Carbamazepine interaction with direct oral anticoagulants: help from the laboratory for the personalized management of oral anticoagulant therapy.

Current guidelines recommend caution in prescribing concomitant use of direct-acting oral anticoagulants (DOACs) and antiepileptic drugs due to drug-drug interactions leading to potential risk of DOACs subtherapeutic concentration and treatment failure. Herein we report a significant interaction between carbamazepine (CZP) and apixaban, causing subtherapeutic concentration of the drug in a patient with atrial fibrillation who had a transient ischemic attack (TIA) episode. Another anti-Xa DOAC, edoxaban, administered to the patient after TIA occurrence did not show significant interaction with CZP. In addition to confirm that cautions should be used when antiepileptic and DOACs are concomitantly prescribed, the present case also demonstrates that, in the management of certain subsets of patients who need anticoagulant treatment, measurement of DOAC plasma concentration can help guide a personalized management and avoid adverse clinical outcomes.

Von Willebrand factor hyperactivity affects the outcome of lower limb revascularization in subjects with type 2 diabetes mellitus complicated by diabetic foot vasculopathy: An observational pilot study.

Aim of this study is to evaluate any differences in VWF antigen, VWF activity and ADAMTS-13 activity before and after successful and non-successful Percutaneous Transluminal Angioplasty (PTA) in subjects with type 2 diabetes (T2DM) complicated by Chronic limb-threatening ischemia (CLTI) in diabetic foot vasculopathy. METHODS: In this prospective observational pilot study, we enrolled 35 T2DM subjects who underwent lower limb PTA. Transcutaneous oximetry was performed in all patients before and 6 weeks after PTA. The change in oxygen partial pressure (TcpO2) before and after PTA was expressed as TcpO2-delta (ΔTcpO2). VWF antigen, VWF activity and ADAMTS-13 activity were measured before and 6 weeks after PTA; changes were expressed as delta and ratio from baseline. RESULTS: Subjects with ∆TcpO2 < 15 mmHg presented higher ΔVWF activity (p = 0.050) and lower ADAMTS-13 activity ratio (p = 0.080). Subjects with ∆TcpO2 < 30 mmHg showed lower ADAMTS-13 activity Δ and ratio (p = 0.028). CONCLUSIONS: VWF antigen levels and VWF activity may potentially affect PTA outcome. Higher levels of VWF could derive from VWF release as consequence of PTA-induced mechanical endothelial damage and/or oxidative stress-induced modifications of VWF structure with impairment of VWF-ADAMTS13 interactions.

Congenital prothrombin deficiency: an update.

Prothrombin (factor II [FII]) deficiency is a rare inherited coagulation disorder, having a prevalence of approximately 1 in 2,000,000. Two phenotypes can be distinguished: (1) true hypoprothrombinemia (type I deficiency), characterized by concomitantly low levels of the zymogen antigen; and (2) dysprothrombinemia (type II deficiency), characterized by the normal or near-normal synthesis of a dysfunctional protein. In the latter case, recent studies showed that particular mutations in the catalytic domain of active thrombin can even impair the enzyme interaction with antithrombin, favoring thromboembolic diseases. In some cases, hypoprothrombinemia associated with dysprothrombinemia was also described in compound heterozygous defects. Prothrombin is essential for the development of mammalian organisms. No living patient with undetectable plasma prothrombin has been reported to date. Prothrombin is encoded by a ≈21 kb gene located on chromosome 11 and containing 14 exons. Thirty-nine different mutations have been identified and characterized in prothrombin deficiency. Many of these are present in the catalytic site, whereas some involve regulatory domains, such as the anion-binding exosite I, the Na+-binding loop, and the light A-chain. Most hypoprothrombinemia-associated mutations are missense, but nonsense mutations leading to stop codons and one single nucleotide deletion have also been identified. Finally, recent developments in the therapy of congenital prothrombin deficiency are presented and discussed.

Oxidation of Met1606 in von Willebrand factor is a risk factor for thrombotic and septic complications in chronic renal failure.

