Acute myeloid leukemia (AML) is a heterogeneous group of blood malignancies with a 5-year survival rate below 30%, highlighting the urgent need for more effective therapeutic strategies. T cell-based immunotherapies have demonstrated remarkable success in solid tumors, yet the role of CD8+ T cells in AML remains unclear. In this study, we analyzed the composition, antigenic specificity, and function of CD8+ T cells in paired blood and bone marrow samples from AML patients. While we did not identify exhausted CD8+ T cells as seen in solid tumors, we observed a distinct population of functional CD69+ CD8+ T cells specifically enriched in the bone marrow. These cells primarily recognized non-tumor antigens, including epitopes derived from Epstein–Barr virus (EBV) and cytomegalovirus (CMV). Notably, this bystander CD8+ T cell population showed high expression of Granzyme K, a cytokine found in the bone marrow of AML patients. Granzyme K did not induce leukemic cell death but instead promoted the secretion of IL-8, a pro-inflammatory cytokine known to play a detrimental role in AML pathology. Rather than mounting an anti-tumor response, these CD8+ T cells contribute to a pro-inflammatory environment that may exacerbate AML progression and severity. These findings provide a rationale for exploring therapeutic strategies aimed at inhibiting pro-inflammatory CD8+ T cells and targeting Granzyme K activity in association with actual therapies. ![Figure][1] ### Competing Interest Statement The authors have declared no competing interest. French Institute for Health and Medical Research (INSERM) French National Cancer Institute, INCA-DGOS-Inserm_12560 “SiRIC CURAMUS, 2017-1-RT-03 “MIF_AML” National Agency for Research (ANR), ANR JCJC DTSTAML Cancer Research for Personalized Medicine (CARPEM) “Inditiatives d'Excellence” (Idex) program, ANR-18-IDEX-0001 Labex Who Am I?, ANR-11-LABX-0071 ITMO Cancer of Aviesan [1]: pending:yes

Thrombocytopenia is a major side effect of a new class of anticancer agents that target histone deacetylase (HDAC). Their mechanism is poorly understood. Here, we show that HDAC6 inhibition and genetic knockdown lead to a strong decrease in human proplatelet formation (PPF). Unexpectedly, HDAC6 inhibition-induced tubulin hyperacetylation has no effect on PPF. The PPF decrease induced by HDAC6 inhibition is related to cortactin (CTTN) hyperacetylation associated with actin disorganization inducing important changes in the distribution of megakaryocyte (MK) organelles. CTTN silencing in human MKs phenocopies HDAC6 inactivation and knockdown leads to a strong PPF defect. This is rescued by forced expression of a deacetylated CTTN mimetic. Unexpectedly, unlike human-derived MKs, HDAC6 and CTTN are shown to be dispensable for mouse PPF in vitro and platelet production in vivo. Our results highlight an unexpected function of HDAC6 – CTTN axis as a positive regulator of human but not mouse MK maturation.

