The antiplatelet activity of PMC, a potent alpha-tocopherol analogue, is mediated through inhibition of cyclo-oxygenase.

PMC, a potent alpha-tocopherol derivative, dose-dependently (5-25 microM) inhibited the ATP-release reaction and platelet aggregation in washed human platelets stimulated by agonists (collagen and ADP). PMC also dose-dependently inhibited the intracellular Ca2+ mobilization, whereas it did not inhibit phosphoinositide breakdown in human platelets stimulated by collagen. PMC (10 and 25 microM) significantly inhibited collagen-stimulated thromboxane A2 (TxA2) formation in human platelets. On the other hand, PMC (25 and 100 microM) did not increase the formation of cyclic AMP or cyclic GMP in platelets. Moreover, PMC (25, 100, and 200 microM) did not affect the thromboxane synthetase activity of aspirin-treated platelet microsomes. PMC (10 and 25 microM) markedly inhibited the exogenous arachidonic acid (100 microM)-induced prostaglandin E2 (PGE2) formation in the presence of imidazole (600 microM) in washed human platelets, indicating that PMC inhibits cyclo-oxygenase activity. We conclude that PMC may exert its anti-platelet aggregation activity by inhibiting cyclooxygenase activity, which leads to reduced prostaglandin formation; this, in turn, is followed by a reduction of TxA2 formation, and finally inhibition of [Ca2+]i mobilization and ATP-release.     

A new insight of anti-platelet effects of sirtinol in platelets aggregation via cyclic AMP phosphodiesterase

Sirtinol, a cell permeable six-membered lactone ring, is derived from naphthol and potent inhibitor of SIR2 and its naphtholic may have the inhibitory effects on platelets aggregation. In this study, platelet function was examined by collagen/epinephrine (CEPI) and collagen/ADP-induced closure times using the PFA-100 system reveal that CEPI-CT and CADP-CT were prolonged by sirtinol. The platelets aggregation regulated by physiological agonists such as: thrombin, collagen and AA and U46619 were significantly inhibited by sirtinol. Increases cAMP level was observed when sirtinol treated with Prostaglandin E1 in washed platelets. Moreover, sirtinol attenuated intracellular Ca²⁺ release and thromboxane B2 formation stimulated by thrombin, collagen, AA and U46619 in human washed platelets. This study indicated that sirtinol could inhibit the platelet aggregation induced by physiological agonists, AA and U46619. The mechanism of action may include an increase of cAMP level with enhanced VASP-Ser157 phosphorylation via inhibition of cAMP phosphodiesterase activity and subsequent inhibition of intracellular Ca²⁺ mobilization, thromboxane A2 formation, and ATP release during the platelet aggregation.     

Inhibitory mechanisms of gabapentin, an antiseizure drug, on platelet aggregation

Gabapentin (Neurontin) is an analogue of gamma-aminobutyric acid (GABA) that is effective against partial seizures. Gabapentin has been reported to modulate serotonin release from platelets, but the effects of gabapentin on platelet activation have not been explored. In this study, gabapentin concentration-dependently (60-240 microM) inhibited platelet aggregation in washed platelets stimulated by collagen (1 microg mL(-1)), ADP (20 microM) and arachidonic acid (60 microM). Gabapentin (120 and 240 microM) also concentration-dependently inhibited collagen (1 microg mL(-1))-induced phosphoinositide breakdown, intracellular Ca(2+) mobilization, thromboxane A(2) formation, and p38 MAPK phosphorylation in human platelets. In conclusion, the most important findings of this study suggest that gabapentin inhibits platelet aggregation, at least in part, through the phospholipase C-inositol 1,4,5-trisphosphate-thromboxane A(2)-Ca(2+) pathway. Thus, it is possible that gabapentin treatment, alone or in combination with other antiplatelet drugs, may induce or potentiate inhibition of platelet aggregation, which may affect haemostasis in-vivo.     

