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.

Eltrombopag for the treatment of inherited thrombocytopenias: a phase II clinical trial.

Patients with inherited thrombocytopenias often require platelet transfusions to raise their platelet count before surgery or other invasive procedures; moreover, subjects with clinically significant spontaneous bleeding may benefit from an enduring improvement of thrombocytopenia. The hypothesis that thrombopoietin-mimetics can increase platelet count in inherited thrombocytopenias is appealing, but evidence is scarce. We conducted a prospective, phase II clinical trial to investigate the efficacy of the oral thrombopoietin-mimetic eltrombopag in different forms of inherited thrombocytopenia. We enrolled 24 patients affected by MYH9-related disease, ANKRD26-related thrombocytopenia, X-linked thrombocytopenia/ Wiskott-Aldrich syndrome, monoallelic Bernard-Soulier syndrome, or ITGB3-related thrombocytopenia. The average pre-treatment platelet count was 40.4 ×109/L. Patients received a 3- to 6-week course of eltrombopag in a dose-escalated manner. Of 23 patients evaluable for response, 11 (47.8%) achieved a major response (platelet count >100 ×109/L), ten (43.5%) had a minor response (platelet count at least twice the baseline value), and two patients (8.7%) did not respond. The average increase of platelet count compared to baseline was 64.5 ×109/L (P<0.001). Four patients with clinically significant spontaneous bleeding entered a program of long-term eltrombopag administration (16 additional weeks): all of them obtained remission of mucosal hemorrhages, with the remission persisting throughout the treatment period. Treatment was globally well tolerated: five patients reported mild adverse events and one patient a moderate adverse event. In conclusion, eltrombopag was safe and effective in increasing platelet count and reducing bleeding symptoms in different forms of inherited thrombocytopenia. Despite these encouraging results, caution is recommended when using thrombopoietinmimetics in inherited thrombocytopenias predisposing to leukemia. ClinicalTrials.gov identifier: NCT02422394.

Acquired von Willebrand syndrome in patients with monoclonal gammopathy of undetermined significance investigated using a mechanistic approach.

BACKGROUND: Acquired von Willebrand syndrome (AVWS) has been reported to occur in association with monoclonal gammopathy, usually of undetermined significance (MGUS). It may present as a type 1 or type 2 von Willebrand factor (VWF) defect depending on the patient's representation of large VWF multimers. MATERIALS AND METHODS: The mathematical model by Galvanin et al., already employed for studying inherited von Willebrand disease (VWD), was used to explore the pathogenic mechanisms behind MGUS-associated AVWS. RESULTS: The patients studied showed significantly reduced VWF levels and function; an increased VWF propeptide to VWF antigen ratio; and all VWF multimers present but in reduced quantities, with the low-molecular-weight VWF forms being significantly more represented than those of higher molecular weight. Our mathematical model revealed a significantly increased VWF elimination rate constant, with values similar to those of type Vicenza VWD. An even more increased VWF proteolysis rate constant was observed, with values one order of magnitude higher than in type 2A VWD but, in contrast, no loss of large multimers. The model predicted the same elimination rate for high- and low-molecular-weight VWF multimers, but proteolysis of the high-molecular-weight forms also contributes to the pool of low-molecular-weight oligomers, which explains why they were relatively over-represented. DISCUSSION: In MGUS-associated AVWS the increase of both clearance and proteolysis contributes to the circulating levels and multimer pattern of VWF, with a phenotype that appears to be a combination of type Vicenza and type 2A VWD. Hence, the mechanisms behind the onset of AVWS seem to differ from those of inherited VWD.

Ferric Carboxymaltose in Patients with Acute Decompensated Heart Failure and Iron Deficiency: A Real-Life Study.

Background: The correction of iron deficiency (ID) with ferric carboxymaltose (FCM) is a recommended intervention in heart failure (HF) with reduced ejection fraction. Our aim is to evaluate, in a real-life setting, the clinical significance of ID screening and FCM treatment in acute decompensated HF (ADHF). Methods: In a cohort of ADHF patients, the prevalence of ID and FCM administration were investigated. Among the 104 patients admitted for ADHF, in n = 90 (median age 84, 53.5% with preserved left ventricular ejection fraction-LVEF), a complete iron status evaluation was obtained. ID was detected in n = 73 (81.1%), 55 of whom were treated with in-hospital FCM. The target dose was reached in n = 13. Results: No significant differences were detected in terms of age, sex, comorbidities, or LVEF between the FCM-supplemented and -unsupplemented patients. During a median follow-up of 427 days (IQR 405-466) among the FCM-supplemented patients, only 14.5% received FCM after discharge; the mortality and rehospitalizations among FCM-supplemented and -unsupplemented patients were similar (p = ns). In a follow-up evaluation, ID was still present in 75.0% of the FCM-supplemented patients and in 69.2% of the unsupplemented patients (p = ns). Conclusions: In this real-life ADHF cohort, FCM was administered at lower-than-prescribed doses, thus having no impact on ID correction. The significance of our findings is that only achieving the target dose of FCM and pursuing outpatient treatment can correct ID and produce long-term clinical benefits.

