Plasma protein regulation of platelet function and metabolism

Summary This reviews summarizes our evidence suggesting that the plasma protein environment influences platelet aggregation potential and metabolic activity.Cationic proteins are capable of restoring the aggregation potential of washed human platelets. The aggregation restoring effect of gamma globulin is inhibited by more anionic proteins in subfractions of Cohn fraction IV and fractions V and VI. Artificial enhancement of the net negative charge of plasma proteins through acylation produces derivatives capable of inhibiting platelet aggregation in platelet rich plasma.The oxygen consumption of washed human platelets is lower than in platelet rich plasma while the lactate production is identical. Autologous plasma, albumin or IgG immunoglobulin restores the oxygen consumption of washed platelets to values comparable to those obtained for platelet rich plasma, while the lactate production is unaffected. Fibrinogen or IgA myeloma protein increases the lactate production, but not the oxygen consumption. Cyclic AMP levels are considerably lower in washed platelets than in platelet rich plasma. Gamma globulin and albumin causes a further decrease, which is progressive with time. Fibrinogen causes no change in platelet cyclic AMP content.It is suggested that these observations may in part be explained by the equilibrium between anionic and cationic proteins in the platelet microenvironment.This hypothesis appears applicable in certain clinical situations.     

Effects of a polyoxyethylene detergent (Brij 58) on platelet aggregation, release and clotting activity

Low concentrations of a polyoxyethylene detergent, Brij 58, inhibited the secondary phase of platelet aggregation induced by ADP in human citrated platelet-rich plasma but had no effect on primary aggregation. Thrombin-induced aggregation of washed human platelets suspended in Tyrode's buffer was inhibited after incubation of cells with 4.10(-6) M detergent. Efflux of [14C]serotonin, 45Ca2+ and labile aorta contracting substance (thromboxane A2) and development of prothrombin-converting activity (platelet factor 3) were abolished concomitantly. Aggregation of washed platelets either by sodium arachidonate or by collagen was also inhibited by the same concentration of Brij 58 which inhibited thrombin aggregation. This concentration did not itself produce any release of a cytoplasmic marker, lactate dehydrogenase, from platelets. Higher concentrations of Brij 58, exceeding 4.10(-5) M, lysed the cells liberating lactate dehydrogenase, serotonin and Ca2+. When albumin was included as a platelet stabilizer in the suspending medium the concentration of detergent required for the inhibitory effects was increased ten-fold. This could be attributed to competitive binding of the detergent to albumin, demonstrated with [14C]acetylated Brij 58. A variety of other polyoxyethylene detergents, at concentrations from 8.10(-4) to 5.10(-3) M, also inhibited platelet aggregation induced by thrombin. It is concluded that low concentrations of Brij 58 stabilize the platelets against the action of aggregating agents, while higher concentrations produce membrane destabilization and cell lysis.     

In vitro effect of trichosanic acid, a major component of Trichosanthes japonica on platelet aggregation and arachidonic acid metabolism in human platelets

The in vitro effect of trichosanic acid (TCA; C18:3, omega-5), a major component of Trichosanthes japonica, on platelet aggregation and arachidonic acid (AA) metabolism in human platelets was studied. TCA dose-dependently suppressed platelet aggregation of platelet rich plasma and washed platelets. TCA decreased collagen (50 micrograms/ml)-stimulated production of thromboxane B2 (TXB2) and 12-hydroxyhepta-decatrienoic acid (HHT) in a dose-dependent manner, while that of 12-hydroxyeicosatetraenoic acid (12-HETE) was rather enhanced. The conversion of exogenously added [14C]AA to [14C]TXB2 and [14C]HHT in washed platelets was dose-dependently reduced by the addition of TCA, while that to [14C]12-HETE was increased. Similar observations were obtained when linolenic acid (LNA; C18:3, omega-3) was used. These results suggest that TCA may decrease TXA2 formation in platelets, probably due to the inhibition of cyclooxygenase pathway, and thereby reduce platelet aggregation.     

Arachidonic acid causes lysis of human platelets in an artificial medium: Protection by plasma

Addition of 1 or 2 mM of arachidonic acid (AA) to a washed platelet suspension (WPS) resulted in an instantaneous rise of light transmission (LT) in the platelet aggregometer. The LT curves showed no evidence of shape change patterns and the recording pen showed no fluctuations. This rise of LT was not affected by the absence of CaCl₂, or the presence of PGI₂ or ASA. However, LT curves, showing shape change patterns and with pen fluctuations, were obtained when plasma, serum or albumin was included in the test system. These latter form of LT curves were also obtained in WPS samples exposed to lower concentrations of AA. Electron microscopic examination of these samples revealed varying degrees of lysis of every platelet in WPS exposed to 1 mM of AA. Numerous lyzed platelets were also found in samples either exposed to 0.5 mM of AA in the absence of added plasma, serum or albumin, or exposed to 1mM of AA in the presence of 5% of added plasma or 2.5 mg/ml albumin. The LT curves of these samples showed shape change patterns and pen fluctuations. These results indicate a pitfall of relying on platelet aggregometry alone in the study of certain AA-induced responses of washed platelets.     

Arachidonic acid causes lysis of human platelets in an artificial medium: protection by plasma.

