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.

[Blood platelets]. / Les plaquettes sanguines.

Studies of inherited platelet disorders have led to major advances in our knowledge of platelet physiology, thus permitting the development of antiplatelet agents that are now widely used to treat vascular diseases. New therapeutic strategies have also resulted from a better understanding of megakaryocytopoiesis, notably including the use of thrombopoietin analogs for immune thrombocytopenias.

PML/RARalpha fusion protein transactivates the tissue factor promoter through a GAGC-containing element without direct DNA association.

A severe coagulopathy is a life-threatening complication of acute promyelocytic leukemia (APL) and is ascribable mainly to the excessive levels of tissue factor (TF) in APL cells regulated in response to the promyelocytic leukemia/retinoic acid receptor alpha (PML/RARalpha) fusion protein. The underlying molecular mechanisms for this regulation remain ill-defined. With U937-PR9 cell lines stably expressing luciferase reporter gene under the control of different mutants of the TF promoter, both luciferase and ChIP data allowed the localization of the PML/RARalpha-responsive sequence in a previously undefined region of the TF promoter at position -230 to -242 devoid of known mammalian transcription factor binding sites. Within this sequence a GAGC motif (-235 to -238) was shown to be crucial because deletion or mutation of these nucleotides impaired both PML/RARalpha interaction and promoter transactivation. However, EMSA results showed that PML/RARalpha did not bind to DNA probes encompassing the -230 to -242 sequences, precluding a direct DNA association. Mutational experiments further suggest that the activator protein 1 (AP-1) sites of the TF promoter are dispensable for PML/RARalpha regulation. This study shows that PML/RARalpha transactivates the TF promoter through an indirect interaction with an element composed of a GAGC motif and the flanking nucleotides, independent of AP-1 binding.

Suppression of tumor growth by viral vector-mediated gene transfer of N-terminal truncated platelet factor 4.

Platelet factor four (PF4), an inhibitor of endothelial cell proliferation in vitro, inhibits angiogenesis and tumor growth in vivo in experimental animals. The present study was designed to determine whether gene therapy-mediated expression of a form of PF4 lacking 16 amino acids of N-terminus from tumor cells could inhibit angiogenesis and tumor growth in vivo. Two replication-defective recombinant retroviral vectors were constructed. One encodes human PF4 (rRV-PF4) and the other encodes the N-truncated peptide (rRVp17-70). These vectors were then used to transduce KB cells, a human head and neck squamous carcinoma cell line. Expression of PF4 and p17-70 transgenes was confirmed by Western blot analysis. In vitro, both rRV-PF4 and rRVp17-70 were able to inhibit selectively the proliferation of human umbilical vascular endothelial cells (HUVEC) but not KB cells. In vivo activity was assessed by injecting 10(7) KB cells subcutaneously into nude mice and by monitoring subsequent tumor growth, xenograft vascular histochemistry, and animal survival. Viral vector-mediated cDNA transfer of PF4 and p17-70 resulted in inhibiting solid tumors through an anti-angiogenic action in vivo. Our data indicate that targeting tumor angiogenesis using viral-mediated gene transfer of full-length and N-terminal truncated PF4 represents a promising strategy for cancer gene therapy.

Adenovirus-mediated gene therapy with an antiangiogenic fragment of thrombospondin-1 inhibits human leukemia xenograft growth in nude mice.

Recent investigations support the idea that angiogenesis is involved in the pathophysiology of leukemia. Within a given microenvironment, the angiogenic response is regulated by a delicate balance of angiogenesis inducers and inhibitors. Thrombospondin-1 (TSP-1) is a multifunctional extracellular glycoprotein showing angiostatic properties in multiple in vitro and in vivo assays. Interestingly, there is also proangiogenic domain in this complex molecule. Development of TSP-1 as an antiangiogenic drug has been hindered by multiplicity of its functional effects, difficulties in its production and its poor pharmacokinetics. The aim of the present study was to establish a recombinant adenovirus (ADV.TSP-1(f)) expressing antiangiogenic fragment of TSP-1 (TSP-1(f)), and to determine the feasibility for use of the adenovirally expressed TSP-1(f) in leukemia gene therapy. The results of this investigation showed that TSP-1(f) was expressed efficiently in adenovirus-transduced human myelogenous leukemia K562 cells. Compared to the controls, although there was almost no effect on proliferation of K562 cells in vitro, adenovirus-mediated TSP-1(f) transduction inhibited the growth of K562 xenografts dramatically. Furthermore, the microvessel density (MVD) was much lower in the ADV.TSP-1(f)-treated tumors compared to the controls. These data support the use of in vivo gene delivery approach to produce antiangiogenic fragment of TSP-1 for leukemia therapy.

Dissociation between fibrinogen and fibrin interaction with platelets in patients with different subtypes of Glanzmann's thrombasthenia: studies in an ex vivo perfusion chamber model.

