Paquita Nurden

Identification of the platelet ADP receptor targeted by antithrombotic drugs

Platelets have a crucial role in the maintenance of normal haemostasis, and perturbations of this system can lead to pathological thrombus formation and vascular occlusion, resulting in stroke, myocardial infarction and unstable angina. ADP released from damaged vessels and red blood cells induces platelet aggregation through activation of the integrin GPIIb–IIIa and subsequent binding of fibrinogen. ADP is also secreted from platelets on activation, providing positive feedback that potentiates the actions of many platelet activators. ADP mediates platelet aggregation through its action on two G-protein-coupled receptor subtypes. The P2Y 1 receptor couples to Gq and mobilizes intracellular calcium ions to mediate platelet shape change and aggregation. The second ADP receptor required for aggregation (variously called P2YADP , P2YAC, P2Ycyc or P2TAC) is coupled to the inhibition of adenylyl cyclase through Gi. The molecular identity of the G i-linked receptor is still elusive, even though it is the target of efficacious antithrombotic agents, such as ticlopidine and clopidogrel and AR-C66096 (ref. 9). Here we describe the cloning of this receptor, designated P2Y12, and provide evidence that a patient with a bleeding disorder has a defect in this gene. Cloning of the P2Y12 receptor should facilitate the development of better antiplatelet agents to treat cardiovascular diseases.

Autologous platelets as a source of proteins for healing and tissue regeneration

Platelets are known for their role in haemostasis where they help prevent blood loss at sites of vascular injury. To do this, they adhere, aggregate and form a procoagulant surface leading to thrombin generation and fibrin formation. Platelets also release substances that promote tissue repair and influence the reactivity of vascular and other blood cells in angiogenesis and inflammation. They contain storage pools of growth factors including PDGF, TGF-beta?and VEGF as well as cytokines including proteins such as PF4 and CD40L. Chemokines and newly synthesised active metabolites are also released. The fact that platelets secrete growth factors and active metabolites means that their applied use can have a positive influence in clinical situations requiring rapid healing and tissue regeneration. Their administration in fibrin clot or fibrin glue provides an adhesive support that can confine secretion to a chosen site. Additionally,the presentation of growth factors attached to platelets and/or fibrin may result in enhanced activity over recombinant proteins. Dental implant surgery with guided bone regeneration is one situation where an autologous platelet-rich clot clearly accelerates ossification after tooth extraction and/or around titanium implants. The end result is both marked reductions in the time required for implant stabilisation and an improved success rate. Orthopaedic surgery, muscle and/or tendon repair, reversal of skin ulcers, hole repair in eye surgery and cosmetic surgery are other situations where autologous plate-lets accelerate healing. Our aim is to review these advances and discuss the ways in which platelets may provide such unexpected beneficial therapeutic effects.

Compound inheritance of a low-frequency regulatory SNP and a rare null mutation in exon-junction complex subunit RBM8A causes TAR syndrome

The exon-junction complex (EJC) performs essential RNA processing tasks. Here, we describe the first human disorder, thrombocytopenia with absent radii (TAR), caused by deficiency in one of the four EJC subunits. Compound inheritance of a rare null allele and one of two low-frequency SNPs in the regulatory regions of RBM8A, encoding the Y14 subunit of EJC, causes TAR. We found that this inheritance mechanism explained 53 of 55 cases (P < 5 × 10−228) of the rare congenital malformation syndrome. Of the 53 cases with this inheritance pattern, 51 carried a submicroscopic deletion of 1q21.1 that has previously been associated with TAR, and two carried a truncation or frameshift null mutation in RBM8A. We show that the two regulatory SNPs result in diminished RBM8A transcription in vitro and that Y14 expression is reduced in platelets from individuals with TAR. Our data implicate Y14 insufficiency and, presumably, an EJC defect as the cause of TAR syndrome.

Exome sequencing identifies NBEAL2 as the causative gene for gray platelet syndrome

Gray platelet syndrome (GPS) is a predominantly recessive platelet disorder that is characterized by mild thrombocytopenia with large platelets and a paucity of α-granules; these abnormalities cause mostly moderate but in rare cases severe bleeding. We sequenced the exomes of four unrelated individuals and identified NBEAL2 as the causative gene; it has no previously known function but is a member of a gene family that is involved in granule development. Silencing of nbeal2 in zebrafish abrogated thrombocyte formation.

An inherited bleeding disorder linked to a defective interaction between ADP and its receptor on platelets. Its influence on glycoprotein IIb-IIIa complex function.

Much discussion has concerned the central role of ADP in platelet aggregation. We now describe a patient (M.L.) with an inherited bleeding disorder whose specific feature is that ADP induces a limited and rapidly reversible platelet aggregation even at high doses. Platelet shape change and other hemostatic parameters were unmodified. A receptor defect was indicated, for, while epinephrine normally lowered cAMP levels of PGE1-treated (M.L.) platelets, ADP was without effect. The binding of [3H]2-methylthio-ADP decreased from 836 +/- 126 molecules/platelet for normals to 30 +/- 17 molecules/platelet for the patient. Flow cytometry confirmed that ADP induced a much lower fibrinogen binding to (M.L.) platelets. Nonetheless, the binding in whole blood of activation-dependent monoclonal antibodies showed that some activation of GP IIb-IIIa complexes by ADP was occurring. Platelets of a patient with type I Glanzmanns thrombasthenia bound [3H]2-methylthio-ADP and responded normally to ADP in the presence of PGE1. Electron microscopy showed that ADP-induced aggregates of (M. L.) platelets were composed of loosely bound shape-changed platelets with few contact points. Thus this receptor defect has a direct influence on the capacity of platelets to bind to each other in response to ADP.

