Safflower Extract Inhibits ADP-Induced Human Platelet Aggregation

Safflower extract is commonly used as a traditional Chinese medicine to promote blood circulation and remove blood stasis. The antioxidant and anticancer properties of safflower extracts have been extensively studied, but their antiaggregative effects have been less analyzed. We found that safflower extract inhibited human platelet aggregation induced by ADP. In addition, we further analyzed several safflower extract compounds, such as hydroxysafflor yellow A, safflower yellow A, and luteolin, which have the same antiaggregative effect. In addition to analyzing the active components of the safflower extract, we also analyzed their roles in the ADP signaling pathways. Safflower extract can affect the activation of downstream conductors of ADP receptors (such as the production of calcium ions and cAMP), thereby affecting the expression of activated glycoproteins on the platelet membrane and inhibiting platelet aggregation. According to the results of this study, the effect of safflower extract on promoting blood circulation and removing blood stasis may be related to its direct inhibition of platelet activation.

Role of ADP receptors on platelets in the growth of ovarian cancer

Key Points P2Y12 is important in the interaction between platelets and cancer cells. A P2Y12 inhibitor or P2Y12 deficiency reduces tumor growth in murine models of ovarian cancer.

Novel Lyn-Mediated Regulation of CLEC-2 Signaling By Gq-Coupled Receptors in Platelets

The CLEC-2 agonist, rhodocytin, elicits powerful platelet activation signals in conjunction with Src family kinases, Syk, and PLCg2. Previous reports have shown that rhodocytin-induced aggregation is dependent on secondary mediators. However, the role of secondary mediators in CLEC-2 signaling is not defined. In this study we report that CLEC-2-induced Syk activation is aspirin-sensitive and that TxA2 plays an important role in the most proximal events such as CLEC-2 phosphorylation and Syk activation. We also show that the activation of other GPCRs, such as the ADP receptors and PARs, can also potentiate CLEC-2 signaling. By using the Gq-inhibitor, UBO-QIC, or P2Y1/P2Y12 antagonists, we show that the Gq-signaling, but not G12/13 or Gi, is essential for GPCR-induced Syk phosphorylation downstream of CLEC-2. We further elucidated the signaling involved in Gq-mediated Syk phosphorylation and identified an important role for PKCs downstream of PLCb regulating SFK activation (Figure 1A). Using Lyn-knock out murine platelets we identified a potential role for Lyn downstream of the Gq-pathway in potentiating CLEC-2 signaling by using. We suggest that, at low concentration of CLEC-2 agonist, the unclustered CLEC-2 receptors are phosphorylated by the Gq-activated Lyn resulting in the potentiation of the initial CLEC-2 signal (Figure 1B). However, Gq receptors by themselves cannot phosphorylate CLEC-2 receptors and require minimal levels of ITAM-containing receptor stimulation in order to initiate unclustered CLEC-2 receptor phosphorylation. Together, these results provide evidence for a novel Lyn-mediated regulation of CLEC-2 signaling by Gq-coupled receptors thereby leading to potentiation of CLEC-2-induced signaling (Figure 1C). Figure 1 A) Western blot showingeffect Gq-pathway inhibitors on CLEC-2 signaling. B) Effect of low concentration of CLEC-2 agonist in Lyn-knock out murine platelets. C) Model showing role of Gq-activated Lyn in CLEC-2 signaling. Figure 1. A) Western blot showingeffect Gq-pathway inhibitors on CLEC-2 signaling. B) Effect of low concentration of CLEC-2 agonist in Lyn-knock out murine platelets. C) Model showing role of Gq-activated Lyn in CLEC-2 signaling. Disclosures No relevant conflicts of interest to declare.

Antipsychotic Drugs Inhibit Platelet Aggregation via P2Y1and P2Y12Receptors

Antipsychotic drugs (APDs) used to treat clinical psychotic syndromes cause a variety of blood dyscrasias. APDs suppress the aggregation of platelets; however, the underlying mechanism remains unknown. We first analyzed platelet aggregation and clot formation in platelets treated with APDs, risperidone, clozapine, or haloperidol, using an aggregometer and rotational thromboelastometry (ROTEM). Our data indicated that platelet aggregation was inhibited, that clot formation time was increased, and that clot firmness was decreased in platelets pretreated with APDs. We also examined the role two major adenosine diphosphate (ADP) receptors, P2Y1and P2Y12, play in ADP-mediated platelet activation and APD-mediated suppression of platelet aggregation. Our results show that P2Y1receptor stimulation with ADP-induced calcium influx was inhibited by APDs in human and rats’ platelets, as assessed byin vitroorex vivoapproach, respectively. In contrast, APDs, risperidone and clozapine, alleviated P2Y12-mediated cAMP suppression, and the release of thromboxane A2 and arachidonic acid by activated platelets decreased after APD treatment in human and rats’ platelets. Our data demonstrate that each APD tested significantly suppressed platelet aggregation via different mechanisms.

