[Cultured platelets]. / Plaquettes sanguines de culture : état de l'art.

Blood platelets are anucleated elements of the blood. With a diameter of 2 to 3 µm, they are the smallest elements of blood. While their main role is to stop or prevent bleeding, they are also involved in other functions, such as immunity, inflammation or tumour progression. The development of biotechnology and the knowledge acquired about the mechanisms regulating the biogenesis of platelets makes the production of cultured platelets a viable option today. Consequently, this type of product could have its place in meeting a number of transfusion challenges such as alloimmunization or refractory states. However, culture yields remain low and many hurdles still need to be overcome before considering an application in transfusion. This article reviews the rationale for the production of cultured platelets for transfusion and summarizes the main advances in the field while highlighting its limitations.

Functional properties of human platelets derived in vitro from CD34+ cells.

The in vitro production of blood platelets for transfusion purposes is an important goal in the context of a sustained demand for controlled products free of infectious, immune and inflammatory risks. The aim of this study was to characterize human platelets derived from CD34+ progenitors and to evaluate their hemostatic properties. These cultured platelets exhibited a typical discoid morphology despite an enlarged size and expressed normal levels of the major surface glycoproteins. They aggregated in response to ADP and a thrombin receptor agonist peptide (TRAP). After infusion into NSG mice, cultured and native platelets circulated with a similar 24 h half-life. Notably, the level of circulating cultured platelets remained constant during the first two hours following infusion. During this period of time their size decreased to reach normal values, probably due to their remodeling in the pulmonary circulation, as evidenced by the presence of numerous twisted platelet elements in the lungs. Finally, cultured platelets were capable of limiting blood loss in a bleeding assay performed in thrombocytopenic mice. In conclusion, we show here that cultured platelets derived from human CD34+ cells display the properties required for use in transfusion, opening the way to clinical trials.

Decreased thrombotic tendency in mouse models of the Bernard-Soulier syndrome.

OBJECTIVE: The platelet glycoprotein (GP)Ib-V-IX complex is a receptor required for normal hemostasis deficient in the Bernard-Soulier bleeding disorder. To evaluate the consequences of GPIb-V-IX deficiency in thrombosis we generated mouse models of the disease by targeting the GPIb beta subunit. METHODS AND RESULTS: Complete deletion (GPIb beta-/-) or an intracellular truncation (GPIb beta deltaIC-/-) reproduced typical and variant forms of Bernard-Soulier, with absent and partial (20%) expression of the complex on the platelet surface. Both strains exhibited thrombocytopenia and enlarged platelets with abnormal microtubular structures but normal granule composition. They exhibited prolonged tail bleeding times, which were less pronounced in GPIb beta deltaIC-/-. Decreased thrombus formation was observed after blood perfusion over a collagen coated surface at high shear. Resistance to vascular occlusion and an abnormal thrombus composition were observed in a model of FeCl3-induced lesion of carotid arteries. In a model of laser-induced lesion of mesenteric arterioles, thrombosis was strongly reduced in GPIb beta-/- mice, while a more modest effect was observed in GPIb beta deltaIC-/- animals. Finally, the two strains were protected against death in a model of systemic thromboembolism. CONCLUSIONS: This study provides in vivo evidence of a decreased thrombotic tendency linked to defective platelet GPIb-V-IX in mouse models of Bernard-Soulier syndrome.

Microtubule plus-end tracking Adenopolyposis Coli negatively regulates proplatelet formation.

Platelets are produced upon profound reorganization of mature megakaryocytes (MK) leading to proplatelet elongation and release into the blood stream, a process termed thrombopoiesis. This highly dynamic process requires microtubules (MT) reorganization by mechanisms that are still incompletely understood. Adenomatous polyposis coli (APC) is a microtubule plus-end tracking protein involved in the regulation of MT in a number of cell systems and its inactivation has been reported to alter hematopoiesis. The aim of our study was to investigate the role of APC in megakaryopoiesis and the final steps of platelet formation. Down-regulation of APC in cultured human MK by RNA interference increased endomitosis and the proportion of cells able to extend proplatelets (68.8% (shAPC1) and 52.5% (shAPC2) vs 28.1% in the control). Similarly an increased ploidy and amplification of the proplatelet network were observed in MK differentiated from Lin- cells of mice with APC-deficiency in the MK lineage. In accordance, these mice exhibited increased platelet counts when compared to wild type mice (1,323 ± 111 vs 919 ± 52 platelets/µL; n = 12 p 0.0033**). Their platelets had a normal size, ultrastructure and number of microtubules coils and their main functions were also preserved. Loss of APC resulted in lower levels of acetylated tubulin and decreased activation of the Wnt signaling pathway. Thus, APC appears as an important regulator of proplatelet formation and overall thrombopoiesis.

