Glanzmann Thrombasthenia 10 Years Later: Progress Made and Future Directions.

Glanzmann thrombasthenia (GT) is the most common inherited platelet disorder (IPD) with mucocutaneous bleeding and a failure of platelets to aggregate when stimulated. The molecular cause is insufficient or defective αIIbß3, an integrin encoded by the ITGA2B and ITGB3 genes. On activation αIIbß3 undergoes conformational changes and binds fibrinogen (Fg) and other proteins to join platelets in the aggregate. The application of next-generation sequencing (NGS) to patients with IPDs has accelerated genotyping for GT; progress accompanied by improved mutation curation. The evaluation by NGS of variants in other hemostasis and vascular genes is a major step toward understanding why bleeding varies so much between patients. The recently discovered role for glycoprotein VI in thrombus formation, through its binding to fibrin and surface-bound Fg, may offer a mechanosensitive back-up for αIIbß3, especially at sites of inflammation. The setting up of national networks for IPDs and GT is improving patient care. Hematopoietic stem cell therapy provides a long-term cure for severe cases; however, prophylaxis by monoclonal antibodies designed to accelerate fibrin formation at injured sites in the vasculature is a promising development. Gene therapy using lentil-virus vectors remains a future option with CRISPR/Cas9 technologies offering a promising alternative route.

A single F153Sß3 mutation causes constitutive integrin αIIbß3 activation in a variant form of Glanzmann thrombasthenia.

This report identifies a novel variant form of the inherited bleeding disorder Glanzmann thrombasthenia, exhibiting only mild bleeding in a physically active individual. The platelets cannot aggregate ex vivo with physiologic agonists of activation, although microfluidic analysis with whole blood displays moderate ex vivo platelet adhesion and aggregation consistent with mild bleeding. Immunocytometry shows reduced expression of αIIbß3 on quiescent platelets that spontaneously bind/store fibrinogen, and activation-dependent antibodies (ligand-induced binding site-319.4 and PAC-1) report ß3 extension suggesting an intrinsic activation phenotype. Genetic analysis reveals a single F153Sß3 substitution within the ßI-domain from a heterozygous T556C nucleotide substitution of ITGB3 exon 4 in conjunction with a previously reported IVS5(+1)G>A splice site mutation with undetectable platelet messenger RNA accounting for hemizygous expression of S153ß3. F153 is completely conserved among ß3 of several species and all human ß-integrin subunits suggesting that it may play a vital role in integrin structure/function. Mutagenesis of αIIb-F153Sß3 also displays reduced levels of a constitutively activated αIIb-S153ß3 on HEK293T cells. The overall structural analysis suggests that a bulky aromatic, nonpolar amino acid (F,W)153ß3 is critical for maintaining the resting conformation of α2- and α1-helices of the ßI-domain because small amino acid substitutions (S,A) facilitate an unhindered inward movement of the α2- and α1-helices of the ßI-domain toward the constitutively active αIIbß3 conformation, while a bulky aromatic, polar amino acid (Y) hinders such movements and restrains αIIbß3 activation. The data collectively demonstrate that disruption of F153ß3 can significantly alter normal integrin/platelet function, although reduced expression of αIIb-S153ß3 may be compensated by a hyperactive conformation that promotes viable hemostasis.

APOLD1 loss causes endothelial dysfunction involving cell junctions, cytoskeletal architecture, and Weibel-Palade bodies, while disrupting hemostasis.

Vascular homeostasis is impaired in various diseases thereby contributing to the progression of their underlying pathologies. The endothelial immediate early gene Apolipoprotein L domain-containing 1 (APOLD1) helps to regulate endothelial function. However, its precise role in endothelial cell biology remains unclear. We have localized APOLD1 to endothelial cell contacts and to Weibel-Palade bodies (WPB) where it associates with von Willebrand factor (VWF) tubules. Silencing of APOLD1 in primary human endothelial cells disrupted the cell junction-cytoskeletal interface, thereby altering endothelial permeability accompanied by spontaneous release of WPB contents. This resulted in an increased presence of WPB cargoes, notably VWF and angiopoietin-2 in the extracellular medium. Autophagy flux, previously recognized as an essential mechanism for the regulated release of WPB, was impaired in the absence of APOLD1. In addition, we report APOLD1 as a candidate gene for a novel inherited bleeding disorder across three generations of a large family in which an atypical bleeding diathesis was associated with episodic impaired microcirculation. A dominant heterozygous nonsense APOLD1:p.R49* variant segregated to affected family members. Compromised vascular integrity resulting from an excess of plasma angiopoietin-2, and locally impaired availability of VWF may explain the unusual clinical profile of APOLD1:p.R49* patients. In summary, our findings identify APOLD1 as an important regulator of vascular homeostasis and raise the need to consider testing of endothelial cell function in patients with inherited bleeding disorders without apparent platelet or coagulation defects.

