Mechanochemistry of von Willebrand factor

AbstractVon Willebrand factor (VWF), a blood multimeric protein with a very high molecular weight, plays a crucial role in the primary haemostasis, the physiological process characterized by the adhesion of blood platelets to the injured vessel wall. Hydrodynamic forces are responsible for extensive conformational transitions in the VWF multimers that change their structure from a globular form to a stretched linear conformation. This feature makes this protein particularly prone to be investigated by mechanochemistry, the branch of the biophysical chemistry devoted to investigating the effects of shear forces on protein conformation. This review describes the structural elements of the VWF molecule involved in the biochemical response to shear forces. The stretched VWF conformation favors the interaction with the platelet GpIb and at the same time with ADAMTS-13, the zinc-protease that cleaves VWF in the A2 domain, limiting its prothrombotic capacity. The shear-induced conformational transitions favor also a process of self-aggregation, responsible for the formation of a spider-web like network, particularly efficient in the trapping process of flowing platelets. The investigation of the biophysical effects of shear forces on VWF conformation contributes to unraveling the molecular mechanisms of many types of thrombotic and haemorrhagic syndromes

Immune and Hereditary Thrombotic Thrombocytopenic Purpura: Can ADAMTS13 Deficiency Alone Explain the Different Clinical Phenotypes?

: Thrombotic thrombocytopenic purpura (TTP) is a thrombotic microangiopathy caused by a hereditary or immune-mediated deficiency of the enzyme ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13). TTPs are caused by the following pathophysiological mechanisms: (1) the presence of inhibitory autoantibodies against ADAMTS13; and (2) hereditary mutations of the ADAMTS13 gene, which is present on chromosome 9. In both syndromes, TTP results from a severe deficiency of ADAMTS13, which is responsible for the impaired proteolytic processing of high-molecular-weight von Willebrand factor (HMW-VWF) multimers, which avidly interact with platelets and subendothelial collagen and promote tissue and multiorgan ischemia. Although the acute presentation of the occurring symptoms in acquired and hereditary TTPs is similar (microangiopathic hemolytic anemia, thrombocytopenia, and variable ischemic end-organ injury), their intensity, incidence, and precipitating factors are different, although, in both forms, a severe ADAMTS13 deficiency characterizes their physiopathology. This review is aimed at exploring the possible factors responsible for the different clinical and pathological features occurring in hereditary and immune-mediated TTPs

Effects of pegylated G-CSF on immune cell number and function in patients with gynecological malignancies

<p>Abstract</p> <p>Background</p> <p>Pegylated granulocyte colony-stimulating factor (G-CSF; pegfilgrastim) is a longer-acting form of G-CSF, whose effects on dendritic cell (DC) and regulatory T cell (Treg) mobilization, and on the <it>in vivo </it>and ex vivo release of immune modulating cytokines remain unexplored.</p> <p>Methods</p> <p>Twelve patients with gynecological cancers received carboplatin/paclitaxel chemotherapy and single-dose pegfilgrastim as prophylaxis of febrile neutropenia. Peripheral blood was collected prior to pegfilgrastim administration (day 0) and on days +7, +11 and +21, to quantify immunoregulatory cytokines and to assess type 1 DC (DC1), type 2 DC (DC2) and Treg cell mobilization. <it>In vitro</it>-differentiated, monocyte-derived DC were used to investigate endocytic activity, expression of DC maturation antigens and ability to activate allogeneic T-cell proliferation.</p> <p>Results</p> <p>Pegfilgrastim increased the frequency of circulating DC1 and DC2 precursors. In contrast, CD4⁺FoxP3⁺<it>bona fide </it>Treg cells were unchanged compared with baseline. Serum levels of hepatocyte growth factor and interleukin (IL)-12p40, but not transforming growth factor-β1 or immune suppressive kynurenines, significantly increased after pegfilgrastim administration. Interestingly, pegfilgrastim fostered <it>in vitro</it> monocytic<it/> secretion of IL-12p40 and IL-12p70 when compared with unconjugated G-CSF. Finally, DC populations differentiated <it>in vitro </it>after clinical provision of pegfilgrastim were phenotypically mature, possessed low endocytic activity, and incited a robust T-cell proliferative response.</p> <p>Conclusions</p> <p>Pegfilgrastim induced significant changes in immune cell number and function. The enhancement of monocytic IL-12 secretion portends favorable implications for pegfilgrastim administration to patients with cancer, a clinical context where the induction of immune deviation would be highly undesirable.</p

Do We Need to Define Therapeutic Ranges for Edoxaban Plasma Concentration?

