P2X1 stimulation promotes thrombin receptor-mediated platelet aggregation

P2X1 receptors are ATP-gated channel demonstrated to be involved in multiple platelet responses, although in vitro analysis has been complicated by the effects of rapid desensitization. To further investigate potential roles of P2X1 receptors in platelet activation, the current study employed methods which maximally preserved P2X1 functionality. In preliminary in vivo studies, P2X1-deficiency reduced thrombus formation following the laser-induced, but not FeCl3-induced injury. Given the multiple potential mechanisms involved in thrombus formation in vivo, including tissue-factor/thrombin generation pathways, subsequent studies were designed to investigate the effects of P2X1 inhibition or stimulation on platelet activation in vitro; specifically, the interaction of P2X1 with thrombin receptor stimulation. Aggregation initiated by low/threshold levels of a protease-activated receptor (PAR)4 agonist was reduced in P2X1-deficient murine platelets, and inhibition of P2X1 in wild-type platelets similarly reduced PAR4-mediated aggregation. In human platelets, aggregation to low/threshold stimulation of PAR1 was inhibited with the P2X1 antagonist MRS2159. In addition, P2X1 stimulation primed human platelet responses, such that subsequent sub-threshold PAR1 responses were converted into significant aggregation. Selective ADP receptor inhibitors attenuated P2X1-mediated priming, suggesting that the synergy between P2X1 and sub-threshold PAR1 stimulation was in part because of enhanced granular release of ADP. Overall, the present study defines a novel interaction between platelet P2X1 and thrombin receptors, with P2X1 functioning to amplify aggregation responses at low levels of thrombin receptor stimulation.     

Potentiation of platelet activation through the stimulation of P2X1 receptors

Summary.  The platelet P2X₁ purinergic receptor is a ligand-gated ion channel that responds to ATP. The precise role of P2X₁ in platelet function is unknown, though stimulation with the P2X₁ agonist α,β-Me-ATP is known to result in platelet shape change through elevation of calcium levels. The aim of the present study was to examine further the effects of P2X₁ stimulation on platelet activation. Stimulation of P2X₁ with α,β-Me-ATP resulted in shape change and small aggregate formation in heparin-anticoagulated platelet preparations. Given the ability of heparin to potentiate platelet activation, subsequent experiments were performed in hirudin. In these platelet preparations, aggregate formation in response to α,β-Me-ATP alone was less than that observed in heparin; however, α,β-Me-ATP significantly potentiated platelet aggregate formation when added in conjunction with other weak platelet agonists [epinephrine or thrombopoietin (TPO)]. Platelet aggregate formation was confirmed by single platelet loss (microaggregate formation), microscopy, and light transmittance studies. Further, the P2X₁ antagonist MRS-2159 inhibited platelet shape change and aggregation responses induced by α,β-Me-ATP. Overall, this study demonstrates that P2X₁ stimulation can induce/potentiate platelet activation in combination with other platelet agonists. These results are the first demonstration of platelet aggregation mediated through direct P2X₁ stimulation, supporting a role for this receptor in regulating platelet activation.     

Levcromakalim decreases vascular tone, cytoplasmic Ca2+ and Ca2+ sensitivity in canine basilar artery

Summary— The involvement of large conductance Ca²⁺-activated K⁺ channels (BK) and ATP-sensitive K⁺ (K_ATP) channels in the regulation of canine basilar arterial tone was estimated in the presence of the agonist and blockers of these channels, by simultaneously measuring the changes in intracellular Ca²⁺ concentration ([Ca²⁺]_i) with the fura-2 microfluorimetric method. In the resting condition, levcromakalim reduced [Ca²⁺]_i and vascular tone. Levcromakalim suppressed the serotonin-induced increases in [Ca²⁺]_i and force of contraction, the maximum effects of which were much greater than those of nicardipine. The inhibitory effects of levcromakalim were blocked by glibenclamide but not by tetraethylammonium (TEA) or iberiotoxin (IbTX). In the presence of levcromakalim, the curve relating [Ca²⁺]_i with force in the presence of serotonin at different extracellular Ca²⁺ concentration ([Ca²⁺]_o) was shifted down- and right-ward compared with that in the absence of levcromakalim, suggesting that levcromakalim may reduce the Ca²⁺-sensitivity of the contractile proteins. Thus, levcromakalim may be a good candidate to suppress delayed cerebral vasospasm after subarachnoid hemorrhage.     