CKD (chronic kidney disease) is a life-threatening pathology, often requiring HD (haemodialysis) and characterized by high OS (oxidative stress), inflammation and perturbation of vascular endothelium. HD patients have increased levels of vWF (von Willebrand factor), a large protein (~240 kDa) released as UL-vWF (ultra large-vWF polymers, molecular mass ~20000-50000 kDa) from vascular endothelial cells and megakaryocytes, and responsible for the initiation of primary haemostasis. The pro-haemostatic potential of vWF increases with its length, which is proteolytically regulated by ADAMTS-13 (a disintegrin and metalloproteinase with thrombospondin motifs 13), a zinc-protease cleaving vWF at the single Tyr1605-Met1606 bond, and by LSPs (leucocyte serine proteases), released by activated PMNs (polymorphonuclear cells) during bacterial infections. Previous studies have shown that in vitro oxidation of Met1606 hinders vWF cleavage by ADAMTS-13, resulting in the accumulation of UL-vWF that are not only more pro-thrombotic than shorter vWF oligomers, but also more efficient in binding to bacterial adhesins during sepsis. Notably, HD patients have increased risk of developing dramatic cardiovascular and septic complications, whose underlying mechanisms are largely unknown. In the present study, we first purified vWF from HD patients and then chemically characterized its oxidative state. Interestingly, HD-vWF contains high carbonyl levels and increased proportion of UL-vWF polymers that are also more resistant to ADAMTS-13. Using TMS (targeted MS) techniques, we estimated that HD-vWF contains >10% of Met1606 in the sulfoxide form. We conclude that oxidation of Met1606, impairing ADAMTS-13 cleavage, results in the accumulation of UL-vWF polymers, which recruit and activate platelets more efficiently and bind more tightly to bacterial adhesins, thus contributing to the development of thrombotic and septic complications in CKD.

Measurement of extended half-life recombinant FVIII molecules: In vitro and ex vivo evidence of relevant assay discrepancies.

Background: Extended half-life recombinant FVIII products (EHL-rFVIIIs) have been engineered to improve the pharmacokinetic profile of FVIII, enabling better hemostatic protection with a reduced number of injections in persons with hemophilia. Previous studies showed several discrepancies in FVIII activity (FVIII:C) measurements for EHL-rFVIIIs comparing one-stage clotting assay (OSA) and chromogenic assay (CSA), although a systematic investigation of this phenomenon is still lacking. Objective: Evaluation of the accuracy and precision of measurement of all available EHL-rFVIIIs with 5 different assays both in vitro and ex vivo. Methods: Damoctocog alfa pegol, rurioctocog alfa pegol, turoctocog alfa pegol, and efmoroctocog alfa were tested with 3 OSA types: (1) aPTT-based commercial reagents with colloidal silica (Synthasil, Werfen-IL); (2) ellagic acid, Synthafax (Werfen-IL); and (3) OSA calibrated with each EHL-rFVIII product and colloidal silica. Measurements were also carried out with 2 different commercially available CSA reagents (Coamatic Factor VIII, Chromogenix-Werfen) and Trinichrom FVIII (Tcoag-Stago). A Bland-Altman analysis was performed to compare all assays. Results: The simple OSA showed significant discrepancies between the expected and measured EHL-rFVIII concentrations as CSA methods, whereas the calibrated OSA assay was accurate and precise in determining the activity of all EHL-rFVIIIs in the in vitro setting. Comparable results were found using ex vivo plasma samples. Conclusion: In this study, only OSA with a calibration curve constructed with each EHL-rFVIII product showed acceptable accuracy and precision in EHL-rFVIIIs measurements.

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.

Oral anticoagulants in fragile patients with percutaneous endoscopic gastrostomy and atrial fibrillation: the ORIGAMI pilot investigation.

