Thrombosis and haemostasis最新文献

Comprehensive characterization of platelets requires various functional assays and analysis techniques, including omics-disciplines, each requiring an individual aliquot of a given sample. Consequently, the sample material per assay is often highly limited rendering downscaling a prerequisite for effective sample exploitation. Here we present a transfer of our recently introduced 96-well-based proteomics workflow (PF96) into the 384-well format (PF384) allowing for a significant increase in sensitivity when processing minute platelet protein amounts. In addition, the 4-fold higher throughput (1500 samples per lab worker per week) allows to easily meet the throughput capacities of modern LC-MS instruments. We determined optimal sample loads followed by highlighting the strengths in comparison to our previous sample preparation approach by processing only 3 µg of purified platelet protein from 22 healthy donors. Major advantages are: (I) improved identification and analyte recovery, especially of low copy number proteins, with signal intensity gains of +130 % and +107 % (peptide and protein level, respectively) (II) substantial intensity gains for key-players in platelet activation including the membrane receptors PAR4, P2X1, GPVI, GPV, GPIX and the downstream mediators AKT, PKA, Rap1, Lyn (III) improved reproducibility with a reduction of technical variance from 22 / 25 % down to 16 / 19 % for detection of lower / higher abundant disease markers and (IV) a 4-fold increase in sample preparation throughput. Taken together, these advantages render PF384 a promising future in clinical proteomics and might pave the way of platelet proteomics with minute sample amounts into molecular diagnostics.

Background:  JAK2V617F-mutated myeloproliferative neoplasms (MPN) exhibit abnormal proliferation of bone marrow progenitors and increased risk of thrombosis, specifically in splanchnic veins (SVT). The contribution of the endothelium to the development of the prothrombotic phenotype was explored.

Material and methods:  Plasma and serum samples from JAK2V617F MPN patients with (n=26) or without (n=7) thrombotic debut and different treatments, were obtained (n=33). Cultured endothelial cells (ECs) were exposed to serum samples from these patients and from healthy donors as controls. Changes in markers of inflammation (VCAM-1, ICAM-1), cell permeability (VE-cadherin), production of VWF, extracellular matrix (ECM) reactivity, and activation of intracellular signaling pathways related to stress, proliferation, inflammation (Akt, p44/42, IkBa), and JAK2/STAT3 pathway, were assessed by immunofluorescence, flow adhesion, SDS-PAGE and immunoblot. Additionally, circulating markers of endothelial activation and damage (VWF, sVCAM-1, sTNFRI, thrombomodulin, angiopoietin-2, a2-antiplasmin activity, PAI-1) were evaluated in Patients' plasma.

Results:  The in vitro studies showed that EC exposure to MPN thrombotic patients' sera resulted in increased VCAM-1 and ICAM-1, and reduced VE-cadherin expression (p<0.05) at the cell surface. Production and release of VWF to the ECM were higher (p<0.05), with increased platelet adhesion after perfusing whole blood, being more noticeable in response to sera from non-treated patients. Furthermore, intracellular activation of Akt, p44/42, IkBa and JAK2/STAT3 was observed. Moreover, plasma levels of VWF, TNF-R1, VCAM-1, thrombomodulin, and angiopoietin-2 were higher in JAK2V617F+ MPN patients with thrombosis.

Conclusion:  The present findings suggest that circulating factors in MPNs with SVT debut induce endothelial proinflammatory and prothrombotic phenotypes, which are modulated in vitro with MPN treatment.

Background:  Fibrinolysis is spatiotemporally well-regulated and greatly influenced by activated platelets and coagulation activity. Our previous real-time imaging analyses revealed that clotting commences on activated platelet surfaces, resulting in uneven-density fibrin structures, and that fibrinolysis initiates in dense fibrin regions and extends to the periphery. Despite the widespread clinical use of direct oral anticoagulants (DOACs), their impact on thrombin-dependent activation of thrombin-activatable fibrinolysis inhibitor (TAFI) and fibrinolysis remains unclear. Here, we investigated the effects of different DOACs on the TAFI-mediated inhibition of fibrinolysis.

Methods:  Using human platelet-containing plasma, we performed turbidimetric assays, thrombin generation assays, and confocal laser scanning microscopy to assess the effects of anticoagulants on fibrinolysis.

Results and conclusion:  Activated platelets-prolonged plasma clot lysis time, shortened by activated TAFI inhibitor (TAFIaI), positively correlated with the amount of thrombin generated. Rivaroxaban (an activated factor X inhibitor) and dabigatran (a direct thrombin inhibitor) dose-dependently shortened lysis time comparably. The highest concentration of DOACs showed no further shortening of lysis time with TAFIaI. The fibrin network structures initiated by activated platelets and the localization of fluorescently labeled plasminogen were unique for these two drugs. Rivaroxaban maintained an uneven fibrin network but promoted faster plasminogen accumulation and fibrinolysis from outside dense fibrin regions. Conversely, dabigatran resulted in a more even fibrin network, with fibrinolysis starting from the activated platelets and propagating to the periphery. Visualizing and analyzing the patterns of fibrin network formation, plasminogen accumulation, and fibrinolysis provide new insights into the specific impact of anticoagulants on coagulation and fibrinolysis.

