Platelet Stimulation Research Articles

Inhibition of platelet aggregation and stimulation of natural killer cells functionality by administration of flavonoids

Platelets play an important role in homeostasis, inflammation and regulation of immune response. Natural Killer cells (NKC) on the other hand are the first line of defense of the immune system against viruses, bacteria and cancer cells. Platelets directly protect tumor cells from NK lysis. Thus, platelet aggregation around migrating cancer cells as well as the response of the immune system constitutes important mechanisms, which influence the progression of malignant diseases. The present study was designed to evaluate the possible effects of flavonoids (genistein, apigenin and quercetin) on the stimulation of the immune system and the inhibition of platelet aggregation. Two experimental protocols were used: a) ex vivo aggregation of human platelets, production of thromboxane A2 (TXA2) and estimation of the expression of the platelet membranic receptor GpIIb/IIIa, and b) functional activity of human NK lymphocytes against K562 target cells in vitro. All substances were found to induce a dose dependent inhibition of platelet aggregation and to reduce production of TXA2 in platelets. There was a decrease in the number of the GpIIb/IIIa receptors on the platelets surface membrane. Flow cytometry analysis revealed that all substances powerfully reinforce cytotoxic activity of NKC against K562 cells. These results show that flavonoids increase the susceptibility of tumor cells to NK cells by decreasing platelet aggregation and stimulating the NK lymphocyte activity.

Stimulation of human platelets by carrageenans.

The rank order of four carrageenans tested as inducers of human platelet aggregation was the same (iota greater than lambda greater than gelcarin greater than kappa) as their relative inflammatory potencies in vivo. All four carrageenans caused some precipitation of plasma proteins, and induced aggregation in platelet-rich plasma or washed platelet suspensions. The second phase of aggregation was citrate-dependent and associated with secretion of 5-hydroxytryptamine and lysosomal enzymes. Platelets could provide a useful model for investigating the actions of carrageenans on cell membranes.

The potentiation of phorbol ester-induced aggregation of human platelets by the prostaglandin endoperoxide analogue, U46619.

It was not possible to desensitize human blood platelets to 12-deoxyphorbol-phenylacetate (DOPP) stimulation in a manner analogous with that to platelet aggregating factor (PAF), prostaglandin-endoperoxide analogue (U46619) or adenosine diphosphate (ADP). Platelets previously desensitized to U46619, when challenged with DOPP and ADP, showed an increased aggregation and release of 5-HT. Sub-threshold aggregating doses of U46619 also caused a potentiation of the platelet response and release reaction to DOPP. The concentration of U46619 used to pretreat platelets affected the extent of potentiation of platelet stimulation induced by DOPP. The degree of potentiation was also affected by the time interval between addition of U46619 and DOPP. U46619 did not potentiate the aggregating effects of PAF, or ionophore A23187. The stimulus potentiation of DOPP by U46619 was abolished by prostacyclin (PGI2) and an antibody to U46619, but was unaffected by indomethacin and CP/CPK.

Critical Role for CD38-mediated Ca2+ Signaling in Thrombin-induced Procoagulant Activity of Mouse Platelets and Hemostasis

CD38, a multifunctional enzyme that catalyzes the synthesis of intracellular Ca(2+) messengers, cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP), is known to be expressed on platelets. However, the role of CD38 in platelets remains unclear. Our present results show that treatment of platelets with thrombin results in a rapid and sustained Ca(2+) signal, resulting from a coordinated interplay of Ca(2+)-mobilizing messengers, inositol 1,4,5-trisphosphate, cADPR, and NAADP. By dissecting the signaling pathway using various agents, we delineated that cADPR and NAADP are sequentially produced through CD38 internalization by protein kinase C via myosin heavy chain IIA following phospholipase C activation in thrombin-induced platelets. An inositol 1,4,5-trisphosphate receptor antagonist blocked the thrombin-induced formation of cADPR and NAADP as well as Ca(2+) signals. An indispensable response of platelets relying on cytosolic calcium is the surface exposure of phosphatidylserine (PS), which implicates platelet procoagulant activity. Scrutinizing this parameter reveals that CD38(+/+) platelets fully express PS on the surface when stimulated with thrombin, whereas this response was decreased on CD38(-/-) platelets. Similarly, PS exposure and Ca(2+) signals were attenuated when platelets were incubated with 8-bromo-cADPR, bafilomycin A1, and a PKC inhibitor. Furthermore, in vivo, CD38-deficient mice exhibited longer bleeding times and unstable formation of thrombus than wild type mice. These results demonstrate that CD38 plays an essential role in thrombin-induced procoagulant activity of platelets and hemostasis via Ca(2+) signaling mediated by its products, cADPR and NAADP.

