Mutations in RASGRP2 gene identified in patients misdiagnosed as Glanzmann thrombasthenia patients.

INTRODUCTION: Glanzmann thrombasthenia (GT) is a severe inherited platelet function disorder (IPFD), presenting with bleeding diathesis and impaired platelet aggregation, is caused by mutations in the genes ITGA2B or ITGB3. AIM: We aimed to study the genetic cause of IPFD mimicking GT. METHODS: During 2017-2019, 16 patients were referred to our tertiary center with bleeding symptoms, impaired platelet aggregation and normal platelet count and size. RESULTS: Using flow cytometry, 13/16 patients were diagnosed with GT, yet three patients displayed normal surface expression of the integrins αIIbß3 and αvß3, as well as normal integrin αIIbß3 activation following incubation with the activating monoclonal antibody anti-LIBS6, while platelet activation following ADP or epinephrine was impaired. Whole exome sequencing detected 2 variants in RASGRP2 gene in all 3 patients. DISCUSSION: Both RASGRP2 mutations predicted frameshift, premature stop codon (p. I427Mfs*92 and p. R494Afs*54, respectively) and truncated calcium-sensing guanine nucleotide exchange factor [CalDAG-GEFI]- the major signaling molecule that regulates integrin-mediated aggregation and granule secretion, causing IPFD-18. CONCLUSION: Patients who suffer from bleeding diathesis without immune dysregulation, may be mistakenly diagnosed as GT. Further studies are required to confirm the diagnosis of specific IPFD.

Treatment tailoring for factor V deficient patients and perioperative management using global hemostatic coagulation assays.

INTRODUCTION: Congenital factor V deficiency (FVD) is a rare bleeding disorder with an estimated incidence of 1 in 1000,000 in the general population. Since the common coagulation tests do not correlate with the bleeding tendency there is an unmet need to predict FVD patients' bleeding hazard prior to surgical interventions. AIM: To optimize treatment prior to surgical interventions, using global coagulation assays, thrombin generation (TG) and rotating thromboelastogram (ROTEM). METHODS: Our cohort included 5 patients with FVD, 4 severe and one mild. Two of them underwent TG and ROTEM prior to surgical interventions, including ex vivo spiking assays using bypass agents and platelets spiking. RESULTS: All five patients exhibited prolonged PT and PTT, non-dependent on their bleeding tendency. Patient 1, who demonstrated severe bleeding phenotype, underwent surgery treated by combination of APCC (FEIBA) and platelet transfusion. Therapy was guided by global tests (TG as well as ROTEM) results. During the pre and post-operative period neither excessive bleeding nor any thrombosis was noted. In contrast, TG and ROTEM analysis of patient 4 has lead us to perform the surgery without any blood products' support. Indeed, the patient did not encounter any bleeding. CONCLUSION: Global coagulation assays may be useful ancillary tools guiding treatment decisions in FVD patients undergoing surgical procedures.

From thrombasthenia to next generation thrombocytopenia: Neonatal alloimmune thrombocytopenia induced by maternal Glanzmann thrombasthenia.

BACKGROUND: Glanzmann thrombasthenia (GT) is a rare autosomal recessive disorder of platelet function caused by mutations in the genes coding for integrin αIIbß3. The aim of this study was to examine the outcome of newborns of GT mothers, with emphasis on thrombocytopenia and bleeding manifestations and their relation to maternal antiplatelet antibodies. PROCEDURE: Medical files of all female patients with GT treated in a single tertiary center from 1999 to 2017 were searched for details on pregnancy and birth. The medical files of their newborns were retrieved, and data on the postnatal course were collected. RESULTS: Nine babies were born to five patients with GT at our center during the study period. Three of the nine newborns had severe thrombocytopenia, and all three were offspring of GT mothers who were positive for antiplatelet antibodies. CONCLUSION: Pregnant GT patients should be examined for platelet antibodies. Assessment and management protocols (including treatment with intravenous immunoglobulins) for fetal and neonatal alloimmune thrombocytopenia should be considered.

Tolerogenic dendritic cells inhibit antiphospholipid syndrome derived effector/memory CD4⁺ T cell response to ß2GPI.

