Heterogeneity of platelets and their responses.

There has been increasing recognition of heterogeneity in blood platelets and their responses, particularly in recent years, where next-generation technologies and advanced bioinformatic tools that interrogate "big data" have enabled large-scale studies of RNA and protein expression across a growing list of disease states. However, pioneering platelet biologists and clinicians were already hypothesizing upon and investigating heterogeneity in platelet (and megakaryocyte) activity and platelet metabolism and aggregation over half a century ago. Building on their foundational hypotheses, in particular Professor Marian A. Packham's pioneering work and a State of the Art lecture in her memoriam at the 2023 International Society on Thrombosis and Haemostasis Congress by Anandi Krishnan, this review outlines the key features that contribute to the heterogeneity of platelets between and within individuals. Starting with important epidemiologic factors, we move stepwise through successively smaller scales down to heterogeneity revealed by single-cell technologies in health and disease. We hope that this overview will urge future scientific and clinical studies to recognize and account for heterogeneity of platelets and aim to apply methods that capture that heterogeneity. Finally, we summarize other exciting new data presented on this topic at the 2023 International Society on Thrombosis and Haemostasis Congress.

The effect of UVA light/8-methoxypsoralen exposure used in Extracorporeal Photopheresis treatment on platelets and extracellular vesicles.

Extracorporeal Photopheresis (ECP) is a leukapheresis based treatment for Cutaneous T-Cell Lymphoma, which takes advantage of the cellular lethal effects of UVA light in combination with a photoactivated drug, 8-methoxypsoralen. 25% of patients treated with ECP do not respond to treatment, however the underlying mechanisms for this lack of response remain unknown. Platelets, a rich source of extracellular vesicles (EVs) and key mediators in thromboinflammatory oncological progression, as well as leukocytes, are both processed through ECP and are subsequently transfused back into the patient, delivering potent immunomodulation. The effect of exposing platelets and their EVs directly to Ultra Violet A light (UVA)/8-methoxypsoralen is currently unknown. Platelet-rich plasma (PRP) was isolated from healthy donors and exposed to UVA light and/or 8-methoxysporalen in vitro and platelet activation and aggregation was assessed. EV size and concentration were also characterised by Nanoparticle Tracking Analysis and Flow Cytometry. We found that UVA light and 8-methoxypsoralen treatment in vitro does not induce platelet aggregation or significantly alter levels of the platelet activation markers, soluble P-selectin or platelet factor 4, with circulating levels of small and large EV size and concentration remaining constant. Therefore, utilising the combination of UVA light and 8-methoxypsoralen used in ECP in vitro does not activate platelets or alter important circulating EVs. Further studies will be needed to validate if our observations are consistent in vivo.

Proteomic analysis of extracellular vesicle cargoes mirror the cardioprotective effects of rivaroxaban in patients with venous thromboembolism.

BACKGROUND: Venous thromboembolism (VTE) remains a significant cause of morbidity and mortality worldwide. Rivaroxaban, a direct oral factor Xa inhibitor, mediates anti-inflammatory and cardiovascular-protective effects besides its well-established anticoagulant properties; yet, these remain poorly characterized. Extracellular vesicles (EVs) are considered proinflammatory messengers regulating a myriad of (patho)physiological processes and may be highly relevant to the pathophysiology of VTE. The effects of Rivaroxaban on circulating EVs in VTE patients remain unknown. We have established that differential EV biosignatures are found in patients with non-valvular atrial fibrillation anticoagulated with Rivaroxaban versus warfarin. Here, we investigated whether differential proteomic profiles of circulating EVs could also be found in patients with VTE. METHODS AND RESULTS: We performed comparative label-free quantitative proteomic profiling of enriched plasma EVs from VTE patients anticoagulated with either Rivaroxaban or warfarin using a tandem mass spectrometry approach. Of the 182 quantified proteins, six were found to be either exclusive to, or enriched in, Rivaroxaban-treated patients. Intriguingly, these proteins are involved in negative feedback regulation of inflammatory and coagulation pathways, suggesting that EV proteomic signatures may reflect both Rivaroxaban's anti-coagulatory and anti-inflammatory potential. CONCLUSIONS: These differences suggest Rivaroxaban may have pleiotropic effects, supporting the reports of its emerging anti-inflammatory and cardiovascular-protective characteristics relative to warfarin.

