Tiziana Fierro

Incidence of a first thromboembolic event in asymptomatic carriers of high-risk antiphospholipid antibody profile: a multicenter prospective study

Persistent antiphospholipid (aPL) antibodies are occasionally found in subjects without prior history of thromboembolic events (TEs), raising the dilemma of whether to initiate or not a primary thromboprophylaxis. A first TE is considered rare in aPL carriers, but previous studies did not consider the aPL profile nor was the test positivity confirmed in a reference laboratory. In this study, 104 subjects with high-risk aPL profile (positive lupus anticoagulant, anticardiolipin, and anti-β(2)-glycoprotein I antibodies, triple positivity) confirmed in a reference laboratory, were followed up for a mean of 4.5 years. There were 25 first TEs (5.3% per year): the cumulative incidence after 10 years was 37.1% (95% confidence interval [CI], 19.9%-54.3%). On multivariate analysis, male sex (hazard ratio = 4.4; 95% CI, 1.5-13.1, P = .007) and risk factors for venous thromboembolism (hazard ratio = 3.3; 95% CI, 1.3-8.5, P = .01) were independent predictors for TEs. Aspirin did not significantly affect the incidence of TE. In conclusion, the occurrence of a first TE in carriers of high-risk aPL profile is considerable; it is more frequent among male subjects and in the presence of additional risk factors for venous TE. These data can help in the decision to initiate primary thromboprophylaxis in these subjects.

Eltrombopag for the treatment of the inherited thrombocytopenia deriving from MYH9 mutations

Platelet transfusion is currently the primary medical treatment for reducing thrombocytopenia in patients with inherited thrombocytopenias. To evaluate whether stimulating megakaryopoiesis could increase platelet count in these conditions, we treated patients with a severe thrombocytopenia induced by MYH9 mutations (MYH9-related disease) with a nonpeptide thrombopoietin receptor agonist, eltrombopag. Twelve adult patients with MYH9-RD and platelet counts of less than 50 × 10(9)/L received 50 mg of eltrombopag orally per day for 3 weeks. Patients who achieved a platelet count higher than 150 × 10(9)/L stopped therapy, those with 100 to 150 platelets × 10(9)/L continued treatment at the same eltrombopag dose for 3 additional weeks, while those with less than 100 platelets × 10(9)/L increased the eltrombopag dose to 75 mg for 3 weeks. Major responses (platelet count of at least 100 × 10(9)/L or 3 times the baseline value) were obtained in 8 patients, minor responses (platelet counts at least twice the baseline value) in 3. One patient did not respond. Bleeding tendency disappeared in 8 of 10 patients with bleeding symptoms at baseline. Mild adverse events were reported in 2 patients. The availability of thrombopoietin mimetics opened new prospects in the treatment of inherited thrombocytopenias. This study is registered at www.clinicaltrials.gov as NCT01133860 (European Union Drug Regulating Authorities Clinical Trials number 2008-001903-42).

Analysis of 339 pregnancies in 181 women with 13 different forms of inherited thrombocytopenia

Pregnancy in women with inherited thrombocytopenias is a major matter of concern as both the mothers and the newborns are potentially at risk of bleeding. However, medical management of this condition cannot be based on evidence because of the lack of consistent information in the literature. To advance knowledge on this matter, we performed a multicentric, retrospective study evaluating 339 pregnancies in 181 women with 13 different forms of inherited thrombocytopenia. Neither the degree of thrombocytopenia nor the severity of bleeding tendency worsened during pregnancy and the course of pregnancy did not differ from that of healthy subjects in terms of miscarriages, fetal bleeding and pre-term births. The degree of thrombocytopenia in the babies was similar to that in the mother. Only 7 of 156 affected newborns had delivery-related bleeding, but 2 of them died of cerebral hemorrhage. The frequency of delivery-related maternal bleeding ranged from 6.8% to 14.2% depending on the definition of abnormal blood loss, suggesting that the risk of abnormal blood loss was increased with respect to the general population. However, no mother died or had to undergo hysterectomy to arrest bleeding. The search for parameters predicting delivery-related bleeding in the mother suggested that hemorrhages requiring blood transfusion were more frequent in women with history of severe bleedings before pregnancy and with platelet count at delivery below 50 × 109/L.

