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Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant vascular dysplasia. Care delivery for HHT patients is impeded by the need for laborious, repeated phenotyping and gaps in knowledge regarding the relationships between causal DNA variants in ENG, ACVRL1, SMAD4 and GDF2, and clinical manifestations. To address this, we analyzed DNA samples from 183 previously uncharacterized, unrelated HHT and suspected HHT cases using the ThromboGenomics high-throughput sequencing platform. We identified 127 rare variants across 168 heterozygous genotypes. Applying modified American College of Medical Genetics and Genomics Guidelines, 106 variants were classified as pathogenic/likely pathogenic and 21 as nonpathogenic (variant of uncertain significance/benign). Unlike the protein products of ACVRL1 and SMAD4, the extracellular ENG amino acids are not strongly conserved. Our inferences of the functional consequences of causal variants in ENG were therefore informed by the crystal structure of endoglin. We then compared the accuracy of predictions of the causal gene blinded to the genetic data using 2 approaches: subjective clinical predictions and statistical predictions based on 8 Human Phenotype Ontology terms. Both approaches had some predictive power, but they were insufficiently accurate to be used clinically, without genetic testing. The distributions of red cell indices differed by causal gene but not sufficiently for clinical use in isolation from genetic data. We conclude that parallel sequencing of the 4 known HHT genes, multidisciplinary team review of variant calls in the context of detailed clinical information, and statistical and structural modeling improve the prognostication and treatment of HHT.

GP1BA encodes the glycoprotein (GP) 1bα subunit of the GP1b/IX/V complex, the platelet receptor for von Willebrand factor (VWF) and a mediator of outside-in signalling that contributes to platelet activation. Biallelic variants in GPIBA that prevent surface expression of GPIB/IX/V cause the recessive disorder Bernard Soulier Syndrome (BSS; OMIM #23100) in which there is often severe bleeding caused by thrombocytopenia and platelet dysfunction. Thrombocytopenia has also been associated with monoallelic missense variants in the GP1bα R loop (residues 227-242) which enhance VWF binding (platelet-type von Willebrand disease (pVWD); OMIM #177820). Other monoallelic GPIBA missense variants such as the Bolzano variant (p.Ala172Val; OMIM #153670) have been associated with dominant thrombocytopenia without altered VWF levels. In order to better describe the relationship between monoallelic GPIBA variants and thrombocytopenia we evaluated data from the NIHR BioResource for Rare Diseases, (NBR-RD) and the Inherited Platelet Disorders programmes at the Universities of Birmingham (UK), Murcia (Spain) and Toronto (Canada) in which patients with unexplained platelet disorders underwent diagnostic next generation sequencing. Using the BeviMed method for genetic association we compared the genotypes at rare nonsynoymous variants of 105 unrelated thrombocytopenia cases (platelet count (PLT) 15. These were present in 29 index cases (18 females, median age 37 years) with unexplained thrombocytopenia and included the variant predicting the p.Asn150Ser substitution which was identified in seven independent index cases. The variants comprised 18 missense, two inframe insertions (one with an additional missense change) and one insertion resulting in a frame shift. Seven had been previously associated with thrombocytopenia. Sixteen of the variants predicted amino acid substitutions or single residue insertions within the GP1bα leucine rich repeat region (LRR; residues 48-200) or in adjacent regions of the LRR N- and C-terminal caps. Cases with variants in the LRR region typically displayed mild mucocutaneous bleeding, abnormal bleeding after minor trauma or surgery and heavy menstrual bleeding. The platelet count was typically 60-100 x109/L and mean platelet volume greater than 11 fL. There were two missense variants in the GP1bα R loop that have been previously associated with pVWD and two further previously unreported missense variants in the cytoplasmic domain. The final variant was a monoallelic single nucleotide insertion predicted to cause a frame shift and absent expression of GP1bα and has been previously reported as a biallelic variant associated with BSS. These data indicate that in a multicentre cross-sectional cohort of cases with otherwise unexplained thrombocytopenia, monoallelic missense GPIBA variants affecting the LRR or adjacent regions were more prevalent than R loop variants associated with pVWD or variants causing absent GP1bα expression associated with biallelic BSS. The LRR region contains the GP1bα-VWF binding site which is predicted to be disrupted by the missense variants observed in this study. The LRR region is also the location of the GP1bα Bolzano variant and six other monoallelic variants previously associated with thrombocytopenia in previous single case reports. These observations extend the repertoire of GPIBA variants associated with thrombocytopenia and although requiring experimental confirmation of pathogenicity, suggest a common molecular pathogenesis for dominant GPIBA-associated thrombocytopenia in which reduced circulating platelet numbers arises from defective GP1bα-VWF interactions. Figure Disclosures Turro: Tachyon Ventures: Other: Partner & Scientific Director.

