Macrophage depletion overcomes human hematopoietic cell engraftment failure in zebrafish embryo.

Zebrafish is widely adopted as a grafting model for studying human development and diseases. Current zebrafish xenotransplantations are performed using embryo recipients, as the adaptive immune system, responsible for host versus graft rejection, only reaches maturity at juvenile stage. However, transplanted primary human hematopoietic stem/progenitor cells (HSC) rapidly disappear even in zebrafish embryos, suggesting that another barrier to transplantation exists before the onset of adaptive immunity. Here, using a labelled macrophage zebrafish line, we demonstrated that engraftment of human HSC induces a massive recruitment of macrophages which rapidly phagocyte transplanted cells. Macrophages depletion, by chemical or pharmacological treatments, significantly improved the uptake and survival of transplanted cells, demonstrating the crucial implication of these innate immune cells for the successful engraftment of human cells in zebrafish. Beyond identifying the reasons for human hematopoietic cell engraftment failure, this work images the fate of human cells in real time over several days in macrophage-depleted zebrafish embryos.

Development of an efficient, ready to use, blood platelet-release device based on two new flow regime parameters: The periodic hydrodynamic loading and the shear stress accumulation.

In vitro production of blood platelets for transfusion purposes is gaining interest. While platelet production is now possible on a laboratory scale, the challenge is to move towards industrial production. Attaining this goal calls for the development of platelet release devices capable of producing large quantities of platelets. To this end, we have developed a continuous-flow platelet release device composed of five spherical chambers each containing two calibrated cones placed in a staggered configuration. Following perfusion of proplatelet-bearing cultured megakaryocytes, the device achieves a high yield of about 100 bona-fide platelets/megakaryocyte, at a flow rate of ∼80 mL/min. Performances and operating conditions comply with the requirements of large-scale platelet production. Moreover, this device enabled an in-depth analysis of the flow regimes through Computational Fluid Dynamics (CFD). This revealed two new universal parameters to be taken into account for an optimal platelet release: i.e. a periodic hydrodynamic load and a sufficient accumulation of shear stress. An efficient 16 Pa.s shear stress accumulation is obtained in our system at a flow rate of 80 mL/min.

ADP receptor P2Y12 is the capstone of the cross-talk between Ca2+ mobilization pathways dependent on Ca2+ ATPases sarcoplasmic/endoplasmic reticulum type 3 and type 2b in platelets.

Background: Blood platelet Ca2+ stores are regulated by 2 Ca2+-ATPases (SERCA2b and SERCA3). On thrombin stimulation, nicotinic acid adenosine dinucleotide phosphate mobilizes SERCA3-dependent stores, inducing early adenosine 5'-diphosphate (ADP) secretion, potentiating later SERCA2b-dependent secretion. Objectives: The aim of this study was to identify which ADP P2 purinergic receptor (P2Y1 and/or P2Y12) is(are) involved in the amplification of platelet secretion dependent on the SERCA3-dependent Ca2+ mobilization pathway (SERCA3 stores mobilization) as triggered by low concentration of thrombin. Methods: The study used the pharmacologic antagonists MRS2719 and AR-C69931MX, of the P2Y1 and P2Y12, respectively, as well as Serca3 -/- mice and mice exhibiting platelet lineage-specific inactivation of the P2Y1 or P2Y12 genes. Results: We found that in mouse platelets, pharmacological blockade or gene inactivation of P2Y12 but not of P2Y1 led to a marked inhibition of ADP secretion after platelet stimulation with low concentration of thrombin. Likewise, in human platelets, pharmacological inhibition of P2Y12 but not of P2Y1 alters amplification of thrombin-elicited secretion through SERCA2b stores mobilization. Finally, we show that early SERCA3 stores secretion of ADP is a dense granule secretion, based on parallel adenosine triphosphate and serotonin early secretion. Furthermore, early secretion involves a single granule, based on the amount of adenosine triphosphate released. Conclusion: Altogether, these results show that at low concentrations of thrombin, SERCA3- and SERCA2b-dependent Ca2+ mobilization pathways cross-talk via ADP and activation of the P2Y12, and not the P2Y1 ADP receptor. The relevance in hemostasis of the coupling of the SERCA3 and the SERCA2b pathways is reviewed.