Critical limb ischemia

Critical limb ischemia (CLI) represents the most advanced clinical stage of peripheral arterial disease. It is usually caused by obstructive atherosclerotic arterial disease and is associated with very high morbidity and mortality. The pathophysiology of CLI is a complex and chronic process affecting the macrovascular and microvascular circulation of the muscle and non-muscle tissues of the lower limbs. In particular, the atherosclerosis-related vascular remodelling, angiogenesis and arteriogenesis are central phenomena in the process. The most common clinical manifestations of CLI are limb pain at rest, with or without trophic skin changes or tissue loss. Diagnosis of CLI is based on physical examination, ankle-brachial index measurement, duplex-ultrasound and angiography; transcutaneous oxygen may also help. Risk factor control is recommended for all patients with CLI. Individuals with minimal or no skin breakdown or in whom comorbid conditions avoid revascularization can be treated with medical therapy (antiplatelet agents, intravenous prostanoids, rheologic agents). Treatment of infection is mandatory to decrease the metabolic demands that hamper wound healing. Therapeutic angiogenesis has been pursued with several approaches ranging from gene therapy to the use of bone marrow-derived progenitor cells, but further phase II and III trials are needed. Finally, the evaluation of the risk, benefit and optimal timing of revascularization lesions or the decision about amputation and its extension is a complex decision that requires a multidisciplinary approach.

Rivaroxaban vs warfarin in high-risk patients with antiphospholipid syndrome

Rivaroxaban is an effective and safe alternative to warfarin in patients with atrial fibrillation and venous thromboembolism. We tested the efficacy and safety of rivaroxaban compared with warfarin in high-risk patients with thrombotic antiphospholipid syndrome. This is a randomized open-label multicenter noninferiority study with blinded end point adjudication. Rivaroxaban, 20 mg once daily (15 mg once daily based on kidney function) was compared with warfarin (international normalized ratio target 2.5) for the prevention of thromboembolic events, major bleeding, and vascular death in patients with antiphospholipid syndrome. Only high-risk patients triple positive for lupus anticoagulant, anti-cardiolipin, and anti-β2-glycoprotein I antibodies of the same isotype (triple positivity) were included in the study. The trial was terminated prematurely after the enrollment of 120 patients (59 randomized to rivaroxaban and 61 to warfarin) because of an excess of events among patients in the rivaroxaban arm. Mean follow-up was 569 days. There were 11 (19%) events in the rivaroxaban group, and 2 (3%) events in the warfarin group. Thromboembolic events occurred in 7 (12%) patients randomized to rivaroxaban (4 ischemic stroke and 3 myocardial infarction), whereas no event was recorded in those randomized to warfarin. Major bleeding occurred in 6 patients: 4 (7%) in the rivaroxaban group and 2 (3%) in the warfarin group. No death was reported. The use of rivaroxaban in high-risk patients with antiphospholipid syndrome was associated with an increased rate of events compared with warfarin, thus showing no benefit and excess risk. This trial was registered at www.clinicaltrials.gov as #NCT02157272.

Nonmuscle Myosin Heavy Chain IIA Mutation Predicts Severity and Progression of Sensorineural Hearing Loss in Patients With MYH9-Related Disease

