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

Abstract

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.

Figures

Figure 1.

Integrin αIIbβ3 and Glanzmann thrombasthenia. The model illustrates the bent resting mature integrin and shows the many interactions between IIb (green) and 3 (violet). Much information has been obtained from variant forms (noted in blue) that block functional sites in the extracellular and cytoplasmic domains while allowing IIb3 expression. Other rare mutations (in red) give rise to activated integrin. Full information on mutations giving rise to Glanzmann thrombasthenia is found in Nurden & Pillois. Fg: fibrinogen; GT: Glanzmann thrombasthenia.

Figure 2.

Loss of CalDAG-GEFI and kindlin-3 function abrogates αIIbβ3 activation. (A) A schematic representation showing examples of agonistic receptors and αIIbβ3 in the resting and activated forms. Intracytoplasmic signaling pathways induced by the binding of appropriate ligands lead to parallel roles of CalDAG-GEFI and kindlin-3 in promoting IIb3 activation. CalDAG-GEFI is critical for RAP1 activation both in the circulation and at sites of vascular injury. It responds to changes in cytoplasmic Ca2+ providing sensitivity and speed to the activation response; its absence leads to a Glanzmann thrombasthenia-like phenotype. Kindlin-3 binds directly to -integrin cytoplasmic tails in platelets but also in white blood cells, explaining the susceptibility to infections and immune disorders in its absence. (B) Key elements of the syndromes resulting from RASGRP2 and FERMT3 defects are summarized. Bleeding in both cases can be very severe but defects in FERMT3 are syndromic and life-threatening. TRAP: thrombin receptor agonist peptid; PAR: protease activated receptor; Cvx: convulxin; GPVI: glycoprotein VI; TXA2: thromboxane A2; PLC/: phospholipase C/γ; DAG: diacylglyerol: CalDAG-GEFI: calcium and diacylglycerol-regulated guanine nucleotide exchange factor I; RAP1: RAS-related protein 1; RD: related disease; AR: autosomal recessive; LTA: light transmission aggregometry; ADP: adenosine triphosphate; Col: collagen; Cvx: convulxin; AA: arachidonic acid; PMA: phorbol 12-myristate 13-acetate.

Figure 3.

G-protein-coupled ADP receptors and signaling pathways highlighting P2RY12 variants and their different subgroups. (A) A schematic representation of ADP-induced signaling pathways. Binding of extracellular ADP to the Gq-coupled P2Y1 receptor initiates aggregation whereas the Gi-coupled P2Y12 receptor enhances and sustains the platelet response. Specific signaling pathways are involved with PLCβ and PI3K enabling both RAP1 activation and RASA3 inactivation; both are required for the formation of a stable hemostatic thrombus. The release of ADP from dense granule storage pools after protein kinase C (PKC) activation contributes to the stabilization of thrombi. (B) The schematic representation shows the structure of P2Y12; variants reported as causal for a bleeding syndrome are highlighted (red spots). (C) Summarizes the genotype/phenotype relationship when the variants are regrouped according to bleeding severity and their mode of transmission. The first group consists of patients with autosomal recessive transmission and platelets showing defects of both the binding of ADP analogs and/or receptor cycling. The p.H187Q variant located in the fifth transmembrane domain has normal receptor expression but suppressed function. Interestingly p.R256 has a side chain that inserts into a hydrophobic pocket and in docking models it potentially makes contact with the phosphate groups of ADP; p.R265W impairs receptor activation and probably alters the conformational state of the receptor. Significantly, a severe phenotype-monoallelic form has a different amino acid substitution on the same residue, p.R265P associated with an increased expression of mutated mRNA and an impaired wild-type homodimer formation, possibly accounting for the bleeding severity. The third subgroup has a mild phenotype and monoallelic expression of mutations that affect receptor function: note that p.P341A located in the PDZ intracellular domain is associated with both abnormal ligand binding and a defect of re-sensitization of the receptor after ADP agonist-induced desensitization. (D) Electron microscopy of platelet aggregates stimulated with 10 M ADP examined at the peak of aggregation. For the control the platelets are in close contact with some of the cells having lost their granule content. For our patient with the p.I240fs*29 mutation, platelets remain loosely bound and their granule contents are still present, as shown by the immunogold (black dots) localization of fibrinogen.

