Thrombosis and haemostasis最新文献

Background:  Platelets prevent extravasation of capillary fluids into the pulmonary interstitial tissue by sealing gaps in inflamed endothelium. This reduces respiratory distress associated with pneumonia. Streptococcus pneumoniae is the leading cause of severe community-acquired pneumonia. Pneumococci produce pneumolysin (PLY), which forms pores in membranes of eukaryotic cells including platelets. Additionally, pneumococci express neuraminidases, which cleave sialic acid residues from eukaryotic glycoproteins. In this study, we investigated the effect of desialylation on PLY binding and pore formation on platelets.

Materials and methods:  We incubated human platelets with purified neuraminidases and PLY, or nonencapsulated S. pneumoniae D39/TIGR4 and isogenic mutants deficient in PLY and/or NanA. We assessed platelet desialylation, PLY binding, and pore formation by flow cytometry. We also analyzed the inhibitory potential of therapeutic immunoglobulin G preparations (IVIG [intravenous immunoglobulin]).

Results:  Wild-type pneumococci cause desialylation of platelet glycoproteins by neuraminidases, which is reduced by 90 to 100% in NanA-deficient mutants. NanC, cleaving only α2,3-linked sialic acid, induced platelet desialylation. PLY binding to platelets then x2doubled (p = 0.0166) and pore formation tripled (p = 0.0373). A neuraminidase cleaving α2,3-, α2,6-, and α2,8-linked sialic acid like NanA was even more efficient. Addition of polyvalent IVIG (5 mg/mL) decreased platelet desialylation induced by NanC up to 90% (p = 0.263) and reduced pore formation >95% (p < 0.0001) when incubated with pneumococci.

Conclusion:  Neuraminidases are key virulence factors of pneumococci and desialylate platelet glycoproteins, thereby unmasking PLY-binding sites. This enhances binding of PLY and pore formation showing that pneumococcal neuraminidases and PLY act in concert to kill platelets. However, human polyvalent immunoglobulin G preparations are promising agents for therapeutic intervention during severe pneumococcal pneumonia.

Background:  Protein Z-dependent protease inhibitor (ZPI) is an anticoagulant serpin that targets factor Xa (FXa) in the presence of protein Z (PZ), and factor XIa (FXIa). In factor-VIII-deficient mice, PZ or ZPI gene knock-out mitigates the bleeding phenotype, and pharmacological inhibition of PZ enhances thrombin generation in plasma from patients with hemophilia.

Aims:  To develop a single-domain antibody (sdAb) directed against ZPI to inhibit its anticoagulant activity.

Methods:  We screened for anti-ZPI sdAbs in a llama-derived phage display immune library of sdAbs. The sdAbs that bound ZPI were produced and purified for characterization. The binding of sdAbs to ZPI or other serpins was evaluated using ELISAs, and ZPI inhibition was measured in an anti-FXa or anti-FXIa chromogenic assay. The sdAbs's procoagulant activity was assessed in a thrombin generation assay in normal plasma, factor VIII- and FXI-deficient plasma.

Results:  Of the four sdAbs found to bind to ZPI, one (referred to as ZPI-sdAb2) dose-dependently inhibited ZPI's anti-FXa and anti-FXIa activities with a mean half-maximal inhibitory concentration of 1.8 and 1.3 µM, respectively. ZPI-sdAb2 did not cross-react with other plasma serpins, such as antithrombin and α1-antitrypsin. ZPI-sdAb2 induced a significant increase in thrombin generation in plasma samples from healthy donors, patients with severe hemophilia A, and patients with FXI deficiency.

Conclusion:  ZPI-sdAb2 is the first specific, direct ZPI inhibitor found to exhibit procoagulant activity in plasma. This sdAb might have potential as a treatment for hemophilia or other bleeding disorders.

Background:  Chronic kidney disease (CKD) is a progressive, irreversible, and incurable condition characterized by high morbidity and mortality, affecting approximately one-tenth of the global population. Rise of urea-derived cyanate levels in CKD patients, severalfold higher in comparison to those found in healthy individuals, leads to an increased rate of carbamylation of lysine residues of proteins and peptides. This posttranslational modification plays an important role in the progression of kidney failure but also in the onset of CKD-related complications, including previously reported coagulopathies. In this study, we have explored the impact of carbamylation on the functionality of von Willebrand factor (vWF), a pivotal player in hemostasis, and its implications for platelet adhesion.

Materials and methods:  We have explored carbamylated vWF's interactions with its partner proteins via ELISA. Mass spectrometry was employed to identify modified lysine residues. Blood platelets isolated from healthy donors were carbamylated, and their activation, binding to endothelium and thromboxane release were evaluated using flow cytometry, adhesion assays and ELISA, respectively.