Moesin, ezrin, and p205 are actin-binding proteins associated with neutrophil plasma membranes.

Actin-binding proteins in bovine neutrophil plasma membranes were identified using blot overlays with 125I-labeled F-actin. Along with surface-biotinylated proteins, membranes were enriched in major actin-binding polypeptides of 78, 81, and 205 kDa. Binding was specific for F-actin because G-actin did not bind. Further, unlabeled F-actin blocked the binding of 125I-labeled F-actin whereas other acidic biopolymers were relatively ineffective. Binding also was specifically inhibited by myosin subfragment 1, but not by CapZ or plasma gelsolin, suggesting that the membrane proteins, like myosin, bind along the sides of the actin filaments. The 78- and 81-kDa polypeptides were identified as moesin and ezrin, respectively, by co-migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoprecipitation with antibodies specific for moesin and ezrin. Although not present in detectable amounts in bovine neutrophils, radixin (a third and closely related member of this gene family) also bound 125I-labeled F-actin on blot overlays. Experiments with full-length and truncated bacterial fusion proteins localized the actin-binding site in moesin to the extreme carboxy terminus, a highly conserved sequence. Immunofluorescence micrographs of permeabilized cells and cell "footprints" showed moesin co-localization with actin at the cytoplasmic surface of the plasma membrane, consistent with a role as a membrane-actin-linking protein.

Synthesis of GPIb beta with novel transmembrane and cytoplasmic sequences in a Bernard-Soulier patient resulting in GPIb-defective signaling in CHO cells.

The molecular defect of a new Bernard-Soulier patient, originating from Morocco and presenting thrombocytopenia with large platelets and an absence of ristocetin-induced platelet agglutination, has been identified and reproduced in transfected heterologous cells. Gene sequencing revealed insertion of a guanine in the domain coding for the transmembrane region of the glycoprotein (GP) Ib beta subunit. This mutation causes a translational frame shift, which creates putative novel transmembrane and cytoplasmic 37 and 125 amino acids domains, respectively. A 34 kDa immunoreactive GPIb beta band, instead of the normal 26 kDa subunit, was detected by Western blotting in lysates from the patient's platelets and from transfected cells and in immunoprecipitates of metabolically labeled cells. The abnormal subunit did not associate with GPIb alpha and was mainly intracellular, although a significant fraction could reach the cell surface. Cells expressing the mutant GPIb-IX complex adhered to a von Willebrand factor matrix but were unable to change shape, unlike cells expressing the wild-type receptor. These results strongly suggest a novel role of the GPIb beta subunit and its transmembrane-intracellular region in GPIb-VWF-dependent signaling, in addition to a role in correct assembly and cell surface targeting of the GPIb-V-IX complex.

Cdc42-dependent F-actin dynamics drive structuration of the demarcation membrane system in megakaryocytes.

UNLABELLED: Essentials Information about the formation of the demarcation membrane system (DMS) is still lacking. We investigated the role of the cytoskeleton in DMS structuration in megakaryocytes. Cdc42/Pak-dependent F-actin remodeling regulates DMS organization for proper megakaryopoiesis. These data highlight the mandatory role of F-actin in platelet biogenesis. SUMMARY: Background Blood platelet biogenesis results from the maturation of megakaryocytes (MKs), which involves the development of a complex demarcation membrane system (DMS). Therefore, MK differentiation is an attractive model for studying membrane remodeling. Objectives We sought to investigate the mechanism of DMS structuration in relationship to the cytoskeleton. Results Using three-dimensional (3D) confocal imaging, we have identified consecutive stages of DMS organization that rely on F-actin dynamics to polarize membranes and nuclei territories. Interestingly, microtubules are not involved in this process. We found that the mechanism underlying F-actin-dependent DMS formation required the activation of the guanosine triphosphate hydrolase Cdc42 and its p21-activated kinase effectors (Pak1/2/3). Förster resonance energy transfer demonstrated that active Cdc42 was associated with endomembrane dynamics throughout terminal maturation. Inhibition of Cdc42 or Pak1/2/3 severely destructured the DMS and blocked proplatelet formation. Even though this process does not require containment within the hematopoietic niche, because DMS structuration was observed upon thrombopoietin-treatment in suspension, integrin outside-in signaling was required for Pak activation and probably resulted from secretion of extracellular matrix by MKs. Conclusions These data indicate a functional link, mandatory for MK differentiation, between actin dynamics, regulated by Cdc42/Pak1/2/3, and DMS maturation.

