A case of acquired transient bleeding diathesis associated with acquired platelet storage pool deficiency and defective thromboxane A2 production.

Acquired disorders of platelet function are an underdiagnosed cause of bleeding tendency. A 14-year-old girl developed moderate mucocutaneous bleeding two weeks after a Mycoplasma pneumoniae infection successfully treated with clarithromycin. The patient was referred to us 7 months later for laboratory investigation of the persisting bleeding diathesis. The patient's personal and family histories were negative for bleeding disorders. Complete blood count, von Willebrand Factor levels and coagulation tests were normal; platelet aggregation, ATP secretion, δ-granules content and serum thromboxane B2 levels were defective. At follow-up visits, laboratory parameters and the bleeding diathesis progressively normalized within 2 years. The patient's condition is compatible with a diagnosis of acquired Storage Pool Deficiency (SPD), associated with defective thromboxane A2 production. To our knowledge, this is the first case of acquired, transient SPD with spontaneous remission. The pathogenic role of Mycoplasma pneumoniae infection or clarithromycin is possible, albeit uncertain.

The complementary roles of VAMP-2, -3, and -7 in platelet secretion and function.

Platelet secretion requires Soluble N-ethylmaleimide Sensitive Attachment Protein Receptors (SNAREs). Vesicle SNAREs/Vesicle-Associated Membrane Proteins (v-SNAREs/VAMPs) on granules and t-SNAREs in plasma membranes mediate granule release. Platelet VAMP heterogeneity has complicated the assessment of how/if each is used and affects hemostasis. To address the importance of VAMP-7 (V7), we analyzed mice with global deletions of V3 and V7 together or platelet-specific deletions of V2, V3, and global deletion of V7. We measured the kinetics of cargo release, and its effects on three injury models to define the context-specific roles of these VAMPs. Loss of V7 minimally affected dense and α granule release but did affect lysosomal release. V3-/-7-/- and V2Δ3Δ7-/- platelets showed partial defects in α and lysosomal release; dense granule secretion was unaffected. In vivo assays showed that loss of V2, V3, and V7 caused no bleeding or occlusive thrombosis. These data indicate a role for V7 in lysosome release that is partially compensated by V3. V7 and V3, together, contribute to α granule release, however none of these deletions affected hemostasis/thrombosis. Our results confirm the dominance of V8. When it is present, deletion of V2, V3, or V7 alone or in combination minimally affects platelet secretion and hemostasis.

What did we know? V8 is the primary VAMP isoform for platelet granule secretion, but V2 and V3 play compensatory roles.V3 is important for platelet endocytosis.V7 plays a minimal role in secretion and does not affect hemostasis.What did we discover? The loss of both V3 and V7 increases α and lysosomal secretion defects.Platelet-specific deletion of V2 and V3 with global V7-deletion causes defective α and lysosomal release.Secretion deficiencies in V3^−/−7^−/− and V2^Δ3^Δ7^−/− have no effect on hemostasis or thrombosis.What is the impact? We show that endosomal v-SNAREs (V3 and V7) play minor roles in secretion.V3^−/−7^−/− and platelet-specific V2^Δ3^Δ7^−/− mice are viable and will be valuable in in vivo studies of membrane trafficking.

Canalicular system reorganization during mouse platelet activation as revealed by 3D ultrastructural analysis.

The canalicular system (CS) has been defined as: 1) an inward, invaginated membrane connector that supports entry into and exit from the platelet; 2) a static structure stable during platelet isolation; and 3) the major source of plasma membrane (PM) for surface area expansion during activation. Recent analysis from STEM tomography and serial block face electron microscopy has challenged the relative importance of CS as the route for granule secretion. Here, We used 3D ultrastructural imaging to reexamine the CS in mouse platelets by generating high-resolution 3D reconstructions to test assumptions 2 and 3. Qualitative and quantitative analysis of whole platelet reconstructions, obtained from immediately fixed or washed platelets fixed post-washing, indicated that CS, even in the presence of activation inhibitors, reorganized during platelet isolation to generate a more interconnected network. Further, CS redistribution into the PM at different times, post-activation, appeared to account for only about half the PM expansion seen in thrombin-activated platelets, in vitro, suggesting that CS reorganization is not sufficient to serve as a dominant membrane reservoir for activated platelets. In sum, our analysis highlights the need to revisit past assumptions about the platelet CS to better understand how this membrane system contributes to platelet function.

Germline heterozygous variants in genes associated with familial hemophagocytic lymphohistiocytosis as a cause of increased bleeding.

