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14 p.-6 fig., Background: The circulating form of human endoglin (sEng) is a cleavage product of membrane-bound endoglin present on endothelial cells. Because sEng encompasses an RGD motif involved in integrin binding, we hypothesized that sEng would be able to bind integrin αIIbβ3, thereby compromising platelet binding to fibrinogen and thrombus stability., Methods: In vitro human platelet aggregation, thrombus retraction, and secretion-competition assays were performed in the presence of sEng. Surface plasmon resonance (SPR) binding and computational (docking) analyses were carried out to evaluate protein-protein interactions. A transgenic mouse overexpressing human sEng (hsEng+) was used to measure bleeding/rebleeding, prothrombin time (PT), blood stream, and embolus formation after FeCl3-induced injury of the carotid artery., Results: Under flow conditions, supplementation of human whole blood with sEng led to a smaller thrombus size. sEng inhibited platelet aggregation and thrombus retraction, interfering with fibrinogen binding, but did not affect platelet activation. SPR binding studies demonstrated that the specific interaction between αIIbβ3 and sEng and molecular modeling showed a good fitting between αIIbβ3 and sEng structures involving the endoglin RGD motif, suggesting the possible formation of a highly stable αIIbβ3/sEng. hsEng+ mice showed increased bleeding time and number of rebleedings compared to wild-type mice. No differences in PT were denoted between genotypes. After FeCl3 injury, the number of released emboli in hsEng+ mice was higher and the occlusion was slower compared to controls., Conclusions: Our results demonstrate that sEng interferes with thrombus formation and stabilization, likely via its binding to platelet αIIbβ3, suggesting its involvement in primary hemostasis control., Promex Stiftung für die Forschung Foundation Consejo Superior de Investigaciones Científicas; Grant/Award Number: 201920E022 Spanish Ministry of Science, Innovation & Universities; Grant/Award Number: RTI2018-102242-B-I00 Comunidad de Madrid; Grant/Award Number: S2022/BMD-7278

Background The SAME device (i-SEP, France) is an innovative filtration-based autotransfusion device able to salvage and wash both red blood cells and platelets. This study evaluated the device performances using human whole blood with the hypothesis that the device will be able to salvage platelets while achieving a erythrocyte yield of 80% and removal ratios of 90% for heparin and 80% for major plasma proteins without inducing signification activation of salvaged cells. Methods Thirty healthy human whole blood units (median volume, 478 ml) were diluted, heparinized, and processed by the device in two consecutive treatment cycles. Samples from the collection reservoir and the concentrated blood were analyzed. Complete blood count was performed to measure blood cell recovery rates. Flow cytometry evaluated the activation state and function of platelets and leukocytes. Heparin and plasma proteins were measured to assess washing performance. Results The global erythrocyte yield was 88.1% (84.1 to 91.1%; median [25th to 75th]) with posttreatment hematocrits of 48.9% (44.8 to 51.4%) and 51.4% (48.4 to 53.2%) for the first and second cycles, respectively. Ektacytometry did not show evidence of erythrocyte alteration. Platelet recovery was 36.8% (26.3 to 43.4%), with posttreatment counts of 88 × 109/l (73 to 101 × 109/l) and 115 × 109/l (95 to 135 × 109/l) for the first and second cycles, respectively. Recovered platelets showed a low basal P-selectin expression at 10.8% (8.1 to 15.2%) and a strong response to thrombin-activating peptide. Leukocyte yield was 93.0% (90.1 to 95.7%) with no activation or cell death. Global removal ratios were 98.3% (97.8 to 98.9%), 98.2% (96.9 to 98.8%), and 88.3% (86.6 to 90.7%) for heparin, albumin, and fibrinogen, respectively. The processing times were 4.4 min (4.2 to 4.6 min) and 4.4 min (4.2 to 4.7 min) for the first and second cycles, respectively. Conclusions This study demonstrated the performance of the SAME device. Platelets and red blood cells were salvaged without significant impact on cell integrity and function. In the meantime, leukocytes were not activated, and the washing quality of the device prevented reinfusion of high concentrations of heparin and plasma proteins. Editor’s Perspective What We Already Know about This Topic What This Article Tells Us That Is New

Background Platelet cytosolic cyclic adenosine monophosphate (cAMP) levels are balanced by synthesis, degradation, and efflux. Efflux can occur via multidrug resistant protein-4 (MRP4; ABCC4) present on dense granule and/or plasma membranes. As lipid rafts have been shown to interfere on cAMP homeostasis, we evaluated the relationships between the distribution and activity of MRP4 in lipid rafts and cAMP efflux. Methods Platelet activation and cAMP homeostasis were analyzed in human and wild-type or MRP4-deleted mouse platelets in the presence of methyl-β-cyclodextrin (MßCD) to disrupt lipid rafts, and of activators of the cAMP signalling pathways. Human platelet MRP4 and effector proteins of the cAMP pathway were analyzed by immunoblots in lipid rafts isolated by differential centrifugation. Results MßCD dose dependently inhibited human and mouse platelet aggregation without affecting per se cAMP levels. An additive inhibitory effect existed between the adenylate cyclase (AC) activator forskolin and MßCD that was accompanied by an overincrease of cAMP, and which was significantly enhanced upon MRP4 deletion. Finally, an efflux of cAMP out of resting platelets incubated with prostaglandin E1 (PGE1) was observed that was partly dependent on MRP4. Lipid rafts contained a small fraction (≈15%) of MRP4 and most of the inhibitory G-protein Gi, whereas Gs protein, AC3, and phosphodiesterases PDE2 and PDE3A were all present as only trace amounts. Conclusion Our results are in favour of part of MRP4 present at the platelet surface, including in lipid rafts. Lipid raft integrity is necessary for cAMP signalling regulation, although MRP4 and most players of cAMP homeostasis are essentially located outside rafts.

Over the last decades, antiplatelet agents, mainly aspirin and P2Y12 receptor antagonists, have significantly reduced morbidity and mortality associated with arterial thrombosis. Their pharmacological characteristics, including pharmacokinetic/pharmacodynamics profiles, have been extensively studied, and a significant number of clinical trials assessing their efficacy and safety in various clinical settings have established antithrombotic efficacy. Notwithstanding, antiplatelet agents carry an inherent risk of bleeding. Given that bleeding is associated with adverse cardiovascular outcomes and mortality, there is an unmet clinical need to develop novel antiplatelet therapies that inhibit thrombosis while maintaining hemostasis. In this review, we present the currently available antiplatelet agents, with a particular focus on their targets, pharmacological characteristics, and patterns of use. We will further discuss the novel antiplatelet therapies in the pipeline, with the goal of improved clinical outcomes among patients with atherothrombotic diseases.