OBJECTIVES: The importance of ß(2)-glycoprotein I (ß(2)GPI)-specific CD4(+) T cells in the development of pathogenic processes in patients with antiphospholipid syndrome (APS) and APS mouse models is well established. Therefore, our objective is to manipulate the ß2GPI specific CD4(+) T cells using tolerogenic dendritic cells (tDCs) to induce tolerance. We aim to evaluate the capability of tDCs to induce antigen-specific tolerance in effector/memory T cells from patients with APS and to elucidate the involved mechanism. METHODS: DCs and tDCs were produced from patients with APS peripheral-blood-monocytes, using specific cytokines. ß(2)GPI-specific tolerance induction was investigated by coculturing control DC (cDC) or tDC, ß(2)GPI-loaded, with autologous effector/memory T cells, evaluating the proliferative response, phenotype, cytokines secretion, viability and regulatory T cells. RESULTS: Human monocyte-derived DCs treated with interleukin (IL)-10 and transforming growth factor ß-1 (10/TGF-DC) induced ß(2)GPI-specific-unresponsiveness in effector/memory CD4(+) T cells (46.5% ± 26.0 less proliferation) in 16 of 20 analysed patients with APS, without affecting the proliferative response to an unrelated candidin. In five analysed patients, 10/TGF-DC-stimulated T cells acquired an IL-2(low)interferon γ(low)IL-10(high) cytokine profile, with just a propensity to express higher numbers of Foxp3(+)CTLA-4(+) cells, but with an evident suppressive ability. In four of 10 analysed patients, 10/TGF-DC-stimulated T cell hyporesponsiveness could not be reverted and showed higher percentages of late apoptosis, p<0.02. CONCLUSIONS: The inherent tolerance induction resistance of activated T cells present during the development of autoimmune diseases has delayed the application of tDC as an alternative therapy. This study highlights the 10/TGF-DC feasibility to induce antigen-specific unresponsiveness in autoreactive T cells generated in patients with APS by inducing apoptosis or T cells with regulatory abilities.

Oxygen uptake kinetics during exercise reveal central and peripheral limitation in patients with iliofemoral venous obstruction.

OBJECTIVE: Pulmonary oxygen uptake (V˙O2) kinetics measured during the initiation of exercise mirror energetic transition during daily activity. The aim of this study was to elucidate the pathophysiological mechanisms of exercise limitation of patients with chronic iliofemoral vein obstruction after deep vein thrombosis by measuring V˙O2 kinetics compared with patients with peripheral arterial disease (PAD) and healthy individuals. METHODS: Eleven patients with iliofemoral vein obstruction (7 men; age, 20-65 years), seven patients with PAD (all men; age 44-60 years) and eight healthy participants (5 men; age 28-58 years) were studied. Participants performed upper and lower limb symptom-limited cardiopulmonary exercise tests on cycle ergometers; and four repeat lower limb tests at a constant work rate corresponding with 90% of the gas exchange threshold for determining V˙O2 kinetics. RESULTS: Phase I V˙O2 amplitude in the constant work rate tests (percent increase over resting V˙O2), representing the initial surge in cardiac output caused by the emptying of leg veins, was 59 ± 19% in the iliofemoral vein obstruction group, 73 ± 22% in PAD, and 85 ± 26% in healthy participants (P = .055 for iliofemoral vein obstruction vs healthy). Phase II V˙O2 kinetics, which largely reflect the kinetics of O2 consumption in the exercising muscles, were slower in iliofemoral vein obstruction (tau = 42 ± 6 seconds), and PAD (tau = 49 ± 19 seconds), compared with healthy participants (23 ± 4 seconds; P < .01). CONCLUSIONS: Slow phase II V˙O2 kinetics reflect a slow onset of muscular aerobic metabolism in both iliofemoral vein obstruction and PAD. The low amplitude phase I of V˙O2 kinetics observed in iliofemoral vein obstruction suggests a damped cardiodynamic phase, consistent with decreased venous return from the obstructed veins. These abnormalities of V˙O2 kinetics may contribute to exercise intolerance in iliofemoral vein obstruction and PAD.

Women with Hemophilia: Case Series of Reproductive Choices and Review of Literature.