Molecular basis of clot retraction and its role in wound healing.

Clot retraction is important for the prevention of bleeding, in the manifestations of thrombosis and for tissue repair. The molecular mechanisms behind clot formation are complex. Platelet involvement begins with adhesion at sites of vessel injury followed by platelet aggregation, thrombin generation and fibrin production. Other blood cells incorporate into a fibrin mesh that is consolidated by FXIIIa-mediated crosslinking and platelet contractile activity. The latter results in the asymmetric redistribution of erythrocytes into a tighter central mass providing the clot with stability and resistance to fibrinolysis. Integrin αIIbß3 on platelets is the key player in these events, bridging fibrin and the platelet cytoskeleton. Glycoprotein VI participates in thrombus formation but not in the retraction. Rheological and environmental factors influence clot construction with retraction driven by the platelet cytoskeleton with actomyosin acting as the motor. Activated platelets provide procoagulant activity stimulating thrombin generation together with the release of a plethora of biologically active proteins and substances from storage pools; many form chemotactic gradients within the fibrin or the underlying matrix. Also released are newly synthesized metabolites and lipid-rich vesicles that circulate within the vasculature and mimic platelet functions. Platelets and their released elements play key roles in wound healing. This includes promoting stem cell and mesenchymal stromal cell recruitment, fibroblast and endothelial cell migration, angiogenesis and matrix formation. These properties have led to the use of autologous clots in therapies designed to accelerate tissue repair while offering the potential for genetic manipulation in both inherited and acquired diseases.

Healing through the lens of immunothrombosis: Biology-inspired, evolution-tailored, and human-engineered biomimetic therapies.

Evolution, from invertebrates to mammals, has yielded and shaped immunoclotting as a defense and repair response against trauma and infection. This mosaic of immediate and local wound-sealing and pathogen-killing mechanisms results in survival, restoration of homeostasis, and tissue repair. In mammals, immunoclotting has been complemented with the neuroendocrine system, platelets, and contact system among other embellishments, adding layers of complexity through interconnecting blood-born proteolytic cascades, blood cells, and the neuroendocrine system. In doing so, immunothrombosis endows humans with survival advantages, but entails vulnerabilities in the current unprecedented and increasingly challenging environment. Immunothrombosis and tissue repair appear to go hand in hand with common mechanisms mediating both processes, a fact that is underlined by recent advances that are deciphering the mechanisms of the repair process and of the biochemical pathways that underpins coagulation, hemostasis and thrombosis. This review is intended to frame both the universal aspects of tissue repair and the therapeutic use of autologous fibrin matrix as a biology-as-a-drug approach in the context of the evolutionary changes in coagulation and hemostasis. In addition, we will try to shed some light on the molecular mechanisms underlying the use of the autologous fibrin matrix as a biology-inspired, evolution-tailored, and human-engineered biomimetic therapy.

Inherited platelet diseases with normal platelet count: phenotypes, genotypes and diagnostic strategy.

Inherited platelet disorders resulting from platelet function defects and a normal platelet count cause a moderate or severe bleeding diathesis. Since the description of Glanzmann thrombasthenia resulting from defects of ITGA2B and ITGB3, new inherited platelet disorders have been discovered, facilitated by the use of high throughput sequencing and genomic analyses. Defects of RASGRP2 and FERMT3 responsible for severe bleeding syndromes and integrin activation have illustrated the critical role of signaling molecules. Important are mutations of P2RY12 encoding the major ADP receptor causal for an inherited platelet disorder with inheritance characteristics that depend on the variant identified. Interestingly, variants of GP6 encoding the major subunit of the collagen receptor GPVI/FcRγ associate only with mild bleeding. The numbers of genes involved in dense granule defects including Hermansky-Pudlak and Chediak Higashi syndromes continue to progress and are updated. The ANO6 gene encoding a Ca2+-activated ion channel required for phospholipid scrambling is responsible for the rare Scott syndrome and decreased procoagulant activity. A novel EPHB2 defect in a familial bleeding syndrome demonstrates a role for this tyrosine kinase receptor independent of the classical model of its interaction with ephrins. Such advances highlight the large diversity of variants affecting platelet function but not their production, despite the difficulties in establishing a clear phenotype when few families are affected. They have provided insights into essential pathways of platelet function and have been at the origin of new and improved therapies for ischemic disease. Nevertheless, many patients remain without a diagnosis and requiring new strategies that are now discussed.

