Diagnosis of inherited platelet disorders on a blood smear: a tool to facilitate worldwide diagnosis of platelet disorders.

Essentials There are many hereditary platelet disorders (HPD) but diagnosing these is challenging. We provide a method to diagnose several HPDs using standard blood smears requiring < 100 µL blood. By this approach, the underlying cause of HPD was characterized in ~25-30% of referred individuals. The method facilitates diagnosis of HPD for patients of all ages around the world. SUMMARY: Background Many hereditary thrombocytopenias and/or platelet function disorders have been identified, but diagnosis of these conditions remains challenging. Diagnostic laboratory techniques are available only in a few specialized centers and, using fresh blood, often require the patient to travel long distances. For the same reasons, patients living in developing countries usually have limited access to diagnosis. Further, the required amount of blood is often prohibitive for pediatric patients. Objectives By a collaborative international approach of four centers, we aimed to overcome these limitations by developing a method using blood smears prepared from less than 100 µL blood, for a systematic diagnostic approach to characterize the platelet phenotype. Methods We applied immunofluorescence labelling (performed centrally) to standard air-dried peripheral blood smears (prepared locally, shipped by regular mail), using antibodies specific for proteins known to be affected in specific hereditary platelet disorders. Results By immunofluorescence labelling of blood smears we characterized the underlying cause in 877/3217 (27%) patients with suspected hereditary platelet disorders (HPD). Currently about 50 genetic causes for HPD are identified. Among those, the blood smear method was especially helpful to identify MYH9 disorders/MYH9-related disease, biallelic Bernard-Soulier syndrome, Glanzmann thrombasthenia and gray platelet syndrome. Diagnosis could be established for GATA1 macrothrombocytopenia, GFI1B macrothrombocytopenia, ß1-tubulin macrothrombocytopenia, filamin A-related thrombocytopenia and Wiskott-Aldrich syndrome. Conclusion Combining basic and widely available preanalytical methods with the immunomorphological techniques presented here, allows detailed characterization of the platelet phenotype. This supports genetic testing and facilitates diagnosis of hereditary platelet disorders for patients of all ages around the world.

Inherited disorders of platelet function: selected updates.

The gene variants responsible for the primary genotype of many platelet disorders have now been identified. Next-generation sequencing technology (NGST), mainly exome sequencing, has highlighted genes responsible for defects in platelet secretion (NBEAL2, gray platelet syndrome), procoagulant activity (STIM1, Stormorken syndrome), and activation pathways (RASGRP2, CalDAG-GEFI deficiency and integrin dysfunction; PRKACG, cyclic adenosine monophosphate-dependent protein kinase deficiency). Often disorders of platelet function are associated with a modified platelet production with changes in platelet number and size and can accompany malfunction of other organs or tissues. Most families have private mutations, and gene variants may prevent protein synthesis, abrogate function, or result in aberrant activated proteins. Nevertheless, bleeding severity is difficult to predict by genotype alone suggesting other factors. A major new challenge of NGST is to identify these factors and help improve patient care. This review concentrates on recent developments and is illustrated from personal observations.

The abnormal proplatelet formation in MYH9-related macrothrombocytopenia results from an increased actomyosin contractility and is rescued by myosin IIA inhibition.

BACKGROUND: Mutations in the MYH9 gene cause autosomal dominant MYH9-related diseases (MYH9-RD) that associate macrothrombocytopenia with various other clinical conditions. The mechanisms giving rise to giant platelets remain poorly understood. OBJECTIVES/PATIENTS: To study the proplatelet formation (PPF) derived from megakaryocytes (MKs) generated in vitro from 11 patients with MYH9-RD with different mutations, compared with controls. METHODS: Proplatelet formation from cultured patients' MKs was evaluated with or without blebbistatin or the ROCK inhibitor Y27632. Myosin IIA and actin distribution were studied in spreading MKs on different surfaces by immunoconfocal analysis. Kinetic studies of contractility were performed on spreading MKs and the impact of blebbistatin on the maturation of the patients' MKs was evaluated by electron microscopy. RESULTS AND CONCLUSIONS: We show that in vitro MKs of 11 patients formed significantly fewer proplatelets than controls. MKs from MYH9-RD displayed an abnormal spreading on polylysine, fibronectin and collagen, with a disorganized actin network and a marked increase in stress fiber formation. Traction force microscopy studies demonstrated an elevated level of contractile forces in adherent mutated MKs. The myosin II inhibitor blebbistatin and the ROCK inhibitor Y27632 both rescued the proplatelet formation defect and normalized the ultrastructural characteristics of MYH9-RD MKs. Altogether, our results show that in MYH9-RD, mutations modify the overall MYH9 function and provoke a proplatelet defect through an excess of actomyosin contractility in spreading MKs. These results may promote new therapeutic strategies aimed at reducing this actomyosin contractility.