Thrombomodulin-associated coagulopathy (TM-AC) is a newly recognized dominant bleeding disorder in which a p.Cys537Stop variant in the thrombomodulin (TM) gene THBD, results in high plasma TM levels and protein C-mediated suppression of thrombin generation. Thrombin in complex with TM also activates thrombin-activatable fibrinolysis inhibitor (TAFI). However, the effect of the high plasma TM on fibrinolysis in TM-AC is unknown. Plasma from TM-AC cases and high-TM model control samples spiked with recombinant soluble TM showed reduced tissue factor-induced thrombin generation. Lysis of plasma clots from TM-AC cases was significantly delayed compared with controls but was completely restored when TM/thrombin-mediated TAFI activation was inhibited. Clots formed in blood from TM-AC cases had the same viscoelastic strength as controls but also showed a TAFI-dependent delay in fibrinolysis. Delayed fibrinolysis was reproduced in high-TM model plasma and blood samples. Partial restoration of thrombin generation with recombinant activated factor VII or activated prothrombin complex concentrate did not alter the delayed fibrinolysis in high-TM model blood. Our finding of a previously unrecognized fibrinolytic phenotype indicates that bleeding in TM-AC has a complex pathogenesis and highlights the pivotal role of TM as a regulator of hemostasis.

The ephrin transmembrane receptor family of tyrosine kinases is involved in platelet function. We report the first EPHB2 variant affecting platelets in 2 siblings (P1 and P2) from a consanguineous family with recurrent bleeding and normal platelet counts. Whole-exome sequencing identified a c.2233C>T variant (missense p.R745C) of the EPHB2 gene. P1 and P2 were homozygous for this variant, while their asymptomatic parents were heterozygous. The p.R745C variant within the tyrosine kinase domain was associated with defects in platelet aggregation, αIIbβ3 activation, and granule secretion induced by G-protein–coupled receptor (GPCR) agonists and convulxin, as well as in thrombus formation on collagen under flow. In contrast, clot retraction, flow-dependent platelet adhesion, and spreading on fibrinogen were only mildly affected, indicating limited effects on αIIbβ3 outside-in signaling. Most importantly, Lyn, Syk, and FcRγ phosphorylation, the initial steps in glycoprotein VI (GPVI) platelet signaling were drastically impaired in the absence of platelet–platelet contact, indicating a positive role for EPHB2 in GPVI activation. Likewise platelet activation by PAR4-AP showed defective Src activation, as opposed to normal protein kinase C activity and Ca2+ mobilization. Overexpression of wild-type and R745C EPHB2 variant in RBL-2H3 (rat basophilic leukemia) cells stably expressing human GPVI confirmed that EPHB2 R745C mutation impaired EPHB2 autophosphorylation but had no effect on ephrin ligand-induced EPHB2 clustering, suggesting it did not interfere with EPHB2-ephrin–mediated cell-to-cell contact. In conclusion, this novel inherited platelet disorder affecting EPHB2 demonstrates this tyrosine kinase receptor plays an important role in platelet function through crosstalk with GPVI and GPCR signaling.