Objectives: MYH9-related disease (MYH9-RD) is an autosomal- dominant disorder deriving from mutations in MYH9, the gene for the nonmuscle myosin heavy chain (NMMHC)-IIA. MYH9-RD has a complex phenotype including congenital features, such as thrombocytopenia, and noncongenital manifestations, namely sensorineural hearing loss (SNHL), nephropathy, cataract, and liver abnormalities. The disease is caused by a limited number of mutations affecting different regions of the NMMHC-IIA protein. SNHL is the most frequent noncongenital manifestation of MYH9-RD. However, only scarce and anecdotal information is currently available about the clinical and audiometric features of SNHL of MYH9-RD subjects. The objective of this study was to investigate the severity and propensity for progression of SNHL in a large series of MYH9-RD patients in relation to the causative NMMHC-IIA mutations. Design: This study included the consecutive patients diagnosed with MYH9-RD between July 2007 and March 2012 at four participating institutions. A total of 115 audiograms were analyzed from 63 patients belonging to 45 unrelated families with different NMMHC-IIA mutations. Cross-sectional analyses of audiograms were performed. Regression analysis was performed, and age-related typical audiograms (ARTAs) were derived to characterize the type of SNHL associated with different mutations. Results: Severity of SNHL appeared to depend on the specific NMMHC-IIA mutation. Patients carrying substitutions at the residue R702 located in the short functional SH1 helix had the most severe degree of SNHL, whereas patients with the p.E1841K substitution in the coiled-coil region or mutations at the nonhelical tailpiece presented a mild degree of SNHL even at advanced age. The authors also disclosed the effects of different amino acid changes at the same residue: for instance, individuals with the p.R702C mutation had more severe SNHL than those with the p.R702H mutation, and the p.R1165L substitution was associated with a higher degree of hearing loss than the p.R1165C. In general, mild SNHL was associated with a fairly flat audiogram configuration, whereas severe SNHL correlated with downsloping configurations. ARTA plots showed that the most progressive type of SNHL was associated with the p.R702C, the p.R702H, and the p.R1165L substitutions, whereas the p.R1165C mutation correlated with a milder, nonprogressive type of SNHL than the p.R1165L. ARTA for the p.E1841K mutation demonstrated a mild degree of SNHL with only mild progression, whereas the ARTA for the mutations at the nonhelical tailpiece did not show any substantial progression. Conclusions: These data provide useful tools to predict the progression and the expected degree of severity of SNHL in individual MYH9-RD patients, which is especially relevant in young patients. Consequences in clinical practice are important not only for appropriate patient counseling but also for development of customized, genotype-driven clinical management. The authors recently reported that cochlear implantation has a good outcome in MYH9-RD patients; thus, stricter follow-up and earlier intervention are recommended for patients with unfavorable genotypes.

A novel congenital dysprothrombinemia leading to defective prothrombin maturation

INTRODUCTION Prothrombin deficiency is a very rare disorder caused by mutations in the F2 gene that generate hypoprothrombinemia or dysprothrombinemia and is characterized by bleeding manifestations that can vary from clinically irrelevant to life-threatening. AIM Here we characterize a patient with a novel missense mutation in F2, c.1090T/A (p.Val322Glu), that causes severe dysprothrombinemia. METHODS Coagulation assays, prothrombin Western Blotting, FII activation by Ecarin, fibrinogen degradation products quantification and thrombin generation assay were carried out to assess prothrombin expression and function. PCR followed by direct sequencing was carried out to characterize the mutation. In silico analysis for missense variant and molecular modeling were applied to predict the mechanism that leads to dysprothrombinemia. RESULTS AND CONCLUSIONS The homozygous patient had a markedly prolonged prothrombin time, strongly reduced FII activity (0.82%) but normal antigen levels. In the thrombin generation assay the lag time and the peak height were unmeasurable, suggesting that the Val322Glu mutation results in the inability of the mutant prothrombin to be fully activated to thrombin. In fact, prothrombin activation by ecarin was defective, with a massive accumulation of the meizothrombin intermediate. Molecular modeling and dynamic simulation studies showed that the Val322Glu mutation interferes with protein flexibility at Arg271 and Arg320. This impairs the switch of the protein from zymogen to proteinase, thus preventing the formation of thrombin. Accumulated meizothrombin, however, maintains some fibrinogen-degrading activity, as shown by the formation of FDPs, and this probably explains the patients mild bleeding phenotype.