C560Rß3 caused platelet integrin αII b ß3 to bind fibrinogen continuously, but resulted in a severe bleeding syndrome and increased murine mortality.

BACKGROUND AND OBJECTIVES: ß(3)-Deficient megakaryocytes were modified by human ß(3)-lentivirus transduction and transplantation to express sufficient levels of a C560Rß(3) amino acid substitution, for investigation of how an activated αII b ß(3) conformation affects platelets in vivo in mice. PATIENT/METHODS: As in our previous report of an R560ß(3) mutation in a patient with Glanzmann thrombasthenia, R560ß(3) murine platelets spontaneously bound antibody that only recognizes activated αII b ß3 bound to its ligand, fibrinogen. RESULTS: With this murine model, we showed that αII b -R560ß3 mutation-mediated continuous binding of fibrinogen occurred in the absence of P-selectin surface expression, indicating that the integrin was in an active conformation, although the platelets circulated in a quiescent manner. Remarkably, only 35% of R560ß(3) 'mutant' mice survived for 6 months after transplantation, whereas 87% of C560ß(3) 'wild-type' mice remained alive. Pathologic examination revealed that R560ß(3) mice had enlarged spleens with extramedullary hematopoiesis and increased hemosiderin, indicating hemorrhage. R560ß(3) megakaryocytes and platelets showed abnormal morphology and irregular granule distribution. Interestingly, R560ß(3) washed platelets could aggregate upon simultaneous addition of fibrinogen and physiologic agonists, but aggregation failed when platelets were exposed to fibrinogen before activation in vitro and in vivo. CONCLUSIONS: The results demonstrate that continuous occupancy of αIIb ß3 with fibrinogen disrupts platelet structure and function, leading to hemorrhagic death consistent with Glanzmann thrombasthenia rather than a thrombotic state.

Recommendations for the Standardization of Light Transmission Aggregometry: A Consensus of the Working Party from the Platelet Physiology Subcommittee of SSC/ISTH.

Light transmission aggregometry (LTA) is the most common method used to assess platelet function. However, there is no universal standard for its performance. The Platelet Physiology Subcommittee of the Scientific and Standardization Committee (SSC) of the International Society on Thrombosis and Haemostasis formed a working party of experts with the aim of producing a series of consensus recommendations for standardizing LTA. Due to a lack of investigations that directly compared different methodologies to perform LTA studies, there were insufficient data to develop evidence-based guidelines. Therefore, the RAND method was used, which obtains a formal consensus among experts about the appropriateness of health care interventions, particularly when scientific evidence is absent, scarce and/or heterogeneous. Using this approach, each expert scored as "appropriate", "uncertain" or "inappropriate" a series of statements about the practice of LTA, which included pre-analytical variables, blood collection, blood processing, methodological details, choice of agonists and the evaluation and reporting of results. After presentation and public discussion at SSC meetings, the assessments were further refined to produce final consensus recommendations. Before delivering the recommendations, a formal literature review was performed using a series of defined search terms about LTA. Of the 1830 potentially relevant studies identified, only 14 publications were considered to be actually relevant for review. Based upon the additional information, 6 consensus statements were slightly modified. The final statements were presented and discussed at the SSC Meeting in Cairo (2010) and formed the basis of a consensus document, which is the subject of the present report. This article is protected by copyright. All rights reserved.