Lentiviral gene rescue of a Bernard-Soulier mouse model to study platelet glycoprotein Ibß function.

UNLABELLED: Essentials A signaling role of glycoprotein (GP)Ibß is postulated but not formally demonstrated in platelets. Lentiviral-mediated rescue in knock-out mice can be used to evaluate GPIbß function in vivo. Transduction of the native subunit corrected the main defects associated with GPIb-IX deficiency Deletion of intracellular 159-170 segment increased thrombosis, 150-160 removal increased bleeding. SUMMARY: Background The platelet glycoprotein (GP)Ib-V-IX complex is required for normal hemostasis and megakaryopoiesis. A role in GPIb-dependent responses has been ascribed to the less well characterized GPIbß subunit using a specific antibody and GPIb-IX transfected cells. Objectives Our aim was to evaluate, in vivo, the role of the GPIbß in hemostasis and thrombosis. Methods GPIbß(null) Sca-1(+) progenitors transduced with viral particles harboring hGPIbß were transplanted into lethally irradiated GPIbß(-/-) recipient mice. Results hGPIbß transplanted into the bone marrow of GPIbß(null) mice rescued GPIb-IX expression in 97% of circulating platelets. These platelets efficiently bound von Willebrand factor (VWF) and extended filopodia on a VWF matrix, demonstrating the restoration of GPIb-dependent adhesive and signaling properties. These mice exhibited less severe macrothrombocytopenia and had normal tail bleeding times as compared with GPIbß(null) mice. This strategy was employed to manipulate and evaluate the role of the GPIbß intracellular domain. Removal of the membrane proximal segment (Δ(150-160) ) decreased GPIb-IX expression by 43%, confirming its involvement in receptor assembly and biosynthesis, and resulted in increased bleeding times and decreased thrombosis in a mechanical injury model in the aorta. On the other hand, deletion of the C-flanking 159-170 segment allowed normal GPIb-IX expression, VWF-dependent responses and bleeding times, but resulted in enhanced arterial thrombosis. Conclusion This pointed to a repressor role of GPIbß in thrombus formation in vivo that was not predicted in studies of heterologous cells. These results highlight the utility of this lentiviral strategy for the structure-function evaluation of GPIb-IX in platelets.

Broader expression of the mouse platelet factor 4-cre transgene beyond the megakaryocyte lineage.

BACKGROUND: Transgenic mice expressing cre recombinase under the control of the platelet factor 4 (Pf4) promoter, in the context of a 100-kb bacterial artificial chromosome, have become a valuable tool with which to study genetic modifications in the platelet lineage. However, the specificity of cre expression has recently been questioned, and the time of its onset during megakaryopoiesis remains unknown. OBJECTIVES/METHODS: To characterize the expression of this transgene, we used double-fluorescent cre reporter mice. RESULTS: In the bone marrow, Pf4-cre-mediated recombination had occurred in all CD42-positive megakaryocytes as early as stage I of maturation, and in rare CD42-negative cells. In circulating blood, all platelets had recombined, along with only a minor fraction of CD45-positive cells. However, we found that all tissues contained recombined cells of monocyte/macrophage origin. When recombined, these cells might potentially modify the function of the tissues under particular conditions, especially inflammatory conditions, which further increase recombination in immune cells. Unexpectedly, a subset of epithelial cells from the distal colon showed signs of recombination resulting from endogenous Pf4-cre expression. This is probably the basis of the unexplained colon tumors developed by Apc(flox/flox) ;Pf4-cre mice, generated in a separate study on the role of Apc in platelet formation. CONCLUSION: Altogether, our results indicate early recombination with full penetrance in megakaryopoiesis, and confirm the value of Pf4-cre mice for the genetic engineering of megakaryocytes and platelets. However, care must be taken when investigating the role of platelets in processes outside hemostasis, especially when immune cells might be involved.