Marginal zone B cells are responsible for the production of alloantibodies following platelet transfusion in mice.

Alloimmunization against platelets remains a potentially serious adverse transfusion event. Alloantibodies produced by the recipient, mainly directed against human leukocyte antigen class I donor antigens, can compromise the therapeutic efficacy of subsequent transfusions, and may lead to refractoriness. Because the mechanism of anti-HLA alloantibody formation is poorly understood, this study aimed to identify the cells involved in the platelet immune response by focusing on the spleen, the main organ that orchestrates this alloimmune response. In the spleen, transfused allogeneic platelets are located in the marginal zone and interact with marginal zone B (MZB) cells, a specialized B-cell population implicated in the capture and follicular delivery of blood-borne antigens. To study the involvement of MZB cells in alloantibody production, we used a murine model reproducing major histocompatibility complex incompatibility between a donor (H2b) and recipient (H2d) that occurs during platelet transfusion. Following weekly H2b platelet transfusions, recipient H2d mice produced anti-H2b immunoglobulin G, which induced a refractory state upon subsequent transfusions. Specific immunodepletion of MZB cells or their displacement from the marginal zone to the B-cell follicles by treatment with an S1P1 antagonist before each transfusion prevented significant alloantibody formation. Under these conditions, transfused platelets were still circulating after 24 hours, whereas they were rapidly removed from circulation in alloimmunized mice. The identification of MZB cells as key players in the platelet alloimmune response opens up new perspectives for minimizing platelet alloimmunization and avoiding the associated refractory state in frequently transfused patients.

Platelet dysfunction and thrombus instability in flow conditions in patients with severe COVID-19.

Severe COVID-19 has been associated with a high rate of thrombotic events but also of bleeding events, particularly when the level of prophylactic anticoagulation was increased. Data on the contribution of platelets to these thrombotic events are discordant between reports, while the involvement of platelets in bleeding events has never been investigated. The objective of the present study was to assess platelet function during the first week of ICU hospitalization in patients with severe COVID-19 pneumonia. A total of 35 patients were prospectively included and blood samples were drawn on day (D) 0, D2 and D7. COVID-19 pneumonia was severe with a median PaO2/FiO2 ratio of 91 [68-119] on D0. Platelets from these patients showed evidence of pre-activation and exhaustion with a significant reduction in the surface expression of GPVI, GPIb and GPIIbIIIa, together with a decrease in serotonin content. Platelets from patients with severe COVID-19 were hyporesponsive with a reduced maximal aggregation response to several platelet agonists and decreased adhesion to immobilized fibrinogen. Aggregation of washed platelets and plasma substitution experiments indicated that a plasma factor was at least partially responsible for this hyporeactivity of platelets. Blood flow experiments showed that severe COVID-19 platelets formed smaller, less stable aggregates on a collagen-coated surface, which could explain why some patients develop bleeding events. These findings should prompt us to carefully evaluate the risks and benefits of high-dose prophylactic anticoagulation, and to decrease the level of anticoagulation once the initial phase of the disease has resolved. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT04359992.

Treatment of congenital thrombocytopenia and decreased collagen reactivity in G6b-B-deficient mice.

Mice lacking the immunoreceptor tyrosine-based inhibition motif-containing co-inhibitory receptor G6b-B (Mpig6b, G6b knockout, KO) are born with a complex megakaryocyte (MK) per platelet phenotype, characterized by severe macrothrombocytopenia, expansion of the MK population, and focal myelofibrosis in the bone marrow and spleen. Platelets are almost completely devoid of the glycoprotein VI (GPVI)-FcRγ-chain collagen receptor complex, have reduced collagen integrin α2ß1, elevated Syk tyrosine kinase activity, and a subset has increased surface immunoglobulins. A similar phenotype was recently reported in patients with null and loss-of-function mutations in MPIG6B. To better understand the cause and treatment of this pathology, we used pharmacological- and genetic-based approaches to rescue platelet counts and function in G6b KO mice. Intravenous immunoglobulin resulted in a transient partial recovery of platelet counts, whereas immune deficiency did not affect platelet counts or receptor expression in G6b KO mice. Syk loss-of-function (R41A) rescued macrothrombocytopenia, GPVI and α2ß1 expression in G6b KO mice, whereas treatment with the Syk kinase inhibitor BI1002494 partially rescued platelet count but had no effect on GPVI and α2ß1 expression or bleeding. The Src family kinase inhibitor dasatinib was not beneficial in G6b KO mice. In contrast, treatment with the thrombopoietin mimetic romiplostim rescued thrombocytopenia, GPVI expression, and platelet reactivity to collagen, suggesting that it may be a promising therapeutic option for patients lacking functional G6b-B. Intriguingly, GPVI and α2ß1 expression were significantly downregulated in romiplostim-treated wild-type mice, whereas GPVI was upregulated in romiplostim-treated G6b KO mice, suggesting a cell intrinsic feedback mechanism that autoregulates platelet reactivity depending on physiological needs.

