The exon-junction complex (EJC) performs essential RNA processing tasks1-5. Here, we describe the first human disorder, Thrombocytopenia with Absent Radii6 (TAR), caused by deficiency in one of the four EJC subunits. A compound inheritance mechanism 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 mechanism explained 53 of 55 cases (P<5×10−228) with the rare congenital malformation syndrome. Fifty-one of those 53 carried a previously associated7 submicroscopic deletion of 1q21.1; two carried a truncation or frameshift null mutation in RBM8A. We show that the two regulatory SNPs result in reduction of RBM8A transcription in vitro and that Y14 expression is reduced in platelets from TAR cases. Our data implicate Y14 insufficiency, and presumably EJC defect, as the cause of TAR syndrome.

SummaryHematopoietic differentiation critically depends on combinations of transcriptional regulators controlling the development of individual lineages. Here, we report the genome-wide binding sites for the five key hematopoietic transcription factors—GATA1, GATA2, RUNX1, FLI1, and TAL1/SCL—in primary human megakaryocytes. Statistical analysis of the 17,263 regions bound by at least one factor demonstrated that simultaneous binding by all five factors was the most enriched pattern and often occurred near known hematopoietic regulators. Eight genes not previously appreciated to function in hematopoiesis that were bound by all five factors were shown to be essential for thrombocyte and/or erythroid development in zebrafish. Moreover, one of these genes encoding the PDZK1IP1 protein shared transcriptional enhancer elements with the blood stem cell regulator TAL1/SCL. Multifactor ChIP-Seq analysis in primary human cells coupled with a high-throughput in vivo perturbation screen therefore offers a powerful strategy to identify essential regulators of complex mammalian differentiation processes.

The collagen type I-derived fragment ␣ 1 (I)CB3 is known to recognize the platelet collagen receptor integrin ␣ 2 ␤ 1 as effectively as the parent collagen, although it lacks platelet-aggregatory activity. We have synthesized the fragment as seven overlapping peptides that spontaneously assemble into triple helices. On the basis of their capacity to bind purified ␣ 2 ␤ 1 and the recombinant ␣ 2 A-domain, and their ability to support ␣ 2 ␤ 1 -mediated cell adhesion, we identified two peptides, CB3(I)-5 and -6, which contain an ␣ 2 ␤ 1 recognition site. Synthesis of the peptide CB3(I)-5/6, containing the overlap sequence between peptides 5 and 6, allowed us to locate the binding site within the 15-residue sequence, GFP*GERGVEGPP*GPA (where P* represents hydroxyproline), corresponding to residues 502-516 of the collagen type I ␣ 1 chain. The Glu and Arg residues in the GER triplet were found to be essential for recognition since substitution of either residue with Ala caused a loss of ␣ 2 A-domain binding. By contrast, substitution of the Glu in GVE did not reduce binding, but rather enhanced it slightly. We were unable to detect significant recognition of ␣ 2 ␤ 1 by the peptide CB3(I)-2 containing the putative ␣ 2 ␤ 1 recognition sequence DGEA. Peptides CB3(I)-1 to -6, together with peptide CB3(I)-5/6, exhibited good platelet-aggregatory activity, in some cases better than collagen. However, peptide CB3(I)-7 was inactive, suggesting the presence of an inhibitory element that might account for the lack of aggregatory activity of the parent ␣ 1 (I)CB3 fragment.Integrins that recognize collagen can modulate cell behavior, including adhesion and spreading, migration, division, metabolism, and the expression of the differentiated phenotype. These important processes are physiologically relevant to growth and development, wound repair, and angiogenesis and in pathological processes such as thrombosis and tumor metastasis.Integrin ␣ 2 ␤ 1 is also an important collagen receptor in hemostasis, where it plays an essential role in the arrest of platelets, under conditions of blood flow, on the collagen fiber surface exposed as a consequence of injury (1-6). Subsequent recognition by the platelet receptor Gp 1 VI of GPP* 2 sequences within the collagen triple helix (7) leads to platelet activation and aggregation with formation of a platelet plug, which serves to stem the loss of blood. Activation of platelets by collagen may also be a cause of thrombosis, especially that associated with rupture of the atherosclerotic plaque, which leads to exposure of underlying collagens (8).Previous fragmentation studies (9) have indicated the presence of a number of integrin ␣ 2 ␤ 1 recognition sites in collagen I, which, with collagen III, represents the main platelet-aggregatory collagen species in the vessel wall and perivascular space (8). In particular, ␣ 2 ␤ 1 -mediated platelet adhesion to fragment ␣ 1 (I)CB3 derived from the ␣ 1 (I) chain of collagen I is as good as to the parent collagen (9, 10). Inhibition studies with short lin...

