Thrombosis and haemostasis最新文献

Leukocyte-platelet aggregates (LPAs) play a crucial role in the pathogenesis of inflammatory diseases, linking pathological immune responses with thrombosis.The levels of LPAs, their composition, and cellular reactivity were determined in patients with distinct inflammatory conditions, namely coronavirus disease 2019 (COVID-19), rheumatoid arthritis (RA), and systemic lupus erythematosus (SLE), compared with healthy controls. Flow cytometry was used to identify cell types and measure LPA levels in the blood. The ability of platelets, neutrophils, and monocytes to form additional LPAs in response to hyperstimulation with phorbol-12-myristate-13-acetate (PMA) was assessed. Coaggregation of isolated neutrophils and platelets in vitro was visualized using scanning electron microscopy. Blood tests included coagulation, hematology, biochemistry, and immunology.LPA levels were significantly higher in all patient groups compared with controls, with variations in the composition: neutrophil-platelet aggregates predominated in the COVID-19 patients, whereas monocyte-platelet aggregates prevailed in the blood of RA and SLE patients. Platelet-to-leukocyte ratios within aggregates varied in a broad range with a substantial prevalence of platelets over leukocytes. Morphological analysis revealed coaggregation of platelets with neutrophils, including relatively large homotypic platelet aggregates associated with one or two neutrophils. In PMA-treated pathological blood samples from COVID-19, RA, and SLE patients, the ability to form additional LPAs over the patients' baseline level was reduced compared with normal blood samples, indicating impaired reactivity (exhaustion) of neutrophils and monocytes in all patient groups.This study highlights distinct changes in the number and composition of LPAs in inflammatory diseases of various etiologies associated with altered functionality of the innate immune cells.

Human platelets express protease-activated receptor (PAR) 1 and 4 on the platelet membrane. PAR1 activation is crucial for hemostasis, while PAR4 activation produces a prolonged pro-inflammatory platelet response. Once activated, the PAR receptors cause platelet activation and aggregation. The PARs are activated by proteolytic cleavage of their N-terminus, revealing a new N-terminus called the tethered ligand. This tethered ligand folds back and activates the receptor. PAR1 and PAR4 differ in their tethered ligand sequence as well as their signaling cascades; however, they are both activated by various serine proteases, specifically thrombin.Much is known about PAR4 activation via thrombin activation; however, little is known about activation by other proteases. Thrombin cleavage of PAR4 and subsequent activation leads to G proteins, Gα_q and Gα_12/13, and β-arrestin signaling. The objective of this study is to characterize trypsin activation of PAR4 on human platelets.Platelets were isolated from the whole blood of healthy human volunteers by centrifugation. Platelets were utilized in flow cytometry assays, calcium mobilization assays, and western blot experiments to characterize trypsin activation of human platelets.Here, we demonstrate that trypsin activates human platelets solely through PAR4. Trypsin activation of PAR4 signaling through both Gα_q and Gα_12/13 is as effective as α-thrombin activation of PAR4.Thrombin may be the main protease to activate PAR4 under typical conditions; however, under pathological conditions such as acute pancreatitis (AP), trypsin activation of PAR4 may be a main driver of platelet activation and disease progression.

Insufficient lipid-lowering therapy is associated with residual cardiovascular risk. Low-density lipoprotein (LDL) is well known to promote atherosclerosis and cardiovascular disease (CVD) and during lipoprotein metabolism, free fatty acids (FFA) are generated. Besides this, residual thrombotic risk occurs in patients with impaired pharmacodynamic response to aspirin-so-called high on-treatment platelet reactivity (HTPR). Until now, it is not known if there is a mutual link.For in vitro analyses, blood from healthy donors was used and incubated with different concentrations of LDL, FFA, and acetylsalicylic acid. Arachidonic acid (AA)-induced light transmittance aggregometry (LTA), thromboxane (TX) formation, and cyclooxygenase (COX)-1 activity were measured. In a cross-sectional analysis, aspirin antiplatelet effects, and LDL and FFA concentrations were measured in 612 aspirin-treated CVD patients. In vitro administration of LDL and FFA impaired aspirin antiplatelet effects. In patients, FFA levels were associated with HTPR to aspirin. FFA levels and plasma LDL correlated with AA-induced platelet aggregation. Statin medication improved aspirin antiplatelet effects. AA-induced platelet aggregation was decreased in patients with statin medication.In this study, we were able to demonstrate a link between residual lipid and thrombotic risk in patients with cardiovascular disease. We could show that LDL and FFA impair pharmacodynamic response to aspirin at the level of COX. Statin therapy improved aspirin antiplatelet effects.