Aim Very little is known regarding reproductive choices, pregnancy, and delivery of women with moderate to severe hemophilia. Our aim was to describe our experience with three hemophiliac women and their journey to achieve motherhood. Methods Medical charts of women with moderate to severe hemophilia A treated at our center were evaluated. Data regarding choices of conception, pregnancy course, mode of delivery, and pregnancy outcomes were obtained. Results Three women are presented. Whereas patient 1 chose to adopt her first child and later had twins through egg donations and a surrogate mother, patient 2 underwent spontaneous pregnancy and delivered via cesarean section. Patient 3 preferred in vitro fertilization and preimplantation genetic diagnosis to avoid hemophilia and hemophilia carriership in her offspring. Conclusion The appropriate means to achieve parenthood for women with moderate to severe hemophilia should be individualized and requires support of a comprehensive multidisciplinary team.

Chronic iliofemoral vein obstruction - an under-recognized cause of exercise limitation.

Local symptoms of chronic venous insufficiency after deep vein thrombosis (DVT) are well described, but little is known about the effect of residual venous obstruction on exercise capacity. We tested our hypothesis that chronic residual iliofemoral vein occlusion (IFVO) after DVT may impair exercise capacity. Nine post-DVT patients with residual IFVO and effort intolerance were studied; a comparison cohort consisted of 11 healthy volunteers. Exercise tolerance was assessed by bimodality incremental symptom-limited cardiopulmonary testing, using leg and arm ergometers. In healthy subjects, leg vein obstruction was modelled by application to the thighs of cuff tourniquets inflated to 30-40 mmHg. Leg exercise tolerance as measured by oxygen uptake at peak exercise (peak ⩒'O2) was reduced in patients (median 50% predicted (range 36-83%) vs. 88% predicted (67-129%) in normal subjects, p < 0.001). Arm exercise tolerance was also reduced in patients, but less severely than in the legs - the median arm: leg ratio of peak ⩒'O2 was 0.95 (0.77-1.43) in patients vs. a normal ratio of 0.73 (0.6-1.0) in healthy subjects (p < 0.003). In healthy subjects, bilateral leg vein obstruction by tourniquets reduced peak ⩒'O2 in leg exercise to 76% predicted (range 55-108%; p < 0.001 vs. standard test). In conclusion, the comparison of arm vs. leg exercise capacity in post-DVT patients with residual IFVO and the effect of experimental venous obstruction (thigh tourniquets) in healthy subjects suggest that reduced exercise capacity in patients was at least partially caused by reduced venous return. Chronic venous obstruction should be recognized as a cause of exercise limitation.

Platelet-rich plasma for bone graft enhancement in sinus floor augmentation with simultaneous implant placement: patient series study.

The use of autologous platelet-rich plasma (PRP) as a source for growth factors in bone grafting is a relatively new and promising technique. Early controlled studies indicate that combining PRP with autologous bone grafts significantly enhances the rate of bone formation and maturation. The study consisted of 105 patients who required sinus augmentation with crestal bone height of less than 5 mm in the posterior maxilla. All patients received a composite bone graft that consisted of 30% to 40% autogenous bone harvested from the lateral wall of the maxilla zygomatic-maxillary buttress and the tuberosity and 60% to 70% xenograft. A total of 50 mL of blood was obtained from each patient before the surgical treatment for preparation of 10 mL of PRP. The graft-PRP mixture was activated by human thrombin. All sinus augmentations were carried out simultaneously with dental implants. At 6 months postoperatively, implants were exposed showing no clinical evidence of crestal bone loss around the implants both clinically and radiographically. All implants were clinically osseointegrated and loaded with fixed porcelain fused to metal prosthesis. The use of PRP in augmenting the severely atrophic posterior maxilla has obvious clinical benefits in terms of reducing the healing period of bone maturation, better graft handling, and accelerated soft tissue healing.

Human T lymphotropic virus type 1 in a seronegative B chronic lymphocytic leukemia patient.

Human T lymphotropic virus type 1 (HTLV-1) is the etiological agent of adult T cell leukemia and HTLV-1-associated myelopathy/tropical spastic paraparesis. HTLV-1 infection in patients with B cell-type chronic lymphocytic leukemia (B-CLL) is rare and has been reported only in areas in which HTLV-1 is endemic. In the present study, we detected HTLV-1 proviral DNA by polymerase chain reaction, using tax primers, in peripheral blood lymphocytes from a B-CLL patient, an immigrant to Israel, where HTLV-1 infection is not endemic. F344 rats injected intravenously with peripheral blood lymphocytes obtained from the patient developed HTLV-1 antibodies. Titers of antibody to HTLV-1 in the rat blood were 1:512 by particle agglutination; enzyme-linked immunosorbent assay and Western blotting were also positive. No antibody against HTLV-1 was demonstrated in the animal model after inoculation of either purified B lymphocytes from the B-CLL patient or peripheral blood mononuclear cells from healthy donors. This is one of the few studies showing the presence of HTLV-1 provirus in T lymphocytes of a B-CLL patient who had multiple infections, and died of salmonella sepsis, and the first report of HTLV-1 antibody induction in an animal model by inoculation of lymphocytes obtained from an HTLV-1-infected B-CLL patient.