The platelet glycoproteins (GPs) Ib, integrin ␣ 2 ␤ 1 , and GPVI are considered central to thrombus formation. Recently, their relative importance has been re-evaluated based on data from murine knockout models. To examine their relationship during human thrombus formation on collagen type I fibers at high shear (1000 s ؊1 ), we tested a novel antibody against GPVI, an immunoglobulin single-chain variable fragment, 10B12, together with specific antagonists for GPIb␣ (12G1 Fab 2 ) and ␣ 2 ␤ 1 (6F1 mAb or GFOGER-GPP peptide).GPVI was found to be crucial for aggregate formation, Ca 2؉ signaling, and phosphatidylserine (PS) exposure, but not for primary adhesion, even with more than 97% receptor blockade. Inhibiting ␣ 2 ␤ 1 revealed its involvement in regulating Ca 2؉ signaling, PS exposure, and aggregate size. Both GPIb␣ and ␣ 2 ␤ 1 contributed to primary adhesion, showing overlapping function. The coinhibition of receptors revealed synergism in thrombus formation: the coinhibition of adenosine diphosphate (ADP) receptors with collagen receptors further decreased adhesion and aggregation, and, crucially, the complete eradication of thrombus formation required the coinhibition of GPVI with either GPIb␣ or ␣ 2 ␤ 1 . In summary, human platelet deposition on collagen depends on the concerted interplay of several receptors: GPIb in synergy with ␣ 2 ␤ 1 mediating primary adhesion, reinforced by activation through GPVI, which further regulates the thrombus formation. IntroductionThe platelet response to exposed subendothelial matrix is fundamental to thrombosis and hemostasis. Uniquely, collagen, the most abundant vessel wall protein, mediates platelet adhesion and activation, localizing and regulating the hemostatic response at sites of injury. Discovering the molecular mechanisms that control platelet-collagen interaction is crucial for understanding the pathogenesis of arteriothrombotic diseases such as stroke and myocardial infarction. Under high shear rate conditions, the glycoprotein (GP) Ib/V/IX complex allows initial platelet rolling over von Willebrand factor (VWF) bound to subendothelial collagen fibers, and subsequently collagen receptors come into contact with their specific binding sequences in the collagen. For the next step, platelet arrest and activation, firm evidence exists of a role for only 2 receptors, integrin ␣ 2 ␤ 1 and immunoglobulin superfamily member GPVI, despite the apparent redundancy in collagen receptors (for a review, see Siljander PR-M and Farndale RW 1 ).According to the 2-site, 2-step model, high-affinity interaction through ␣ 2 ␤ 1 stops the platelet, allowing low-affinity binding of GPVI, which generates signaling required for the subsequent thrombus formation. Platelet deposition under flow was found to be dependent on GPIb/V/IX and ␣ 2 ␤ 1 , 2-4 whereas no platelet deposition occurred on the GPVI-specific substrate collagen-related peptide (CRP), even under low shear rates. 5 The limited number of studies with human platelets deficient in either GPVI or ␣ 2 ␤ 1 support the 2-site, ...

Gray platelet syndrome (GPS) is a predominantly recessive platelet disorder characterized by a 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 cases and identified as the causative gene NBEAL2, a gene with previously unknown function but a member of a gene family involved in granule development. Silencing of nbeal2 in zebrafish abrogated thrombocyte formation.