TO THE EDITOR: We have identified 9 affected individuals from 3 unrelated families with macrothrombocytopenia and mild to moderate bleeding diathesis inherited due to missense mutations in the glucosamine (UDP- N -acetyl)-2-epimerase/ N -acetylmannosamine kinase ( GNE ) gene. Previous reports have

The von Willebrand receptor complex, which is composed of the glycoproteins Ibα, Ibβ, GPV, and GPIX, plays an essential role in the earliest steps in hemostasis. During the last 4 decades, it has become apparent that loss of function of any 1 of 3 of the genes encoding these glycoproteins (namely, GP1BA, GP1BB, and GP9) leads to autosomal recessive macrothrombocytopenia complicated by bleeding. A small number of variants in GP1BA have been reported to cause a milder and dominant form of macrothrombocytopenia, but only 2 tentative reports exist of such a variant in GP1BB. By analyzing data from a collection of more than 1000 genome-sequenced patients with a rare bleeding and/or platelet disorder, we have identified a significant association between rare monoallelic variants in GP1BB and macrothrombocytopenia. To strengthen our findings, we sought further cases in 2 additional collections in the United Kingdom and Japan. Across 18 families exhibiting phenotypes consistent with autosomal dominant inheritance of macrothrombocytopenia, we report on 27 affected cases carrying 1 of 9 rare variants in GP1BB.

Inherited defects of platelet function disorders are rare and difficult to diagnose due to lack of standardized platelet tests. An aspirin-like platelet defect is characterised by reduced thromboxane A2 (TXA2) signalling due to a defect in the arachidonic acid (AA) pathway in platelets. Patients with aspirin-like defect present with mild to moderate bleeding symptoms and impaired platelet aggregation responses to AA and ADP. This is similar to the irreversible effect of aspirin on platelets, which is mediated through inhibition of prostaglandin H synthase-1 also known as cyclooxygenase-1 (PTGS1/COX1). We for the first time report platelet function disorders due to autosomal recessive inheritance of variants in PTGS1. In a total of 3563 cases with bleeding disorders, comprising 1169 whole genome sequenced probands of the BRIDGE-BPD study and 2394 panel sequenced index cases of the ThromboGenomics cohort, we identified 15 unrelated cases of each cohort with an aspirin-like platelet function defect. Two of these cases had rare a variants in PTGS1, the gene encoding COX-1, which catalyses the conversion of arachidonic acid to prostaglandin H2. The first case presented with epistaxis and peri-operative bleeding. She had reduced platelet aggregation responses to arachidonic acid, ADP, collagen and epinephrine. Incubation of control blood with collagen resulted in enhanced levels of thromboxane B2, PGD2, PGE2, 11-HETE and 15-HETE which was absent in the index case.We identified a homozygous missense variant in PTGS1, p.Trp322Ser with a Combined Annotation Dependant Depletion Score (CADD) of 31.0. This variant was absent from GnomAD. The variant co-segregated in an autosomal recessive inheritance mode, with aspirin-like defect phenotype in the seven family members who were investigated. PTGS1 was not expressed on the platelets by western blot. Immunophenotyping demonstrated absence on the platelet surface but presence on neutrophils. The second case of the presented with menorrhagia, nosebleeds, easy bleeding and bruising. She had reduced aggregation responses to arachidonic acid, ADP, collagen and epinephrine. We identified two variants in cis: a splice-donor variant (g. 125133553 T>A), CADD 24.3; and an upstream non-coding variant (g. 125132069 C>G), CADD 16.63. The frequency of these variants were respectively; 1.7 x 10-5 and 8 x 10-3 in GnomAD. Platelet RNA and protein expression studies in the propositus revealed alternative splicing with the generation of a smaller protein due the splice variant. In contrast, the non coding variant had no effect on promoter or enhancer activity and therefore, is likely benign. In this case, the mode of inheritance is autosomal dominant with a dominant negative effect, which has been reported previously. For the other 13 cases of the Bridge-BPD study we also interrogated the non-coding space and interactors in the arachidonic acid pathway, none of which had genetic variants explaining the phenotype. For the 15 ThromboGenomics cohort cases because they were sequenced on targeted platform similar investigations could not take place. These cases could have a non inherited cause for the platelet defect or it is also permissible that variation in a hitherto undefined pathways unique to individual cases might be causal. In conclusion, we for the first time report autosomal recessive inheritance of variants in PTGS1 as cause for a rare inherited bleeding disorder. The effect of the mutation are selective loss of expression of PTGS1 within platelets and decreased enzyme function. Two previous reports demonstrated autosomal dominant inheritance. The first demonstrated autosomal dominant inheritance of variants in PTGS1 as modifier in a well characterized family with haemophilia A and platelet function disorder (Nance et al JTH 2016). The second reported rare heterozygous variants in PTGS1 in two cases with a bleeding tendency which was not further specified in the report (Bastida et al Haematologica 2018). Disclosures Laffan: Pfizer: Honoraria; Roche: Consultancy, Speakers Bureau.

