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The ephrin transmembrane receptor family of tyrosine kinases is involved in platelet function. We report the first EPHB2 variant affecting platelets in 2 siblings (P1 and P2) from a consanguineous family with recurrent bleeding and normal platelet counts. Whole-exome sequencing identified a c.2233C>T variant (missense p.R745C) of the EPHB2 gene. P1 and P2 were homozygous for this variant, while their asymptomatic parents were heterozygous. The p.R745C variant within the tyrosine kinase domain was associated with defects in platelet aggregation, αIIbβ3 activation, and granule secretion induced by G-protein–coupled receptor (GPCR) agonists and convulxin, as well as in thrombus formation on collagen under flow. In contrast, clot retraction, flow-dependent platelet adhesion, and spreading on fibrinogen were only mildly affected, indicating limited effects on αIIbβ3 outside-in signaling. Most importantly, Lyn, Syk, and FcRγ phosphorylation, the initial steps in glycoprotein VI (GPVI) platelet signaling were drastically impaired in the absence of platelet–platelet contact, indicating a positive role for EPHB2 in GPVI activation. Likewise platelet activation by PAR4-AP showed defective Src activation, as opposed to normal protein kinase C activity and Ca2+ mobilization. Overexpression of wild-type and R745C EPHB2 variant in RBL-2H3 (rat basophilic leukemia) cells stably expressing human GPVI confirmed that EPHB2 R745C mutation impaired EPHB2 autophosphorylation but had no effect on ephrin ligand-induced EPHB2 clustering, suggesting it did not interfere with EPHB2-ephrin–mediated cell-to-cell contact. In conclusion, this novel inherited platelet disorder affecting EPHB2 demonstrates this tyrosine kinase receptor plays an important role in platelet function through crosstalk with GPVI and GPCR signaling.

The ephrin transmembrane receptor family of tyrosine kinases is involved in platelet function. We report the first EPHB2variant affecting platelets in 2 siblings (P1 and P2) from a consanguineous family with recurrent bleeding and normal platelet counts. Whole-exome sequencing identified a c.2233C>T variant (missense p.R745C) of the EPHB2gene. P1 and P2 were homozygous for this variant, while their asymptomatic parents were heterozygous. The p.R745C variant within the tyrosine kinase domain was associated with defects in platelet aggregation, αIIbβ3 activation, and granule secretion induced by G-protein–coupled receptor (GPCR) agonists and convulxin, as well as in thrombus formation on collagen under flow. In contrast, clot retraction, flow-dependent platelet adhesion, and spreading on fibrinogen were only mildly affected, indicating limited effects on αIIbβ3 outside-in signaling. Most importantly, Lyn, Syk, and FcRγ phosphorylation, the initial steps in glycoprotein VI (GPVI) platelet signaling were drastically impaired in the absence of platelet–platelet contact, indicating a positive role for EPHB2 in GPVI activation. Likewise platelet activation by PAR4-AP showed defective Src activation, as opposed to normal protein kinase C activity and Ca2+mobilization. Overexpression of wild-type and R745C EPHB2 variant in RBL-2H3 (rat basophilic leukemia) cells stably expressing human GPVI confirmed that EPHB2 R745C mutation impaired EPHB2 autophosphorylation but had no effect on ephrin ligand-induced EPHB2 clustering, suggesting it did not interfere with EPHB2-ephrin–mediated cell-to-cell contact. In conclusion, this novel inherited platelet disorder affecting EPHB2 demonstrates this tyrosine kinase receptor plays an important role in platelet function through crosstalk with GPVI and GPCR signaling.

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The role of the sarco-endoplasmic reticulum calcium (Ca2+) adenosine triphosphatase (ATPase) 3 (SERCA3) in platelet physiology remains poorly understood. Here, we show that SERCA3 knockout (SERCA3−/−) mice exhibit prolonged tail bleeding time and rebleeding. Thrombus formation was delayed both in arteries and venules in an in vivo ferric chloride–induced thrombosis model. Defective platelet adhesion and thrombus growth over collagen was confirmed in vitro. Adenosine 5′-diphosphate (ADP) removal by apyrase diminished adhesion and thrombus growth of control platelets to the level of SERCA3−/− platelets. Aggregation, dense granule secretion, and Ca2+ mobilization of SERCA3−/− platelets induced by low collagen or low thrombin concentration were weaker than controls. Accordingly, SERCA3−/− platelets exhibited a partial defect in total stored Ca2+ and in Ca2+ store reuptake following thrombin stimulation. Importantly ADP, but not serotonin, rescued aggregation, secretion, and Ca2+ mobilization in SERCA3−/− platelets, suggesting specificity. Dense granules appeared normal upon electron microscopy, mepacrine staining, and total serotonin content, ruling out a dense granule defect. ADP induced normal platelet aggregation, excluding a defect in ADP activation pathways. The SERCA3-specific inhibitor 2,5-di-(tert-butyl)-1,4-benzohydroquinone diminished both Ca2+ mobilization and secretion of control platelets, as opposed to the SERCA2b inhibitor thapsigargin. This confirmed the specific role of catalytically active SERCA3 in ADP secretion. Accordingly, SERCA3-dependent Ca2+ stores appeared depleted in SERCA3−/− platelets. Finally, αIIbβ3 integrin blockade did not affect SERCA3-dependent secretion, therefore proving independent of αIIbβ3 engagement. Altogether, these results show that SERCA3-dependent Ca2+ stores control a specific ADP secretion pathway required for full platelet secretion induced by agonists at low concentration and independent of αIIbβ3.

