Ent-16β,17-dihydroxy-kauran-19-oic acid,a kaurane diterpene acid from Siegesbeckia pubescens,presents antiplatelet and antithrombotic effects in rats

The antiplatelet and antithrombotic effects of ent-16β,17-dihydroxy-kauran-19-oic acid (DDKA) isolated from Siegesbeckia pubescens were investigated with different methods both in vitro and in vivo. We tested the antithrombotic activity of DDKA in arterio-venous shunt model. The effects of DDKA on adenosine diphosphate (ADP)-, Thrombin-, Arachidonic acid-induced rat platelets aggregation were tested in vitro. We also assessed its bleeding side effect by measuring coagulation parameters after intravenous administration for 5 days and investigated the potential mechanisms underlying such activities. In vivo, DDKA significantly reduced thrombus weight in the model of arterio-venous shunt. Meanwhile, DDKA increased plasma cAMP level determined by radioimmunoassay in the same model. Notably, DDKA prolonged PT and APTT in rats after intravenous administration DDKA for successive 5 days. In vitro, pretreatment with DDKA on washed rat platelets significantly inhibited various agonists stimulated platelet aggregation and caused an increase in cAMP level in platelets activated by ADP. These findings support our hypothesis that DDKA possesses antiplatelet and antithrombotic activities. The mechanisms underlying such activities may involve the anticoagulatory effect and cAMP induction.     

Quercitrin inhibits platelet activation in arterial thrombosis

BackgroundThe ingestion of flavonoids has been reported to be associated with reduced cardiovascular disease risk. Quercitrin is a common flavonoid in nature, and it exhibits antioxidant properties. Although the process of thrombogenesis is intimately related to cardiovascular disease risk, it is unclear whether quercitrin plays a role in thrombogenesis.PurposeThe aim of this study was to examine the antiplatelet effect of quercitrin in platelet activation.MethodsPlatelet aggregation, granule secretion, calcium mobilization, and integrin activation were used to assess the antiplatelet activity of quercitrin. Antithrombotic effect was determined in mouse using ferric chloride (FeCl₃)-induced arterial thrombus formation in vivo and thrombus formation on collagen-coated surfaces under arteriolar shear in vitro. Transection tail bleeding time was used to evaluate whether quercitrin inhibited primary hemostasis.ResultsQuercitrin significantly impaired collagen-related peptide-induced platelet aggregation, granule secretion, reactive oxygen species generation, and intracellular calcium mobilization. Outside-in signaling of αIIbβ3 integrin was significantly inhibited by quercitrin in a concentration-dependent manner. The inhibitory effect of quercitrin resulted from inhibition of the glycoprotein VI-mediated platelet signal transduction during cell activation. Further, the antioxidant effect is derived from decreased phosphorylation of components of the TNF receptor-associated factor 4/p47^phox/Hic5 axis signalosome. Oral administration of quercitrin efficiently blocked FeCl₃-induced arterial thrombus formation in vivo and thrombus formation on collagen-coated surfaces under arteriolar shear in vitro, without prolonging bleeding time. Studies using a mouse model of ischemia/reperfusion-induced stroke indicated that treatment with quercitrin reduced the infarct volume in stroke.ConclusionsOur results demonstrated that quercitrin could be an effective therapeutic agent for the treatment of thrombotic diseases.     

Molecular docking‐assisted screening reveals tannic acid as a natural protein disulphide isomerase inhibitor with antiplatelet and antithrombotic activities

Protein disulphide isomerase (PDI) promotes platelet activation and constitutes a novel antithrombotic target. In this study, we reported that a PDI‐binding plant polyphenol, tannic acid (TA), inhibits PDI activity, platelet activation and thrombus formation. Molecular docking using plant polyphenols from dietary sources with cardiovascular benefits revealed TA as the most potent binding molecule with PDI active centre. Surface plasmon resonance demonstrated that TA bound PDI with high affinity. Using Di‐eosin‐glutathione disulphide fluorescence assay and PDI assay kit, we showed that TA inhibited PDI activity. In isolated platelets, TA inhibited platelet aggregation stimulated by either GPVI or ITAM pathway agonists. Flow cytometry showed that TA inhibited thrombin‐ or CRP‐stimulated platelet activation, as reflected by reduced granule secretion and integrin activation. TA also reduced platelet spreading on immobilized fibrinogen and platelet adhesion under flow conditions. In a laser‐induced vascular injury mouse model, intraperitoneal injection of TA significantly decreased the size of cremaster arteriole thrombi. No prolongation of mouse jugular vein and tail‐bleeding time was observed after TA administration. Therefore, we identified TA from natural polyphenols as a novel inhibitor of PDI function. TA inhibits platelet activation and thrombus formation, suggesting it as a potential antithrombotic agent.     