Endoglin (Eng) is an endothelial cell (EC) transmembrane glycoprotein involved in adhesion and angiogenesis. Eng mutations result in vessel abnormalities as observed in hereditary hemorrhagic telangiectasia of type 1. The role of Eng was investigated in endothelial functions and permeability under inflammatory conditions, focusing on the actin dynamic signaling pathway. Endothelial Colony-Forming Cells (ECFC) from human cord blood and mouse lung/aortic EC (MLEC, MAEC) from Eng+/+ and Eng+/− mice were used. ECFC silenced for Eng with Eng-siRNA and ctr-siRNA were used to test tubulogenesis and permeability +/− TNFα and +/− LIM kinase inhibitors (LIMKi). In silico modeling of TNFα–Eng interactions was carried out from PDB IDs 5HZW and 5HZV. Calcium ions (Ca2+) flux was studied by Oregon Green 488 in epifluorescence microscopy. Levels of cofilin phosphorylation and tubulin post-translational modifications were evaluated by Western blot. F-actin and actin–tubulin distribution/co-localization were evaluated in cells by confocal microscopy. Eng silencing in ECFCs resulted in a decrease of cell sprouting by 50 ± 15% (p &lt, 0.05) and an increase in pseudo-tube width (41 ± 4.5%, p &lt, 0.001) compared to control. Upon TNFα stimulation, ECFC Eng–siRNA displayed a significant higher permeability compared to ctr-siRNA (p &lt, 0.01), which is associated to a higher Ca2+ mobilization (p &lt, 0.01). Computational analysis suggested that Eng mitigated TNFα activity. F-actin polymerization was significantly increased in ECFC Eng-siRNA, MAEC+/−, and MLEC+/− compared to controls (p &lt, 0.001, p &lt, 0.01, and p &lt, 0.01, respectively) as well as actin/tubulin distribution (p &lt, 0.01). Furthermore, the inactive form of cofilin (P-cofilin at Ser3) was significantly decreased by 36.7 ± 4.8% in ECFC Eng-siRNA compared to ctr-siRNA (p &lt, 0.001). Interestingly, LIMKi reproduced the absence of Eng on TNFα-induced ECFC-increased permeability. Our data suggest that Eng plays a critical role in the homeostasis regulation of endothelial cells under inflammatory conditions (TNFα), and loss of Eng influences ECFC-related permeability through the LIMK/cofilin/actin rearrangement-signaling pathway.

Introduction Clinical benefit-risk balance of direct oral anticoagulants (DOAC) in atherothrombosis prevention differs between anti-Xa and anti-IIa drugs and their specific effect on platelet functions remains controversial. We hence investigated rivaroxaban and dabigatran effect on platelets in identical experimental conditions. Materials and methods Blood of fifteen healthy volunteers was spiked with DOAC which plasma concentrations were measured by specific anti-Xa or anti-IIa assays. Light transmission aggregometry measured in platelet-rich plasma used low doses of agonists: 0.5 mM arachidonic acid, 2.5 μM ADP, 0.5 μM epinephrine, 0.8 μg/ml collagen, 7.5 μM TRAP-6 and 0.5 pM tissue factor in the presence of H-Gly-Pro-Arg-Pro-OH to prevent fibrin polymerization. Platelet adhesion on collagen fibres was evaluated in whole blood under flow. Same experiments were reproduced in the presence of aspirin. Results Median [95% CI] plasma concentrations were of 28 [23–36], 128 [119–144] and 321 [293–361] ng/ml for rivaroxaban and 39 [34–45], 171 [166–193] and 353 [349–382] ng/ml for dabigatran. DOAC did not modify platelet aggregation or adhesion on collagen fibres at any tested concentrations. However, they delayed platelet aggregation secondary to coagulation activation with a more potent effect with dabigatran (p Conclusion Efficacy of combining DOAC and aspirin in atherothrombosis prevention would not stem from a direct antiplatelet effect of the formers but to their additive inhibitory effect on platelet aggregation secondary to coagulation activation. This effect differs according to DOAC molecules and may also result from the pleiotropic roles of the different coagulation factors targeted by DOAC.

Lowe syndrome (LS) is an oculocerebrorenal syndrome of Lowe (OCRL1) genetic disorder resulting in a defect of the OCRL protein, a phosphatidylinositol-4,5-bisphosphate 5-phosphatase containing various domains including a Rho GTPase-activating protein (RhoGAP) homology domain catalytically inactive. We previously reported surgery-associated bleeding in patients with LS, suggestive of platelet dysfunction, accompanied with a mild thrombocytopenia in several patients. To decipher the role of OCRL in platelet functions and in megakaryocyte (MK) maturation, we conducted a case-control study on 15 patients with LS (NCT01314560). While all had a drastically reduced expression of OCRL, this deficiency did not affect platelet aggregability, but resulted in delayed thrombus formation on collagen under flow conditions, defective platelet spreading on fibrinogen and impaired clot retraction. We evidenced alterations of the myosin light chain phosphorylation (P-MLC), with defective Rac1 activity and, inversely, elevated active RhoA. Altered cytoskeleton dynamics was also observed in cultured patient MKs showing deficient proplatelet extension with increased P-MLC that was confirmed using control MKs transfected with OCRL-specific small interfering(si)RNA (siOCRL). Patients with LS also had an increased proportion of circulating barbell-shaped proplatelets. Our present study establishes that a deficiency of the OCRL protein results in a defective actomyosin cytoskeleton reorganisation in both MKs and platelets, altering both thrombopoiesis and some platelet responses to activation necessary to ensure haemostasis.

International audience; Extracorporeal membrane oxygenation (ECMO) is an extracorporeal circulation used to manage patients with severe circulatory or respiratory failure. It is associated with both high bleeding and thrombosis risks, mainly as a result of biomaterial/blood interface phenomena, high shear stress, and complex inflammatory response involving the activation of coagulation and complement systems, endothelial cells, leukocytes, and platelets. Besides their critical role in hemostasis, platelets are important players in inflammatory reactions, especially due to their ability to bind and activate leukocytes. Hence, we reviewed studies on platelet function of ECMO patients. Moreover, we addressed the issue of platelet-leukocyte aggregates (PLAs), which is a key step in both platelet and leukocyte activation, and deserves to be investigated in these patients. A reduced expression of GPIb and GPVI was found under ECMO therapy, due to the shedding processes. However, defective platelet aggregation is inconsistently reported and is still not clearly defined. Due to the high susceptibility of PLAs to pre-analytical conditions, defining and strictly adhering to a rigorous laboratory methodology is essential for reliable and reproducible results, especially in the setting of complex inflammatory situations like ECMO. We provide results on sample preparation and flow cytometric whole blood evaluation of circulating PLAs.