Impact of Tenofovir Versus Abacavir on HIV-Related Endothelial Dysfunction

Ischemic cardiovascular events are a frequent complication of long-lasting HIV infection and have been attributed either to the infection itself or to antiretroviral therapy (ART). Some cohort studies suggested that in particular abacavir may be associated with an increased cardiovascular risk, but the pathogenic mechanisms remain unknown. Recently, abacavir use was significantly associated with incident cardiovascular disease in HIV-infected veterans and it was related to higher risk of acute myocardial infarction (AMI). Endothelial dysfunction is a central mechanism in atherosclerosis and a marker of cardiovascular risk. We have previously shown that HIVinfected patients present an endothelial dysfunction that tends to improve upon ART, suggesting that the infection itself rather than therapy is responsible for endothelial damage. In the present study we compared treatment with abacavir (ABC) and tenofovir (TDF) for their impact on endothelial dysfunction. In a retrospective, case-control study, plasma levels of soluble vascular cell adhesion molecule-1 (sVCAM-1) and monocyte chemoattractant protein-1 (MCP-1), two markers of endothelial dysfunction, were measured by flow cytometry using a bead-based assay (FlowCytomix, Bender MedSystems, Vienna, Austria) in 69 HIV-infected patients, before starting therapy and after 6–12 months of either ABC (n = 35, 18 females, age 44 – 12 years) or TDF (n = 34, 17 females, age 43 – 11 years). The two groups were similar for baseline immune-virologic status and all patients showed an increase of CD4 + count and a lowering of viral load after therapy, without differences between ABC and TDF. Twenty-one patients were recalled 28–34 months after the beginning of therapy (11 ABC and 10 TDF) for the measurement of peripheral vascular endothelial function by finger arterial pulse wave amplitude with the Endo-PAT2000 system, and of circulating endothelial cells (CECs) by flow cytometry. Two control groups were included: 20 HIV-infected patients (6 females, age 42 – 9 years), from whom blood samples were collected at diagnosis and after 6–12 months without therapy, and 10 healthy ageand gender-matched controls. All results are expressed as means – standard error of the mean (SEM). Differences between groups were assessed by using one-way analysis of variance (ANOVA) with the Kruskal-Wallis post hoc test, using the GraphPad Prism version 4.00 for Windows software (GraphPad, San Diego, CA). At diagnosis endothelial activation markers were significantly higher in HIV-infected patients than in healthy controls (sVCAM-1: 783 – 157 versus 518 – 53 ng/mL, p < 0.05; MCP-1: 293 – 22 versus 228 – 20 pg/mL, p < 0.05). After 6–12 months of treatment, sVCAM-1 and MCP-1 decreased significantly in the TDF group, although not to normal levels, but not in the ABC group, while in untreated patients endothelial dysfunction did not change, or even slightly worsened, at followup (Fig. 1A and B). Upon reevaluation after 28–34 months of treatment endothelial function, as assessed by peripheral arterial tonometry and CECs, was still significantly altered in HIV-infected patients as compared with healthy controls (Fig. 1C), with ABC-treated patients showing significantly higher CECs than controls and tendentially higher than TDF-treated patients (Fig. 1D); moreover, in ABC-treated but not in TDFtreated patients, CECs inversely correlated with endothelial function (Fig. 1E and F). Endothelial dysfunction detected by impaired peripheral arterial tonometry and increased CECs, two parameters not previously measured in HIV-infected patients, is a reliable marker of vascular damage and predicts late cardiovascular ischemic events. Therefore, our data confirm that chronic HIV-infection impairs endothelial function and show that antiretroviral treatment with TDF, but less with ABC, improves it. Very recent data showing that TDF selectively decreases the production of cardiovascular disease-related inflammatory cytokines are supportive of our conclusions. Whether persistent endothelial dysfunction in ABC-treated patients contributes to the suggested increased cardiovascular risk associated with ABC use needs to be assessed in prospective studies.

Apparent genotype–phenotype mismatch in a patient with MYH9-related disease: When the exception proves the rule

Apparent genotype–phenotype mismatch in a patient with MYH9-related disease: When the exception proves the rule -

Coinheritance of three novel FV gene mutations in a patient with a severe FV deficiency

V. BAFUNNO,* G. FAVUZZI , T. FIERRO, M. CHETTA,* E. MASTRODICASA,§ E. CHINNI, E. GRANDONE, M. MARGAGLIONE* and P. GRESELE *Medical Genetics, Department of Biomedical Sciences, University of Foggia, Foggia; Atherosclerosis and Thrombosis Unit, Research Department, Istituto di Ricovero e Cura a Carattere Scientifico, Casa Sollievo della Sofferenza, S. Giovanni Rotondo, Foggia; Department of Internal Medicine, Section of Internal and Cardiovascular Medicine, University of Perugia, Perugia; and §Pediatric Onco-Hematology, S. Maria della Misericordia Hospital, Perugia, Italy