Mutations in MYH9 result in the May-Hegglin anomaly, and Fechtner and Sebastian syndromes. The May-Heggllin/Fechtner Syndrome Consortium.

The autosomal dominant, giant-platelet disorders, May-Hegglin anomaly (MHA; MIM 155100), Fechtner syndrome (FTNS; MIM 153640) and Sebastian syndrome (SBS), share the triad of thrombocytopenia, large platelets and characteristic leukocyte inclusions ('Döhle-like' bodies). MHA and SBS can be differentiated by subtle ultrastructural leukocyte inclusion features, whereas FTNS is distinguished by the additional Alport-like clinical features of sensorineural deafness, cataracts and nephritis. The similarities between these platelet disorders and our recent refinement of the MHA (ref. 6) and FTNS (ref. 7) disease loci to an overlapping region of 480 kb on chromosome 22 suggested that all three disorders are allelic. Among the identified candidate genes is the gene encoding nonmuscle myosin heavy chain 9 (MYH9; refs 8-10), which is expressed in platelets and upregulated during granulocyte differentiation. We identified six MYH9 mutations (one nonsense and five missense) in seven unrelated probands from MHA, SBS and FTNS families. On the basis of molecular modelling, the two mutations affecting the myosin head were predicted to impose electrostatic and conformational changes, whereas the truncating mutation deleted the unique carboxy-terminal tailpiece. The remaining missense mutations, all affecting highly conserved coiled-coil domain positions, imparted destabilizing electrostatic and polar changes. Thus, our results suggest that mutations in MYH9 result in three megakaryocyte/platelet/leukocyte syndromes and are important in the pathogenesis of sensorineural deafness, cataracts and nephritis.

Altered cytoskeleton organization in platelets from patients with MYH9-related disease.

MYH9-related disease (MYH9-RD) is an autosomal dominant disorder deriving from mutations in the MYH9 gene encoding for the heavy chain of non-muscle myosin IIA, and characterized by thrombocytopenia and giant platelets. Isoform IIA of myosin is the only one expressed in platelets, but the possibility that MYH9 mutations affect the organization of contractile structures in these blood elements has never been investigated. In this work we have analyzed the composition and the agonist-induced reorganization of the platelet cytoskeleton from seven MYH9-RD patients belonging to four different families. We found that an increased amount of myosin was constitutively associated with actin in the cytoskeleton of resting MYH9-RD platelets. Upon platelet stimulation, an impaired increase in the total cytoskeletal proteins was observed. Moreover, selected membrane glycoproteins, tyrosine kinases, and small GTPases failed to interact with the cytoskeleton in agonist-stimulated MYH9-RD platelets. These results demonstrate for the first time that mutations of MYH9 result in an alteration of the composition and agonist-induced reorganization of the platelet cytoskeleton. We suggest that these abnormalities may represent the biochemical basis for the previously reported functional alterations of MYH9-RD platelets, and for the abnormal platelet formation from megakaryocytes, resulting in thrombocytopenia and giant platelets.

Factors influencing post-transfusional platelet increment in pediatric patients given hematopoietic stem cell transplantation.

Patients undergoing allogeneic hematopoietic stem cell transplantation (HSCT) always require platelet transfusions, but the increase in platelet count is often less than expected. Since factors responsible for poor response to platelet transfusions in this clinical setting are largely unknown, we performed a prospective study in 87 consecutive children transplanted in a single institution. The mean 16-h corrected count increment (CCI) of 598 platelet transfusions was 5.76 +/- 8.32 x 10(9)/l. Both before and after HSCT, 13.8% of patients had antibodies against HLA and/or platelet-specific antigens. Univariate analysis identified 12 factors significantly associated with a lower post-transfusion CCI, but only four reached statistical significance in the multivariate analysis. These four factors were concomitant therapy with vancomycin, alloimmunization, use of an Autopheresis cell separator for preparation of platelet concentrates and cytomegalovirus infection. We, therefore, suggest that a better response to platelet transfusions could be obtained by choosing a suitable cell separator, by avoiding the use of vancomycin and by adopting measures that reduce alloimmunization and CMV infection. Moreover, screening patients for HLA and platelet-specific antibodies before HSCT would identify the majority of subjects who will develop alloimmune refractoriness after transplantation and would allow the search for a compatible donor in advance.