Inherited thrombocytopenias: history, advances and perspectives.

Over the last 100 years the role of platelets in hemostatic events and their production by megakaryocytes have gradually been defined. Progressively, thrombocytopenia was recognized as a cause of bleeding, first through an acquired immune disorder; then, since 1948, when Bernard-Soulier syndrome was first described, inherited thrombocytopenia became a fascinating example of Mendelian disease. The platelet count is often severely decreased and platelet size variable; associated platelet function defects frequently aggravate bleeding. Macrothrombocytopenia with variable proportions of enlarged platelets is common. The number of circulating platelets will depend on platelet production, consumption and lifespan. The bulk of macrothrombocytopenias arise from defects in megakaryopoiesis with causal variants in transcription factor genes giving rise to altered stem cell differentiation and changes in early megakaryocyte development and maturation. Genes encoding surface receptors, cytoskeletal and signaling proteins also feature prominently and Sanger sequencing associated with careful phenotyping has allowed their early classification. It quickly became apparent that many inherited thrombocytopenias are syndromic while others are linked to an increased risk of hematologic malignancies. In the last decade, the application of next-generation sequencing, including whole exome sequencing, and the use of gene platforms for rapid testing have greatly accelerated the discovery of causal genes and extended the list of variants in more common disorders. Genes linked to an increased platelet turnover and apoptosis have also been identified. The current challenges are now to use next-generation sequencing in first-step screening and to define bleeding risk and treatment better.

Platelet glycoprotein VI promotes metastasis through interaction with cancer cell-derived galectin-3.

Increasing evidence suggests that platelets play a predominant role in colon and breast cancer metastasis, but the underlying molecular mechanisms remain elusive. Glycoprotein VI (GPVI) is a platelet-specific receptor for collagen and fibrin that triggers platelet activation through immunoreceptor tyrosine-based activation motif (ITAM) signaling and thereby regulates diverse functions, including platelet adhesion, aggregation, and procoagulant activity. GPVI has been proposed as a safe antithrombotic target, because its inhibition is protective in models of arterial thrombosis, with only minor effects on hemostasis. In this study, the genetic deficiency of platelet GPVI in mice decreased experimental and spontaneous metastasis of colon and breast cancer cells. Similar results were obtained with mice lacking the spleen-tyrosine kinase Syk in platelets, an essential component of the ITAM-signaling cascade. In vitro and in vivo analyses supported that mouse, as well as human GPVI, had platelet adhesion to colon and breast cancer cells. Using a CRISPR/Cas9-based gene knockout approach, we identified galectin-3 as the major counterreceptor of GPVI on tumor cells. In vivo studies demonstrated that the interplay between platelet GPVI and tumor cell-expressed galectin-3 uses ITAM-signaling components in platelets and favors the extravasation of tumor cells. Finally, we showed that JAQ1 F(ab')2-mediated inhibition of GPVI efficiently impairs platelet-tumor cell interaction and tumor metastasis. Our study revealed a new mechanism by which platelets promote the metastasis of colon and breast cancer cells and suggests that GPVI represents a promising target for antimetastatic therapies.

A Glanzmann thrombasthenia family associated with a TUBB1-related macrothrombocytopenia.

BACKGROUND: Macrothrombocytopenia (MTP) is a rare but enigmatic complication of Glanzmann thrombasthenia (GT), an inherited bleeding disorder caused by the absence of platelet aggregation due to deficiencies of the αIIbß3 integrin. OBJECTIVES: We report a family with type I GT and a prolonged bleeding time but unusually associated with congenital mild thrombocytopenia and platelet size heterogeneity with giant forms. METHODS AND RESULTS: Sanger sequencing of DNA from the propositus identified 2 heterozygous ITGB3 gene mutations: p.P189S and p.C210S both of which prevent αIIbß3 expression and are causative of GT but without explaining the presence of enlarged platelets. High-throughput screening led to the detection of a predicted disease-causing heterozygous mutation in the TUBB1 gene: p.G146R, encoding ß1-tubulin, a component of the platelet cytoskeleton and a gene where mutations are a known cause of MTP. CONCLUSIONS: Family screening confirmed that this rare phenotype results from oligogenic inheritance while suggesting that the GT phenotype dominates clinically.