The P2Y12 receptor is a key player in platelet activation and a major target for antithrombotic drugs. The beneficial effects of P2Y12 receptor antagonists might, however, not be restricted to the primary and secondary prevention of arterial thrombosis. Indeed, it has been established that platelet activation also has an essential role in inflammation. Additionally, nonplatelet P2Y12 receptors present in immune cells and vascular smooth muscle cells might be effective players in the inflammatory response. This review will investigate the biological and clinical impact of P2Y12 receptor inhibition beyond its platelet-driven antithrombotic effects, focusing on its anti-inflammatory role. We will discuss the potential molecular and cellular mechanisms of P2Y12-mediated inflammation, including cytokine release, platelet−leukocyte interactions and neutrophil extracellular trap formation. Then we will summarize the current evidence on the beneficial effects of P2Y12 antagonists during various clinical inflammatory diseases, especially during sepsis, acute lung injury, asthma, atherosclerosis, and cancer.

16 p.-7 fig. Rossi. E. et al., Complex interactions between platelets and activated endothelium occur during the thrombo-inflammatory reaction at sites of vascular injuries and during vascular hemostasis. The endothelial receptor endoglin is involved in inflammation through integrin-mediated leukocyte adhesion and transmigration; and heterozygous mutations in the endoglin gene cause hereditary hemorrhagic telangiectasia type 1. This vascular disease is characterized by a bleeding tendency that is postulated to be a consequence of telangiectasia fragility rather than a platelet defect, since platelets display normal functions in vitro in this condition. Here, we hypothesize that endoglin may act as an adhesion molecule involved in the interaction between endothelial cells and platelets through integrin recognition. We find that the extracellular domain of human endoglin promotes specific platelet adhesion under static conditions and confers resistance of adherent platelets to detachment upon exposure to flow. Also, platelets adhere to confluent endothelial cells in an endoglin-mediated process. Remarkably, Chinese hamster ovary cells ectopically expressing the human αIIbβ3 integrin acquire the capacity to adhere to myoblast transfectants expressing human endoglin, whereas platelets from Glanzmann’s thrombasthenia patients lacking the αIIbβ3 integrin are defective for endoglin-dependent adhesion to endothelial cells. Furthermore, the bleeding time, but not the prothrombin time, is significantly prolonged in endoglin-haplodeficient (Eng+/−) mice compared to Eng+/+ animals. These results suggest a new role for endoglin in αIIbβ3 integrin-mediated adhesion of platelets to the endothelium, and may provide a better understanding on the basic cellular mechanisms involved in hemostasis and thrombo-inflammatory events., This work was supported by Grants from Ministerio de Economia y Competitividad of Spain (SAF2013-43421-R to C. B., SAF2013-45784-R to J. M. L.-N., and BFU2010-15237 to C. G.-M.), Centro de Investigacion Biomedica en Red de Enfermedades Raras (CIBERER; ISCIII-CB06/07/0038 and ER16PIAC707 to C. B.), Red de Investigacion Cooperativa en Enfermedades Renales (REDINREN to J. M. L.-N.), The Conny-Maeva Charitable Foundation (to D. M. S.), and Fundación Renal Iñigo Alvarez de Toledo to J. M. L.-N. CIBERER and REDINREN are initiatives of the Instituto de Salud Carlos III (ISCIII) of Spain supported by FEDER funds.

Antiplatelet agents are at risk for bleeding complications, the management of which differs depending on the clinical situation and on the antiplatelet agent itself. Neutralization of antiplatelets is sometimes necessary, most often leading to platelet transfusion, although the benefit of this strategy is poorly documented. In addition, if platelet transfusion corrects the platelet inhibition induced by aspirin and probably by clopidogrel and prasugrel, it does not neutralize ticagrelor, as a consequence of its pharmacological properties. The clinical benefit of platelet transfusion is limited, and the most recent studies are challenging it. However, it is indicated on a perioperative basis for surgeries with high hemorrhagic risk and is discussed in severe hemorrhages. The neutralization of ticagrelor is a concern and the antidote currently under development may be a solution. In all cases, other therapeutic solutions may be considered, such as administration of desmopressin, tranexamic acid or activated factor VII.

αIIbβ3, the major platelet integrin, plays a central role in hemostasis and thrombosis. Upon platelet activation, conformation of αIIbβ3 changes and allows fibrinogen binding and, subsequently, platelet aggregation. It was previously shown that a lipid-modified platelet permeable peptide, which corresponds to the intracellular acidic membrane distal sequence 1000LEEDDEEGE1008 of αIIb (pal-K-LEEDDEEGE or pal-K-1000-1008), inhibits thrombin-induced human platelet aggregation, by inhibiting talin association with the integrin. YMESRADR, a peptide corresponding to the extracellular sequence 313-320 of αIIb, is also a potent platelet aggregation inhibitor by mimicking the effect of a clasp between the head domains of αIIb and β3. The aim of the present study was to investigate the synergistic effect of the intra- and extracellular- peptide inhibitors on platelet aggregation, as well as on the phosphorylation of two signaling proteins, focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK). Platelet preincubation with Pal-K-LEEDDEGE followed by YMESRADR showed a synergistic inhibitory activity on platelet aggregation. Platelet incubation with threshold inhibitory concentrations of both peptides provoked almost the total inhibition of aggregation, PAC-1 binding, and fibrinogen binding, but not P-selectin exposure on activated platelets' surface. Like RGDS peptide, this mixture inhibits FAK phosphorylation whose phosphorylation is well known to be consecutive to the aggregation (postoccupancy events). However, in contrast to RGDS peptide that enhances ERK phosphorylation and activation, the mixture of threshold inhibitory concentrations of Pal-K-LEEDDEEGE and YMESRADR inhibits ERK phosphorylation. We suggest that the use of the intracellular in combination with the extracellular peptide inhibitor, acting with a non-RGD-like mechanism, may provide an alternative way to antagonize integrin αIIbβ3 activation.

AimManagement of ticagrelor-induced bleeding is challenging as platelet transfusion is ineffective. An effective strategy is needed. This study aimed to investigatein vitrothe efficacy of four haemostatic drugs (HDs), namely recombinant activated factor VII (rFVIIa), fibrinogen concentrate (Fib), tranexamic acid (TXA) and factor XIII concentrate (FXIII) to improve the haemostatic capacity in the presence of ticagrelor.MethodsBlood was spiked with ticagrelor then supplemented by either HD or control. Several assays were performed: ADP-induced platelet aggregation measured by impedance aggregometry, light transmission and two global assays, thrombolastography with the platelet mapping device (TEG-PM) and a platelet-dependent thrombin generation assay (TGA).ResultsTicagrelor inhibited ADP-induced platelet aggregation and decreased the clot strength maximum amplitude (MA) in TEG-PMADP. None of the HDs corrected these parameters. However, rFVIIa shortened the coagulation time R using TEG-PMthrombinand the time to peak prolonged by ticagrelor in TGA. Fib increased MAthrombinand FXIII decreased LY30. TXA had no effects.ConclusionsWhereas none of the HDs corrected ticagrelor-induced platelet inhibition, rFVIIa shortened coagulation times, Fib increased clot firmness and FXIII decreased fibrinolysis. Consequently, they may bypass ticagrelor effects by acting on fibrin formation or fibrinolysis. Further studies are needed to confirm these datain vivo.