Traumatic vessel injuries initiating hemostasis generate high shear conditions.

Blood flow is a major regulator of hemostasis and arterial thrombosis. The current view is that low and intermediate flows occur in intact healthy vessels, whereas high shear levels (>2000 s-1) are reached in stenosed arteries, notably during thrombosis. To date, the shear rates occurring at the edge of a lesion in an otherwise healthy vessel are nevertheless unknown. The aim of this work was to measure the shear rates prevailing in wounds in a context relevant to hemostasis. Three models of vessel puncture and transection were developed and characterized for a study that was implemented in mice and humans. Doppler probe measurements supplemented by a computational model revealed that shear rates at the edge of a wound reached high values, with medians of 22 000 s-1, 25 000 s-1, and 7000 s-1 after puncture of the murine carotid artery, aorta, or saphenous vein, respectively. Similar shear levels were observed after transection of the mouse spermatic artery. These results were confirmed in a human venous puncture model, where shear rates in a catheter implanted in the cubital vein reached 2000 to 27 000 s-1. In all models, the high shear conditions were accompanied by elevated levels of elongational flow exceeding 1000 s-1. In the puncture model, the shear rates decreased steeply with increasing injury size. This phenomenon could be explained by the low hydrodynamic resistance of the injuries as compared with that of the downstream vessel network. These findings show that high shear rates (>3000 s-1) are relevant to hemostasis and not exclusive to arterial thrombosis.

A gain-of-function variant in the Wiskott-Aldrich syndrome gene is associated with a MYH9-related disease-like syndrome.

While loss-of-function variants in the WAS gene are associated with Wiskott-Aldrich syndrome and lead to microthrombocytopenia, gain-of-function variants of WAS are associated with X-linked neutropenia (XLN) and the absence of microthrombocytopenia. Only a few XLN families have been reported so far, and their platelet phenotype was not described in detail. To date, no renal involvement was described in XLN. In the present study, we report exome sequencing of individuals from 3 generations of a family with a dominant disease combining neutropenia, macrothrombocytopenia, and renal failure. We identified a heterozygous missense gain-of-function variant in the WAS gene (c.881T>C, p.I294T) that segregates with the disease and is already known to cause XLN. There was no pathogenic variant in MYH9, TUBB1, or ACTN1. This is the first report of a WAS gain-of-function variant associated with both the hematological phenotype of XLN (neutropenia, macrothrombocytopenia) and renal disease (proteinuria, renal failure) with glomerular tip lesion hyalinosis and actin condensations in effaced podocytes foot processes.

Rasa3 deficiency minimally affects thrombopoiesis but promotes severe thrombocytopenia due to integrin-dependent platelet clearance.