BACKGROUND: Extensive data support the superior safety without any trade-off in efficacy of direct oral anticoagulants (DOACs) compared to vitamin K antagonists (VKA) in patients with nonvalvular atrial fibrillation, deep venous thrombosis or pulmonary embolism. Whether DOACs may be successfully used to treat complex and fragile patients with percutaneous endoscopic gastrostomy (PEG) remains to be proven. The purpose of this pilot study was to evaluate the feasibility, anticoagulant effect, and preliminary safety/efficacy profile of edoxaban administered via PEG in patients with an indication for long-term oral anticoagulation. METHODS: In this prospective, single-arm, pilot study, 12 patients with PEG and guideline-recommended indication for anticoagulation for nonvalvular atrial fibrillation were prospectively enrolled. Crushed edoxaban at approved doses was administered via PEG. Quantitative measures of edoxaban's antifactor Xa activity were performed at steady state. Thromboembolic and bleeding events were assessed at one-month follow-up. RESULTS: Steady state edoxaban plasma levels were at therapeutic range in all patients; mean plasma concentration was 208.5 (±78.6) ng/mL. At one month follow-up, none had suffered a thromboembolic event; one developed minor bleeding, and one died from non-cardiovascular death, owing to sudden worsening of a pre-existing underlying severe condition. CONCLUSIONS: In this pilot investigation, we report for the first time that crushed edoxaban, administered at approved doses through PEG in fragile and complex patients, is feasible, results in therapeutic edoxaban concentrations, and is apparently effective and safe.

The dominant-negative von Willebrand factor gene deletion p.P1127_C1948delinsR: molecular mechanism and modulation.

Understanding molecular mechanisms in the dominant inheritance of von Willebrand disease would improve our knowledge of pathophysiologic processes underlying its prevalence. Cellular models of severe type 2 von Willebrand disease, caused by a heterozygous deletion in the von Willebrand factor (VWF) gene, were produced to investigate the altered biosynthesis. Coexpression of the wild-type and in-frame deleted (p.P1127_C1948delinsR) VWF forms impaired protein secretion, high molecular weight multimer formation and function (VWF collagen-binding 1.9% ± 0.5% of wild-type), which mimicked the patient's phenotype. mRNA, protein, and cellular studies delineated the highly efficient dominant-negative mechanism, based on the key role of heterodimers as multimer terminators. The altered VWF, synthesized in large amounts with the correctly encoded "cysteine knot" domain, formed heterodimers and heterotetramers with wild-type VWF, in addition to deleted homodimers. Impaired multimerization was associated with reduced amounts of VWF in late endosomes. Correction of the dominant-negative effect was explored by siRNAs targeting the mRNA breakpoint, which selectively inhibited the in-frame deleted VWF expression. Although the small amount of the deleted protein synthesized after inhibition still exerted dominant, even though weakened, negative effects, the siRNA treatment restored secretion of large multimers with improved function (VWF collagen-binding 28.0% ± 3.3% of wild-type).

The p.P1127S pathogenic variant lowers von Willebrand factor levels through higher affinity for the macrophagic scavenger receptor LRP1: Clinical phenotype and pathogenic mechanisms.

BACKGROUND: The index case is a 21-year-old Italian woman with a mild hemorrhagic syndrome and von Willebrand factor antigen (VWF:Ag) = 34.3 U/dl, VWF recombinant glycoprotein Ib (VWF:GpIbR) = 32.8 U/dl, and factor VIII (FVIII) = 55.3 IU/dl. AIMS: The aim of this study is to characterize from a genetic and biochemical standpoint this low VWF phenotype. METHODS: Coagulation and biochemical methods were used to study the structural and functional pattern of VWF multimers in the index case's plasma. Recombinant wild-type and p.P1127S VWF variants were produced using human embryonic kidney (HEK)-293 cells. In addition, genetic screening was carried out to detect single nucleotide variants of some scavenger VWF/FVIII receptor genes such as CLEC4M, STAB2, and ASGR2. RESULTS: Genetic investigation revealed that the index case inherited from her mother the heterozygous missense mutation c.3379C > T (VWF exon 25), causing the p.P1127S substitution in the VWF D'D3 domain. The index case was also homozygous for the scavenger receptor ASGR2 c.-95 CC-genotype. Desmopressin normalized the VWF level of the patient, although its clearance was faster (t1/2  = 6.7 h) than in normal subjects (t1/2  = 12 ± 0.7 h). FVIII-VWF interaction, A Disintegrin And Metalloprotease with ThromboSpondin type 1 motif-13 levels, ristocetin-induced-platelet-aggregation, and VWF multimeric pattern were normal. The p.P1127S variant was normally synthesized and secreted by HEK-293 cells, and molecular modeling predicts a conformational change showing higher affinity for the macrophagic scavenger receptor lipoprotein receptor-related protein 1 (LRP1), as also experimentally verified. CONCLUSIONS: The p.P1127S variant may cause a low VWF phenotype, stemming from an increased VWF affinity for the scavenger receptor LRP1 and, consequently, an accelerated clearance of VWF.