The role of the sarco-endoplasmic reticulum calcium (Ca2+) adenosine triphosphatase (ATPase) 3 (SERCA3) in platelet physiology remains poorly understood. Here, we show that SERCA3 knockout (SERCA3−/−) mice exhibit prolonged tail bleeding time and rebleeding. Thrombus formation was delayed both in arteries and venules in an in vivo ferric chloride–induced thrombosis model. Defective platelet adhesion and thrombus growth over collagen was confirmed in vitro. Adenosine 5′-diphosphate (ADP) removal by apyrase diminished adhesion and thrombus growth of control platelets to the level of SERCA3−/−platelets. Aggregation, dense granule secretion, and Ca2+mobilization of SERCA3−/−platelets induced by low collagen or low thrombin concentration were weaker than controls. Accordingly, SERCA3−/−platelets exhibited a partial defect in total stored Ca2+and in Ca2+store reuptake following thrombin stimulation. Importantly ADP, but not serotonin, rescued aggregation, secretion, and Ca2+mobilization in SERCA3−/−platelets, suggesting specificity. Dense granules appeared normal upon electron microscopy, mepacrine staining, and total serotonin content, ruling out a dense granule defect. ADP induced normal platelet aggregation, excluding a defect in ADP activation pathways. The SERCA3-specific inhibitor 2,5-di-(tert-butyl)-1,4-benzohydroquinone diminished both Ca2+mobilization and secretion of control platelets, as opposed to the SERCA2b inhibitor thapsigargin. This confirmed the specific role of catalytically active SERCA3 in ADP secretion. Accordingly, SERCA3-dependent Ca2+stores appeared depleted in SERCA3−/−platelets. Finally, αIIbβ3integrin blockade did not affect SERCA3-dependent secretion, therefore proving independent of αIIbβ3engagement. Altogether, these results show that SERCA3-dependent Ca2+stores control a specific ADP secretion pathway required for full platelet secretion induced by agonists at low concentration and independent of αIIbβ3.

Macrothrombocytopenias are the most important subgroup of inherited thrombocytopenias. This subgroup is particularly heterogeneous because the affected genes are involved in various functions such as cell signaling, cytoskeleton organization, and gene expression. Herein we describe the clinical and hematological features of a consanguineous family with a severe autosomal recessive macrothrombocytopenia associated with a thrombocytopathy inducing a bleeding tendency in the homozygous mutated patients. Platelet activation and cytoskeleton reorganization were impaired in these homozygous patients. Exome sequencing identified a c.222C>G mutation (missense p.74Ile>Met) in PRKACG, a gene encoding the γ-catalytic subunit of the cyclic adenosine monophosphate-dependent protein kinase, the mutated allele cosegregating with the macrothrombocytopenia. We demonstrate that the p.74Ile>Met PRKACG mutation is associated with a marked defect in proplatelet formation and a low level in filamin A in megakaryocytes (MKs). The defect in proplatelet formation was rescued in vitro by lentiviral vector-mediated overexpression of wild-type PRKACG in patient MKs. We thus conclude that PRKACG is a new central actor in platelet biogenesis and a new gene involved in inherited thrombocytopenia with giant platelets associated with a thrombocytopathy.