Congenital macrothrombocytopenias are a heterogeneous group of rare inherited disorders characterized by decreased platelets count with enlarged platelet size. However, patients with isolated macrothrombocytopenia are often misdiagnosed with idiopathic thrombocytopenic purpura (ITP) and wrongly treated with immunoglobulin injections, steroids and splenectomy. Bernard-Soulier Syndrome (BSS) is a bleeding disorder caused by defects in the platelet glycoprotein GPIb/IX/V complex, a receptor for von Willebrand factor (VWF) and thrombin. Patients present with macrothrombocytopenia and their platelets do not agglutinate in response to ristocetin, while maintaining a normal aggregation in response to a variety of aggregating agents. GPIb/IX/V complex consists of two GPIbα and four GPIbβ subunits stabilized by disulfide bonds (Luo et al, 2007). This heterodimer is non-covalently associated with two GPIX and one GPV subunits. The N-terminal residues of GPIbα form seven leucine-rich repeats (LRRs) and include the binding sites for VWF and thrombin. BSS is due to homozygous mutations in GP1BA, GP1BB or GP9 genes, encoding the components of the GPIb/IX/V complex (Savoia et al, 2014). However, some families with hereditary macrothrombocytopenia and mild or no bleeding diathesis were described with heterozygous mutations in both the GP1BA (Savoia et al, 2001; Vettore et al, 2008) and/or GP1BB genes (Kunishima et al, 2001; Savoia et al, 2014). These patients show mild thrombocytopenia with slightly increased mean platelet volume (MPV), a variable percentage of giant platelets, a modest decrease of cell-surface expression of the GPIb/IX/V complex, sometimes leading to a reduced aggregation in response to ristocetin. However, due to the absence or paucity of biological signs, the use of gene sequencing is mandatory to pinpoint the GPIb complex involvement. The International Consortium for the Study of Clinical and Molecular Aspects of BSS described 60 gene variations in GP1BA (28%), 59 in GP1BB (28%) and 92 in GP9 (44%) (Savoia et al, 2014). Most of these variations (85%) were homozygous and most cases were products of consanguineous marriages. The most common reported variation is a GP1BA missense mutation, leading to an amino acid substitution (Ala172Val), known as the Bolzano variant, located in the sixth LRR (Savoia et al, 2001). Here, we describe a French family with a new mutation in GP1BA gene located in the fifth LRR. The propositus presented with mild thrombocytopenia (89 × 109/l) with large platelets (MPV: 13·1 fl; Advia®, Siemens, Munchen, Germany) without bleeding diathesis. Platelets aggregated normally with ADP and collagen but agglutination response to 1·25 mg/ml ristocetin and the GPIb/IX/V complex platelet surface expression were modestly decreased (Table 1). RIPA by 1·25 mg/ml (Intensity, %) Normal range: [85–94] RIPA by 1·25 mg/ml (Velocity, %) Normal range: [85–212] GPIb expression (% of control) The propositus' sister exhibited more pronounced bleeding symptoms with menorrhagia, epistaxis, postoperative bleeding that did not require transfusion (Table 1). Both patients had a slightly elevated MPV with 17% large platelets, but no giant platelets on the peripheral blood smear (Table 1, Fig 1A). Pedigree analysis revealed an autosomal dominant inheritance pattern, as several members of different generations exhibit macrothrombocytopenia (Supplementary data). Morphologically, bone marrow cells from the propositus revealed small megakaryocytes with reduced and vacuolated cytoplasm (Fig 1B). In vitro study of megakaryocytes derived from peripheral blood CD34+ cells in presence of thrombopoietin and stem cell factor (also termed KIT ligand) revealed a reduced percentage of mature CD41+CD42+ cells in the propositus as compared to control whereas the ploidy level was not affected (Fig 1C). Sanger sequencing of the patient's GP1BA, GP1BB and GP9 genes revealed a novel single nucleotide mutation located in GP1BA (Fig 1D). The substituted residue was highly conserved in GP1BA orthologues from ten distantly related species. This variation was called deleterious by diverse prediction algorithms (Polyphen-2 score 1, SIFT score 0) and was not reported in the ExAC and 1000G databases (Supplementary data). The first reported thrombocytopenic patients with large platelets, slightly reduced aggregation to ristocetin and GPIb/IX/V platelet surface expression carried a monoallelic Ala172Val substitution in GPIbα (Savoia et al, 2001), known as the Bolzano variant. Other monoallelic mutations in the GP1BA gene have since been identified (Savoia et al, 2001; Vettore et al, 2008) and some were also discovered in the GP1BB gene that were associated with macrocytosis though not always with thrombocytopenia (Kunishima et al, 2001; Savoia et al, 2014). Macrothrombocytopenia due to haploinsufficiency of the GP1BB gene is also a finding frequently observed in patients with the diGeorge syndrome (del22q11) (Liang et al, 2007) but these patients are mainly diagnosed via extra-haematological symptoms. In our laboratory, we sequenced GP1BA, GP1BB and GP9 genes in all patients with mild macrothrombocytopenia and found only one family carrying the substitution of asparagine by a serine at position 150 that has not yet been listed by the International Consortium for the Study of Clinical and Molecular Aspects of BSS. Thus, in contrast to the Bolzano variant, our results do not support a mutational founder effect. Like the Bolzano variant, we have observed a variable phenotype among the mutation carriers despite similar platelet phenotype. A combination of genetic, environmental and lifestyle factors may explain the discrepancies, such as gender. Indeed, the individual with the highest bleeding score predominantly suffered from gynaecological and obstetrical bleeding. Balduini et al (2009) showed that megakaryocyte maturation was not affected in subjects with monoallelic Bolzano mutation while proplatelet formation was severely reduced in vitro. In this study, the maturation was evaluated according to the morphology of CD34+ derived megakaryocytes at day 12. We observed fewer megakaryocytes (CD41+CD42+ cells) at day 13 of culture without reduction of modal ploidy. Those results are consistent with those described in a murine model of BSS (Ware et al, 2000). We reported here a new monoallelic mutation in the GP1BA gene that slows down megakaryocyte differentiation without reducing polyploidization. A diagnosis of monoallelic BSS should be suspected in cases of rather moderate thrombocytopenia with a dominant transmission pattern. The important element is the presence of macroplatelets (>10%) with only a few, even no, giant platelets (<5%). These two criteria, associated with discrete quantitative and qualitative alterations of the GPIb/IX/V complex, may help to orient the diagnosis towards a defect in the GPIb/IX/V complex. Dorsaf Ghalloussi: performed the research, contributed essential reagents or tools, analysed the data and wrote the paper. Noémie Saut: performed the research, contributed essential reagents or tools and analysed the data. Denis Bernot: contributed essential reagents or tools and analysed the data. Xavier Pillois: performed the research, contributed essential reagents or tools and analysed the data. Philippe Rameau: contributed essential reagents or tools and analysed the data. Gérard Sébahoun: analysed the data and wrote the paper. Marie-Christine Alessi: analysed the data and wrote the paper. Hana Raslova: contributed essential reagents or tools, analysed the data and wrote the paper. Véronique Baccini: designed the research study, performed the research, contributed essential reagents or tools, analysed the data and wrote the paper. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