Antiplatelet effect of marchantinquinone, isolated from Reboulia hemisphaerica, in rabbit washed platelets

Platelet activation is involved in serious pathological situations, including atherosclerosis and restenosis. It is important to find efficient antiplatelet medicines to prevent fatal thrombous formation during the course of these diseases. Marchantinquinone, a natural compound isolated from Reboulia hemisphaerica, inhibited platelet aggregation and ATP release stimulated by thrombin (0.1 units mL(-1)), platelet-activating factor (PAF; 2 ng mL(-1)), collagen (10 microg mL(-1)), arachidonic acid (100 microM), or U46619 (1 microM) in rabbit washed platelets. The IC50 values of marchantinquinone on the inhibition of platelet aggregation induced by these five agonists were 62.0 +/- 9.0, 86.0 +/- 7.8, 13.6 +/- 4.7, 20.9 +/- 3.1 and 13.4 +/- 5.3 microM, respectively. Marchantinquinone inhibited thromboxane B2 (TxB2) formation induced by thrombin, PAF or collagen. However, marchantinquinone did not inhibit TxB2 formation induced by arachidonic acid, indicating that marchantinquinone did not affect the activity of cyclooxygenase and thromboxane synthase. Marchantinquinone did inhibit the rising intracellular Ca2+ concentration stimulated by the five platelet-aggregation inducers. The formation of inositol monophosphate induced by thrombin was inhibited by marchantinquinone. Platelet cAMP and cGMP levels were unchanged by marchantinquinone. The results indicate that marchantinquinone exerts antiplatelet effects by inhibiting phosphoinositide turnover.     

Thromboxane synthase inhibition potentiates washed platelet activation by endogenous and exogenous arachidonic acid

The effect of the thromboxane (TX) synthase inhibitors dazoxiben and imidazole on platelet activation by endogenous and exogenous arachidonic acid (AA) was tested with human washed platelets. Dazoxiben (1-20 microM) inhibited the formation of TXB2 and markedly enhanced the shape change, aggregation, and (3H)serotonin release induced by added AA or when prostaglandin synthesis from endogenous AA was triggered by collagen, hydrogen peroxide or methyl mercury chloride (methyl-Hg). Platelet activation by hydrogen peroxide (20-1200 microM) or methyl-Hg (1-5 microM) was entirely dependent on endogenous prostaglandin (PG) synthesis since acetylsalicylic acid (ASA), indomethacin or the cyclic endoperoxide/TXA2-antagonist BM 13.177 counteracted these stimulants with and without dazoxiben. Apparently, the potentiation is due to accumulating cyclic endoperoxides which during TX synthase inhibition reach greater platelet-activating potency than TXA2. Albumin or human platelet-poor plasma inhibited the platelet activation by hydrogen peroxide and methyl-Hg and suppressed the potentiation by dazoxiben. The latter effect of albumin may result from its PGD isomerase activity which redirects the cyclic endoperoxide metabolism to the platelet-inhibitory PGD2. The results show that non-platelet factors such as albumin are necessary to prevent a potentiating effect of TX synthase inhibitors on platelet activation.     

三乙酰莽草酸对血小板聚集的抑制作用

目的：研究三乙酰莽草酸（TSA）对血小板聚集功能的抑制作用及其作用机理。方法：用比浊法测定血小板聚集功能,分光光度法测定MDA的含量,放免法测定TXB₂,6-酮-PGF_1α,cAMP和cGMP的含量。结果：TSA 12.5,25,50,100和200 mg.kg^-1 ig明显抑制ADP和胶原诱导的大鼠血小板聚集;TSA 12.5,50和200　mg.kg^-1 ig显著增加大鼠血小板内cAMP水平,但不影响cGMP水平。TSA 200 mg.kg^-1对AA诱导的血小板中MDA的生成,ADP诱导的血小板中TXB2和腹主动脉壁6-酮-PGF_1α的生成有轻度抑制作用。结论：TSA抑制血小板聚集作用部分与血小板内cAMP水平升高有关。     

Ginkgolide C inhibits platelet aggregation in cAMP- and cGMP-dependent manner by activating MMP-9