International audience; Ticagrelor, an antagonist of the platelet adenosine diphosphate (ADP)-P2Y(12) receptor is recommended for patients with acute coronary syndromes. However, ticagrelor exposes to a risk of bleeding, the management of which is challenging because platelet transfusion is ineffective, and no antidote is yet available. We hypothesized that the vasopressor drug epinephrine could counter the antiplatelet effects of ticagrelor and restore platelet functions. We assessed in vitro the efficiency of epinephrine in restoring platelet aggregation inhibited by ticagrelor and investigated the underlying mechanisms. Washed platelet aggregation and secretion were measured upon stimulation by epinephrine alone or in combination with ADP, in the presence or absence of ticagrelor. Mechanistic investigations used P2Y(1) and phosphoinositide 3-kinase (PI3K) inhibitors and included vasodilator-stimulated phosphoprotein (VASP) and Akt phosphorylation assays as well as measurement of Ca2+ mobilisation. We found that epinephrine restored ADP-induced platelet aggregation, but not dense granule release. Epinephrine alone failed to induce aggregation whereas it fully induced VASP dephosphorylation and Akt phosphorylation regardless of the presence of ticagrelor. In the presence of ticagrelor, blockage of the P2Y(1) receptor prevented restoration of platelet aggregation by the combination of epinephrine and ADP, as well as intracellular Ca2+ mobilisation. In combination with ADP, epinephrine induced platelet aggregation of ticagrelor-treated platelets through inhibition of the CAMP pathway and activation of the PI3K pathway, thus enabling the P2Y(1) receptor signalling and subsequent Ca2+ mobilisation. This proof-of-concept study needs to be challenged in vivo for the management of bleeding in ticagrelor-treated patients.

International audience; Key points • The vascular S1Pgradient is dispensablefor platelet formationin mice• Instead, local S1P production restrains megakaryopoiesis via S1P 1 and can further suppress platelet production via S1P 2 when deregulated. AbstractThe bioactive lipid mediator sphingosine 1-phosphate (S1P) was recently assigned critical roles in platelet biology: whereas S1P 1 receptor-mediated S1P gradient sensing was reported to be essential for directing proplatelet extensions from megakaryocytes (MKs) toward bone marrow sinusoids, MK sphingosine kinase 2 (Sphk2)-derived S1P was reported to further promote platelet shedding through receptor-independent intracellular actions, and platelet aggregation through S1P 1. Yet clinical use of S1P pathway modulators including fingolimod has not been associated with risk of bleeding or thrombosis. We therefore revisited the role of S1P in platelet biology in mice. Surprisingly, no reduction in platelet counts was observed when the vascular S1P gradient was ablated by impairing S1P provision to plasma or S1P degradation in interstitial fluids, nor when gradient sensing was impaired by S1pr1 deletion selectively in MKs. Moreover, S1P 1 expression and signaling were both undetectable in mature MKs in situ, and MK S1pr1 deletion did not affect platelet aggregation or spreading. When S1pr1 deletion was induced in hematopoietic progenitor cells, platelet counts were instead significantly elevated. Isolated global Sphk2 deficiency was associated with thrombocytopenia, but this was not replicated by MK-restricted Sphk2 deletion and was reversed by compound deletion of either Sphk1 or S1pr2, suggesting that this phenotype arises from increased S1P export and S1P 2 activation secondary to redistribution of sphingosine to Sphk1. Consistent with clinical observations, we thus observe no essential role for S1P 1 in facilitating platelet production or activation. Instead, S1P restricts megakaryopoiesis through S1P 1 , and can further suppress thrombo-poiesis through S1P 2 when aberrantly secreted in the hematopoietic niche.

PG and CBL share equal senior authorship.; International audience; Discrepancies in preclinical studies of aspirin (ASA) antiplatelet activity in mouse models of bleeding and arterial thrombosis led us to evaluate commonly reported methods in order to propose a procedure for reliably measuring the effects of single dose ASA on mouse hemostasis. FVB and C57Bl6 mice received 100 mg/kg of ASA or vehicle orally 30 min or 3 h prior to investigate either hemostasis using the tail bleeding assay or carotid thrombosis induced by FeCl3, or to blood sampling for isolated platelet aggregation and TXB2 generation. Expected inhibition of COX1 by ASA was ascertained by a strong decrease in TXB2 production, and its effect on platelet function and hemostasis, by decreased collagen-induced aggregation and increased bleeding time, respectively. Strikingly, we determined that anti-hemostatic effects of ASA were more predictable 30 min after administration than 3 h later. Conversely, ASA did not alter time to arterial occlusion of the carotid upon FeCl3-induced thrombosis, suggesting ASA not to be used as reference inhibitor drug in this model of arterial thrombosis.

This review focuses on multidrug resistance protein 4 (MRP4 or ABCC4) that has recently been shown to play a role in cAMP homeostasis, a key-pathway in vascular biology and in platelet functions. In vascular system, recent data provide evidence that inhibition of MRP4 prevents human coronary artery smooth muscle cell proliferation in vitro and in vivo, as well as human pulmonary artery smooth muscle cell proliferation in vitro and pulmonary hypertension in mice in vivo. In the heart, MRP4 silencing in adult rat ventricular myocytes results in an increase in intracellular cAMP levels leading to enhanced cardiomyocyte contractility. However, a prolonged inhibition of MRP4 can promote cardiac hypertrophy. In addition, secreted cAMP, through its metabolite adenosine, prevents adrenergically induced cardiac hypertrophy and fibrosis. Finally, MRP4 inhibition in platelets induces a moderate thrombopathy. The localization of MRP4 underlines the emerging concept of cAMP compartmentalization in platelets, which is a major regulatory mechanism in other cells. cAMP storage in platelet dense granules might limit the cAMP cytosolic concentration upon adenylate cyclase activation, a necessary step to induce platelet activation. In this review, we discuss the therapeutic potential of direct pharmacological inhibition of MRP4 in atherothrombotic disease, via its vasodilating and antiplatelet effects.

This prospective observational study was designed to analyze platelet functions across time in 50 patients scheduled for liver transplantation (LT) secondary to decompensated cirrhosis or hepatocellular carcinoma. Platelet functions were assessed before LT (pre-LT), one week (D7) and 1 month (D28) after LT. Platelet count significantly increased from pre-LT time to day 28 as well as circulating CD34+hematopoietic stem cells. To avoid any influence of platelet count on assays, platelet function was evaluated on platelet-rich-plasma adjusted to pre-LT platelet count. Although platelet secretion potential did not differ between time-points, as evaluated by the expression of CD62P upon strong activation, platelet aggregation in response to various agonists significantly increased along time, however with no concomitant increase of circulating markers of platelet activation: platelet microvesicles, platelet-leukocyte complexes, soluble CD40L and soluble CD62P. In the multivariate analysis, hepatic function was associated with platelet count and function. A lower platelet aggregation recovery was correlated with Child C score. History of thrombosis or bleeding was associated with respective higher or lower values of platelet aggregation. This longitudinal analysis of platelet functions in LT patients showed an improvement of platelet functions along time together with platelet count increase, with no evidence of platelet hyperactivation at any time-point.