Platelet proteo-transcriptomic profiling validates mediators of thrombosis and proteostasis in patients with myeloproliferative neoplasms.

Patients with chronic Myeloproliferative Neoplasms (MPN) including polycythemia vera (PV) and essential thrombocythemia (ET) exhibit unique clinical features, such as a tendency toward thrombosis and hemorrhage, and risk of disease progression to secondary bone marrow fibrosis and/or acute leukemia. Although an increase in blood cell lineage counts (quantitative features) contribute to these morbid sequelae, the significant qualitative abnormalities of myeloid cells that contribute to vascular risk are not well understood. Here, we address this critical knowledge gap via a comprehensive and untargeted profiling of the platelet proteome in a large (n= 140) cohort of patients (from two independent sites) with an established diagnosis of PV and ET (and complement prior work on the MPN platelet transcriptome from a third site). We discover distinct MPN platelet protein expression and confirm key molecular impairments associated with proteostasis and thrombosis mechanisms of potential relevance to MPN pathology. Specifically, we validate expression of high-priority candidate markers from the platelet transcriptome at the platelet proteome (e.g., calreticulin (CALR), Fc gamma receptor (FcγRIIA) and galectin-1 (LGALS1) pointing to their likely significance in the proinflammatory, prothrombotic and profibrotic phenotypes in patients with MPN. Together, our proteo-transcriptomic study identifies the peripherally-derived platelet molecular profile as a potential window into MPN pathophysiology and demonstrates the value of integrative multi-omic approaches in gaining a better understanding of the complex molecular dynamics of disease.

Case Report: Hypergranular Platelets in Vaccine-Induced Thrombotic Thrombocytopenia After ChAdOx1 nCov-19 Vaccination.

BACKGROUND: Vaccine-induced thrombotic thrombocytopenia (VITT) post SARS-CoV-2 vaccination is characterized by thrombocytopenia and severe thrombosis. Platelet function during patient recovery in the medium-/long-term has not been investigated fully. Here, we undertook a 3-month study, assessing the recovery of a VITT patient and assessing platelet morphology, granule content and dense-granule release at two distinct time points during recovery. CASE PRESENTATION: A 61 year-old female was admitted to hospital 15 days post ChAdOx1 nCov-19 vaccination. Hematological parameters and peripheral blood smears were monitored over 3 months. Platelet morphology and granule populations were assessed using transmission electron microscopy (TEM) at two distinct time points during recovery, as was agonist-induced platelet dense-granule release. Upon admission, the patient had reduced platelet counts, increased D-dimer and high anti-PF4 antibodies with multiple sites of cerebral venous sinus thrombosis (CVST). Peripheral blood smears revealed the presence of large, hypergranular platelets. Following treatment, hematological parameters returned to normal ranges over the study period. Anti-PF4 antibodies remained persistently high up to 90 days post-admission. Two days after admission, VITT platelets contained more granules per-platelet when compared to day 72 and healthy platelets. Additionally, maximal ATP release (marker of dense-granule release) was increased on day 2 compared to day 72 and healthy control platelets. CONCLUSION: This study highlights a previously unreported observation of platelet hypergranularity in VITT which may contribute to the thrombotic risk associated with VITT. Optimal approaches to monitoring recovery from VITT over time remains to be determined but our findings may help inform therapeutic decisions relating to anticoagulation treatment in this novel pathology.

Non-severe COVID-19 is associated with endothelial damage and hypercoagulability despite pharmacological thromboprophylaxis.