In this report, we investigated the effect of ginkgolide C (GC) from Ginkgo biloba leaves in collagen (10 mug/ml)-stimulated platelet aggregation. It has been known that matrix metalloproteinase-9 (MMP-9) is released from human platelets, and that it significantly inhibited platelet aggregation stimulated by collagen. Zymographic analysis confirmed that pro-MMP-9 (92-kDa) was activated by GC to form an activated MMP-9 (86-kDa) on gelatinolytic activities. And then, GC dose-dependently inhibited platelet aggregation, intracellular Ca(2+) mobilization, and thromboxane A(2) (TXA(2)) formation in collagen-stimulated platelets. In addition, GC significantly increased the formation of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), which have an anti-platelet function in both resting and collagen-stimulated platelets. Therefore, we demonstrate that the inhibitory effect of GC on platelet aggregation might be involved into the following pathways. GC may increase intracellular cAMP and cGMP production and MMP-9 activity, inhibit intracellular Ca(2+) mobilization and TXA(2) production, thereby leading to inhibition of platelet aggregation. These results strongly indicate that GC is a potent inhibitor of collagen-stimulated platelet aggregation. It may be a suitable tool for a negative regulator during platelet activation.     

Effect of a potent cyclooxygenase inhibitor, 5-ethyl-4-methoxy-2-phenylquinoline (KTC-5), on human platelets

Because the metabolites of arachidonic acid participate in many physiopathological responses, including inflammation and platelet aggregation, cyclooxygenase inhibitors are important in the treatment of associated diseases. A biologically active compound, 5-ethyl-4-methoxy-2-phenylquinoline (KTC-5), selectively and concentration dependently inhibited aggregation of platelets from man and ATP release caused by arachidonic acid (200 microM) and collagen (10 microg mL(-1)) without affecting the aggregation caused by thrombin (0.1 U mL(-1)) and U46619 (2 microM). The IC50 value (drug concentration inhibiting maximum response by 50%) of KTC-5 for aggregation induced by arachidonic acid and collagen was 0.11+/-0.04 microM and 0.20+/-0.03 microM, respectively. This inhibitory effect of KTC-5 was reversible and time dependent. KTC-5 specifically inhibited intracellular calcium mobilization initiated by arachidonic acid or collagen without affecting that caused by thrombin or U46619 in human platelets. Furthermore, KTC-5 inhibited thromboxane B2 and prostaglandin D2 formation provoked by arachidonic acid. The IC50 value of KTC-5 for arachidonic-acid-induced thromboxane B2 formation was 0.07+/-0.02 microM. Based on these observations, the data indicated that KTC-5 potently inhibited human platelet aggregation and ATP release mainly via the inhibition of the cyclooxygenase-1 activity. Moreover, KTC-5 inhibited lipopolysaccharide-induced prostaglandin E2 formation in RAW264.7 cells in the presence of external arachidonic acid with an IC50 value of 0.17+/-0.06 microM. Immunoblot analysis showed that KTC-5 did not affect the cyclooxygenase-2 expression in the presence of lipopolysaccharide on RAW264.7 cells. This result indicated that KTC-5 affects the activity of cyclooxygenase-2. According to these data, we concluded that KTC-5 is a cyclooxygenase inhibitor for both subtypes.     

Pharmacological characterization of cinnamophilin, a novel dual inhibitor of thromboxane synthase and thromboxane A2 receptor.

1. The pharmacological effects of cinnamophilin, a new lignan, isolated from Cinnamomum philippinense, was determined in vitro in human platelet, rat isolated aorta and guinea-pig isolated trachea and in vivo in mice and guinea-pigs. 2. Cinnamophilin inhibited dose-dependently human platelet-rich plasma (PRP) aggregation induced by arachidonic acid (AA), collagen and U-46619 with IC50 of 5.0 +/- 0.4, 5.6 +/- 0.6 and 3.0 +/- 0.4 microM, respectively. The second wave of ADP- or adrenaline-induced platelet aggregation was inhibited by cinnamophilin, while the first wave was only slightly inhibited by cinnamophilin above 30 microM. 3. Cinnamophilin was found to be a thromboxane A2 (TXA2) receptor blocking agent in human platelet, rat aorta and guinea-pig trachea as revealed by its competitive antagonism of U-46619-induced aggregation of human-PRP, contraction of rat aortic rings and guinea-pig tracheal rings with pA2 values of 7.3 +/- 0.2, 6.3 +/- 0.1 and 5.2 +/- 0.2, respectively. 4. [3H]-inositol monophosphate formation and the rise of intracellular Ca2+ caused by U-46619 in human platelet was suppressed by cinnamophilin (10 microM). 5. Cinnamophilin induced a dose-dependent inhibition of thromboxane B2 (TXB2) formation, while the prostaglandin E2 (PGE2) formation was increased. Cinnamophilin did not affect unstimulated platelet adenosine 3':5'-cyclic monophosphate (cyclic AMP) levels. When the platelets were challenged with AA, a dose-dependent rise in cyclic AMP was observed. Dazoxiben (a pure TX synthase inhibitor) and SQ 29548 (a pure TXA2 receptor antagonist) did not affect cyclic AMP levels in AA-treated platelets.(ABSTRACT TRUNCATED AT 250 WORDS)     