Results:  Using mass spectrometry we detected the vWF's lysine residue smost susceptible to carbamylation. This modification has in turn affected vWF's interactions with its key binding partners: decreased binding to collagen types I/III but increased the affinity to factor FVIII, while its binding to fibrinogen remained unchanged. Carbamylation of vWF impeded vWF-blood platelet binding, but carbamylation of platelets led to their increased thrombin-dependent activation as observed by enhanced phosphatidylserine exposure, improved their binding to vascular endothelium, at the same time decreasing the production of the prothrombotic mediator, thromboxane A2.

Conclusion:  Our findings highlight the multifaceted impact of carbamylation on vWF and platelets, disturbing the delicate balance of coagulation cascade. These alterations could contribute to the complex hemostatic imbalance in ESKD, underscoring the need for further research to fully understand these mechanisms and their clinical implications.

Background:  Disruptions in the pathways for activating and deactivating proteases in the bloodstream can lead to thrombosis and bleeding issues. Leucine aminopeptidases (LAPs), which are exopeptidases essential for regulating protein and peptide activities, are recognized as clinical biomarkers for liver diseases. However, the relationship between serum LAP activity and the risks of bleeding or thrombosis, as well as the identification of the specific tissues or organs that control LAP levels, is not well understood.

Methods:  We performed a retrospective study to evaluate serum LAP activities in 149,360 patients with 47 different diseases and 9,449 healthy individuals. The analysis was conducted using SPSS V2.6, RStudio V.1.3.1073, and libraries in Python 3.8.

Results:  Our research revealed that 21 of the 47 diseases studied showed increased median serum LAP activities, while 26 diseases were associated with significantly lower activities, especially those related to thrombosis. Furthermore, most diseases were found to have an increased risk of bleeding and thrombosis, indicated by higher Q25 and lower Q75 LAP activities compared to the control group. Receiver operating characteristic curve analysis confirmed the effectiveness of LAP activities as biomarkers for specific conditions like hepatic encephalopathy, liver cancer, pancreatitis, and pancreatic cancer. Diseases were categorized into clusters with similar bleeding or thrombotic tendencies through principal component analysis.

Conclusion:  This study highlighted regulatory influence of the liver and pancreas on LAP levels. The established link between serum LAP concentrations and the risk of bleeding or thrombosis paved the way for the development of diagnostic and preventative approaches for various medical conditions.

Background:  Given the current debate in clinical research about the relationship between tobacco smoking and the risk of venous thromboembolism (VTE), a Mendelian randomization (MR) study was conducted aimed at elucidating the causal associations of current and past tobacco smoking with the risk of VTE, from the perspective of genetics.

Methods:  Two-sample univariate and multivariable MR analyses were designed, using summary-level data from large genome-wide association studies involving European individuals. Causality was primarily assessed using multiplicative fixed-effects or random-effects model and inverse variance weighting, supplemented by MR-Egger regression, MR-PRESSO, Cochran's Q test, and leave-one-out for sensitivity analysis to test the reliability of the results.

Results:  In the univariate MR analysis, no significant causal effects were found between current tobacco smoking and the risk of VTE, deep vein thrombosis (DVT), and pulmonary embolism (PE). Similarly, no significant causal effects were found between past smoking and VTE, DVT, and PE. As for the multivariable MR analysis, results were consistent with univariate MR analysis, with no significant causal effect of either current or past tobacco smoking on the risk of VTE, DVT, and PE.

Conclusion:  Evidence from both univariate and multivariable MR analyses demonstrated no significant causal relationships between current and past tobacco smoking and VTE, DVT, and PE. This contradicts positive correlations reported in some previous observational studies, which may be explained by other confounding factors. This provided genetic evidence for the conclusion reported in other observational studies that smoking did not affect VTE risk.

Introduction:  Current guideline suggests a switch from vitamin K antagonist (VKA) to direct oral anticoagulant (DOAC) in patients with low time in therapeutic range (TTR < 70%). Poor international normalized ratio (INR) control may be the result of poor compliance, and might therefore be associated with subsequent DOAC intake. Therefore, this study evaluates the effect of previous TTR and other measures of INR control on DOAC nonadherence and nonpersistence, in patients who switched from VKA to DOAC.

Methods:  A total of 437 patients who switched from VKA to DOAC between 2012 and 2019 were included using data from Certe Thrombosis Service, IADB.nl pharmacy community database University Groningen, and Statistics Netherlands. DOAC prescriptions were used to determine nonadherence and nonpersistence. INR control (i.e., TTR, time under therapeutic range [TUR], and INR variability) was assessed during the last 180 days of VKA use. Multivariable regression models were applied to determine the association between INR control and DOAC nonpersistence/nonadherence.