ß-1 tubulin R307H SNP alters microtubule dynamics and affects severity of a hereditary thrombocytopenia.

BACKGROUND: Single nucleotide polymorphisms (SNPs) in platelet-associated genes partly explain inherent variability in platelet counts. Patients with monoallelic Bernard Soulier syndrome due to the Bolzano mutation (GPIBA A156V) have variable platelet counts despite a common mutation for unknown reasons. OBJECTIVES: We investigated the effect of the most common SNP (R307H) in the hematopoietic-specific tubulin isotype ß-1 in these Bernard Soulier patients and potential microtubule-based mechanisms of worsened thrombocytopenia. PATIENTS/METHODS: Ninety-four monoallelic Bolzano mutation patients were evaluated for the R307H ß-1 SNP and had platelet counts measured by three methods; the Q43P SNP was also evaluated. To investigate possible mechanisms underlying this association, we used molecular modeling of ß-1 tubulin with and without the R307H SNP. We transfected SNP or non-SNP ß-1 tubulin into MCF-7 and CMK cell lines and measured microtubule regrowth after nocodazole-induced depolymerization. RESULTS: We found that patients with at least one R307H SNP allele had significantly worse thrombocytopenia; manual platelet counting revealed a median platelet count of 124 in non-SNP patients and 76 in SNP patients (both ×10(9)  L(-1) ; P < 0.01). The Q43P SNP had no significant association with platelet count. Molecular modeling suggested a structural relationship between the R307H SNP and microtubule stability via alterations in the M-loop of ß tubulin; in vitro microtubule recovery assays revealed that cells transfected with R307H SNP ß-1 had significantly impaired microtubule recovery. CONCLUSIONS: Our data show that the R307H SNP is significantly associated with the degree of thrombocytopenia in congenital and acquired platelet disorders, and may affect platelets by altering microtubule behavior.

Thrombocytopenia, giant platelets, and leukocyte inclusion bodies (May-Hegglin anomaly): clinical and laboratory findings.

PURPOSE: May-Hegglin anomaly is a rare hereditary condition characterized by the triad of thrombocytopenia, giant platelets, and inclusion bodies in leukocytes. Clinical features and the pathogenesis of bleeding in this disease are poorly defined. PATIENTS AND METHODS: From 1988 to 1996 we studied 15 new May-Hegglin anomaly patients from 7 unrelated Italian families. In addition to clinical examination and routine laboratory testing, we measured bleeding time, platelet aggregation and release reaction, and platelet staining for tubulin, and performed ultrastructural study of polymorphonuclear leukocytes. RESULTS: Although the mean age of our patients was 33 years, May-Hegglin anomaly had not been previously recognized in any of them. Bleeding diatheses ranged from severe to absent, and platelet count from 26 to 178 x 10(9)/L. No correlation was found between bleeding tendency and platelet count. Previous therapy with corticosteroids, high-dose immunoglobulins, and splenectomy had no effect on platelet count or bleeding diathesis. Desmopressin infusion greatly shortened the bleeding time in the most severely affected patient. The in vitro function of platelets was normal except for the absence of shape change in all subjects and defective response to epinephrine in 8 of 15 patients. Platelet tubulin was distributed unevenly instead of being organized in a circumferential band at the cell periphery. CONCLUSION: The diagnosis of May-Hegglin is easily missed, and its frequency is probably underestimated. A qualitative defect of platelets may be responsible for mild bleeding diathesis even in the absence of thrombocytopenia, while severe bleeding results from both qualitative and quantitative platelet defects. May-Hegglin anomaly should be suspected whenever a patient has a low platelet count or a bleeding diathesis of unknown origin.