Platelet homeostasis is dependent on a tight regulation of both platelet production and clearance. The small GTPase Rap1 mediates platelet adhesion and hemostatic plug formation. However, Rap1 signaling is also critical for platelet homeostasis as both Rap1 deficiency and uninhibited Rap1 signaling lead to marked thrombocytopenia in mice. Here, we investigated the mechanism by which deficiency in Rasa3, a critical negative regulator of Rap1, causes macrothrombocytopenia in mice. Despite marked morphological and ultrastructural abnormalities, megakaryocytes in hypomorphic Rasa3hlb/hlb (R3hlb/hlb) or Rasa3-/- mice demonstrated robust proplatelet formation in vivo, suggesting that defective thrombopoiesis is not the main cause of thrombocytopenia. Rather, we observed that R3hlb/hlb platelets became trapped in the spleen marginal zone/red pulp interface, with evidence of platelet phagocytosis by macrophages. Clearance of mutant platelets was also observed in the liver, especially in splenectomized mice. Platelet count and platelet life span in Rasa3-mutant mice were restored by genetic or pharmacological approaches to inhibit the Rap1/talin1/αIIbß3 integrin axis. A similar pattern of splenic clearance was observed in mice injected with anti-αIIbß3 but not anti-glycoprotein Ibα platelet-depleting antibodies. In summary, we describe a potentially novel, integrin-based mechanism of platelet clearance that could be critical for our understanding of select inherited and acquired thrombocytopenias.

Transfusion of fresh washed platelets does not prevent experimental polymicrobial-induced septic shock in mice.

INTRODUCTION: The specific role of platelets during sepsis is not yet fully understood, probably related to the paradox of platelets being potentially beneficial but also deleterious via their thrombotic functions. OBJECTIVE: To evaluate the impact of thrombocytopenia on septic shock in mice and to investigate whether transfusion of fresh washed platelets, either fully functional or with impaired hemostatic properties, might have beneficial effects. METHODS: Septic shock was induced by cecal ligation and puncture (CLP). Experimental depletion of circulating platelets was induced with a rat anti-mouse GPIbα monoclonal antibody. Transfusion of either wild-type washed platelets, platelets treated with the antiplatelet drugs acetylsalicylic acid (ASA) and clopidogrel, or GPIIbIIIa-deficient washed platelets treated with ASA and clopidogrel was performed 4 h after CLP surgery. RESULTS: Depletion of circulating platelets negatively affected septic shock, worsening systemic inflammation, coagulopathy, organ damage, and mortality, raising the question of whether a higher platelet count could be protective. Transfusion of fully functional platelets or platelets with combined treatment with ASA and clopidogrel, with or without additional GPIIbIIIa deficiency, afforded an immediate return of circulating platelet counts to their initial values before surgery. However, transfusion of each of the three types of platelets did not prevent arterial hypotension, inflammatory response, coagulopathy, and organ damage during septic shock. CONCLUSION: Depletion of circulating platelets negatively affects septic shock, while transfusion of washed platelets has no significant beneficial effect in mice.

Characterization of the Role of Integrin α5ß1 in Platelet Function, Hemostasis, and Experimental Thrombosis.

OBJECTIVE: Integrins are key regulators of various platelet functions. The pathophysiological importance of most platelet integrins has been investigated, with the exception of α5ß1, a receptor for fibronectin. The aim of this study was to characterize the role of α5ß1 in megakaryopoiesis, platelet function, and to determine its importance in hemostasis and arterial thrombosis. APPROACH AND RESULTS: We generated a mouse strain deficient for integrin α5ß1 on megakaryocytes and platelets (PF4Cre-α5-/-). PF4Cre-α5-/- mice were viable, fertile, and presented no apparent signs of abnormality. Megakaryopoiesis appears unaltered as evidence by a normal megakaryocyte morphology and development, which is in agreement with a normal platelet count. Expression of the main platelet receptors and the response of PF4Cre-α5-/- platelets to a series of agonists were all completely normal. Adhesion and aggregation of PF4Cre-α5-/- platelets under shear flow on fibrinogen, laminin, or von Willebrand factor were unimpaired. In contrast, PF4Cre-α5-/- platelets displayed a marked decrease in adhesion, activation, and aggregation on fibrillar cellular fibronectin and collagen. PF4Cre-α5-/- mice presented no defect in a tail-bleeding time assay and no increase in inflammatory bleeding in a reverse passive Arthus model and a lipopolysaccharide pulmonary inflammation model. Finally, no defects were observed in three distinct experimental models of arterial thrombosis based on ferric chloride-induced injury of the carotid artery, mechanical injury of the abdominal aorta, or laser-induced injury of mesenteric vessels. CONCLUSION: In summary, this study shows that platelet integrin α5ß1 is a key receptor for fibrillar cellular fibronectin but is dispensable in hemostasis and arterial thrombosis.