In-hospital case fatality related to acute pulmonary embolism (PE) has been falling since the beginning of this century. However, annual incidence rates continue to climb, and an increasing number of PE survivors need long-term follow-up, chronic anticoagulation treatment, and readmission(s) to the hospital. In European countries, median reimbursed hospital costs for acute PE are still moderate compared with the United States but can increase several-fold in patients with comorbidities and those necessitating potentially life-saving reperfusion treatment. The use of catheter-directed treatment (CDT) has constantly increased in the United States since the past decade, and it has now entered a rapid growth phase in Europe as well, estimated to reach an annual penetration rate of up to 31% among patients with intermediate-high- or high-risk PE by 2030. Ongoing randomised controlled trials are currently investigating the clinical efficacy and safety of these devices. In addition, they will deliver data permitting calculation of their cost-effectiveness in different health care reimbursement systems, by revealing the extent to which they can reduce complications and consequently the need for intensive care and the overall length of hospital stay. After discharge, key cost drivers are related to chronic cardiopulmonary diseases (other than PE itself) leading to frequent readmissions, persistent symptoms, and functional limitations which result in poor quality of life, productivity loss, and substantial indirect costs. Implementation of structured outpatient programmes with a holistic approach to post-PE care, targeting overall cardiovascular health and the patient's well-being, bears the potential to cost-effectively reduce the overall socioeconomic burden of PE.

Background:  Clot waveform analysis (CWA) provides a global assessment of hemostasis and may be useful for patients with cirrhosis with complex hemostatic abnormalities. This study aimed to assess the association between prothrombin time (PT-) and activated partial thromboplastin time (aPTT-) based CWA parameters and cirrhosis severity and prospectively evaluate the role of CWA in predicting mortality and acute decompensation (AD) over 1 year.

Methods:  This prospective study included adult patients with cirrhosis between June 2021 and December 2023 at Chulalongkorn University Hospital. The PT- and aPTT-based CWA parameters were obtained using an automated coagulation analyzer.

Results:  A total of 560 patients with cirrhosis were included; 165 (29.5%) and 47 (11.5%) had Child-Turcotte-Pugh (CTP) B and C cirrhosis, respectively. The PT- and aPTT-based CWA parameters, including maximum velocity (min1), maximum acceleration (min2), and maximum deceleration (max2), were significantly lower (p ≤ 0.05) in patients with decompensated cirrhosis than in those with compensated cirrhosis. Additionally, CWA values were significantly higher in patients with higher CTP and Model for End-Stage Liver Disease (MELD) scores. Multivariable analysis revealed that liver stiffness (LS) and max2 of PT-based CWA assay were independently associated with CTP B/C. In addition, min2 and max2 of PT-based CWA assay were independently associated with 1-year mortality. No significant differences in CWA parameters were observed between patients with and without portal vein thrombosis. CWA parameters were not related to AD during the 1-year follow-up.

Conclusion:  A hypocoagulable profile based on CWA parameters is associated with advanced-stage cirrhosis. CWA may be a useful objective marker for assessing cirrhosis severity and predicting 1-year mortality.

This is an executive summary of the recent guidance produced by the Scottish Intercollegiate Guidelines Network (SIGN) dementia guideline group with regards to the investigation of suspected dementia. This is a sub-section of the broader SIGN 168 guideline released in November 2023. The guideline group included clinicians with expertise in Old Age Psychiatry, Neurology, Radiology, and Nuclear Medicine supported by colleagues from the SIGN and Healthcare Improvement Scotland teams. There was representation from carers and support organizations with experience of dementia, to ensure the recommendations were appropriate from the perspective of the people being assessed for possible dementia and their carers. As the 2018 National Institute for Health and Clinical Excellence (NICE) dementia review included a review of the evidenced investigation of dementia, the SIGN guideline development group decided to focus on a review on the up-to-date evidence regarding the role of imaging and fluid biomarkers in the diagnosis of dementia. To give context to the consideration of more advanced diagnostic biomarker investigations, the guideline and this summary include the NICE guidance on the use of standard investigations as well as more specialist investigations. The evidence review supports consideration of the use of structural imaging, nuclear medicine imaging, and established Alzheimer's cerebrospinal fluid biomarkers (amyloid and tau) in the diagnosis of dementia. Although routine use of amyloid positron emission tomography imaging was not recommended, its potential use, under specialist direction, in patients with atypical or young-onset presentations of suspected Alzheimer's dementia was included as a clinical good practice point.

Introduction:  Prethrombin-1 is a Gla-domain lacking enzymatically inactive split product that results from the cleavage of fragment 1 from prothrombin by thrombin in a feedback reaction.

Methods:  A prethrombin-1 preparation derived from human plasma was tested for its hemostatic and thrombogenic properties. Animal models of nail clipping (for rabbits) and tail clipping (for mice) were developed to measure blood loss in FVIII-inhibitor or rivaroxaban anticoagulated rabbits and mice, respectively. A modified Wessler test was used in rabbits to assess the thrombogenic potential by Wessler score and clot weight. Studies were performed in groups of three to six for prethrombin-1 dose escalation and comparison with prothrombin, Beriplex®, FEIBA®, and saline as a control. Data were analyzed using t-statistics or the Mann Whitney U test as applicable.

Results:  Prethrombin-1 has excellent hemostatic properties in anticoagulated mouse and rabbit bleeding models. Wessler tests suggest that in contrast to activated and nonactivated prothrombin complexes, prethrombin-1 has negligible thrombogenic potential.

Conclusion:  The thrombin zymogen prethrombin-1 promotes hemostasis with reduced risk of thrombosis. Prethrombin-1 may have potential to become a life-saving treatment for patients who bleed or are at risk of bleeding.