The platelet glycoprotein GPIbbeta intracellular domain participates in von Willebrand factor induced-filopodia formation independently of the Ser 166 phosphorylation site.

SUMMARY BACKGROUND: Circulating platelets are initially recruited at the site of vessel injury by von Willebrand factor (VWF) immobilized on collagen fibers. This process, mediated by the GPIb-V-IX complex, is accompanied by specific intracellular signaling leading to reorganization of the platelet actin cytoskeleton and extension of filopodia. OBJECTIVES/METHODS: To evaluate the GPIbalpha and GPIbbeta intracellular domains contribution to this signaling, we generated Chinese hamster ovary (CHO) cells expressing a GPIb-IX complex with mutant forms of the two subunits and we measured their ability to extend filopodia upon adhesion on a VWF matrix. RESULTS: Complete intracellular deletion or elimination of the filamin or the 14-3-3zeta binding sites in GPIbalpha did not prevent filopodia extension. In contrast, deletion of the juxtamembrane (Leu(150)-Arg(160)) or central (Ala(159)-Pro(170)) intracellular segment of GPIbbeta resulted in a 21% and 23% reduction in the number of cells extending filopodia, respectively. This occurred without decreasing adhesion efficiency or GPIb-IX association with filamin A or 14-3-3zeta. Alanine scanning mutagenesis of the Leu(150)-Pro(170) segment identified Arg(164), Leu(165), Leu(167), Thr(168) and Pro(170) as important residues for efficient filopodia formation. Surprisingly, mutation of the Ser(166) PKA phosphorylation site did not alter adhesion and shape change. A role for the GPIbbeta subunit was reinforced by the decreased capacity to extend filopodia upon adhesion on VWF of platelets from knock-in mice expressing a GPIbbeta intracellular deletion mutant. CONCLUSIONS: Altogether, our results strongly support participation of GPIbbeta and its intracellular region in GPIb-dependent platelet activation and shape change triggered by a VWF matrix.

Biosynthesis and intracellular post-translational processing of normal and mutant platelet glycoprotein GPIb-IX.

The multisubunit leucine-rich glycoprotein (GP) Ib-IX-V complex mediates von Willebrand factor-dependent platelet adhesion at sites of blood-vessel injury. Molecular defects of this receptor are reported to cause the Bernard-Soulier haemorrhagic disorder. To gain insight into the mechanisms controlling expression of normal and defective receptors, we performed pulse-chase metabolic studies and detailed analysis of intracellular processing in GPIb-IX-transfected Chinese-hamster ovary cells. In the native complex, after early subunit association, sugars N-linked to the three subunits are trimmed and sialylated in the Golgi compartment and GPIbalpha undergoes extensive O-glycosylation. Surface biotinylation during chase demonstrated that only fully processed complexes reach the cell surface. Tunicamycin treatment revealed that early N-glycosylation is not required for O-glycosylation of GPIbalpha and surface expression of the complex. Biosynthetic studies were then performed on a Bernard-Soulier variant based on previous description of abnormal GPIbalpha size and decreased surface expression. The mutant complex associated normally, but displayed defective processing of its N-linked sugars and abnormal O-glycosylation of GPIbalpha. Confocal immunofluorescence microscopy revealed that the mutant complexes could reach the cell surface but also accumulated intracellularly, while use of compartment specific markers showed strong co-localization in the endoplasmic reticulum (ER) and ER-to-Golgi intermediate compartments ('ERGIC') and only slight labelling of the cis-Golgi. Blockade before the Golgi was confirmed by brefeldin A treatment, which restored O-glycosylation and processing of N-linked sugars. The present study has shown that transfer from the ER to the Golgi represents an important step for controlling post-translational processing and surface expression of normal GPIb-IX-V complex.

Cloning, characterization, and chromosomal localization of human superillin (SVIL).