STIM1 and STIM2 Are Located in the Acidic Ca2+ Stores and Associates with Orai1 upon Depletion of the Acidic Stores in Human Platelets

Mammalian cells accumulate Ca2+ into agonist-sensitive acidic organelles, vesicles that possess a vacuolar proton-ATPase. Acidic Ca2+ stores include secretory granules and lysosome-related organelles. Current evidence clearly indicates that acidic Ca2+ stores participate in cell signaling and function, including the activation of store-operated Ca2+ entry in human platelets upon depletion of the acidic stores, although the mechanism underlying the activation of store-operated Ca2+ entry controlled by the acidic stores remains unclear. STIM1 has been presented as the endoplasmic reticulum Ca2+ sensor, but its role sensing intraluminal Ca2+ concentration in the acidic stores has not been investigated. Here we report that STIM1 and STIM2 are expressed in the lysosome-related organelles and dense granules in human platelets isolated by immunomagnetic sorting. Depletion of the acidic Ca2+ stores using the specific vacuolar proton-ATPase inhibitor, bafilomycin A1, enhanced the association between STIM1 and STIM2 as well as between these proteins and the plasma membrane channel Orai1. Depletion of the acidic Ca2+ stores also induces time-dependent co-immunoprecipitation of STIM1 with the TRPC proteins hTRPC1 and hTRPC6, as well as between Orai1 and both TRPC proteins. In addition, bafilomycin A1 enhanced the association between STIM2 and SERCA3. These findings demonstrate the location of STIM1 and STIM2 in the acidic Ca2+ stores and their association with Ca2+ channels and ATPases upon acidic stores discharge.

Arrestin-2 Differentially Regulates PAR4 and ADP Receptor Signaling in Platelets

Arrestins can facilitate desensitization or signaling by G protein-coupled receptors (GPCR) in many cells, but their roles in platelets remain uncharacterized. Because of recent reports that arrestins can serve as scaffolds to recruit phosphatidylinositol-3 kinases (PI3K)s to GPCRs, we sought to determine whether arrestins regulate PI3K-dependent Akt signaling in platelets, with consequences for thrombosis. Co-immunoprecipitation experiments demonstrate that arrestin-2 associates with p85 PI3Kα/β subunits in thrombin-stimulated platelets, but not resting cells. The association is inhibited by inhibitors of P2Y12 and Src family kinases (SFKs). The function of arrestin-2 in platelets is agonist-specific, as PAR4-dependent Akt phosphorylation and fibrinogen binding were reduced in arrestin-2 knock-out platelets compared with WT controls, but ADP-stimulated signaling to Akt and fibrinogen binding were unaffected. ADP receptors regulate arrestin recruitment to PAR4, because co-immunoprecipitates of arrestin-2 with PAR4 are disrupted by inhibitors of P2Y1 or P2Y12. P2Y1 may regulate arrestin-2 recruitment to PAR4 through protein kinase C (PKC) activation, whereas P2Y12 directly interacts with PAR4 and therefore, may help to recruit arrestin-2 to PAR4. Finally, arrestin2(-/-) mice are less sensitive to ferric chloride-induced thrombosis than WT mice, suggesting that arrestin-2 can regulate thrombus formation in vivo. In conclusion, arrestin-2 regulates PAR4-dependent signaling pathways, but not responses to ADP alone, and contributes to thrombus formation in vivo.