ADAMTS-13/von Willebrand factor ratio: A prognostic biomarker for portal vein thrombosis in compensated cirrhosis. A prospective observational study.

BACKGROUND AND AIMS: In cirrhosis, decreased portal flow velocity, thrombophilia factors, and portal hypertension are considered risk factors for portal vein thrombosis (PVT). In cirrhosis, the transformation of the stellate cells causes a progressive decrease of ADAMTS-13, while VWF multimers secretion by endothelial cells is strongly enhanced. This imbalance leads to an accumulation of ultra-large VWF multimers that in sinusoidal circulation could favor PVT both in intra- and extra-hepatic branches, mostly in decompensated cirrhosis. This prospective study was aimed at identifying possible clinical, biochemical, and hemostatic factors predictive for non-tumoral PVT in a cohort of patients with compensated cirrhosis. METHODS: Seventynine compensated cirrhosis patients were prospectively followed for 48 months, receiving a periodic Doppler-ultrasound liver examination associated with an extensive evaluation of clinical, biochemical, and hemostatic profile. RESULTS: Five patients developed PVT (cumulative prevalence = 6.3%), occurring 4-36 months after enrollment. In logistic regression analysis, the ADAMTS-13/VWF:GpIbR ratio < 0.4 was the only independent variable significantly associated with PVT (OR 14.6, 95% C.I.:1.36-157.2, p = 0.027). A Cox-regression-analysis confirmed this finding (HR = 7.7, p = 0.027). CONCLUSIONS: The ADAMTS-13/VWF ratio < 0.4 measured in compensated cirrhosis could be a reliable predictive biomarker for PVT development, paving the way to novel therapeutic strategies to prevent and treat PVT in this clinical setting.

Direct oral anticoagulants and therapeutic adherence: do not let your guard down.

BACKGROUND: Direct oral anticoagulants (DOAC) and vitamin K antagonist drugs (VKA) are recommended for stroke prevention in atrial fibrillation and for treatment of venous thromboembolism. Undoubtedly, DOAC have contributed to improve quality of life of these patients, but unfortunately, available 'real world' data show a very high variable compliance to DOAC. AIMS AND OBJECTIVES: to evaluate predictors that adversely affect therapeutic adherence in patients naive naïve  to DOAC. METHODS AND POPULATION: this study was conducted on an outpatient population in oral anticoagulant therapy in a period between January 2019 and February 2020. Patients naiveto DOAC and treated for at least 6 months were enrolled. Non-Italian-speaking patients, cognitive or psychiatric disorders, refusal to participate or non-consent to the interview were exclusion criteria. A socio-demographic scale and the 8-item Morisky scale (MMAS-8) questionnaire assessed therapeutic adherence. RESULTS: One hundred two DOAC-naïve patients were selected from a population of 407 patients on the first visit at our centre. The population was homogeneously represented for gender (males 48%). The mean age was 79.5 years. Atrial fibrillation (65.7%) resulted the main reason for DOAC prescription and a polypharmacy was detected in 47.1% of the patients. Moreover, an optimal adherence to DOAC therapy was assessed in less than 30% of patients. CONCLUSIONS: Polypharmacy, patient's isolation, such as a low education level were statistically associated with a low therapeutic adherence. Therapeutic adherence remains an unsolved problem for anticoagulated patient. To identify patients at higher risk of poor compliance and therapeutic failure and establish targeted care pathways is a priority.