Supervillin is a 205-kDa F-actin binding protein originally isolated from bovine neutrophils. This protein is tightly associated with both actin filaments and plasma membranes, suggesting that it forms a high-affinity link between the actin cytoskeleton and the membrane. Human supervillin cDNAs cloned from normal human kidney and from the cervical carcinoma HeLa S3 predict a bipartite structure with three potential nuclear localization signals in the NH2-terminus and three potential actin-binding sequences in the COOH-terminus. In fact, throughout its length, the COOH-terminal half of supervillin is similar to segments 2-6 plus the COOH-terminal "headpiece" of villin, an actin-binding protein in intestinal microvilli. A comparison of the bovine and human sequences indicates that supervillin is highly conserved at the amino acid level, with 79.2% identity of the NH2-terminus and conservation of three of the four nuclear localization signals found in bovine supervillin. The COOH-terminus is even more conserved, with 95.1% amino acid identity overall and 100% conservation of the villin-like headpiece. Supervillin mRNAs are expressed in all human tissue tested, bu are most abundant in muscle, bone marrow, thyroid gland, and salivary gland; comparatively little message is found in brain. Human supervillin mRNA is approximately 7.5 kb; this message is especially abundant in HeLa S3 cervical carcinoma, SW480 adenocarcinoma, and A549 lung carcinoma cell lines. The human supervillin gene (SVIL) is localized to a single chromosomal locus at 10p11.2, a region that is deleted in some prostate tumors.

Platelet glycoprotein V binds to collagen and participates in platelet adhesion and aggregation.

Glycoprotein V (GPV) is a subunit of the platelet GPIb-V-IX receptor for von Willebrand factor and thrombin. GPV is cleaved from the platelet surface during activation by thrombin, but its role in hemostasis is still unknown. It is reported that GPV knockout mice had a decreased tendency to form arterial occluding thrombi in an intravital thrombosis model and abnormal platelet interaction with the subendothelium. In vitro, GPV-deficient platelets exhibited defective adhesion to a collagen type I-coated surface under flow or static conditions. Aggregation studies demonstrated a decreased response of the GPV-deficient platelets to collagen, reflected by an increased lag phase and reduced amplitude of aggregation. Responses to adenosine diphosphate, arachidonic acid, and the thromboxane analog U46619 were normal but were enhanced to low thrombin concentrations. The defect of GPV null platelets made them more sensitive to inhibition by the anti-GPVI monoclonal antibody (mAb) JAQ1, and this was also the case in aspirin- or apyrase-treated platelets. Moreover, an mAb (V.3) against the extracellular domain of human GPV selectively inhibited collagen-induced aggregation in human or rat platelets. V.3 injected in rats as a bolus decreased the ex vivo collagen aggregation response without affecting the platelet count. Finally, surface plasmon resonance studies demonstrated binding of recombinant soluble GPV on a collagen-coupled matrix. In conclusion, GPV binds to collagen and appears to be required for normal platelet responses to this agonist. (Blood. 2001;98:1038-1046)

Actin-binding membrane proteins identified by F-actin blot overlays.

Actin and associated proteins at the cytoskeleton-plasma membrane interface stabilize the membrane bilayer, control cell shape, and delimit specialized membrane domains. To identify membrane proteins that bind directly to F-actin, we have developed a blot overlay assay with 125I-labeled F-actin. In the soil amoebae, Dictyostelium discoideum, the major proteins reactive in this assay are p30a, a 34-kD peripheral membrane protein that is concentrated in filopodia and at sites of cell-cell adhesion, and ponticulin, a 17-kD transmembrane glycoprotein required for efficient chemotaxis and for control of pseudopod dynamics. Proteins with apparent molecular masses of approximately 34- and approximately 17-kD also are observed on F-actin blot overlays of many mammalian cell lines. However, in mammalian cells, the most prominent F-actin binding proteins in this assay exhibit apparent molecular masses of 78-, 80-, 81-, approximately 120-, and 205-kD. Bovine neutrophils contain the 78-, 81-, and 205-kD proteins, all of which co-isolate with a plasma membrane-enriched fraction. We have previously identified the 78-, 80-, and 81-kD proteins as moesin, radixin, and ezrin, respectively. These proteins, which are members of the protein 4.1 superfamily, colocalize with actin in cell surface extensions and have been implicated in the protrusion of microvilli, filopodia, and membrane ruffles. The 205-kD protein (p205) appears to be absent from current databases, and its characteristics are still under investigation. We here report that the 120-kD protein is drebrin, a submembranous actin-binding protein originally identified as a developmentally regulated brain protein. Thus, it appears that F-actin blot overlays provide an efficient assay for simultaneous monitoring of a subset of F-actin binding proteins, including p30a, ponticulin, moesin, radixin, ezrin, p205, and drebrin.