CHANGES IN PLATELET FREE Ca2+ CONCENTRATION AFTER CHRONIC DIGOXIN TREATMENT

Summary— Cell Na+ and Ca²⁺ concentrations control each other by various mechanisms. In excitable cells from various origins, Ca²⁺ extrusion from the cell and its entry are dependent for a large part on the activity of the Na+, Ca²⁺-countertransport system. Cytosolic free Ca²⁺ concentration is also controlled by the Na+–H+ exchange activity. To analyze the changes in cytosolic Ca²⁺ concentration accompanying the reduction of the membrane Na+ gradient, cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) was measured by fluorescent dyes in platelets and erythrocytes from healthy subjects, before and during digoxin treatment (0.25 mg/day for 6 days). [Ca²⁺]_i was increased in platelets from 169±30 to 321±61 nmol/l ( n = 7, P <0.02) and unchanged in erythrocytes (121±6 and 104±7 nmol/l). This increase in platelet [Ca²⁺]_i was not accompanied by a change in serotonin content (5.43±0.67 vs 5.49±0.61 10⁻⁷ mol per 10¹¹ cells) and could not be reproduced by in vitro addition of 10⁻⁴ mol/l ouabain (198±33 vs 186±73 nmol/l). The enhanced [Ca²⁺]_i in platelets is thus not a short-term consequence of a reduced membrane Na+ gradient, but reflects either the overload of intracellular Ca²⁺ stores or an enhanced in vivo stimulation by hormones or neurotransmitters.     

Effects of plasma membrane Ca2+-ATPase tyrosine phosphorylation on human platelet function

BACKGROUND: The plasma membrane Ca(2+)-ATPase (PMCA) plays an essential role in maintaining low intracellular Ca(2+) ([Ca(2+)](i)) in resting platelets. Earlier studies demonstrated that platelet activation by thrombin results in tyrosine phosphorylation of PMCA, which inhibits pump activity. OBJECTIVES: The objective was to determine the functional consequences of PMCA tyrosine phosphorylation. METHODS: A decapeptide including the tyrosine phosphorylation site of PMCA and a scrambled version were synthesized and introduced into human platelets using saponin. Fura-2 calcium monitoring and aggregometry were used to characterize the effects of inhibition of tyrosine phosphorylation. RESULTS: Western blot analysis of immunoprecipitates showed that introduction of the inhibitory peptide decreased tyrosine phosphorylation of PMCA by nearly 60% in saponin-permeabilized, thrombin-treated platelets as compared with the scrambled control peptide. Concomitant with inhibition of PMCA tyrosine phosphorylation was a significant decrease in [Ca(2+)](i) during thrombin-mediated platelet activation. The functional consequence of reduced PMCA tyrosine phosphorylation and decreased [Ca(2+)](i) was a significant delay in the onset of thrombin-mediated platelet aggregation. CONCLUSIONS: The results demonstrate that PMCA tyrosine phosphorylation regulates [Ca(2+)](i) during platelet activation, which affects downstream events in the activation process. Moreover, PMCA tyrosine phosphorylation and resultant inhibition of PMCA activity produces a positive feedback loop mechanism by enhancing the increase in [Ca(2+)](i) accompanying platelet activation.     

