Illustrated Abstracts of the 5th EUPLAN International Conference.

These illustrated capsules have been prepared by some speakers of State-of-the-Art talks and of original investigations, presented at the 5th European Platelet Network (EUPLAN) International Conference, which was held at the Università degli Studi di Milano (Italy) on September 28-30, 2022. The programme featured various state-of-the-art lectures and a selection of oral presentations covering a broad range of topics in platelet and megakaryocyte biology, from basic science to recent advances in clinical studies. As usual, the meeting brought together senior scientists and trainees in an informal atmosphere to discuss platelet science in person.

Megakaryocyte NLRP3 hyperactivation induces mild anemia and potentiates inflammatory response in mice.

Background: The NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasome has been described in both immune cells and platelets, but its role in the megakaryocyte (MK) lineage remains elusive. Objective: The aim of this study was to explore the role of NLRP3 inflammasome in megakaryocytes and platelets. Methods: We generated Nlrp3 A350V/+/Gp1ba-CreKI/+ mice carrying a mutation genetically similar to the one observed in human Muckle-Wells syndrome, which leads to hyperactivity of NLRP3 specifically in MK and platelets. Results: Platelets from the mutant mice expressed elevated levels of both precursor and active form of caspase-1, suggesting hyperactivity of NLRP3 inflammasome. Nlrp3 A350V/+/Gp1ba-CreKI/+ mice developed normally and had normal platelet counts. Expression of major platelet receptors, platelet aggregation, platelet deposition on collagen under shear, and deep vein thrombosis were unchanged. Nlrp3 A350V/+/Gp1ba-CreKI/+ mice had mild anemia, reduced Ter119+ cells in the bone marrow, and splenomegaly. A mild increase in MK TGF-ß1 might be involved in the anemic phenotype. Intraperitoneal injection of zymosan in Nlrp3 A350V/+/Gp1ba-CreKI/+ mice induced increased neutrophil egression and elevated levels of a set of proinflammatory cytokines, alongside IL-10 and G-CSF, in the peritoneal fluid as compared with control animals. Conclusion: MK/platelet NLRP3 inflammasome promotes the acute inflammatory response and its hyperactivation in mice leads to mild anemia and increased extramedullary erythropoiesis.

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.

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.

Platelet Src family kinases: A tale of reversible phosphorylation.

Sarcoma (Src) family kinases (SFKs) have occupied a central place in platelet research for over 40 years. Discovered by virologists and oncologists as the proto proto-oncogene, Src tyrosine kinase spurred a phenomenal burst of research on reversible tyrosine phosphorylation and signal transduction. For a time, platelets were adopted as the model of choice for studying the biological functions of Src, owing to their ease of isolation, high Src expression, and lack of a nucleus, only to be abandoned due to challenges of culturing and manipulating using common molecular biology-based techniques. For platelet biologists, SFKs have remained an important area of investigation, initiating and amplifying signals from all major adhesion, activation, and inhibitory receptors, including the integrin αIIbß3, the collagen receptor complex glycoprotein VI-Fc receptor γ-chain, the G protein-coupled ADP receptor P2Y12 and the inhibitory receptors platelet endothelial cell adhesion molecule-1 and G6b-B. The vital roles of SFKs in platelets is highlighted by the severe phenotypes of null and gain-of-function mutations in SFKs in mice and humans, and effects of pharmacologic inhibitors on platelet activation, thrombosis, and hemostasis. The recent description of critical regulators of SFKs in platelets, namely, C-terminal Src kinase (Csk), Csk homologous kinase (Chk), the receptor-type protein-tyrosine phosphatase receptor type J (PTPRJ) helps explain some of the bleeding side effects of tyrosine kinase inhibitors and are novel therapeutic targets for regulating the thrombotic and hemostatic capacity of platelets. Recent findings from Chk, Csk, and PTPRJ knockout mouse models highlighted that SFKs are able to autoinhibit by phosphorylating their C-terminal tyrosine residues, providing fundamental insights into SFK autoregulation.

Enhanced BCR signaling inflicts early plasmablast and germinal center B cell death.

