Increase In Platelet Cyclic AMP Research Articles

Inhibition of the platelet P2Y12 receptor for adenosine diphosphate potentiates the antiplatelet effect of prostacyclin.

Activation of two receptors for adenosine diphosphate (ADP), P2Y(1) and P2Y(12), is necessary for ADP-induced platelet aggregation (PA). It is generally believed that the antithrombotic effects of drugs inhibiting P2Y(12), such as clopidogrel, are uniquely mediated by inhibition of P2Y(12)-dependent PA. However, as P2Y(12) is negatively coupled to adenylyl cyclase (AC), its inhibition may also exert antithrombotic effects through the potentiation of prostacyclin (PGI(2)), which inhibit PA by stimulating AC. To test whether inhibition of P2Y(12) potentiates the antiplatelet effects of PGI(2). We measured the effects of PGI(2) (0.01-10 microm) on PA of washed human platelets induced by thrombin (0.5 U mL(-1)) in the presence or absence of ARC69931MX (anti-P2Y(12)) or MRS2500 (anti-P2Y(1)). PGI(2) inhibited PA in the presence of anti-P2Y(12), but not in the presence of anti-P2Y(1) or in the absence of inhibitors. In contrast, dibutyryl-cyclicAMP inhibited PA both in the presence and absence of anti-P2Y(1) or anti-P2Y(12). PGI(2) increased platelet cyclicAMP levels only in the absence of thrombin or in the presence of thrombin plus anti-P2Y(12). PGI(2) did not inhibit PA induced by thrombin, because its effect on AC was prevented by released ADP interacting with P2Y(12). Anti-P2Y(12) drugs, by rescuing AC activity, potentiate the antiplatelet effect of PGI(2), which may contribute to their antithrombotic effect.

Effects of 5‘-Phosphate Derivatives of 2-Hexynyl Adenosine and 2-Phenylethynyl Adenosine on Responses of Human Platelets Mediated by P2Y Receptors

Newly synthesized mono-, di-, and triphosphate of 2-alkynyl adenosines showed very different behavior in human platelet P2Y receptor models, according to the different alkynyl chains. In fact, 2-hexynyladenosine di- (5) and triphosphate (7) induced platelet shape change and aggregation and inhibited PGE(1)-induced increase in platelet cyclic AMP. On the contrary, the corresponding 2-phenylethynyladenosine di- (6) and triphosphate (8) did not induce platelet shape change or aggregation, but inhibited platelet aggregation induced by ADP.

Roles for both cyclic GMP and cyclic AMP in the inhibition of collagen-induced platelet aggregation by nitroprusside.

In studies on human platelets, nitroprusside (NP) alone at 1-10 micromol/l increased platelet cyclic AMP (cAMP) by 40-70%, whereas increases in cyclic GMP (cGMP) were much larger in percentage though not in concentration terms. Collagen enhanced these increases in cAMP up to fourfold, without affecting cGMP. This effect was partly prevented by indomethacin or aspirin, indicating that platelet cyclo-oxygenase products acted synergistically with NP to increase cAMP. ADP released from the platelets by collagen tended to restrict this cAMP accumulation. Addition of 2',5'-dideoxyadenosine (DDA), an inhibitor of adenylyl cyclase, decreased both the inhibition of collagen-induced platelet aggregation by NP and the associated accumulation of cAMP without affecting cGMP, indicating that cAMP mediates part of the inhibitory effect of NP. Unlike DDA, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), an inhibitor of guanylyl cyclase, blocked all increases in both cGMP and cAMP caused by NP, as well as the inhibition of platelet aggregation, suggesting that cAMP accumulation was secondary to that of cGMP. Human platelet cGMP-dependent protein kinase (PKG) coelectrophoresed with the purified bovine type Ibeta isoenzyme. An inhibitor of this enzyme (Rp)-beta-phenyl-1,N2-etheno-8-bromoguanosine 3',5'-cyclic-monophosphorothioate, diminished the inhibition of collagen-induced platelet aggregation by NP, but had little additional effect when DDA was present. This showed that both PKG and cAMP participate in the inhibition of collagen-induced platelet aggregation by NP. Moreover, selective activators of PKG and cAMP-dependent protein kinases had supra-additive inhibitory effects, suggesting that an optimal inhibitory effect of NP requires simultaneous activation of both enzymes.

