[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.

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.

[Comparative study on platelet transfusion in Lorraine-Champagne and Alsace in 2015]. / Étude comparative des transfusions de concentrés plaquettaires en Alsace et en Lorraine-Champagne en 2015.

OBJECTIVES: To find explanations for the observed differences of platelets concentrates (PC) deliveries between 2 French regions, Lorraine-Champagne (LOCH) and Alsace (ALSA). METHODS: This is a non-interventional prospective study, performed during 30 days in 2015 in intensive care, cardiovascular surgery and onco-hematological wards of 8 LOCH and ALSA hospitals. Questionnaires listing clinical and biological parameters were attached to the prescription forms and filled in at each PC prescription. RESULTS: In all, 290 patients, 1093 prescriptions and 1093 deliveries of PC were analyzed. The pre-transfusional context (patient weight, prophylactic or curative situation, pre-transfusional platelet count) were homogenous. The phasing of the prescription forms wording had a direct impact on the doses' formulation : 100 % of the LOCH forms were expressed in platelet quantity (PQ), vs 22 % in ALSA. The mean interval between 2 transfusions was 2.9 days in ALSA vs 4.9 days in LOCH. The mean PQ/delivery was higher in ALSA (5.6.1011 vs 4.0.1011 in LOCH). The delivered PQs were compared to the 2003 French recommendations that were in force in 2015. Twenty-seven percent of the LOCH delivered PQs were in the recommended interval, vs Forty-nine percent in ALSA. Due to the systematic delivery of a single PC unit, including weights>80Kg, LOCH presented 63 % insufficient PQ deliveries. Twenty-two percent of the deliveries in ALSA were over the recommended interval, mostly linked with the simultaneous delivery of a second PC. CONCLUSION: This study identifies disparities in terms of prescription and delivery between LOCH and ALSA, which may explain their respective PC consumption levels.

Platelet transfusion: Current challenges.

Since the late sixties, platelet concentrates are transfused to patients presenting with severe thrombocytopenia, platelet function defects, injuries, or undergoing surgery, to prevent the risk of bleeding or to treat actual hemorrhage. Current practices differ according to the country or even in different hospitals and teams. Although crucial advances have been made during the last decades, questions and debates still arise about the right doses to transfuse, the use of prophylactic or therapeutic strategies, the nature and quality of PC, the storage conditions, the monitoring of transfusion efficacy and the microbiological and immunological safety of platelet transfusion. Finally, new challenges are emerging with potential new platelet products, including cold stored or in vitro produced platelets. The most debated of these points are reviewed.

[Adaptation of platelet concentrates delivery to HAS/ANSM requirements evolution in EFS Grand-Est]. / Adaptation de la délivrance des concentrés plaquettaires aux recommandations HAS/ANSM 2015 à l'EFS Grand-Est.

Evolution of French HAS/ANSM guidelines in 2015 about optimal platelet transfusion dose for patients brought us to review about our practices for platelet concentrates delivery in EFS Grand-Est region. In addition, pathogen inactivation in platelet concentrates has been implemented all over the country and transfusion centers have merged. All these changes required harmonization. In this context, our major issue was to answer patient's requirements, according to the new guidelines keeping in mind the cost effectiveness on public finances. We report here on the changes in our practices and their impacts.

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.

Platelets are dispensable for antibody-mediated transfusion-related acute lung injury in the mouse.