Only three recognition motifs, GFOGER, GLOGER, and GASGER, all present in type I collagen, have been identified to date for collagen-binding integrins, such as ␣ 2 ␤ 1 . Sequence alignment was used to investigate the occurrence of related motifs in other human fibrillar collagens, and located a conserved array of novel GER motifs within their triple helical domains. We compared the integrin binding properties of synthetic triple helical peptides containing examples of such sequences (GLSGER, GMOGER, GAOGER, and GQRGER) or the previously identified motifs. Recombinant inserted (I) domains of integrin subunits ␣ 1 , ␣ 2 , and ␣ 11 bound poorly to all motifs other than GFOGER and GLOGER. Similarly, ␣ 2 ␤ 1 -containing resting platelets adhered well only to GFOGER and GLOGER, while ADP-activated platelets, HT1080 cells and two active ␣ 2 I domain mutants (E318W, locked open) bound all motifs well, indicating that affinity modulation determines the sequence selectivity of integrins. GxO/SGER peptides inhibited platelet adhesion to collagen monomers with order of potency F > L > M > A. These results establish GFOGER as a high affinity sequence, which can interact with the ␣ 2 I domain in the absence of activation and suggest that integrin reactivity of collagens may be predicted from their GER content.Collagen, the most abundant structural protein of the vertebrate organism, currently has 27 reported family members (1). As either a mechanical support or as a bioactive surface, collagen plays a crucial role in processes as diverse as morphogenesis, wound repair, inflammation, tumor metastasis, hemostasis, and thrombosis. The tensile strength of the fibrillar collagens, types I-III, V, XI, and XXVII, is crucial to the function of connective tissues including the blood vessel wall. The non-fibrillar collagens, such as types IV and VI, provide a flexible support for endothelial and epithelial cell attachment and development. Integrins, heterodimeric adhesion molecules, form an important subgroup of receptors, which mediate interaction between collagen and cells. Four ␣-subunits, ␣ 1 , ␣ 2 , ␣ 10 , and ␣ 11 , which associate non-covalently with ␤ 1 , constitute the native collagen-binding integrin family (2).Integrin ␣ 2 ␤ 1 is a well characterized and widespread receptor for collagen, laminin, and other non-matrix ligands among nucleated cells including epithelial and endothelial cells, smooth muscle cells, fibroblasts, leukocytes, and mast cells (3, 4), mediating a wide range of cellular activities. ␣ 2 ␤ 1 is the only collagen binding integrin in platelets, and is crucial for deposition on collagens exposed in damaged arterial walls (5, 6). Accordingly, the expression levels of ␣ 2 ␤ 1 have been associated with myocardial infarction and stroke (7). Recently, knockout studies suggested that the platelet activatory collagen receptor glycoprotein VI (GPVI) is mandatory for the initiation of integrin-mediated adhesion (8), but this concept was challenged by further mouse studies (9). Moreover, in vitro studies suggest that ...

Key Points Developed a targeted sequencing platform covering 63 genes linked to heritable bleeding, thrombotic, and platelet disorders. The ThromboGenomics platform provides a sensitive genetic test to obtain molecular diagnoses in patients with a suspected etiology.

Glycoprotein (GP) VI is the major receptor responsible for platelet activation by collagen, but the collagen-binding surface of GPVI is unknown. To address this issue we expressed, from insect cells, the immunoglobulin (Ig)-like ectodomains (residues 1-185) of human and murine GPVI, called hD1D2 and mD1D2, respectively. Both proteins bound specifically to collagen-related peptide (CRP), a GPVIspecific ligand, but hD1D2 bound CRP more strongly than did mD1D2. Molecular modeling and sequence comparison identified key differences between hD1D2 and mD1D2. Ten mutant hD1D2s were expressed, of which 4 had human residues replaced by their murine counterpart, and 6 had replacements by alanine. CRP binding studies with these mutants demonstrated that the exchange of lysine at position 59 for the corresponding murine glutamate substantially reduced binding to CRP. The position of lysine59 on the apical surface of GPVI suggests a mode of CRP binding analogous to that used by the related killer cell Ig-like receptors to bind HLA. This surface was confirmed as critical for collagen binding by epitope mapping of an inhibitory phage antibody against GPVI. This anti-GPVI, clone 10B12, gave dose-dependent inhibition of the hD1D2-collagen interaction. Clone 10B12 inhibited activation of platelets by CRP and collagen in aggregometry and thrombus formation by the latter in whole blood perfusion. Antibody 10B12 showed significantly reduced binding to the hD1D2-E59, and, on that basis, the GPVI:10B12 interface was modeled. IntroductionDamage to blood vessels exposes circulating platelets to the extracellular matrix. Here, collagen supports adhesion and stimulates platelet activation by acting as a ligand for a number of platelet receptors. 1 Platelets are first tethered by the interaction of glycoprotein (GP) Ib␣ with the A1 domain of von Willebrand factor (VWF), 2 a plasma protein that binds to exposed collagen. 3 Firm platelet adhesion results from the concerted action of the collagen receptor GPIaIIa (integrin ␣2␤1) and the fibrinogen receptor GPIIbIIIa (␣IIb␤3) which also binds immobilized VWF. 4 Collagen-mediated activation of platelets is dependent on the engagement and clustering of GPVI, 1,5 an immunoglobulin (Ig) superfamily member with homology to killer cell Ig-like receptors (KIRs) and the Fc␣RI. 6 We and others have described the ligandbinding sites of ␣2 integrin 7 and GPIb␣ 2 at the structural level.Recent work has demonstrated the key role played by GPVI in arterial thrombus formation in mice and provides a clear basis for the development of potentially therapeutic GPVI inhibitors. 8 Indeed, blockade of GPVI is attractive for several reasons. First, the expression of GPVI appears to be restricted to platelets and megakaryocytes. 9 Second, collagen is required for prothrombotic "COAT" platelet formation, 10 for which blockade of GPVI may provide a specific control point. Third, patients with congenital or acquired autoantibody-mediated GPVI deficiency have only a mild bleeding disorder, despite having a significantly ...