Involvement of platelet cyclic GMP but not cyclic AMP suppression in leukocyte-dependent platelet adhesion to endothelial cells induced by platelet-activating factor in vitro.

1. Incubation of endothelial cells with platelets in the absence or the presence of PAF (10 nM) markedly increased platelet cyclic AMP levels, which were significantly decreased by indomethacin (3 microM). Co-incubation of endothelial cells and platelets with polymorphonuclear leukocytes (PMNs) did not change the platelet cyclic AMP levels. 2. Incubation of endothelial cells with platelets in the absence of PAF increased platelet cyclic GMP levels, which were increased 3.5 fold by PAF. These cyclic GMP levels were significantly decreased by NG-nitro-L-arginine (100 microM), and completely by methylene blue (10 microM). When endothelial cells and platelets were co-incubated with PMNs, the cyclic GMP level in the cell mixture was 42.5 and 65.3% lower than that in endothelial cells and platelets without and with PAF stimulation, respectively. 3. PAF induced platelet adhesion to endothelial cells only when PMNs were present. Methylene blue dose-dependently potentiated the PMN-dependent platelet adhesion induced by PAF, although it had no effect in the absence of PMNs. 4. Sodium nitroprusside and 8-bromo cyclic GMP but not dibutyryl cyclic AMP significantly, although partially, inhibited the platelet adhesion. Inhibition of cyclic GMP-specific phosphodiesterase by zaprinast slightly inhibited the PMN-induced platelet adhesion and potentiated the inhibitory effect of 8-bromo cyclic GMP, while these drugs markedly inhibited the adhesion of platelet aggregates induced by PMN sonicates. 5. These results suggest that the impairment by activated PMNs of EDRF-induced platelet cyclic GMP formation is involved in part in the mechanism of PMN-dependent platelet adhesion to endothelial cells induced by PAF in vitro. The precise mechanism still remains to be clarified.

Combined Inhibitory Effects of Ethanol and Adenosine on the Responses of Rabbit Platelets to Thrombin

We have previously shown that acutely administered ethanol, resulting in blood alcohol concentrations of 40-90 mM, inhibits experimentally induced arterial thrombosis in rabbits. This inhibition by ethanol in vivo is more pronounced than that observed on stimulated platelets in vitro, when a similar concentration of ethanol is added before an aggregating agent. It may be, then, that ethanol has combined effects in vivo with other inhibitors of platelet function. Adenosine has been found to be an important mediator of some of the in vivo effects of ethanol, and we investigated whether ethanol has combined inhibitory effects with adenosine on thrombin-stimulated platelet responses in vitro. Aggregation and secretion of [14C]serotonin from washed, prelabeled rabbit platelets, pretreated with aspirin, were studied. Maximal aggregation induced by 0.15 units thrombin/ml was slightly inhibited by 87 mM ethanol; secretion of serotonin was reduced from 24% to 12%. However, when thrombin-induced aggregation was significantly reduced by 1 microM adenosine, ethanol, at 44 and 87 mM, further inhibited aggregation. Secretion of [14C]serotonin was reduced to < 3%, with the combination of adenosine and the higher concentration of ethanol. Ethanol did not increase platelet cyclic AMP (cAMP) above basal levels, nor did it affect the increase in cAMP caused by adenosine. The adenosine receptor antagonist, 8-phenyltheophylline, at 1 microM, blocked the inhibitory effects of adenosine on platelet responses and prevented the adenosine-induced increase in cAMP. Unexpectedly, however, 8-phenyltheophylline (1-2 microM) did not completely block the combined inhibitory effects of ethanol and adenosine; this incomplete reversal was not associated with increases in cAMP over basal levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of cyclooxygenase activity and increase in platelet cyclic AMP by girinimbine, isolated from Murraya euchrestifolia