Mutations in the most divergent α-tubulin isotype, α8-tubulin, cause defective platelet biogenesis.

BACKGROUND: In the panel of genes commonly associated with inherited macrothrombocytopenia, an important fraction encodes key cytoskeletal proteins such as tubulin isotypes, the building blocks of microtubules. Macrothrombocytopenia-causing mutations have been identified in the TUBB1 and TUBA4A genes, emphasizing their importance in the formation of platelets and their marginal band, a unique microtubule ring-like structure that supports the platelet typical disc-shaped morphology. This raised the hypothesis that other tubulin isotypes normally expressed in platelets could play a similar role in their formation. OBJECTIVES: To assess whether tubulin isotype genes other than TUBA4A and TUBB1 could be implicated in inherited macrothrombocytopenia. METHODS: We used high throughput sequencing to screen a cohort of 448 French blood donors with mild thrombocytopenia for mutations in a panel of selected genes known or suspected to be involved in platelet biogenesis. RESULTS: We identified six distinct novel mutations in TUBA8, which encodes the most-divergent α-tubulin, as the causative determinant of macrothrombocytopenia and platelet marginal band defects. Functionally, all TUBA8 mutations were found to fully or partially inhibit the incorporation of the mutated α8-tubulin in the microtubule network. CONCLUSION: This study provides strong support for a key role of multiple tubulin genes in platelet biogenesis by discovering variants in a tubulin gene that was previously not known to be important for platelets.

The Effects of Micro-vessel Curvature Induced Elongational Flows on Platelet Adhesion.

The emerging profile of blood flow and the cross-sectional distribution of blood cells have far reaching biological consequences in various diseases and vital internal processes, such as platelet adhesion. The effects of several essential blood flow parameters, such as red blood cell free layer width, wall shear rate, and hematocrit on platelet adhesion were previously explored to great lengths in straight geometries. In the current work, the effects of channel curvature on cellular blood flow are investigated by simulating the accurate cellular movement and interaction of red blood cells and platelets in a half-arc channel for multiple wall shear rate and hematocrit values. The results show significant differences in the emerging shear rate values and distributions between the inner and outer arc of the channel curve, while the cell distributions remain predominantly uninfluenced. The simulation predictions are also compared to experimental platelet adhesion in a similar curved geometry. The inner side of the arc shows elevated platelet adhesion intensity at high wall shear rate, which correlates with increased shear rate and shear rate gradient sites in the simulation. Furthermore, since the platelet availability for binding seems uninfluenced by the curvature, these effects might influence the binding mechanics rather than the probability. The presence of elongational flows is detected in the simulations and the link to increased platelet adhesion is discussed in the experimental results.

In Situ Exploration of Murine Megakaryopoiesis using Transmission Electron Microscopy.

Differentiation and maturation of megakaryocytes occur in close association with the cellular and extracellular components of the bone marrow. These processes are characterized by the gradual appearance of essential structures in the megakaryocyte cytoplasm such as a polyploid and polylobulated nucleus, an internal membrane network called demarcation membrane system (DMS) and the dense and alpha granules that will be found in circulating platelets. In this article, we describe a standardized protocol for the in situ ultrastructural study of murine megakaryocytes using transmission electron microscopy (TEM), allowing for the identification of key characteristics defining their maturation stage and cellular density in the bone marrow. Bone marrows are flushed, fixed, dehydrated in ethanol, embedded in plastic resin, and mounted for generating cross-sections. Semi-thin and thin sections are prepared for histological and TEM observations, respectively. This method can be used for any bone marrow cell, in any EM facility and has the advantage of using small sample sizes allowing for the combination of several imaging approaches on the same mouse.

Megakaryocyte Culture in 3D Methylcellulose-Based Hydrogel to Improve Cell Maturation and Study the Impact of Stiffness and Confinement.