Endocannabinoids Control Platelet Activation and Limit Aggregate Formation under Flow

Background

The endocannabinoid system has previously been implicated in the regulation of neurons and inflammatory cells. Additionally, it has been reported that endocannabinoid receptors are present on circulating platelets, but there has been conflicting evidence on their contribution to platelet function.

Objectives

Our aim was to examine the role of endocannabinoids in platelet function in vitro and in vivo.

Methods and Results

We studied the effects of the well-characterized endogenous endocannabinoid anandamide on platelet aggregation in suspension, α-granule release, calcium mobilization, Syk phosphorylation, as well as platelet spreading and aggregate formation under flow. Anandamide inhibits platelet aggregation and α-granule release by collagen, collagen-derived peptide CRP-XL, ADP, arachidonic acid and thromboxane A2 analogue U46619. However, activation via thrombin receptor PAR-1 stays largely unaffected. Calcium mobilization is significantly impaired when platelets are stimulated with collagen or CRP-XL, but remains normal in the presence of the other agonists. In line with this finding, we found that anandamide prevents collagen-induced Syk phosphorylation. Furthermore, anandamide-treated platelets exhibit reduced spreading on immobilized fibrinogen, have a decreased capacity for binding fibrinogen in solution and show perturbed platelet aggregate formation under flow over collagen. Finally, we investigated the influence of Cannabis sativa consumption by human volunteers on platelet activation. Similar to our in vitro findings with anandamide, ex vivo collagen-induced platelet aggregation and aggregate formation on immobilized collagen under flow were impaired in whole blood of donors that had consumed Cannabis sativa.

Conclusions

Endocannabinoid receptor agonists reduce platelet activation and aggregate formation both in vitro and ex vivo after Cannabis sativa consumption. Further elucidation of this novel regulatory mechanism for platelet function may prove beneficial in the search for new antithrombotic therapies.     

In vitro activity of juglone (5-hydroxy-1,4-naphthoquinone) against both fluconazole-resistant and susceptible Candida isolates

BackgroundThe rise in antifungal resistance and drug class limitations are causing higher morbidity and mortality rates all over the world. This issue highlights the urgent need for new and improved antifungal drugs with a novel target.AimsIn order to evaluate whether juglone can be served as an alternative antifungal to cure drug-resistant Candida infections, we studied the in vitro susceptibility of juglone against fluconazole-susceptible and -resistance Candida isolates, alone and in combination.MethodsAntifungal susceptibility testing was performed according to the CLSI (Clinical and Laboratory Standards Institute) guidelines.ResultsJuglone exhibited the highest minimal inhibitory concentration (MIC) values, followed by fluconazole and nystatin. Voriconazole showed significantly better antifungal activity than juglone, fluconazole, and nystatin, with MIC₅₀ and MIC₉₀ of 0.031 and 0.5 μg/mL. There were significant differences in MICs of fluconazole (p < 0.001) and juglone (p < 0.0003) between Candida albicans and the rest of the species. Combination of juglone with fluconazole revealed insignificant effects against fluconazole-susceptible and -resistant Candida isolates. Juglone increased the antifungal activity of fluconazole; however, no synergism effects were observed for any combination, and only an insignificant effect was found against all tested Candida species.ConclusionsAlthough obtaining new antifungal drugs is a critical point, a completely novel approach should be implemented.     

