Daytime plasma cortisol and cortisol response to dexamethasone suppression are associated with a prothrombotic state in hypertension.

Background and aims: A prothrombotic state was demonstrated in patients with Cushing's syndrome and is involved in the development and progression of cardiovascular and renal damage in hypertensive patients. This study was designed to examine the relationships between cortisol secretion and the hemostatic and fibrinolytic systems in hypertension. Methods: In 149 middle-aged, nondiabetic, essential hypertensive patients free of cardiovascular and renal complications, we measured hemostatic markers that express the spontaneous activation of the coagulation and fibrinolytic systems and assessed daily cortisol levels (8 AM, 3 PM, 12 AM; area under the curve, AUC-cortisol) together with the cortisol response to dexamethasone overnight suppression (DST-cortisol). Results: Plasma levels of D-dimer (D-dim), prothrombin fragment 1 + 2 (F1 + 2), and von Willebrand factor (vWF) were progressively and significantly higher across tertiles of AUC-cortisol and DST-cortisol, whereas no differences were observed in fibrinogen, tissue plasminogen activator, plasminogen activator inhibitor-1, antithrombin III, protein C, and protein S. D-dim, F1 + 2, and vWF were significantly and directly correlated with age and both AUC-cortisol and DST-cortisol. Multivariate regression analysis showed that both AUC-cortisol and DST-cortisol were related to plasma D-dim, F1 + 2, and vWF independently of age, body mass index, blood pressure, and renal function. Conclusion: Greater daily cortisol profile and cortisol response to overnight suppression are independently associated with a prothrombotic state in hypertensive patients and might contribute to the development of organ damage and higher risk of cardiovascular complications.

Removal of endothelial surface-associated von villebrand factor suppresses accelerate datherosclerosis after myocardial infarction.

BACKGROUND: Thromboinflammation involving platelet adhesion to endothelial surface-associated von Willebrand factor (VWF) has been implicated in the accelerated progression of non-culprit plaques after MI. The aim of this study was to use arterial endothelial molecular imaging to mechanistically evaluate endothelial-associated VWF as a therapeutic target for reducing remote plaque activation after myocardial infarction (MI). METHODS: Hyperlipidemic mice deficient for the low-density lipoprotein receptor and Apobec-1 underwent closed-chest MI and were treated chronically with either: (i) recombinant ADAMTS13 which is responsible for proteolytic removal of VWF from the endothelial surface, (ii) N-acetylcysteine (NAC) which removes VWF by disulfide bond reduction, (iii) function-blocking anti-factor XI (FXI) antibody, or (iv) no therapy. Non-ischemic controls were also studied. At day 3 and 21, ultrasound molecular imaging was performed with probes targeted to endothelial-associated VWF A1-domain, platelet GPIbα, P-selectin and vascular cell adhesion molecule-1 (VCAM-1) at lesion-prone sites of the aorta. Histology was performed at day 21. RESULTS: Aortic signal for P-selectin, VCAM-1, VWF, and platelet-GPIbα were all increased several-fold (p < 0.01) in post-MI mice versus sham-treated animals at day 3 and 21. Treatment with NAC and ADAMTS13 significantly attenuated the post-MI increase for all four molecular targets by > 50% (p < 0.05 vs. non-treated at day 3 and 21). On aortic root histology, mice undergoing MI versus controls had 2-4 fold greater plaque size and macrophage content (p < 0.05), approximately 20-fold greater platelet adhesion (p < 0.05), and increased staining for markers of platelet transforming growth factor-ß1 signaling. Accelerated plaque growth and inflammatory activation was almost entirely prevented by ADAMTS13 and NAC. Inhibition of FXI had no significant effect on molecular imaging signal or plaque morphology. CONCLUSIONS: Plaque inflammatory activation in remote arteries after MI is strongly influenced by VWF-mediated platelet adhesion to the endothelium. These findings support investigation into new secondary preventive therapies for reducing non-culprit artery events after MI.

The Utility of Thromboelastography in Monitoring Von Willebrand Disease: A Case Report.