The 3D environment leading to both confinement and mechanical constraints is increasingly recognized as an important determinant of cell behavior. 3D culture has thus been developed to better approach the in vivo situation. Megakaryocytes differentiate from hematopoietic stem and progenitor cells (HSPCs) in the bone marrow (BM). The BM is one of the softest tissues of the body, confined inside the bone. The bone being poorly extensible at the cell scale, megakaryocytes are concomitantly subjected to a weak stiffness and high confinement. This protocol presents a method for the recovery of mouse lineage negative (Lin-) HSPCs by immuno-magnetic sorting and their differentiation into mature megakaryocytes in a 3D medium composed of methylcellulose. Methylcellulose is non-reactive towards megakaryocytes and its stiffness may be adjusted to that of normal bone marrow or increased to mimic a pathological fibrotic marrow. The process to recover the megakaryocytes for further cell analyses is also detailed in the protocol. Although proplatelet extension is prevented within the 3D milieu, it is described below how to resuspend the megakaryocytes in liquid medium and to quantify their capacity to extend proplatelets. Megakaryocytes grown in 3D hydrogel have a higher capacity to form proplatelets compared to those grown in a liquid milieu. This 3D culture allows i) to differentiate progenitors towards megakaryocytes reaching a higher maturation state, ii) to recapitulate phenotypes that may be observed in vivo but go unnoticed in classical liquid cultures, and iii) to study transduction pathways induced by the mechanical cues provided by a 3D environment.

Platelet FcγRIIA-induced serotonin release exacerbates the severity of transfusion-related acute lung injury in mice.

Transfusion-related acute lung injury (TRALI) remains a major cause of transfusion-related fatalities. The mechanism of human antibody-mediated TRALI, especially the involvement of the Fcγ receptors, is not clearly established. Contrary to mice, human platelets are unique in their expression of the FcγRIIA/CD32A receptor, suggesting that our understanding of the pathogenesis of antibody-mediated TRALI is partial, as the current murine models incompletely recapitulate the human immunology. We evaluated the role of FcγRIIA/CD32A in TRALI using a humanized mouse model expressing the FcγRIIA/CD32A receptor. When challenged with a recombinant chimeric human immunoglobulin G1/mouse anti-major histocompatibility complex class I monoclonal antibody, these mice exhibited exacerbated alveolar edema and higher mortality compared with wild-type (WT) mice. Unlike in WT mice, monocytes/macrophages in CD32A+ mice were accessory for TRALI initiation, indicating the decisive contribution of another cell type. Platelet activation was dramatically increased in CD32A+ animals, resulting in their increased consumption and massive release of their granule contents. Platelet depletion prevented the exacerbation of TRALI in CD32A+ mice but did not affect TRALI in WT animals. By blocking platelet serotonin uptake with fluoxetine, we showed that the severity of TRALI in CD32A+ mice resulted from the serotonin released by the activated platelets. Furthermore, inhibition of 5-hydroxytryptamine 2A serotonin receptor with sarpogrelate, before or after the induction of TRALI, abolished the aggravation of lung edema in CD32A+ mice. Our findings show that platelet FcγRIIA/CD32A activation exacerbates antibody-mediated TRALI and provide a rationale for designing prophylactic and therapeutic strategies targeting the serotonin pathway to attenuate TRALI in patients.

In Vivo Two-photon Imaging of Megakaryocytes and Proplatelets in the Mouse Skull Bone Marrow.

Platelets are produced by megakaryocytes, specialized cells located in the bone marrow. The possibility to image megakaryocytes in real time and their native environment was described more than 10 years ago and sheds new light on the process of platelet formation. Megakaryocytes extend elongated protrusions, called proplatelets, through the endothelial lining of sinusoid vessels. This paper presents a protocol to simultaneously image in real time fluorescently labeled megakaryocytes in the skull bone marrow and sinusoid vessels. This technique relies on a minor surgery that keeps the skull intact to limit inflammatory reactions. The mouse head is immobilized with a ring glued to the skull to prevent movements from breathing. Using two-photon microscopy, megakaryocytes can be visualized for up to a few hours, enabling the observation of cell protrusions and proplatelets in the process of elongation inside sinusoid vessels. This allows the quantification of several parameters related to the morphology of the protrusions (width, length, presence of constriction areas) and their elongation behavior (velocity, regularity, or presence of pauses or retraction phases). This technique also allows simultaneous recording of circulating platelets in sinusoid vessels to determine platelet velocity and blood flow direction. This method is particularly useful to study the role of genes of interest in platelet formation using genetically modified mice and is also amenable to pharmacological testing (study the mechanisms, evaluating drugs in the treatment of platelet production disorders). It has become an invaluable tool, especially to complement in vitro studies as it is now known that in vivo and in vitro proplatelet formation rely on different mechanisms. It has been shown, for example, that in vitro microtubules are required for proplatelet elongation per se. However, in vivo, they rather serve as a scaffold, elongation being mainly promoted by blood flow forces.

