Transplant-associated thrombotic microangiopathy and immune haematological complications following intestine-containing organ transplantation: experience from over 100 consecutive cases.

Descriptions of passenger lymphocyte syndrome (PLS), immune cytopenias and transplant-associated thrombotic microangiopathy (TA-TMA) after intestine-containing transplants remain scarce. We describe our centre's experience of these complications from 2007 to 2019. Ninety-six patients received 103 transplants. PLS occurred in 9 (9%) patients (median 12 days post-transplant); all due to ABO antibodies. There were 31 minor ABO mismatch transplants. No patient required change in immunosuppression. Immune cytopenias (excluding PLS) occurred in six patients at an incidence of 1·7/100 patient years; three immune haemolysis, one immune thrombocytopenia, one acquired Glanzmann's and one immune neutropenia; 50% occurred with other cytopenias. All cases eventually responded to treatment, with a median of four treatments (range 1-8) and 5/6 were treated with rituximab. One patient with immune haemolysis required bortezomib. Complications were common in patients with immune cytopenias; 4/6 with infection needing intravenous antibiotics and 3/6 with venous thromboembolism. In 3/6 cases, a secondary cause for the immune cytopenia was evident. Switching from tacrolimus to ciclosporin was not necessary. There were five cases of transplant-associated thrombotic microangiopathy (TA-TMA; 1·5/100 patient years) requiring calcineurin inhibitor withdrawal; two cases associated with acute rejection. Two cases were managed with plasma exchange, one with plasma infusions and one with eculizumab. Further research in this patient group is required.

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.

Congenital platelet disorders and understanding of platelet function.

Genetic defects of platelets constitute rare diseases that include bleeding syndromes of autosomal dominant, recessive or X-linked inheritance. They affect platelet production, resulting in a low circulating platelet count and changes in platelet morphology, platelet function, or a combination of both with altered megakaryopoiesis and a defective platelet response. As a result, blood platelets fail to fulfil their haemostatic function. Most studied of the platelet function disorders are deficiencies of glycoprotein mediators of adhesion and aggregation while defects of primary receptors for stimuli include the P2Y12 ADP receptor. Studies on inherited defects of (i) secretion from storage organelles (dense granules, α-granules), (ii) the platelet cytoskeleton and (iii) the generation of pro-coagulant activity have identified genes indirectly controlling the functional response. Signalling pathway defects leading to agonist-specific modifications of platelet aggregation are the current target of exome-sequencing strategies. We now review recent advances in the molecular characterization of platelet function defects.

A novel Pro126His beta propeller mutation in integrin alphaIIb causes Glanzmann thrombasthenia by impairing progression of pro-alphaIIbbeta3 from endoplasmic reticulum to Golgi.

BACKGROUND: Glanzmann thrombasthenia (GT) is an autosomal recessive bleeding disorder characterized by lack of platelet aggregation in response to most physiological agonists and caused by either a lack or dysfunction of the platelet integrin alphaIIbbeta3 (glycoprotein IIb/IIIa). PATIENTS: Mucocutaneous bleeding manifestations and platelet dysfunction consistent with GT were observed in a 20-year-old proband of a Chinese family. OBJECTIVES: To determine the molecular basis of GT and characterize the mutation by in vitro expression studies. RESULTS: Analysis of the patient's platelets by fluorescence-activated cell sorting demonstrated the presence of trace amounts of beta3, exposed on her platelet surface, but a complete absence of alphaIIbbeta3. Sequence analysis revealed a novel C470A transversion in exon 4 of the alphaIIb gene predicting a Pro126His alteration in the blade 2 of the alphaIIb beta propeller domain. The proband was homozygous for the mutation, the mother and the father were heterozygous, whereas 100 healthy subjects lacked this transversion. Chinese hamster ovary cells cotransfected with cDNAs of mutated alphaIIb and wild-type beta3 failed to express alphaIIbbeta3 on the cell surface as shown by FACS. Western blot analysis of the cell lysates showed no detectable mature alphaIIb. Immunoprecipitation with antibody against beta3 demonstrated pro-alphaIIb in the cells expressing the mutant alphaIIbbeta3, indicating pro-alphaIIbbeta3 complex formation. Intracellular immunofluorescence studies demonstrated the pro-alphaIIbbeta3 complex that co-localized with an ER marker, but showed minimal co-localization with a Golgi marker. CONCLUSIONS: A novel Pro126His mutation in alphaIIb compromised transport of the pro-alphaIIbbeta3 complex from the endoplasmic reticulum to the Golgi, leading to intracellular retention. The impaired alphaIIbbeta3 transport is responsible for the thrombasthenia in this patient.