Results:  On VKA, 67.7% of the patients had a TTR below 70%. DOAC nonpersistence was 39.8% (95% confidence interval [CI]: 33.4-45.5%) during a median follow-up of 34.4 months (interquartile range: 19.1-49.2). Approximately 80% of persistent patients were DOAC-adherent. Low TTR was not associated with DOAC nonpersistence (hazard ratio: 1.14, 95% CI: 0.69-1.87) and DOAC nonadherence (odds ratio: 1.38, 95% CI: 0.67-2.84), nor were TUR and INR variability.

Conclusion:  Previous INR control during VKA therapy is not associated with subsequent DOAC nonadherence and nonpersistence. This study suggests that INR control on VKA cannot, and therefore should not, be used for predicting DOAC adherence or persistence.

The management of anticoagulant therapy in pregnant women with mechanical heart valves (MHVs) is difficult and often challenging even for clinicians experienced in the field. These pregnancies, indeed, are burdened with higher rates of complications for both the mother and the fetus, compared to those in women without MHVs. The maternal need for an optimal anticoagulation as provided by vitamin K antagonists is counterbalanced by their teratogen effect on the embryo and fetus. On the other hand, several concerns have been raised about the efficacy of heparins in pregnant women with MHVs, considering the high risk of thrombotic complications in these patients. Therefore, numerous clinical issues about the management of pregnant women with MHVs remain unanswered, such as the selection of the best anticoagulant agent, the optimal anticoagulation levels to be achieved and maintained, and the evaluation of long-term effects for both the mother and the fetus. Based on a comprehensive review of the current literature, the Italian Federation of the Centers for the Diagnosis and the Surveillance of the Antithrombotic Therapies (FCSA) proposes experience-based suggestions and expert opinions. Particularly, this consensus document aims at providing practical guidance for clinicians dealing with pregnant women with MHVs, to optimize maternal and fetal outcomes while guaranteeing adequate anticoagulation. Finally, FCSA highlights the need for the creation of multidisciplinary teams experienced in the management of pregnant women with MHVs during pregnancy, delivery, and postpartum, in order to better deal with such complex clinical issues and provide a comprehensive counseling to these patients.

Objective:  Platelet plays a key role in thrombosis formation, especially that the alteration of platelet function may influence the thrombosis development. This study aimed to investigate platelet function alterations in the formation of portal vein thrombosis (PVT) in cirrhosis.

Methods:  Cirrhotic patients admitted to The First Affiliated Hospital of Soochow University between October 2021 and April 2023 were recruited and divided into PVT and non-PVT groups according to radiological results. Clinical parameters and prognosis were also collected and assessed to identify potential risk factors. Flow cytometry was used to detect the expression of CD62p, CD63, monocyte-platelet aggregates (MPAs), neutrophil-platelet aggregates (NPAs), and von Willebrand factor antigen (vWF-Ag) to evaluate platelet activation and adhesion function.

Results:  A total of 145 subjects were enrolled in our study including 60 cirrhotic PVT patients, 60 cirrhotic non-PVT patients, and 25 healthy volunteers. Multivariate analysis suggested that esophageal gastric varices, splenectomy, and D-dimer were independent risk factors for PVT pathogenesis in cirrhosis. The vWF-Ag expression level was reduced in the PVT group compared with the non-PVT group (p = 0.046) but was not an independent risk factor for PVT formation pathogenesis. The expression of CD41⁺CD62p⁺ and CD41⁺CD63⁺ platelets in the PVT group was significantly elevated compared with that in the non-PVT group (p < 0.05). There were no significant differences in MPAs and NPAs between the two cirrhotic groups. Subgroup analysis showed that the mean fluorescence intensity (MFI) of CD62p and CD63 was associated with portal hypertension-related complications (p = 0.008, p < 0.001), and CD63 MFI was significantly associated with thrombosis burden (p = 0.019). CD41⁺CD62p⁺ and CD41⁺CD63⁺ platelets as well as MPAs and NPAs were highly expressed in the splenectomy group compared with those in the nonsplenectomy group in cirrhotic patients (p < 0.05). Positive correlations were found between CD62p MFI and CD63 MFI, MPAs and NPAs (r = 0.642, p < 0.001; r = 0.378, p = 0.003; r = 0.430, p < 0.001). In addition, platelet counts were also correlated with MPAs (r = 0.556, p < 0.001) and NPAs (r = 0.467, p < 0.001). Cirrhotic patients with PVT had higher mortality and were more likely to experience portal hypertension-related complications in the prognostic analysis (p < 0.05).

Conclusion:  Highly activated platelet function exists in patients with cirrhosis, and platelet activation was elevated during PVT formation, suggesting that activated platelets may participate in the formation of PVT in patients with cirrhosis.