PAR-1-dependent pp60src activation is dependent on protein kinase C and increased [Ca2+]i: evidence that pp60src does not regulate PAR-1-dependent Ca2+ entry in human platelets

Summary. Background: The role of the tyrosine kinase pp60^src in PAR‐1‐dependent Ca²⁺ entry was investigated in human platelets. pp60^src plays a role in thapsigargin (TG)‐evoked store‐operated Ca²⁺ entry (SOCE), which is thought to be a major component of thrombin‐evoked Ca²⁺ entry. Methods: pp60^src tyr⁴¹⁶ phosphorylation was used to assess pp60^src activation. Fura‐2‐loaded platelets were used to monitor intracellular Ca²⁺ concentration ([Ca²⁺]_i). Results: Activation of PAR‐1 with the specific agonist SFLLRN increased pp60^src activation within 10 s. This required phospholipase C (PLC) activity, Ca²⁺ release and a rise in intracellular Ca²⁺. PP2, an inhibitor of Src‐family tyrosine kinases, inhibited SFLLRN‐evoked Ca²⁺ entry, but also inhibited Ca²⁺ release and the extrusion of Ca²⁺ by the plasma membrane Ca²⁺ ATPase. Actin polymerization and conventional protein kinase C (cPKC) activity were required for TG‐ and SFLLRN‐evoked pp60^src activation. Although Gö6976, an inhibitor of cPKCs, inhibited TG‐evoked SOCE, it had little effect on SFLLRN‐ or thrombin‐evoked Ca²⁺ entry. Conclusions: These data indicate that stimulation of PAR‐1 leads to activation of pp60^src in human platelets, through PLC and cPKC activation, Ca²⁺ release and actin polymerization. However, as PKC and actin polymerization are not needed for SFLLRN‐evoked Ca²⁺ entry, we suggest that pp60^src is also not required. The apparent inhibition of SFLLRN‐evoked Ca²⁺ entry by PP2 is likely to be secondary to reduced Ca²⁺ release. These data argue against a contribution of this SOCE pathway to PAR‐1‐dependent Ca²⁺ entry.     

L-ARGININE METHYLESTER REDUCES Ca2+/Cl− -DEPENDENT L-[3H]GLUTAMATE BINDING AND Ca2+-ACTIVATED NEUTRAL PROTEASE ACTIVITY IN RAT HIPPOCAMPAL MEMBRANES

Specific binding of L-[3H]glutamate was measured in Tris-HCl buffer in rat hippocampal membranes. In these experimental conditions 1 mM CaCl2 induced an increase in binding due to an increase in Bmax. L-Arginine methylester did not modify the Cl(-)-dependent binding of L-[3H]glutamate, but it decreased Ca2+/Cl(-)-stimulated binding in a dose-dependent manner, decreasing Bmax without changing KD. L-Arginine methylester reduced calcium-activated neutral protease activity in a dose-dependent manner. Serine protease inhibitors (aprotinin and di-isopropylfluorophosphate) did not affect L-[3H]glutamate binding, whereas leupeptin reduced it in a dose-dependent manner. L-Arginine did not mimic the effect of L-arginine methylester in either model.     

The cytoplasmic Ca2+ response to glucose as an indicator of impairment of the pancreatic β-cell function

Abstract. The effects of glucose on the cytoplasmic Ca²⁺ concentration (Ca²⁺_i) regulating insulin release were investigated using pancreatic β-cells representative for the normal and diabetic situations. Increase of the glucose concentration resulted in a slight lowering of Ca²⁺_i followed by a rise, often manifested as high amplitude oscillations. The Ca²⁺_i-lowering component in the glucose action associated with suppression of insulin release became particularly prominent when the β-cells were already depolarized by tolbutamide. Glucose-induced inhibition of insulin release was observed also in experiments with rats made diabetic with streptozotocin or alloxan. Other studies indicated lowering of plasma insulin after intravenous glucose administration in patients with insulin- and noninsu-lin-dependent diabetes mellitus. Brief exposure of β-cells to 2–2 mmol 1^-1 streptozotocin resulted in impairment of the response to glucose, manifested as disappearance of the cyclic variation of Ca²⁺_i. The results indicate that glucose-induced depolarisation is a vulnerable process, the disturbance of which may contribute to insulin secretory defects in diabetes mellitus.     