Acquired Glanzmann's thrombasthenia caused by glycoprotein IIb/IIIa autoantibodies of the immunoglobulin G1 (IgG1), IgG2 or IgG4 subclass: a study in six cases.

BACKGROUND: Acquired Glanzmann's thrombasthenia (GT) is an uncommon bleeding disorder caused by glycoprotein (GP) IIb/IIIa-specific autoantibodies. Covering of the fibrinogen binding site of GPIIb/IIIa results in a moderate-to-severe bleeding tendency. MATERIALS AND METHODS: We performed a diagnostic evaluation and evaluated the underlying risk factors in six patients with a bleeding tendency caused by acquired GT. RESULTS: One patient, with GPIIb/IIIa autoantibodies of the immunoglobulin G2 (IgG2) subclass, used diclophenac and recovered after discontinuation of this drug. A second patient was primarily diagnosed with multiple angiodysplastic lesions. In this patient, the acquired GT was caused by GPIIb/IIIa autoantibodies of the IgG4 subclass that was treated with DDAVP and platelet transfusions. A third patient with Hodgkin's lymphoma and anti-GPIIb/IIIa of the IgG2 subclass was treated for haemorrhagic diathesis with corticosteroids and azathioprin. A fourth patient, with IgG2 anti-GPIIb/IIIa autoantibodies, diagnosed with mantle cell lymphoma, responded well to treatment of an axillary mass with local radiotherapy. The fifth and sixth patients, with IgG1 anti-GPIIb/IIIa autoantibodies, appeared to have GT after splenectomy because of autoimmune thrombocytopenia. They were treated with corticosteroids, intravenous immunoglobulin and Rituximab. CONCLUSION: Although it might be a rare event, one should be aware of acquired GT as a cause of an unexpected primary disorder of haemostasis in patients with lymphoma or autoimmune disease. The lack of platelet destruction in these cases of acquired GT can be explained, either by the subclass of the autoantibodies (i.e. IgG2 or IgG4) or by the arrested platelet destruction by IgG1 (or IgG3) autoantibodies after splenectomy.

Mutations in GPIIIa molecule as a cause for Glanzmann thrombasthenia in Indian patients.

BACKGROUND: Glanzmann thrombasthenia (GT) results from a quantitative or qualitative defect of GPIIb-IIIa complex, the fibrinogen receptor on platelets, which plays a very important role in platelet aggregation. In this report we describe the molecular studies on 22 patients with Glanzmann Thrombasthenia at our institute. OBJECTIVES: The main objective was to identify the mutations present in our GT population in order to establish a strategy for genetic counseling and antenatal diagnosis. METHODS: Twenty-two patients with GT were included in the present study. Complete blood count (CBC), platelet aggregation, flow cytometry, Western blot, single strand conformation polymorphism (SSCP) and denaturing gradient gel electrophoresis (DGGE) were performed in all the patients. The patients showing an abnormal migration pattern in SSCP or DGGE were sequenced further on an automated sequencer. RESULTS: Of the 22 patients studied, mutations were detected in 12 individuals. Of these, 11 were novel mutations and one mutation Y115C was reported earlier. Flow cytometric analysis showed the absence of receptors in type I GT, highly reduced levels in type II GT and normal levels in type III GT. The DGGE analysis and SSCP analysis of the patients showed different migration patterns. Sequencing was performed in all patients showing an abnormal migration pattern. Of the 22 cases studied mutations could be detected in 12 cases of GT. We could detect six patients with point mutations, four patients with insertions and five patients with deletion mutations. Exon 4 has been found to be the most common site for mutations in our patients. CONCLUSION: This study has shown a wide array of mutations present in our GT patients which would be extremely useful in genetic counseling and prenatal diagnosis, essential in preventing these disorders in succeeding generations.

Insertion of a C in the exon 28 of integrin alphaIIb gene leading to a frameshift mutation is responsible for Glanzmann thrombasthenia in a Japanese case.