A novel platelet glycoprotein Ib-binding protein with human platelet aggregation-inhibiting activity from Trimeresurus jerdonii venom

Platelet glycoprotein Ib (GPIb) is a primary adhesion receptor and involved in platelet-related disorders. However, it is difficult to study GPIb-specific platelet stimulation using physiological ligands in vivo. GPIb-binding snake C-type lectins (snaclecs) are useful tools for exploring GPIb in vitro because they act on platelets differently. In the present study, a novel GPIb-binding snaclec, named jerdonibitin, was purified, molecular cloned and characterized from Trimeresurus jerdonii venom. On SDS-polyacrylamide gel electrophoresis, it showed a single band with an apparent molecular weight of 25 kDa under non-reducing conditions and two distinct bands with apparent molecular weights of 15 kDa (α-subunit) and 13 kDa (β-subunit) under reducing conditions. The cDNA sequences of each subunit of jerdonibitin were identified and both deduced amino acid sequences were confirmed by N-terminal protein sequencing and trypsin-digested peptide mass fingerprinting of MALDI-TOF. Sequence alignment showed that jerdonibitin is a snaclec and has sequence similarity with TSV-GPIb-BP (a GPIb-inhibitory snaclec). Jerdonibitin dose-dependently inhibited platelet aggregation induced by ristocetin or low-dose thrombin, but not by high-dose thrombin. The GPIbα was detected by affinity chromatography on jerdonibitin. In vivo, jerdonibitin also dose-dependently induced thrombocytopenia of mice and platelet counts remained at very low level after 18 h intravenous injection. In summary, a novel GPIb-inhibitory snaclec was molecular cloned and characterized, which might provide insights into investigation of how GPIb-inhibitory snaclecs work and development of new antiplatelet agents.

Chlamydia pneumoniae Induces Chemokine Expression by Platelets in Patients with Atherosclerosis

Objective: In this study, the role of Chlamydia pneumoniae in triggering platelets to induce the inflammatory potential chemokines CCL3, CCL5, CCL7 and CXCL8 in atherosclerotic patients was investigated. Subjects and Methods: Venous blood from control subjects (n = 35) and atherosclerotic patients (n = 35) was collected in tubes with and without EDTA. Platelets from controls and patients were separated from whole blood and then stimulated with lipopolysaccharide (LPS), live C. pneumoniae and heat-treated C. pneumoniae. The ability of C. pneumoniae and its LPS to stimulate platelets and expression of CCL3, CCL5, CCL7 and CXCL8 was assessed with immunofluorescence. Immunosorbent assays were used to detect anti-C. pneumoniae antibodies in sera from patients and healthy subjects. Results: Nonstimulated platelets from patients showed significant expression of CCL3, CCL5, CCL7 and CXCL8 compared to controls (p < 0.0001). Stimulation of platelets from patients with live and heat-treated C. pneumoniae and its LPS demonstrated significant induction of chemokines compared to similarly stimulated platelets from controls (p < 0.01). After stimulation with heat-treated C. pneumoniae chemokine expression in platelets from controls was significantly lower than after stimulation with live C. pneumoniae (p < 0.01), which was not the case when platelets from patients were stimulated. Increased levels of anti-C. pneumoniae antibodies were detected in sera from patients compared to healthy subjects, suggesting prior C. pneumoniae exposure. Conclusion: Our data demonstrated an interactive link between C. pneumoniae and platelets in atherosclerotic patients, leading to induction of potential chemokines and possibly disease development.

Epinephrine-mediated protein kinase C and Rap1b activation requires the co-stimulation of Gz-, Gq-, and Gi-coupled receptors

We have recently shown that ADP-induced activation of protein kinase C (PKC) requires the co-stimulation of both P2Y1 and P2Y12 receptors. In this work, we show that inhibition of ADP-mediated phosphorylation of pleckstrin, the main PKC substrate, caused by antagonists of the P2Y12 receptor can be reversed by stimulation of the α2-adrenergic receptor by epinephrine. However, we also observed that addition of epinephrine alone caused a marked phosphorylation of pleckstrin. This effect occurred in the absence of Gq stimulation, as it was not associated to intracellular Ca2+ release. Epinephrine-induced pleckstrin phosphorylation was time- and dose-dependent, and was inhibited by the α2-adrenergic antagonist yohimbin. Phosphorylation of pleckstrin did not occur when platelet stimulation with epinephrine was performed in the presence of the ADP scavenger apyrase, and was suppressed by antagonists of both P2Y1 and P2Y12 ADP receptors. Importantly, no release of dense granules was measured in epinephrine-treated platelets. Addition of epinephrine to platelets was also able to stimulate Rap1b activation. Similarly to pleckstrin phosphorylation, however, this effect was prevented in the presence of apyrase or upon pharmacologic blockade of either P2Y1 or P2Y12 receptors. These results indicate that sub-threshold amounts of ADP in the medium are essential to allow epinephrine stimulation of α2-adrenergic receptor to elicit platelet responses, and reveal a novel synergism among strong stimulation of Gz and sub-threshold stimulation of both Gq and Gi, able to dissociate PKC activation from intracellular Ca2+ mobilisation.