Possible role of recombinant activated factor VII (rFVIIa) in the control of hemorrhage associated with massive trauma.

PURPOSE: To report our experience with recombinant activated factor VII (rFVIIa) to control hemorrhage in trauma patients with profound multifactorial coagulopathy. rFVIIa forms a complex with tissue factor exposed at sites of tissue damage and induces activation of coagulation limited to the site of injury. It is approved for use in hemophilia patients, however, its use in trauma is still controversial due to the theoretical risk of thromboembolic complications. CLINICAL FEATURES: Nineteen critically ill, multi-transfused patents with trauma (ten blunt and nine penetrating), aged 25 + 17 yr,were treated with rFVIIa after all conventional hemostatic measures had failed. After one to three doses of rFVIIa, hemorrhaging ceased within minutes in 15/19 (78.9%) patients. The total dose of rFVIIa required to control bleeding was 195 +/- 112.7 microg x kg(-1). Shortening of prothrombin time and partial thromboplastin time was observed within 15-30 min from 22.7 +/- 7.9 to 10.4 +/- 2.6 sec and 71 +/- 38.9 to 42.2 +/- 24 sec respectively, (P < 0.05). Transfusion requirements decreased from 30 +/- 18.3 units used within 5.6 +/- 3.4 hr of admission to 2.8 +/- 2.5 within the following 24 hr (P < 0.05). One patient developed clinical deep vein thrombosis. No systemic activation of coagulation was observed clinically. Thirteen patients (68.4%) survived and recovered. Four patents did not respond to rFVIIa treatment and exsanguinated within 24 hr. Two patients died after one week, one from sepsis and one from multi-organ failure. CONCLUSIONS: rFVIIa is a promising adjunctive hemostatic treatment for trauma patients suffering from massive bleeding. Controlled trials are warranted to evaluate the safety and efficacy of this drug.

Sustained integrin ligation involves extracellular free sulfhydryls and enzymatically catalyzed disulfide exchange.

Studies have suggested a pivotal role for free sulfhydryls in platelet integrin function, and enzyme-mediated reduction of disulfide bonds on platelets has been implicated. The platelet fibrinogen receptor alpha(IIb)beta(3) is the best-studied platelet integrin and serves as a model system for studying the structure-function relation in this family of adhesion receptors. The demonstration of free sulfhydryls on the exofacial domain of purified alpha(IIb)beta(3), specifically in its activated conformation, prompted us to explore the potential for activation-dependent, enzymatically catalyzed thiol expression on intact platelets and the possible role of surface-associated protein disulfide isomerase (PDI) in alpha(IIb)beta(3) ligation. Using the membrane-impermeant sulfhydryl blocker para-chloromercuriphenyl sulfonate, the inhibitor of disulfide exchange bacitracin, and the monoclonal anti-PDI antibody RL90, we examined fibrinogen binding to alpha(IIb)beta(3) as well as ligation-induced allosteric changes in the conformation of alpha(IIb)beta(3). We sought to distinguish the possible involvement of disulfide exchange in agonist-induced platelet stimulation from its role in integrin ligation. Analysis of the role of free thiols in platelet aggregation suggested a thiol-independent initial ligation followed by a thiol-dependent stabilization of binding. Flow cytometric analysis showed that sustained binding of fibrinogen, as well as expression of ligand-induced binding site epitopes and ligand-bound conformation, depended on free thiols and disulfide exchange. Expression of P-selectin was minimally affected, even with complete inhibition of alpha(IIb)beta(3) function. These data indicate that although agonist-induced platelet stimulation is independent of ecto-sulfhydryls, engagement of integrin alpha(IIb)beta(3) on the intact platelet depends totally on their enzymatically catalyzed surface expression.