The Future of Laboratory-Developed Tests in Hemostasis Laboratories.

Laboratory-developed tests (LDTs) are widely used in clinical hemostasis laboratories. The extent to which LDTs are regulated varies greatly around the world, and proposed changes to regulations have raised concerns about the future of LDTs in clinical laboratories. It is increasingly difficult to justify the use of an LDT where a commercially available method with regulatory approval is available. Conversely, where there is no suitable test with regulatory approval and there is a compelling clinical need, using an LDT outweighs the risk associated with not performing the test. We argue that LDTs are still required in specialist clinical laboratories to fulfill unmet clinical needs, and in lower middle-income countries where they are a vital resource.

Automated measurement of coagulation and fibrinolytic activation markers: Outcomes in coronavirus disease 2019 (COVID-19) patients.

BACKGROUND: Severe coronavirus disease 2019 (COVID-19) is characterized by marked hypoxaemia and lung oedema, often accompanied by disordered blood coagulation and fibrinolytic systems, endothelial damage and intravascular fibrin deposition. PATIENTS/METHODS: We present a retrospective observational study of 104 patients admitted to hospital with COVID-19. Plasma samples were collected within 72 h of admission. In addition to routine coagulation and haematology testing, soluble thrombomodulin (sTM), thrombin-antithrombin (TAT), tissue plasminogen activator-plasminogen activator inhibitor 1 complex (tPAI-C) and plasmin-α2 antiplasmin complex (PIC) were performed by automated chemiluminescent enzyme immunoassays. RESULTS: Significantly higher levels of D-dimer, TAT, sTM and tPAI-C were observed in non-survivors compared to survivors. To confirm which parameters were independent risk factors for mortality, multiple logistic regression was performed on D-dimer, TAT. sTM, tPAI-C and PIC data. Only increasing sTM was significantly associated with mortality, with an odds ratio of 1.065 for each 1.0 TU/mL increment (95% CI 1.025-1.115). CONCLUSIONS: Of the haemostatic variables measured, sTM, which can be rapidly assayed, is the best independent predictor of mortality in patients hospitalized with COVID-19, and this suggests that endothelial dysfunction plays an important role in disease progression.

A performance evaluation of chemiluminescence enzyme immunoassays on the Sysmex CN-6500 haemostasis analyser.

BACKGROUND: The Sysmex CN-6500 is a new haemostasis analyser with an integrated immunoassay module that performs chemiluminescence enzyme assay (CLEIA) in addition to coagulation, turbidimetric, chromogenic and platelet aggregation tests. AIMS: To evaluate the analytical performance of the CN-6500 against the predicate device (Sysmex HISCL-800) for soluble thrombomodulin (TM), thrombin-antithrombin (TAT), tissue plasminogen activator/plasminogen activator inhibitor 1 complex (tPAI-C) and plasmin α2 plasmin inhibitor complex (PIC) assays. METHODS: Imprecision was assessed by testing two levels of quality control plasmas 10 times on 5 separate days. Comparability was studied in 230 plasmas from normal donors (n = 30), patients with suspected disseminated intravascular coagulation (DIC, n = 100), sepsis (n = 20) or liver disease (n = 20), lipaemic (n = 20), haemolysed (n = 20) and icteric samples (n = 20). Limit of detection, limit of quantitation and linearity were determined by testing serial dilutions of normal plasma. Sample carryover was assessed by testing samples with high and low normal levels of the analytes concerned. RESULTS: The CN-6500 performed 21 CLEIA tests per hour, while simultaneously performing coagulation tests. Acceptable between-run imprecision was obtained using commercial controls with normal and high activity for each analyte (%CV <4%), for all four assays. Excellent linearity was observed (slope 0.89-1.03; r2 >0.99) across the measurement range. The lower limits of detection and quantitation were as follows: TM <0.3/0.6 TU/ml, TAT >0.1/<0.2 ng/ml, PIC <0.004/<0.008 µg/ml and tPAI-C < 0.01/<0.1 ng/ml, respectively. All four assays showed excellent correlation between analysers and were unaffected by haemolysis, icterus or lipaemia. No carryover was observed. CONCLUSIONS: Our data demonstrate that the performance of the CLEIA assays on the CN-6500 is comparable to that of a stand-alone immunoassay analyser.

Pathological lesions of the digestive tract in free-ranging mountain gorillas (Gorilla beringei beringei).