Girinimbine is an antiplatelet agent isolated from Murraya euchrestifolia. In washed rabbit platelets, it inhibited arachidonic acid (AA)-, collagen-, U46619- and platelet-activating factor (PAF)- induced aggregation and ATP release in a concentration-dependent manner with ic 50 values of 9.1 ± 1.5, 16.7 ± 1.7, 60.0 ± 5.1 and 71.9 ± 5.6 μM, respectively. However, it did not apparently affect thrombin-induced aggregation and ATP release even when a concentration of 80 μM was used. In citrated human platelet-rich plasma (PRP), girinimbine selectively inhibited secondary aggregation and ATP release without appearing to affect the primary aggregation induced by epinephrine and ADP. The formation of both platelet thromboxane B 2 (TxB 2) and prostaglandin D 2 (PGD 2) caused by AA was inhibited by girinimbine concentration dependently, with a maximal effect at 20 μM. Girinimbine also inhibited cyclooxygenase activity as reflected by the attenuation of prostaglandin E 2 (PGE 2) formation after incubation of sheep vesicular gland microsomes with arachidonic acid. In myo-[ 3H]inositol-labeled and fura-2-loaded platelets, [ 3H]inositol monophosphate generation and the increase in intracellular Ca 2+ ([Ca 2+] i) stimulated by AA and collagen, but not that stimulated by U46619, PAF and thrombin, were inhibited by girinimbine (20 μM). Platelet cyclic AMP levels were elevated by high concentrations of girinimbine (20 and 80 μM). These data indicate that the antiplatelet effect of girinimbine is due to the inhibition of cyclooxygenase activity and elevation of the cyclic AMP level.

Synthesis of some flavone derivatives: potent inhibitors of human platelet aggregation

A series of 20 benzodioxan derivatives of flavone were synthesized and characterised each bearing an ester or amide side chain of variable length. The activity of the compounds on washed human platelet aggregation induced by ADP (5 μM), collagen (2.5 μg/ml) and thrombin (0.1 U/ml) was evaluated. The ethoxycarbonyl side chain derivative inhibited all 3 types of aggregation agents with an IC 50 of 1, 2, and 24 μM respectively, making it one of the most potent platelet aggregation inhibitors among the flavonoids. Its mode of action should involve an increase in platelet cyclic AMP.

Ticlopidine Selectively Inhibits Human Platelet Responses to Adenosine Diphosphate

Platelet aggregation and fibrinogen binding were studied in 15 individuals before and 7 days after the oral administration of ticlopidine (250 mg b.i.d.). Ticlopidine significantly inhibited platelet aggregation induced by adenosine diphosphate (ADP), the endoperoxide analogue U46619, collagen or low concentrations of thrombin, but did not inhibit platelet aggregation induced by epinephrine or high concentrations of thrombin. Ticlopidine inhibited 125I-fibrinogen binding induced by ADP, U46619 or thrombin (1 U/ml). The ADP scavengers apyrase or CP/CPK, added in vitro to platelet suspensions obtained before ticlopidine, caused the same pattern of aggregation and 125I-fibrinogen binding inhibition as did ticlopidine. Ticlopidine did not inhibit further platelet aggregation and 125I-fibrinogen binding induced in the presence of ADP scavengers. After ticlopidine administration, thrombin or U46619, but not ADP, increased the binding rate of the anti-GPII b/III a monoclonal antibody 7E3 to platelets. Ticlopidine inhibited clot retraction induced by reptilase plus ADP, but not that induced by thrombin or by reptilase plus epinephrine, and prevented the inhibitory effect of ADP, but not that of epinephrine, on the PGE1-induced increase in platelet cyclic AMP. The number of high- and low-affinity binding sites for 3H-ADP on formalin-fixed platelets and their Kd were not modified by ticlopidine. These findings indicate that ticlopidine selectively inhibits platelet responses to ADP.