Extended coagulation profile of children with Long Covid: a prospective study.

Emerging data suggests that endotheliopathy changes can be associated with post covid condition (PCC) in adults. Research on the matter in children is lacking. We analyzed an extended coagulation profile including biomarkers of endothelial damage in children with PCC and compared it with a control group of children that fully recovered post- SARS-CoV-2 infection. A case-control study enrolling children below 18 years of age with previous microbiologically confirmed SARS-CoV-2 infection in a pediatric post-covid unit in Italy ≥ 8 weeks after the initial infection. Samples were taken at 8 and 12 weeks after the SARS-CoV-2 diagnosis and analyzed for coagulation profiling (fibrinogen, prothrombin time, international normalized ratio, activated partial thromboplastin time, d-dimers, factor VIII coagulant activity, plasma von Willebrand factor (VWF) antigen and VWF ristocetin cofactor (RC)). We compared coagulation profiles in samples from children identified with PCC (at least one, or three or more symptoms, which could not be explained by an alternative diagnosis, at the 8- and 12-week follow-up assessment using the pediatric Long Covid International Severe Acute Respiratory and Emerging Infection Consortium (ISARIC) survey. Seventy-five children were enrolled, 49.3% were females, the median age was 10.2 (IQR 4.9) years. Forty-six (61%) of the children had at least one persisting symptom at the eight weeks post-onset, (PCC8); 39/75 (52%) had persistent symptoms for more than 12 weeks (PCC12) and 15/75(32%) had at least three persisting symptoms (PCC ≥ 3) at 12 weeks. Children with PCC presented more frequently with abnormal D-Dimer levels above the reference range compared to children that had fully recovered at the 8-12 weeks (39.1% vs. 17.2%, p = 0.04), and 12 week follow up or more (41% vs. 17.2%, p = 0.05), and in children with three or more symptoms at 12 weeks follow up compared to those that had recovered (64.3% vs. 22.2%, p = 0.002). For the other coagulation profiles, there were abnormal values detected for VWF, FVIII, RC and Fibrinogen but no significant differences between children with PCC compared to controls. Although the majority of children in our cohort showed coagulation profile within or close to normal ranges, we found that a higher proportion of children with PCC, and specifically those with a more severe spectrum characterized with three or more persisting symptoms, had abnormal D-dimer levels compared to other children that fully recovered from an acute SARS-CoV-2 infection.

Prostaglandin E2 differentially modulates human platelet function through the prostanoid EP2 and EP3 receptors.

Activated human platelets synthesize prostaglandin (PG) E(2), although at lower rate than thromboxane A(2). PGE(2) acts through different receptors (EP1-4), but its role in human platelet function remains poorly characterized compared with thromboxane. We studied the effect of PGE(2) and its analogs on in vitro human platelet function and platelet and megakaryocyte EP expression. Platelets preincubated with PGE(2) or its analogs were stimulated with agonists and studied by optical aggregometry. Intraplatelet calcium mobilization was investigated by the stopped flow method; platelet vasodilator-stimulated phosphoprotein (VASP), P-selectin, and microaggregates were investigated by flow cytometry. PGE(2) at nanomolar concentrations dose-dependently increased the slope (velocity) of the secondary phase of ADP-induced platelet aggregation (EC(50), 25.6 ± 6 nM; E(max) of 100 ± 19% increase versus vehicle-treated), without affecting final maximal aggregation. PGE(2) stabilized reversible aggregation induced by low ADP concentrations (EC(50), 37.7 ± 9 nM). The EP3 agonists, 11-deoxy-16,16-dimethyl PGE(2) (11d-16dm PGE(2)) and sulprostone enhanced the secondary wave of ADP-induced aggregation, with EC(50) of 48.6 ± 10 nM (E(max), 252 ± 51%) and 5 ± 2 nM (E(max), 300 ± 35%), respectively. The EP2 agonist butaprost inhibited ADP-induced secondary phase slopes (IC(50), 40 ± 20 nM). EP4 stimulation had minor inhibitory effects. 11d-16dm PGE(2) alone raised intraplatelet Ca(2+) and enhanced ADP-induced Ca(2+) increase. 11d-16dm PGE(2) and 17-phenyltrinor PGE(2) (EP3 > EP1 agonist) at nanomolar concentrations counteracted PGE(1)-induced VASP phosphorylation and induced platelet microaggregates and P-selectin expression. EP1, EP2, EP3, and EP4 were expressed on human platelets and megakaryocytes. PGE(2) through different EPs finely modulates human platelet responsiveness. These findings should inform the rational selection of novel antithrombotic strategies based on EP modulation.