NPP4 is a procoagulant enzyme on the surface of vascular endothelium

Ap3A is a platelet-dense granule component released into the extracellular space during the second wave of platelet aggregation on activation. Here, we identify an uncharacterized enzyme, nucleotide pyrophosphatase/phosphodiesterase-4 (NPP4), as a potent hydrolase of Ap3A capable of stimulating platelet aggregation and secretion. We demonstrate that NPP4 is present on the surface of vascular endothelium, where it hydrolyzes Ap3A into AMP and ADP, and Ap4A into AMP and ATP. Platelet aggregation assays with citrated platelet-rich plasma reveal that the primary and secondary waves of aggregation and dense granule release are strongly induced by nanomolar NPP4 in a concentration-dependent manner in the presence of Ap3A, while Ap3A alone initiates a primary wave of aggregation followed by rapid disaggregation. NPP2 and an active site NPP4 mutant, neither of which appreciably hydrolyzes Ap3A, have no effect on platelet aggregation and secretion. Finally, by using ADP receptor blockade we confirm that NPP4 mediates platelet aggregation via release of ADP from Ap3A and activation of ADP receptors. Collectively, these studies define the biologic and enzymatic basis for NPP4 and Ap3A activity in platelet aggregation in vitro and suggest that NPP4 promotes hemostasis in vivo by augmenting ADP-mediated platelet aggregation at the site of vascular injury.

Classical PKCs Regulate ADP-Induced Thromboxane Generation by Modulating Tyrosine Phosphorylation On Novel PKC Isoform Delta Through Shptp-1

Abstract 1064 Introduction: Adenosine Di-phosphate (ADP) is stored in dense granules of platelets and is released upon platelet activation acting as a feedback activator by binding to G-protein coupled P2Y1 and P2Y12 receptors. ADP stimulation causes platelets to change shape, aggregate, release dense and a-granule contents and synthesize thromboxane A2 that can further act as a feedback activator potentiating platelet responses by binding to thromboxane receptor (TP). Protein kinase C is a serine threonine specific kinase that regulates multiple platelet functional responses. Specific PKC isoforms regulating platelet responses downstream of ADP receptors are not completely known. Aim: The aim of the current study is to elucidate the role of PKC isoforms in regulating ADP-induced platelet functional responses in platelets. Methods: We sought to delineate the mechanism of ADP-induced platelet responses by performing platelet aggregation (aggregometry), ATP secretion (luciferin-luciferase reaction) and thromboxane generation (ELISA kit measuring TxB2) in human or murine platelets by pre-incubating the platelets with control (DMSO) or inhibitors wherever mentioned. We also evaluated the role of PKCd to ADP-induced platelet responses by using murine platelets lacking PKCd. Background and Results: Murugappan et al have shown that PKCd was not activated downstream of ADP receptors based on the inability of ADP to cause threonine 507 phosphorylation on PKCd in platelets. However, studies from other labs have shown that PKCd can be activated when it is phosphorylated on its tyrosine residues. In the current study we show that, upon stimulation with 2MeSADP, PKCd is phosphorylated on tyrosine residue 311 in a time-dependent manner in platelets (Fig A). Also, ADP-induced thromboxane generation (Fig B) and ADP-induced thromboxane-mediated dense granule secretion were significantly inhibited in PKCd knockout murine platelets compared to those of wild type platelets. Similarly, thromboxane generation downstream of ADP receptors in human platelets pre-incubated with a PKCd inhibitor is significantly inhibited compared to control indicating a role for PKCd in mediating ADP-induced responses in platelets. Bynagari et al have shown that ADP-induced thromboxane generation is potentiated in the presence of the pan-PKC inhibitor, GF 109203X and the isoform regulating this effect is PKCe. We observed that pre-incubation of PKCe knockout murine platelets with GF 109203X further potentiated ADP-induced thromboxane generation suggesting that there are other PKC isoforms negatively regulating ADP-induced thromboxane generation. We show that this potentiating effect of thromboxane generation with GF 109203X in WT or PKCe KO murine platelets correlate with an increase in the phosphorylation of Y311 on PKCd (Fig C) suggesting that ADP-induced thromboxane generation is regulated through PKCd Y311 phosphorylation. Tyrosine phosphorylation on PKCd is mediated by Src family kinases (SFKs) as the phosphorylation is abolished with PP2, a SFK inhibitor and is only partially inhibited in Fyn knockout murine platelets suggesting that other SFKs also mediate this tyrosine phosphorylation. Surprisingly, pre-incubation of platelets with LY-333531, a classical PKC isoform (a/b) inhibitor potentiated PKCd Y311 phosphorylation (Fig D) as well as thromboxane generation (Fig E) downstream of ADP receptors suggesting a role for classical PKCs. Also, platelets pre-incubated with LY-333531 showed a decrease in the phosphorylation of SHPTP-1 (Fig F), a tyrosine phosphatase, rendering it active. The active SHPTP-1 phosphatase may dephosphorylate and activate SFKs, which can now phosphorylate PKCd on Y311 in platelets. Conclusions: In the current study, we report for the first time that the novel PKC isoform d is tyrosine phosphorylated downstream of ADP receptors through which it mediates ADP-induced thromboxane generation. We also show a novel role for classical PKC isoforms a/b in regulating tyrosine phosphorylation on novel isoform, PKCd possibly through the tyrosine phosphatase SHPTP-1 and Src family kinases in platelets. Disclosures: No relevant conflicts of interest to declare.

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

SummaryWe 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.