Figure 4.

Hermansky-Pudlak syndrome. Syndromic platelet defect resulting from the abnormal biogenesis of dense granules with an autosomal recessive inheritance. (A) Summarizes the genotype/phenotype relationship of different types of Hermansky-Pudlak syndrome (HPS) regrouped according to the four multi-protein complexes involved in different steps of lysosome-related organelle (LRO) biogenesis including vesicle formation and/or trafficking: BLOC1, 2 and 3 and AP-3 with each containing several subunits. Variants in HPS 7, 8 and 9 are causal in BLOC1 but with mild clinical consequences. For BLOC2, variants in HPS 3, 5 or 6 give syndromes of moderate severity; they are rare but a variant of HPS 3 occurs in Puerto Rican communities. The most frequent causes of HPS are BLOC3 defects (variants in HPS 1 or 4). The syndromes are severe and are associated with lung fibrosis and gastro-intestinal defects; in Puerto Rico a community is affected by a common HPS1 variant. In the last group, defects of AP-3 are associated with neutropenia and infections. (B) A scheme illustrates the major steps of granule biogenesis. Dense granules may derive from the endolysosomal system, including either early or late endosomes. As secretory granules the membrane constituents including membrane transporters come from the Golgi complex. A network of interconnected and functionally distinct tubular subdomains transports their cargoes along microtubule tracks from the endosomes. Tubules ferry contents from the trans-Golgi-network and the plasma membrane to the LRO, a system that requires the coordination of numerous effectors. Components stored in dense granules or associated with their membranes are shown. (C) Platelets from a control and a patient with HPS3 examined by electron microscopy as whole mounts are shown. The dense granules observed in controls as black spots are absent from the patient’s platelet. AP-3: adaptor protein-3; BLOC: biogenesis of lysosome-related organelles complex; LRO: lysosome-related organelle; OCA: oculocutaneous albinism; MVB: multivesicular body.

Figure 5.

Schema with management options for patients with inherited defects of platelet function. It is important to limit risks in daily life. It is also important to provide each patient with a card detailing the type of disorder and proposing the medication to be used according to the bleeding risk, in particular when platelet expert medical centers are unavailable to take charge of the patient’s needs. While the main therapeutic approaches for all disorders, including those patients with Glanzmann thrombasthenia (GT), RASGRP2 (CalDAG-GEFI)-related disease, and leukocyte adhesion deficiency III (LAD-III) syndrome for whom bleeding can be severe, are similar, some situations (e.g., isoantibody formation in GT) or infections in LAD-III syndrome need special care. Surgery requires a multidisciplinary consensus to evaluate the risk of bleeding, the benefit-risk ratio of prophylaxis, and to assess therapeutic efficiency. Pregnancy is always a challenge, from women with mild bleeding risks for whom minimal measures are needed to those with a severe bleeding history and for whom maximal prophylactic care is required. Here again, the management must be planned between obstetricians, anesthetists and hematologists. The contraindication of epidural or spinal anesthesia for those with mild risk remains a difficult problem, as is the choice of vaginal delivery or Cesarean section for the most severe forms. In the figure, it is noted that for mild bleeding risk, an antifibrinolytic medication or desmopressin is preferred as the first line of bleeding prevention and that platelet transfusions should be considered only when these approaches are insufficient. There is a risk of allo- or iso-immunization which, if it occurs, can be a lifelong handicap, and recombinant activated factor VII is increasingly recommended. Finally, stem cell transplantation is reserved for young patients with recurrent, severe bleeding that fails to respond to standard treatments and when the survival of the patient is at risk. Gene therapy is not yet an option for the disorders covered in this review but will probably become available in the future.