It is still not clear how B cell receptor (BCR) signaling intensity affects plasma cell (PC) and germinal center (GC) B cell differentiation. We generated Cγ1 Cre/wt Ptpn6 fl/fl mice where SHP-1, a negative regulator of BCR signaling, is deleted rapidly after B cell activation. Although immunization with T-dependent antigens increased BCR signaling, it led to PC reduction and increased apoptosis. Dependent on the antigen, the early GC B cell response was equally reduced and apoptosis increased. At the same time, a higher proportion of GC B cells expressed cMYC, suggesting GC B cell-Tfh cell interactions may be increased. GC B cell numbers returned to normal at later stages, whereas affinity maturation was suppressed in the long term. This confirms that BCR signaling not only directs affinity-dependent B cell selection but also, without adequate further stimulation, can inflict cell death, which may be important for the maintenance of B cell tolerance.

Severity of Megakaryocyte-Driven Osteosclerosis in Mpig6b-Deficient Mice Is Sex-Linked.

Patients with chronic myelofibrosis often suffer from osteosclerosis, which is associated with bone pain and may lead to bone marrow failure. The pathogenesis of myelofibrosis is linked to aberrant megakaryocyte development and function. Null and loss-of-function mutations in MPIG6B, which codes for the inhibitory heparan sulfate receptor G6b-B, result in severe macrothrombocytopenia, large megakaryocyte clusters, and focal primary myelofibrosis in mice and humans. We investigated the development of osteosclerosis in Mpig6b null (Mpig6b-/- ) mice. Although male and female Mpig6b-/- mice presented with elevated bone marrow megakaryocyte number and macrothrombocytopenia, female Mpig6b-/- mice developed progressive splenomegaly starting at 8 weeks of age. Micro-computed tomography (µCT) of femurs showed that female Mpig6b-/- mice had increased cortical thickness and reduced bone marrow area starting at 8 weeks of age and developed occlusion of the medullary cavity by trabeculae by 16 weeks of age. In contrast, male Mpig6b-/- mice developed only a small number of trabeculae in the medullary cavity at the proximal diaphysis and demonstrated a temporary decrease in bone volume fraction and trabecular thickness at 16 weeks. Ovariectomy of 10-week-old female Mpig6b-/- mice prevented the development of medullary cavity osteosclerosis, whereas orchiectomy of male Mpig6b-/- mice did not exacerbate their disease. Importantly, ovariectomized female Mpig6b-/- mice also demonstrated improvement in spleen weight compared to sham-operated Mpig6b-/- mice, establishing estrogen as a contributing factor to the severity of the megakaryocyte-driven osteosclerosis. © 2021 American Society for Bone and Mineral Research (ASBMR).

Lymphatic blood filling in CLEC-2-deficient mouse models.

C-type lectin-like receptor 2 (CLEC-2) is considered as a potential drug target in settings of wound healing, inflammation, and infection. A potential barrier to this is evidence that CLEC-2 and its ligand podoplanin play a critical role in preventing lymphatic vessel blood filling in mice throughout life. In this study, this aspect of CLEC-2/podoplanin function is investigated in more detail using new and established mouse models of CLEC-2 and podoplanin deficiency, and models of acute and chronic vascular remodeling. We report that CLEC-2 expression on platelets is not required to maintain a barrier between the blood and lymphatic systems in unchallenged mice, post-development. However, under certain conditions of chronic vascular remodeling, such as during tumorigenesis, deficiency in CLEC-2 can lead to lymphatic vessel blood filling. These data provide a new understanding of the function of CLEC-2 in adult mice and confirm the essential nature of CLEC-2-driven platelet activation in vascular developmental programs. This work expands our understanding of how lymphatic blood filling is prevented by CLEC-2-dependent platelet function and provides a context for the development of safe targeting strategies for CLEC-2 and podoplanin.

Single-Cell Analyses Reveal Megakaryocyte-Biased Hematopoiesis in Myelofibrosis and Identify Mutant Clone-Specific Targets.