An apparently silent nucleotide substitution (c.7056C>T) in the von Willebrand factor gene is responsible for type 1 von Willebrand disease.

BACKGROUND: Nucleotide variations not changing protein sequences are considered silent mutations; accumulating data suggest that they can, however, be important in human diseases. DESIGN AND METHODS: We report an altered splicing process induced by a silent substitution (c.7056C>T) in the von Willebrand factor gene in a case of type 1 von Willebrand disease originally classified as lacking von Willebrand factor mutations. RESULTS: The c.7056C>T synonymous substitution introduces a new donor splice site within exon 41, leading to messenger RNA lacking nucleotides 7055-7081 (c.7055_7081del). The encoded von Willebrand factor protein is predicted to lack amino acids 2352-2360 in the B2 domain. The patient's von Willebrand disease phenotype was characterized by reduced plasma and platelet von Willebrand factor, which was normal in function and multimer structure. In vitro expression studies demonstrated that co-transfection of equimolar c.7055_7081del and wild-type von Willebrand factor (mimicking the patient's heterozygous state) induced a 50% lower von Willebrand factor secretion than the wild type, while almost no von Willebrand factor secretion was seen with the mutated von Willebrand factor alone. The secreted von Willebrand factor was structurally and functionally normal, suggesting that the c.7056C>T substitution behaves like a loss-of-function allele. CONCLUSIONS: This is the first report of a synonymous von Willebrand factor substitution being responsible for von Willebrand disease. Our findings suggest the need to reconsider the role of von Willebrand factor polymorphisms in von Willebrand disease.

Focus on Key Issues in Immune Thrombotic Thrombocytopenic Purpura: Italian Experience of Six Centers.

Immune-mediated thrombotic thrombocytopenic purpura is a rare and challenging hematological disease caused by the antibody anti-ADAMTS13. Though the mortality rate has decreased considerably in recent years, fatalities still remain unacceptable. This study aimed at further adding to the existing knowledge of this medical challenge. We enrolled 89 consecutive patients observed in six Italian centers (from 8 August 2013 to 28 May 2021) with a diagnosis of immune-mediated thrombotic thrombocytopenic purpura. Clinical information and blood parameters were collected for all patients. We describe clinical manifestations and laboratory data, possible risk factors and the therapeutic management of first episodes or relapses. A total of 74 first episodes and 19 relapses (median 3 years (interquartile range (IQR): 2-7)) were recorded. Seventy percent of patients enrolled at the first episode showed neurological signs and/or symptoms. All the patients enrolled at the first episode were treated with plasma exchange (median = 12; IQR: 8-19.5) and methylprednisolone (1 mg/kg/day). Rituximab (375 mg/m2 weekly for four weeks) and caplacizumab were given to 15 (20.2%) and 2 patients (2.6%), respectively. We observed an overall mortality of 5.4% in the follow-up (median 60 months; IQR: 36.0-103.5). All fatalities occurred after a diagnostic delay. Present data point to the importance of the early detection of factors mostly associated with poor outcomes. It is likely that use of caplacizumab could improve the prognosis in those patients.

Reduced survival of type 2B von Willebrand factor, irrespective of large multimer representation or thrombocytopenia.

