity (HPR) associates with ischemic risk in patients after percutaneous intervention (PCI). This study aimed to evaluate the association of HPR as assessed by multiple electrode aggregometry (MEA) with ischemic, thromboembolic, and bleeding risk in patients with atrial fibrillation (AF) undergoing PCI. Patients with AF and an indication for oral anticoagulation (OAC) were included in this prospective cohort study on day 1-3 after PCI. Platelet aggregation [U] was analyzed by MEA. HPR and low platelet reactivity (LPR) were defined as ADP-induced aggregation ≥ 46 U and ≤ 18 U, respectively. TRAP-6-induced aggregation reference was 94-156 U. The primary outcome was time to all-cause death, myocardial infarction, or stroke at 6 months. The secondary outcome was time to non-major clinically relevant bleedings or major bleedings. 159 patients were enrolled between May 2020 and May 2021. The median age was 78 years (interquartile range 72-82) and 111 (70%) were male. Median ADP- and TRAP-induced aggregation were 12 (6-17) and 49 (35-68) U, respectively. 147 (93%) patients had a low overall aggregability. HPR was detected in 2 patients (1%) and 125 (79%) had LPR. ADP-induced aggregation did not significantly associate with the primary outcome (r = 0.081, p = 0.309) but correlated inversely with bleeding risk (r = - 0.201, p = 0.011). HPR status as assessed by MEA among patients with AF after PCI was rare and overall aggregability was low. Conventional cut-off values for HPR might be inappropriate for these patients. ADP-induced aggregation might be helpful to identify patients at risk for bleeding.