Myelofibrosis is a severe myeloproliferative neoplasm characterized by increased numbers of abnormal bone marrow megakaryocytes that induce fibrosis, destroying the hematopoietic microenvironment. To determine the cellular and molecular basis for aberrant megakaryopoiesis in myelofibrosis, we performed single-cell transcriptome profiling of 135,929 CD34+ lineage- hematopoietic stem and progenitor cells (HSPCs), single-cell proteomics, genomics, and functional assays. We identified a bias toward megakaryocyte differentiation apparent from early multipotent stem cells in myelofibrosis and associated aberrant molecular signatures. A sub-fraction of myelofibrosis megakaryocyte progenitors (MkPs) are transcriptionally similar to healthy-donor MkPs, but the majority are disease specific, with distinct populations expressing fibrosis- and proliferation-associated genes. Mutant-clone HSPCs have increased expression of megakaryocyte-associated genes compared to wild-type HSPCs, and we provide early validation of G6B as a potential immunotherapy target. Our study paves the way for selective targeting of the myelofibrosis clone and illustrates the power of single-cell multi-omics to discover tumor-specific therapeutic targets and mediators of tissue fibrosis.

Interplay between the tyrosine kinases Chk and Csk and phosphatase PTPRJ is critical for regulating platelets in mice.

The Src family kinases (SFKs) Src, Lyn, and Fyn are essential for platelet activation and also involved in megakaryocyte (MK) development and platelet production. Platelet SFKs are inhibited by C-terminal Src kinase (Csk), which phosphorylates a conserved tyrosine in their C-terminal tail, and are activated by the receptor-type tyrosine phosphatase PTPRJ (CD148, DEP-1), which dephosphorylates the same residue. Deletion of Csk and PTPRJ in the MK lineage in mice results in increased SFK activity, but paradoxically hypoactive platelets resulting from negative feedback mechanisms, including upregulation of Csk homologous kinase (Chk) expression. Here, we investigate the role of Chk in platelets, functional redundancy with Csk, and the physiological consequences of ablating Chk, Csk, and PTPRJ in mice. Platelet count was normal in Chk knockout (KO) mice, reduced by 92% in Chk;Csk double KO (DKO) mice, and partially rescued in Chk;Csk;Ptprj triple KO (TKO) mice. Megakaryocyte numbers were significantly increased in both DKO and TKO mice. Phosphorylation of the inhibitory tyrosine of SFKs was almost completely abolished in DKO platelets, which was partially rescued in Src and Fyn in TKO platelets. This residual phosphorylation was abolished by Src inhibitors, revealing an unexpected mechanism in which SFKs autoinhibit their activity by phosphorylating their C-terminal tyrosine residues. We demonstrate that reduced inhibitory phosphorylation of SFKs leads to thrombocytopenia, with Csk being the dominant inhibitor in platelets and Chk having an auxiliary role. PTPRJ deletion in addition to Chk and Csk ameliorates the extent of thrombocytopenia, suggesting targeting it may have therapeutic benefits in such conditions.

Catalytic dysregulation of SHP2 leading to Noonan syndromes affects platelet signaling and functions.

Src homology 2 domain-containing phosphatase 2 (SHP2), encoded by the PTPN11 gene, is a ubiquitous protein tyrosine phosphatase that is a critical regulator of signal transduction. Germ line mutations in the PTPN11 gene responsible for catalytic gain or loss of function of SHP2 cause 2 disorders with multiple organ defects: Noonan syndrome (NS) and NS with multiple lentigines (NSML), respectively. Bleeding anomalies have been frequently reported in NS, but causes remain unclear. This study investigates platelet activation in patients with NS and NSML and in 2 mouse models carrying PTPN11 mutations responsible for these 2 syndromes. Platelets from NS mice and patients displayed a significant reduction in aggregation induced by low concentrations of GPVI and CLEC-2 agonists and a decrease in thrombus growth on a collagen surface under arterial shear stress. This was associated with deficiencies in GPVI and αIIbß3 integrin signaling, platelet secretion, and thromboxane A2 generation. Similarly, arterial thrombus formation was significantly reduced in response to a local carotid injury in NS mice, associated with a significant increase in tail bleeding time. In contrast, NSML mouse platelets exhibited increased platelet activation after GPVI and CLEC-2 stimulation and enhanced platelet thrombotic phenotype on collagen matrix under shear stress. Blood samples from NSML patients also showed a shear stress-dependent elevation of platelet responses on collagen matrix. This study brings new insights into the understanding of SHP2 function in platelets, points to new thrombopathies linked to platelet signaling defects, and provides important information for the medical care of patients with NS in situations involving risk of bleeding.

Heparan sulfates are critical regulators of the inhibitory megakaryocyte-platelet receptor G6b-B.