BACKGROUND: Type 2B von Willebrand factor (VWF) is characterized by gain of function mutations in the A1 domain inducing a greater affinity for platelet GPIb, possibly associated with the disappearance of large VWF multimers and thrombocytopenia. DESIGN AND METHODS: VWF survival was explored using 1-desamino-8-D-arginine vasopressin (DDAVP) in 18 patients with type 2B von Willebrand disease (VWD) and compared with their platelet count and large VWF multimer representation. RESULTS: A similarly significant shorter VWF survival, expressed as T(1/2)elimination (T(1/2)el), was observed in patients lacking large VWF multimers (type 2B) and in those with a normal multimer pattern (atypical type 2B) (4.47+/-0.41 h and 4.87+/-0.9 h, respectively, vs. normal 15.53+/-2.17 h) due mainly to a greater VWF clearance. The half-life of large VWF multimers, explored by VWF collagen binding (VWF:CB) activity, was likewise reduced. The similarly reduced VWF half-life was also confirmed by the increase in the VWF propeptide ratio (a useful tool for exploring VWF survival) which was found to be the same in type 2B and atypical type 2B patients. The post-DDAVP drop in platelet count occurred in all patients lacking large multimers but not in those with a normal multimer pattern. A correlation was always found between pre- and/or post-DDAVP thrombocytopenia and the lack of large VWF multimers in type 2B VWD while these were unrelated to the reduced VWF half-life. CONCLUSIONS: In addition to demonstrating that a shorter VWF survival contributes to the type 2B and atypical type 2B VWD phenotype, our findings suggest that VWF clearance and proteolysis are independent phenomena.

Platelet-Derived Microparticles Bearing PF4 and Anti-GAGS Immunoglobulins in Patients with Sepsis.

PF4 is a megakaryocyte-derived cationic chemokine that plays a part in innate immunity through its activity on the macrophages. In bacterial sepsis, PF4 binds to glycosaminoglycans (GAGs) on the surface of aerobic bacteria, giving rise to an antigenic complex that induces the early formation of anti-PF4 IgG-IgA-IgM. This triggers the immune response in patients receiving heparin therapy who develop heparin-induced thrombocytopenia (HIT). These antibodies have also been identified in patients with chronic Gram-negative infections. Given the complexity of this innate immune response network, our study on 45 patients with sepsis focused on the immune response mediated by platelet PF4. We analyzed the role of IgG-IgA-IgM against PF4-GAGs, and the presence of specific PF4-bearing platelet microparticles (PMPs). Anti-GAGs/PF4 IgG-IgA-IgM levels were significantly higher in septic patients than in control groups (healthy controls or acute patients without sepsis, p < 0.001). PF4-bearing PMP levels were only significantly higher in septic patients (p < 0.001). The occurrence of IgG-IgA-IgM against PF4-GAGs and PF4+ PMPs correlated with an improvement in patients' sepsis. In conclusion, we demonstrated that, in the course of bacterial sepsis, platelet activation leads to the formation of specific PF4-bearing PMPs. These specific microparticles bind to polyanionic sequences on the surface of aerobic bacteria, giving rise to an antigenic complex that induces the early formation of IgG-IgA-IgM against PF4-GAGs as an innate immune response to infection.

Link between von Willebrand factor multimers, relapses and coronary microcirculation in patients with thrombotic thrombocytopenic purpura in remission.

INTRODUCTION: ADAMTS13 deficiency results in unusually large von Willebrand factor (ULVWF) multimers in the circulation and a higher risk of microthrombi due to high shear stress. In patients treated for acquired thrombotic thrombocytopenic purpura (TTP), a persistently severe ADAMTS13 deficiency (<10%) in remission is associated with more relapses. A reduced plasma ADAMTS13 activity and increased VWF levels are associated with a higher risk of myocardial infarction. Assessing coronary flow reserve (CFR) enables a better cardiovascular risk stratification: a lower CFR correlates inversely with cardiovascular risk. The aim of the study was to establish whether patients with TTP in remission have an impaired coronary microcirculation, in terms of a lower CFR, and whether there is any correlation between ADAMTS13 activity, the presence of ULVWF multimers, and the occurrence of relapses. METHODS: The clinical information and hemostatic parameters of 24 patients with TTP in remission managed at our center were analyzed. The CFR was assessed in a subgroup of the TTP patients and compared with a control group consisting of 50 healthy volunteers. RESULTS: The CFR was statistically lower in patients in remission of TTP than in controls, but there were no differences between TTP patients with normal and lower CFR. The occurrence of relapses correlated with the presence of ULVWF multimers and with a residual ADAMTS13 activity. CONCLUSIONS: When compared with healthy controls, TTP patients in remission have an impaired coronary microcirculation and the occurrence of relapses in the former reveal the presence of ULVWF multimers.

A phase III study comparing secondary long-term prophylaxis versus on-demand treatment with vWF/FVIII concentrates in severe inherited von Willebrand disease.