Ristocetin-induced platelet agglutination stimulates GPIIb/IIIa-dependent calcium influx.

We found that intracellular Ca2+ concentration ([Ca2+]i) increased during ristocetin-induced agglutination of aequorin loaded platelets resuspended in plasma. Chelation of extracellular Ca2+ had no effect on platelet clumping, but delayed and greatly reduced Ca2+ increase, indicating that it derived for the most part from Ca2+ influx. Nine monoclonal antibodies (MA) against glycoprotein (GP) Ib largely prevented ristocetin-induced platelet clumping and [Ca2+]i increase, while three anti-GPIb MA with no effect on platelet clumping did not interfere with Ca2+ movement. In unstirred samples platelet agglutination was greatly reduced and [Ca2+]i increase was abolished, suggesting that close platelet-to-platelet contact, in addition to von Willebrand factor (vWF) binding to GPIb, is necessary for Ca2+ transient. Nine MA against GPIIb/IIIa, the gly-arg-gly-asp-ser (GRGDS) peptide and GPIIb/IIIa complex dissociation had no effect on platelet agglutination, but significantly reduced Ca2+ increase. Our results suggest that platelet clumping induced by vWF binding to GPIb is responsible for GPIIb-IIIa dependent Ca2+ influx.

Defect of platelet aggregation and adhesion induced by autoantibodies against platelet glycoprotein IIIa.

A young patient developed chronic idiopathic thrombocytopenic purpura. Prednisone therapy normalized platelet number, but bleeding symptoms did not disappear. Platelet function was severely impaired, since platelet aggregation, ATP release and adhesion to collagen and subendothelial matrix were significantly reduced. Plasma and purified immunoglobulins of the patient reproduced the functional defects in normal platelets. Immunoblotting revealed that patient's plasma contained an antibody reacting with a component of platelets with the same electrophoretic mobility of glycoproteins IIIa of normal platelets. Moreover, patient's plasma inhibited the binding of an anti-GPIIb/IIIa monoclonal antibody to platelet surface. Additional immunosuppressive therapy with prednisone and azathioprine normalized platelet function and induced the disappearance of bleeding symptoms.

Platelet function after in vivo and in vitro treatment with thrombolytic agents.

Whereas in vitro studies showed that plasmin may induce both inhibition and activation of platelets, in vivo and ex vivo investigations suggested that thrombolytic agents are responsible for platelet stimulation. To gain further information on this topic, ex vivo platelet function was studied in 24 subjects with acute myocardial infarction treated with streptokinase or recombinant tissue-type plasminogen activator (rt-PA). Ten patients with acute myocardial infarction who did not receive thrombolytic treatment were also investigated. The data shows that at the end of thrombolytic infusion, the maximal extent of platelet aggregation and adenosine triphosphate release was reduced in treated patients compared with that in untreated ones. In subjects treated with streptokinase, the defect in platelet aggregation derived from both cellular and plasmatic defects. Plasmatic beta-thromboglobulin concentration was significantly reduced after streptokinase, but unchanged after rt-PA. Three days after thrombolytic treatment, platelet aggregation of patients receiving streptokinase or rt-PA was not significantly different from that of untreated subjects. A similar defect in platelet function was obtained in vitro, incubating normal platelet-rich plasma with pharmacologic concentrations of streptokinase. Again, platelet function defect derived from both cellular and plasmatic damages. It cannot be excluded that platelet activation occurs in patients with acute myocardial infarction during the very early phases of thrombolytic treatment. However, it is suggested that a transient defect in platelet function follows both streptokinase and rt-PA infusion.

Platelet aggregation in platelet-rich plasma and whole blood in 120 patients with myeloproliferative disorders.