UNLABELLED: Essentials Role of platelets in immunological transfusion-related acute lung injury (TRALI) is debated. Immunological TRALI was tested in mice exhibiting severe thrombocytopenia or platelet dysfunction. Platelets are required to prevent lung hemorrhage but not edema formation and respiratory distress. Platelets are dispensable for the initiation and development of TRALI. SUMMARY: Background Transfusion-related acute lung injury (TRALI) is a serious transfusion-related complication. Previous conflicting studies have indicated that platelets are either crucial or dispensable for TRALI. Objectives To evaluate the role of platelets in major histocompatibility complex (MHC) I-induced-TRALI. Methods Antibody-mediated TRALI was experimentally induced in mice by lipopolysaccharide priming followed by the administration of an anti-MHC I mAb. Results TRALI was tested in the context of severe thrombocytopenia provoked by the administration of diphtheria toxin (DT) in transgenic iDTR mice selectively expressing DT receptor in megakaryocytes. The pathologic responses occurring within the first 10 min following the injection of the anti-MHC I mAb, i.e. the severity of lung edema and the drop in aortic blood oxygenation, were similar in severely thrombocytopenic DT-iDTR and control mice. At later times, mortality was nevertheless increased in DT-iDTR mice, owing to lung hemorrhages. When less severe thrombocytopenia was induced with an antiplatelet mAb, TRALI started and developed similarly as in control mice, but hemorrhages were absent. Furthermore, when platelet functions were defective because of administration of aspirin or clopidogrel, or because of glycoprotein (GP)IIbIIIa deficiency, TRALI still developed but no lung hemorrhages were observed. In contrast, when GPVI was immunodepleted, TRALI still occurred, but was occasionally accompanied by hemorrhages. Conclusions Platelets are dispensable for the initiation and development of MHC I-induced TRALI. Although they do not protect against the disruption of the vascular endothelial cell barrier and the subsequent plasma leakage and edema formation, platelets are essential to prevent more serious damage resulting in hemorrhages in alveoli.

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.

Platelets in cancer. From basic research to therapeutic implications.

Platelets are well-known for their major role in primary hemostasis and thrombosis. Cancer patients frequently manifest thrombotic events and present abnormalities in blood coagulation which appear to be linked to altered platelet function and turnover. Moreover, numerous studies indicate an intimate cross-talk between platelets and tumor growth, angiogenesis and metastatic dissemination. Finally, several experimental data and clinical trials suggest possible benefits of anti-platelet drugs on some cancers. Here, we will review the current state of basic biological research regarding the role of platelets in cancer progression. We also critically review the possible clinical applicability of some anti-platelet therapies to limit tumor growth and prevent metastatic dissemination.

Antiplatelet drugs: which targets for which treatments?

The current standard care for acute coronary syndromes is dual antiplatelet therapy combining the COX1 inhibitor aspirin with a drug targeting the P2Y12 receptor, together with anticoagulation during and after early revascularization by percutaneous intervention. In very high-risk patients, glycoprotein (GP) IIb/IIIa antagonists may also be used. Secondary prevention of ischemic events requires dual antiplatelet therapy for several months followed by lifelong low-dose aspirin. The duration of treatment and the drugs to combine nevertheless remain matters of debate and the focus of ongoing research. Despite great progress, there is still room for improved efficacy and this could involve new targets for both antiplatelet drugs (like the thrombin receptor PAR1) and anticoagulants. However, improved efficacy is offset by an increased risk of bleeding. Stroke patients are still waiting for better treatment, their bleeding risk being particularly high. New targets including the collagen receptor, glycoprotein VI (GPVI), and the GPIb-von Willebrand factor axis, governing platelet interaction with the diseased vessel wall, should enable us to complete the armamentarium of antiplatelet drugs.

Distinct localizations and roles of non-muscle myosin II during proplatelet formation and platelet release.

BACKGROUND: At the end of maturation, megakaryocytes (MKs) form long cytoplasmic extensions called proplatelets (PPT). Enormous changes in cytoskeletal structures cause PPT to extend further, to re-localize organelles such as mitochondria and to fragment, leading to platelet release. Two non-muscle myosin IIs (NMIIs) are expressed in MKs; however, only NMII-A (MYH9), but not NMII-B (MYH10), is expressed in mature MKs and is implicated in PPT formation. OBJECTIVES: To provide in vivo evidence on the specific role of NMII-A and IIB in MK PPT formation. METHODS: We studied two transgenic mouse models in which non-muscle myosin heavy chain (NMHC) II-A was genetically replaced either by II-B or by a chimeric NMHCII that combined the head domain of II-A with the rod and tail domains of II-B. RESULTS AND CONCLUSIONS: This work demonstrates that the kinetic properties of NM-IIA, depending on the N-terminal domain, render NMII-A the better NMII candidate to control PPT formation. Furthermore, the carboxyl-terminal domain determines myosin II localization in the constriction region of PPT and is responsible for the specific role of NMII in platelet release.