The finding of parasites and bacterial pathogens in mountain gorilla feces and oral lesions in gorilla skeletal remains has not been linked to pathological evidence of morbidity or mortality. In the current study, we conducted a retrospective study of digestive tracts including oral cavity, salivary glands, esophagus, stomach, intestines (gastrointestinal tract [GI]), liver, and pancreas of 60 free-ranging mountain gorillas from Uganda, Rwanda, and the Democratic Republic of Congo that died between 1985 and 2007. We reviewed clinical histories and gross pathology reports and examined histological sections. On histology, enteritis (58.6%), gastritis (37.3%), and colitis (29.3%) were the commonest lesions in the tracts. Enteritis and colitis were generally mild, and judged likely to have been subclinical. Gastritis was often chronic and proliferative or ulcerative, and associated with nematodiasis. A gastro-duodenal malignancy (carcinoid) was present in one animal. A number of incidental lesions were identified throughout the tract and cestodes and nematodes were frequently observed grossly and/or histologically. Pigmentation of teeth and tongue were a common finding, but periodontitis and dental attrition were less common than reported from past studies of skeletal remains. Despite observing numerous GI lesions and parasites in this study of deceased free-living mountain gorillas, we confirmed mortality attributable to gastroenteritis in just 8% (5/60) cases, which is less than that described in captive gorillas. Other deaths attributed to digestive tract lesions included cleft palate in an infant, periodontal disease causing systemic infection in an older adult and gastric cancer. Of all the parasitic infections observed, only hepatic capillariasis and gastric nematodiasis were significantly associated with lesions (hepatitis and gastritis, respectively). Understanding GI lesions in this endangered species is key in the management of morbidity associated with GI ailments.

Parasitic encephalitis caused by Stephanurus dentatus in a pig in Brazil.

A pig was in left lateral recumbency with limb spasticity, accentuated prostration, and strabismus, and was euthanized. During autopsy, yellowing of the leptomeninges at the ventral pons to medulla oblongata was noted. In the cerebellar peduncles, there was a focally extensive black-to-yellow area at the level of the vestibular nuclei. Histologic examination revealed a cross-section of a nematode larva, consistent with Stephanurus dentatus, bordered by edema and marked infiltration of mononuclear cells, plasma cells, and a few eosinophils. Vacuolation of the neuropil, with rare gitter cells and axonal spheroids, was also observed. We diagnosed parasitic encephalitis caused by S. dentatus migration based on the pathology findings and characterization of the parasite.

Platelet-enhanced plasma: Characterization of a novel candidate resuscitation fluid's extracellular vesicle content, clotting parameters, and thrombin generation capacity.

BACKGROUND: Platelet transfusion is challenging in emergency medicine because of short platelet shelf life and stringent storage conditions. Platelet-derived extracellular vesicles (PEV) exhibit platelet-like properties. A plasma generated from expired platelet units rich in procoagulant PEV may be able to combine the benefits of plasma and platelets for resuscitation while increasing shelf life and utilizing an otherwise wasted resource. STUDY DESIGN AND METHODS: Freeze-thaw cycling of platelet-rich plasma (PRP) followed by centrifugation to remove platelet remnants was utilized to generate platelet-enhanced plasma (PEP). An in vitro model of dilutional coagulopathy was also designed and used to test PEP. Rotational thromboelastometry and calibrated automated thrombography were used to assess clotting and extracellular vesicles (EV) procoagulant activity. Capture arrays were used to specifically measure EV subpopulations of interest (ExoView™, NanoView Biosciences). Captured vesicles were quantified and labeled with Annexin-V-FITC, CD41-PE, and CD63-AF647. Platelet alpha granule content (platelet-derived growth factor AB, soluble P-selectin, vascular endothelial growth factor A, and neutrophil activating peptide 2-chemokine (C-X-C motif) ligand 7) was measured. Commercially available platelet lysates were also characterized. RESULTS: PEP is highly procoagulant, rich in growth factors, exhibits enhanced thrombin generation, and restores hemostasis within an in vitro model of dilutional coagulopathy. The predominant vesicle population were PEV with 7.0 × 109 CD41+PS+ EV/ml compared to 4.7 × 107 CD41+PS+ EV/ml in platelet-free plasma (p = .0079). Commercial lysates show impaired but rescuable clotting. DISCUSSION: PEP is a unique candidate resuscitation fluid containing high PEV concentration with preliminary evidence, indicating a potential for upscaling the approach using platelet concentrates. Commercial lysate manufacturer workflows may be suitable for this, but further optimization and characterization of PEP is required.