Glycaemic variability affects ischaemia-induced angiogenesis in diabetic mice.

The aim of the present study was to investigate the role of GV (glycaemic variability) in diabetic vascular complications and to explore the molecular pathways modulated by glycaemic 'swings'. We developed a murine model. A total of 30 diabetic mice received once daily basal insulin administration plus two oral boluses of glucose solution (GV group, named 'V') and 30 diabetic mice received once daily basal insulin plus two oral boluses of saline solution (stable hyperglycaemia group, named 'S') for a period of 30 days. Glycaemia was measured eight times daily to detect GV. Finally, postischaemic vascularization, induced by hindlimb ischaemia 30 days after diabetes onset, was evaluated. We found that GV was significantly different between S and V groups, whereas no significant difference in the mean glycaemic values was detected. Laser Doppler perfusion imaging and histological analyses revealed that the ischaemia-induced angiogenesis was significantly impaired in V mice compared with S group, after ischaemic injury. In addition, immunostaining and Western blot analyses revealed that impaired angiogenic response in V mice occurred in association with reduced VEGF (vascular endothelial growth factor) production and decreased eNOS (endothelial nitric oxide synthase) and Akt (also called protein kinase B) phosphorylation. In conclusion, we describe a murine model of GV. GV causes an impairment of ischaemia-induced angiogenesis in diabetes, likely to be independent of changes in average blood glucose levels, and this impaired collateral vessel formation is associated with an alteration of the VEGF pathway.

Cystic fibrosis transmembrane conductance regulator (CFTR) expression in human platelets: impact on mediators and mechanisms of the inflammatory response.

Inflammatory lung disease is a primary cause of morbidity and mortality in cystic fibrosis (CF). Mechanisms of unresolved acute inflammation in CF are not completely known, although the involvement of cystic fibrosis transmembrane conductance regulator (CFTR) in nonrespiratory cells is emerging. Here we examined CFTR expression and function in human platelets (PLTs) and found that they express a biologically active CFTR. CFTR blockade gave an ∼50% reduction in lipoxin A(4) (LXA(4)) formation during PLT/polymorphonuclear leukocytes (PMN) coincubations by inhibiting the lipoxin synthase activity of PLT 12-lipoxygenase. PLTs from CF patients generated ∼40% less LXA(4) compared to healthy subject PLTs. CFTR inhibition increased PLT-dependent PMN viability (33.0±5.7 vs. 61.2±8.2%; P=0.033), suppressed nitric oxide generation (0.23±0.04 vs. 0.11±0.002 pmol/10(8) PLTs; P=0.004), while reducing AKT (1.02±0.12 vs. 0.71±0.007 U; P=0.04), and increasing p38 MAPK phosphorylation (0.650±0.09 vs. 1.04±0.24 U; P=0.03). Taken together, these findings indicate that PLTs from CF patients are affected by the molecular defect of CFTR. Moreover, this CF PLT abnormality may explain the failure of resolution in CF.