Inherited bleeding, thrombotic and platelet disorders (BPDs) affect approximately 3M people worldwide and an appreciable portion have a disorder of megakaryopoiesis and the production and function of platelets, including the formation of granules. While genetic variants in 76 genes have been implicated in BPDs, many patients remain without a molecular diagnosis (Lentaigne et al, Simeoni et al). We hypothesised that some of these disorders may be caused by compound inheritance of variants in two different genes, a mode of inheritance thus far never implicated in BPDs. For the pilot phase of the 100 000 Genomes Project we have sequenced the whole genomes of 10,000 individuals consisting of probands with molecularly unexplained rare disorders and their close relatives, with 3,000 having inherited disorders of the blood and immune system. We searched for causal variants in known BPD-related genes and employed a new statistical method for Bayesian evaluation of rare variants in Mendelian disease (BeviMed) (Greene et al) to identify novel marginal associations between rare variants and disease status. Where the identified variants could not individually explain the phenotype in full within the pedigrees, we searched for additional variants affecting other BPD-related genes or novel genes identified using BeviMed. First, we have identified a large pedigree in which certain members with mild thrombocytopenia (lower 10th percentile of the population distribution) are affected by a single variant encoding a premature stop at residue 69 in the major isoform of the tropomyosin gene TPM4 expressed in megakaryocytes (Pleines et al). Other members of the family have distinctly more severe macrothrombocytopenia (with platelet counts as low as 24x109/l) and this is due to inheritance of a second variant in the actinin gene ACTN1 leading to a Thr340Met substitution, demonstrating a compound additive effect of variants in two genes encoding cytoskeletal proteins important for actin polymerization and thereby causing inadequate platelet formation. It is noteworthy that both of these genes have been identified in a genome wide association study of the count and mean volume of platelets (Gieger et al). Second, we used BeviMed to identify genes containing variants that are marginally associated with a syndrome defined by a platelet granule defect combined with familial autism (Bijl et al). The strongest association is due to splice variants in a granule-related gene on Chr17p13 but in all four unrelated cases at least one additional variant is required to explain the observed segregation patterns. Two of these unrelated cases harbor variants in two other marginally associated genes at Chr9q32 and Chr4q31 and a third harbors a de novo copy number variant. These additional variants likely explain why the children in three of the families are affected by this syndrome while their parents are not. In conclusion, we have used whole genome sequencing, pedigree building, detailed platelet phenotyping and new association approaches to identify the first cases of digenic inheritance of BPDs. Our results illustrate the cooperative role of different cytoskeletal proteins in platelet formation and cement the role of granule biology in the function of both platelets and neurons. References: Lentaigne et al (2016) Inherited platelet disorders: towards DNA-based diagnosis. Blood127(23) 2814-2823.Simeoni et al (2016) A high-throughput sequencing test for diagnosing inherited bleeding, thrombotic, and platelet disorders. Blood127(23) 2793-2803.Greene et al (2016) Bayesian evaluation of variant involvement in Mendelian disease. http://cran.r-project.org/web/packages/BeviMed.Pleines et al (2016) Tropomyosins regulate platelet biogenesis. (Under submission).Gieger et al (2011) New gene functions in megakaryopoiesis and platelet formation. Nature480(7376) 201-208Bijl et al (2015) Platelet studies in autism spectrum disorder patients and first-degree relatives. Molecular Autism6:57. Disclosures No relevant conflicts of interest to declare.