Von Willebrand disease (VWD) is the most common inherited bleeding disorder. It is primarily attributed to malfunctioning or deficient Von Willebrand factor (VWF). Thromboelastography (TEG) has emerged as a valuable tool for assessing coagulation dynamics and guiding transfusion therapy in bleeding patients. Given this, we present a case study of a 23-year-old pregnant female with a past medical history of type 2B VWD, wherein TEG was employed to optimize disease screening and therapy monitoring while minimizing costs and preventing complications associated with low platelet counts. This case underscores the potential utility of TEG in enhancing the care of VWD patients, particularly in unique critical settings such as pregnancy.

Neovascularization by DPSC-ECs in a Tube Model for Pulp Regeneration Study.

The process of neovascularization during cell-based pulp regeneration is difficult to study. Here we developed a tube model that simulates root canal space and allows direct visualization of the vascularization process in vitro. Endothelial-like cells (ECs) derived from guiding human dental pulp stem cells (DPSCs) into expressing endothelial cell markers CD144, vWF, VEGFR1, and VEGFR2 were used. Human microvascular endothelial cells (hMVECs) were used as a positive control. DPSC-ECs formed tubules on Matrigel similar to hMVECs. Cells were mixed in fibrinogen/thrombin or mouse blood and seeded into wells of 96-well plates or injected into a tapered plastic tube (14 mm in length and 1 or 2 mm diameter of the apex opening) with the larger end sealed with MTA to simulate root canal space. Cells/gels in wells or tubes were incubated for various times in vitro and observed under the microscope for morphological changes. Samples were then fixed and processed for histological analysis to determine vessel formation. Vessel-like networks were observed in culture from 1 to 3 d after cell seeding. Cells/gels in 96-well plates were maintained up to 25 d. Histologically, both hMVECs and DPSC-ECs in 96-well plates or tubes showed intracellular vacuole formation. Some cells showed merged large vacuoles indicating the lumenization. Tubular structures were also observed resembling blood vessels. Cells appeared healthy throughout the tube except some samples (1 mm apical diameter) in the coronal third. Histological analysis also showed pulp-like soft tissue throughout the tube samples with vascular-like structures. hMVECs formed larger vascular lumen size than DPSC-ECs while the latter tended to have more lumen and tubular structure counts. We conclude that DPSC-ECs can form vascular structures and sustained in the 3-dimensional fibrin gel system in vitro. The tube model appears to be a proper and simple system simulating the root canal space for vascular formation and pulp regeneration studies.

Binding Mechanism between Platelet Glycoprotein and Cyclic Peptide Elucidated by McMD-Based Dynamic Docking.

The cyclic peptide OS1 (amino acid sequence: CTERMALHNLC), which has a disulfide bond between both termini cysteine residues, inhibits complex formation between the platelet glycoprotein Ibα (GPIbα) and the von Willebrand factor (vWF) by forming a complex with GPIbα. To study the binding mechanism between GPIbα and OS1 and, therefore, the inhibition mechanism of the protein-protein GPIbα-vWF complex, we have applied our multicanonical molecular dynamics (McMD)-based dynamic docking protocol starting from the unbound state of the peptide. Our simulations have reproduced the experimental complex structure, although the top-ranking structure was an intermediary one, where the peptide was bound in the same location as in the experimental structure; however, the ß-switch of GPIbα attained a different conformation. Our analysis showed that subsequent refolding of the ß-switch results in a more stable binding configuration, although the transition to the native configuration appears to take some time, during which OS1 could dissociate. Our results show that conformational changes in the ß-switch are crucial for successful binding of OS1. Furthermore, we identified several allosteric binding sites of GPIbα that might also interfere with vWF binding, and optimization of the peptide to target these allosteric sites might lead to a more effective inhibitor, as these are not dependent on the ß-switch conformation.

[Mechanism of astragaloside â £ combined with Panax notoginseng saponins in regulating angiogenesis to treat cerebral ischemia based on network pharmacology and experimental verification].