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.

Diagnosis of suspected inherited platelet function disorders: results of a worldwide survey.

BACKGROUND: Diagnosis of inherited platelet function disorders (IPFDs) is important for appropriate management and to improve epidemiologic and clinical knowledge. However, there remains a lack of consensus on the diagnostic approach. OBJECTIVES: To gain knowledge on the current practices for the diagnosis of IPFD worldwide. METHODS: A 67-item questionnaire was distributed to the ISTH members and to the members of several national hemostasis and thrombosis societies. RESULTS: A total of 202 laboratories from 37 countries participated in the survey. The most frequent criterion to define patients with a suspected IPFD was a history of mucocutaneous bleeding and no acquired cause, but heterogeneity on the identification criteria was evident. Only 64.5% of respondents performed a direct clinical interview. On average, each laboratory studied 72 patients per year. The most commonly used laboratory equipment were the light-transmission aggregometer, the Platelet Function Analyzer-100, and the flow cytometer. Screening tests were platelet count, peripheral blood smear, light-transmission aggregometry, and Platelet Function Analyzer-100. Second-step tests were flow cytometry, molecular genetic analysis, and electron microscopy. Methodologies varied widely. In total, ~ 14,000 patients were investigated yearly and 60% turned out to not have a defect. Of the remaining 40%, only 8.7% received a diagnosis at a molecular level. CONCLUSIONS: Many laboratories worldwide are involved in the diagnosis of IPFD. A large fraction of the patients studied remain without a diagnosis. A high variability in the diagnostic approaches is evident.

The future of glycoprotein VI as an antithrombotic target.

The treatment of acute coronary syndromes has been considerably improved in recent years with the introduction of highly efficient antiplatelet drugs. However, there are still significant limitations: the recurrence of adverse vascular events remains a problem, and the improvement in efficacy is counterbalanced by an increased risk of bleeding, which is of particular importance in patients at risk of stroke. One of the most attractive targets for the development of new molecules with potential antithrombotic activity is platelet glycoprotein (GP)VI, because its blockade appears to ideally combine efficacy and safety. This review summarizes current knowledge on GPVI regarding its structure, its function, and its role in physiologic hemostasis and thrombosis. Strategies for inhibiting GPVI are presented, and evidence of the antithrombotic efficacy and safety of GPVI antagonists is provided.

ß-arrestin-1 participates in thrombosis and regulates integrin aIIbß3 signalling without affecting P2Y receptors desensitisation and function.

ß-arrestin-1 (ß-arr1) and ß-arrestin-2 (ß-arr2) are cytosolic proteins well-known to participate in G protein-coupled receptor desensitisation and signalling. We used genetically-inactivated mice to evaluate the role of ß-arr1 or ß-arr2 in platelet function, P2Y receptor desensitisation, haemostasis and thrombosis. Platelet aggregation, soluble fibrinogen binding and P-selectin exposure induced by various agonists were near normal in ß-arr1-/- and ß-arr2-/- platelets. In addition, deficiency in ß-arr1 or ß-arr2 was not critical for P2Y receptors desensitisation. A functional redundancy between ß-arr1 and ß-arr2 may explain these unchanged platelet responses. Interestingly, ß-arr1-/- but not ß-arr2-/- mice were protected against laser- and FeCl3-induced thrombosis. The tail bleeding times, number of rebleeds and volume of blood loss were unchanged in ß-arr1-/- and ß-arr2-/- mice, suggesting no defect in haemostasis. ß-arr1-/- platelet activation upon adhesion to immobilised fibrinogen was inhibited, as attested by a 37 ± 5% (n = 3, p<0.0001) decrease in filopodia extension, suggesting defective signalling through integrin αIIbß3. ß-arr1 appeared to be located downstream of Src family kinases and to regulate αIIbß3 signalling by increasing Akt phosphorylation. Overall, this study supports a role for ß-arr1 in promoting thrombus formation, in part through its participation in αIIbß3 signalling, and no role of ß-arr1 and ß-arr2 in agonist-induced platelet activation and P2Y receptors desensitisation.