[Epidural labor analgesia and parturient with type 2B von Willebrand disease]. / Analgésie péridurale obstétricale et maladie de Willebrand de type 2B.

Type 2B von Willebrand disease (vWD) is an inherited bleeding syndrome resulting from a qualitative abnormality of von Willebrand Factor with an increased affinity for the glycoprotein Ib platelet receptor. Pregnancy increases the severity of this disease by decreasing the platelet count restricting epidural anaesthesia because of adverse risk of spinal bleeding. There is a phenotypic variability of Type 2B vWD depending of the von Willebrand Factor mutation. We report here the strategy we used to administer epidural anaesthesia for a patient with Type 2B vWD resulting from the P1337L mutation of von Willebrand Factor.

Mean platelet volume: comparison of three analysers towards standardization of platelet morphological phenotype.

INTRODUCTION: The aim of this study was to show variability in the measurement of the mean platelet volume (MPV) depending on the instrument used. METHODS: This prospective analysis was carried out to measure MPV with three instruments, in 30 healthy controls and 113 hospital patients. RESULTS: Firstly, for values in the normal range, the values obtained with the Siemens Advia(®) 2120 are lower than those given by the Beckman Coulter LH750(®) (-0.89), which are in turn lower than those obtained with the Sysmex XE-2100D(®) (-1.11), which represents a 20-25% variation in the measurement. These results emphasize the lack of universal external calibration for MPV analysis and thus make any intercentre comparison of MPV impossible unless the automated haematology analyser used is indicated. Secondly, we stress the differences in behaviour of the instruments in the presence of abnormally large platelets, i.e. an underestimate of the platelet count and the MPV may be provided because instruments using impedance technology may fail to take into account these platelets, but they rightly flag them. CONCLUSION: To harmonize our procedures, we propose definitions of platelet size (normal size, macroplatelets and giant platelets) based on the coordinated interpretation of the MPV, the distribution of platelet volume and the morphological appearance.

Natural history of platelet antibody formation against αIIbß3 in a French cohort of Glanzmann thrombasthenia patients.

Treatment of the bleeding syndrome in Glanzmann thrombasthenia (GT) is often complicated by naturally occurring isoantibodies directed against the αIIbß3 integrin that cause the removal of or render ineffective transfused donor platelets. Such antibodies are produced after transfusion or pregnancy when the patient's immune system comes into contact with normal platelets. Despite many reports of anti-αIIbß3 antibodies in GT patients, there is no consensus pertaining to their frequency, their long-term evolution in the circulation, or their formation in relation to either (i) the extent of the αIIbß3 deficiency in the patient's platelets or (ii) the nature of the genetic defect (ITGA2B or ITGB3 genes). Antibody screening was performed on a large series of 24 GT patients in South-West France dividing the patients into two cohorts: (i) 16 patients with the French gypsy mutation (c.1544 + 1G>A) within ITGA2B that gives platelets totally lacking αIIbß3 and (ii) 8 patients carrying other defects of ITGA2B or ITGB3 with different expression levels of αIIbß3. Our results confirm that patients with premature termination mutations resulting in platelets lacking αIIbß3 are the most susceptible to form isoantibodies, a finding that may be useful in deciding the choice of therapy between platelet transfusion and the use of recombinant factor VIIa (FVIIa).

Advances in our understanding of the molecular basis of disorders of platelet function.