Familial hemophagocytic lymphohistiocytosis (FHL) is caused by biallelic variants in genes regulating granule secretion in cytotoxic lymphocytes. In FHL3-5, the affected genes UNC13D, STX11 and STXBP2 have further been shown to regulate the secretion of platelet granules, giving rise to compromised platelet function. Therefore, we aimed to investigate platelet degranulation in patients heterozygous for variants in UNC13D, STX11 and STXBP2. During the work-up of patients referred to the Coagulation Unit, Skåne University Hospital, Malmö, Sweden and the Department of Hematology, Rigshospitalet, Copenhagen, Denmark due to bleeding tendencies, 12 patients harboring heterozygous variants in UNC13D, STX11 or STXBP2 were identified using targeted whole exome sequencing. Transmission electron microscopy (TEM) was used to assess the secretion of platelet dense granules following thrombin stimulation. Platelet degranulation, activation and aggregation were further assessed by flow cytometry (FC) and light transmission aggregometry (LTA) with lumi-aggregometry. In total, eight out of twelve (67%) patients showed impaired degranulation by at least one of the assays (TEM, FC and LTA). In the 12 patients, eight different heterozygous variants were identified. One variant was strongly associated with impaired degranulation, while four of the variants were associated with impaired granule secretion to a slightly lesser extent. One additional variant was found in six out of the twelve patients, and was associated with varying degrees of degranulation impairment. Accordingly, six out of the eight (75%) identified variants were associated with impaired platelet degranulation. Our results suggest that heterozygous variants in UNC13D, STX11 and STXBP2 are sufficient to cause platelet secretion defects resulting in increased bleeding.

Expert opinion on the use of platelet secretion assay for the diagnosis of inherited platelet function disorders: Communication from the ISTH SSC Subcommittee on Platelet Physiology.

Assessment of platelet secretion is crucial for diagnosing suspected inherited platelet function disorders (IPFD). A previous survey of the SSC on Platelet Physiology of the ISTH and a comprehensive review highlighted that most of the platelet secretion assays (PSAs) lack standardization and validation. The aim of this study was to provide expert consensus guidance on the use of PSAs for IPFD diagnosis. We surveyed 26 experts from 10 different countries using the RAND/UCLA methodology, to attain a consensus on sensitivity, specificity, feasibility, time to readout, and cost of most PSAs. Answers were then graded in three categories: appropriate, uncertain, and inappropriate. Equivocal or misinterpretable statements required a second and third round survey involving 14 of the original 26 experts. We report here the consolidated results of the entire procedure. There was uniform agreement on several general statements, including that PSAs should be performed in hemostasis laboratories as first line diagnostic tests even in patients with normal platelet aggregation, and should include a δ-granule secretion marker. Among the specific assays examined, lumiaggregometry, other luciferin/luciferase-based assays, high-performance liquid chromatography methods, radiolabeled-serotonin based assays, and whole-mount transmission electron microscopy were rated as appropriate for the measurement of δ-granule release, and platelet P-selectin expression by flow cytometry and released proteins by ELISA for α-granule release. For most of the other PSAs, the expert opinions were widely dispersed. Lack of expert consensus on many PSAs clearly indicates an unmet need for rigorous standardization, multicenter comparison of results, and validation of PSAs for clinical laboratory practice.

Oral Squamous Cell Carcinoma Is Associated with a Low Thrombosis Risk Due to Storage Pool Deficiency in Platelets.

Venous thrombo-embolism (VTE) disease is the second most common cause of mortality in cancer patients, and evaluation and prevention of thrombosis risk is essential. VTE-associated risk varies according to the type of tumor disease. Oral cancer is the most frequent type of head and neck cancer, and it represents approximately 2.1% of all cancers worldwide. Most tumors are squamous cell carcinomas and are mainly due to tobacco and alcohol abuse. VTE risk associated with oral squamous cell carcinoma (OSCC) is low. However, many studies have shown that OSCC has the following biological features of cancers associated with a high thrombosis risk: modified thrombosis and fibrinolysis mechanisms; strong expression of procoagulant proteins; secretion of procoagulant microparticles; and production of procoagulant cytokines. Using an original mouse model of tongue squamous cell carcinoma, our study aimed to clarify this paradoxical situation. First, we showed that OSCC tumors have a pro-aggregatory phenotype and a high local thrombosis risk. Second, we found that tongue tumor mice do not have an elevated systemic thrombosis risk (the risk of an "at distance" thrombosis event such as lower extremity deep venous thrombosis or pulmonary embolism) and even show a reduction in risk. Third, we demonstrated that tongue tumor mice show a reduction in platelet reactivity, which explains the low systemic thrombosis risk. Finally, we found that tongue tumor mice present granule pool deficiency, thereby explaining the reduction in platelet reactivity and systemic thrombosis risk.