BACKGROUND: Glanzmann thrombasthenia (GT) is a hereditary bleeding disorder caused by a qualitative or quantitative defect in the integrin alphaIIbbeta3. OBJECTIVE: Our objective is to identify the gene mutation that resulted in GT. PATIENTS AND METHODS: The patient was a 66-year-old male with a history of frequent bleeding. The expression levels of the integrin proteins in the platelets were determined by flow cytometry and Western blot analysis. The sequences of genomic DNA and mRNA encoding for alphaIIb and beta3 were analyzed by the dye-terminator cycle sequencing method. For transfection experiments, expression vectors encoding for wild-type alphaIIb, mutated alphaIIb, beta3, green fluorescent protein (GFP) fusion wild-type alphaIIb, GFP fusion mutated alphaIIb and DsRed fusion beta3 were constructed. These vectors were transfected to COS-7 cells, and the expression levels were determined. RESULTS: The alphaIIb protein was remarkably reduced in the patient's platelets, and gene analysis showed that the patient possessed compound heterozygous mutations in the alphaIIb gene. One was a C --> G substitution at the splice acceptor site (- 3) of exon 26 (CAG -->GAG) and the other was the insertion of an additional C at the region including six C bases between 2911 and 2916 in exon 28 (InsC). Transfection experiments using COS-7 cells showed that alphaIIb containing InsC had expressed and formed a complex with beta3, but had not been transported to the Golgi apparatus. CONCLUSIONS: In the present study the novel mutation InsC, leading to a frameshift that affects the transmembrane domain and the cytoplasmic tail, was found to be responsible for GT.

Type I Glanzmann thrombasthenia caused by an apparently silent beta3 mutation that results in aberrant splicing and reduced beta3 mRNA.

We report a novel genetic defect in a patient with type I Glanzmann thrombasthenia. Flow cytometry analysis revealed undetectable levels of platelet glycoproteins alphaIIb and beta3, although residual amounts of both proteins were detectable in immunoblotting analysis. Sequence analysis of reversely transcribed platelet beta3 mRNA showed a 100-base pair deletion in the 3'-boundary of exon 11, that results in a frame shift and appearance of a premature STOP codon. Analysis of the corresponding genomic DNA fragment revealed the presence of a homozygous C1815T transition in exon 11. The mutation does not change the amino acid residue but it creates an ectopic consensus splice donor site that is used preferentially, causing splicing out of part of exon 11. The parents of the proband, heterozygous for this mutation, were asymptomatic and had reduced platelet content of alphaIIbbeta3. PCR-based relative quantification of beta3 mRNA failed to detect the mutant transcript in the parents and showed a marked reduction in the patient. The results suggest that the thrombasthenic phenotype is, mainly, the result of the reduced availability of beta3-mRNA, most probably due to activation of the nonsense-mediated mRNA decay mechanism. They also show the convenience of analyzing both genomic DNA and mRNA, in order to ascertain the functional consequences of single nucleotide substitutions.

Molecular basis of Glanzmann's Thrombasthenia and current strategies in treatment.

Glanzmann Thrombasthenia, an exceptional inherited platelet disorder is characterized by a complete lack of platelet aggregation due to a defect in the alpha(IIb)beta(3) complex or to a qualitative abnormality of this complex. Advances in molecular biology have permitted to precise the molecular abnormality on alpha(IIb) or beta(3) genes responsible for the disease and have also contributed to a better knowledge of normal platelet physiology. Hemorrhages are the main clinical problem. Current principles of therapeutic management are proposed, with special reference to the risk of platelet alloimmunisation.

A novel Phe171Cys mutation in integrin alpha causes Glanzmann thrombasthenia by abrogating alphabeta complex formation.

BACKGROUND: Glanzmann thrombasthenia (GT) is an autosomal recessive bleeding disorder characterized by lack of platelet aggregation induced by most agonists. The disease is caused by mutations in either alpha(IIb)[glycoprotein (GP) IIb] or beta(3) (GPIIIa) genes that lead to a lack or dysfunction of the integrin alpha(IIb)beta(3) which serves as a fibrinogen receptor. PATIENTS: Mucocutaneous bleeding manifestations and platelet dysfunction consistent with GT were observed in three members of a Cypriot family: a 3-year-old proband, her father and her paternal uncle. OBJECTIVE: To determine the molecular basis of GT in this family and to characterize possible biochemical and structural defects. RESULTS: Analysis of the patients' platelets by fluorescence-activated cell sorting demonstrated trace amounts of beta(3), no alpha(IIb) and no alpha(IIb)beta(3) on the membrane. Sequence analysis revealed a novel T607G transversion in exon 5 of the alpha(IIb) gene predicting a Phe171Cys alteration that created a PstI recognition site. All three patients were homozygous for the mutation, the mother and paternal grandparents of the proband were heterozygous, whereas 110 healthy subjects lacked this transversion. Chinese hamster ovary cells cotransfected with cDNAs of mutated alpha(IIb) and wild-type beta(3) failed to express alpha(IIb)beta(3) as shown by immunoprecipitation and immunohistochemistry experiments. Structural analysis of the alpha(IIb)beta(3) model, which was based on the crystal structure of alpha(v)beta(3), indicated that Phe171 plays an essential role in the interface between the beta-propeller domain of alpha(IIb) and the betaA domain of beta(3). CONCLUSIONS: A novel Phe171Cys mutation in the alpha(IIb) gene of patients with GT is associated with abrogation of alpha(IIb)beta(3) complex formation.