Hydrophoretic high-throughput selection of platelets in physiological shear-stress range

A gentle, but fast means for low-stress, high-throughput platelet purification is of significant clinical and biotechnological utility. Current implementations to sort platelets, however, require an external physical field, specialized buffer, or the harsh separation condition of high shear stress that tends to cause platelet stimulation. Here we report the use of hydrophoretic size separation in a wider channel and its parallelization to augment its throughput capability, maintaining physiological shear-stress range. We demonstrate a parallelized device comprising 10 stacks of the wide-channel hydrophoresis device, yielding a throughput of 2.9 million cells s(-1) and a platelet purity of 76.8%. The use of the wide channel for hydrophoresis also facilitates clogging-free separation by sorting blood clots and plaques. The wide-channel hydrophoresis offers the potential for gentle, fast, clogging-free sorting of rare blood cells with extreme throughput capabilities.

Inhibitory Effects of Resveratrol on Platelet Activation Induced by Thromboxane A2Receptor Agonist in Human Platelets

Resveratrol (RSVL), a polyphenolic compound found in red wine is believed to be a contributor in decreasing the incidence of coronary heart disease. Although its primary target is unknown, it blocks platelet aggregation by an ill-defined mechanism. Protein kinase C (PKC), which would redistribute from the cytosol to the platelet membrane upon platelet stimulation, plays a key role in the signal transduction system of platelets in human. In this study, we investigated the effect of RSVL and a PKC inhibitor (DL-erythro-1,3-Dihydroxy-2-aminooctadecane, PKCI) on platelet aggregation induced by a thromboxane A(2) receptor agonist (U46619, 9,11-Dideoxy-11α, 9α-epoxymethanoprostaglandin F(2α)) using a platelet aggregometer. We also studied the platelet membranebound fibrinogen (PFig) content and the activity of protein kinase C (PKC) in platelets from healthy volunteers using flow cytometry, and a phosphorimaging system, respectively. Our results showed that RSVL blocked platelet aggregation and PFig content induced by U46619 in a concentration-dependent manner. PKCI and RSVL had an additive effect in inhibiting platelet aggregation and PFig content. Furthermore, RSVL (final concentration 50 μM) remarkably depressed the activity of PKC in the membrane of platelets and the percentage of membrane PKC activity in total PKC activity. Taken together, these results suggested that RSVL suppressed U46619-induced platelet aggregation and PFig content partially through the inhibition of the activity of PKC in platelets.

Tranexamic acid partially improves platelet function in patients treated with dual antiplatelet therapy

Although the impact of tranexamic acid on platelet function remains controversial, tranexamic acid is part of clinical algorithms for the management of platelet dysfunction. The goal of our prospective, observational study was to examine the effects of tranexamic acid on platelet function in patients treated with dual antiplatelet therapy compared to those who ceased antiplatelet therapy for at least 7 days. Forty patients scheduled for cardiac surgery were enrolled in this study. Group 1 consisted of 20 patients who ceased antiplatelet therapy with aspirin and clopidogrel at least 7 days before surgery. Group 2 consisted of 20 patients who were treated with aspirin and clopidogrel until the day before surgery. Using the Multiplate device (Dynabyte, Munich, Germany), multiple electrode aggregometry (MEA) was performed following platelet stimulation with thrombin receptor activating peptide-6 (TRAP-6), arachidonic acid or ADP on blood collected 20 min before and after application of 2 g tranexamic acid. Compared with group 1, platelet aggregation was statistically significantly reduced in ASPItest and ADPtest in group 2, whereas there were no significant differences in the TRAPtest. In group 1, platelet aggregation did not differ significantly before and after tranexamic acid treatment. In contrast, in group 2, we observed a significant increase in arachidonic acid-induced [295 (280/470) arbitrary aggregation units × min [AU*min; median (25th/75th percentile) vs. 214 (83/409) AU*min, P = 0.01] and ADP-induced platelet aggregation [560 AU*min (400/760 AU*min) vs. 470 AU*min (282/550 AU*min), P = 0.013], whereas platelet aggregation following stimulation with TRAP-6 did not change significantly [980 (877/1009) AU*min, median (25th/75th percentile) after tranexamic acid vs. 867 (835/961) AU*min before tranexamic acid, P = 0.464]. The results of this study indicate that tranexamic acid potentially corrects defects in arachidonic acid-induced and ADP-induced platelet aggregation imposed by dual antiplatelet therapy. However, platelet aggregation in response to arachidonic acid or ADP in the blood of patients who have not received aspirin and clopidogrel is unaffected by tranexamic acid. These results support the use of tranexamic acid to partially reverse platelet aggregation dysfunction due to antiplatelet therapy.