International Council for Standardization in Haematology (ICSH) laboratory guidance for the verification of haemostasis analyser-reagent test systems. Part 2: Specialist tests and calibrated assays.

Before a new method is used for clinical testing, it is essential that it is evaluated for suitability for its intended purpose. This document gives guidance for the performance, verification and implementation processes required by regulatory and accreditation bodies. It covers the planning and verification of specialist haemostatic tests, including factor assays, D-dimers, direct anticoagulants and thrombophilia testing.

International Council for Standardization in Haematology (ICSH) laboratory guidance for the evaluation of haemostasis analyser-reagent test systems. Part 1: Instrument-specific issues and commonly used coagulation screening tests.

Before a new method is used for clinical testing, it is essential that it is evaluated for suitability for its intended purpose. This document gives guidance for the performance of verification, validation and implementation processes required by regulatory and accreditation bodies. It covers the planning and execution of an evaluation of the commonly performed screening tests (prothrombin time, activated partial thromboplastin time, thrombin time and fibrinogen assay), and instrument-specific issues. Advice on selecting an appropriate haemostasis analyser, planning the evaluation, and assessing the reference, interval, precision, accuracy, and comparability of a haemostasis test system are also given. A second companion document will cover specialist haemostasis testing.

Histopathology and microscopic morphology of protozoan and metazoan parasites of free ranging armadillos in Brazil

ABSTRACT: This study assessed microscopic morphology of protozoan and metazoan parasites, as well as parasite-associated histopathologic changes in five Brazilian free-ranging armadillos. Three armadillos had intra sarcolemmal cysts of Sarcocystis sp. in skeletal muscles without microscopic changes. One Dasypus novemcinctus was found parasitized with a nematode morphologically compatible with an oxyurid in the small intestine. One Dasypus sp. had neutrophilic enteritis associated with adult and larval stages of Strongyloides sp. and one D. novemcinctus had multiple embryonated eggs free in the lumen of the small intestine with mild neutrophilic enteritis. These findings represent a contribution for expanding our knowledge on parasitic diseases of armadillos.

RESUMO: Este estudo avaliou a morfologia microscópica de parasitos protozoários e metazoários, bem como lesões associadas ao parasitismo em cinco tatus de vida livre no Brasil. Três tatus tinham cistos de Sarcocystis sp. Intra-sarcolemal em músculos esqueléticos sem alterações microscópicas. Um Dasypus novemcinctus estava parasitado com um nematodo morfologicamente compatível com oxiurideo no intestino delgado. Um Dasypus sp. apresentou enterite neutrofílica associada com estágios larvais de Strongyloides sp. e um D. novemcinctus apresentou múltiplos ovos embrionados livres no lúmen do intestino delgado, associado a enterite neutrofílica discreta. Estes achados representam uma contribuição para a expansão do conhecimento sobre doenças parasitárias de tatus.

Histopathology and microscopic morphology of protozoan and metazoan parasites of free ranging armadillos in Brazil / Histopatologia e morfologia microscópica de parasitos protozoários e metazoários de tatus de vida livre no Brasil

This study assessed microscopic morphology of protozoan and metazoan parasites, as well as parasite-associated histopathologic changes in five Brazilian free-ranging armadillos. Three armadillos had intra sarcolemmal cysts of Sarcocystis sp. in skeletal muscles without microscopic changes. One Dasypus novemcinctus was found parasitized with a nematode morphologically compatible with an oxyurid in the small intestine. One Dasypus sp. had neutrophilic enteritis associated with adult and larval stages of Strongyloides sp. and one D. novemcinctus had multiple embryonated eggs free in the lumen of the small intestine with mild neutrophilic enteritis. These findings represent a contribution for expanding our knowledge on parasitic diseases of armadillos.(AU)

Este estudo avaliou a morfologia microscópica de parasitos protozoários e metazoários, bem como lesões associadas ao parasitismo em cinco tatus de vida livre no Brasil. Três tatus tinham cistos de Sarcocystis sp. Intra-sarcolemal em músculos esqueléticos sem alterações microscópicas. Um Dasypus novemcinctus estava parasitado com um nematodo morfologicamente compatível com oxiurideo no intestino delgado. Um Dasypus sp. apresentou enterite neutrofílica associada com estágios larvais de Strongyloides sp. e um D. novemcinctus apresentou múltiplos ovos embrionados livres no lúmen do intestino delgado, associado a enterite neutrofílica discreta. Estes achados representam uma contribuição para a expansão do conhecimento sobre doenças parasitárias de tatus.(AU)

Pathology of Free-Ranging and Captive Brazilian Anteaters.