For the BRIDGE-Bleeding and Platelet Disorder Consortium. We here present a pedigree with nine cases that present with a new syndrome characterized by early onset myelofibrosis, thrombocytopenia, bleeding and bone pathologies. The syndrome is caused by a dominant gain-of-function mutation in universal tyrosine kinase SRC and it is the first example of a pathogenic germline mutation in this extensively studied kinase. The genetic discovery has been made possible by making use of our collection of 1,115 patients with inherited bleeding and platelet disorders of unknown molecular etiology enrolled by us at 25 tertiary referral centers as part of the NIHR BioResource - Rare Diseases pilot phase of the 100,000 Genomes Project. The analysis of genome sequencing data for 2 affected individuals was empowered by comparison of Human Phenotype Ontology (HPO; Westbury et al., Genome Medicine, 2015) terms of the patients with OMIM and the Mouse Phenotype Ontology terms from 7,541 strains. The selection of the SRC gene as the most likely candidate gene was also informed by the use of our Blueprint blood cell progenitor RNA-seq data (Chen et al., Science 2014). Modeling of the E527K substitution predicts loss of SRC’s self-inhibitory capacity, which we confirmed with in vitro studies showing increased SRC kinase activity and enhanced Tyr419 phosphorylation in COS7 cells overexpressing E527K SRC. The active form of SRC predominates in patients’ platelets, resulting in enhanced overall tyrosine phosphorylation and reduced platelet activity. Patients with myelofibrosis have a hypercellular bone marrow with trilineage dysplasia and their stem cells grown in vitro form more myeloid and megakaryocyte (MK) colonies than control cells. Patient MKs generate platelets which are dysmorphic, low in number, highly variable in size and with a paucity of α-granules. Over-active SRC in patient-derived MKs causes a reduction in proplatelet formation, which can be rescued by SRC kinase inhibition. Stem cells transduced with lentiviral E527K SRC form MKs with enhanced tyrosine phosphorylation levels and with reduced ploidy compared to cells transduced with wild-type SRC. Patient-derived and E527K-transduced MKs show Y419 SRC positive stained podosomes that induce a strongly altered organization of filamentous actin. Expression of mutated src in zebrafish recapitulates patients’ blood and bone phenotypes. Our findings reveal an unexpected and wide-ranging pathobiology for this universal kinase Src with podosome formation. In hematopoiesis constitutive active Src primarily affects megakaryopoiesis with ineffective formation of platelets. Its inadequate function is associated with bleeding. The kinase Src is activated frequently in human cancers hence new SFK inhibitors are undergoing clinical trials and side effects with severe bleeding are observed frequently. It remains to be determined whether this new class of SFK inhibitors inadvertently causes inadequate formation of platelets, which do not function properly. Disclosures: No relevant conflicts of interest to declare.

Authors contributed equally to this work. ~ Currently at Genomics England Ltd, London, United Kingdom Next generation sequencing is transforming our understanding of human genetic variation and is becoming a routine part of human genetic analysis. The identification of millions of new variants, which are mainly rare and assessing their implications for human health presents new challenges to researchers and clinicians. We have analyzed missense variants in the ITGB2A and ITGB3 genes obtained from whole exome and whole genome sequencing (WES & WGS) data from 5 databases: The Human Genome Mutation Database, the 1000 Genomes project, the UK10K Whole Exome Sequencing project, the UK10K Whole Genome Sequencing project, and The National Heart, Lung and Blood Institute Exome Sequencing Project. Together, these encompass variants of the platelet αIIbβ3 integrin receptor from ~32,000 alleles derived from 16,108 individuals. We identified 111 missense variants that have previously been associated with Glanzmann thrombasthenia (GT), 20 variants associated with alloimmune thrombocytopenia, and 5 variants associated with aniso/macrothrombocytopenia. None of the GT variants were found in the last four databases, indicating that they have minor allele frequencies (MAF) less than ~0.01%, attesting to both their rarity and the likelihood that they entered the population within the last ~2,500 years. We also identified 114 novel missense variants in ITGB2A affecting ~11% of the amino acids and 68 novel missense variants in ITGB3 affecting ~9% of the amino acids. 