A New Anti-Platelet Drug, E5510, Has Multiple Suppressive Sites during Receptor-Mediated Signal Transduction in Human Platelets

The mode of action of E5510, 4-cyano-5,5-bis(4-methoxyphenyl)-4-pentenoic acid, which has very potent anti-platelet activities, was investigated by examining its effects on the biochemical responses in the process of human platelet activation. In a whole-cell system, E551Ü inhibited the increased turnover of inositol phospholipids arising from phospholipase C activation, arachidonic acid release from phospholipids by phospholipase A2, mobilization of intracellular free Ca2+, protein kinase C activation, and thromboxane A2 production. In a cell-free system, E5510 inhibited cyclooxygenase activity and cyclic AMP-dependent phosphodiesterase activity in a dose-dependent manner. An elevation of cyclic AMP in platelets was also observed at a relatively high concentration of E5510. It was suggested that receptor-mediated turnover of inositol phospholipids, intracellular Ca2+ increase, arachidonic acid release from phospholipids and protein kinase C activation might be indirectly inhibited by the increased cyclic AMP level in platelets. Thromboxane A2 production in the whole-cell system was very strongly inhibited by E5510, and the IC50 for this effect was 100 times lower than that of direct inhibition of cyclooxygenase in the cell-free system. It was concluded that although the primary mode of action of E5510 is the inhibition of the cyclooxygenase pathway of positive signal transduction in platelets, E5510 has another mode of action by increasing platelet cyclic AMP, which can act as a negative messenger in platelet signal transduction, and these multiple sites of action synergistically antagonize platelet cellular activation.

A new anti-platelet drug, E5510, has multiple suppressive sites during receptor-mediated signal transduction in human platelets.

The mode of action of E5510, 4-cyano-5,5-bis(4-methoxyphenyl)-4-pentenoic acid, which has very potent anti-platelet activities, was investigated by examining its effects on the biochemical responses in the process of human platelet activation. In a whole-cell system, E5510 inhibited the increased turnover of inositol phospholipids arising from phospholipase C activation, arachidonic acid release from phospholipids by phospholipase A2, mobilization of intracellular free Ca2+, protein kinase C activation, and thromboxane A2 production. In a cell-free system, E5510 inhibited cyclooxygenase activity and cyclic AMP-dependent phosphodiesterase activity in a dose-dependent manner. An elevation of cyclic AMP in platelets was also observed at a relatively high concentration of E5510. It was suggested that receptor-mediated turnover of inositol phospholipids, intracellular Ca2+ increase, arachidonic acid release from phospholipids and protein kinase C activation might be indirectly inhibited by the increased cyclic AMP level in platelets. Thromboxane A2 production in the whole-cell system was very strongly inhibited by E5510, and the IC50 for this effect was 100 times lower than that of direct inhibition of cyclooxygenase in the cell-free system. It was concluded that although the primary mode of action of E5510 is the inhibition of the cyclooxygenase pathway of positive signal transduction in platelets, E5510 has another mode of action by increasing platelet cyclic AMP, which can act as a negative messenger in platelet signal transduction, and these multiple sites of action synergistically antagonize platelet cellular activation.

DN 9693: A Phosphodiesterase Inhibitor with a Platelet Membrane Effect

SummaryWe have examined the in vitro effects of DN 9693 (piperidinylimidazo-quinazolinone) on various aspects of platelet reactivity. Our results are consistent with its known function as a phosphodiesterase inhibitor in that it increased platelet cyclic AMP, particularly in conjunction with an adenylate cyclase stimulator, and exerted a profound inhibitory effect on platelet aggregation responses to a variety of agonists. DN 9693 also inhibited ristocetin-induced platelet agglutination (RIPA). We therefore examined its effect on ristocetin co-factor assays and on the binding of a monoclonal antibody (McAb) to platelet membrane glycoprotein lb (GPIb). The drug inhibited the binding of the monoclonal antibody in a dose-dependent manner. This suggests an effect of the drug on the platelet surface membrane with reduced expression of GPIb. Our results indicate that in addition to its anticipated inhibitory effect on platelet aggregation, DN 9693 may also inhibit platelet adhesion.