BACKGROUND: Hypercoagulability and endothelial dysfunction are hallmarks of coronavirus disease 2019 (COVID-19) and appear to predict disease severity. A high incidence of thrombosis despite thromboprophylaxis is reported in patients with moderate to severe COVID-19. Recent randomized clinical trials suggest that therapeutic-intensity heparin confers a survival benefit in moderate-severity COVID-19 compared to standard-intensity heparin, potentially by harnessing heparin-mediated endothelial-stabilizing and anti-inflammatory effects. OBJECTIVE: We hypothesized that patients with moderate-severity COVID-19 exhibit enhanced hypercoagulability despite standard-intensity thromboprophylaxis with low molecular weight heparin (LMWH) compared to non-COVID-19 hospitalized patients. METHODS: Patients with moderate COVID-19 and a control group (severe acute respiratory syndrome coronavirus 2 [SARS-CoV-2]-negative hospitalized patients) receiving LMWH thromboprophylaxis were recruited. Markers of endothelial damage and plasma thrombin generation parameters were assessed. RESULTS: Tissue plasminogen activator levels were significantly increased in the COVID-19 group (8.3 ± 4.4 vs. 4.9 ± 2.4 ng/ml; P = .02) compared to non-COVID-19-hospitalized patients. Despite thromboprophylaxis, mean endogenous thrombin potential was significantly increased among COVID-19 patients (1929 ± 448 vs. 1528 ± 460.8 nM*min; P = .04) but lag time to thrombin generation was significantly prolonged (8.1 ± 1.8 vs. 6.2 ± 1.8 mins; P = .02). While tissue factor pathway inhibitor (TFPI) levels were similar in both groups, in the presence of an inhibitory anti-TFPI antibody, the difference in lag time between the groups was abrogated. CONCLUSIONS: Collectively, these data demonstrate that COVID-19 of moderate severity is associated with increased plasma thrombin generation and endothelial damage, and that hypercoagulability persists despite standard LMWH thromboprophylaxis. These findings may be of clinical interest given recent clinical trial data which suggest escalated heparin dosing in non-severe COVID-19 may be associated with improved clinical outcomes.

Ruling out Pulmonary Embolism in Patients with (Suspected) COVID-19-A Prospective Cohort Study.

Background Diagnostic strategies for suspected pulmonary embolism (PE) have not been prospectively evaluated in COVID-19 patients. Methods Prospective, multicenter, outcome study in 707 patients with both (suspected) COVID-19 and suspected PE in 14 hospitals. Patients on chronic anticoagulant therapy were excluded. Informed consent was obtained by opt-out approach. Patients were managed by validated diagnostic strategies for suspected PE. We evaluated the safety (3-month failure rate) and efficiency (number of computed tomography pulmonary angiographies [CTPAs] avoided) of the applied strategies. Results Overall PE prevalence was 28%. YEARS was applied in 36%, Wells rule in 4.2%, and "CTPA only" in 52%; 7.4% was not tested because of hemodynamic or respiratory instability. Within YEARS, PE was considered excluded without CTPA in 29%, of which one patient developed nonfatal PE during follow-up (failure rate 1.4%, 95% CI 0.04-7.8). One-hundred seventeen patients (46%) managed according to YEARS had a negative CTPA, of whom 10 were diagnosed with nonfatal venous thromboembolism (VTE) during follow-up (failure rate 8.8%, 95% CI 4.3-16). In patients managed by CTPA only, 66% had an initial negative CTPA, of whom eight patients were diagnosed with a nonfatal VTE during follow-up (failure rate 3.6%, 95% CI 1.6-7.0). Conclusion Our results underline the applicability of YEARS in (suspected) COVID-19 patients with suspected PE. CTPA could be avoided in 29% of patients managed by YEARS, with a low failure rate. The failure rate after a negative CTPA, used as a sole test or within YEARS, was non-negligible and reflects the high thrombotic risk in these patients, warranting ongoing vigilance.

Routine Hematological Parameters May Be Predictors of COVID-19 Severity.

To date, coronavirus disease 2019 (COVID-19) has affected over 100 million people globally. COVID-19 can present with a variety of different symptoms leading to manifestation of disease ranging from mild cases to a life-threatening condition requiring critical care-level support. At present, a rapid prediction of disease severity and critical care requirement in COVID-19 patients, in early stages of disease, remains an unmet challenge. Therefore, we assessed whether parameters from a routine clinical hematology workup, at the time of hospital admission, can be valuable predictors of COVID-19 severity and the requirement for critical care. Hematological data from the day of hospital admission (day of positive COVID-19 test) for patients with severe COVID-19 disease (requiring critical care during illness) and patients with non-severe disease (not requiring critical care) were acquired. The data were amalgamated and cleaned and modeling was performed. Using a decision tree model, we demonstrated that routine clinical hematology parameters are important predictors of COVID-19 severity. This proof-of-concept study shows that a combination of activated partial thromboplastin time, white cell count-to-neutrophil ratio, and platelet count can predict subsequent severity of COVID-19 with high sensitivity and specificity (area under ROC 0.9956) at the time of the patient's hospital admission. These data, pending further validation, indicate that a decision tree model with hematological parameters could potentially form the basis for a rapid risk stratification tool that predicts COVID-19 severity in hospitalized patients.