In vitro platelet aggregation in platelet-rich plasma (PRP) and in whole blood (WB) was assessed in 31 patients with idiopathic myelofibrosis, 32 with essential thrombocytosis, 23 with polycythemia vera, and 34 with chronic myelogenous leukemia. In PRP most subjects showed normal or reduced platelet aggregation, whereas in WB the majority of patients showed increased platelet function. Spontaneous platelet aggregation (SPA) was observed frequently in WB, whereas it was seldom observed in PRP. SPA in WB was inhibited by in vitro addition of aspirin and apyrase, and SPA was only partially dependent on high platelet count because it also occurred in samples with normal platelet content (at variance with 13 subjects with reactive thrombocytosis, in which SPA was observed only in samples with high platelet concentration). Platelets from patients with idiopathic myelofibrosis had the highest tendency to undergo SPA.

Platelet-Independent defect in hemostasis associated with sirolimus use.

Sirolimus is currently used to prevent rejection of solid organ transplant, and sirolimus-eluting stents have shown promise for the prevention of coronary artery restenosis. Thrombocytopenia is a well-known adverse effect of sirolimus limiting its use. Herein we report on a patient in whom sirolimus caused a platelet-independent hemostasis defect. The patient was a 52-year-old woman who underwent renal transplant with consequent normal kidney function. The immunosuppressive regimen included basiliximab, steroids, and cyclosporine induction later shifted to sirolimus and mycophenolate due to biopsy findings of tubular necrosis on day 6 posttransplantation. At discharge the serum creatinine was 0.7 mg/dL. Four months after transplantation the patient was admitted to our hospital because of fever (37.5 degrees C to 38 degrees C), anorexia, and asthenia. Blood analysis showed: creatinine 1.7 mg/dL, Hb 9.6 g/dL, WBC 6 x 10(3)/microL, PLT 123 x 10(3)/microL, liver function tests normal, LDH 720 mU/mL, fibrinogen 628 mg/dL, d-dimer 0.42 ng/mL, FDP > 40 ng/mL, INR 1.10, PT 87%, aPTT 40 seconds. Cultures and tests for infection were negative. Serum sirolimus level was 25.9 ng/mL. The following day the serum creatinine rose to 2.3 mg/dL and diuresis fell to 20 mL/h. Multiple bleeding times (Ivy test) performed before the renal biopsy were repeatedly over 30 minutes (normal 3 to 5 minutes), despite normal platelet count and platelet function studies. There was no spontaneous aggregation and in vitro aggregation was normal (collagen, ADP, adrenalin, and ristocetin induced). Coagulation studies showed a defect in fibrin formation and a reduction of fibrinolysis. Suspension of sirolimus treatment was followed by remission of fever, improvement of renal function (serum creatinine 1.2 mg/dL), and normalization of bleeding time.

[Results of splenectomy in 45 cases of idiopathic thrombocytopenic purpura: search of prognostic indices]. / Risultati della splenectomia in 45 casi di porpora trombocitopenica idiopatica: ricerca di indici prognostici.

We report on the effects of splenectomy in 45 consecutive patients affected by idiopathic thrombocytopenia who did not obtain lasting remission with prednisone therapy. None of the patients had serious complications after surgery, and only one died a year later from an infectious disease. Two years after surgery, 89% of the patients were in remission; 79% had no therapy after splenectomy, while 10% achieved remission in response to additional medical treatment. A significantly better response to splenectomy was obtained in the subjects under 50 years old (90% remission) and by the younger patients who had had a transient response to initial prednisone therapy (100% remission). All patients with thrombocytosis 15 days after surgery were in remission two years later. We were unable to identify parameters constantly associated with a poor response to splenectomy, since at least 25% of the patients with the worst prognostic features obtained lasting remission.

Inherited thrombocytopenias: the evolving spectrum.