Network pharmacology and animal and cell experiments were employed to explore the mechanism of astragaloside Ⅳ(AST Ⅳ) combined with Panax notoginseng saponins(PNS) in regulating angiogenesis to treat cerebral ischemia. The method of network pharmacology was used to predict the possible mechanisms of AST Ⅳ and PNS in treating cerebral ischemia by mediating angiogenesis. In vivo experiment: SD rats were randomized into sham, model, and AST Ⅳ(10 mg·kg~(-1)) + PNS(25 mg·kg~(-1)) groups, and the model of cerebral ischemia was established with middle cerebral artery occlusion(MCAO) method. AST Ⅳ and PNS were administered by gavage twice a day. the Longa method was employed to measure the neurological deficits. The brain tissue was stained with hematoxylin-eosin(HE) to reveal the pathological damage. Immunohistochemical assay was employed to measure the expression of von Willebrand factor(vWF), and immunofluorescence assay to measure the expression of vascular endothelial growth factor A(VEGFA). Western blot was employed to determine the protein levels of vascular endothelial growth factor receptor 2(VEGFR2), VEGFA, phosphorylated phosphatidylinositol 3-kinase(p-PI3K), and phosphorylated protein kinase B(p-AKT) in the brain tissue. In vitro experiment: the primary generation of rat brain microvascular endothelial cells(rBEMCs) was cultured and identified. The third-generation rBMECs were assigned into control, model, AST Ⅳ(50 µmol·L~(-1)) + PNS(30 µmol·L~(-1)), LY294002(PI3K/AKT signaling pathway inhibitor), 740Y-P(PI3K/AKT signaling pathway agonist), AST Ⅳ + PNS + LY294002, and AST Ⅳ + PNS + 740Y-P groups. Oxygen glucose deprivation/re-oxygenation(OGD/R) was employed to establish the cell model of cerebral ischemia-reperfusion injury. The cell counting kit-8(CCK-8) and scratch assay were employed to examine the survival and migration of rBEMCs, respectively. Matrigel was used to evaluate the tube formation from rBEMCs. The Transwell assay was employed to examine endothelial cell permeability. Western blot was employed to determine the expression of VEGFR2, VEGFA, p-PI3K, and p-AKT in rBEMCs. The results of network pharmacology analysis showed that AST Ⅳ and PNS regulated 21 targets including VEGFA and AKT1 of angiogenesis in cerebral infarction. Most of these 21 targets were involved in the PI3K/AKT signaling pathway. The in vivo experiments showed that compared with the model group, AST Ⅳ + PNS reduced the neurological deficit score(P<0.05) and the cell damage rate in the brain tissue(P<0.05), promoted the expression of vWF and VEGFA(P<0.01) and angiogenesis, and up-regulated the expression of proteins in the PI3K/AKT pathway(P<0.05, P<0.01). The in vitro experiments showed that compared with the model group, the AST Ⅳ + PNS, 740Y-P, AST Ⅳ + PNS + LY294002, and AST Ⅳ + PNS + 740Y-P improved the survival of rBEMCs after OGD/R, enhanced the migration of rBEMCs, increased the tubes formed by rBEMCs, up-regulated the expression of proteins in the PI3K/AKT pathway, and reduced endothelial cell permeability(P<0.05, P<0.01). Compared with the LY294002 group, the AST Ⅳ + PNS + LY294002 group showed increased survival rate, migration rate, and number of tubes, up-regulated expression of proteins in the PI3K/AKT pathway, and decreased endothelial cell permeability(P<0.05,P<0.01). Compared with the AST Ⅳ + PNS and 740Y-P groups, the AST Ⅳ + PNS + 740Y-P group presented increased survival rate, migration rate, and number of tubes and up-regulated expression of proteins in the PI3K/AKT pathway, and reduced endothelial cell permeability(P<0.01). This study indicates that AST Ⅳ and PNS can promote angiogenesis after cerebral ischemia by activating the PI3K/AKT signaling pathway.

Twenty-Four week Taichi training improves pulmonary diffusion capacity and glycemic control in patients with Type 2 diabetes mellitus.