Synthesis of Novel 3-Butylphthalide Derivatives Containing Isopentenylphenol Moiety as Potential Antiplatelet Agents for the Treatment of Ischemic Stroke

In order to find novel antiplatelet drugs for the treatment of ischemic stroke, a series of 3-butylphthalide derivatives containing isopentenylphenol moiety were designed, synthesized and characterized with spectroscopic analyses. The in vitro antiplatelet activity results indicated that compound 3 better inhibited the arachidonic acid (AA) induced platelet aggregation than aspirin (ASP) and 3-butylphthalide (NBP). Additionally, compared with precursor NBP, compound 3 possessed outstanding antithrombotic activity in the animal experiment model, which could effectively alleviate the formation of tail thrombus and carotid artery thrombus in mice. More importantly, intraperitoneal administration of compound 3 can well protected the rats against ischemia/reperfusion-induced brain injury. Further pharmacokinetic (PK) assay indicated that compound 3 had good absorption characteristics and metabolic stability in vivo. Overall, the present research provides a new candidate compound for the treatment of ischemic stroke caused by platelet aggregation.     

A novel fibrinolytic metalloproteinase,barnettlysin-I from Bothrops barnetti (barnett´s pitviper) snake venom with anti-platelet properties

BackgroundViperid snake venoms contain active components that interfere with hemostasis. We report a new P-I class snake venom metalloproteinase (SVMP), barnettlysin-I (Bar-I), isolated from the venom of Bothrops barnetti and evaluated its fibrinolytic and antithrombotic potential.MethodsBar-I was purified using a combination of molecular exclusion and cation-exchange chromatographies. We describe some biochemical features of Bar-I associated with its effects on hemostasis and platelet function.ResultsBar-I is a 23.386 kDa single-chain polypeptide with pI of 6.7. Its sequence (202 residues) shows high homology to other members of the SVMPs. The enzymatic activity on dimethylcasein (DMC) is inhibited by metalloproteinase inhibitors e.g. EDTA, and by α2-macroglobulin. Bar-I degrades fibrin and fibrinogen dose- and time-dependently by cleaving their α-chains. Furthermore, it hydrolyses plasma fibronectin but not laminin nor collagen type I. In vitro Bar-I dissolves fibrin clots made either from purified fibrinogen or from whole blood. In contrast to many other P-I SVMPs, Bar-I is devoid of hemorrhagic activity. Also, Bar-I dose- and time-dependently inhibits aggregation of washed human platelets induced by vWF plus ristocetin and collagen (IC₅₀ = 1.3 and 3.2 μM, respectively), presumably Bar-I cleaves both vWF and GPIb. Thus, it effectively inhibits vWF-induced platelet aggregation. Moreover, this proteinase cleaves the collagen-binding α2-A domain (160 kDa) of α2β1-integrin. This explains why it additionally inhibits collagen-induced platelet activation.ConclusionA non-hemorrhagic but fibrinolytic metalloproteinase dissolves fibrin clots in vitro and impairs platelet function.General significanceThis study provides new opportunities for drug development of a fibrinolytic agent with antithrombotic effect.     

A novel antithrombotic effect of sulforaphane via activation of platelet adenylate cyclase: ex vivo and in vivo studies

Sulforaphane is a naturally occurring isothiocyanate, which can be found in cruciferous vegetables such as broccoli and cabbage. Sulforaphane was found to have very potent inhibitory effects on tumor growth through regulation of diverse mechanisms. However, no data are available concerning the effects of sulforaphane on platelet activation and its relative issues. Activation of platelets caused by arterial thrombosis is relevant to a variety of cardiovascular diseases. Hence, the aim of this study was to examine the in vivo antithrombotic effects of sulforaphane and its possible mechanisms in platelet activation. Sulforaphane (0.125 and 0.25 mg/kg) was effective in reducing the mortality of ADP-induced acute pulmonary thromboembolism in mice. Other in vivo studies also revealed that sulforaphane (0.25 mg/kg) significantly prolonged platelet plug formation in mice. In addition, sulforaphane (15–75 μM) exhibited more-potent activity of inhibiting platelet aggregation stimulated by collagen. Sulforaphane inhibited platelet activation accompanied by inhibiting relative Ca²⁺ mobilization; phosphorylation of phospholipase C (PLC)γ2, protein kinase C (PKC), mitogen-activated protein kinases (MAPKs) and Akt; and hydroxyl radical (OH^●) formation. Sulforaphane markedly increased cyclic (c)AMP, but not cyclic (c)GMP levels, and stimulated vasodilator-stimulated phosphoprotein (VASP) phosphorylation. SQ22536, an inhibitor of adenylate cyclase, but not ODQ (1H-[1,2,4]Oxadiazolo[4,3-a]quinoxal in-1-one), an inhibitor of guanylate cyclase, obviously reversed the sulforaphane-mediated effects on platelet aggregation; PKC activation, p38 MAPK, Akt and VASP phosphorylation; and OH^● formation. Furthermore, a PI3-kinase inhibitor (LY294002) and a p38 MAPK inhibitor (SB203580) both significantly diminished PKC activation and p38 MAPK and Akt phosphorylation; in contrast, a PKC inhibitor (RO318220) did not diminish p38 MAPK or Akt phosphorylation stimulated by collagen. This study demonstrates for the first time that in addition to it originally being considered as an agent for prevention of tumor growth, sulforaphane possesses potent antiplatelet activity which may initially activate adenylate cyclase/cAMP, followed by inhibiting intracellular signals (such as the PI3-kinase/Akt and PLCγ2-PKC-p47 cascades) and ultimately inhibiting platelet activation. Therefore, this novel role of sulforaphane may represent a high therapeutic potential for treatment or prevention of cardiovascular diseases.     