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Essentials Thromboelastography (TEG)-platelet mapping (PM) assay is used for antiplatelet-drug monitoring. The TEG-PM was evaluated for dypiridamole and aspirin monitoring in 4 BH-implanted children. Some TEG-PM tracings were atypical leading to wrong results. The TEG-PM has to be improved for antiplatelet-drug monitoring during ventricular assistance. Introduction The complex hemostatic changes associated with Berlin Heart (BH) implantation in children require a challenging antithrombotic treatment. The aim of this retrospective analysis was to evaluate the thromboelastography (TEG)-platelet mapping (PM) assay to monitor antiplatelet therapy in children implanted with a BH. Methods TEG-PM was performed in 4 BH-implanted patients receiving dipyridamole and aspirin, and 9 healthy volunteers. Patients’ antiplatelet therapy was adjusted to TEG-PM results. Light transmission aggregometry (LTA) was also available for 2 of these patients. Results Between 2009 and 2014, 4 BH-implanted patients received a dual antiplatelet therapy monitored by TEG-PM. In 2 patients, 18 of 34 tracings were atypical, because the maximum amplitude due to fibrin never stabilized, which made difficult antiplatelet therapy adjustment as recommended by BH's guidelines. To overcome this difficulty, TEG-PM and LTA were next performed in parallel. However, both methods led to different decisions to adjust antiplatelet therapy in 57% of the cases. In order to better understand this atypical tracing, TEG-PM was also performed in 9 volunteers and surprisingly 3 of them had the same atypical tracing. This atypical tracing was corrected by adding apyrase, suggesting that adenosine diphosphate (ADP) participates to spontaneous platelet activation in heparinized samples. In addition, we evidenced a high variability in the responses of TEG-PM with ADP in volunteers. Conclusions Antiplatelet therapy monitoring in BH-implanted children remains challenging, as TEG-PM is sensitive to several preanalytical and analytical conditions.

Le syndrome de Lowe (SL) est une maladie genetique rare, egalement appelee syndrome oculo-cerebro-renal en reference aux organes majoritairement touches. Il est provoque par des mutations du gene OCRL1, qui code pour OCRL, enzyme ubiquitaire de type phosphatidylinositolbisphosphate 5-phosphatase de type II qui participe au controle du taux intracellulaire de PI(4,5)P2. Le PI(4,5)P2 est implique dans de nombreux mecanismes cellulaires en relation avec le trafic membranaire et l’organisation du cytosquelette d’actine. OCRL est une proteine ubiquitaire presente dans les plaquettes. Nous avons recemment observe une augmentation du risque hemorragique associee a une anomalie de l’hemostase primaire. Les differents domaines fonctionnels d’OCRL et les mecanismes intracellulaires controlant l’activation plaquettaire permettent d’emettre plusieurs hypotheses quant aux consequences de l’absence d’OCRL dans les plaquettes. Les resultats d’une etude cas/controles ayant permis d’etudier de maniere approfondie les fonctions plaquettaires dans le SL sont en cours d’analyse. Ils devraient permettre de verifier certaines de ces hypotheses.

Background Ticagrelor (TICA), a potent P2Y12 receptor antagonist is recommended to patients with acute coronary syndromes. However, TICA exposes to a risk of bleeding, which management is challenging because platelet transfusion is ineffective and no antidote is available. We hypothesized that in a milieu containing ADP, epinephrine (EPI), a current vasopressor drug, could counter the antiplatelet effects of TICA and restore platelet functions, via α2A-adrenergic receptors and Gz-protein pathway. Objective To assess in vitro the efficacy of EPI in restoring platelet aggregation inhibited by TICA and investigate the underlying mechanisms. Method Using lumi-aggregometry of washed platelets, we evaluated aggregation and secretion induced by EPI alone or in combination with ADP. VASP dephosphorylation and Akt phosphorylation were quantified to determine AMPc and PI3k pathway contribution, respectively. The role of P2Y1 receptor in restoration of platelet aggregation was determined using MRS2179, a specific inhibitor, and calcium mobilization measurement. Results TICA inhibited ADP-induced platelet aggregation (5 [2–17] vs 59 [54–73]%, P Conclusion In combination with ADP, EPI restored platelet aggregation inhibited by TICA trough inhibition of cAMP pathway and activation of PI3 K pathway, thus allowing the P2Y1 receptor signaling and subsequent calcium mobilization by ADP. This proof-of-concept study must be challenged in vivo for the management of bleeding in ticagrelor-treated patients.

The αIIb cytoplasmic domain of platelet integrin αIIbβ3 contains an unorganized acidic membrane-distal (1000)LEEDDEEGE(1008) region. We have shown that a platelet permeable peptide corresponding to the above region the palmitoyl-K-LEEDDEEGE (pal-K-1000-1008) inhibits platelet aggregation induced by thrombin or by pal-K-989-995, a palmitoylated peptide corresponding to the membrane-proximal αIIb cytoplasmic domain (989)KVGFFKR(995). We now tested the anti-aggregatory activity of (i) a lipid-modified scrambled acidic peptide (pal-K-GDDEELEEE), (ii) two smaller peptides derived from the acidic amino sequence: palmitoyl-K-(1000)LEEDDE(1005) (pal-K-1000-1005) and palmitoyl-K-(1005)EEGE(1008) (pal-K-1005-1008) and (iii) lipid-modified palmitoyl-acidic peptides with alanine (Ala) substitution at residues 1001, 1003, 1004 and 1005 and one peptide with a double Ala substitution at residues 1001 and 1004 of the 1000-1008 sequence. All the peptides tested showed an inhibitory activity, however, the palmitoylated peptide with the natural and the whole acidic sequence, being the most active. Our results suggest that the whole acidic sequence, rather than some specific amino acids, contributes to the aggregation inhibitory activity. The inhibitory peptide, pal-K-1000-1008, inhibited the association of talin with αIIbβ3 in thrombin-activated platelets, as demonstrated by co-immunoprecipitation experiments, while the scrambled peptide was inefficient. We suggest that, by interacting with αIIb cytoplasmic domain, pal-K-1000-1008 has an anti-aggregatory inhibitory activity due to a specific inhibition of talin binding to αIIbβ3.