Comparison of soluble CD40L concentrations and release capacities in apheresis and prestorage pooled platelet concentrates

Soluble CD40L (sCD40L) is expressed by platelets and is involved in the stabilization of arterial thrombi. Additionally, it was shown that sCD40L accumulation occurred in stored blood products triggering adverse transfusion reactions like TRALI. To study the influence of the preparation technique on sCD40L accumulation and platelet function we examined CD40L concentrations in prestorage pooled platelet concentrates compared to apheresis products. In addition, sCD40L release capacity was determined as a marker for platelet viability. sCD40L concentrations were determined in prestorage pooled platelet concentrates (n = 8) and in platelet apheresis concentrates (n = 8) before and after platelet stimulation (recalcification and clot formation) at day 1, 3 and 5 under routine storage conditions. sCD40L concentrations were determined by a commercially available ELISA kit. sCD40L concentrations in storage medium increased over time in prestorage pooled platelet concentrates (from 1,185 pg/mL ± 87 pg/mL at day 1 to 4,464 pg/mL ± 212 pg/mL at day 5) as well as in apheresis products (from 581 pg/mL ± 124 pg/mL at day 1 to 2,718 pg/mL ± 154 pg/mL at day 5) in a hyperbolic manner. Recalcification and clot formation caused an increase in sCD40L concentrations (for example 3,842 pg/mL ± 769 pg/mL before platelet activation to 31,219 pg/mL ± 2,063 pg/mL after platelet activation at day 3), and we observed comparable release capacities for both preparation techniques, however, decreasing over storage time up to 50% (day 5) of the respective control value (day 1). Amounts of sCD40L accumulation and release capacity during storage of platelet concentrates were dependent on storage duration, but showed no relevant differences regarding the preparation technique. After 5 days of storage, CD40L basal levels were increased, in contrast sCD40L release capacity was decreased. By recalcification and clot formation sCD40L release capacity could be easily induced and is assumed to be used as a marker for platelet viability.

Collagen stimulation of platelets induces a rapid spatial response of cAMP and cGMP signaling scaffolds

Intracellular communication is tightly regulated in both space and time. Spatiotemporal control is important to achieve a high level of specificity in both dimensions. For instance, cAMP-dependent kinase (PKA) attains spatial resolution by interacting with distinct members of the family of A-kinase anchoring proteins (AKAPs) that position PKA at specific loci within the cell. To control the cAMP induced signal in time, distinct signal terminators such as phosphodiesterases and phosphatases are often co-localized at the AKAP scaffold. In platelets, high levels of cAMP/cGMP maintain the resting state to allow free circulation. Exposure to collagen, for instance when the vessel is damaged, triggers platelet activation through initiation of the GPVI (glycoprotein VI)/FcRγ-chain forming the onset of a plethora of signaling pathways. Consequently overall intra-platelet cAMP and cGMP levels drop, however detail on how PKA, but also cGMP-dependent protein kinase (PKG) respond in relation to their localized signaling scaffolds is currently missing. To investigate this, we employed a quantitative chemical proteomics approach in activated human platelets enabling the specific enrichment of cAMP/cGMP signaling nodes. Our data reveal that within a few minutes several specific PKA and PKG signaling nodes respond significantly to the activating signal, whereas others do not, suggesting a rapid adaption of specific localized cAMP and cGMP pools to the stimulus. Using protein phosphorylation data gathered we touch upon the potential cross-talk between protein phosphorylation and signaling scaffold function as a general theme in platelet spatiotemporal control.