High concentrations of exogenous arachidonate inhibit calcium mobilization in platelets by stimulation of adenylate cyclase

1. Exposure of platelets to exogenous arachidonic acid results in aggregation and secretion, which are inhibited at high arachidonate concentrations. The mechanisms for this have not been elucidated fully. In our studies in platelet suspensions, peak aggregation and secretion occurred at 2-5 microM-sodium arachidonate, with complete inhibition around 25 microM. 2. In platelets loaded with quin2 or fura-2, the cytoplasmic Ca2+ concentration, [Ca2+]i, rose in the presence of 1 mM-CaCl2 from 60-80 nM to 300-500 nM at 2-5 microM-arachidonate, followed by inhibition to basal values at 25-50 microM. Thromboxane production was not inhibited at 25 microM-arachidonate. Cyclic AMP increased in the presence of theophylline, from 3.5 pmol/10(8) platelets in unexposed platelets to 8 pmol/10(8) platelets at 50 microM-arachidonate; all platelet responses were inhibited with doubling of cyclic AMP contents. 3. The adenylate cyclase inhibitor 2',5'-dideoxyadenosine attenuated the inhibitory effect of arachidonate, suggesting that it is mediated by increased platelet cyclic AMP and that it is unlikely to be due to irreversible damage to platelets. 4. Aspirin or the combined lipoxygenase/cyclo-oxygenase inhibitor BW 755C did not prevent the inhibition by arachidonate of either [Ca2+]i signals or aggregation induced by U46619. 5. Thus high arachidonate concentrations inhibit Ca2+ mobilization in platelets, and this is mediated by stimulation of adenylate cyclase. High arachidonate concentrations influence platelet responses by modulating intracellular concentrations of two key messenger molecules, cyclic AMP and Ca2+.

Prostaglandin E1 and forskolin antagonize C-kinase activation in the human platelet.

In human platelets, prostaglandin E1 and forskolin inhibit 5-hydroxytryptamine-induced phospholipase C, C-kinase and myosin light-chain kinase activity in a concentration-dependent way. Phospholipase C activation, however, was only partly inhibited, and this at higher concentrations than the protein kinases. Direct activation of the C kinase either by exogenous synthetic diacylglycerol or by 12-O-tetradecanoylphorbol 13-acetate was antagonized by prostaglandin E1 and forskolin. Since C-kinase activation is one of the key events in excitatory signal transduction in the platelets, we suggest that the inhibitory effect of agents that increase platelet cyclic AMP on platelet secretion and aggregation might reside in their capacity to antagonize C-kinase activity.

Platelet protein phosphorylation, elevation of cytosolic calcium, and inositol phospholipid breakdown in platelet activation induced by plasmin.

Studies have been performed on the biochemical mechanism of platelet activation induced by the fibrinolytic protease plasmin. In washed human platelets, greater than or equal to 1.0 caseinolytic units (CU/ml plasmin induced aggregation. Platelet [14C]serotonin release was stimulated by 1.0 CU/ml plasmin to an extent comparable to that induced by 1.0 U/ml thrombin. A dose- and time-dependent phosphorylation of the platelet 47,000- and 20,000-kD proteins was noted in 32PO4-labeled platelets incubated with plasmin; phosphorylation was not affected by extracellular Ca2+, but was completely inhibited by an increase in platelet cyclic AMP. Phosphorylation of these platelet proteins suggested that plasmin may act on platelets by stimulating a rise in cytosolic calcium concentration ([Cai2+]) and activating inositol phospholipid-dependent phospholipase C and protein kinase C. Using both quin2 fluorescence and aequorin luminescence as indicators, plasmin was found to elevate platelet [Cai2+] in the presence or absence of extracellular Ca2+. Phospholipase C activation was shown by the generation of [3H]diglyceride in [3H]arachidonic acid-labeled platelets and [32P]phosphatidic acid in 32PO4 labeled platelets exposed to plasmin. Plasmin did not induce formation of thromboxane A2 (TXA2). Only small amounts of this eicosanoid were detected late in the time course after plasmin stimulation. Our results indicate that plasmin causes platelet aggregation and secretion associated with phosphorylation of the 47,000- and 20,000-kD proteins, Ca2+ mobilization, and phospholipase C and protein kinase C activation.