The dopamine D(1) receptor agonist SKF-82958 serves as a discriminative stimulus in the rat

The antiplatelet effect of the pyridazinone analogue, 4, 5-dihydro-6-[4-[2-hydroxy-3-(3,4 dimethoxybenzylamino)propoxy]naphth-1-yl]-3(2H)-pyridazinone (HCL-31D), was investigated in vitro with rabbit platelets. HCL-31D dose-dependently inhibited the platelet aggregation and ATP release induced by collagen (10 microg/ml), arachidonic acid (100 microM) or thrombin (0.1 U/ml) with an IC(50) of about 0.95-5.41 microM. HCL-31D (0.5-5 microM) increased the platelet cyclic AMP level in a dose-dependent manner. Furthermore, HCL-31D potentiated cyclic AMP formation caused by prostaglandin E(1) but not that caused by 3-isobutyl-1-methylxanthine (IBMX). HCL-31D also attenuated phosphoinositide breakdown and intracellular Ca(2+) elevation induced by collagen, arachidonic acid or thrombin. HCL-31D inhibited the formation of thromboxane B(2) induced by collagen or thrombin but not by arachidonic acid. In addition, HCL-31D did not affect platelet cylooxygenase and thromboxane synthase activity. These data indicate that HCL-31D is an inhibitor of phosphodiesterase and that its antiplatelet effect is mainly mediated by elevation of cyclic AMP levels.     

Inhibition of human platelet cyclic AMP phosphodiesterase and of platelet aggregation by a hemisynthetic flavonoid, amentoflavone hexaacetate

Amentoflavone hexaacetate (AmAc) was synthesized from natural amentoflavone (Am), a biflavonoid extracted from Viburnum lantana L. Am does not inhibit aggregation of intact platelets up to a concentration of 100 microM but inhibits human platelet cAMP phosphodiesterase (IC50 = 22.0 microM). AmAc is a potent inhibitor of the aggregation of washed human platelets induced by ADP (IC50 = 2.3 microM) or collagen (IC50 = 4.7 microM). AmAc inhibits crude (IC50 = 8.6 microM) or partially purified (IC50 = 42.2 microM) human platelet cAMP phosphodiesterase. In the presence of prostaglandin E1, AmAc (10 microM) induces a 3.7-fold increase in total platelet cAMP. The characteristics of this action suggest a role for cAMP in the mechanism of action of AmAc. The incubation of AmAc with intact platelets for 5 min is necessary for its activity.     

Inhibitory effect of 8-bromo cyclic GMP on an extracellular Ca2+-dependent arachidonic acid liberation in collagen-stimulated rabbit platelets

The inhibitory effect of cyclic GMP on collagen-induced platelet activation was studied using 8-bromo cyclic GMP (8brcGMP) in washed rabbit platelets. Addition of collagen (1 micrograms/ml) to platelet suspension caused shape change and aggregation associated with thromboxane (TX) A2 formation. 8brcGMP (10-1000 microM) inhibited collagen-induced platelet aggregation and TXA2 formation in a concentration-dependent manner. 8brcGMP did not affect platelet cyclooxygenase pathways, but markedly inhibited collagen-induced arachidonic acid (AA) liberation from membrane phospholipids in [3H]AA-prelabeled platelets, indicating that the inhibitory effect of 8brcGMP on collagen-induced aggregation is due to an inhibition of AA liberation. In [32P]orthophosphate-labeled platelets, collagen stimulated phosphorylation of a 20,000 dalton (20-kD) and 40-kD proteins. 8BrcGMP stimulated phosphorylation of a specific protein having molecular weight of 46-kD and inhibited collagen-induced both 20- and 40-kD protein phosphorylation. Collagen could stimulate the AA liberation without activation of phospholipase C or Na+-H+ exchange, but could not in the absence of extracellular Ca2+. These findings suggest that cyclic GMP inhibits collagen-induced AA liberation which is mediated by an extracellular Ca2+-dependent phospholipase A2. However, cyclic GMP seems to inhibit the Ca2+-activated phospholipase A2 indirectly, since 8brcGMP had no effect on Ca2+ ionophore A23187-induced platelet aggregation or AA liberation. It is therefore suggested that cyclic GMP may regulate collagen-induced increase in an availability of extracellular Ca2+ which is responsible for phospholipase A2 activation in rabbit platelets.     