Glanzmann's thrombasthenia due to a point mutation within intron 10 results in aberrant splicing of the beta3 gene.

Glanzmann's thrombasthenia (GT) is an hereditary bleeding disorder caused by a quantitative or qualitative defect in the integrin alphaIIbbeta3. We attempted to identify genetic defects responsible for a case of GT in Korea. The patient was a 6-year-old boy who had suffered from hemorrhage and purpura. The cDNAs of alphaIIb and beta3 were amplified by reverse transcription (RT)-PCR and were sequenced. RT-PCR of COS7 cells transfected with Exontrap vectors containing the beta3 genomic DNA fragments were performed to verify an aberrant splicing. Transient expression of alphaIIbbeta3 on transfected-COS7 cells was determined by flow cytometry, and the presence of alphaIIbbeta3 was confirmed by immunoprecipitation. We discovered an abnormality with the insertion of 38 bp between exon 10 and exon 11, including a stop codon, in beta3 cDNA. Sequence analysis of genomic DNA showed a point mutation, G-->T, at the position 29 107 of intron 10. RT-PCR analyses of COS7 cells transfected with Exontrap vector containing the mutant beta3 gene revealed that the point mutation at the position 29 107 of intron 10, G-->T, was responsible for an aberrant splicing in the beta3 gene. The transfection experiments and immunoprecipitation revealed the absence of alphaIIbbeta3 in the COS7 cells transfected with mutant gene. The mutation at the position 29 107 of intron 10, G-->T, in the beta3 genomic DNA was found to induce an aberrant splicing and to be responsible for GT in this patient.

Two different beta3 cysteine substitutions alter alphaIIb beta3 maturation and result in Glanzmann thrombasthenia.

We report the defects responsible for Glanzmann thrombasthenia in two patients showing traces of abnormally migrating platelet beta3 in immunoblotting. Using PCR-SSCP and direct sequencing, we identified a novel homozygous mutation in exon 10 of the beta3 gene of patient 1 which gave a C457 to Y amino acid substitution. A C542 to R substitution in beta3 of patient 2 was previously reported by us. These cysteines are present in EGF-domains 1 and 3 respectively of beta3. We therefore constructed mutants carrying substitutions on cysteine residues in each of the first three EGF domains of beta3, C457, C495 and C542 respectively. Transient expression of these mutants in COS-7 cells, including the C542 and C547 double mutant, proved that disulfide disruption directly affects cell surface expression of the integrin. We then showed by metabolic (35S) labeling and Endo-H glycosidase treatment that these substitutions strongly affected complex maturation within the cell.

Acquired disorder of platelet function associated with autoantibodies against membrane glycoprotein IIb-IIIa complex--1. Glycoprotein analysis.

A patient with idiopathic thrombocytopenic purpura developed after splenectomy a thrombasthenia-like severe haemorrhagic diathesis characterized by a normal or subnormal platelet count, prolonged bleeding time, strongly reduced platelet adhesion to glass and defective platelet aggregation in response to ADP and collagen. In contrast to hereditary thrombasthenia membrane glycoproteins (GP) IIb and IIIa were normally present in the patient's platelets. Immunoelectrophoretic analysis revealed an abnormal behaviour of the patient's GP IIb-IIIa complex. Autoantibodies against GP IIb-IIIa were detected in Triton-extracted washed platelets. Incubation of normal platelets with plasma from the patient resulted in a similar immunoelectrophoretic abnormality of the GP IIb-IIIa complex indicating that bound autoantibodies (IgG) are responsible for the abnormal immunoelectrophoretic behaviour of the patient's GP IIb-IIIa complex. Platelet fibrinogen was severely reduced similar to classical thrombasthenia suggesting that the GP IIb-IIIa complex is involved in platelet fibrinogen storage.

Platelet abnormalities in hepatobiliary diseases.