Direct Evidence for the Inhibition of Platelet Aggregation and Release by Intracellular Cyclic AMP Produced with a New Photoactivatable Derivative

An increase in platelet cyclic AMP (cAMP) via stimulation of adenylate cyclase is thought to be the underlying mechanism by which potent prostaglandins i.e. PGD2, PGI2, inhibit platelet functions. We report here new and direct evidence for the inhibitory effects of cAMP on platelet aggregation and serotonin release. Washed platelets from rat were incubated with a new photoactivatable cAMP analogue (4,5-dimethoxy-2-nitrobenzyl ester); this compound is almost physiologically inert before irradiation and liberates free cAMP ("cAMP jumps") following light flashes. A single flash, delivered after 2 min incubation in 100-200 microM of the analogue, dramatically inhibited thrombin-induced aggregation, as compared with controls. Endogenous serotonin release, measured in the same samples by means of an electrochemically treated carbon electrode was undetectable after the cAMP jump. Pre-irradiated solutions added to platelets had no effect. The kinetics of the flash-induced effects were also studied. From these results we can conclude that: i) the photoactivatable cAMP derivative has to permeate through the platelet membrane; ii) the analogue remains photolabile; and, iii) intracellular cAMP, resulting from photolysis dramatically inhibits platelet aggregation and serotonin release. It is possible that cAMP exerts its effects by regulating cytoplasmic free calcium concentration and/or other actions affecting platelet activation.

Mechanism of action of the platelet function inhibitor from Vipera russelli siamensis snake venom

Y.-S. Li, K.-F. Liu and Q.-C. Wang. Mechanism of action of the platelet function inhibitor from Vipera russelli siamensis snake venom. Toxicon 24, 875 – 883, 1986. — Human platelet aggregation induced by ADP, adrenaline, collagen or thrombin was inhibited by the venom inhibitor. Heating reduced both its phospholipase A 2 enzymatic and anti-aggregatory activities, although not in parallel. The inhibitor caused significant dose-related inhibitory effects on the clot retraction of rabbit platelet-rich plasma caused by thrombin, while platelet malondialdehyde formation stimulated by thrombin was not affected. Furthermore, the venom inhibitor increased basal cyclic AMP levels in platelets, while cyclic GMP content was slightly lowered, but not in a dose-dependent manner. In addition, microscopic study revealed that the cytoskeleton was disordered after treatment of platelets with the venom inhibitor. The platelets lost their discoid form, while the ultrastructural changes of platelet aggregation induced by ADP were blocked. It is concluded that increasing platelet cyclic AMP and the disorder of the cytoskeleton may be the mechanism of action of the venom inhibitor on platelet function.

Two mechanisms for inhibition of ADP-induced platelet shape change by 5'-p-fluorosulfonylbenzoyladenosine. Conversion to adenosine, and covalent modification at an ADP binding site distinct from that which inhibits adenylate cyclase.

The interaction of ADP with platelets leads to shape change, exposure of fibrinogen binding sites, and aggregation, all of which have been shown to be inhibited by 5'-p-fluorosulfonylbenzoyladenosine (FSBA), an alkylating analogue of adenine nucleotides which binds covalently to a 100-kDa polypeptide in intact platelet membranes (Figures, W. R., Niewiarowski, S., Morinelli, T., Colman, R. F., and Colman, R. W. (1981) J. Biol. Chem. 256, 7789-7795). In plasma, FSBA can break down to adenosine which stimulates adenylate cyclase. To distinguish between direct effects of FSBA and the actions of adenosine, we have used washed platelet suspensions and adenosine deaminase. We studied the effects of FSBA on shape change and cyclic AMP metabolism, and on the binding of 2-methylthio-ADP, which mimics the effects of ADP on cyclic AMP metabolism at concentrations too low to activate platelets. Inhibition of ADP-induced shape change of platelets incubated with FSBA for 2 min in platelet-rich plasma was greatly reduced by adenosine deaminase. In the presence of a phosphodiesterase inhibitor, 100 microM FSBA increased platelet cyclic AMP to the same extent as did 10 microM adenosine. These effects were inhibited by theophylline, an adenosine receptor antagonist, and by adenosine deaminase. Incubation of washed platelets for 60 min with FSBA and adenosine deaminase caused a concentration-dependent inhibition of ADP-induced shape change. Inhibition closely paralleled the covalent incorporation of 3H from tritiated FSBA into platelet membranes. Under these conditions, FSBA did not block inhibition of cyclic AMP accumulation by ADP, nor did it block the binding of 2-methylthio-ADP. We conclude that part of the inhibition of shape change caused by brief exposure to FSBA is due to adenosine, but at longer times shape change is inhibited in association with covalent incorporation of sulfonylbenzoyladenosine. This effect of FSBA is independent of adenosine and occurs at a site distinct from that at which ADP inhibits adenylate cyclase.