Clinical significance of altered collagen-receptor functioning in platelets with emphasis on glycoprotein VI.

Much interest surrounds the receptors α2ß1 and glycoprotein VI (GPVI) whose synchronized action mediates the attachment and activation of platelets on collagen, essential for preventing blood loss but also the most thrombogenic component of the vessel wall. Subject to density variations on platelets through natural polymorphisms, the absence of α2ß1 or GPVI uniquely leads to a substantial block of hemostasis without causing major bleeding. Specific to the megakaryocyte lineage, GPVI and its signaling pathways are most promising targets for anti-thrombotic therapy. This review looks at the clinical consequences of the loss of collagen receptor function with emphasis on both the inherited and acquired loss of GPVI with brief mention of mouse models when necessary. A detailed survey of rare case reports of patients with inherited disease-causing variants of the GP6 gene is followed by an assessment of the causes and clinical consequences of acquired GPVI deficiency, a more frequent finding most often due to antibody-induced platelet GPVI shedding. Release of soluble GPVI is brought about by platelet metalloproteinases; a process induced by ligand or antibody binding to GPVI or even high shear forces. Also included is an assessment of the clinical importance of GPVI-mediated platelet interactions with fibrin and of the promise shown by the pharmacological inhibition of GPVI in a cardiovascular context. The role for GPVI in platelet function in inflammation and in the evolution and treatment of major illnesses such as rheumatoid arthritis, cancer and sepsis is also discussed.

Acquired Glanzmann thrombasthenia: From antibodies to anti-platelet drugs.

In contrast to the inherited platelet disorder given by mutations in the ITGA2B and ITGB3 genes, mucocutaneous bleeding from a spontaneous inhibition of normally expressed αIIbß3 characterizes acquired Glanzmann thrombasthenia (GT). Classically, it is associated with autoantibodies or paraproteins that block platelet aggregation without causing a fall in platelet count. However, inhibitory antibodies to αIIbß3 are widely associated with primary immune thrombocytopenia (ITP), occur in secondary ITP associated with leukemia and related disorders, solid cancers and myeloma, other autoimmune diseases, following organ transplantation while cytoplasmic dysregulation of αIIbß3 function features in myeloproliferative and myelodysplastic syndromes. Antibodies to αIIbß3 occur during viral and bacterial infections, while drug-dependent antibodies reacting with αIIbß3 are a special case. Direct induction of acquired GT is a feature of therapies that block platelets in coronary artery disease. This review looks at these conditions, emphasizing molecular mechanisms, therapy, patient management and future directions for research.

Autologous fibrin scaffolds: When platelet- and plasma-derived biomolecules meet fibrin.

The healing of vascularized mammalian tissue injuries initiate with hemostasis and clotting as part of biological defense system leading to the formation of a fibrin clot in which activated platelets are trapped to quickly stop bleeding and destroy microbials. In order to harness the therapeutic potential of biomolecules secreted by platelets and stemmed from plasma, blood deconstruction has allowed to yield autologous platelet-and plasma-derived protein fibrin scaffold. The autologous growth factors and microparticles stemmed from platelets and plasma, interact with fibrin, extracellular matrix, and tissue cells in a combinatorial, synergistic, and multidirectional way on mechanisms governing tissue repair. This interplay will induce a wide range of cell specifications during inflammation and repair process including but not limited to fibrogenesis, angiogenesis, and immunomodulation. As biology-as-a-drug approach, autologous platelet-and plasma-derived protein fibrin scaffold is emerging as a safe and efficacious natural human-engineered growth factor delivery system to repair musculoskeletal tissues, and skin and corneal ulcers and burns. In doing so, it acts as therapeutic agent not perfect but close to biological precision. However, this autologous, biocompatible, biodegradable, and long in vivo lasting strategy faces several challenges, including its non-conventional single dose-response effect, the lack of standardization in its preparation and application, and the patient's biological features. In this review, we give an account of the main events of tissue repair. Then, we describe the procedure to prepare autologous platelet-and plasma-derived protein fibrin scaffolds, and the rationale behind these biomaterials, and finally, we highlight the significance of strategic accuracy in their application.