BF066, a novel dual target antiplatelet agent without significant bleeding

In this study, we report BF066, a novel adenine derivative, inhibits platelet activation and thrombosis via the adenosine receptor (A(2A)) activation and phosphodiesterase (PDE) inhibition. BF066 inhibits platelet aggregation and ATP releasing induced by multiple platelet agonists in a dose-dependent manner. The inhibition of BF066 on ADP-induced aggregation is potentiated by adenosine and can be dramatically antagonized by the A(2A) antagonist SCH58261. BF066 also inhibits the PDE activity and platelet spreading on fibrinogen. In FeCl(3)-injured mouse mesenteric arterial thrombosis model, BF066 prevents thrombus formation effectively, similar to clopidogrel. Intriguingly, at dose achieving similar antithrombotic effect compared to clopidogrel, BF066 does not increase bleeding significantly. Taken together, these results suggest that BF066 may be an effective and safe antiplatelet agent targeting both PDE and A(2A). Considering the successful use of combined antiplatelet therapy, BF066 may be further developed as a novel dual target antiplatelet agent.     

Studies on the antiplatelet and antithrombotic profile of anti-inflammatory coumarin derivatives

Abstract

The interest towards coumarin-based structures stems from their polypharmacological profile. Herein, we present a series of Mannich bases and 7-azomethine-linked coumarin derivatives exhibiting antiplatelet and antithrombotic activities, in addition to the already known anti-inflammatory and antioxidant activities. Among others, compounds 15 and 16 were found to be the most potent and selective inhibitors of platelet aggregation whereas compound 3 also proved to be the most potent in the clot retraction assay. Structure–activity relationship studies were conducted to elucidate the molecular determinants responsible for the herein observed activities. The chance of inhibiting cyclooxygenase-1 was also investigated for evaluating the platelet aggregation induced by arachidonic acid. Taken together, these results suggest that the investigation of other targets connected to the antiplatelet activity, such as phosphodiesterase-3 (PDE3), could be a viable strategy to shed light on the polypharmacological profile of coumarin-based compounds. Docking simulations towards PDE3 were also carried out.     

Platelet microparticle-associated protein disulfide isomerase promotes platelet aggregation and inactivates insulin

Platelet-derived microparticles (pMP) have been shown to be pro-aggregatory and retain most of their platelet membrane markers. Recent studies have correlated elevated pMP levels with pathogenesis of diabetes mellitus and cardiovascular disease. The pro-aggregatory effect of pMP has been largely attributed to their negatively charged outer surface and activation of factor X by membrane associated Tissue factor (TF). Here we sought to investigate whether, like platelets, protein disulfide isomerase (PDI) is present on the surface of pMP and, if so, to analyze its contribution to platelet hyperaggregability and insulin degradation. Using a fluorescent assay based upon a novel pseudo-substrate of PDI, flow cytometry and immunological techniques, we have demonstrated the presence of PDI on the surface of pMP (termed msPDI) and its ability to influence insulin-mediated Akt phosphorylation (Thr308) in 3T3-L1 fibroblasts. Moreover, pMP are shown to contain catalytically active PDI, capable of both promoting platelet aggregation and disrupting insulin signaling. pMP increased initial rates of aggregation by 4-fold and the pro-aggregatory activity of pMPs could be attenuated with an anti-PDI antibody. The pMP insulin-reductase activity was further attributed to PDI based on the ability of anti-PDI antibodies to block the degradation of insulin, thereby restoring insulin signaling. Plasma pMP counts were also obtained from diabetic (n=10) and non-diabetic individuals (n=10) and found to be elevated in the diabetic state. Detection of increased levels of PDI-containing microparticles in patients with T2D raises the possibility that platelet hypersensitivity and insulin desensitization observed in diabetes can partially be attributed to msPDI activity.     