Genetic defects of platelet function give rise to mucocutaneous bleeding of varying severity because platelets fail to fulfil their haemostatic role after vessel injury. Abnormalities of pathways involving glycoprotein (GP) mediators of adhesion (Bernard-Soulier syndrome, platelet-type von Willebrand disease) and aggregation (Glanzmann thrombasthenia) are the most studied and affect the GPIb-IX-V complex and integrin αIIbß3, respectively. Leukocyte adhesion deficiency-III combines Glanzmann thrombasthenia with infections and defects of kindlin-3, a mediator of integrin activation. Agonist-specific deficiencies in platelet aggregation relate to mutations of primary receptors for ADP (P2Y(12)), thromboxane A(2) (TXA2R) and collagen (GPVI); however, selective abnormalities of intracellular signalling pathways remain better understood in mouse models. Defects of secretion from δ-granules are accompanied by pigment defects in the Hermansky-Pudlak and Chediak-Higashi syndromes; they concern multiple genes and protein complexes involved in secretory organelle biogenesis and function. Quebec syndrome is linked to a tandem duplication of the urokinase plasminogen activator (PLAU) gene while locus assignment to chromosome 3p has advanced the search for the gene(s) responsible for α-granule deficiency in the gray platelet syndrome. Defects of α-granule biosynthesis also involve germline VPS33B mutations in the ARC (arthrogryposis, renal dysfunction and cholestasis) syndrome. A mutation in transmembrane protein 16F (TMEM16F) has been linked to a defective procoagulant activity and phosphatidylserine expression in the Scott syndrome. Cytoskeletal dysfunction (with platelet anisotrophy) occurs not only in the Wiskott-Aldrich syndrome but also in filamin A deficiency or MYH9-related disease while GATA1 mutations or RUNX1 haploinsufficiency can affect expression of multiple platelet proteins.

Rare homozygous status of P43 ß1-tubulin polymorphism causes alterations in platelet ultrastructure.

ß1-tubulin is the main constituent of the platelet marginal band and studies with deficient mice showed that it maintains discoid shape and it is required for normal platelet formation. TUBB1 Q43P polymorphism is associated with decreased ß1-tubulin expression, diminished platelet reactivity, and partial loss of discoid shape in heterozygous carriers. However, to date no studies have been carried out on homozygous PP individuals. Our study included 19 subjects genotyped for TUBB1 Q43P polymorphism (4 QQ, 4 QP, and 2 PP). The two PP individuals were recruited after genotyping of 2073 individuals. Biochemical, microscopy, and molecular studies were performed. Real-time PCR showed a ~40% decrease in TUBB1 mRNA in the two PP individuals compared to four QQ subjects. Western blot analysis confirmed this reduction. Electron microscopy revealed a majority of normal discoid platelets in PP individuals, although platelets with loose, re-orientated or invaginated protofilaments, and an over-developed open canalicular system were observed. Such abnormalities were not observed in QQ subjects. Morphometric analyses showed no differences between PP and QQ individuals. Immunofluorescence confirmed the presence of a normal marginal band in a majority of platelets from PP subjects. Interestingly, both PP subjects had a 40% lower platelet count than QP and QQ. TUBB1 Q43P polymorphism in homozygosity mildly affects platelet ultrastructure and our data further suggest that high levels of ß1-tubulin might not be critical to sustain platelet discoid shape.

Use of autologous platelet-rich clots for the prevention of local injury bleeding in patients with severe inherited mucocutaneous bleeding disorders.

Stopping or preventing local bleeding in patients with inherited bleeding disorders linked to abnormal platelet function is traditionally treated by transfusion of blood cell products or recombinant factor VIIa. We now report the use in such patients of autologous platelet-rich clots as an aid to preventing bleeding and to facilitating tissue regeneration at superficial sites. Two patients with von Willebrand's disease (VWD) type 2B and one patient with type I Glanzmann thrombasthenia were treated after tooth extraction and dental surgery. A fourth patient with platelet-type VWD underwent a skin biopsy. Whereas all four patients had a lifelong history of bleeding complications, the application of an autologous platelet-rich clot immediately after surgery combined with tranexamic acid intake to slow fibrinolysis prevented blood loss and resulted in rapid and normal healing. This new procedure is simple, safe and inexpensive; it provides extra security for patients with a bleeding risk undergoing dentistry or superficial surgery.

An acquired inhibitor to the GPVI platelet collagen receptor in a patient with lupus nephritis.