The immunoreceptor tyrosine-based inhibition motif (ITIM)-containing receptor G6b-B is critical for platelet production and activation. Loss of G6b-B results in severe macrothrombocytopenia, myelofibrosis and aberrant platelet function in mice and humans. Using a combination of immunohistochemistry, affinity chromatography and proteomics, we identified the extracellular matrix heparan sulfate (HS) proteoglycan perlecan as a G6b-B binding partner. Subsequent in vitro biochemical studies and a cell-based genetic screen demonstrated that the interaction is specifically mediated by the HS chains of perlecan. Biophysical analysis revealed that heparin forms a high-affinity complex with G6b-B and mediates dimerization. Using platelets from humans and genetically modified mice, we demonstrate that binding of G6b-B to HS and multivalent heparin inhibits platelet and megakaryocyte function by inducing downstream signaling via the tyrosine phosphatases Shp1 and Shp2. Our findings provide novel insights into how G6b-B is regulated and contribute to our understanding of the interaction of megakaryocytes and platelets with glycans.

Comparative Analysis of Microfluidics Thrombus Formation in Multiple Genetically Modified Mice: Link to Thrombosis and Hemostasis.

Genetically modified mice are indispensable for establishing the roles of platelets in arterial thrombosis and hemostasis. Microfluidics assays using anticoagulated whole blood are commonly used as integrative proxy tests for platelet function in mice. In the present study, we quantified the changes in collagen-dependent thrombus formation for 38 different strains of (genetically) modified mice, all measured with the same microfluidics chamber. The mice included were deficient in platelet receptors, protein kinases or phosphatases, small GTPases or other signaling or scaffold proteins. By standardized re-analysis of high-resolution microscopic images, detailed information was obtained on altered platelet adhesion, aggregation and/or activation. For a subset of 11 mouse strains, these platelet functions were further evaluated in rhodocytin- and laminin-dependent thrombus formation, thus allowing a comparison of glycoprotein VI (GPVI), C-type lectin-like receptor 2 (CLEC2) and integrin α6ß1 pathways. High homogeneity was found between wild-type mice datasets concerning adhesion and aggregation parameters. Quantitative comparison for the 38 modified mouse strains resulted in a matrix visualizing the impact of the respective (genetic) deficiency on thrombus formation with detailed insight into the type and extent of altered thrombus signatures. Network analysis revealed strong clusters of genes involved in GPVI signaling and Ca2+ homeostasis. The majority of mice demonstrating an antithrombotic phenotype in vivo displayed with a larger or smaller reduction in multi-parameter analysis of collagen-dependent thrombus formation in vitro. Remarkably, in only approximately half of the mouse strains that displayed reduced arterial thrombosis in vivo, this was accompanied by impaired hemostasis. This was also reflected by comparing in vitro thrombus formation (by microfluidics) with alterations in in vivo bleeding time. In conclusion, the presently developed multi-parameter analysis of thrombus formation using microfluidics can be used to: (i) determine the severity of platelet abnormalities; (ii) distinguish between altered platelet adhesion, aggregation and activation; and (iii) elucidate both collagen and non-collagen dependent alterations of thrombus formation. This approach may thereby aid in the better understanding and better assessment of genetic variation that affect in vivo arterial thrombosis and hemostasis.

The Gp1ba-Cre transgenic mouse: a new model to delineate platelet and leukocyte functions.

Conditional knockout (KO) mouse models are invaluable for elucidating the physiological roles of platelets. The Platelet factor 4-Cre recombinase (Pf4-Cre) transgenic mouse is the current model of choice for generating megakaryocyte/platelet-specific KO mice. Platelets and leukocytes work closely together in a wide range of disease settings, yet the specific contribution of platelets to these processes remains unclear. This is partially a result of the Pf4-Cre transgene being expressed in a variety of leukocyte populations. To overcome this issue, we developed a Gp1ba-Cre transgenic mouse strain in which Cre expression is driven by the endogenous Gp1ba locus. By crossing Gp1ba-Cre and Pf4-Cre mice to the mT/mG dual-fluorescence reporter mouse and performing a head-to-head comparison, we demonstrate more stringent megakaryocyte lineage-specific expression of the Gp1ba-Cre transgene. Broader tissue expression was observed with the Pf4-Cre transgene, leading to recombination in many hematopoietic lineages, including monocytes, macrophages, granulocytes, and dendritic and B and T cells. Direct comparison of phenotypes of Csk, Shp1, or CD148 conditional KO mice generated using either the Gp1ba-Cre or Pf4-Cre strains revealed similar platelet phenotypes. However, additional inflammatory and immunological anomalies were observed in Pf4-Cre-generated KO mice as a result of nonspecific deletion in other hematopoietic lineages. By excluding leukocyte contributions to phenotypes, the Gp1ba-Cre mouse will advance our understanding of the role of platelets in inflammation and other pathophysiological processes in which platelet-leukocyte interactions are involved.