Macrothrombocytopenias are the most important subgroup of inherited thrombocytopenias. This subgroup is particularly heterogeneous because the affected genes are involved in various functions such as cell signaling, cytoskeleton organization, and gene expression. Herein we describe the clinical and hematological features of a consanguineous family with a severe autosomal recessive macrothrombocytopenia associated with a thrombocytopathy inducing a bleeding tendency in the homozygous mutated patients. Platelet activation and cytoskeleton reorganization were impaired in these homozygous patients. Exome sequencing identified a c.222C>G mutation (missense p.74Ile>Met) in PRKACG,a gene encoding the γ-catalytic subunit of the cyclic adenosine monophosphate-dependent protein kinase, the mutated allele cosegregating with the macrothrombocytopenia. We demonstrate that the p.74Ile>Met PRKACG mutation is associated with a marked defect in proplatelet formation and a low level in filamin A in megakaryocytes (MKs). The defect in proplatelet formation was rescued in vitro by lentiviral vector-mediated overexpression of wild-type PRKACG in patient MKs. We thus conclude that PRKACG is a new central actor in platelet biogenesis and a new gene involved in inherited thrombocytopenia with giant platelets associated with a thrombocytopathy.

Filaminopathies A caused by mutations in the X-linked FLNAgene are responsible for a wide spectrum of rare diseases including 2 main phenotypes, the X-linked dominant form of periventricular nodular heterotopia (FLNA-PVNH) and the otopalatodigital syndrome spectrum of disorders. In platelets, filamin A (FLNa) tethers the principal receptors ensuring the platelet–vessel wall interaction, glycoprotein Ibα and integrin αIIbβ3, to the underlying cytoskeleton. Hemorrhage, coagulopathy, and thrombocytopenia are mentioned in several reports on patients with FLNA-PVNH. Abnormal platelet morphology in 2 patients with FLNA-PVNH prompted us to examine a third patient with similar platelet morphology previously diagnosed with immunologic thrombocytopenic purpura. Her enlarged platelets showed signs of FLNa degradation in Western blotting, and a heterozygous missense mutation in FLNAwas detected. An irregular distribution of FLNa within the total platelet population was shown by confocal microscopy for all 3 patients. In vitro megakaryocyte cultures showed an abnormal differentiation, including an irregular distribution of FLNa with a frayed aspect, the presence of enlarged α-granules, and an abnormal fragmentation of the cytoplasm. Mutations in FLNAmay represent an unrecognized cause of macrothrombocytopenia with an altered platelet production and a modified platelet–vessel wall interaction.

Filaminopathies A caused by mutations in the X-linked FLNA gene are responsible for a wide spectrum of rare diseases including 2 main phenotypes, the X-linked dominant form of periventricular nodular heterotopia (FLNA-PVNH) and the otopalatodigital syndrome spectrum of disorders. In platelets, filamin A (FLNa) tethers the principal receptors ensuring the platelet–vessel wall interaction, glycoprotein Ibα and integrin αIIbβ3, to the underlying cytoskeleton. Hemorrhage, coagulopathy, and thrombocytopenia are mentioned in several reports on patients with FLNA-PVNH. Abnormal platelet morphology in 2 patients with FLNA-PVNH prompted us to examine a third patient with similar platelet morphology previously diagnosed with immunologic thrombocytopenic purpura. Her enlarged platelets showed signs of FLNa degradation in Western blotting, and a heterozygous missense mutation in FLNA was detected. An irregular distribution of FLNa within the total platelet population was shown by confocal microscopy for all 3 patients. In vitro megakaryocyte cultures showed an abnormal differentiation, including an irregular distribution of FLNa with a frayed aspect, the presence of enlarged α-granules, and an abnormal fragmentation of the cytoplasm. Mutations in FLNA may represent an unrecognized cause of macrothrombocytopenia with an altered platelet production and a modified platelet–vessel wall interaction.

The adhesion of cells to the extracellular matrix plays a major role in cell migration. Pretreatment with platelet-derived growth factor (PDGF) inhibited the adhesion of smooth muscle cells to fibronectin by 80%. This inhibition decreased as concentrations of fibronectin increased. In the presence of 200 µmGRGDS peptide, only 45% of PDGF-treated cells adhered to fibronectin compared with 80% of control cells. This indicates that a decrease in integrin avidity was induced by PDGF. Cell adhesion was partially restored when the activation of the extracellular signal-regulated kinase (ERK) was inhibited with PD98059. The remaining inhibition of adhesion (50%) was independent of the fibronectin concentration, suggesting that the ERK pathway is involved in the decrease in integrin avidity. This was confirmed by depleting ERK protein levels by treatment with ERK antisense oligonucleotide. The adhesion of ERK control oligonucleotide-treated cells decreased by 41% when the concentration of GRGDS peptide was increased from 50 to 200 µmbut only decreased by 11% in ERK antisense oligonucleotide-treated cells. Treatment with PDGF also delayed focal complex assembly and inhibited stress fiber formation. Consistent with a delay in tyrosine phosphorylation of paxillin, PDGF treatment caused a lag in focal complex formation, although this was not associated with any change in Src family tyrosine kinase activity. Our results indicate that PDGF inhibits smooth muscle cells adhesion by two pathways. The first involves an ERK-dependent decrease in integrin avidity; the second involves the ERK-independent inhibition of focal complex assembly.