OBJECTIVE:Facioscapulohumeral muscular dystrophy (FSHD) is linked to either contraction of D4Z4 repeats on chromosome 4 or to mutations in the SMCHD1 gene, both of which result in the aberrant expression of the transcription factor DUX4. However, it is still difficult to correlate these genotypes with the phenotypes observed in patients. Because we have recently shown that mice with disrupted Fat1 functions exhibit FSHD-like phenotypes, we have investigated the expression of the human FAT1 gene in FSHD.METHODS:We first analyzed FAT1 expression in FSHD adult muscles and determined whether FAT1 expression was driven by DUX4. We next determined FAT1 expression levels in 64 muscles isolated from 16 control fetuses. These data were further complemented with analysis of Fat1 expression in developing mouse embryos.RESULTS:We demonstrated that FAT1 expression is independent of DUX4. Moreover, we observed that (1) in control fetal human biopsies or in developing mouse embryos, FAT1 is expressed at lower levels in muscles that are affected at early stages of FSHD progression than in muscles that are affected later or are nonaffected; and (2) in adult muscle biopsies, FAT1 expression is lower in FSHD muscles compared to control muscles.INTERPRETATION:We propose a revised model for FSHD in which FAT1 levels might play a role in determining which muscles will exhibit early and late disease onset, whereas DUX4 may worsen the muscle phenotype.

The plethora of knowledge gained on myelodysplastic syndromes (MDS), a heterogeneous pre-malignant disorder of hematopoietic stem cells, through sequencing of several pathway genes has unveiled molecular pathogenesis and its progression to AML. Evolution of phenotypic classification and risk-stratification based on peripheral cytopenias and blast count has moved to five-tier risk-groups solely concerning chromosomal aberrations. Increased frequency of complex abnormalities, which is associated with genetic instability, defines the subgroup of worst prognosis in MDS. However, the independent effect of monosomal karyotype remains controversial. Recent discoveries on mutations in RNA-splicing machinery (SF3B1, SRSF2, ZRSR2, U2AF1, U2AF2); DNA methylation (TET2, DNMT3A, IDH1/2); chromatin modification (ASXL1, EZH2); transcription factor (TP53, RUNX1); signal transduction/kinases (FLT3, JAK2); RAS pathway (KRAS, NRAS, CBL, NF1, PTPN11); cohesin complex (STAG2, CTCF, SMC1A, RAD21); DNA repair (ATM, BRCC3, DLRE1C, FANCL); and other pathway genes have given insights into the independent effects and interaction of co-occurrence of mutations on disease-phenotype. RNA-splicing and DNA methylation mutations appeared to occur early and are reported as ‘founder’ mutations in over 50% MDS patients. TET2 mutation, through altered DNA methylation, has been found to have independent prognostic response to hypomethylating agents. Moreover, presence of DNMT3A, TET2 and ASXL1 mutations in normal elderly individuals forms the basis of understanding that accumulation of somatic mutations may not cause direct disease-development; however, cooperation with other mutations in the genes that are frequently mutated in myeloid and other hematopoietic cancers might result in clonal expansion through self-renewal and/or proliferation of hematopoietic stem cells. Identification of small molecules as inhibitors of epigenetic mutations has opened avenues for tailoring targeted drug development. The recommendations of a Clinical Advisory Committee is being considered by WHO for a revised classification of risk-groups of MDS, which is likely to be published in mid 2016, based on the new developments and discoveries of gene mutations.