Potent antiplatelet activity of sesamol in an in vitro and in vivo model: pivotal roles of cyclic AMP and p38 mitogen-activated protein kinase

Sesamol is a potent phenolic antioxidant which possesses antimutagenic, antihepatotoxic and antiaging properties. Platelet activation is relevant to a variety of acute thrombotic events and coronary heart diseases. There have been few studies on the effect of sesamol on platelets. Therefore, the aim of this study was to systematically examine the detailed mechanisms of sesamol in preventing platelet activation in vitro and in vivo. Sesamol (2.5?5 μM) exhibited more potent activity of inhibiting platelet aggregation stimulated by collagen than other agonists. Sesamol inhibited collagen-stimulated platelet activation accompanied by [Ca²⁺]_i mobilization, thromboxane A₂ (TxA₂) formation, and phospholipase C (PLC)γ2, protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) phosphorylation in washed platelets. Sesamol markedly increased cAMP and cGMP levels, endothelial nitric oxide synthase (eNOS) expression and NO release, as well as vasodilator-stimulated phosphoprotein (VASP) phosphorylation. SQ22536, an inhibitor of adenylate cyclase, markedly reversed the sesamol-mediated inhibitory effects on platelet aggregation and p38 MAPK phosphorylation, and sesamol-mediated stimulatory effects on VASP and eNOS phosphorylation, and NO release. Sesamol also reduced hydroxyl radical (OH^●) formation in platelets. In an in vivo study, sesamol (5 mg/kg) significantly prolonged platelet plug formation in mice. The most important findings of this study demonstrate for the first time that sesamol possesses potent antiplatelet activity, which may involve activation of the cAMP-eNOS/NO-cGMP pathway, resulting in inhibition of the PLCγ2-PKC-p38 MAPK-TxA₂ cascade, and, finally, inhibition of platelet aggregation. Sesamol treatment may represent a novel approach to lowering the risk of or improving function in thromboembolism-related disorders.     

Quercetin-3-rutinoside Inhibits Protein Disulfide Isomerase by Binding to Its b′x Domain

Quercetin-3-rutinoside inhibits thrombus formation in a mouse model by inhibiting extracellular protein disulfide isomerase (PDI), an enzyme required for platelet thrombus formation and fibrin generation. Prior studies have identified PDI as a potential target for novel antithrombotic agents. Using a fluorescence enhancement-based assay and isothermal calorimetry, we show that quercetin-3-rutinoside directly binds to the b′ domain of PDI with a 1:1 stoichiometry. The binding of quercetin-3-rutinoside to PDI induces a more compact conformation and restricts the conformational flexibility of PDI, as revealed by small angle x-ray scattering. The binding sites of quercetin-3-rutinoside to PDI were determined by studying its interaction with isolated fragments of PDI. Quercetin-3-rutinoside binds to the b′x domain of PDI. The infusion of the b′x fragment of PDI rescued thrombus formation that was inhibited by quercetin-3-rutinoside in a mouse thrombosis model. This b′x fragment does not possess reductase activity and, in the absence of quercetin-3-rutinoside, does not affect thrombus formation in vivo. The isolated b′ domain of PDI has potential as an antidote to reverse the antithrombotic effect of quercetin-3-rutinoside by binding and neutralizing quercetin-3-rutinoside.     