Inhibition of Platelet Thromboxane Formation and Phosphoinositides Breakdown by Diisoeugenol

Abstract— Diisoeugenol inhibited the platelet aggregation and ATP release of rabbit platelets caused by ADP, arachidonic acid, platelet-activating factor (PAF), collagen and thrombin. Prolongation of the incubation time of platelets with diisoeugenol did not cause further inhibition and the aggregability of platelets could not be restored after washing. In human platelet-rich plasma, diisoeugenol inhibited the biphasic aggregation and ATP release induced by adrenaline and ADP in a concentration-dependent manner. Thromboxane B₂ formation caused by arachidonic acid, collagen and thrombin was markedly inhibited by diisoeugenol in a concentration-dependent manner. Diisoeugenol also inhibited the formation of inositol monophosphate caused by collagen, PAF and thrombin. The cAMP level of washed platelets was not changed by diisoeugenol. It is concluded that the antiplatelet effect of diisoeugenol is due to the inhibition of thromboxane formation and phosphoinositides breakdown.     

Studies on the antiplatelet effect of the stable epoprostenol analogue beraprost sodium and its mechanism of action in rats

Baraprost sodium (sodium (+/-)-(1R*,2R*,3aS*,8bS*)-2,3,3a.8b- tetrahydro-2-hydroxy-1-[(E)-(3S*)-3-hydroxy-4-methyl-1-octen-6- 1H-cyclopenta[b]benzo-furan-5-butyrate, TRK-100) is a novel stable epoprostenol (prostaglandin I2, PGI2) analogue having antiplatelet and vasodilating actions. Its effect on platelet aggregation in whole blood ex vivo and platelet suspension in vitro, formation of cyclic AMP(cAMP), production of malondialdehyde(MDA), and 45Ca++-influx into platelets were studied in rats. Oral administration of TRK-100 (0.3-1 mg/kg) showed a dose-dependent inhibition of platelet aggregation induced by ADP and collagen in whole blood and also inhibited in vitro thrombin-induced aggregation of platelet suspension in the presence or absence of external Ca++. Oral TRK-100 (0.3-3 mg/kg) dose-dependently increased plasma cAMP levels and this action was confirmed in vitro with platelet rich plasma in the presence or absence of theophylline. 45Ca++-influx into platelets stimulated by thrombin was dose-dependently inhibited by TRK-100 (3-100 nmol/l). TRK-100 (3-100 nmol/l) also suppressed MDA production induced by thrombin in platelet suspension but not that induced by arachidonic acid. From these results, TRK-100 which is orally active was suggested to exert its antiplatelet action through the increase of cAMP in platelets by activation of adenylate cyclase, concomitantly followed by the inhibition of Ca++-influx and thromboxane A2 formation.     

The antiplatelet activity of Geiji-Bokryung-Hwan,Korean traditional formulation,is mediated through inhibition of phospholipase C and inhibition of TxB(2) synthetase activity

Geiji-Bokryung-Hwan (GBH), consisting of herbes of Cinnamomi ramulus (Geiji), Poria cocos (Bokryun), Mountan cortex radicis (Mokdanpi), Paeoniae radix (Jakyak), and Persicae semen (Doin), on antiplatelet activity in human platelet suspensions was studied. The mechanism involved in the antiplatelet activity of GBH in human platelet suspensions was investigated. GBH did not significantly affect the thromboxane synthetase activity of aspirin-treated platelet microsomes and GBH (15 and 30 microg/ml) significantly inhibited [3H]arachidonic acid released in collagen-activated platelets but not in unactivated-platelets. Nitric oxide (NO) production in human platelets was measured by a chemiluminesence detection method in this study. GBH did not significantly affect nitrate production in collagen (10 microg/ml)-induced human platelet aggregation. Various concentrations of GBH (0, 5, 10, 15, and 30 microg/ml) dose-dependently inhibited [3H]inositol monophosphate formation stimulated by collagen (10 microg/ml) in [3H]myoinositol-loaded platelets at different incubation times (1, 2, 3, and 5 min). These results indicated that the antiplatelet activity of GBH may possibly be due to the inhibition of phospholipase C (PLC) activity, leading to reduce phosphoinositide breakdown, followed by the inhibition of thromboxane A(2) formation, and then inhibition of [Ca(2+)](i) mobilization of platelet aggregation stimulated by agonists. In conclusion, GBH suppressed PLC in a dose-dependent manner, and may have pharmaceutical applications. These data suggest that GBH extracts merit investigation as a potential anti-atherosclerogenic agent in humans.     