BACKGROUND: There is a lack of prospective clinical trials specifically designed to evaluate the benefits of prophylaxis with vWF/FVIII concentrates in patients with inherited von Willebrand disease (vWD). The aim of the study was to compare efficacy of secondary long-term prophylaxis (PRO) with vWF/FVIII in the prevention of bleeding episodes in severe vWD patients to standard of care (on-demand treatment; ODT). MATERIALS AND METHODS: In this 12-month, phase III, open-label study (PRO.WILL), vWD patients (aged ≥6 years) were randomised to PRO (n=9; 5 completed) or ODT (n=10; 7 completed) treatment with Fanhdi®/Alphanate® (Grifols) according to current licensing status for use in vWD. We assessed the proportion of patients who did not present any spontaneous bleeding episode, adverse events (AEs) or thrombotic events. RESULTS: All patients on ODT had vWD type 2 or 3 vs 70% of patients on PRO. All ODT patients experienced bleeds vs 60% on PRO. PRO patients showed fewer bleeds (n=32 vs n=172 [112 in the same patient, mostly mucosal]; p<0.0001) and lower risk of bleeding (relative attributable risk estimate: -0.667; 95% CI: -2.374, -0.107; p<0.001). Most frequent bleeds in ODT and PRO groups were, respectively, epistaxis (n=52 vs n=15) and gastrointestinal (n=13 [9 in the same patient] vs n=1). While most bleeds lasted one day under ODT (31/32), only epistaxis did so in PRO group (14/15). No AEs due to study medication were observed. DISCUSSION: Despite the small sample size and the heterogeneity of the study population, patients on vWF/FVIII prophylaxis showed a reduction in bleeding risk and rate compared to on-demand treatment.

A novel von Willebrand factor mutation (I1372S) associated with type 2B-like von Willebrand disease: an elusive phenotype and a difficult diagnosis.

Mutations in the A1 domain of von Willebrand factor (VWF) may be associated with gain of function in the VWF-platelet GPIb interaction and consumption of large VWF multimers, as seen in type 2B von Willebrand disease (VWD). We report a new VWF abnormality associated with greater VWF-GPIb interaction in the presence of all VWF multimers. The index case is a woman with a lifelong history of bleeding, found hyperresponsive to ristocetin with spontaneous platelet aggregation (SPA). She had normal factor VIII, VWF:Ag, VWF:RCo and VWF:CB levels, normal VWF:RCo/VWF:Ag and VWF:CB/VWF:Ag ratios, and a full panel of plasma and platelet VWF multimers. A missense mutation (4115T>G) was found in exon 28 of the VWF gene, which replaced a isoleucine with a serine at position 1372 of pre-pro-VWF (I1372S) at heterozygous level. Recombinant VWF carrying the I1372S mutation and showing a normal VWF multimer organisation was capable of inducing SPA on normal platelet-rich plasma (unlike wild-type VWF), as well as a hyper-response to ristocetin in the same platelets (0.6 mg/ml ristocetin vs. 1.2 of wild-type VWF). The new I1372S VWF mutation, characterized by SPA and hyper-responsiveness to ristocetin thus has some of the features of type 2B VWD, but not the lack of large VWF multimers, so we defined this variant as type 2B-like VWD. Why I1372S VWF is associated with bleeding symptoms, despite normal VWF levels and multimer organisation, remains to be seen.

Type IIb von Willebrand disease: role of qualitative defects in atherosclerosis and endothelial dysfunction.

OBJECTIVE: To verify whether a hereditary bleeding tendency, such as von Willebrand disease (vWD) type IIB, protects against the onset of atherosclerosis. PARTICIPANTS AND METHODS: Twenty-four patients with vWD type IIB and 24 healthy controls, matched for common atherosclerotic risk factors. All patients were evaluated by color Doppler ultrasound of the common carotid, carotid bifurcation, common femoral artery, brachial artery, and abdominal aorta, investigating intima-media thickness (IMT) and presence of plaques in each arterial district. Flow mediated dilation (FMD) of the brachial artery was used to test endothelial function. RESULTS: vWD type IIB patients presented no significant difference in IMT in any arterial district. FMD showed no differences between the 2 groups. CONCLUSIONS: The quantitative clotting defect characteristic of vWD type IIB does not seem to protect against atherosclerosis.

Type 2B von Willebrand disease with or without large multimers: A distinction of the two sides of the disorder is long overdue.