Platelet abnormalities associated with hepatobiliary diseases include increased (thrombocytosis) and decreased (thrombocytopenia) numbers of platelets as well as abnormalities in function (thrombocytopathy or thrombasthenia). Hepatic diseases that are accompanied by platelet abnormalities include hepatitis, cirrhosis, portal hypertension, and neoplastic disorders both benign and malignant. The objective of this work is to examine the platelet abnormalities that occur with a variety of hepatobiliary disorders. Thrombocytosis is seen as a reactive entity following splenectomy. Thrombocytopenia is associated with hypersplenism, dysproteinemias and liver disease related disseminated intravascular coagulation (DIC). Qualitative platelet abnormalities are found in hepatic failure, liver diseases associated with high or low levels of lipid, and with medications given for a variety of hepatocellular diseases. Clinically common and significant platelet abnormalities associated with liver disease are thrombocytopenia secondary to portal hypertension and the thrombasthenias following metabolic changes and/or therapeutic interventions of liver disease.

[Glanzmann's thrombasthenia: a rare example of an integrin deficit]. / La tromboastenia di Glanzmann: un raro esempio di deficit di un'integrina.

Glanzmann's thrombasthenia is an autosomal recessive life-long bleeding disorder, originated from a quantitative or qualitative defect of the major platelet membrane receptor: the GPIIb/IIIa complex. The GPIIb/IIIa complex is a calcium-dependent heterodimer, belonging to the integrin superfamily. The complex of activated platelets can bind fibrinogen, von Willebrand factor, fibronectin and vitronectin, which are proteins playing an important role in platelet adhesion and aggregation. Thrombasthenic platelets are deficient in GPIIb, GPIIIa and GPIIb/IIIa complex; however, platelets from few thrombasthenic patients content near-normal amounts of functionally abnormal complex. The GPIIb/IIIa quantitative or functional defect leads to defective platelet hemostatic plug formation. Hemorrhagic symptoms consist of purpura, gingival hemorrhage, menorrhagia and epistaxis. Some cases of Glanzmann's thrombasthenia have been characterized at the molecular genetic level. Molecular abnormalities include: GPIIb or GPIIIa partial gene deletion, GPIIIa gene insertion, a point mutation resulting in an amino acid substitution within the GPIIIa molecule. In spite of the contemporary reduction of both GPIIb and GPIIIa in most cases of Glanzmann's thrombasthenia, it appears that a molecular abnormality affecting only one of the glycoprotein genes may result in a thrombasthenic phenotype.

[Thrombocytic diseases in generalized myasthenia gravis and their correction by using plasmapheresis and administration of anti- immunoglobulin antisera]. / Trombotsitarnye narusheniia pri generalizovannoi miastenii i ikh korrektsiia s pomo'shchiu plazmafereza i vvedeniia anti- syvorotok k immunoglobulinam.

Platelet adhesion and aggregation studies were performed in different test systems in 25 patients with plasmapheresis-treated myasthenia patients. In vivo studies with plasmapheresis and in vitro investigation with anti-immunoglobulin antisera revealed the normalization of the impaired platelet functions.

[Platelet membrane glycoproteins and the molecular mechanisms of hereditary platelet dysfunction].

Glycoproteins, present on the outer surface of platelets, function in the aggregating and releasing reactions of these cells. Some of the hereditary diseases of platelet dysfunction, such as, thrombasthenia and Bernard-Soulier syndrome, have been shown to result from defects in some of these glycoproteins. Recent advances in molecular biology have enabled us to analyze the genes which encode the defective glycoproteins in these patients. The information from these studies also helps us to understand in detail the function of the glycoproteins. At present, the major glycoproteins, GP II b/IIIa and GP I b/IX, have been studied extensively and we know the details of the mechanisms through which they function. However, as to the minor glycoproteins, GP I a/II a, GP IV and GP VI, details remain to be elucidated.

[Anti-platelet antibody and platelet function].

The effect of anti-platelet antibodies, including murine monoclonal antibodies, autoantibodies and alloantibodies, on platelet function was analyzed. The target antigen of these antiplatelet antibodies, investigated in the present study, was a glycoprotein IIb/IIIa, which is a receptor of fibrinogen and plays an important role in platelet aggregation. Some of these antibodies inhibited agonist-induced platelet aggregation. The target antigen of one murine monoclonal antibodies, designated OP-G2, was a glycoprotein IIb/IIIa and interestingly, this antibody induced platelet aggregation, which required divalent cation and fibrinogen. We compared the epitope of these antibodies by inhibition assay and found the epitope of these antibodies to be very close. The binding of OP-G2 to the platelets required Ca2+. These data suggest that OP-G2 recognizes an epitope at or in very close proximity to the fibrinogen binding site of GPIIb/IIIa, as compared with other antibodies.