This study evaluated the effect of 24-week Taichi training and Taichi plus resistance band training on pulmonary diffusion capacity and glycemic control in patients with Type 2 diabetes mellitus (T2DM). Forty-eight patients with T2DM were randomly divided into three groups: Group A-Taichi training: practiced Taichi 60 min/day, 6 days/week for 24 weeks; Group B-Taichi plus resistance band training: practiced 60-min Taichi 4 days/week plus 60-min resistance band training 2 days/week for 24 weeks; and Group C-controls: maintaining their daily lifestyles. Stepwise multiple regression analysis was applied to predict diffusion capacity of the lungs for carbon monoxide (DLCO) by fasting blood glucose, insulin, glycosylated hemoglobin (HbA1c), tumour necrosis factor alpha (TNF-α), von Willebrand Factor (vWF), interleukin-6 (IL-6), intercellular adhesion molecule 1 (ICAM-1), endothelial nitric oxide synthase (eNOS), nitric oxide (NO), endothelin-1 (ET-1), vascular endothelial growth factor, and prostaglandin I-2. Taichi with or without resistance band training significantly improved DLCO, increased insulin sensitivity, eNOS and NO, and reduced fasting blood glucose, insulin, HbA1c, TNF-α, vWF, IL-6, ICAM-1, and ET-1. There was no change in any of these variables in the control group. DLCO was significantly predicted (R2 = 0.82) by insulin sensitivity (standard-ß = 0.415, P<0.001), eNOS (standard-ß = 0.128, P = 0.017), TNF-α (standard-ß = -0.259, P = 0.001), vWF (standard-ß = -0.201, P = 0.007), and IL-6 (standard-ß = -0.175, P = 0.032) in patients with T2DM. The impact of insulin sensitivity was the most important predictor for the variation of DLCO based on the multiple regression modeling. This study demonstrates that 24-week Taichi training and Taichi plus resistance band training effectively improve pulmonary diffusion capacity and blood glycemic control in patients with T2DM. Variation of DLCO is explained by improved insulin sensitivity and endothelial function, and reduced inflammatory markers, including TNF-α, vWF, and IL-6.

Optimization of plasma-based BioID identifies plasminogen as a ligand of ADAMTS13.

ADAMTS13, a disintegrin and metalloprotease with a thrombospondin type 1 motif, member 13, regulates the length of Von Willebrand factor (VWF) multimers and their platelet-binding activity. ADAMTS13 is constitutively secreted as an active protease and is not inhibited by circulating protease inhibitors. Therefore, the mechanisms that regulate ADAMTS13 protease activity are unknown. We performed an unbiased proteomics screen to identify ligands of ADAMTS13 by optimizing the application of BioID to plasma. Plasma BioID identified 5 plasma proteins significantly labeled by the ADAMTS13-birA* fusion, including VWF and plasminogen. Glu-plasminogen, Lys-plasminogen, mini-plasminogen, and apo(a) bound ADAMTS13 with high affinity, whereas micro-plasminogen did not. None of the plasminogen variants or apo(a) bound to a C-terminal truncation variant of ADAMTS13 (MDTCS). The binding of plasminogen to ADAMTS13 was attenuated by tranexamic acid or ε-aminocaproic acid, and tranexamic acid protected ADAMTS13 from plasmin degradation. These data demonstrate that plasminogen is an important ligand of ADAMTS13 in plasma by binding to the C-terminus of ADAMTS13. Plasmin proteolytically degrades ADAMTS13 in a lysine-dependent manner, which may contribute to its regulation. Adapting BioID to identify protein-interaction networks in plasma provides a powerful new tool to study protease regulation in the cardiovascular system.

Acquired bleeding disorders.