3D ultrastructural analysis of α-granule, dense granule, mitochondria, and canalicular system arrangement in resting human platelets.

BACKGROUND: State-of-the-art 3-dimensional (3D) electron microscopy approaches provide a new standard for the visualization of human platelet ultrastructure. Application of these approaches to platelets rapidly fixed prior to purification to minimize activation should provide new insights into resting platelet ultrastructure. OBJECTIVES: Our goal was to determine the 3D organization of α-granules, dense granules, mitochondria, and canalicular system in resting human platelets and map their spatial relationships. METHODS: We used serial block face-scanning electron microscopy images to render the 3D ultrastructure of α-granules, dense granules, mitochondria, canalicular system, and plasma membrane for 30 human platelets, 10 each from 3 donors. α-Granule compositional data were assessed by sequential, serial section cryo-immunogold electron microscopy and by immunofluorescence (structured illumination microscopy). RESULTS AND CONCLUSIONS: α-Granule number correlated linearly with platelet size, while dense granule and mitochondria number had little correlation with platelet size. For all subcellular compartments, individual organelle parameters varied considerably and organelle volume fraction had little correlation with platelet size. Three-dimensional data from 30 platelets indicated only limited spatial intermixing of the different organelle classes. Interestingly, almost 70% of α-granules came within ≤35 nm of each other, a distance associated in other cell systems with protein-mediated contact sites. Size and shape analysis of the 1488 α-granules analyzed revealed no more variation than that expected for a Gaussian distribution. Protein distribution data indicated that all α-granules likely contained the same major set of proteins, albeit at varying amounts and varying distribution within the granule matrix.

VAMP8 and serotonin transporter levels are associated with venous thrombosis risk in a Spanish female population. Results from the RETROVE Project.

INTRODUCTION: Platelet hyper-reactivity has been associated with thrombosis and high levels of human vesicle-associated membrane protein 8 (VAMP8) and serotonin transporter (SERT). Two polymorphisms (rs1010 of VAMP8 gene and in SERT gene (SLC6A4)) are associated with arterial thrombosis. AIM: To determine if levels of serotonin, SERT and/or VAMP8 and these polymorphisms are associated with the risk of venous thrombosis. MATERIAL AND METHODS: A total of 324 individuals were included in the RETROVE Study (Riesgo de Enfermedad TROmboembólica VEnosa). VAMP8, SERT and serotonin were determined by ELISA; polymorphisms of SLC6A4 and VAMP8 by polymerase chain reaction (PCR) and real time PCR. The venous thrombotic risk was calculated by a logistic regression method to estimate the crude and adjusted OR (adjusted for sex, age, body mass index and venous thrombosis risk co-factors). RESULTS: Statistically significant high levels of VAMP8 and SERT were found in patients, but not in controls. In contrast, serotonin showed lower levels in patients than in controls. When individuals were studied by gender, only women exhibited a statistically significant difference: the OR for VAMP8 was 3.25 (1.61-6.56 95% CI). The adjusted OR did not change. The OR for SERT was 2.76 (1.36-5.60 95% CI), the adjusted OR was maintained also. For serotonin with OR of 2.62 (1.40-4.92 95% CI), the adjusted OR was not significant. In contrast males did not show significant differences. No statistically differences between patients and controls were found for both polymorphisms. CONCLUSIONS: VAMP8 and SERT levels are associated with venous thrombosis in a female Spanish population.

Sorting machineries: how platelet-dense granules differ from α-granules.

Platelets respond to vascular injury via surface receptor stimulation and signaling events to trigger aggregation, procoagulant activation, and granule secretion during hemostasis, thrombosis, and vascular remodeling. Platelets contain three major types of secretory granules including dense granules (or δ-granules, DGs), α-granules (AGs), and lysosomes. The contents of platelet granules are specific. Platelet DGs store polyphosphate and small molecules such as ADP, ATP, Ca2+, and serotonin, while AGs package most of the proteins that platelets release. The platelet DGs and AGs are regarded as being budded from the endosomes and the trans-Golgi network (TGN), respectively, and then matured from multivesicular bodies (MVBs). However, the sorting machineries between DGs and AGs are different. Inherited platelet disorders are associated with deficiency of DGs and AGs, leading to bleeding diathesis in patients with Hermansky-Pudlak syndrome (HPS), gray platelet syndrome (GPS), and arthrogryposis, renal dysfunction, and cholestasis syndrome (ARC). Here, we reviewed the current understanding about how DGs differ from AGs in structure, biogenesis, and function. In particular, we focus on the sorting machineries that are involved in the formation of these two types of granules to provide insights into their diverse biological functions.