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.

Diagnosis of suspected inherited platelet function disorders: results of a worldwide survey.

BACKGROUND: Diagnosis of inherited platelet function disorders (IPFDs) is important for appropriate management and to improve epidemiologic and clinical knowledge. However, there remains a lack of consensus on the diagnostic approach. OBJECTIVES: To gain knowledge on the current practices for the diagnosis of IPFD worldwide. METHODS: A 67-item questionnaire was distributed to the ISTH members and to the members of several national hemostasis and thrombosis societies. RESULTS: A total of 202 laboratories from 37 countries participated in the survey. The most frequent criterion to define patients with a suspected IPFD was a history of mucocutaneous bleeding and no acquired cause, but heterogeneity on the identification criteria was evident. Only 64.5% of respondents performed a direct clinical interview. On average, each laboratory studied 72 patients per year. The most commonly used laboratory equipment were the light-transmission aggregometer, the Platelet Function Analyzer-100, and the flow cytometer. Screening tests were platelet count, peripheral blood smear, light-transmission aggregometry, and Platelet Function Analyzer-100. Second-step tests were flow cytometry, molecular genetic analysis, and electron microscopy. Methodologies varied widely. In total, ~ 14,000 patients were investigated yearly and 60% turned out to not have a defect. Of the remaining 40%, only 8.7% received a diagnosis at a molecular level. CONCLUSIONS: Many laboratories worldwide are involved in the diagnosis of IPFD. A large fraction of the patients studied remain without a diagnosis. A high variability in the diagnostic approaches is evident.

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.

An adenine insertion in exon 6 of human GP6 generates a truncated protein associated with a bleeding disorder in four Chilean families.

BACKGROUND: Glycoprotein VI (GPVI), 60-65 kDa, is a major collagen receptor on platelet membranes involved in adhesive and signaling responses. Mice lacking GPVI have impaired platelet response to collagen and defective primary adhesion and subsequent thrombus formation. Complete or partial deficiency of GPVI in humans is a rare condition presenting as a mild bleeding disorder. The defect in most of the reported patients is acquired and associated with other diseases. To date, only two patients have been characterized at the molecular level who carry different compound heterozygous mutations in the GP6 gene. OBJECTIVE: To report four unrelated patients from non-consanguineous families who presented with mucocutaneous bleeding. They had absent platelet aggregation and (14) C-5-HT secretion with collagen, convulxin and collagen-related peptide. RESULTS: Flow cytometry and immunofluorescence-confocal microscopy showed an absence of GPVI in non-permeabilized platelets. All the patients had an adenine insertion in exon 6 (c.711_712insA), changing the reading frame and generating a premature 'stop codon' in site 242 of the protein. The mutation predicts the synthesis of the truncated protein before the trans-membrane domain, corresponding to a band of ≈49 kDa observed in western blots and in permeabilized platelets by immunofluorescence. Platelet mRNA from all the patients was sequenced and contained the corresponding adenine insertion. Heterozygous relatives had no pathological bleeding, normal response to collagen and convulxin and intermediate membrane expression of GPVI. CONCLUSIONS: The identification of four unrelated homozygous patients with an identical defect suggests that inherited GPVI deficiency is more frequent than previously suspected, at least in Chile.

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.

Inherited platelet disorders.

Inherited diseases of the megakaryocyte lineage give rise to bleeding when platelets fail to fulfill their hemostatic function upon vessel injury. Platelet defects extend from the absence or malfunctioning of adhesion (GPIb-IX-V, Bernard-Soulier syndrome) or aggregation receptors (integrin αIIbß3, Glanzmann thrombasthenia) to defects of primary receptors for soluble agonists, secretion from storage organelles, activation pathways and the generation of procoagulant activity. In disorders such as the Chediak-Higashi, Hermansky-Pudlak, Wiskott-Aldrich and Scott syndromes the molecular lesion extends to other cells. In familial thrombocytopenia (FT), platelets are produced in insufficient numbers to assure hemostasis. Some FT affect platelet morphology and give rise to the 'giant platelet' syndromes (e.g. MYH9-related diseases) with changes in megakaryocyte maturation within the bone marrow and premature release of platelets. Diseases of platelet production may also affect other cells and in some cases interfere with development and/or functioning of major organs. Diagnosis of platelet disorders requires platelet function testing, studies often aided by the quantitative analysis of receptors by flow cytometry and fluorescence and electron microscopy. New generation DNA-based procedures including whole exome sequencing offer an exciting new perspective. Transfusion of platelets remains the most common treatment of severe bleeding, management with desmopressin is often used for mild disorders. Substitute therapies are available including rFVIIa and the potential use of thrombopoietin analogues for FT. Stem cell or bone marrow transplantation has been successful for several diseases while gene therapy shows promise in the Wiskott-Aldrich syndrome.

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.

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.

Platelet-released growth factors enhance the secretion of hyaluronic acid and induce hepatocyte growth factor production by synovial fibroblasts from arthritic patients.

OBJECTIVES: Autologous platelet-secreted growth factors (GFs) may have therapeutic effects in osteoarthritis (OA) capsular joints via multiple mechanisms. Our aim was to examine the effect of a platelet-derived preparation rich in growth factors (PRGFs) in OA synovial cell biology. METHODS: Synovial cells were isolated from 10 osteoarthritic patients and cultured in serum-free media (basal conditions) and exposed to either a platelet-poor preparation or PRGF for 72 h. Cells activated with interleukin-1beta (IL-1beta) for 48 h were also exposed to PRGF. Changes in several events relevant to joint homeostasis including (i) hyaluronic acid (HA) secretion, (ii) the balance between metalloproteinase-1, -3 and -13 (MMP-1, MMP-3 and MMP-13) and tissue inhibitor-1 (TIMP-1) and (iii) the secretion of transforming growth factor-beta1(TGF-beta1), vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF), were all assessed. RESULTS: PRGF significantly enhanced HA secretion compared with platelet-poor preparations, P < 0.05; at the same time release of TIMP-1, MMP-1, MMP-3 and MMP-13 were not affected. An increased HGF production was observed (P < 0.05) but VEGF and TGF-beta1 levels remained unchanged. PRGF significantly enhanced the secretion of HA induced by IL-1beta activation, P < 0.05, but it did not modify the IL-1beta-induced rise in MMP-1, MMP-3 and VEGF. In contrast, PRGF-induced HGF production was abolished by the presence of IL-1beta during PRGF treatment, P < 0.05. CONCLUSIONS: Intra-articular administration of PRGF might be beneficial in restoring HA concentration and switching angiogenesis to a more balanced status but does not halt the effects of IL-1beta on synovial cells.