P2Y12 receptor-mediated potentiation of thrombin-induced thromboxane A2 generation in platelets occurs through regulation of Erk1/2 activation

BACKGROUND: Thromboxane A2 (TXA2) is a positive feedback lipid mediator that is generated upon stimulation of platelets with various agonists. Aspirin works as an antithrombotic drug by blocking the generation of TXA2. The aim of this study was to evaluate the role of the purinergic P2Y receptors in thrombin-induced TXA2 generation. RESULTS: PAR1-activating peptide (SFLLRN), PAR4-activating peptide (AYPGKF), and thrombin, induced the activation of cytosolic phospholipase A2 (cPLA2), release of arachidonic acid (AA) from membrane-bound phospholipids, and subsequent TXA2 generation in human platelets. The actions of these agonists were significantly inhibited in the presence of the P2Y12 receptor antagonist, AR-C69931MX, but not the P2Y1 receptor antagonist, MRS2179. In addition, AYPGKF- and thrombin-induced TXA2 generation was significantly reduced in platelets from mice dosed with clopidogrel, confirming the results obtained with the human platelets. Also, Pearl mouse platelets that lack releasable nucleotides generated significantly less TXA2 when compared with the wild-type littermates in response to PAR stimulation. Inhibition of extracellular signal-regulated protein kinase 1/2 (Erk 1/2) activation using U0126, an inhibitor of MAP kinase kinase (MEK), suppressed PAR-mediated cPLA2 phosphorylation and TXA2 generation. Further, platelets that were pretreated with AR-C69931MX, as well as Pearl mouse platelets, displayed the reduced levels of Erk1/2 phosphorylation upon stimulation with the PAR agonists. CONCLUSIONS: Based on these findings, we conclude that thrombin-induced Erk1/2 activation is essential for PAR-mediated TXA2 generation, which is potentiated by the P2Y12 receptor-mediated signaling pathway but not the P2Y1 receptor-mediated signaling pathway. Finally, using selective inhibitors of Src kinases, we show that PAR-mediated Src activation precedes Erk1/2 activation.     

Activation of GPVI by collagen is regulated by α2β1 and secondary mediators

Summary.  We have compared the roles of adenosine diphosphate (ADP), thromboxanes and the integrin α₂β₁ in the activation of washed platelets by collagen in the presence of the α_IIbβ₃ antagonist lotrafiban. The stimulation of protein tyrosine phosphorylation by a collagen suspension is markedly delayed in the presence of the above inhibitors but shows substantial recovery with time. In comparison, activation of phospholipase C (PLC), Ca²⁺ elevation and dense granule secretion are more severely suppressed by the above inhibitors. α₂β₁ blockade has a slightly greater inhibitory effect on all of the above responses than a combination of ADP receptor antagonists and cyclooxygenase inhibitor. Platelets exposed to a collagen monolayer show robust elevation of Ca²⁺ that is delayed in the presence of the above inhibitors and which is accompanied by α-granule secretion. These results demonstrate that secondary mediators and α₂β₁ modulate collagen-induced intracellular signaling but have negligible effect on GPVI signaling induced by the specific agonist convulxin. This work supports the postulate that the major role of α₂β₁ is to increase the avidity of collagen for the platelet surface and by doing so enhance activation of GPVI. Therefore we propose an important role of secondary mediators in collagen-induced signaling is the indirect regulation of GPVI signaling via activation of α₂β₁.     

Lineage-specific overexpression of the P2Y1 receptor induces platelet hyper-reactivity in transgenic mice