Summary Background Genetic variations that affect the structure of the thromboxane A2 receptor (TP receptor) provide insights into the function of this key platelet and vascular receptor, but are very rare in unselected populations. Objectives To determine the functional consequences of the TP receptor Trp29Cys (W29C) substitution. Patients/Methods We performed a detailed phenotypic analysis of an index case (P1) with reduced platelet aggregation and secretion responses to TP receptor pathway activators, and a heterozygous TP receptor W29C substitution. An analysis of the variant W29C TP receptor expressed in heterologous cells was performed. Results Total TP receptor expression in platelets from P1 was similar to that of controls, but there was reduced maximum binding and reduced affinity of binding to the TP receptor antagonist [3H]SQ29548. HEK293 cells transfected with W29C TP receptor cDNA showed similar total TP receptor expression to wild-type (WT) controls. However, the TP receptor agonist U46619 was less potent at inducing rises in cytosolic free Ca2+ in HEK293 cells expressing the W29C TP receptor than in WT controls, indicating reduced receptor function. Immunofluorescence microscopy and cell surface ELISA showed intracellular retention and reduced cell surface expression of the W29C TP receptor in HEK293 cells. Consistent with the platelet phenotype, both maximum binding and the affinity of binding of [3H]SQ29548 to the W29C TP receptor were reduced compared to WT controls. Conclusion These findings extend the phenotypic description of the very rare disorder TP receptor deficiency, and show that the W29C substitution reduces TP receptor function by reducing surface receptor expression and by disrupting ligand binding.

SummaryClot retraction is an essential step during primary haemostasis, thereby promoting thrombus stability and wound healing. Integrin αIIbβ3 plays a critical role in clot retraction, by inducing acto-myosin interactions that allow platelet cytoskeleton reorganisation. However, the signalling pathways that lead to clot retraction are still misunderstood. In this study, we report the first data on the kinetics of myosin II light chain (MLC) phosphorylation during clot retraction. We found an early phosphorylation peak followed by a second peak. By using specific inhibitors of kinases and small G proteins, we showed that MLC kinase (MLCK), RhoA/ROCK, and Rac-1 were involved in clot retraction and in the early MLC phosphorylation peak. Only Rac-1 and actin polymerisation, controlled by outside-in signalling, were crucial to the second MLC phosphorylation peak.

Rationale: Sphingosine-1-phosphate (S1P) signaling is essential for vascular development and postnatal vascular homeostasis. The relative importance of S1P sources sustaining these processes remains unclear. Objective: To address the level of redundancy in bioactive S1P provision to the developing and mature vasculature. Methods and Results: S1P production was selectively impaired in mouse platelets, erythrocytes, endothelium, or smooth muscle cells by targeted deletion of genes encoding sphingosine kinases -1 and -2. S1P deficiency impaired aggregation and spreading of washed platelets and profoundly reduced their capacity to promote endothelial barrier function ex vivo. However, and in contrast to recent reports, neither platelets nor any other source of S1P was essential for vascular development, vascular integrity, or hemostasis/thrombosis. Yet rapid and profound depletion of plasma S1P during systemic anaphylaxis rendered both platelet- and erythrocyte-derived S1P essential for survival, with a contribution from blood endothelium observed only in the absence of circulating sources. Recovery was sensitive to aspirin in mice with but not without platelet S1P, suggesting that platelet activation and stimulus–response coupling is needed. S1P deficiency aggravated vasoplegia in this model, arguing a vital role for S1P in maintaining vascular resistance during recovery from circulatory shock. Accordingly, the S1P 2 receptor mediated most of the survival benefit of S1P, whereas the endothelial S1P 1 receptor was dispensable for survival despite its importance for maintaining vascular integrity. Conclusions: Although source redundancy normally secures essential S1P signaling in developing and mature blood vessels, profound depletion of plasma S1P renders both erythrocyte and platelet S1P pools necessary for recovery and high basal plasma S1P levels protective during anaphylactic shock.

Management of ticagrelor-induced bleeding is challenging as platelet transfusion is ineffective. An effective strategy is needed. This study aimed to investigateBlood was spiked with ticagrelor then supplemented by either HD or control. Several assays were performed: ADP-induced platelet aggregation measured by impedance aggregometry, light transmission and two global assays, thrombolastography with the platelet mapping device (TEG-PM) and a platelet-dependent thrombin generation assay (TGA).Ticagrelor inhibited ADP-induced platelet aggregation and decreased the clot strength maximum amplitude (MA) in TEG-PMWhereas none of the HDs corrected ticagrelor-induced platelet inhibition, rFVIIa shortened coagulation times, Fib increased clot firmness and FXIII decreased fibrinolysis. Consequently, they may bypass ticagrelor effects by acting on fibrin formation or fibrinolysis. Further studies are needed to confirm these data

BACKGROUND AND PURPOSE New antithrombotic agents with the potential to prevent atherothrombotic complications are being developed to target receptors on platelets and other cells involved in plaque growth. The aim of this study was to investigate the antiplatelet effects of F 16618, a new non-peptidic PAR1 (thrombin receptor) antagonist. EXPERIMENTAL APPROACH We investigated the inhibitory effect of F 16618 on human platelet aggregation ex vivo, in whole blood and washed platelets, by using a multiple-electrode platelet aggregometer based on impedance and an optical aggregometer, respectively. Its effects on whole-blood haemostasis (clot parameters) were analysed with the ROTEM thromboelastometry device and the platelet function analyser PFA-100. A guinea-pig model of arterial thrombosis was used to investigate its effects on thrombus formation in vivo. KEY RESULTS F 16618 inhibited PAR1 agonist peptide (SFLLR-peptide)-induced washed platelet aggregation ex vivo. This effect was concentration-dependent and exhibited a competitive inhibition profile. Washed platelet aggregation, as well as P-selectin expression induced by thrombin, were significantly inhibited by 10 µM F 16618. In whole-blood experiments, 20 µM F 16618 inhibited SFLLR-induced platelet aggregation by 49%. In contrast, it had no effect on whole-blood haemostasis. In the guinea-pig model of carotid thrombosis, 0.32 mg·kg−1 F 16618 doubled the occlusion time. CONCLUSIONS AND IMPLICATIONS F 16618 was shown to have strong antithrombotic activity in vivo and moderate antiplatelet effects ex vivo. As these effects were not associated with major effects on physiological haemostasis, this molecule is a good antiplatelet drug candidate for use either alone or in combination with current treatments.

Lowe syndrome (LS) is a rare X-linked disorder caused by mutations in the oculocerebrorenal gene (OCRL), encoding OCRL, a phosphatidylinositol 5-phosphatase with a RhoGAP domain. An abnormal rate of haemorrhagic events was found in a retrospective clinical survey. Herein, we report the results of exploration of haemostasis in six LS patients. All patients had normal coagulation tests but prolonged closure times (CTs) in the PFA-100 system. Healthy donors' blood samples incubated with a RhoA kinase inhibitor had prolonged CTs. This suggests that an aberrant RhoA pathway in platelets contributes to CT prolongation and primary haemostasis disorders in LS.