Acquired bleeding disorders can develop in previously healthy people irrespective of age or gender but are particularly common in patients with certain underlying conditions. Here, we review recent advances in the management of acquired haemophilia A (AHA), acquired von Willebrand syndrome (AVWS), and patients with hemostatic abnormalities due to chronic liver disease (CLD). Patients with AHA can now benefit from prophylaxis with emicizumab, a therapeutic antibody that mimics the function of activated coagulation factor VIII. The treatment of AVWS remains challenging in many situations and requires careful consideration of the underlying condition. Haemostatic abnormalities in CLD are often compensated by proportional reduction in pro and anti-haemostatic factors resulting in sustained or even increased thrombin generation. Consequently, bleeding in CLD is rarely caused by haemostatic failure and infusion of plasma or coagulation factor concentrates may not be effective.

COVID-19 increases extracorporeal coagulation during hemodialysis associated with upregulation of vWF/FBLN5 signaling in patients with severe/critical symptoms.

BACKGROUND: COVID-19 has been shown to increase the risk of extracorporeal coagulation during hemodialysis in patients, but the underlying mechanism remains unclear. This study aimed to investigate the effect and mechanism of COVID-19 on the risk of extracorporeal coagulation in patients with chronic kidney disease undergoing hemodialysis. METHODS: A retrospective analysis of the extracorporeal coagulation status of 339 hemodialysis patients at our center before and after COVID-19 infection was performed, including subgroup analyses. Post-infection blood composition was analyzed by protein spectrometry and ELISA. RESULTS: Compared to the pre-COVID-19 infection period, COVID-19-induced extracorporeal coagulation predominantly occurred in patients with severe/critical symptoms. Further proteomic analysis demonstrated that in patients with severe/critical symptoms, the coagulation cascade reaction, platelet activation, inflammation, and oxidative stress-related pathways were significantly amplified compared to those in patients with no/mild symptoms. Notably, the vWF/FBLN5 pathway, which is associated with inflammation, vascular injury, and coagulation, was significantly upregulated. CONCLUSIONS: Patients with severe/critical COVID-19 symptoms are at a higher risk of extracorporeal coagulation during hemodialysis, which is associated with the upregulation of the vWF/FBLN5 signaling pathway. These findings highlight the importance of early anticoagulant therapy initiation in COVID-19 patients with severe/critical symptoms, particularly those undergoing hemodialysis. Additionally, vWF/FBLN5 upregulation may be a novel mechanism for virus-associated thrombosis/coagulation.

Cost-effectiveness of prophylaxis with recombinant vs plasma-derived VWF in severe von Willebrand disease in the United States.

ABSTRACT: We evaluated the cost-effectiveness of prophylaxis with recombinant von Willebrand factor (rVWF) vs with plasma-derived von Willebrand factor (pdVWF) for patients with severe Von Willebrand disease. We found that rVWF is a cost-saving factor replacement compared with pdVWF across all willingness-to-pay thresholds in the United States.

Endothelial PTP1B Deletion Promotes VWF Exocytosis and Venous Thromboinflammation.