Summary: Smooth muscle cells were cultured from pig aorta. Changes in both the growth and the properties of sulfated proteoglycans were observed during passage. The population doubling time during log phase growth was 34 h from Passages 3 to 7–8 but 20 h at the Passage 11, and the cell density at the stationary phase, was 86 000 and 136 000 cells/cm2 at Passages 3 and 11, respectively. Structural characteristics of sulfated proteoglycans secreted into the medium were investigated after metabolic labeling with [35S]-sulfate. Significant differences were observed with age in vitro: a) [35S]proteoglycan complexes were in a greater amount at Passage 10 than at Passage 3; b) the hydrodynamic size of at least 45% of subunits and about 90% of monomers decreased with in vitro aging; c) this decrease in the size of proteoglycans was partly due to a decrease in the size of their glycanic chains; d) an increase of 15% in the proportion of dermatan sulfate was observed when cells were subjected to 10 passages.

U46619, a thromboxane A2 analogue, and basic fibroblast growth factor (FGF-2) both induced the expression of the inducible cyclo-oxygenase (Cox)-2 in porcine aortic smooth-muscle cells. This induction was dose-dependent (submaximal at 300 nM for U46619 and 1 ng/ml for FGF-2) and time-dependent, with similar intensity and maximal expression at 2 h. Under these conditions, both inducers stimulated rapid activation of extracellular signal-regulated kinase (ERK2) at 5–10 min, a transient and lower intensity being induced by U46619 whereas that induced by FGF-2 was sustained (> 1 h). PD98059, an inhibitor of the ERK pathway, inhibited the expression of Cox-2. In contrast, activation of Jun-N-terminal kinase (JNK1) was sustained with U46619 but poorly induced by FGF-2. Cox-2 expression induced by U46619 or FGF-2 was similarly reduced by prostaglandin (PGE2), forskolin or dibutyryl-cAMP, suggesting a regulatory effect of adenylate cyclase on Cox-2 expression. However, activation of ERK2 by FGF-2 was not affected by PGE2 whereas that of JNK1 by U46619 was inhibited, suggesting that inhibition of COX-2 expression by cAMP may be downstream of ERK2. The effects of PGE2 and forskolin on Cox-2 and phosphorylation of JNK1 were reversed with the protein kinase A inhibitor H89. In addition, endogenous PGE2 down-regulated the expression of Cox-2 by the two inducers, as stimulation of the cells in the presence of different Cox inhibitors increased the expression of the protein. Overall, these results suggest that exogenous and endogenous PGE2 exert negative inhibitory effects on Cox-2 expression mediated by stimulation of protein kinase A.

Stimulation of smooth muscle cells with basic fibroblast growth factor (bFGF) results in the activation of the mitogen-activated protein kinase (MAP kinase) cascade and leads to cell proliferation. We show that transforming growth factor β1 (TGF-β1), at concentrations that completely inhibited bFGF-induced mitogenic activity, decreased bFGF-induced MAP kinase activity. Under these conditions, tyrosine and threonine phosphorylations of MAP kinase were differentially affected depending on the time period of TGF-β1 pretreatment. After a short (30 min) TGF-β1 pretreatment, the bFGF-mediated increase in phosphorylation of p42mapk on threonine was inhibited, with no effect on the level of phosphotyrosine or decrease in the electrophoretic mobility of p42mapk. This suggests that TGF-β1 inhibited MAP kinase activity through the action of a serine/threonine phosphatase. In contrast, a longer TGF-β1 pretreatment (4 h) partly inhibited the bFGF-induced MAP kinase mobility shift and correlated with the inhibition of phosphorylation on both threonine and tyrosine, suggesting that long-term TGF-β1 treatment prevented activation of the MAP kinase cascade or directly blocked MAP kinase. The ability of long-term (4 h) but not short-term (30 min) TGF-β1 pretreatment to inhibit MAP kinase activity was completely dependent on protein synthesis and suggests that TGF-β1 inhibits MAP kinase activity by two distinct mechanisms. These findings provide a molecular basis for the growth-inhibitory action of TGF-β1 on bFGF-induced mitogenic activity.

No relevant conflicts of interest to declare.

Abstract 1152This icon denotes a clinically relevant abstract