SummaryPlatelet dysfunction after cardiopulmonary bypass (CPB) can contribute to excessive post-operative bleeding. Most trials of the protective effect of aprotinin in this setting have involved hypothermic CPB, which is more deleterious for platelets than normothermic CPB.Here we investigated the effect of aprotinin on platelet function during normothermic CPB in pediatric patients. Twenty patients (9 newborns [

Management of ticagrelor-induced bleeding is challenging, as no antidote is currently available. Platelet transfusion, usually proposed to reverse antiplatelet drugs, has been suggested to be ineffective but few data are available.To assess the efficacy of platelet supplementation to restore platelet aggregation inhibited by ticagrelor.In vitro study.Blood samples were obtained from the French Blood Bank Institute.Healthy blood donors.Whole blood from healthy donors was spiked with ticagrelor or aspirin (used as a positive control).Platelet aggregation was investigated with impedance aggregometry on whole blood [expressed in ohms (V)] and light transmission aggregometry (expressed in %) on platelet-rich plasma using ADP or arachidonic acid as agonists for ticagrelor or aspirin, respectively. Platelet supplementation was defined as the addition of washed platelet suspension increasing at least 60% of whole blood platelet count.Ticagrelor (3.25 mM) inhibited ADP-induced platelet aggregation compared with control either in whole blood (2 vs. 13 V, P 0.05) or in platelet-rich plasma (15 vs. 75% P 0.05). Aspirin (25 mM) inhibited arachidonic acid-induced aggregation (1 vs. 7.5 V, P 0.05 in whole blood and 5 vs. 77.5%, P = 0.01 in platelet-rich plasma). Platelet supplementation completely restored arachidonic acid-induced platelet aggregation in whole blood (10 vs. 1 V, P = 0.008) and platelet-rich plasma (73 vs. 5%, P 0.01) in aspirin-treated samples, whereas it failed to correct ADP-induced aggregation (2 vs. 2 V in whole blood and 13.5 vs. 15% in platelet-rich plasma, P 0.05) in ticagrelor-treated samples. We also report a case of a ticagrelor-treated patient in whom platelet transfusion failed to restore ADP-induced platelet aggregation.Platelet supplementation restored platelet aggregation in aspirin-spiked but not in ticagrelor-spiked samples. These results do not support the use of platelet transfusion to reverse the effects of ticagrelor.

Molecules that reduce the level of cyclic adenosine 5'-monophosphate (cAMP) in the platelet cytosol, such as adenosine 5'-diphosphate (ADP) secreted from dense granules, trigger platelet activation. Therefore, any change in the distribution and/or availability of cyclic nucleotides or ADP may interfere with platelet reactivity. In this study, we evaluated the role of multidrug resistance protein 4 (MRP4, or ABCC4), a nucleotide transporter, in platelet functions in vivo and in vitro by investigating MRP4-deficient mice. MRP4 deletion resulted in a slight increase in platelet count but had no impact on platelet ultrastructure. In MRP4-deficient mice, the arterial occlusion was delayed and the tail bleeding time was prolonged. In a model of platelet depletion and transfusion mimicking a platelet-specific knockout, mice injected with MRP4(-/-) platelets also showed a significant increase in blood loss compared with mice injected with wild-type platelets. Defective thrombus formation and platelet activation were confirmed in vitro by studying platelet adhesion to collagen in flow conditions, integrin αIIbβ3 activation, washed platelet secretion, and aggregation induced by low concentrations of proteinase-activated receptor 4-activating peptide, U46619, or ADP. We found no role of MRP4 in ADP dense-granule storage, but MRP4 redistributed cAMP from the cytosol to dense granules, as confirmed by increased vasodilator-stimulated phosphoprotein phosphorylation in MRP4-deficient platelets. These data suggest that MRP4 promotes platelet aggregation by modulating the cAMP-protein kinase A signaling pathway, suggesting that MRP4 might serve as a target for novel antiplatelet agents.

Agonist-stimulated platelet activation triggers conformational changes of integrin αIIbβ3, allowing fibrinogen binding and platelet aggregation. We have previously shown that an octapeptide, p1YMESRADR8, corresponding to amino acids 313–320 of the β-ribbon extending from the β-propeller domain of αIIb, acts as a potent inhibitor of platelet aggregation. Here we have performed in silico modelling analysis of the interaction of this peptide with αIIbβ3 in its bent and closed (not swing-out) conformation and show that the peptide is able to act as a substitute for the β-ribbon by forming a clasp restraining the β3 hybrid and βI domains in a closed conformation. The involvement of species-specific residues of the β3 hybrid domain (E356 and K384) and the β1 domain (E297) as well as an intrapeptide bond (pE315-pR317) were confirmed as important for this interaction by mutagenesis studies of αIIbβ3 expressed in CHO cells and native or substituted peptide inhibitory studies on platelet functions. Furthermore, NMR data corroborate the above results. Our findings provide insight into the important functional role of the αIIb β-ribbon in preventing integrin αIIbβ3 head piece opening, and highlight a potential new therapeutic approach to prevent integrin ligand binding.

Shear-induced platelet aggregation (SIPA) involves the sequential interaction of von Willebrand factor (VWF) with both glycoprotein Ib (GPIb) and αIIbβ3 receptors. Type 2B recombinant VWF (2B-rVWF), characterized by an increased affinity for GPIb, induces strong SIPA at a high shear rate (4000 s–1). Despite the increased affinity of 2B-rVWF for GPIb, patients with type 2B von Willebrand disease have a paradoxical bleeding disorder, which is not well understood. The purpose of this study was to determine if SIPA induced by 2B-rVWF was associated with αIIbβ3-dependent platelet activation. To this end, we have addressed the influence of 2B-rVWF (Val553Met substitution) on SIPA-dependent variations of tyrosine protein phosphorylation (P-Tyr) and the effect of αIIbβ3 blockers. At a high shear rate, 2B-rVWF induced a strong SIPA, as shown by a 92.7% ± 0.4% disappearance of single platelets (DSP) after 4.5 minutes. In these conditions, increased P-Tyr of proteins migrating at positions 64 kd, 72 kd, and 125 kd were observed. The band at 125 kd was identified as pp125FAK using anti–phospho-FAK antibody. This effect, which required a high level of SIPA (> 70% DSP), was observed at 4000 s–1 but not at 200 s–1. Monoclonal antibodies (MoAbs) 6D1 (anti-GPIb) and 328 (anti-VWF A1 domain), completely abolished SIPA and p125FAK phosphorylation mediated by 2B-rVWF. In contrast, neither RGDS peptide nor MoAb 7E3, both known to block αIIbβ3 engagement, had any effect on SIPA and pp125FAK. The size of aggregates formed at a high shear rate in the presence of 2B-rVWF was decreased by genistein, demonstrating the biologic relevance of pp125FAK. These findings provide a unique mechanism whereby the enhanced interaction of 2B-rVWF with GPIb, without engagement of αIIbβ3, is sufficient to induce SIPA but does not lead to stable thrombus formation.