Most, but not all patients with type 2B von Willebrand disease (VWD)-which features gain-of-function mutations in the A1 domain of von Willebrand factor (VWF)-have no circulating large VWF multimers. Similarities and differences were analysed in 33 type 2B patients, 12 with a normal and 21 with an abnormal multimer pattern, to see whether they should be considered separately. The minimum aggregating dose of ristocetin was similarly reduced in both patient groups, and modulated by their underlying VWF mutations. Platelet VWF content was normal in all patients lacking in large multimers, but sometimes reduced in those with a normal multimer pattern. All the former patients and none of the latter had persistent or transient thrombocytopenia. A short VWF half-life (affecting plasma VWF levels) was seen in both groups, but more pronounced in patients without large multimers. Bleeding scores were also high in all patients, but more so in those without large multimers, apparently regardless of their platelet count. The marked phenotypic heterogeneity of type 2B VWD concerns not only patients' VWF multimer pattern, but also their bleeding risk, and consequently their appropriate treatment too. Hence the need to clearly distinguish between type 2B VWD with normal or abnormal VWF multimers.

Combined partial exon skipping and cryptic splice site activation as a new molecular mechanism for recessive type 1 von Willebrand disease.

We describe the complex picture associated with a mutated splice junction in intron 13 of von Willebrand factor (VWF) gene. The proband, characterized by a marked decrease in plasma and platelet VWF and near normal multimer organization, was classified as recessive type 1 von Willebrand disease (VWD). Genetic analysis demonstrated that he was homozygous for the 1534-3C > A mutation in the consensus sequence of the acceptor splicing site of intron 13 of the VWF gene. Platelet mRNA analysis documented three VWF transcripts: a wild type generated by the correct recognition of the mutated splice site, a smaller transcript not containing exon 14, and a longer one that, in addition to exons 13 and 14, included a 62bp fragment corresponding to the end of intron 13. The small transcript derives from the skipping of exon 14, the long one from the activation of a cryptic splice site in intron 13; both show a premature stop codon in VWF propeptide, so the proband VWF derives entirely from the correct splice site recognition. Combined incomplete exon skipping and cryptic splice site activation are first recognized in VWD. Since the 1534-3C > A mutation does not abolish the normal processing of mRNA, it is unlikely to be found in type 3 VWD. This mutation therefore appears to be peculiar to type 1 VWD.

Diagnostic Value of Measuring Platelet Von Willebrand Factor in Von Willebrand Disease.

Von Willebrand disease (VWD) may be caused by an impaired von Willebrand factor (VWF) synthesis, its increased clearance or abnormal function, or combinations of these factors. It may be difficult to recognize the different contributions of these anomalies. Here we demonstrate that VWD diagnostics gains from measuring platelet VWF, which can reveal a defective VWF synthesis. Measuring platelet VWF revealed that: severe type 1 VWD always coincided with significantly lower platelet and plasma VWF levels, whereas mild forms revealed low plasma VWF levels associated with low or normal platelet VWF levels, and the latter were associated with a slightly shorter VWF survival; type Vicenza (the archetype VWD caused by a reduced VWF survival) featured normal platelet VWF levels despite significantly reduced plasma VWF levels; type 2B patients could have either normal platelet VWF levels associated with abnormal multimer patterns, or reduced platelet VWF levels associated with normal multimer patterns; type 2A patients could have reduced or normal platelet VWF levels, the former associated mainly with type 2A-I, the latter with type 2A-II; plasma and platelet VWF levels were normal in type 2N, except when the defect was associated with a quantitative VWF mutation. Our findings show that measuring platelet VWF helps to characterize VWD, especially the ambiguous phenotypes, shedding light on the mechanisms underlying the disorder.

Fainting induces an acute increase in the concentration of plasma factor VIII and von Willebrand factor.

BACKGROUND AND OBJECTIVES: von Willebrand factor (VWF) is stored in the Weibel-Palade bodies of endothelial cells and may be released in response to different secretion stimuli such as stress, physical exercise, adrenaline, DDAVP and thrombin. DESIGN AND METHODS: We found that fainting can also induce an acute increase in plasma VWF and factor FVIII (FVIII) concentrations, following observations in two patients with von Willebrand's disease (VWD) who experienced a fainting episode during venipuncture for blood collection. RESULTS: One patient was classified as having type Vicenza VWD, the other as type 1 VWD; both had normal platelet VWF content. After the fainting episode, FVIII and VWF levels were significantly higher than the levels in blood samples collected without stress; mean increases were 4.35-fold for FVIII, 4-fold for VWF:Ag and 5.3-fold for VWF:RCo, with values overshooting the upper limit of the normal range. Moreover, the post-fainting plasma VWF multimer pattern was characterized by a significant increase in all oligomers with the appearance of unusually large VWF multimers, similar to those observed following DDAVP infusion. INTERPRETATION AND CONCLUSIONS: These findings demonstrate that fainting acts as a stimulus capable of inducing the release of VWF from endothelial cells, and further highlight the role of stress in determining hemostatic states potentially favorable to the development of thrombotic complications.