Summary.  In order to investigate the role of the platelet P2Y₁ receptor in several aspects of platelet activation and thrombosis, transgenic (TG) mice overexpressing this receptor specifically in the megakaryocytic/platelet lineage were generated using the promoter of the tissue-specific platelet factor 4 gene. Studies of the saturation binding of [³³P]2MeSADP in the presence or absence of the selective P2Y₁ antagonist MRS2179 indicated that wild-type (WT) mouse platelets bore 150 ± 31 P2Y₁ receptors and TG platelets 276 ± 34, representing an 84% increase in P2Y₁ receptor density. This led to a well defined phenotype of platelet hyper-reactivity in vitro, as shown by increased aggregations in response to adenosine 5'-diphosphate (ADP) and low concentration of collagen in TG as compared with WT platelets. Moreover, overexpression of the P2Y₁ receptor enabled ADP to induce granule secretion, unlike in WT platelets, which suggests that the level of P2Y₁ expression is critical for this event. Our results further suggest that the weak responses of normal platelets to ADP are due to a limited number of P2Y₁ receptors rather than to activation of a specific transduction pathway. TG mice displayed a shortened bleeding time and an increased sensitivity to in vivo platelet aggregation induced by infusion of a mixture of collagen and epinephrine. Overall, these findings emphasize the importance of the P2Y₁ receptor in hemostasis and thrombosis and suggest that variable expression levels of this receptor on platelets might play a role in thrombotic states in human, which remains to be assessed.     

Ticagrelor binds to human P2Y12 independently from ADP but antagonizes ADP-induced receptor signaling and platelet aggregation

Summary. Background: P2Y₁₂ plays an important role in regulating platelet aggregation and function. This receptor is the primary target of thienopyridine antiplatelet agents, the active metabolites of which bind irreversibly to the receptor, and of newer agents that can directly and reversibly modulate receptor activity. Objective: To characterize the receptor biology of the first reversibly binding oral P2Y₁₂ antagonist, ticagrelor (AZD6140), a member of the new cyclopentyltriazolopyrimidine (CPTP) class currently in phase III development. Methods: Ticagrelor displayed apparent non‐competitive or insurmountable antagonism of ADP‐induced aggregation in human washed platelets. This was investigated using competition binding against [³H]ADP, [³³P]2MeS‐ADP and the investigational CPTP compound [¹²⁵I]AZ11931285 at recombinant human P2Y₁₂. Functional receptor inhibition studies were performed using a GTPγS‐binding assay, and further binding studies were performed using membranes prepared from washed human platelets. Results: Radioligand‐binding studies demonstrated that ticagrelor binds potently and reversibly to human P2Y₁₂ with K_on and K_off of (1.1 ± 0.2) × 10^?4 nm ^?1 s^?1 and (8.7 ± 1.4) × 10^?4 s^?1, respectively. Ticagrelor does not displace [³H]ADP from the receptor (K_i > 10 μm ) but binds competitively with [³³P]2MeS‐ADP (K_i = 4.3 ± 1.3 nm ) and [¹²⁵I]AZ11931285 (K_i = 0.33 ± 0.04 nm ), and shows apparent non‐competitive inhibition of ADP‐induced signaling but competitive inhibition of 2MeS‐ADP‐induced signaling. Binding studies on membranes prepared from human washed platelets demonstrated similar non‐competitive binding for ADP and ticagrelor. Conclusions: These data indicate that P2Y₁₂ is targeted by ticagrelor via a mechanism that is non‐competitive with ADP, suggesting the existence of an independent receptor‐binding site for CPTPs.     

Pharmacological control of the human gastric histamine H2 receptor by famotidine: comparison with H1, H2 and H3 receptor agonists and antagonists

Histamine 0.1 microM-0.1 mM increased adenylate cyclase activity five- to ten-fold in human fundic membranes, with a potency Ka = 3 microM. The histamine dose-response curve was mimicked by the H3 receptor agonist (R) alpha-MeHA, but at 100 times lower potency, Ka = 0.3 mM. Histamine-induced adenylate cyclase activation was abolished by H2, H1 and H3 receptor antagonists, according to the following order of potency IC50: famotidine (0.3 microM) greater than triprolidine (0.1 mM) thioperamide (2 mM), respectively. Famotidine has no action on membrane components activating the adenylate cyclase system, including the Gs subunit of the enzyme stimulated by forskolin and cell surface receptors sensitive to isoproterenol (beta 2-type), PGE2 and VIP. The Schild plot was linear for famotidine (P less than 0.01) with a regression coefficient r = 0.678. The slope of the regression line was 0.64 and differs from unity. Accordingly, famotidine showed a slow onset of inhibition and dissociation from the H2 receptor in human cancerous HGT-1 cells. The results demonstrate that famotidine is a potent and selective H2 receptor antagonist with uncompetitive actions in human gastric mucosa. Consequently, famotidine might be a suitable drug with long-lasting actions in the treatment of Zollinger-Ellison syndrome. The results also confirm and extend the previous observations that (R) alpha-MeHA and thioperamide are two selective ligands at histamine H3 receptor sites. In the human gastric mucosa, these drugs are respectively 330 and 6700 times less potent than histamine and famotidine on the adenylate cyclase system. The possible involvement of histamine H3 receptors in the regulation of gastric secretion is proposed.     