The chapter of inherited thrombocytopenias has expanded greatly over the last decade and many "new" forms deriving from mutations in "new" genes have been identified. Nevertheless, nearly half of patients remain without a definite diagnosis because their illnesses have not yet been described. The diagnostic approach to these diseases can still take advantage of the algorithm proposed by the Italian Platelet Study Group in 2003, although an update is required to include the recently described disorders. So far, transfusions of platelet concentrates have represented the main tool for preventing or treating bleedings, while haematopoietic stem cell transplantation has been reserved for patients with very severe forms. However, recent disclosure that an oral thrombopoietin mimetic is effective in increasing platelet count in patients with MYH9-related thrombocytopenia opened new therapeutic perspectives. This review summarizes the general aspects of inherited thrombocytopenias and describes in more detail MYH9-related diseases (encompassing four thrombocytopenias previously recognized as separate diseases) and the recently described ANKRD26-related thrombocytopenia, which are among the most frequent forms of inherited thrombocytopenia.

International collaboration as a tool for diagnosis of patients with inherited thrombocytopenia in the setting of a developing country.

BACKGROUND: Inherited thrombocytopenias (ITs) are heterogeneous genetic disorders that frequently represent a diagnostic challenge. The requirement of highly specialized tests for diagnosis represents a particular problem in resource-limited settings. To overcome this difficulty, we applied a diagnostic algorithm and developed a collaboration program with a specialized international center in order to increase the diagnostic yield in a cohort of patients in Argentina. METHODS: Based on the algorithm, initial evaluation included collection of clinical data, platelet size, blood smear examination and platelet aggregation tests. Confirmatory tests were performed according to diagnostic suspicion, which included platelet glycoprotein expression, immunofluorescence for myosin-9 in granulocytes and platelet thrombospondin-1 and molecular screening of candidate genes. RESULTS: Thirty-one patients from 14 pedigrees were included; their median age was 32 (4-72) years and platelet count 72 (4-147)×10(9) L(-1). Autosomal dominant inheritance was found in nine (64%) pedigrees; 10 (71%) had large platelets and nine (29%) patients presented with syndromic forms. A definitive diagnosis was made in 10 of 14 pedigrees and comprised MYH9-related disease in four, while classic and monoallelic Bernard-Soulier syndrome, gray platelet syndrome, X-linked thrombocytopenia, thrombocytopenia 2 (ANKRD26 mutation) and familial platelet disorder with predisposition to acute myelogenous leukemia were diagnosed in one pedigree each. CONCLUSIONS: Adoption of an established diagnostic algorithm and collaboration with an expert referral center proved useful for diagnosis of IT patients in the setting of a developing country. This initiative may serve as a model to develop international networks with the goal of improving diagnosis and care of patients with these rare diseases.

Safety and efficacy of low-dose fondaparinux (1.5 mg) for the prevention of venous thromboembolism in acutely ill medical patients with renal impairment: the FONDAIR study.

BACKGROUND: Renal impairment is common, affecting around 40% of acutely ill medical patients, and is associated with an increased risk of both venous thromboembolism (VTE) and bleeding. The clinical benefit of effective thromboprophylactic strategies may be outweighed in these patients by an excessive rate of hemorrhage. OBJECTIVE: To assess the safety and efficacy of lower prophylactic doses of fondaparinux in acutely ill medical patients with renal impairment. PATIENTS/METHODS: We carried out a multicenter, investigator-initiated, prospective cohort study. Patients at risk of VTE with a creatinine clearance between 20 and 50 mL min(-1) were treated with fondaparinux 1.5 mg qd for a minimum of 6 to a maximum of 15 days. The primary outcome was the incidence of major bleeding; secondary outcomes were clinically relevant non-major bleeding (CRNMB) and symptomatic VTE. RESULTS: We enrolled 206 patients with a mean age of 82 years, mean creatinine clearance of 33 mL min(-1) , and a mean Charlson co-morbidity index of 8.2. One patient had major bleeding (0.49%, 95% confidence interval [CI] 0.03-3.10), eight had CRNMB (3.88%, 95% CI 1.81-7.78) and three developed symptomatic VTE (1.46%, 0.38-4.55). Twenty-three patients (11.17%, 7.36-16.48) died. No independent predictors of bleeding were found at univariate analysis. CONCLUSIONS: The addition of moderate to severe renal impairment to patients with traditional risk factors for VTE identified a population of very elderly acutely ill medical patients potentially at high risk of both VTE and bleeding complications. The recently approved lower prophylactic dose of fondaparinux appears to be a safe and relatively effective strategy in these patients.