170 Does atrial differences in endothelium damage, leukocyte and platelet activation contribute to chamber specific thrombogenic status in patients with atrial fibrillation?

In atrial fibrillation (AF), the reasons why most of the thrombi form in the left atrium are mainly unknown. In the vasculature, endothelial damage together with platelet activation and inflammation contribute to initiation of blood coagulation and thrombus growth. The purpose of this study was to investigate whether atrial-specific differences in endothelial damage, leukocyte activation, platelet stimulation occur in patients with AF. Twenty patients (15 men, 5 women; age 55 ± 8 years, 15 paroxystic AF, 5 persistent AF) with AF undergoing ablation were investigated. Blood samples from the left and right atrium were obtained at the start of the procedure. Procoagulant microparticles (MPs), reliable markers of vascular damage were measured by capture assays. Their procoagulant abilities were quantified by functional prothrombinase assay and their cellular origin were determined (endothelium, platelet, leukocyte). In addition, platelet reactivity was evaluated by whole blood flow cytometry for expression of platelet Pselectin (CD62P), active glycoprotein IIbIIIa receptor (PAC-1). Platelet aggregation was evaluated using Arachidonic acid (AA), ADP, TRAP and collageninduced whole blood aggregometry. No atrial-specific differences in the levels of total procoagulant MP, leukocyte-derived-MP and platelet-derived MP could be evidenced. Conversely, endothelial-derived MPs (CD105+) were slightly elevated in the right atrium (RA 0.96 ± 0.53 vs. LA 0.80 ± 0.45 nm PhtdSer Eq.; p = 0.041). Likewise, collagen-induced platelet aggregation was evidenced in the right atrium (Collagen 1 mg/l RA: 48 ± 33% vs LA 37 ± 29%; p 0.035; collagen 2,5 mg/l RA: 76 ± 25% vs LA: 60 ± 29%; p = 0.001). In patients with AF, endothelial damage and collageninduced platelet aggregation appear slightly more pronounced in the right atrium. Our data did not substantiate the view that chamber specific enhanced thrombogenic status could be a reliable explanation for the increased propensity for thrombus formation observed in the left atrium in AF patients.

169 Does atrial differences in endothelium damage, leukocyte and platelet activation contribute to chamber specific thrombogenic status in patients with atrial fibrillation?

In atrial fibrillation (AF), the reasons why most of the thrombi form in the left atrium are mainly unknown. In the vasculature, endothelial damage together with platelet activation and inflammation contribute to initiation of blood coagulation and thrombus growth. The purpose of this study was to investigate whether atrial-specific differences in endothelial damage, leukocyte activation, platelet stimulation occur in patients with AF. Twenty patients (15 men, 5 women; age 55 ± 8 years, 15 paroxystic AF, 5 persistent AF) with AF undergoing ablation were investigated. Blood samples from the left and right atrium were obtained at the start of the procedure. Procoagulant microparticles (MPs), reliable markers of vascular damage were measured by capture assays. Their procoagulant abilities were quantified by functional prothrombinase assay and their cellular origin were determined (endothelium, platelet, leukocyte). In addition, platelet reactivity was evaluated by whole blood flow cytometry for expression of platelet Pselectin (CD62P), active glycoprotein IIbIIIa receptor (PAC-1). Platelet aggregation was evaluated using Arachidonic acid (AA), ADP, TRAP and collageninduced whole blood aggregometry. No atrial-specific differences in the levels of total procoagulant MP, leukocyte-derived-MP and platelet-derived MP could be evidenced. Conversely, endothelial-derived MPs (CD105+) were slightly elevated in the right atrium (RA 0.96 ± 0.53 vs. LA 0.80 ± 0.45 nm PhtdSer Eq.; p = 0.041). Likewise, collagen-induced platelet aggregation was evidenced in the right atrium (Collagen 1 mg/l RA: 48 ± 33% vs LA 37 ± 29%; p 0.035; collagen 2,5 mg/l RA: 76 ± 25% vs LA: 60 ± 29%; p = 0.001). In patients with AF, endothelial damage and collageninduced platelet aggregation appear slightly more pronounced in the right atrium. Our data did not substantiate the view that chamber specific enhanced thrombogenic status could be a reliable explanation for the increased propensity for thrombus formation observed in the left atrium in AF patients.