BACKGROUND: Endothelial activation promotes the release of procoagulant extracellular vesicles and inflammatory mediators from specialized storage granules. Endothelial membrane exocytosis is controlled by phosphorylation. We hypothesized that the absence of PTP1B (protein tyrosine phosphatase 1B) in endothelial cells promotes venous thromboinflammation by triggering endothelial membrane fusion and exocytosis. METHODS: Mice with inducible endothelial deletion of PTP1B (End.PTP1B-KO) underwent inferior vena cava ligation to induce stenosis and venous thrombosis. Primary endothelial cells from transgenic mice and human umbilical vein endothelial cells were used for mechanistic studies. RESULTS: Vascular ultrasound and histology showed significantly larger venous thrombi containing higher numbers of Ly6G (lymphocyte antigen 6 family member G)-positive neutrophils in mice with endothelial PTP1B deletion, and intravital microscopy confirmed the more pronounced neutrophil recruitment following inferior vena cava ligation. RT2 PCR profiler array and immunocytochemistry analysis revealed increased endothelial activation and adhesion molecule expression in primary End.PTP1B-KO endothelial cells, including CD62P (P-selectin) and VWF (von Willebrand factor). Pretreatment with the NF-κB (nuclear factor kappa B) kinase inhibitor BAY11-7082, antibodies neutralizing CD162 (P-selectin glycoprotein ligand-1) or VWF, or arginylglycylaspartic acid integrin-blocking peptides abolished the neutrophil adhesion to End.PTP1B-KO endothelial cells in vitro. Circulating levels of annexin V+ procoagulant endothelial CD62E+ (E-selectin) and neutrophil (Ly6G+) extracellular vesicles were also elevated in End.PTP1B-KO mice after inferior vena cava ligation. Higher plasma MPO (myeloperoxidase) and Cit-H3 (citrullinated histone-3) levels and neutrophil elastase activity indicated neutrophil activation and extracellular trap formation. Infusion of End.PTP1B-KO extracellular vesicles into C57BL/6J wild-type mice most prominently enhanced the recruitment of endogenous neutrophils, and this response was blunted in VWF-deficient mice or by VWF-blocking antibodies. Reduced PTP1B binding and tyrosine dephosphorylation of SNAP23 (synaptosome-associated protein 23) resulting in increased VWF exocytosis and neutrophil adhesion were identified as mechanisms, all of which could be restored by NF-κB kinase inhibition using BAY11-7082. CONCLUSIONS: Our findings show that endothelial PTP1B deletion promotes venous thromboinflammation by enhancing SNAP23 phosphorylation, endothelial VWF exocytosis, and neutrophil recruitment.

[ADAMTS13-Mediated Proteolytic Cleavage of Unusually Large von Willebrand Factor Polymers on Endothelial Cells in the Absence of Fluid Shear Stress].

OBJECTIVE: To investigate the molecular mechanism of proteolytic cleavage of unusually large von Willebrand Factor(ULVWF) on endothelial cells by ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 repeats-13) in the absence of fluid shear stress, so as to provide a theoretical basis for the pathogenesis of thrombotic thrombocytopenic purpura (TTP) and other thrombotic disorders. METHODS: The ADAMTS13-mediated proteolysis of ULVWF on the surface of endothelial cells in the absence of fluid shear stress was observed through immunofluorescence microscopy. The variation in VWF antigen levels in the conditioned media were determined by ELISA assay. The levels of VWF and the proteolytic fragments released into the conditioned media were determined by ELISA assay and Western blot in the absence and presence of fluid shear stress or FVIII. The effect of ADAMTS13-mediated ULVWF cleavage on the normal distribution of plasma VWF multimers was evaluated by multimer analysis. Histamine stimulated human umbilical vein endothelial cells (HUVECs) were incubated with ADAMTS13 and various N- and C-terminally truncated mutants. Then the ULVWF that maintained binding to the cells were observed through immunofluorescence microscopy and the soluble ULVWF released from endothelial cells was determined by ELISA, so as to demonstrate the domains of ADAMTS13 required for proteolysis of ULVWF on endothelial cells. RESULTS: The ULVWF strings on the endothelial cell surface were rapidly proteolyzed by recombinant and plasma ADAMTS13 in the absence of fluid shear stress. This proteolytic processing of ULVWF depended on incubation time and ADAMTS13 concentration, but not shear stress and FVIII. The distribution of VWF releaseded by ADAMTS13-mediated proteolysis was quite similar to that secreted by endothelial cells under histamine stimulation, suggesting the ULVWF cleavage occured at the cell surface. The proteolysis of the ULVWF on endothelial cells required the Cys-rich(CysR) and spacer domains, but not the TSP1 2-8 and CUB domains of ADAMTS13. CONCLUSION: The ULVWF polymers on endothelial cells are sensitive to ADAMTS13-mediated cleavage even in the absence of fluid shear stress. The findings provide novel insight into the molecular mechanism of ADAMTS13-mediated ULVWF cleavage at the cellular level and may contribute to understanding of the pathogenesis of TTP and other thrombotic disorders.

O-glycan determinants regulate VWF trafficking to Weibel-Palade bodies.