BACKGROUND: GPVI is a major platelet collagen signaling receptor. In rare cases of immune thrombocytopenic purpura (ITP), autoantibodies to GPVI result in receptor shedding. OBJECTIVES: To investigate a possible pathogenic role of plasma anti-GPVI antibody located in a woman with lupus nephritis. METHODS: Measured were (i) platelet aggregation to collagen and convulxin, (ii) platelet GPVI expression (flow cytometry and western blotting), (iii) plasma soluble GPVI (sGPVI, dual antibody ELISA), and (iv) plasma anti-GPVI antibody (ELISA using recombinant sGPVI). RESULTS: In 2006 and early 2007, the patient had a normal platelet count but a virtual absence of platelet aggregation to collagen and convulxin. Her platelets responded normally to other agonists including cross-linking ITAM-dependent FcgammaRIIA by monoclonal antibody, IV.3. Flow cytometry and western blotting showed a platelet deficiency of GPVI. Plasma sGPVI levels were undetectable whereas ELISA confirmed the presence of anti-GPVI antibody. Sequencing revealed a normal GPVI cDNA structure. The patient's plasma and the isolated IgG3 fraction activated and induced GPVI shedding from normal platelets. A deteriorating clinical condition led to increasingly strict immunosuppressive therapy. This was globally associated with a fall in plasma anti-GPVI titres, the restoration of platelet GPVI and the convulxin response, and the loss of her nephrotic syndrome. CONCLUSIONS: Our results show that this patient acquired a potent anti-GPVI IgG3 antibody with loss of GPVI and collagen-related platelet function. Further studies are required to determine whether anti-GPVI antibodies occur in other lupus patients with nephritis.

Altered megakaryocytopoiesis in von Willebrand type 2B disease.

Type 2B von Willebrand disease (VWD2B) is caused by gain-of-function amino acid substitutions in the von Willebrand factor (VWF) A1 domain. These allow facilitated binding of mutated VWF to platelet GPIbalpha with prolonged lifetimes of VWF bonds and enhanced ADAMTS-13 cleavage of large VWF multimers. A bleeding rather than prothrombotic syndrome is due to: (i) decreased large VWF multimers in plasma; (ii) limited thrombus formation; and (iii) thrombocytopenia affecting some but not all patients. Accumulating evidence points to an altered megakaryocytopoiesis in VWD2B with the production of enlarged or giant platelets showing an abnormal ultrastructure and, in a cohort of patients, the presence of circulating platelet agglutinates. In fact, evidence from in vitro cultures and marrow aspirates suggests that the upregulated VWF function can lead to abnormal VWF trafficking in megakaryocytes, a modified platelet production with interacting proplatelets, and the presence or even release of platelet agglutinates in the bone marrow.

Induction of megakaryocytes to synthesize and store a releasable pool of human factor VIII.

von Willebrand factor (VWF) is a complex plasma glycoprotein that modulates platelet adhesion at the site of a vascular injury, and it also serves as a carrier protein for factor (F)VIII. As megakaryocytes are the only hematopoietic lineage to naturally synthesize and store VWF within alpha-granules, this study was performed to determine if expression of a FVIII transgene in megakaryocytes could lead to trafficking and storage of FVIII with VWF in platelet alpha-granules. Isolex selected CD34+ cells from human G-CSF mobilized peripheral blood cells (PBC) and murine bone marrow were transduced with a retrovirus encoding the B-domain deleted form of human FVIII (BDD-FVIII). Cells were then induced with cytokines to form a population of multiple lineages including megakaryocytes. Chromogenic analysis of culture supernatant from FVIII-transduced human cells demonstrated synthesis of functional FVIII. Treatment of cells with agonists of platelet activation (ADP, epinephrine, and thrombin receptor-activating peptide) resulted in the release of VWF antigen and active FVIII into the supernatant from transduced cells. Immunofluorescence analysis of cultured human and murine megakaryocytes revealed a punctate pattern of staining for FVIII that was consistent with staining for VWF. Electron microscopy of transduced megakaryocytes using immunogold-conjugated antibodies colocalized FVIII and VWF within the alpha-granules. FVIII retained its association with VWF in human platelets isolated from the peripheral blood of NOD/SCID mice at 2-6 weeks post-transplant of transduced human PBC. These results suggest feasibility for the development of a locally inducible secretory pool of FVIII in platelets of patients with hemophilia A.