Calmodulin regulates the non-amyloidogenic metabolism of amyloid precursor protein in platelets

A balance between the proteolytic processing of amyloid precursor protein APP through the amyloidogenic and the non-amyloidogenic pathways controls the production and release of amyloid β-protein, whose accumulation in the brain is associated to the onset of Alzheimer Disease. APP is also expressed on circulating platelets. The regulation of APP processing in these cells is poorly understood. In this work we show that platelets store considerable amounts of APP fragments, including sAPPα, that can be released upon stimulation of platelets. Moreover, platelet stimulation also promotes the proteolysis of intact APP expressed on the cell surface. This process is supported by an ADAM metalloproteinase, and causes the release of sAPPα. Processing of intact platelet APP is promoted also by treatment with calmodulin antagonist W7. W7-induced APP proteolysis occurs through the non-amyloidogenic pathway, is mediated by a metalloproteinase, and causes the release of sAPPα. Co-immunoprecipitation and pull-down experiments revealed a physical association between calmodulin and APP. These results document a novel role of calmodulin in the regulation of non-amyloidogenic processing of APP.

A novel P2Y12-Independent Signaling Pathway Mediating Akt Phosphorylation In Response to Thrombin Receptors

AbstractAbstract 3191The serine-threonine kinase Akt plays an important role in regulating platelet activation. Stimulation of platelets with various agonists results in Akt activation as indicated by Akt phosphorylation. However, the mechanisms of Akt phosphorylation in platelets are not completely understood. It has been previously shown that Akt phosphorylation in response to thrombin receptors is dependent on Gi signaling activated primarily by secreted ADP through its receptor P2Y12. By using P2Y12 knockout mice combined with recombinant CHO models, we demonstrate a Gi-independent pathway for Akt phosphorylation in response to thrombin. Although Akt phosphorylation in response to low dose thrombin or the PAR4 thrombin receptor peptide AYPGKF was abolished in P2Y12 deficient platelets, at high concentrations, they stimulated substantial phosphorylation of both Akt residues Thr308 and Ser473 in P2Y12 deficient platelets, demonstrating that there are P2Y12-dependent and -independent pathways contributing to Akt phosphorylation in response to thrombin receptors. Thrombin or AYPGKF repressed forskolin-induced cAMP production in the wild type mouse platelets but not in the P2Y12 deficient platelets, suggesting that Gi activation by thrombin or AYPGKF is dependent on ADP receptor P2Y12. Therefore, thrombin- or AYPGKF-induced Akt phosphorylation in P2Y12 deficient platelets is Gi independent. AYPGKF-induced Akt phosphorylation was enhanced by expression of recombinant PAR4 cDNA in CHO cells, demonstrating that stimulation of thrombin receptor PAR4 is able to elicit Akt phosphorylation in the absence of platelet secretion. It is unlikely that PAR4-induced Akt phosphorylation in CHO cells involves the P2Y12 pathway, because CHO cells apparently do not express functional P2Y12. This conclusion is supported by the observation that ADP failed to stimulate Akt phosphorylation and inhibit forskolin-induced cAMP production in CHO cells. Unlike thrombin, U46619-induced Akt phosphorylation was dramatically decreased by P2Y12 deficiency, suggesting that TXA2-induced Akt phosphorylation is largely P2Y12 dependent. In addition, P2Y12-independent Akt phosphorylation was not inhibited by the integrin inhibitor peptide RGDS or integrin β3 deficiency, demonstrating that integrin αIIbβ3 outside-in signaling is not required for thrombin-induced, P2Y12-independent, Akt phosphorylation. Furthermore, Akt phosphorylation in response to thrombin or AYPGKF in P2Y12 deficient platelets was inhibited by the calcium chelator dimethyl-BAPTA, the Src family kinase inhibitor PP2, or PI3K inhibitors, but was not affected by PKC inhibitors. Thus, our results reveal a novel P2Y12-independent signaling pathway mediating Akt phosphorylation in response to thrombin receptors. Disclosures:No relevant conflicts of interest to declare.