Posttransplant Lymphoproliferative Disorder After Solid Organ Transplant: A Heterogeneous, Aggressive Disorder.

Posttransplant lymphoproliferative disorder (PTLD) is a rare complication of solid organ transplant. We identified 40 patients diagnosed with PTLD between 2009 and 2020 and analyzed their presentation, treatment strategies, and outcomes. Median age at diagnosis was 52.5 years (range 21.3 to 79). Median duration of immunosuppression was 95 months (range 4 to 292). Diffuse large B cell lymphoma (n = 16, 40%) and Burkitt lymphoma (n = 6, 15%) were the most common histological subtypes. First-line therapy varied. The median number of treatment lines was 1 (range 0 to 4). Sixteen patients (40%) achieved complete response after first-line therapy. Nineteen patients (47.5%) relapsed or progressed and received salvage therapy; 45% were alive at the end of the study period (median survival 52 months; range 1 to 266; 95% confidence interval 0 to 104). Causes of death included lymphoma-related (45.5%), therapy-related (27.3%), and other (27.3%). Five (22.7%) died within 3 months of diagnosis. Pearson's r test identified disease stage (P = .045) and proliferation index (P = .005) as negative predictors of response to frontline therapy. Bone marrow involvement (P = .033) and increased age (P = .018) were significant predictors of survival. Early mortality and poor response to frontline therapy are common, outlining the need for improved treatment strategies.

Nonvalvular atrial fibrillation patients anticoagulated with rivaroxaban compared with warfarin exhibit reduced circulating extracellular vesicles with attenuated pro-inflammatory protein signatures.

BACKGROUND: Rivaroxaban, a direct oral factor Xa inhibitor, mediates anti-inflammatory and cardiovascular-protective effects besides its well-established anticoagulant properties; however, these remain poorly characterized. Extracellular vesicles (EVs) are important circulating messengers regulating a myriad of biological and pathological processes and may be highly relevant to the pathophysiology of atrial fibrillation as they reflect alterations in platelet and endothelial biology. However, the effects of rivaroxaban on circulating pro-inflammatory EVs remain unknown. OBJECTIVES: We hypothesized that rivaroxaban's anti-inflammatory properties are reflected upon differential molecular profiles of circulating EVs. METHODS: Differences in circulating EV profiles were assessed using a combination of single vesicle analysis by Nanoparticle Tracking Analysis and flow cytometry, and proteomics. RESULTS: We demonstrate, for the first time, that rivaroxaban-treated non-valvular atrial fibrillation (NVAF) patients (n=8) exhibit attenuated inflammation compared with matched warfarin controls (n=15). Circulating EV profiles were fundamentally altered. Moreover, quantitative proteomic analysis of enriched plasma EVs from six pooled biological donors per treatment group revealed a profound decrease in highly pro-inflammatory protein expression and complement factors, together with increased expression of negative regulators of inflammatory pathways. Crucially, a reduction in circulating levels of soluble P-selectin was observed in rivaroxaban-treated patients (compared with warfarin controls), which negatively correlated with the patient's time on treatment. CONCLUSION: Collectively, these data demonstrate that NVAF patients anticoagulated with rivaroxaban (compared with warfarin) exhibit both a reduced pro-inflammatory state and evidence of reduced endothelial activation. These findings are of translational relevance toward characterizing the anti-inflammatory and cardiovascular-protective mechanisms associated with rivaroxaban therapy.

COVID-19 induces a hyperactive phenotype in circulating platelets.