Marked von Willebrand factor and factor VIII elevations in severe acute respiratory syndrome coronavirus-2-positive, but not severe acute respiratory syndrome coronavirus-2-negative, pneumonia: a case-control study.

Patients with novel coronavirus pneumonia show increased thrombotic risk. Although hemostatic alterations have been described in novel coronavirus pneumonia patients, case-control studies of von Willebrand factor (VWF), factor VIII (FVIII), and a disintegrin-like and metalloprotease with thrombospondin type I motif, member 13 (ADAMTS13) are lacking. VWF, ADAMTS13, FVIII, d-dimer, C-reactive protein, and routine blood cells and chemistry were measured in 10 novel coronavirus pneumonia patients and 10 non-novel coronavirus pneumonia controls. Hemostatic factors were measured less than 48 h of hospital admission in patients without invasive ventilation. d-Dimer, C-reactive protein, and fibrinogen concentrations, high in both groups, did not differ significantly in novel coronavirus pneumonia vs. non-novel coronavirus pneumonia patients. Median VWF-antigen (324 vs. 153 IU/dl, P < 0.0001), VWF-Rco (342 vs. 133 IU/dl, P < 0.001), and FVIII-activity levels (203 vs. 123 IU/dl, P < 0.0001) were significantly higher in novel coronavirus pneumonia cases vs. controls ADAMTS13-activity was normal in both groups. Coronavirus pneumonia cases vs. non-novel coronavirus pneumonia controls showed marked VWF/FVIII elevation, suggesting specific virus-induced endothelial activation causing VWF/FVIII hypersecretion, which may represent a therapeutic target in novel coronavirus pneumonia.

Case Report: Two Cases of Pediatric Thrombotic Thrombocytopenic Purpura Treated With Combined Therapy.

Thrombotic thrombocytopenic purpura (TTP) is a thrombotic microangiopathy caused by a severely reduced activity of the von Willebrand factor-cleaving protease ADAMTS13. Over 95% of TTPs are acquired, due to autoantibody inhibitors. In children, acquired TTP is a very rare, life-threatening disease. To date, no consensus exists on the treatment strategy of pediatric TTP. We report the cases of two pediatric patients with a diagnosis of TTP, successfully treated with a combination of various therapeutic approaches. Although the patients complained of different sets of symptoms, laboratory data showed Coombs negative hemolytic anemia, renal impairment, and low platelet count in both cases. The diagnosis of acquired TTP was supported by the PLASMIC score and confirmed by the reduction of the ADAMTS13 activity and the presence of anti-ADAMTS13 antibodies. Intravenous immunoglobulin, corticosteroids, and plasma exchange (PEX) were performed without delay. As soon as available, caplacizumab was added to the therapy, with a prompt normalization of platelet count. Nevertheless, ADAMTS13 activity was persistently low, and anti-ADAMTS13 antibodies level was high; thus, a course of rituximab was administered, with persistent normalization of laboratory findings. No adverse events were observed during the treatment. In our experience, the combined use of PEX, caplacizumab, and immunosuppressive therapy during the acute phase of the disease is safe and may have a significant impact on the prognosis with successful clinical outcome and decrease in life-threatening events.

Ristocetin-induced self-aggregation of von Willebrand factor.

Von Willebrand factor (VWF) is a large multimeric adhesive glycoprotein, with complex roles in thrombosis and hemostasis, present in circulating blood and in secretory granules of endothelial cells and platelets. High shear stress triggers conformational changes responsible for both binding to the platelet receptor glycoprotein GpIb and its self-association, thus supporting the formation of platelet plug under flow. Ristocetin also promotes the interaction of VWF with GpIb and is able to induce platelet aggregation, and thus is largely used to mimic this effect in vitro. In this research paper, we followed the time course of VWF self-association in solution induced by ristocetin binding by light scattering and at the same time we collected atomic force microscopy images to clarify the nature of the assembly that is formed. In fact, this process evolves initially through the formation of fibrils that subsequently interact to form supramolecular structures whose dimensions would be capable of trapping platelets even in the absence of any degree of shear stress or interaction with external surfaces. This intrinsic property, that is the ability to self-aggregate, may be involved in some pathological settings that have been revealed in clinical practice.