A mutation of the human EPHB2 gene leads to a major platelet functional defect.

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, αIIbß3 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 αIIbß3 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.

Personal reflections on the early contributions of Gus Born to platelet research.

Professor GVR Born, Gus to his friends, was one of the great pioneers of platelet research. My early memories of him have enabled me to look back at his early years in Oxford and London. A brilliant and generous man with always the time to discuss and advise he was instrumental in deciphering the principle stages of the aggregation of blood platelets by ADP, a path aided by his development and use of the platelet aggregometer. He applied his knowledge to the real time analysis of platelet and leukocyte involvement in thrombus formation in animal models and to the development of atherosclerosis and thrombosis and their pharmacological inhibition. What follows is a personal account of the major steps in this early work and of the actors involved.

Acquired Antibodies to αIIbß3 in Glanzmann Thrombasthenia: From Transfusion and Pregnancy to Bone Marrow Transplants and Beyond.

Patients with the inherited bleeding disorder Glanzmann thrombasthenia (GT) possess platelets that lack αIIbß3 integrin and fail to aggregate, and have moderate to severe mucocutaneous bleeding. Many become refractory to platelet transfusions due to the formation of isoantibodies to αIIbß3 with the rapid elimination of donor platelets and/or a block of function. Epitope characterization has shown isoantibodies to be polyclonal and to recognize different epitopes on the integrin with ß3 a major site and αvß3 on endothelial and vascular cells a newly recognized target. Pregnancy in GT can also lead to isoantibody formation when fetal cells with ß3 integrins pass into the circulation of a mother lacking them; a consequence is neonatal thrombocytopenia and a high risk of mortality. Antibody removal prior to donor transfusions can provide transient relief, but all evidence points to recombinant FVIIa as the first choice for GT patients either to stop bleeding or as prophylaxis. Promoting thrombin generation by rFVIIa favors GT platelet interaction with fibrin, and the risk of deep vein thrombosis also associated with prolonged immobilization and catheter use requires surveillance. Although having a high risk, allogeneic bone marrow transplantation associated with different stem cell sources and conditioning regimens has proved successful in many cases of severe GT with antibodies, and often, the associated conditioning and immunosuppressive therapy leads to loss of isoantibody production. Animal models of gene therapy for GT show promising results, but isoantibody production can be stimulated and CRISPR/Cas9 technology has yet to be applied. Up-to-date consensus protocols for dealing with isoantibodies in GT are urgently required, and networks providing patient care should be expanded.

In silico analysis of structural modifications in and around the integrin αIIb genu caused by ITGA2B variants in human platelets with emphasis on Glanzmann thrombasthenia.

BACKGROUND: Studies on the inherited bleeding disorder, Glanzmann thrombasthenia (GT), have helped define the role of the αIIbß3 integrin in platelet aggregation. Stable bent αIIbß3 undergoes conformation changes on activation allowing fibrinogen binding and its taking an extended form. The αIIb genu assures the fulcrum of the bent state. Our goal was to determine how structural changes induced by missense mutations in the αIIb genu define GT phenotype. METHODS: Sanger sequencing of ITGA2B and ITGB3 in the index case followed by in silico modeling of all known GT-causing missense mutations extending from the lower part of the ß-propeller, and through the thigh and upper calf-1 domains. RESULTS: A homozygous c.1772A>C transversion in exon 18 of ITGA2B coding for a p.Asp591Ala substitution in an interconnecting loop of the lower thigh domain of αIIb in a patient with platelets lacking αIIbß3 led us to extend our in silico modeling to all 16 published disease-causing missense variants potentially affecting the αIIb genu. Modifications of structuring H-bonding were the major cause in the thigh domain although one mutation gave mRNA decay. In contrast, short-range changes induced in calf-1 appeared minor suggesting long-range effects. All result in severe to total loss of αIIbß3 in platelets. The absence of mutations within a key Ca2+-binding loop in the genu led us to scan public databases; three potential single allele variants giving major structural changes were identiffied suggesting that this key region is not protected from genetic variation. CONCLUSIONS: It appears that the αIIb genu is the object of stringent quality control to prevent platelets from circulating with activated and extended integrin.