96% of the novel variants had MAF To estimate the percentage of the 114 novel identified variants that are likely to be deleterious we used 3 different algorithms, CADD, Polyphen 2-HDVI, and SIFT. The algorithms showed moderate concordance in their rankings of the likelihood that a variant is deleterious. To compare their predictive powers, we performed receiver operating characteristic (ROC) analysis based on their ability to discriminate confirmed GT missense variants (positive controls) from alloantigens (negative controls); the area under the curve (AUC) values were 0.91, 0.88, and 0.90, respectively. At cutoff values that achieved greater than 95% sensitivity for each algorithm: 1) the specificity values were 75%, 65%, and 60%, and 2) the percentages of novel αIIb+β3 missense variants predicted to be deleterious were 43%, 56%, and 58%. Polyphen 2-HDVI and SIFT identified αIIb P176H and β3 C547G as highly likely to be deleterious and αIIb P943A as much less likely to be deleterious, whereas CADD did not differentiate them in the same way. We conclude that ~1.1% of individuals in the populations studied carry at least one missense variant in αIIb or β3 and that 0.6% carry a variant that might be deleterious and therefore may result in a hemorrhagic GT-like phenotype. The rarity of almost all of the novel missense variants identified indicates that they entered the population recently. Despite having detailed knowledge of the structure and function of αIIbβ3, it is difficult to predict with certainty the impact of any single missense variant. This will pose serious challenges as more individuals undergo WES and WGS; we anticipate that linkage to health record data, as will happen for the UK 100,000 Genomes project, will aid clinical interpretation. Finally, “hypomorphic” gene variants that produce only a partial decrease in expression, such as αIIb P943A, may contribute to the wide variation in αIIbβ3 surface expression observed in the healthy population. Disclosures No relevant conflicts of interest to declare.

A genome-wide scan in nearly 70,000 individuals showed that the common SNP rs8109288 in the first intron of the human TPM4 gene exerts an effect on the volume and count of platelets (Gieger et al. Nature 2011). We isolated a mouse line with an ENU-induced missense mutation in Tpm4. Mice carrying this mutation exhibited dose-dependent macrothrombocytopenia, while other blood cell counts were normal. Bone marrow transplant experiments demonstrated that the phenotype is intrinsic to hematopoietic cells. Notably, Tpm4 insufficiency did not affect the life span or in vitro function of mutant platelets, and there was no evidence of an increased propensity to bleeding. Megakaryocyte numbers in the bone marrow were increased, although maturation as measured by ploidy appeared normal. Mutant megakaryocytes displayed altered morphology indicating fragmentation, and markedly decreased proplatelet formation in vitro. Based on Gieger et al., we examined the functional requirement for TPM4 in human megakaryocytes. We found that the localisation of TPM4 in proplatelet-forming megakaryocytes was extremely similar to the localisation in their mouse counterparts, suggesting an identical role. Furthermore, knock down of TPM4by shRNA in human megakaryocytes did not affect maturation as measured by CD41 and CD42 expression, but significantly reduced the number of proplatelet-forming cells. The occasional megakaryocyte that did form proplatelets did not exhibit the typical “beads-on-a-string” phenotype. Typically, one large bulb at the end of a protrusion or a string with no clearly distinguishable beads was observed. We therefore performed a look-up in the BRIDGE consortium database, which enrolled 542 cases with inherited bleeding and platelet disorders of unknown aetiology in the NIHR BioResource for exome sequencing. Calling of variants revealed single nucleotide variants with consequences in TPM4which were absent from ~30,000 control haplotypes, in three BRIDGE cases. Two cases with a stop codon at residue 105 and R91H variant presented with macrothrombocytopenia and mild bleeding symptoms with platelet counts of 103 and 128 x10e9/L and volumes of 15.10 and 14.00 fl, respectively. The remaining case with variant D20N (not conserved, genomic evolutionary rate profiling score of 2.68 compared to 4.62 for the other variants) had a count in the normal range (232 x10e9/L) and a reduced platelet volume of 7.20 fl. Together, these results provide compelling evidence that Tropomyosin 4 is a crucial regulator of platelet production in mice and humans, being specifically required for the terminal stages of platelet formation. Our studies demonstrate that the common intronic variant exerts a subtle effect, whilst two extremely rare variants have a more robust effect on platelet formation leading to counts and volumes at the tails of the population distribution. The lack of concordance between mice and humans with regard to bleeding may be explained by strong modifiers at loci other than TPM4. Disclosures No relevant conflicts of interest to declare.