There is little information on the anatomical pathology of Brazilian anteaters. Considering the relevance of knowledge of diseases of these species for their conservation, the aim of this study was to describe pathological changes in 99 captive and free-ranging anteaters from the Brazilian states of São Paulo, Minas Gerais, Bahia, Mato Grosso do Sul and Amazonas. Forty-two animals were killed on roads and 10 died from burns injuries. Other significant conditions included the metabolic diseases of iron storage disease, tissue mineralization and taurine deficiency, protozoan and metazoan infections, candidiasis, sporotrichosis, clostridiosis and proliferative disorders including squamous cell carcinoma.

A comparative evaluation of the CN-6000 haemostasis analyser using coagulation, amidolytic, immuno-turbidometric and light transmission aggregometry assays.

BACKGROUND: The CN-6000 (Sysmex Corp.) is a new haemostasis analyser with blood coagulation, amidolytic, immuno-turbidometric and light transmission aggregometry (LTA) capabilities. Transmitted light is monitored at multiple wavelengths (340, 405, 575, 660, 800 nm), from an LED light source. AIMS: To evaluate the performance of the CN-6000 against a predicate device. METHODS: The CN-6000 was evaluated against the CS-5100 (Sysmex) for 14 different tests, using 880 samples from normal subjects, anticoagulated patients, critically ill patients, plasmas with high or low fibrinogen content or abnormal levels of interfering substances. Between-day assay imprecision was assessed using commercial QC materials (n = 10 replicates on each of 5 days). RESULTS: Acceptable levels of imprecision were obtained for all assays. Agreement between the two analysers was excellent for all assays. Throughput was 35% higher using the CN-6000 (337 vs 250 tests per hour for PT, aPTT and fibrinogen). The CN-6000 also demonstrated improved clot detection in plasmas with high levels of interfering substances as demonstrated by a 29% reduction in "vote-outs" due to low light transmission (24 vs 34). CONCLUSIONS: The CN-6000 demonstrated excellent comparability with the predicate instrument and acceptable levels of imprecision in all assays. Improvements in throughput and clot detection in the presence of interfering substances were also shown.

A practical method for reducing the interference due to lipaemia in coagulation tests.

INTRODUCTION: Plasma samples with gross lipaemia present a challenge for coagulation laboratories using optical analysers. High-speed centrifugation may be used to remove excess lipids but it has not established whether this affects haemostasis tests. The aims were to determine whether the removal of lipid by centrifugation affects PT, APTT, fibrinogen, D-dimer and von Willebrand factor activity measurements. METHODS: Twenty-six lipaemic samples (median [range]): triglyceride 4.6 mmol/L [0.5-17.0]; cholesterol: 4.06 mmol/L [2.20-9.41] and 20 plasmas spiked with Intralipid 20 or lipid isolated from patient plasmas (median triglyceride of 11.95 mmol/L [5.0-17.0] and cholesterol 4.33 [3.22-7.06]), were tested before and after the removal of the lipid layer by centrifugation (10000 g for 10 minutes). Tests were performed using the CS-5100 (Sysmex) coagulation analyser. RESULTS: Thirteen, 9, 3 and 1 of the lipaemic or spiked samples failed to give PT, APTT, fibrinogen and D-dimer results, respectively. Centrifugation significantly reduced triglyceride (median 2.7, [0-6.1 mmol/L]) and cholesterol (median 0.52 [0-3.5]), allowing clot detection in all tests. There were no statistically significant differences in fibrinogen, D-dimer or VWF levels in samples before and after lipid removal. A small but clinically insignificant change in PT and APTT was observed after lipid removal. CONCLUSION: High-speed centrifugation reduces lipaemia sufficiently to allow testing on an optical coagulation analyser without introducing clinically significant differences PT, APTT, fibrinogen, D-dimer or VWF activity values.

A randomised controlled trial of extended anticoagulation treatment versus standard treatment for the prevention of recurrent venous thromboembolism (VTE) and post-thrombotic syndrome in patients being treated for a first episode of unprovoked VTE (the ExACT study).