Glaucocalyxin A Inhibits Platelet Activation and Thrombus Formation Preferentially via GPVI Signaling Pathway

Platelets play a pivotal role in atherothrombosis and the antiplatelet agents have been proved to be useful in preventing onset of acute clinical events including myocardial infarction and stroke. Increasing number of natural compounds has been identified to be potential antiplatelet agents. Here we report the antiplatelet effect of glaucocalyxin A (GLA), an ent-diterpenoid that we isolated and purified from the aerial parts of Rabdosia japonica (Burm. f.) var. glaucocalyx (Maxim.) Hara, and investigate the molecular mechanisms by which GLA inhibits platelet activation and thrombus formation. The effect of GLA on platelet activation was measured using platelets freshly isolated from peripheral blood of healthy donors. Results showed that pretreatment of human platelets with lower concentrations of GLA (0.01μg/ml, 0.1μg/ml) significantly inhibited platelet aggregation induced by collagen (P<0.001) and CRP (P<0.01), a synthetic GPVI ligand, but not by ADP and U46619. Accordingly, GLA inhibited collagen-stimulated tyrosine phosphorylation of Syk, LAT, and phospholipase Cγ2, the signaling events in collagen receptor GPⅥ pathway. GLA also inhibited platelet p-selectin secretion and integrin activation by convulxin, a GPVI selective ligand. Additionally, GLA was found to inhibit low-dose thrombin-induced platelet activation. Using a flow chamber device, GLA was found to attenuate platelet adhesion on collagen surfaces in high shear condition. In vivo studies showed that GLA administration increased the time for complete occlusion upon vascular injury in mice, but did not extend tail-bleeding time when mice were administered with relatively lower doses of GLA. Therefore, the present results provide the molecular basis for the inhibition effect of GLA on platelet activation and its in vivo effect on thrombus formation, suggesting that GLA could potentially be developed as an antiplatelet and antithrombotic agent.     

In vitro and in vivo antithrombotic and cytotoxicity effects of ferulic acid

We discovered recently in vitro and in vivo antithrombotic and cytotoxicity effects of ferulic acid. The cytotoxicity assays showed that ferulic acid (～300 μg/mL) did not cause any significant toxicity on three cell lines, platelets, leukocytes, and erythrocytes. In vitro assays showed inhibitory effects of ferulic acid on thrombin (THR)‐ or collagen/epinephrine‐stimulated platelet activation by inhibiting platelet aggregation, and decreasing clot retraction activity. The in vitro effect of ferulic acid on THR‐stimulated platelet activation was proved by the decrease in the secretion of serotonin from the platelets. The anticoagulant effects of ferulic acid were confirmed by the prolongation of the intrinsic or/and extrinsic pathways and the delay of recalcification time in plasma coagulation. Ferulic acid had antithrombotic effect in acute thromboembolism model in vivo, and decreased the expression of α_IIbβ₃/FIB and phosphorylation of AKT in THR‐stimulated platelet activation in vivo, and their antithrombotic efficacies hold promise for therapeutic targeting in our ongoing studies.     

Juglone,isolated from Juglans mandshurica Maxim,induces apoptosis via down-regulation of AR expression in human prostate cancer LNCaP cells

Juglone is a natural compound which has been isolated from Juglans mandshurica Maxim. Recent studies have shown that juglone had various pharmacological effects such as anti-viral, anti-bacterial and anti-cancer. However, its anti-cancer activity on human prostate cancer LNCaP cell has not been examined. Thus, the current study was designed to elucidate the molecular mechanism of apoptosis induced by juglone in androgen-sensitive prostate cancer LNCaP cells. MTT assay was performed to examine the anti-proliferative effect of juglone. Occurrence of apoptosis was detected by Hoechst 33342 staining and flow cytometry in LNCaP cells treated with juglone for 24 h. The result shown that juglone inhibited the growth of LNCaP cells in a dose-dependent manner. Morphological changes of apoptotic body formation after juglone treatment were observed by Hoechst 33342 staining. This apoptotic induction was associated with loss of mitochondrial membrane potential, and caspase-3, -9 activation. Moreover, we found that juglone significantly inhibited the expression levels of androgen receptor (AR) and prostate-specific antigen (PSA) in a dose-dependent manner, as well as abrogated up-regulation of AR and PSA genes with and/or without dihydrotestosterone (DHT). Take together, our results demonstrated that juglone might induce the apoptosis in LNCaP cell via down-regulation of AR expression. Therefore, our results indicated that juglone may be a potential candidate of drug for androgen-sensitive prostate cancer.     

Mice Lacking the SLAM Family Member CD84 Display Unaltered Platelet Function in Hemostasis and Thrombosis

Background

Platelets are anuclear cell fragments derived from bone marrow megakaryocytes that safeguard vascular integrity by forming thrombi at sites of vascular injury. Although the early events of thrombus formation—platelet adhesion and aggregation—have been intensively studied, less is known about the mechanisms and receptors that stabilize platelet-platelet interactions once a thrombus has formed. One receptor that has been implicated in this process is the signaling lymphocyte activation molecule (SLAM) family member CD84, which can undergo homophilic interactions and becomes phosphorylated upon platelet aggregation.