Coronavirus Disease 2019 (COVID-19), caused by the novel Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), has affected over 30 million globally to date. Although high rates of venous thromboembolism and evidence of COVID-19-induced endothelial dysfunction have been reported, the precise aetiology of the increased thrombotic risk associated with COVID-19 infection remains to be fully elucidated. Therefore, we assessed clinical platelet parameters and circulating platelet activity in patients with severe and nonsevere COVID-19. An assessment of clinical blood parameters in patients with severe COVID-19 disease (requiring intensive care), patients with nonsevere disease (not requiring intensive care), general medical in-patients without COVID-19, and healthy donors was undertaken. Platelet function and activity were also assessed by secretion and specific marker analysis. We demonstrated that routine clinical blood parameters including increased mean platelet volume (MPV) and decreased platelet:neutrophil ratio are associated with disease severity in COVID-19 upon hospitalisation and intensive care unit (ICU) admission. Strikingly, agonist-induced ADP release was 30- to 90-fold higher in COVID-19 patients compared with hospitalised controls and circulating levels of platelet factor 4 (PF4), soluble P-selectin (sP-selectin), and thrombopoietin (TPO) were also significantly elevated in COVID-19. This study shows that distinct differences exist in routine full blood count and other clinical laboratory parameters between patients with severe and nonsevere COVID-19. Moreover, we have determined all COVID-19 patients possess hyperactive circulating platelets. These data suggest abnormal platelet reactivity may contribute to hypercoagulability in COVID-19 and confirms the role that platelets/clotting has in determining the severity of the disease and the complexity of the recovery path.

Pathophysiology of the Venous Thromboembolism Risk in Preeclampsia.

Preeclampsia complicates up to 8% of pregnancies and is a leading cause of fetomaternal morbidity andmortality. Treatment options are limited, with supportive care and delivery of the placenta representing the cornerstone of current management strategies. Derangements in blood coagulation are wellrecognised in this disorder and appear to favour an increased risk of venous thromboembolism among affected women. This risk appears to be most significant in the postpartum period. The mechanisms underlying this increased thrombosis risk remain to be fully elucidated although increased expression of procoagulant factors, endothelial dysfunction, attenuation of endogenous anticoagulant activity and increased platelet activity have been implicated in the prothrombotic tendency. Preeclampsia is also occasionally complicated by life-threatening haemorrhagic events and current evidence suggests that in some severe manifestations of this disease a coagulopathy with a clinical bleeding tendency may be the predominant haemostatic abnormality. Identifying affected women at significant risk of thrombosis and managing the competing thrombotic and haemorrhagic risks continue to be a significant clinical challenge. Derangements in blood coagulation are also implicated in the pathogenesis of preeclampsia; however, the role of antiplatelet or anticoagulant drugs in the prevention and treatment of this disorder remains a source of considerable debate. In addition, the potential role of specific haemostatic markers as diagnostic or screening tools for preeclampsia has also yet to be determined. Further characterisation of the underlying molecular mechanisms would likely be of major translational relevance and could provide insights into the pathogenesis of this disease as well as the associated haemostatic dysfunction.

Implementation of an acute DVT ambulatory care pathway in a large urban centre: current challenges and future opportunities.

BACKGROUND: Ambulatory management of isolated acute deep venous thrombosis (DVT) is the recommended standard of care in selected populations. However, in practice a significant number of patients continue to be managed as in-patients. OBJECTIVES: In this study we aimed to evaluate acute DVT treatment pathways in our emergency department (ED) in practice and to identify barriers to outpatient management. METHODS: This study was a cross-sectional analysis of prospectively collected data pertaining to consecutive patients presenting to the ED of a large, city center, academic teaching hospital over a 46 week period who were diagnosed with DVT. RESULTS: Implementation of an outpatient care pathway led to the majority of patients presenting with DVT in our institution being treated without hospital admission. Forty percent (31/78) of patients with DVT were treated with a direct oral anticoagulant (DOAC) as an outpatient in line with international best practice guidelines. CONCLUSION: The study provides a clear picture of the clinical profile and management of patients in clinical practice. Due to the lack of resources and supported infrastructure it is difficult to effectively implement outpatient venous thromboembolism (VTE) management to its full potential. Directing resources towards strategies which facilitate outpatient DVT treatment among vulnerable patient groups could represent a means of reducing hospital admissions for DVT in urban centers. Our study highlights the success and clinical limitations of the outpatient treatment model, which should become standard as part of wider VTE care.