丁基苯酞对大鼠血栓形成及血小板功能的影响

目的研究消旋、左旋和右旋丁基苯酞(dl-,l-和d-NBP)对血栓形成及血小板功能的影响。方法利用半体外血栓形成术及比浊法,观察dl-,l-和d-NBP及阿司匹林(Asp)对大鼠血栓湿重和血小板聚集率的影响,并用放免法、荧光分光光度法测定其对血小板内cAMP和TXB2的水平以及血小板5-HT释放率的影响。结果ip,dl-NBP和l-NBP可剂量依赖性地抑制大鼠血栓形成,且l-NBP作用与Asp相似,d-NBP对半体外血栓形成无显著作用;dl-,d-和l-NBP可显著抑制胶原、ADP、花生四烯酸诱导的血小板聚集。结论NBP有抗血栓作用,l-NBP作用最强,dl-NBP作用较弱,其抗栓作用与升高血小板内cAMP的含量及抑制5-HT释放有关。     

Amyloid beta peptide-activated signal pathways in human platelets

Amyloid beta peptide (amyloid-beta), which accumulates in the cerebral microvessels in an age-dependent manner, plays a key role in the pathogenesis of cerebral amyloid angiopathy. Platelets are an important cellular element in vasculopathy of various causes. Amyloid-beta may activate or potentiate platelet aggregation. The present study explored the signaling events that underlie amyloid-beta activation of platelet aggregation. Platelet aggregometry, immunoblotting and assays to detect activated cellular events were applied to examine the signaling processes of amyloid-beta activation of platelets. Exogenous amyloid-beta (1-2 microM) potentiated platelet aggregation caused by collagen and other agonists. At higher concentrations (5-10 microM), amyloid-beta induced platelet aggregation which was accompanied by an increase in thromboxane A2 (TxA2) formation. These amyloid-beta actions on platelets were causally related to amyloid-beta activation of p38 mitogen-activated protein kinase (MAPK). Inhibitors of p38 MAPK and its upstream signaling pathways including proteinase-activated receptor 1 (PAR1), Ras, phosphoinositide 3-kinase (PI3-kinase), or Akt, but not extracellular signal-regulated kinase 2 (ERK2)/c-Jun N-terminal kinase 1 (JNK1), blocked amyloid-beta-induced platelet activation. These findings suggest that the p38 MAPK, but not ERK2 or JNK1 pathway, is specifically activated in amyloid-beta-induced platelet aggregation with the following signaling pathway: PAR1 --> Ras/Raf --> PI3-kinase --> Akt --> p38 MAPK --> cytosolic phospholipase A2 (cPLA2)--> TxA2. In conclusion, this study demonstrates amyloid-beta activation of a p38 MAPK signaling pathway in platelets leading to aggregation. Further studies are needed to define the specific role of amyloid-beta activation of platelets in the pathogenesis of vasculopathy including cerebral amyloid angiopathy.     