ABSTRACT: von Willebrand factor (VWF) undergoes complex posttranslational modification within endothelial cells (ECs) before secretion. This includes significant N- and O-linked glycosylation. Previous studies have demonstrated that changes in N-linked glycan structures significantly influence VWF biosynthesis. In contrast, although abnormalities in VWF O-linked glycans (OLGs) have been associated with enhanced VWF clearance, their effect on VWF biosynthesis remains poorly explored. Herein, we report a novel role for OLG determinants in regulating VWF biosynthesis and trafficking within ECs. We demonstrate that alterations in OLGs (notably reduced terminal sialylation) lead to activation of the A1 domain of VWF within EC. In the presence of altered OLG, VWF multimerization is reduced and Weibel-Palade body (WPB) formation significantly impaired. Consistently, the amount of VWF secreted from WPB after EC activation was significantly reduced in the context of O-glycosylation inhibition. Finally, altered OLG on VWF not only reduced the amount of VWF secreted after EC activation but also affected its hemostatic efficacy. Notably, VWF secreted after WPB exocytosis consisted predominantly of low molecular weight multimers, and the length of tethered VWF string formation on the surface of activated ECs was significantly reduced. In conclusion, our data therefore support the hypothesis that alterations in O-glycosylation pathways directly affect VWF trafficking within human EC. These findings are interesting given that previous studies have reported altered OLG on plasma VWF (notably increased T-antigen expression) in patients with von Willebrand disease.

Impact of rise and fall phases of shear on platelet activation and aggregation using microfluidics.

Blood flow disorders are often the result of the non-physiological narrowing of blood arteries caused by atherosclerosis and thrombus. The blood then proceeds through rising-peak-decreasing phases as it passes through the narrow area. Although abnormally high shear is known to activate platelets, the shear process that platelets undergo in small arteries is complex. Thus, understanding how each shear phase affects platelet activation can be used to improve antiplatelet therapy and decrease the risk of side effects like bleeding. Blood samples were sheared (68.8 ms,5200 s-1) in vitro by the microfluidic technique, and platelet activation levels (P-selectin and integrin αIIbß3) and von Willebrand factor (vWF) binding to platelets were analyzed by flow cytometry. Post-stenosis platelet aggregation was dynamically detected using microfluidic technology. We studied TXA2, P2Y12-ADP, and integrin αIIbß3-fibrinogen receptor pathways by adding antiplatelet drugs, such as acetylsalicylic acid (ASA, an active ingredient of aspirin that inhibits platelet metabolism), ticagrelor (hinders platelet activation), and tirofiban (blocks integrin αIIbß3 receptor) in vitro, respectively, to determine platelet activation function mediated by transient non-physiological high shear rates. We demonstrated that platelets can be activated under transient pathological high shear rates. The shear rise and fall phases influenced shear-induced platelet activation by regulating the binding of vWF to platelets. The degree of platelet activation and aggregation increased with multiple shear rise and fall phases. ASA did not inhibit shear-mediated platelet activation, but ticagrelor and tirofiban effectively inhibited shear-mediated platelet activation. Our data demonstrated that the shear rise and fall phases play an important role in shear-mediated platelet activation and promote platelet activation and aggregation in a vWF-dependent manner. Blocking integrin αIIbß3 receptor and hindering P2Y12-ADP were beneficial to reducing shear-mediated platelet activation.

Diagnosis and treatment of von Willebrand disease in 2024 and beyond.

MANUSCRIPT BACKGROUND AND AIM: The diagnosis and clinical care of patients with von Willebrand disease (VWD) has continued to evolve since the characterization of the von Willebrand factor (VWF) gene in 1985. This condition is almost certainly the most common inherited bleeding disorder, and the major symptomatic burden of the disease is experienced by females during their reproductive years. Diagnosis relies on the identification of a personal and family history of excessive mucocutaneous bleeding, and laboratory features consistent with quantitative and/or qualitative abnormalities of VWF. This review focuses on three aspects of VWD management, with current updates and a look into the future. MANUSCRIPT THEMES: First, we will address the role of genetics in the diagnosis and possible therapies for VWD. With current technologies, VWD genetic diagnosis is usually confined to the confirmation of type 2 subtypes of the disease and type 3 VWD analysis for family planning. While type 3 VWD is a potential candidate for the application of gene therapy, no treatments are currently close to entering the clinic. Second, the peri-procedural management of patients with VWD remains an important element of care. The choice of product, its dose and schedule all require careful consideration depending upon the type and disruptive nature of the planned procedure. Lastly, in addition to gene therapy, several other novel therapeutic interventions are also being developed for bleeding and prophylaxis in VWD. These include a VWF aptamer interfering with VWF clearance and bioengineered forms of VWF.