The facio scapulo humeral dystrophy (FSHD) is the third most prevalent muscular dystrophy. The common clinical signs usually appear during the second decade of life but when the first molecular dysregulations occur is still unknown. Our aim was to determine whether molecular dysregulations can be identified during FSHD fetal muscle development. We compared 2 muscle biopsies coming from one FSHD fetus and the cells derived from these biopsies with biopsies and cells coming from control fetuses. We mainly focus on DUX4 isoform expression since the expression of DUX4 has been confirmed in both FSHD cells and biopsies by several laboratories. We measured by qRT-PCR DUX4 isoforms expression in fetal FSDH myotubes treated or not with a shRNA directed against DUX4 mRNA. We also analyzed DUX4 downstream genes expression in myotubes and fetal or adult FSDH and control quadriceps biopsies. We show that DUX4-FL is not expressed in control myotubes whereas it is expressed FSHD myotubes. Interestingly, DUX4-FL expression level is much lower in trapezius than in quadriceps myotubes which is confirmed by the level of expression of DUX4 downstream genes: We observed that TRIM43 and MBD3L2 are already overexpressed in FSHD fetal quadriceps biopsies, at similar levels at those observed in adult FSHD quadriceps biopsies. These results indicate that molecular markers of the disease are already expressed during fetal life, raising the question as to the role of DUX4 in the onset and progression of FSHD. The facio scapulo humeral dystrophy (FSHD) is the third most prevalent muscular dystrophy. The common clinical signs usually appear during the second decade of life but when the first molecular dysregulations occur is still unknown. Our aim was to determine whether molecular dysregulations can be identified during FSHD fetal muscle development. We compared 2 muscle biopsies coming from one FSHD fetus and the cells derived from these biopsies with biopsies and cells coming from control fetuses. We mainly focus on DUX4 isoform expression since the expression of DUX4 has been confirmed in both FSHD cells and biopsies by several laboratories. We measured by qRT-PCR DUX4 isoforms expression in fetal FSDH myotubes treated or not with a shRNA directed against DUX4 mRNA. We also analyzed DUX4 downstream genes expression in myotubes and fetal or adult FSDH and control quadriceps biopsies. We show that DUX4-FL is not expressed in control myotubes whereas it is expressed FSHD myotubes. Interestingly, DUX4-FL expression level is much lower in trapezius than in quadriceps myotubes which is confirmed by the level of expression of DUX4 downstream genes: We observed that TRIM43 and MBD3L2 are already overexpressed in FSHD fetal quadriceps biopsies, at similar levels at those observed in adult FSHD quadriceps biopsies. These results indicate that molecular markers of the disease are already expressed during fetal life, raising the question as to the role of DUX4 in the onset and progression of FSHD.

SummaryBackgroundThe molecular bases of the cellular changes that occur during human megakaryocyte (MK) ontogeny remain unknown, and may be important for understanding the significance of MK differentiation from human embryonic stem cells (hESCs)MethodsWe optimized the differentiation of MKs from hESCs, and compared these with MKs obtained from primary human hematopoietic tissues at different stages of development.ResultsTranscriptome analyses revealed a close relationship between hESC-derived and fetal liver-derived MKs, and between neonate-derived and adult-derived MKs. Major changes in the expression profiles of cell cycle and transcription factors (TFs), including MYC and LIN28b, and MK-specific regulators indicated that MK maturation progresses during ontogeny towards an increase in MK ploidy and a platelet-forming function. Important genes, including CXCR4, were regulated by an on-off mechanism during development.DiscussionOur analysis of the pattern of TF network and signaling pathways was consistent with a growing specialization of MKs towards hemostasis during ontogeny, and support the idea that MKs derived from hESCs reflect primitive hematopoiesis.