Objective

The role of CD84 in platelet physiology and thrombus formation was investigated in CD84-deficient mice.

Methods and Results

We generated CD84-deficient mice and analyzed their platelets in vitro and in vivo. Cd84^−/− platelets exhibited normal activation and aggregation responses to classical platelet agonists. Furthermore, CD84 deficiency did not affect integrin-mediated clot retraction and spreading of activated platelets on fibrinogen. Notably, also the formation of stable three-dimensional thrombi on collagen-coated surfaces under flow ex vivo was unaltered in the blood of Cd84^−/− mice. In vivo, Cd84^−/− mice exhibited unaltered hemostatic function and arterial thrombus formation.

Conclusion

These results show that CD84 is dispensable for thrombus formation and stabilization, indicating that its deficiency may be functionally compensated by other receptors or that it may be important for platelet functions different from platelet-platelet interactions.     

Another “String to the Bow” of PJ34, a Potent Poly(ADP-Ribose)Polymerase Inhibitor: An Antiplatelet Effect through P2Y12 Antagonism?

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.     

A Protein Disulfide Isomerase Gene Fusion Expression System That Increases the Extracellular Productivity of Bacillus brevis

We have developed a versatile Bacillus brevis expression and secretion system based on the use of fungal protein disulfide isomerase (PDI) as a gene fusion partner. Fusion with PDI increased the extracellular production of heterologous proteins (light chain of immunoglobulin G, 8-fold; geranylgeranyl pyrophosphate synthase, 12-fold). Linkage to PDI prevented the aggregation of the secreted proteins, resulting in high-level accumulation of fusion proteins in soluble and biologically active forms. We also show that the disulfide isomerase activity of PDI in a fusion protein is responsible for the suppression of the aggregation of the protein with intradisulfide, whereas aggregation of the protein without intradisulfide was prevented even when the protein was fused to a mutant PDI whose two active sites were disrupted, suggesting that another PDI function, such as chaperone-like activity, synergistically prevented the aggregation of heterologous proteins in the PDI fusion expression system.     

Oxidative stress-related mechanisms affecting response to aspirin in diabetes mellitus

Type 2 diabetes mellitus (T2DM) is a major cardiovascular risk factor. Persistent platelet activation plays a key role in atherothrombosis in T2DM. However, current antiplatelet treatments appear less effective in T2DM patients vs nondiabetics at similar risk. A large body of evidence supports the contention that oxidative stress, which characterizes DM, may be responsible, at least in part, for less-than-expected response to aspirin, with multiple mechanisms acting at several levels. This review discusses the pathophysiological mechanisms related to oxidative stress and contributing to suboptimal aspirin action or responsiveness. These include: (1) mechanisms counteracting the antiplatelet effect of aspirin, such as reduced platelet sensitivity to the antiaggregating effects of NO, due to high-glucose-mediated oxidative stress; (2) mechanisms interfering with COX acetylation especially at the platelet level, e.g., lipid hydroperoxide-dependent impaired acetylating effects of aspirin; (3) mechanisms favoring platelet priming (lipid hydroperoxides) or activation (F₂-isoprostanes, acting as partial agonists of thromboxane receptor), or aldose-reductase pathway-mediated oxidative stress, leading to enhanced platelet thromboxane A₂ generation or thromboxane receptor activation; (4) mechanisms favoring platelet recruitment, such as aspirin-induced platelet isoprostane formation; (5) modulation of megakaryocyte generation and thrombopoiesis by oxidative HO-1 inhibition; and (6) aspirin–iron interactions, eventually resulting in impaired pharmacological activity of aspirin, lipoperoxide burden, and enhanced generation of hydroxyl radicals capable of promoting protein kinase C activation and platelet aggregation. Acknowledgment of oxidative stress as a major contributor, not only of vascular complications, but also of suboptimal response to antiplatelet agents in T2DM, may open the way to designing and testing novel antithrombotic strategies, specifically targeting oxidative stress-mediated mechanisms of less-than-expected response to aspirin.