Chromogenic anti-Xa assay is currently used in the management of patients on unfractionated heparin (UFH). It has been shown that inter-assay variability in anti-Xa levels can be explained in part by the presence or absence of dextran sulfate (DXS) in the reagents. DXS has the ability to dissociate UFH from neutralizing proteins, including platelet factor 4 (PF4).Investigate whether PF4 plasma levels along with the presence/absence of DXS in anti-Xa reagents are associated with variations in UFH anti-Xa levels in different clinical situations.In the prospective multicenter study DEXHEP-NCT04700670, critically ill patients on UFH therapy (four groups) were recruited. Blood was collected into citrate and CTAD tubes. Chromogenic anti-Xa levels were assessed using seven reagent/analyzer combinations including two without DXS. Plasma PF4 was measured by ELISA (Zymutest-PF4-Hyphen-Biomed).A total of 144 patients were analyzed: average PF4 levels in citrate plasma samples were consistently higher than in CTAD ones (206 vs. 46 ng/mL, p < 10^-4), regardless of the patient group. Using a linear mixed-effect model, we found a significant effect of both DXS and PF4 on anti-Xa level, with a significant interaction term (p < 10^-4). Considering the 0.3 to 0.7 IU/mL therapeutic range, agreement between anti-Xa values (Liquid-anti-Xa/DXS-free vs. Biophen-LRT/DXS) was observed in roughly two-thirds of the patients.PF4 levels slightly affects anti-Xa levels, the use of CTAD tubes minimizing the effect. However, PF4 levels do not fully explain the differences of anti-Xa levels observed in the presence or absence of DXS, which has a greater effect. Anti-Xa assays require better standardization.

Inflammation and thrombosis are traditionally considered two separate entities of acute host responses to barrier breaks. While inciting inflammatory responses is a prerequisite to fighting invading pathogens and subsequent restoration of tissue homeostasis, thrombus formation is a crucial step of the hemostatic response to prevent blood loss following vascular injury. Though originally designed to protect the host, excessive induction of either inflammatory signaling or thrombus formation and their reciprocal activation contribute to a plethora of disorders, including cardiovascular, autoimmune, and malignant diseases. In this state-of-the-art review, we summarize recent insights into the intricate interplay of inflammation and thrombosis. We focus on the protective aspects of immunothrombosis as well as evidence of detrimental sequelae of thromboinflammation, specifically regarding recent studies that elucidate its pathophysiology beyond coronavirus disease 2019 (COVID-19). We introduce recently identified molecular aspects of key cellular players like neutrophils, monocytes, and platelets that contribute to both immunothrombosis and thromboinflammation. Further, we describe the underlying mechanisms of activation involving circulating plasma proteins and immune complexes. We then illustrate how these factors skew the inflammatory state toward detrimental thromboinflammation across cardiovascular as well as septic and autoimmune inflammatory diseases. Finally, we discuss how the advent of new technologies and the integration with clinical data have been used to investigate the mechanisms and signaling cascades underlying immunothrombosis and thromboinflammation. This review highlights open questions that will need to be addressed by the field to translate our mechanistic understanding into clinically meaningful therapeutic targeting.

Thrombocytopenia is one of the most common manifestations of the antiphospholipid syndrome (APS). However, its causes are still poorly defined. We have shown recently that antiphospholipid antibodies (aPL) directed against β2-glycoprotein I (β2GPI) of the IgG isotype induced platelet activation and aggregation while aPL directed against cardiolipin and anti-β2GPI IgM had no effect. Since platelet activation by anti-β2GPI might lead to platelet consumption and lower platelet count or overt thrombocytopenia, we analyzed the association of aPL with platelet count.Data of consecutive patients with test orders for anticardiolipin IgG/IgM and anti-β2GPI IgG/IgM and full blood count in our laboratory from August 2015 to April 2019 were analyzed.We identified 2,815 individual patients (mean age 45.7 years; 71.1% women) with complete data on aPL and platelet count, of which 445 individuals (mean age 41.0 years; 75.3% women) showed increased aPL titers. Patients with anti-β2GPI of the IgG isotype had significantly lower platelet count (220 ± 84 versus 264 ± 90 G/L, p < 0.0001) and higher frequency of thrombocytopenia (platelet count <100 G/L; 12.2% versus 2.4%, p < 0.005) than patients negative for all four aPL. These differences could not be explained by comorbidities or medications. Neither anticardiolipin IgG nor aPL of the IgM isotype was associated with lower platelet count or thrombocytopenia.The exclusive association of anti-β2GPI IgG aPL with low platelet count coincides with its unique ability to activate platelets and induce aggregation in vitro. This supports the hypothesis that anti-β2GPI IgG aPL may induce thrombocytopenia by chronic platelet consumption in vivo.