Glanzmann thrombasthenia: a review of ITGA2B and ITGB3 defects with emphasis on variants, phenotypic variability, and mouse models

Characterized by mucocutaneous bleeding arising from a lack of platelet aggregation to physiologic stimuli, Glanzmann thrombasthenia (GT) is the archetype-inherited disorder of platelets. Transmitted by autosomal recessive inheritance, platelets in GT have quantitative or qualitative deficiencies of the fibrinogen receptor, αIIbβ3, an integrin coded by the ITGA2B and ITGB3 genes. Despite advances in our understanding of the disease, extensive phenotypic variability with respect to severity and intensity of bleeding remains poorly understood. Importantly, genetic defects of ITGB3 also potentially affect other tissues, for β3 has a wide tissue distribution when present as αvβ3 (the vitronectin receptor). We now look at the repertoire of ITGA2B and ITGB3 gene defects, reexamine the relationship between phenotype and genotype, and review integrin structure in the many variant forms. Evidence for modifications in platelet production is assessed, as is the multifactorial etiology of the clinical expression of the disease. Reports of cardiovascular disease and deep vein thrombosis, cancer, brain disease, bone disorders, and pregnancy defects in GT are discussed in the context of the results obtained for mouse models where nonhemostatic defects of β3-deficiency or nonfunction are being increasingly described.

Megakaryocytes possess a functional intrinsic apoptosis pathway that must be restrained to survive and produce platelets

Deletion of Bak and Bax, the effectors of mitochondrial apoptosis, does not affect platelet production, however, loss of prosurvival Bcl-xL results in megakaryocyte apoptosis and failure of platelet shedding.

A prototypic platelet septin and its participation in secretion

Studies are presented characterizing platelet CDCrel-1, a protein expressed to high levels by megakaryocytes and belonging to a family of conserved proteins, termed septin. Septin filaments originally were identified in yeast as essential for budding but have become increasingly associated with processes in higher eukaryotic cells involving active membrane movement such as cytokinesis and vesicle trafficking. Direct proof of an in vivo function for septins in higher eukaryotes is limited to the characterization of the Drosophila septin, termed PNUT. We present studies identifying platelet CDCrel-1 as a protein kinase substrate in the presence of known platelet agonists. The immunopurification of CDCrel-1 revealed it to be part of a macromolecular complex containing a protein involved in platelet secretion, syntaxin 4. Moreover, CDCrel-1 was localized in situ to areas surrounding platelet-storage granules. The relevance of CDCrel-1 to normal platelet function was established with the characterization of platelets from a CDCrel-1Null mouse. As compared with platelets from wild-type littermates, CDCrel-1Null platelets aggregate and release stored [14C]serotonin in the presence of subthreshold levels of collagen. These results provide new insights into the mechanisms regulating platelet secretion and identify platelet septins as a protein family contributing to membrane trafficking within the megakaryocyte and platelet.

Thrombocytopenia resulting from mutations in filamin A can be expressed as an isolated syndrome

Filaminopathies A caused by mutations in the X-linked FLNA gene are responsible for a wide spectrum of rare diseases including 2 main phenotypes, the X-linked dominant form of periventricular nodular heterotopia (FLNA-PVNH) and the otopalatodigital syndrome spectrum of disorders. In platelets, filamin A (FLNa) tethers the principal receptors ensuring the platelet-vessel wall interaction, glycoprotein Ibα and integrin αIIbβ3, to the underlying cytoskeleton. Hemorrhage, coagulopathy, and thrombocytopenia are mentioned in several reports on patients with FLNA-PVNH. Abnormal platelet morphology in 2 patients with FLNA-PVNH prompted us to examine a third patient with similar platelet morphology previously diagnosed with immunologic thrombocytopenic purpura. Her enlarged platelets showed signs of FLNa degradation in Western blotting, and a heterozygous missense mutation in FLNA was detected. An irregular distribution of FLNa within the total platelet population was shown by confocal microscopy for all 3 patients. In vitro megakaryocyte cultures showed an abnormal differentiation, including an irregular distribution of FLNa with a frayed aspect, the presence of enlarged α-granules, and an abnormal fragmentation of the cytoplasm. Mutations in FLNA may represent an unrecognized cause of macrothrombocytopenia with an altered platelet production and a modified platelet-vessel wall interaction.

Clopidogrel: a review of its mechanism of action

The search for active antiplatelet drugs within the original chemical class of the thienopyridines, led to the discovery of clopidogrel, a novel ADP-selective agent whose antiaggregating properties are several times higher than those of ticlopidine. The antiaggregating properties of this compound are well known and, very recently, new results have clarified its mechanism of action. Clopidogrel is active only after intravenous or oral administration, and no circulating activity has been found in the plasma of treated animals or human volunteers. Experiments in rats have demonstrated that the antiaggregating activity was caused by a shortlasting metabolite generated in the liver by a cytochrome P450-dependent pathway. The antiaggregating property of clopidogrel is caused by an inhibition of the binding of ADP to its platelet receptors, and more specifically to the low affinity receptors, the high affinity binding sites being unaffected by clopidogrel. Several events in the ADP activation process, including adenylyl cyclase down-regulation, protein tyrosine phosphorylation, activation of the GPIIb-IIIa complex, fibrinogen binding, aggregation and release, were inhibited by clopidogrel and indicate their close relationship with the activation of a low affinity receptor by ADP. In contrast, binding of ADP to its high affinity binding sites (clopidogrel-resistant receptors) induced shape change, cytosolic calcium increase and phosphorylations of several other proteins, some events which were clopidogrel-sensitive. Thus, clopidogrel not only constitutes a potent antithrombotic drug in humans but also a good tool to study the effect of ADP on platelets.