Increased von Willebrand factor levels in polycythemia vera and phenotypic differences with essential thrombocythemia.

BACKGROUND: Acquired von Willebrand factor (VWF) deficiency was described in Philadelphia-negative myeloproliferative neoplasms, especially in essential thrombocythemia (ET). VWF phenotype in contemporary patients with polycythemia vera (PV) remains less explored. OBJECTIVES: To characterize the VWF phenotype in PV and to compare VWF phenotype in PV with matched healthy subjects and ET patients. PATIENTS/METHODS: We studied 48 PV patients, treated according to current recommendations (hematocrit ≤ 45%, on low-dose aspirin prophylaxis); 48 healthy and 41 subjects with ET, all sex, age, and blood group matched. We measured VWF antigen, activity, multimeric pattern, ADAMTS-13, and factor VIII (FVIII) antigen. RESULTS: In patients with PV, VWF antigen and activity were significantly higher than in healthy subjects (antigen: 119[96-137] vs 93[79-107] IU/dL; activity: 114[95-128] vs 90[79-107] IU/dL, respectively, medians and interquartile, P < 0.01), with normal multimeric distribution. ADAMTS-13 levels were similar between patients with PV and healthy subjects. FVIII levels were higher in PV than in healthy subjects (141[119-169] versus 98[88-123] IU/dL, respectively, P < 0.01). By multivariable analysis, JAK2-p.V617F allelic burden, erythrocyte count, and male sex significantly predicted VWF antigen and activity levels. As compared to patients with ET, patients with PV showed similar VWF antigen levels but approximately 40% higher activity (79[49-104] vs 112[93-125] IU/dL, respectively, P < 0.01). CONCLUSIONS: Patients with PV show increased VWF and FVIII levels, predicted by JAK2-p.V617F burden and erythrocyte count. At variance with ET, acquired VWF defect was not observed in PV. High VWF/FVIII levels may sustain the thrombotic diathesis of PV and may be investigated as biomarkers for risk stratification.

Noncanonical type 2B von Willebrand disease associated with mutations in the VWF D'D3 and D4 domains.

We observed a 55-year-old Italian man who presented with mucosal and cutaneous bleeding. Results of his blood analysis showed low levels of von Willebrand factor (VWF) antigen and VWF activity (both VWF ristocetin cofactor and VWF collagen binding), mild thrombocytopenia, increased ristocetin-induced platelet aggregation, and a deficiency of high-molecular-weight multimers, all typical phenotypic hallmarks of type 2B von Willebrand disease (VWD). The analysis of the VWF gene sequence revealed heterozygous in cis mutations: (1) c.2771G>A and (2) c.6532G>T substitutions in the exons 21 and 37, respectively. The first mutation causes the substitution of an Arg residue with a Gln at position 924, in the D'D3 domain. The second mutation causes an Ala to Ser substitution at position 2178 in the D4 domain. The patient's daughter did not present the same fatherly mutations but showed only the heterozygous polymorphic c.3379C>T mutation in exon 25 of the VWF gene causing the p.P1127S substitution, inherited from her mother. The in vitro expression of the heterozygous in cis VWF mutant rVWFWT/rVWF924Q-2178S confirmed and recapitulated the ex vivo VWF findings. Molecular modeling showed that these in cis mutations stabilize a partially stretched and open conformation of the VWF monomer. Transmission electron microscopy and atomic force microscopy showed in the heterozygous recombinant form rVWFWT/rVWF924Q-2178S a stretched conformation, forming strings even under static conditions. Thus, the heterozygous in cis mutations 924Q/2178S promote conformational transitions in the VWF molecule, causing a type 2B-like VWD phenotype, despite the absence of typical mutations in the A1 domain of VWF.