The duration of anticoagulation treatment for venous thromboembolism (VTE) depends on whether the event was provoked or unprovoked. Major surgery or trauma are well-established major provoking factors associated with a low risk of recurrence, but the magnitude of risk with VTE after minor surgery or trauma is uncertain.To compare the rate of recurrence in patients with VTE provoked by minor surgery or trauma with that in patients with VTE provoked by major surgery or trauma.Nested, case-control study of patients with a first VTE diagnosed within 90 days after major or minor surgery or trauma. Patients with unprovoked VTE or VTE provoked by cancer or nonsurgical risk factors were excluded. Crude and adjusted odds ratios with 95% confidence intervals (CI) were calculated and results were adjusted for potential confounders.A total of 319 patients with recurrent VTE (cases) were matched to 974 patients without recurrence (controls). The incidence of recurrence after VTE provoked by minor surgery (6.5%; 95% CI: 5.6-7.6) was more than double that after VTE provoked by major surgery (3.0%; 95% CI: 2.4-3.6), a difference that remained even after adjustment for known VTE risk factors. There were no differences in recurrence rates between VTE provoked by minor and major trauma.The risk of recurrence is higher in patients with VTE provoked by minor surgery than major surgery. These findings support the concept that the risk of recurrence is higher with minimally provoked VTE than with VTE provoked by major transient risk factors.

Patient care pathways provide an integrated care approach to atrial fibrillation (AF) management. International guidelines propose various patient care pathways, each emphasizing different strategies for assessing stroke and bleeding risk. Due to different ethnicities and susceptibility to stroke or bleeding risk, caution should be taken during application of Western cohorts-derived patient care pathways to Asian cohorts. Evidence-based rather than eminence-based strategies should be adopted for AF patient care. In this clinical focus, we summarize and compare patient care pathways, using evidence on the implementation in real-world registries, and strategies for assessing stroke and bleeding risk across Asian and non-Asian guidelines.

Portal vein system-specific risk factors contributing to portal vein thrombosis in cirrhosis are poorly investigated.This study aimed to quantify contact system and intrinsic pathway activation in the peripheral compared to portal venous blood in patients with decompensated cirrhosis.Adult patients with cirrhosis undergoing transjugular intrahepatic portosystemic shunt underwent simultaneous blood sampling from a peripheral vein and the portal vein. Complexes of serine proteases with their respective inhibitors were measured by ELISA to quantify contact system (PKa:C1-INH [plasma kallikrein:C1-esterase inhibitor] and FXIIa:C1-INH) and intrinsic pathway activation (FXIa:C1-INH, FXIa:α1at [α-1 antitrypsin], FXIa:AT [antithrombin], and FIXa:AT).Twenty patients with cirrhosis (mean age 55 ± 7 years, M = 58%, Child-Pugh A/B/C 6/11/3) and 25 healthy controls (mean age 45 ± 12 years, M = 60%) were enrolled. The etiology of cirrhosis was primarily alcohol abuse, followed by chronic viral infection. Log-transformed peripheral levels of all the complexes were significantly higher in patients compared with controls. While levels of PKa:C1-INH, FXIIa:C1-INH, FXIa:C1-INH and FXIa:α1at were similar in peripheral and portal venous blood in cirrhotic patients, FXIa:AT and FIXa:AT levels were significantly higher in portal blood (p = 0.013 and 0.011, respectively). FXIa:C1-INH significantly correlated with both contact system complexes (FXIIa:C1-INH and PKa:C1-INH) and with FIX:AT.Markers of contact system and intrinsic pathway activation in the systemic circulation were significantly higher in cirrhosis versus controls. Complexes of FXIa and FIXa with AT were significantly higher in the portal than in peripheral plasma in cirrhosis, possibly indicating a unique heparin-like effect in portal venous blood.