Platelet P2Y 12 Receptor Deletion or Pharmacological Inhibition does not Protect Mice from Sepsis or Septic Shock.

Introduction Platelets are increasingly appreciated as key effectors during sepsis, raising the question of the usefulness of antiplatelet drugs to treat patients with sepsis. Objective Evaluate the potential contribution of the platelet P2Y 12 receptor in the pathogenesis of polymicrobial-induced sepsis and septic shock in mice. Methods The effects of P2Y 12 inhibition using clopidogrel treatment and of platelet-specific deletion of the P2Y 12 receptor in mice were examined in two severity grades of cecal ligation and puncture (CLP) leading to mild sepsis or septic shock. Results Twenty hours after induction of the high grade CLP, clopidogrel- and vehicle-treated mice displayed a similar 30% decrease in mean arterial blood pressure (MAP) characteristic of shock. Septic shock-induced thrombocytopenia was not modified by clopidogrel treatment. Plasma concentrations of inflammatory cytokines and myeloperoxidase (MPO) were similarly increased in clopidogrel- and vehicle-treated mice, indicating comparable increase in systemic inflammation. Thrombin-antithrombin (TAT) complexes and the extent of organ damage were also similar. In mild-grade CLP, clopidogrel- and vehicle-treated mice did not display a significant decrease in MAP, while thrombocytopenia and plasma concentrations of TNFα, IL6, IL10, MPO, TAT and organ damage reached similar levels in both groups, although lower than those reached in the high grade CLP. Similarly, mice with platelet-specific deletion of the P2Y 12 receptor were not protected from CLP-induced sepsis or septic shock. Conclusion The platelet P2Y 12 receptor does not contribute to the pathogenesis of sepsis or septic shock in mice, suggesting that P2Y 12 receptor antagonists may not be beneficial in patients with sepsis or septic shock.

Respective roles of Glycoprotein VI and FcγRIIA in the regulation of αIIbß3-mediated platelet activation to fibrinogen, thrombus buildup, and stability.

BACKGROUND: The interplay between platelets and fibrinogen is the cornerstone of thrombus formation. Integrin αIIbß3 is the main platelet adhesion receptor for fibrinogen and mediates an outside-in signal upon ligand binding that reinforces platelet activation. In addition, FcγRIIA and glycoprotein VI (GPVI) contribute to platelet activation on fibrinogen, thereby participating in thrombus growth and stability. To date, the relative importance of these two immunoreceptor tyrosine-based activation motif-bearing receptors in these processes remains unknown. OBJECTIVE: The aim of this study was to evaluate the relative contributions of FcγRIIA and GPVI to platelet activation on fibrinogen and subsequent thrombus growth and stability. METHODS: We evaluated human and mouse platelet adhesion to fibrinogen in static assays and a flow-based approach to evaluate the contribution of FcγRIIA and GPVI to thrombus growth and stability. RESULTS: We first confirmed that integrin αIIbß3 is the key receptor supporting platelet adhesion and spreading on fibrinogen. Using human platelets treated with pharmacological blocking agents and transgenic mouse platelets expressing human receptors, data indicate that GPVI, but not FcγRIIA, plays a prominent role in platelet activation on fibrinogen. Moreover, using a flow-based assay, we observed that blockade of GPVI with 1G5, but not FcγRIIA with IV.3, prevents thrombus growth. Finally, we observed that 1G5, but not IV.3, promotes the disaggregation of thrombi formed on collagen in vitro. CONCLUSION: This study provides evidence that GPVI, but not FcγRIIA, induces platelet activation and spreading on fibrinogen, and promotes thrombus buildup and stability.