Defective Production, Turnover, and Secretion of the Platelet α-Granule Protein P-Selectin In Mice with Disrupted FOG1-NuRD Interaction

AbstractAbstract 547We reported recently that point mutations in the GATA-1 co-factor FOG-1 that disrupt binding to the NuRD chromatin remodeling complex display defects in the erythroid and megakaryocyte (MK) lineages. Homozygous FOG-1 mutant (ki/ki) mice display a Gray Platelet Syndrome (GPS)-like macrothrombocytopenia. Specifically, ki/ki platelets exhibit a paucity of α-granules and increased numbers of lysosomal-like vacuoles, reminiscent of the platelet morphology observed in human patients with GPS due to GATA-1 mutations. To understand the molecular basis for the phenotype in ki/ki platelets, we compared mRNA and protein expression of α-granule proteins in wildtype (wt) and ki/ki MK. P-selectin mRNA in ki/ki MKs was 3–5 fold reduced when compared to controls, while platelet factor 4 (Pf4), platelet basic protein (PBP) and von Willebrand factor (Vwf), were only mildly reduced. Strikingly, P-selectin protein levels were more severely reduced in ki/ki MK and platelets being nearly undetectable by Western blot and immunohistochemistry. In contrast PF4, PBP, and vWF protein levels were normal. P-selectin loss was MK-specific as it was expressed at normal levels in endothelial cells. The dramatic decrease in P-selectin protein in ki/ki MK relative to the more moderate reduction in mRNA was due to a >5-fold decrease in protein half-life, as measured by metabolic pulse-chase and immunoprecipitation experiments. This suggests that P-selectin in ki/ki MKs is largely mistargeted to lysosomes. Nevertheless, immunofluorescence microscopy and volume de-convolution analyses of ki/ki platelets showed that the remaining P-selectin resided in vesicular compartments that were distinct from Lamp1 (a lysosomal marker)-containing granules, indicating that a fraction of P-selectin remains within α-like granules. Interestingly, the reduction in P-selectin levels in ki/ki platelets was accompanied by a defect in agonist-stimulated degranulation. While stimulation of wt platelets with AYP, thrombin or phrobol myristate acetate induced α-granule, dense granule and lysosome degranulation, ki/ki platelets failed to do so or undergo platelet aggregation. In summary, the GPS-like phenotype in FOG-1/NuRD interaction-defective MKs involves reduced P-selectin mRNA production, accelerated turnover of P-selectin, abnormal granule formation, and defective degranulation in response to diverse agonists. These findings may partially explain patients having platelet α-granule deficiency associated with decreased P-selectin and selective impairment of thrombin-induced activation with GPS. Future work will determine whether similar defects occur in human GPS. Disclosures:No relevant conflicts of interest to declare.

Pharmacodynamics-Mediated Drug Disposition (PDMDD) and Precursor Pool Lifespan Model for Single Dose of Romiplostim in Healthy Subjects

The objective of this study was to characterize the pharmacokinetics and pharmacodynamics (PK-PD) of romiplostim after single-dose administration in healthy subjects. The mean serum romiplostim concentrations (PK data) and mean platelet counts (PD data) collected from 32 subjects receiving a single intravenous (0.3, 1 and 10 μg/kg) or subcutaneous (0.1, 0.3, 1, and 2 μg/kg) dose were fitted simultaneously to a mechanistic PK-PD model based on pharmacodynamics-mediated drug disposition (PDMDD) and a precursor pool lifespan concept. The two-compartment PK model incorporated receptor-mediated endocytosis and linear mechanisms as parallel elimination pathways. The maximal concentration of receptors (assumed to be proportional to the platelet count), the equilibrium dissociation constant, and the first-order internalization rate constant for endocytosis of the drug-receptor complex were 0.022 fg/platelet, 0.131 ng/mL, and 0.173 h⁻¹, respectively. Romiplostim concentration stimulates the production of platelet precursors via the Hill function, where the SC₅₀ was 0.052 ng/mL and S (max) was 11.2. The estimated precursor cell and platelet lifespans were 5.9 and 10.5 days, respectively. Model-based simulations revealed that the romiplostim exposure and the platelet response are both dependent on the dose administered and the baseline platelet counts. Also, weekly dosing produced a sustained PD response while dosing intervals ≥2 weeks resulted in fluctuating platelet counts. Thus, the mechanistic PK-PD model was suitable for describing the romiplostim PK-PD interplay (PDMDD), the dose-dependent platelet stimulation, and the lifespans of thrombopoietic cell populations.