(N)-methanocarba-2MeSADP (MRS2365) is a subtype-specific agonist that induces rapid desensitization of the P2Y1 receptor of human platelets

Adenosine diphosphate (ADP) initiates and maintains sustained aggregation of platelets through simultaneous activation of both the Gq-coupled P2Y1 receptor and the Gi-coupled P2Y12 receptor. We recently described the synthesis and P2Y1 receptor-specific agonist activity of (N)-methanocarba-2MeSADP (MRS2365). Consequences of selective activation of the P2Y1 receptor by MRS2365 have been further examined in human platelets. Whereas MRS2365 alone only induced shape change, addition of MRS2365 following epinephrine treatment, which activates the Gi/z-linked, alpha2A-adrenergic receptor, resulted in sustained aggregation that was indistinguishable from that observed with ADP. Conversely, the platelet shape change promoted by ADP in the presence of the GPIIb/IIIa antagonist eptifibatide was similar to that promoted by MRS2365. Preaddition of the high affinity P2Y1 receptor antagonist MRS2500 inhibited the effect of MRS2365, whereas addition of MRS2500 subsequent to MRS2365 reversed the MRS2365-induced shape change. Preactivation of the P2Y1 receptor with MRS2365 for 2 min resulted in marked loss of capacity of ADP to induce aggregation as evidenced by a greater than 20-fold rightward shift in the concentration effect curve of ADP. This inhibitory effect of P2Y1 receptor activation was dependent on the concentration of MRS2365 (EC50 = 34 nm). The inhibitory effect of preincubation with MRS2365 was circumvented by activation of the Gq-coupled 5-HT2A receptor suggesting that MRS2365 induces loss of the ADP response as a consequence of desensitization of the Gq-coupled P2Y1 receptor. The time course of MRS2365-induced loss of aggregation response to epinephrine was similar to that observed with ADP. These results further demonstrate the P2Y1 receptor selectivity of MRS2365 and illustrate the occurrence of agonist-induced desensitization of the P2Y1 receptor of human platelets studied in the absence of P2Y12 receptor activation .     

The regulation of integrin-linked kinase in human platelets: evidence for involvement in the regulation of integrin α2β1

BACKGROUND: Activation of the platelet integrin alpha 2 beta 1 is closely regulated due to the high thrombogenicity of its ligand. As a beta 1 interacting kinase, ILK represents a candidate intracellular regulator of alpha 2 beta 1 in human platelets. OBJECTIVES: We investigated the regulation of ILK in human platelets and the role of ILK in regulating alpha 2 beta 1 activation in HEL cells, a megakaryocytic cell line. METHODS: An in-vitro kinase assay was used to determine the effect of platelet agonists on ILK kinase activity together with the contribution of PI3K and PKC on ILK activation. Interaction of ILK with beta 1-integrin subunits was investigated by coimmunoprecipitation and the role of ILK in regulating alpha 2 beta 1 function assessed by overexpression studies in HEL cells. RESULTS: We report that collagen and thrombin modulate ILK kinase activity in human platelets in an aggregation-independent manner. Furthermore, ILK activity is dually regulated by PI3K and PKC in thrombin-stimulated platelets and regulated by PI3K in collagen-stimulated cells. ILK associates with the beta 1-integrin subunits immunoprecipitated from platelet cell lysates, an association which increased upon collagen stimulation. Overexpression of ILK in HEL cells enhanced alpha 2 beta 1-mediated adhesion whereas overexpression of kinase-dead ILK reduced adhesion, indicating a role for this kinase in the positive regulation of alpha 2 beta 1. CONCLUSIONS: Our findings that ILK regulates alpha 2 beta 1 in HEL cells, is activated in platelets and associates with beta 1-integrins, raise the possibility that it may play a key role in adhesion events upon agonist stimulation of platelets.     