Lipid levels in the second year of life among HIV-infected and HIV-exposed uninfected Latin American children.

BACKGROUND: Dyslipidemia is observed among older children and adults with HIV. We examined nonfasting cholesterol and triglycerides in two groups of 12-23-month-old Latin American children - HIV-infected vs. HIV-exposed but uninfected (HEU). METHODS: HIV-infected and HEU children in Latin America and Jamaica were enrolled in an observational cohort. Eligibility for this analysis required having cholesterol and triglyceride results available during the second year of life. RESULTS: HIV-infected (n = 83) children were slightly older at the time of lipid testing than the HEU (n = 681). Forty percent of the HIV-infected children were on protease inhibitor-based antiretroviral therapy (ART); 41% were not on ART. There was no statistically significant difference in mean cholesterol concentrations (mg/dl) by HIV status; however, the HIV-infected children had higher mean triglyceride concentrations. The prevalence of high cholesterol (>200  mg/dl) and high triglycerides (>110 mg/dl) was higher among the HIV-infected vs. HEU. Among the HIV-infected children, mean cholesterol and triglyceride concentrations varied by ART. Children receiving no ART had a significantly lower mean cholesterol concentration. Those receiving protease inhibitor-containing ART had a significantly higher mean triglyceride concentration compared to the other two antiretroviral regimen groups. CONCLUSION: A greater proportion of HIV-infected children at 12-23 months have hyperlipidemia when compared to HEU children, with the highest triglyceride concentrations observed among those receiving protease inhibitor-containing ART, and the lowest cholesterol levels among those not receiving ART. Implications of these findings will require continued follow-up of HIV-infected children who initiate therapy early in life.

Proplatelet formation in heterozygous Bernard-Soulier syndrome type Bolzano.

BACKGROUND: Although mutations of GPIb alpha are among the most frequent causes of inherited platelet disorders, the mechanisms for the onset of thrombocytopenia and platelet macrocytosis are still poorly defined. OBJECTIVE: In this work we analyzed in vitro megakaryocyte differentiation and proplatelet formation in six subjects heterozygous for the Ala156Val mutation in the GPIb alpha (Bolzano mutation). METHODS: Human megakaryocytes were obtained by differentiation of patient cord blood-derived CD34(+) cells and peripheral blood-derived CD45(+) cells. Proplatelet formation was evaluated by phase contrast and fluorescence microscopy. RESULTS: Megakaryocyte differentiation from both cord blood (one patient) and peripheral blood (five patients) was comparable to controls. However, proplatelet formation was reduced by about 50% with respect to controls. An identical defect of proplatelet formation was observed when megakaryocytes were plated on fibrinogen, von Willebrand factor or grown in suspension. Morphological evaluation of proplatelet formation revealed an increased size of proplatelet tips, which was consistent with the increased diameters of patients' blood platelets. Moreover, alpha-tubulin distribution within proplatelets was severely deranged. CONCLUSIONS: Megakaryocytes from patients carrying a Bolzano allele of GPIb alpha display both quantitative and qualitative abnormalities of proplatelet formation in vitro. These results suggest that a defect of platelet formation contributes to macrothrombocytopenia associated to the Bolzano mutation, and indicate a key role for GPIb alpha in proplatelet formation.