Catherine Léon

Defective platelet aggregation and increased resistance to thrombosis in purinergic P2Y(1) receptor-null mice.

ADP is a key agonist in hemostasis and thrombosis. ADP-induced platelet activation involves the purinergic P2Y(1) receptor, which is responsible for shape change through intracellular calcium mobilization. This process also depends on an unidentified P2 receptor (P2cyc) that leads to adenylyl cyclase inhibition and promotes the completion and amplification of the platelet response. P2Y(1)-null mice were generated to define the role of the P2Y(1) receptor and to determine whether the unidentified P2cyc receptor is distinct from P2Y(1). These mice are viable with no apparent abnormalities affecting their development, survival, reproduction, or the morphology of their platelets, and the platelet count in these animals is identical to that of wild-type mice. However, platelets from P2Y(1)-deficient mice are unable to aggregate in response to usual concentrations of ADP and display impaired aggregation to other agonists, while high concentrations of ADP induce platelet aggregation without shape change. In addition, ADP-induced inhibition of adenylyl cyclase still occurs, demonstrating the existence of an ADP receptor distinct from P2Y(1). P2Y(1)-null mice have no spontaneous bleeding tendency but are resistant to thromboembolism induced by intravenous injection of ADP or collagen and adrenaline. Hence, the P2Y(1) receptor plays an essential role in thrombotic states and represents a potential target for antithrombotic drugs.

The P2Y1 receptor is an ADP receptor antagonized by ATP and expressed in platelets and megakaryoblastic cells

© 1997 Federation of European Biochemical Societies.

The P2Y12 receptor induces platelet aggregation through weak activation of the αIIbβ3 integrin – a phosphoinositide 3-kinase-dependent mechanism

High concentrations of adenosine‐5′‐diphosphate ADP are able to induce partial aggregation without shape change of P2Y1 receptor‐deficient mouse platelets through activation of the P2Y12 receptor. In the present work we studied the transduction pathways selectively involved in this phenomenon. Flow cytometric analyses using R‐phycoerythrin‐conjugated JON/A antibody (JON/A‐PE), an antibody which recognizes activated mouse αIIbβ3 integrin, revealed a low level activation of αIIbβ3 in P2Y1 receptor‐deficient platelets in response to 100 μM ADP or 1 μM 2MeS‐ADP. Adrenaline induced no such activation but strongly potentiated the effect of ADP in a dose‐dependent manner. Global phosphorylation of 32P‐labeled platelets showed that P2Y12‐mediated aggregation was not accompanied by an increase in the phosphorylation of myosin light chain (P20) or pleckstrin (P47) and was not affected by the protein kinase C (PKC) inhibitor staurosporine. On the other hand, two unrelated phosphoinositide 3‐kinase inhibitors, wortmannin and LY294002, inhibited this aggregation. Our results indicate that (i) the P2Y12 receptor is able to trigger a P2Y1 receptor‐independent inside‐out signal leading to αIIbβ3 integrin activation and platelet aggregation, (ii) ADP and adrenaline use different signaling pathways which synergize to activate the αIIbβ3 integrin, and (iii) the transduction pathway triggered by the P2Y12 receptor is independent of PKC but dependent on phosphoinositide 3‐kinase.

Differential Involvement of the P2Y1 and P2Y12 Receptors in Platelet Procoagulant Activity

Objective—In vivo, activated platelets contribute to the initiation of thrombin generation through the exposure of phosphatidylserine to form a procoagulant catalytic surface and through platelet-leukocyte interactions, which lead to the exposure of leukocyte tissue factor (TF). On the basis of observations that the platelet P2Y1 and P2Y12 receptors both contribute to thrombosis and thrombin formation in an in vivo model of TF-induced thromboembolism, we further characterized the role of these receptors in thrombin generation. Methods and Results—By using the selective P2 antagonists MRS2179 and AR-C69931MX, the P2Y12 receptor was found to be involved in thrombin-induced exposure of PS on isolated platelets and consequently in TF-induced thrombin formation in platelet-rich plasma. By contrast, the P2Y1 receptor was not involved in phosphatidylserine exposure nor in thrombin generation in platelet-rich plasma. In addition, both receptors were found to contribute to the interactions between platelets and leukocytes mediated by platelet P-selectin exposure, which result in TF exposure at the surface of leukocytes. Conclusions—Overall, these results point to a differential involvement of the 2 platelet ADP receptors in the generation of thrombin and provide further evidence for the relevance of molecules targeting these receptors as antithrombotic agents.