Phorbol ester‐evoked Ca2+signaling in human platelets is via autocrine activation of P2X1 receptors,not a novel non‐capacitative Ca2+entry

Summary. Background: Platelets are reported to possess a protein kinase C (PKC)‐dependent non‐capacitative Ca²⁺entry (NCCE) pathway. The phorbol ester, phorbol, 12‐myristate, 13‐acetate (PMA) has been suggested to stimulate platelet NCCE. Recently we demonstrated important roles in store‐operated Ca²⁺entry in human platelets for Na⁺/Ca²⁺ exchangers (NCXs) and autocrine signaling between platelets after dense granule secretion. As PMA evokes dense granule secretion, we have investigated the role of NCXs and autocrine signaling in PMA‐evoked Ca²⁺entry. Objectives: To investigate the roles of NCXs and dense granule secretion in PMA‐evoked Ca²⁺signaling in human platelets. Methods: Fura‐2‐ or sodium‐binding benzofuran isophthalate (SBFI)‐loaded platelets were used to monitor cytosolic Ca²⁺or Na⁺ concentrations. Dense granule secretion was monitored as ATP release using luciferin–luciferase. Results: The NCX inhibitors KB‐R7943 or SN‐6, and removal of extracellular Na⁺, significantly reduced PMA‐evoked Ca²⁺entry. PMA‐evoked dense granule secretion was almost abolished by pretreatment with the PKC inhibitor Ro‐31‐8220 and significantly slowed by KB‐R7943. The P_2X1 antagonists Ro‐0437626 or MRS‐2159, or desensitization of P_2X1 receptors by prior treatment with α,β‐Methylene‐ATP or omitting apyrase from the medium, reduced PMA‐evoked Ca²⁺entry. Ro‐0437626 or chelation of extracellular Ca²⁺ slowed but did not abolish PMA‐evoked ATP release, indicating that PMA‐evoked dense granule secretion does not require P_2X1 receptor activation but is accelerated by P_2X1‐mediated Ca²⁺entry. The presence of NCX3 in human platelets was demonstrated by Western blotting. Conclusion: PMA‐evoked Ca²⁺entry results from an NCX3‐dependent dense granule secretion and subsequent P_2X1 receptor activation by secreted ATP, rather than activation of a novel NCCE pathway.     

Critical role of ADP interaction with P2Y12 receptor in the maintenance of αIIbβ3 activation: association with Rap1B activation

OBJECTIVE: Platelet integrin alpha(IIb)beta3 plays a crucial role in platelet aggregation, and the affinity of alpha(IIb)beta3 for fibrinogen is dynamically regulated. Employing modified ligand-binding assays, we analyzed the mechanism by which alpha(IIb)beta3 maintains its high-affinity state. METHODS AND RESULTS: Washed platelets adjusted to 50 x 10(3) microL(-1) were stimulated with 0.2 U mL(-1) thrombin or 5 microm U46619 under static conditions. After the completion of alpha(IIb)beta3 activation and granule secretion, different kinds of antagonists were added to the activated platelets. The activated alpha(IIb)beta3 was then detected by fluorescein isothiocyanate (FITC)-labeled PAC1. The addition of 1 mum AR-C69931MX (a P2Y12 antagonist) or 1 mm A3P5P (a P2Y1 antagonist) disrupted the sustained alpha(IIb)beta3 activation by approximately 92% and approximately 38%, respectively, without inhibiting CD62P or CD63 expression. Dilution of the platelet preparation to 500 microL(-1) also disrupted the sustained alpha(IIb)beta3 activation, and the disruption by such dilution was abrogated by the addition of exogenous adenosine 5'-diphosphate (ADP) in a dose-dependent fashion. The amounts of ADP released from activated platelets determined by high-performance liquid chromatography were compatible with the amounts of exogenous ADP required for the restoration. We next examined the effects of antagonists on protein kinase C (PKC) and Rap1B activation induced by 0.2 U mL(-1) thrombin. Thrombin induced long-lasting PKC and Rap1B activation. AR-C69931MX markedly inhibited Rap1B activation without inhibiting PKC activation. CONCLUSIONS: Our data indicate that the continuous interaction between released ADP and P2Y12 is critical for the maintenance of alpha(IIb)beta3 activation.     