Abexinostat is a pan histone deacetylase inhibitor (HDACi) that demonstrates efficacy in malignancy treatment. Like other HDACi, this drug induces a profound thrombocytopenia whose mechanism is only partially understood. We have analyzed its effect at doses reached in patient plasma on in vitro megakaryopoiesis derived from human CD34+ cells. When added at day 0 in culture, abexinostat inhibited CFU-MK growth, megakaryocyte (MK) proliferation and differentiation. These effects required only a short incubation period. Decreased proliferation was due to induction of apoptosis and was not related to a defect in TPO/MPL/JAK2/STAT signaling. When added later (day 8), the compound induced a dose-dependent decrease (up to 10-fold) in proplatelet (PPT) formation. Gene profiling from MK revealed a silencing in the expression of DNA repair genes with a marked RAD51 decrease at protein level. DNA double-strand breaks were increased as attested by elevated γH2AX phosphorylation level. Moreover, ATM was phosphorylated leading to p53 stabilization and increased BAX and p21 expression. The use of a p53 shRNA rescued apoptosis, and only partially the defect in PPT formation. These results suggest that HDACi induces a thrombocytopenia by a p53-dependent mechanism along MK differentiation and a p53-dependent and -independent mechanism for PPT formation.

The release of transforming growth factor-beta1 (TGF-beta1) in the bone marrow microenvironment is one of the main mechanisms leading to myelofibrosis in murine models and probably in the human idiopathic myelofibrosis (IMF). The regulation of TGF-beta1 synthesis is poorly known but seems regulated by nuclear factor kappaB (NF-kappaB). We previously described the overexpression of an immunophilin, FK506 binding protein 51 (FKBP51), in IMF megakaryocytes. Gel shift and gene assays show that FKBP51's overexpression in a factor-dependent hematopoietic cell line, induces a sustained NF-kappaB activation after cytokine deprivation. This activation correlates with a low level of IkappaBalpha. A spontaneous activation of NF-kappaB was also detected in proliferating megakaryocytes and in circulating CD34(+) patient cells. In normal cells, NF-kappaB activation was only detected after cytokine treatment. The expression of an NF-kappaB superrepressor in FKBP51 overexpressing cells and in derived megakaryocytes from CD34(+) of IMF patients revealed that NF-kappaB activation was not involved in the resistance to apoptosis after cytokine deprivation of these cells but in TGF-beta1 secretion. These results highlight the importance of NF-kappaB's activation in the fibrosis development of this disease. They also suggest that FKBP51's overexpression in IMF cells could play an important role in the pathogenesis of this myeloproliferative disorder.

Objective. To examine whether the in vitro model of embryonic stem (ES) cell hematopoietic differentiation is suitable to study the function of intracytoplasmic regions of cytokine receptors, we used the thrombopoietin receptor Mpl as a typical cytokine receptor.Materials and Methods. ES cells deficient in c-mpl (mpl(-/-)) were transfected with genes encoding the full-length or two mutated forms of the intracytoplasmic domain of Mpl using the pEF-BOS expression vector. The mutated forms lack box1 or box2.Results. pEF-BOS was able to maintain protein production during ES cell differentiation. Reintroduction of full-length-c-mpl into mpl(-/-) ES cells restored the response of megakaryocyte progenitors to a truncated form of human Mpl-ligand conjugated to polyethylene glycol (PEG-rhuMGDF) and the formation of platelets, for which mpl(-/-) ES cells are defective. In addition, enforced expression of Mpl resulted in the development of all myeloid progenitors and mature cells in the presence of PEG-rhuMGDF. Blast colony-forming cells, the in vitro equivalent of the hemangioblast, also generated blast cell colonies with a hematopoietic potential equivalent to that of the wild type in the presence of PEG-rhuMGDF, although its growth is normally dependent on vascular endothelial cell growth factor (VEGF). Thus, Mpl acts as a substitute for other cytokine receptors and for a tyrosine kinase receptor, Flk-1, indicating that Mpl has no instructive role in hematopoietic cell commitment and differentiation. The Mpl mutant forms lacking box1 or box2 prevented response of ES cell-derived blast colony-forming cells or progenitors to PEG-rhuMGDF. Therefore, these two regions, essential for signaling by cytokine receptors, are required for the responses of ES cell-derived hematopoietic cells to PEG-rhuMGDF.Conclusions. These results show that the in vitro hematopoietic differentiation of ES cells is suitable for studying the role of various intracytoplasmic regions of cytokine receptors. (C) 2000 International Society for Experimental Hematology. Published by Elsevier Science Inc.