Background: Glanzmann thrombasthenia (GT) is a moderate to severe inherited bleeding syndrome associating a lack of platelet aggregation with quantitative or qualitative deficiencies of the αIIbβ3 integrin. Rare patients with mutations in ITGA2B or ITGB3 genes show a variable GT-like phenotype associated with macrothrombocytopenia (MTP). Gain-of-function mutations that localize to the integrin cytoplasmic domains cause autosomal dominant MTP. We now report a novel heterozygous ΔLeu in ITGB3 causing a structural modification of the cytoplasmic domain of β3 in a family with moderate MTP. In silico modeling was performed to obtain structural insights into this and other known mutations affecting cytoplasmic domains of αIIb and β3 and impacting megakaryopoiesis. Aim: To help define structural changes in αIIbβ3 linked to MTP. Results: A novel in-frame heterozygous 3bp deletion (c.2230_2232delCTC) was identified by exome sequencing performed as part of the BRIDGE-BPD consortium project in a French pedigree with lifelong mucocutaneous bleeding. The consequence is the loss of β3 Leu718 (ΔL718) the second L of a LLITIHD motif of the mature protein (nomenclature used throughout). Platelets of the proband have (i) moderate expression of αIIbβ3, (ii) a major reduction in fibrinogen (Fg) and PAC-1 binding after ADP stimulation yet (iii) spontaneous Fg or PAC-1 binding to platelets was not seen. Western blotting showed a slightly slower migration of β3 and normal αIIb. The proband had a moderate MTP with platelet size heterogeneity and frequent enlarged α-granules regrouping several of normal size with Fg bound to the membrane of the α-granules as revealed by immunogold staining and electron microscopy. Platelet aggregation was much reduced with all physiologic agonists including TRAP with a reduced surface expression of P-selectin and diminished secretion. The same 3bp del in ITGB3 co-segregates with the platelet phenotype and bleeding across 3 generations in the family. We then performed molecular modeling (PyMol Molecular Graphics System version 1.3) to compare the structural effects of this mutation on αIIbβ3 with other cytoplasmic tail mutations described in the literature giving GT-like phenotypes and/or MTP. Firstly αIIb-R995Q/W or β3-D723H were confirmed to disrupt a salt bridge between the integrin subunits and induce the separation of their tails. PyMol modeling showed that the DL718 results in D723 being no longer in phase with R995 thereby disrupting the salt bridge. Significantly, introduction of a β-turn by an activating β3 L718P substitution again results in the spontaneous separation of the tails, once more through a conformational change of the mutated β3. In contrast, a previously reported ITGB3 C2268T variant resulting in Arg724*, a truncation coming after the deletion in our patient, or a β3 S752P substitution that completely blocks platelet aggregation and PAC-1 binding (and specific loss of kindlin-3 binding) despite a capacity to secrete a storage pool of Fg bound α-granule αIIbβ3 failed to give MTP despite resulting in a GT-like syndrome. Conclusions: The novel ITGB3 ΔL718 of β3 showed an intermediate phenotype with MTP, a much decreased platelet aggregation, a reduced activity of αIIbβ3 at the platelet surface but a progressive fusion of α-granules. This is despite the theoretical retention of the talin and kindlin-3 binding sites suggesting that transmembrane signaling is the principle defect. Our studies highlight how considerable phenotypic heterogeneity exists in patients with genetic variants affecting αIIb and β3 cytoplasmic domains and suggest different signaling mechanisms for surface and intracellular αIIbβ3 pools. *PN and ET; WHO and ATN made equal contributions, On behalf of the BRIDGE-Bleeding and Platelet Disorder Consortium Disclosures No relevant conflicts of interest to declare.