Venous thromboembolism (VTE) is prevalent and impactful, with a risk of death, morbidity and recurrence. Post-thrombotic syndrome (PTS) is a common consequence and associated with impaired quality of life (QoL). The ExACT study was a non-blinded, prospective, multicentred randomised controlled trial comparing extended versus limited duration anticoagulation following a first unprovoked VTE (proximal deep vein thrombosis or pulmonary embolism). Adults were eligible if they had completed ≥3 months anticoagulation (remaining anticoagulated). The primary outcome was time to first recurrent VTE from randomisation. The secondary outcomes included PTS severity, bleeding, QoL and D-dimers. Two-hundred and eighty-one patients were recruited, randomised and followed up for 24 months (mean age 63, male:female 2:1). There was a significant reduction in recurrent VTE for patients receiving extended anticoagulation [2·75 vs. 13·54 events/100 patient years, adjusted hazard ratio (aHR) 0·20 (95% confidence interval (CI): 0·09 to 0·46, P < 0·001)] with a non-significant increase in major bleeding [3·54 vs. 1·18 events/100 patient years, aHR 2·99 (95% CI: 0·81-11·05, P = 0·10)]. Outcomes of PTS and QoL were no different between groups. D-dimer results (on anticoagulation) did not predict VTE recurrence. In conclusion, extended anticoagulation reduced VTE recurrence but did not reduce PTS or improve QoL and was associated with a non-significant increase in bleeding. Results also suggest very limited clinical utility of D-dimer testing on anticoagulated patients.

Considerations towards a roadmap for collection, handling and storage of blood extracellular vesicles.

There is an increasing interest in exploring clinically relevant information that is present in body fluids, and extracellular vesicles (EVs) are intrinsic components of body fluids ("liquid biopsies"). In this report, we will focus on blood. Blood contains not only EVs but also cells, and non-EV particles including lipoproteins. Due to the high concentration of soluble proteins and lipoproteins, blood, plasma and serum have a high viscosity and density, which hampers the concentration, isolation and detection of EVs. Because most if not all studies on EVs are single-centre studies, their clinical relevance remains limited. Therefore, there is an urgent need to improve standardization and reproducibility of EV research. As a first step, the International Society on Extracellular Vesicles organized a biomarker workshop in Birmingham (UK) in November 2017, and during that workshop several working groups were created to focus on a particular body fluid. This report is the first output of the blood EV work group and is based on responses by work group members to a questionnaire in order to discover the contours of a roadmap. From the answers it is clear that most respondents are in favour of evidence-based research, education, quality control procedures, and physical models to improve our understanding and comparison of concentration, isolation and detection methods. Since blood is such a complex body fluid, we assume that the outcome of the survey may also be valuable for exploring body fluids other than blood.

Systemic Exosomal Delivery of shRNA Minicircles Prevents Parkinsonian Pathology.

The development of new therapies to slow down or halt the progression of Parkinson's disease is a health care priority. A key pathological feature is the presence of alpha-synuclein aggregates, and there is increasing evidence that alpha-synuclein propagation plays a central role in disease progression. Consequently, the downregulation of alpha-synuclein is a potential therapeutic target. As a chronic disease, the ideal treatment will be minimally invasive and effective in the long-term. Knockdown of gene expression has clear potential, and siRNAs specific to alpha-synuclein have been designed; however, the efficacy of siRNA treatment is limited by its short-term efficacy. To combat this, we designed shRNA minicircles (shRNA-MCs), with the potential for prolonged effectiveness, and used RVG-exosomes as the vehicle for specific delivery into the brain. We optimized this system using transgenic mice expressing GFP and demonstrated its ability to downregulate GFP protein expression in the brain for up to 6 weeks. RVG-exosomes were used to deliver anti-alpha-synuclein shRNA-MC therapy to the alpha-synuclein preformed-fibril-induced model of parkinsonism. This therapy decreased alpha-synuclein aggregation, reduced the loss of dopaminergic neurons, and improved the clinical symptoms. Our results confirm the therapeutic potential of shRNA-MCs delivered by RVG-exosomes for long-term treatment of neurodegenerative diseases.

Tspan18 is a novel regulator of the Ca2+ channel Orai1 and von Willebrand factor release in endothelial cells.