RUNX1-induced silencing of non-muscle myosin heavy chain IIB contributes to megakaryocyte polyploidization

Megakaryocytes are unique mammalian cells that undergo polyploidization (endomitosis) during differentiation, leading to an increase in cell size and protein production that precedes platelet production. Recent evidence demonstrates that endomitosis is a consequence of a late failure in cytokinesis associated with a contractile ring defect. Here we show that the non-muscle myosin IIB heavy chain (MYH10) is expressed in immature megakaryocytes and specifically localizes in the contractile ring. MYH10 downmodulation by short hairpin RNA increases polyploidization by inhibiting the return of 4N cells to 2N, but other regulators, such as of the G1/S transition, might regulate further polyploidization of the 4N cells. Conversely, re-expression of MYH10 in the megakaryocytes prevents polyploidization and the transition of 2N to 4N cells. During polyploidization, MYH10 expression is repressed by the major megakaryocyte transcription factor RUNX1. Thus, RUNX1-mediated silencing of MYH10 is required for the switch from mitosis to endomitosis, linking polyploidization with megakaryocyte differentiation.

Cloning and sequencing of a human cDNA encoding endothelial P2Y1 purinoceptor

We have cloned and sequenced a 3055-bp human placenta cDNA encoding a P2 purinoceptor. An identical receptor has also been found in human saphenous vein endothelial cells. Comparison of the deduced amino acid (aa) sequence to those of previously described P2Y1 purinoceptors revealed greater than 95% aa identity with the rat and mouse counterparts, and 83% with the chick P2Y1 purinoceptor. Northern blot experiments revealed two mRNAs of 7.5 and 4.6 kb whose expression varies according to the tissue examined.

Abnormal megakaryocyte morphology and proplatelet formation in mice with megakaryocyte-restricted MYH9 inactivation

Mutations in the MYH9 gene encoding nonmuscle myosin IIA lead to macrothrombocytopenia as observed in MYH9-related disorders. We used mice with megakaryocyte-restricted MYH9 inactivation to explore the role of myosin in thrombopoiesis. In situ, bone marrow MYH9Delta megakaryocytes were irregularly shaped, appearing leaky with poorly defined limits. The demarcation membranes were abnormally organized and poorly developed, pointing to an insufficient reservoir for the future formation of platelets. The cytoskeletal-rich peripheral zone was lacking due to the absence of the myosin filament network that normally surrounds the granular zone in wild-type cells. In vitro studies of cultured cells showed that MYH9Delta megakaryocytes were unable to form stress fibers upon adhesion to collagen, suggesting that the leaky shape results from defects in internal tension and anchorage to the extracellular environment. Surprisingly, the proportion of cells extending proplatelets was increased in MYH9Delta megakaryocytes and the proplatelet buds were larger. Overall, this study provides evidence for a role of myosin in different steps of megakaryocyte development through its participation in the maintenance of cell shape, formation and organization of the demarcation membranes and the peripheral zone, anchorage to the extracellular matrix, and proplatelet formation.

Contractile Forces Sustain and Polarize Hematopoiesis from Stem and Progenitor Cells

Self-renewal and differentiation of stem cells depend on asymmetric division and polarized motility processes that in other cell types are modulated by nonmuscle myosin-II (MII) forces and matrix mechanics. Here, mass spectrometry-calibrated intracellular flow cytometry of human hematopoiesis reveals MIIB to be a major isoform that is strongly polarized in hematopoietic stem cells and progenitors (HSC/Ps) and thereby downregulated in differentiated cells via asymmetric division. MIIA is constitutive and activated by dephosphorylation during cytokine-triggered differentiation of cells grown on stiff, endosteum-like matrix, but not soft, marrow-like matrix. In vivo, MIIB is required for generation of blood, while MIIA is required for sustained HSC/P engraftment. Reversible inhibition of both isoforms in culture with blebbistatin enriches for long-term hematopoietic multilineage reconstituting cells by 5-fold or more as assessed in vivo. Megakaryocytes also become more polyploid, producing 4-fold more platelets. MII is thus a multifunctional node in polarized division and niche sensing.