Protease-activated receptor 1 (PAR-1), the main thrombin receptor on vascular cells, plays a key role in platelet activation. We examined the range of PAR-1 expression on platelets, obtained twice, 1 week apart, from 100 healthy subjects and found a 2-fold interindividual variation in receptor numbers (95% CI = 858-1700). Because PAR-1 density was stable with time (r2 = 76%,P

AIMS: To investigate functional platelet recovery after preoperative withdrawal of aspirin and clopidogrel and platelet function 5 days after treatment resumption. METHODS/RESULTS: We conducted an observational study, which prospectively included consecutive patients taking aspirin, taking clopidogrel, and untreated controls (15 patients in each group). The antiplatelet drugs were withdrawn five days before surgery (baseline) and were reintroduced two days after surgery. Platelet function was evaluated by optical aggregation in the presence of collagen, arachidonic acid (aspirin) and ADP (clopidogrel) and by VASP assay (clopidogrel). Platelet-leukocyte complex (PLC) level was quantified at each time-point. At baseline, platelet function was efficiently inhibited by aspirin and had recovered fully in most patients 5 days after drug withdrawal. PLC levels five days after aspirin reintroduction were similar to baseline (+4±10%; p = 0.16), in line with an effective platelet inhibition. Chronic clopidogrel treatment was associated with variable platelet inhibition and its withdrawal led to variable functional recovery. PLC levels were significantly increased five days after clopidogrel reintroduction (+10±15%; p = 0.02), compared to baseline. CONCLUSIONS: Aspirin withdrawal 5 days before high-bleeding-risk procedures was associated with functional platelet recovery, and its reintroduction two days after surgery restored antiplaletet efficacy five days later. This was not the case of clopidogrel, and further work is therefore needed to define its optimal perioperative management.

Background Neuro- and vasoprotective effects of poly(ADP-ribose)polymerase (PARP) inhibition have been largely documented in models of cerebral ischemia, particularly with the potent PARP inhibitor PJ34. Furthermore, after ischemic stroke, physicians are faced with incomplete tissue reperfusion and reocclusion, in which platelet activation/aggregation plays a key role. Data suggest that certain PARP inhibitors could act as antiplatelet agents. In that context, the present in vitro study investigated on human blood the potential antiplatelet effect of PJ34 and two structurally different PARP inhibitors, DPQ and INO-1001. Methods and results ADP concentrations were chosen to induce a biphasic aggregation curve resulting from the successive activation of both its receptors P2Y1 and P2Y12. In these experimental conditions, PJ34 inhibited the second phase of aggregation; this effect was reduced by incremental ADP concentrations. In addition, in line with a P2Y12 pathway inhibitory effect, PJ34 inhibited the dephosphorylation of the vasodilator stimulated phosphoprotein (VASP) in a concentration-dependent manner. Besides, PJ34 had no effect on platelet aggregation induced by collagen or PAR1 activating peptide, used at concentrations inducing a strong activation independent on secreted ADP. By contrast, DPQ and INO-1001 were devoid of any effect whatever the platelet agonist used. Conclusions We showed that, in addition to its already demonstrated beneficial effects in in vivo models of cerebral ischemia, the potent PARP inhibitor PJ34 exerts in vitro an antiplatelet effect. Moreover, this is the first study to report that PJ34 could act via a competitive P2Y12 antagonism. Thus, this antiplatelet effect could improve post-stroke reperfusion and/or prevent reocclusion, which reinforces the interest of this drug for stroke treatment.

In the vitamin K-dependent protein family, only protein S (PS) contains a thrombin-sensitive region (TSR), located between the domain containing the γ-carboxyglutamic acid and the first epidermal growth factor-like domain. To better define the role of TSR in the PS molecule, we expressed a recombinant human PS (rHPS) and its analogue lacking TSR (rTSR-less), and prepared factor Xa- and thrombin-cleaved rHPS. A peptide reproducing TSR (TSR-peptide) was also synthesized in an attempt to obtain direct evidence of the domain involvement in PS anticoagulant activity. In a coagulation assay, both rTSR-less and factor Xa-cleaved PS were devoid of activated protein C cofactor activity. The TSR-peptide did not inhibit rHPS activity, showing that TSR must be embedded in the native protein to promote interaction with activated protein C. The binding of rHPS to activated platelets and to phospholipid vesicles was not modified after factor Xa- or thrombin-mediated TSR cleavage, whereas the binding of rTSR-less was markedly reduced. This suggested a role for TSR in conferring to PS a strong affinity for phospholipid membranes. TSR-peptide did not directly bind to activated platelets or compete with rHPS for phospholipid binding. The results of the present study show that TSR may not interact directly with membranes, but probably constrains the γ-carboxyglutamic acid-rich domain in a conformation allowing optimal interaction with phospholipids.

Agonist-induced platelet activation triggers 'inside-out' signalling which activates alpha IIb-beta 3, the most abundant integrin in platelet membranes. The engagement of activated alpha IIb-beta 3 integrin by linking fibrinogen is necessary for platelet aggregation, and this induces subsequent outside-in signalling, which enhances platelet activation. Here we studied the involvement of Cbl during alpha IIb-beta 3-integrin-mediated signal transduction. During thrombin-induced platelet activation, Cbl was tyrosine phosphorylated, and phosphoinositide 3-kinase (PI 3-kinase) activity measured in Cbl immunoprecipitates was increased. Both Cbl phosphorylation and its association with PI 3-kinase were dependent on alpha IIb-beta 3 engagement by linking fibrinogen. The P256 and anti-LIBS6 (ligand-induced binding site 6) antibodies, which activate platelets directly through alpha IIb-beta 3, induced Cbl phosphorylation and increased the PI 3-kinase activity associated with Cbl. Both thrombin and antibodies to alpha IIb-beta 3 induced association of Cbl with the tyrosine kinase, Syk. Experiments performed with inhibitors of tyrosine kinases indicated that both Src-family kinases and Syk contribute to phosphorylation of Cbl and its consequent association with PI 3-kinase. The results show that, following integrin alpha IIb-beta 3 engagement, Cbl is tyrosine phosphorylated, recruits PI 3-kinase to this integrin signalling pathway and possibly enhances PI 3-kinase activity, downstream of Src-family tyrosine kinases and Syk activation.

Protein phosphorylation was studied during platelet stimulation in two ranges of ionized [Ca2+]. At ionized [Ca2+]ior = 1 microM, proteins were phosphorylated. At ionized [Ca2+]ior = 4 microM, phosphoproteins disappeared. Protein dephosphorylation was prevented by the combined action of calpeptin and phosphatase inhibitors. Protein tyrosine phosphatase activity was stimulated regardless of the ionized [Ca2+] level. Protein tyrosine kinase activity was stimulated at ionized [Ca2+]ior =1 microM, whereas at ionized [Ca2+]ior =4 microM, no protein tyrosine kinase activity was observed except in the presence of calpeptin. Thus, the massive tyrosine phosphoprotein disappearance observed at a high ionized [Ca2+]i resulted not only in protein tyrosine phosphatase activation, but also in calpain-induced protein tyrosine kinase inactivation.