The thrombocytopenia and absent radii (TAR) syndrome is a rare disease associating bilateral radial agenesis and congenital thrombocytopenia. Here, we investigated in vitro megakaryocyte (MK) differentiation and expression of c-mpl in 6 patients. Using blood or marrow CD34(+) cells, the colony-forming unit (CFU)-MK number was markedly reduced. CD34(+) cells were also cultured in liquid medium in the presence of a combination of 3 cytokines (stem cell factor, interleukin-3, and interleukin-6) or megakaryocyte growth and development factor (PEG-rHuMGDF) with or without SCF. In the presence of PEG-rHuMGDF, the majority of mature megakaryocytes (CD41 high, CD42 high) underwent apoptosis. This phenomenon was also observed in cultures stimulated by three cytokines. However, this last combination of cytokines allowed a more complete terminal MK differentiation. Surprisingly, a homogeneous population of CD34(-)CD41(+)CD42(-) cells accumulated during the cultures. This population was unable to differentiate along the myeloid pathways. This result suggests that a fraction of MK cells is unable to differentiate in the TAR syndrome. We subsequently investigated whether this could be related to an abnormality in c-mpl. No mutation or rearrangement in the c-mpl gene was found by Southern blots or by sequencing of the c-mpl coding region and its promoter in any of the patients. Using Western blot analysis, a decreased level of Mpl was found in patient platelets. A decreased level of c-mpl messenger RNA in TAR platelets was also detected with a lower c-mpl-P to c-mpl-K ratio in comparison to adult platelets. Altogether, these results demonstrate that the thrombocytopenia of the TAR syndrome is associated with a dysmegakaryocytopoiesis characterized by cells blocked at an early stage of differentiation. (Blood. 2000;95:1633-1641)

The Wiskott-Aldrich syndrome (WAS) is an X-linked hereditary disease characterized by thrombocytopenia with small platelet size, eczema, and increased susceptibility to infections. The gene responsible for WAS was recently cloned. Although the precise function of WAS protein (WASP) is unknown, it appears to play a critical role in the regulation of cytoskeletal organization. The platelet defect, resulting in thombocytopenia and small platelet size, is a consistent finding in patients with mutations in the WASP gene. However, its exact mechanism is unknown. Regarding WASP function in cytoskeletal organization, we investigated whether these platelet abnormalities could be due to a defect in proplatelet formation or in megakaryocyte (MK) migration. CD34(+) cells were isolated from blood and/or marrow of 14 WAS patients and five patients with hereditary X-linked thrombocytopenia (XLT) and cultured in serum-free liquid medium containing recombinant human Mpl-L (PEG-rHuMGDF) and stem-cell factor (SCF) to study in vitro megakaryocytopoiesis. In all cases, under an inverted microscope, normal MK differentiation and proplatelet formation were observed. At the ultrastructural level, there was also no abnormality in MK maturation, and normal filamentous MK were present. Moreover, the in vitro produced platelets had a normal size, while peripheral blood platelets of the same patients exhibited an abnormally small size. However, despite this normal platelet production, we observed that F-actin distribution was abnormal in MKs from WAS patients. Indeed, F-actin was regularly and linearly distributed under the cytoplasmic membrane in normal MKs, but it was found concentrated in the center of the WAS MKs. After adhesion, normal MKs extended very long filopodia in which WASP could be detected. In contrast, MKs from WAS patients showed shorter and less numerous filopodia. However, despite this abnormal filopodia formation, MKs from WAS patients normally migrated in response to stroma-derived factor-1alpha (SDF-1alpha), and actin normally polymerized after SDF-1alpha or thrombin stimulation. These results suggest that the platelet defect in WAS patients is not due to abnormal platelet production, but instead to cytoskeletal changes occuring in platelets during circulation.