357 TOLAZOLINE INHIBITION OF PLATELET AGGREGATION

Tolazoline (TZ), an imidazoline compound with vasodilatory and histaminic properties, is used in the therapy of neonatal persistent pulmonary hypertension (PPH). Inhibition of thromboxane A2 (TBX-A) synthesis by TZ has been reported. The in-vitro effects of TZ on platelet aggregation were studied. Platelet rich plasma obtained from 6 healthy adults and from cord blood samples of 6 healthy term infants was incubated with TZ and aggregated with ADP (10μM). At increasing concentrations of TZ, decreases in maximal aggregation occurred, followed by disaggregation (mean±SEM): The relationship between TZ concentration and both maximal and 10-minute aggregation was linear up to 103 μg/ml (r≥0.88). The inhibition of primary aggregation at the highest TZ concentrations is inconsistent with TBX-A inhibition alone. Preliminary data, using a radioimmunoassay, suggest an increase in platelet cyclic AMP when incubated with TZ, perhaps mediated by hist-amine receptors. In-vivo inhibition of platelet aggregation and TBX-A release by TZ may be benefical in some forms of PPH.

Effect of forskolin on platelet deaggregation and cyclic AMP generation.

The diterpene, forskolin, induced a partial deaggregation of ADP- or collagen-aggregated human platelets in vitro. An increase in platelet cyclic AMP by forskolin was assumed to mediate the platelet deaggregation. PGE1 also deaggregated these platelets, and a combination of forskolin and PGE1 produced deaggregation greater than the maximum which could be obtained with each agent alone. A greater than additive effect was observed on the platelet cyclic AMP level in the presence of both forskolin and PGE1. No additive effect was observed in the phosphorylation of molecular weight (Mr) 21K polypeptide using forskolin (0.1 mmol/l) and PGE1 (5 mumol/l) suggesting that although cyclic AMP is responsible for the deaggregation process a mechanism other than phosphorylation through cyclic AMP-dependent protein kinase may be responsible for the effect of forskolin on platelet deaggregation.

Phorbol myristate acetate stimulates formation of phosphatidyl inositol 4-phosphate and phosphatidyl inositol 4,5-bisphosphate in human platelets

The tumor-promoting phorbol ester PMA increased the incorporation of 32P-phosphate into PIP (150%) and PIP2 (50%) in human platelets over the same range of concentrations that stimulate protein kinase C activity (i.e. 1–10 ng/ml). PMA also increased the total content of PIP (2.5-fold) and PIP2 (1.5-fold). The increase in 32P-PIP and 32P-PIP2 was 50% completed at 2 min after 10 ng/ml PMA, and was maximal by 20 min. The increase in PIP and PIP2 was accompanied by a fall of 32P-PI and PI mass over the same time period and concentration range of PMA, but no 32P-PA was fourmed, indicating that phosphoinositide hydrolysis by phospholipase C was not stimulated. Inhibition of phospholipase C activity by increasing platelet cyclic AMP did not duplicate the effects of PMA. We conclude that PMA may directly affect inositol lipid kinases and/or phosphatases, or that PMA stimulation of protein kinase C provides feedback regulation of the enzymes that determine the levels of polyphosphoinositides involved in transmembrane stimulus-response coupling.