Ca2+ entry via Orai1 store-operated Ca2+ channels in the plasma membrane is critical to cell function, and Orai1 loss causes severe immunodeficiency and developmental defects. The tetraspanins are a superfamily of transmembrane proteins that interact with specific 'partner proteins' and regulate their trafficking and clustering. The aim of this study was to functionally characterize tetraspanin Tspan18. We show that Tspan18 is expressed by endothelial cells at several-fold higher levels than most other cell types analyzed. Tspan18-knockdown primary human umbilical vein endothelial cells have 55-70% decreased Ca2+ mobilization upon stimulation with the inflammatory mediators thrombin or histamine, similar to Orai1-knockdown. Tspan18 interacts with Orai1, and Orai1 cell surface localization is reduced by 70% in Tspan18-knockdown endothelial cells. Tspan18 overexpression in lymphocyte model cell lines induces 20-fold activation of Ca2+ -responsive nuclear factor of activated T cell (NFAT) signaling, in an Orai1-dependent manner. Tspan18-knockout mice are viable. They lose on average 6-fold more blood in a tail-bleed assay. This is due to Tspan18 deficiency in non-hematopoietic cells, as assessed using chimeric mice. Tspan18-knockout mice have 60% reduced thrombus size in a deep vein thrombosis model, and 50% reduced platelet deposition in the microcirculation following myocardial ischemia-reperfusion injury. Histamine- or thrombin-induced von Willebrand factor release from endothelial cells is reduced by 90% following Tspan18-knockdown, and histamine-induced increase of plasma von Willebrand factor is reduced by 45% in Tspan18-knockout mice. These findings identify Tspan18 as a novel regulator of endothelial cell Orai1/Ca2+ signaling and von Willebrand factor release in response to inflammatory stimuli.

Towards mechanisms and standardization in extracellular vesicle and extracellular RNA studies: results of a worldwide survey.

The discovery that extracellular vesicles (EVs) can transfer functional extracellular RNAs (exRNAs) between cells opened new avenues into the study of EVs in health and disease. Growing interest in EV RNAs and other forms of exRNA has given rise to research programmes including but not limited to the Extracellular RNA Communication Consortium (ERCC) of the US National Institutes of Health. In 2017, the International Society for Extracellular Vesicles (ISEV) administered a survey focusing on EVs and exRNA to canvass-related views and perceived needs of the EV research community. Here, we report the results of this survey. Overall, respondents emphasized opportunities for technical developments, unraveling of molecular mechanisms and standardization of methodologies to increase understanding of the important roles of exRNAs in the broader context of EV science. In conclusion, although exRNA biology is a relatively recent emphasis in the EV field, it has driven considerable interest and resource commitment. The ISEV community looks forward to continuing developments in the science of exRNA and EVs, but without excluding other important molecular constituents of EVs.

Soluble GPVI is elevated in injured patients: shedding is mediated by fibrin activation of GPVI.

Soluble glycoprotein VI (sGPVI) is shed from the platelet surface and is a marker of platelet activation in thrombotic conditions. We assessed sGPVI levels together with patient and clinical parameters in acute and chronic inflammatory conditions, including patients with thermal injury and inflammatory bowel disease and patients admitted to the intensive care unit (ICU) for elective cardiac surgery, trauma, acute brain injury, or prolonged ventilation. Plasma sGPVI was measured by enzyme-linked immunosorbent assay and was elevated on day 14 after thermal injury, and was higher in patients who developed sepsis. sGPVI levels were associated with sepsis, and the value for predicting sepsis was increased in combination with platelet count and Abbreviated Burn Severity Index. sGPVI levels positively correlated with levels of D-dimer (a fibrin degradation product) in ICU patients and patients with thermal injury. sGPVI levels in ICU patients at admission were significantly associated with 28- and 90-day mortality independent of platelet count. sGPVI levels in patients with thermal injury were associated with 28-day mortality at days 1, 14, and 21 when adjusting for platelet count. In both cohorts, sGPVI associations with mortality were stronger than D-dimer levels. Mechanistically, release of GPVI was triggered by exposure of platelets to polymerized fibrin, but not by engagement of G protein-coupled receptors by thrombin, adenosine 5'-diphosphate, or thromboxane mimetics. Enhanced fibrin production in these patients may therefore contribute to the observed elevated sGPVI levels. sGPVI is an important platelet-specific marker for platelet activation that predicts sepsis progression and mortality in injured patients.