Expression of matrix metalloproteinase-9 in human platelets: regulation of platelet activation in in vitro and in vivo studies

1. The aim of this study was to identify the presence of matrix metalloproteinase-9 (MMP-9) in human platelets and systematically examine its inhibitory mechanisms of platelet activation. 2. In this study, we report on an efficient method for the quantitative analysis of pro-MMP-9 in human platelets using capillary zone electrophoresis (CZE). To elucidate subcellular localization of MMP-9 in human platelets, we investigated intraplatelet MMP-9 by immunogold labeling and visualized it using electron microscopy. In an in vivo thrombotic study, platelet thrombus formation was induced by irradiation of mesenteric venules with filtered light in mice pretreated with fluorescein sodium. 3. MMP-9-gold labeling was observed on the plasma membrane, alpha-granules, open canalicular system, and within the cytoplasma both in resting and activated platelets. Furthermore, activated MMP-9 concentration-dependently (15-90 ng ml(-1)) inhibited platelet aggregation stimulated by agonists. Activated MMP-9 (21 and 90 ng ml(-1)) inhibited phosphoinositide breakdown, intracellular Ca(2+) mobilization, and thromboxane A(2) formation in human platelets stimulated by collagen (1 microg ml(-1)). In addition, activated MMP-9 (21 and 90 ng ml(-1)) significantly increased the formation of nitric oxide/cyclic GMP. 4. Rapid phosphorylation of a platelet protein of Mr 47,000 (P47), a marker of protein kinase C activation, was triggered by phorbol-12, 13-dibutyrate (PDBu) (60 nm). This phosphorylation was markedly inhibited by activated MMP-9 (21 and 90 ng ml(-1)). Activated MMP-9 (1 microg g(-1)) significantly prolonged the latency period of inducing platelet plug formation in mesenteric venules. 5. These results indicate that the antiplatelet activity of activated MMP-9 may be involved in the following pathways. (1) Activated MMP-9 may inhibit the activation of phospholipase C, followed by inhibition of phosphoinositide breakdown, protein kinase C activation, and thromboxane A(2) formation, thereby leading to inhibition of intracellular Ca(2+) mobilization. (2) Activated MMP-9 also activated the formation of nitric oxide/cyclic GMP, resulting in inhibition of platelet aggregation. These results strongly indicate that MMP-9 is a potent inhibitor of aggregation. It may play an important role as a negative feedback regulator during platelet activation.     

Mechanisms involved in the antiplatelet activity of 8-methyl-4-(1-piperazinyl)-7-(3-pyridinylmethoxy)-2H-1-benzopyran-2-one (RC414)

The effect on human platelets of 8-methyl-4-(1-piperazinyl)-7-(3-pyridinylmethoxy)-2H-1-benzopyran-2-one (RC414) was tested in vitro by measuring aggregation induced by several agonists, cAMP and cGMP levels, cAMP phosphodiesterase and PKC activities and [Ca2+]i. The RC414 effect on nitric oxide production was also evaluated. RC414 in a dose-dependent manner inhibited aggregation both in platelet rich plasma and in washed platelets. It was particularly effective in platelets challenged by collagen, ADP and thrombin: IC50 values are 0.51 +/- 0.12 microM, 0.98 +/- 0.36 microM and 1.00 +/- 0.15 microM, respectively. RC414 increased cAMP levels, through the specific inhibition of the cAMP high affinity phosphodiesterase (IC50 = 1.73 +/- 0.35 microM). RC414 reduced [Ca2+]i transients and PKC activation induced by thrombin. In addition RC414 was able to increase nitric oxide formation involving the stimulation of constitutive nitric oxide synthase enzyme. In conclusion, RC414 exerts its powerful anti-platelet activity by increasing cAMP intracellular levels and nitric oxide formation.     

Hispidulin, a natural flavone, inhibits human platelet aggregation by increasing cAMP levels

Hispidulin, a natural flavone, and theophylline inhibited platelet aggregation triggered by adenosine-5'-monophosphate, arachidonic acid, paf-acether and collagen. Hispidulin was 100-fold more potent than theophylline. A threshold concentration of PGE1 did not modify the anti-aggregatory effect of hispidulin but potentiated the effect of theophylline. A threshold concentration of hispidulin had no effect on the inhibitory action of theophylline. Hispidulin (100 microM) and theophylline (10 mM) increased the control cAMP level in platelets 4-fold. A threshold concentration of PGE1 had a small effect on hispidulin-induced cAMP levels but increased the theophylline-induced cAMP levels 3-fold. Theophylline (10 mM)-induced cAMP levels were not modified by hispidulin. We demonstrate a correlation between the inhibition of platelet aggregation and the increase in cAMP levels induced by hispidulin. These data suggest that hispidulin could inhibit platelet aggregation by elevating cAMP levels by a mechanism different from that of theophylline or PGE1.