Evaluation of the Total Thrombus-Formation System (T-TAS): application to human and mouse blood analysis.

The Total Thrombus-formation Analyser System (T-TAS) is a whole blood flow chamber system for the measurement of in vitro thrombus formation under variable shear stress conditions. Our current study sought to evaluate the potential utility of the T-TAS for the measurement of thrombus formation within human and mouse whole blood. T-TAS microchips (collagen, PL chip; collagen/tissue thromboplastin, AR chip) were used to analyze platelet (PL) or fibrin-rich thrombus formation, respectively. Blood samples from humans (healthy and patients with mild bleeding disorders) and wild-type (WT), mice were tested. Light transmission lumi-aggregometer (lumi-LTA) was performed in PRP using several concentrations of ADP, adrenaline, arachidonic acid, collagen, PAR-1 peptide and ristocetin. Thrombus growth (N = 22) increased with shear within PL (4:40 ± 1.11, 3:25 ± 0.43 and 3:12 ± 0.48 mins [1000, 1500 and 2000s-1]) and AR chips (3:55 ± 0.42 and 1:49 ± 0.19 [240s-1 and 600s-1]). The area under the curve (AUC) on the PL chip was also reduced at 1000s-1 compared to 1500/2000s-1 (260 ± 51.7, 317 ± 55.4 and 301 ± 66.2, respectively). In contrast, no differences in the AUC between 240s-1 and 600s-1 were observed in the AR chip (1593 ± 122 and 1591 ± 158). The intra-assay coefficient of variation (CV) (n = 10) in the PL chip (1000s-1) and AR chip (240s-1) were T1014.1%, T6016.7%, T10-6022.8% and AUC1024.4% or T10 9.03%, T808.64%, T10-8023.8% and AUC305.1%. AR chip thrombus formation was inhibited by rivaroxaban (1 µM), but not with ticagrelor (10 µM). In contrast, PL chip thrombus formation was totally inhibited by ticagrelor. T-TAS shows an overall agreement with lumi-LTA in 87% of patients (n = 30) with normal PL counts recruited into the genotyping and phenotyping of platelet (GAPP) study and suspected to have a PL function defect. The onset (T10) of thrombus formation in WT mice (N = 4) was shorter when compared to humans e.g. PL chip (1000s-1) T10 were 02:02 ± 00:23 and 03:30 ± 0:45, respectively). T-TAS measures in vitro thrombus formation and can be used for monitoring antithrombotic therapy, investigating patients with suspected PL function defects and monitoring PL function within mice.

Collagen type I degradation fragments act through the collagen receptor LAIR-1 to provide a negative feedback for osteoclast formation.

The major organic component of bone is collagen type I. Osteoclasts are terminally differentiated multinucleated cells of hematopoietic origin that are essential for physiological development of bone and teeth. We examined if osteoclast differentiation from murine bone marrow precursors is affected by collagen type I, or by its degradation products produced by human recombinant cathepsin K. Osteoclasts formation was dose-dependently inhibited in the presence of full length collagen type I or its 30-75 kDa degradation products added to the osteoclast differentiation media for the duration of an experiment. Collagen degradation fragments signaled through SH-2 phosphatases, inhibiting calcium signaling and NFATc1 translocation in osteoclast precursors. Osteoclasts and their precursors expressed a collagen receptor of leukocyte receptor complex family, LAIR-1. Importantly, collagen fragments failed to inhibit osteoclast formation from LAIR-1 deficient murine osteoclast precursors. This study demonstrates that collagen degradation fragments inhibit osteoclast formation acting through LAIR-1, providing a novel mechanism for the physiologically-relevant negative control of osteoclastogenesis.