Podocyte-Specific Deletion of Myh9 Encoding Nonmuscle Myosin Heavy Chain 2A Predisposes Mice to Glomerulopathy

ABSTRACT Genome-wide association studies linked single-nucleotide polymorphisms (SNPs) at the MYH9 locus to chronic kidney disease among African-Americans, particularly glomerular diseases such as HIV nephropathy and idiopathic focal and segmental glomerulosclerosis (FSGS). However, these MYH9 SNPs are intronic, and despite extensive sequencing, a causal variant remains elusive. To investigate the role of MYH9 in kidney disease, we selectively deleted Myh9 from mouse podocytes and found that mutant C57BL/6 mice did not develop renal insufficiency or proteinuria compared to control littermates, even when the mice were aged for 9 months. To explain the surprisingly normal phenotype, we considered genetic redundancy with the paralog Myh10 in podocytes, but we found that Myh10 was not expressed in podocytes in Myh9-deficient or control mice. We tested whether Myh9 podocyte deletion predisposed mice to glomerulopathy in response to injury by doxorubicin hydrochloride (Adriamycin), and we found that Myh9 podocyte-deleted mice developed proteinuria and glomerulosclerosis, while control mice were resistant. In summary, Myh9 podocyte deletion in C57BL/6 mice results in susceptibility to experimental doxorubicin hydrochloride glomerulopathy. We review evidence that MYH9 dysfunction in humans results in similar susceptibility and place our data, the first examination of Myh9 kidney disease in experimental animals, in the context of recent findings in human kidney disease, including the role of APOL1.

Intrinsic impaired proplatelet formation and microtubule coil assembly of megakaryocytes in a mouse model of Bernard-Soulier syndrome

Bernard-Soulier syndrome is caused by a deficiency of the platelet surface glycoprotein Ib-IX-V complex resulting in a severe platelet function defect. However this is compounded by a reduction in platelet numbers and the platelets in the circulation are abnormally large. As for many other platelet disorders, recent evidence and findings of this study suggest that the defective morphology has its origins in megakaryocyte function. See related perspective article on page 756. Background Giant platelets and thrombocytopenia are invariable defects in the Bernard-Soulier syndrome caused by deficiency of the GPIb-V-IX complex, a receptor for von Willebrand factor supporting platelet adhesion to the damaged arterial wall. Various properties of this receptor may be considered potential determinants of the macrothrombocytopenia. Design and Methods To explore the underlying mechanisms of the disease, megakaryopoiesis was studied in a mouse model deficient in GPIbβ. Megakaryocytes were initially characterized in situ in the bone marrow of adult mice, after which their capacity to differentiate into proplatelet-bearing cells was evaluated in cultured fetal liver cells. Results The number of megakaryocyte progenitors, their differentiation and progressive maturation into distinct classes and their level of endoreplication were normal in GPIbβ−/− bone marrow. However, the more mature cells exhibited ultrastructural anomalies with a thicker peripheral zone and a less well developed demarcation membrane system. GPIbβ−/− megakaryocytes could be differentiated in culture from Lin− fetal liver cells in normal amounts but the proportion of cells able to extend proplatelets was decreased by 41%. Moreover, the GPIbβ−/− cells extending proplatelets displayed an abnormal morphology characterized by fewer pseudopodial extensions with thicker shaft sections and an increased diameter of the terminal coiled elements. GPIbβ−/− released platelets were larger but retained a typical discoid shape. Proplatelet formation was similarly affected in bone marrow explants from adult mice examined by videomicroscopy. The marginal microtubular ring contained twice as many tubulin fibers in GPIbβ−/− proplatelet buds in cultured and circulating platelets. Conclusions Altogether, these findings point to a role of the GPIb-V-IX complex intrinsic to megakaryocytes at the stage of proplatelet formation and suggest a functional link with the underlying microtubular cytoskeleton in platelet biogenesis.