Do We Need to Define Therapeutic Ranges for Edoxaban Plasma Concentration

Pathogenesis Of Portal Vein Thrombosis In Liver Cirrhosis: The Role of the ADAMTS13/VWF Unbalance

Increasing evidence shows a potential role of ADAMTS13 deficiency as a risk factor for the high prevalence of portal vein thrombosis (PVT) in cirrhotic patients. This deficiency, due to myofibroblastic transformation of hepatic stellate cells (HSCs), the source of ADAMTS13, is responsible for the prevalence of ultra large molecular weight multimers of von Willebrand factor (UL-VWF) in the hepatic microcirculation. This phenomenon would favor the prohaemostatic function of VWF, which, together with an elevation of coagulation FVIII, which is associated to VWF, could sustain microcirculatory thrombosis in the liver. These phenomena, triggering an increase of the intra-hepatic pressure, would cause a slowdown of the portal flow, favoring the occurrence of PVT. Although this scenario is justified by retrospective observational clinical studies, it will be mandatory to clarify the ADAMTS13 expression in HSCs associated with the activity of plasma ADAMTS13 in different stage of liver diseases. Hence, a prospective clinical trial (ClinicalTrials.gov Identifier: NCT03322696) is ongoing to unravel the linkage between all the actors involved in the complex phenomenon of PVT occurring in cirrhosi

Detection of Platelet-Activating Antibodies Associated with Vaccine-Induced Thrombotic Thrombocytopenia by Flow Cytometry: An Italian Experience

Rare cases of thrombocytopenia and thrombosis after anti-COVID-19 adenovirus-associated mRNA vaccines (VITT) due to platelet-activating anti-platelet-factor 4 (PF4)/polyanion antibod-ies have been reported. VITT laboratory diagnosis, similarly to heparin-induced thrombocytope-nia (HIT) diagnosis, requires immunoassays for anti-PF4/polyanion antibodies identification, such as ELISA assays and platelet-activating functional tests, such as heparin-induced platelet activation test (HIPA), to confirm their pathogenicity. We compared the flow cytometry (FC) measurement of platelet p-selectin exposure to the gold standard functional test HIPA for diagno-sis confirmation in 13 patients with a clinical VITT syndrome (6M/7F; median age 56 (33–78)) who resulted positive to anti-PF4/polyanion antibodies ELISA assays (12/13). FC and HIPA sim-ilarly identified three different patterns: (1) a typical non-heparin-dependent VITT pattern (seven and six patients by FC and HIPA, respectively); (2) low/no platelet activation in patients under IvIg therapy (five out of five and two out of four patients by FC and HIPA, respectively); (3) a HIT pattern. Antibodies investigated by FC became negative after 7, 17, and 24 days of therapy in three patients. FC measurement of P-selectin exposure was as sensitive as HIPA but simpler to de-tect anti-PF4/polyanion antibodies in VITT patients. FC could reliably discriminate VITT from HIT, thus helping for the choice of the anticoagulant

ORal anticoagulants In fraGile patients with percutAneous endoscopic gastrostoMy and atrIal fibrillation: the (ORIGAMI) study

BACKGROUND: Randomized trials support the safety and efficacy of direct oral anticoagulants (DOACs) versus vitamin K antagonists (VKAs) in patients with nonvalvular atrial fibrillation, leading to increased use of these compounds. Crushed forms of DOACs have been shown to be reliable, but evidence supporting percutaneous endoscopic gastrostomy (PEG) delivery is lacking. PEG is a long-term option for enteral food and drug delivery in patients unable to maintain oral intake, bypassing the risks and disadvantages of parenteral nutrition.AIMS: The ORal anticoagulants In fraGile patients with percutAneous endoscopic gastrostoMy and atrIal fibrillation (ORIGAMI) study investigates the safety and efficacy of Edoxaban administered via PEG in patients with atrial fibrillation and a clinical indication for a long-term anticoagulation.DESIGN: In this prospective, single-centre observational study, 12 PEG-treated patients with indication to anticoagulation will receive edoxaban via PEG and will be followed up to 6 months. Plasma antifactor Xa activity and edoxaban concentrations will be assessed. Thromboembolic (ischaemic stroke, systemic embolism, venous thromboembolism) and bleeding events (Bleeding Academic Research Consortium and Thrombolysis in Myocardial Infarction) will be recorded at 1 and 6 months.PRELIMINARY CASES: A retrospective analysis of five atrial fibrillation cases undergoing PEG implantation at our Institution who received edoxaban via PEG showed plasma anti-FXa levels at a steady state of 146 ± 15 ng/ml, without major adverse event at a mean follow-up of 6 months.CONCLUSION: ORIGAMI prospectively investigates PEG-administration of edoxaban in PEG-treated patients requiring long-term anticoagulation. Our preliminary retrospective data support this route of DOAC administration. CLINICALTRIALS.GOV IDENTIFIER: NCT04271293