Early-onset indicators of a hypercoagulable state and clinical complications in a cohort of children with sickle cell trait.

Adults with sickle cell trait (SCT) have a procoagulant state with increased risk of thromboembolism, but limited data are available for children. We compared the coagulation profile of children with SCT, different sickle cell disease (SCD) genotypes, and healthy controls. Compared to controls and similarly to HbSC patients, 41 SCT children (mean age 6.85 years; 20 males; 88% Africans) had a characteristic procoagulant profile: higher levels of factor VIII, von Willebrand factor (VWF) Ag and CBA, D-dimer; lower levels of ADAMTS 13 activity, ADAMTS13 activity: VWFAg, plasminogen activator inhibitor, tissue plasminogen activator. Moreover, 13/41 had clinical complications of SCD, five requiring hospitalization.

Vascular study of decellularized porcine long bones: Characterization of a tissue engineering model.

INTRODUCTION: Massive bone allografts enable the reconstruction of critical bone defects in numerous conditions (e.g. tumoral, infection or trauma). Unfortunately, their biological integration remains insufficient and the reconstruction may suffer from several postoperative complications. Perfusion-decellularization emerges as a tissue engineering potential solution to enhance osseointegration. Therefore, an intrinsic vascular study of this novel tissue engineering tool becomes essential to understand its efficacy and applicability. MATERIAL AND METHODS: 32 porcine long bones (humeri and femurs) were used to assess the quality of their vascular network prior and after undergoing a perfusion-decellularization protocol. 12 paired bones were used to assess the vascular matrix prior (N = 6) and after our protocol (N = 6) by immunohistochemistry. Collagen IV, Von Willebrand factor and CD31 were targeted then quantified. The medullary macroscopic vascular network was evaluated with 12 bones: 6 were decellularized and the other 6 were, as control, not treated. All 12 underwent a contrast-agent injection through the nutrient artery prior an angio CT-scan acquisition. The images were processed and the length of medullary vessels filled with contrast agent were measured on angiographic cT images obtained in control and decellularized bones by 4 independent observers to evaluate the vascular network preservation. The microscopic cortical vascular network was evaluated on 8 bones: 4 control and 4 decellularized. After injection of gelatinous fluorochrome mixture (calcein green), non-decalcified fluoroscopic microscopy was performed in order to assess the perfusion quality of cortical vascular lacunae. RESULTS: The continuity of the microscopic vascular network was assessed with Collagen IV immunohistochemistry (p-value = 0.805) while the decellularization quality was observed through CD31 and Von Willebrand factor immunohistochemistry (p-values <0.001). The macroscopic vascular network was severely impaired after perfusion-decellularization; nutrient arteries were still patent but the amount of medullary vascular channels measured was significantly higher in the control group compared to the decellularized group (p-value <0.001). On average, the observers show good agreement on these results, except in the decellularized group where more inter-observer discrepancies were observed. The microscopic vascular network was observed with green fluoroscopic signal in almost every canals and lacunae of the bone cortices, in three different bone locations (proximal metaphysis, diaphysis and distal metaphysis). CONCLUSION: Despite the aggressiveness of the decellularization protocol on medullary vessels, total porcine long bones decellularized by perfusion retain an acellular cortical microvascular network. By injection through the intact nutrient arteries, this latter vascular network can still be used as a total bone infusion access for bone tissue engineering in order to enhance massive bone allografts prior implantation.