The reversible P2Y12 antagonist cangrelor influences the ability of the active metabolites of clopidogrel and prasugrel to produce irreversible inhibition of platelet function

Summary.  Background:  Agents that act as antagonists at P2Y₁₂ ADP receptors on platelets are in use (clopidogrel), and in development for use (cangrelor and prasugrel), in patients with cardiovascular disease. Cangrelor is a direct-acting reversible antagonist being developed for short-term infusion; clopidogrel and prasugrel are oral prodrugs that provide irreversible inhibition via transient formation of active metabolites. At the cessation of cangrelor infusion, patients are likely to receive clopidogrel or prasugrel as a means of maintaining antiplatelet therapy. Objectives:  To apply an experimental in vitro approach to investigate the possibility that cangrelor influences the ability of the active metabolites of clopidogrel and prasugrel to inhibit ADP-mediated platelet function. Methods:  The effects of cangrelor and the active metabolites of clopidogrel (C-AM) and prasugrel (P-AM) on platelet function were assessed by ADP-induced platelet P-selectin expression in whole blood. The method involved rapid removal of the antagonists by dilution, and measurement of residual platelet inhibition. Results:  Cangrelor, C-AM and P-AM markedly inhibited P-selectin expression. The effect of cangrelor, but not of C-AM and P-AM, was reversible following antagonist removal. Preincubation of blood with cangrelor prior to addition of C-AM or P-AM reduced the ability of metabolites to irreversibly antagonize P2Y₁₂. Irreversible inhibition was maintained when blood was preincubated with metabolites prior to cangrelor. Conclusions:  Cangrelor influences the ability of the active metabolites of clopidogrel or prasugrel to inhibit platelet function irreversibly. Careful consideration should be given to the timing of administration of an oral P2Y₁₂ antagonist following cangrelor infusion.     

A mechanism to safeguard platelet adhesion under high shear flow: von Willebrand factor–glycoprotein Ib and integrin α2β1–collagen interactions make complementary, collagen-type-specific contributions to adhesion

BACKGROUND: Blood vessels contain different types of collagen, with types I and III being the major components of vascular collagen. Platelet adhesion under high shear stress has been suggested to depend on the binding of von Willebrand factor (VWF) to collagen. OBJECTIVE: We analyzed the collagen type specificity for the interaction with VWF and high shear stress platelet adhesion. METHODS: VWF binding to different types of immobilized collagen and effects of antibodies against glycoprotein Ib (gpIb) and integrin alpha(2)beta(1) on platelet adhesion to type I and III collagens under high shear were analyzed. RESULTS: VWF showed high-affinity, selective binding to human and bovine type III collagens, but weak or no affinity for types I, II, IV and V under static conditions. Anti-integrin alpha(2)beta(1) markedly inhibited adhesion to type I collagen, but did not affect that to type III collagen. Anti-gpIb antibody significantly inhibited adhesion to type III collagen. Adding both antibodies abrogated the adhesion to either type I or III collagen. CONCLUSIONS: Both the gpIb-VWF interaction and the integrin alpha(2)beta(1)-collagen interaction contribute to platelet adhesion to collagen under high shear stress, and integrin alpha(II)beta(1) makes a greater contribution to adhesion to type I collagen because less VWF is bound to it.