To explore the possible role of a residual or variant alphaIIbbeta3 integrin (alphaIIbbeta3) in thrombogenesis, we used a new ex vivo perfusion chamber model to examine blood from patients with different subtypes of Glanzmann's thrombasthenia (GT). Non-anticoagulated blood was perfused through capillaries coated with type III collagen for 4.5 min (shear rate: 1600/s). Platelet deposition was quantified as platelet adhesion and mean thrombus size volume; fibrin and von Willebrand Factor (VWF) were specifically revealed by immunohistochemistry. In two patients with variant and in one patient with type II GT, platelet adhesion was maximal and we observed an unexpected formation of thrombi that were smaller than normal in size. These thrombi were surrounded by a thick meshwork that displayed a strong staining for fibrin and VWF. In two patients with heterozygous GT, platelet adhesion and thrombogenesis were normal. In two patients with type I GT, there was no thrombus formation, although platelet adhesion was also maximal. These data suggest the existence of a substitute pathway for thrombogenesis mediated by fibrin and possibly alphaIIbbeta3 (alphaIIbbeta3 at a reduced level, as in type II, and/or abnormal) as this fibrin network was not observed in type I GT with no alphaIIbbeta3. These interactions might facilitate haemostasis and even lead to thrombosis under certain favourable conditions. Furthermore, these data might have pharmacological relevance to the development of anti-alphaIIbbeta3 antithrombotic agents.

A Ser752-->Pro substitution in the cytoplasmic domain of beta3 in a Glanzmann thrombasthenia variant fails to prevent interactions between the alphaIIbbeta3 integrin and the platelet granule pool of fibrinogen.

A Glanzmann thrombasthenia variant with a beta3 Ser752-->Pro cytoplasmic domain substitution has platelets that fail to aggregate or bind soluble fibrinogen (Fg) after activation. Despite this, Fg is normally present in the alpha-granules. We have used immunoelectron microscopy to examine the reactivity of Fg with the different pools of alphaIIbbeta3 in the patient's platelets. Immunogold labelling was performed on cryosections using an anti-ligand-induced binding site (LIBS) monoclonal antibody (mAb), which binds to alphaIIbbeta3 only when Fg is bound, or a mixture of two anti-receptor-induced binding site (RIBS) mAbs that specifically recognize receptor-bound Fg. Labelling of the alpha-granule membrane and channels of the surface-connected canalicular system in unstimulated platelets confirmed that the mutated alphaIIbbeta3 retains the capacity to transport Fg. When the patient's platelets were stimulated with ADP in the presence of Fg, as expected there was a much-decreased activation of surface-exposed alphaIIbbeta3. However, thrombin-induced activation was associated with both secretion and a rapid increase in the labelling of internal membrane systems by anti-RIBS and anti-LIBS mAbs, with mobilization of the internal Fg pool. Yet labelling on the surface of the patient's platelets was transient. Our studies implied that alphaIIbbeta3 in platelets may bind fibrinogen in different activation states and that this patient specifically lacked high-affinity binding.

A Leu7Pro mutation in the signal peptide of platelet glycoprotein (GP)IX in a case of Bernard-Soulier syndrome abolishes surface expression of the GPIb-V-IX complex.

This paper describes the molecular defect of the second case of Bernard-Soulier syndrome, initially reported in 1957. Analysis of the patient's platelets by flow cytometry and Western blotting failed to detect surface expression of any of the four subunits of the glycoprotein (GP)Ib-V-IX complex and revealed small amounts of intracellular GPIbalpha, GPIbbeta and GPV but no GPIX. DNA sequencing revealed a novel missense mutation in the GPIX gene which replaced Leu (CTG) by Pro (CCG) at position 7 of the signal peptide. This mutation is, to date, the only known example of a leader sequence defect in Bernard-Soulier syndrome. The change occurred in a prototypic alpha-helical hydrophobic core region, typically enriched in leucine and devoid of proline residues. Co-transfection of GPIXPro7 with normal GPIbalpha and GPIbbeta into Chinese hamster ovary cells reproduced the platelet phenotype, resulting in no detectable GPIX, low intracellular levels of GPIbalpha and GPIbbeta, and an absence of surface expression. This mutation presumably leads to an abnormal conformation and, hence, incorrect insertion of GPIX into the endoplasmic reticulum and/or to defective signal peptide cleavage, both of which are required for correct transport to the cell membrane. This provides further evidence for a critical role of GPIX in controlling biosynthesis of the GPIb-IX complex.

Erythropoietin and granulocyte colony-stimulating factor increase plasminogen activator inhibitor-1 release in HUVEC culture.

UNLABELLED: Recombinant human erythropoietin (rHuEPO) is considered to be a mitogenic and chemotactic agent in cultured endothelial cells (ECs). The effect of recombinant granulocyte-macrophage (rGM-CSF) and granulocyte (rG-CSF) colony-stimulating factors on the proliferation of human umbilical vein endothelial cells (HUVECs) has been controversial. METHODS: HUVEC proliferation and the release of endothelial markers in HUVEC culture stimulated by rHuEPO, rGM-CSF and G-CSF were measured. RESULTS: We found the dose-dependent stimulating effect of rHuEPO and rGM-CSF on HUVEC proliferation, but we did not achieve this with rG-CSF. rHuEPO like rG-CSF was an effective agent in stimulating the plasminogen activator inhibitor (PAI)-1 release from HUVECs to a cultured medium, while rGM-CSF failed. CONCLUSION: We suggest that rHuEPO showed prothrombotic changes in HUVEC culture. Our results support the idea of suspected rHuEPO direct prothrombotic role in haemodialysed patients treated with rHuEPO.