Addition of 1 or 2 mM of arachidonic acid (AA) to a washed platelet suspension (WPS) resulted in an instantaneous rise of light transmission (LT) in the platelet aggreagameter. The LT curves showed no evidence ofshape change patterns and the recording pen showed no fluctuations. This rise of LT was not affected by the absence of CaCl2, or the presence of PGI2 or ASA. However, LT curves, showing shape change patterns and with pen fluctuations, were obtained when plasma, serum or albumin was included in the test system. These latter form of LT curves were also obtained in WPS samples exposed to lower concentrations of AA. Electron microscopic examination of these samples revealed varying degrees of lysis of every platelet in WPS exposed to 1 mM of AA. Numerous lyzed platelets were also found in samples either exposed to 0.5 mM of AA in the absence of added plasma, serum or albumin, or exposed to 1 mM of AA in the presence of 5% of added plasma or 2.5 mg/ml albumin. The LT curves of these samples showed shape change patterns and pen fluctuations. These results indicate a pitfall of relying on platelet aggregometry alone in the study of certain AA-induced responses of washed platelets.     

Binding of an [125I] labelled thromboxane A2/prostaglandin H2 receptor agonist to baboon platelets

To characterize the thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptor on baboon platelets the binding of [125I]BOP was studied. [125I]BOP bound to washed baboon platelets in a saturable manner. Scatchard analysis of binding isotherms revealed a Kd of 1.12 +/- 0.08 nM and a binding capacity of 54 +/- 5 fmoles/10(8) platelets (326 sites/platelet). Several TXA2/PGH2 agonists and antagonists displaced [125I]BOP from its baboon platelet binding site with a rank order of potency similar to human platelets: I-BOP greater than SQ29548 greater than U46619 = I-PTA-OH greater than PTA-OH. I-BOP aggregated washed baboon platelets with an EC50 of 10 +/- 4 nM. The results indicate that [125I]BOP binds to the TXA2/PGH2 receptor on baboon platelets and that this receptor is similar to its human counterpart.     

Interactions among white spruce tannins, Bacillus thuringiensis subsp. kurstaki, and spruce budworm (Lepidoptera: Tortricidae), on larval survival, growth, and development

The interactions among white spruce, Picea glauca (Moench) Voss, purified acetone tannin extracts (hydrolyzable and condensed tannin), Bacillus thuringiensis subsp. kurstaki Cry1A(c) delta-endotoxin strain HD-73 (Btk), and spruce budworm, Choristoneura fumiferana (Clemens) (Lepidoptera: Tortricidae) on larval survival, growth, and development were investigated over the whole larval feeding period by using artificial diet supplemented with three concentrations of Btk toxins per milliliter of diet (0, 0.021, and 1.72 microg/ml) and three concentrations of foliar tannin extract (0, 8, and 15% dry mass basis). At high Btk concentration, tannin antagonized Btk potency against spruce budworm by lowering Btk-related larval mortality from 83 to 43%. At moderate Btk concentration tannin did not affect Btk potency. Host tree tannins antagonized not only the lethal effects of Btk toxin but also sublethal Btk-related impacts in terms of larval development, pupal weight, relative consumption rate, and growth rate. When alone in the diet, tannin negatively affected larval survival, growth, and development. Maximum potency of tannins against spruce budworm larvae (60% mortality) was reached at dietary concentrations corresponding to what is found in the plant (8% dry mass). The addition of Btk toxin in food containing tannin reduced percentage of larval mortality by one-third, indicating that Btk toxin can antagonize tannin potency against the insect. Development of Btk transgenic spruce trees should consider the antagonistic effect the toxin may have on the resistance conferred by tannins that have evolved naturally in spruce trees.     

Characterization of the biosynthetic pathway of prostaglandin D2 in human platelet-rich plasma

The biosynthetic mechanism of prostaglandin D2 in human platelet-rich plasma has been investigated. Platelet-rich plasma was separated into washed platelets and platelet-poor plasma, and [1-14C]prostaglandin H2 was incubated with each fraction. The enzymatic conversion of the endoperoxide to prostaglandin D2 was found only in platelet-poor plasma and not in washed platelets or platelet lysate. This prostaglandin D synthetase activity was purified to homogeneity and identified as serum albumin by sodium dodecyl sulfate polyacrylamide gel electrophoresis, isoelectric focusing, and immunoelectrophoresis. The optimal pH and Km value for prostaglandin H2 were 9.0 and 6 microM, respectively. Glutathione was not required for the activity. Although prostaglandin H2 ws converted to prostaglandin D2 and E2 in the reaction, only the prostaglandin D2 formation was dependent on the protein amount and abolished by prior boiling. The action of this activity under physiological conditions was examined in a model system constituted of serum albumin and washed platelets. Prostaglandin D2 formation was observed in association with thrombin-evoked platelet aggregation in this system and was proportional to the number of platelets and the concentration of serum albumin, suggesting that thrombin-stimulated platelets released prostaglandin H2, and the latter compound was then converted to prostaglandin D2 by the action of serum albumin. Consistent with this interpretation, prostaglandin H2 added to platelet-rich plasma was converted in part to prostaglandin D2, and the aggregation caused by this endoperoxide was greatly enhanced by neutralizing the action of prostaglandin D2 with anti-prostaglandin D2 antiserum.     

Thrombocytin, a serine protease from Bothrops atrox venom. 2. Interaction with platelets and plasma-clotting factors.

Thrombocytin, a serine protease from Bothrops atrox venom, caused platelet aggregation and release of platelet constituents at a concentration of 10(-7) M and clot retraction at a concentration of 2 x 10(-9) M. Thrombocytin was slightly more active when tested on platelets in plasma than on washed platelets suspended in Tyrode--albumin solution. Thrombin was 5 times more active than thrombocytin when tested on platelets in plasma and 50 times more active when tested on washed platelets. The patterns or release induced by thrombocytin and thrombin were similar. Prostaglandin E1 (10(-5) M) produced complete inhibition of platelet release induced by thrombocytin and thrombin. Indomethacin (10(-4) M) was without any effect. Antithrombin III, in the presence of heparin, inhibited the action of thrombocytin on platelets and on a synthetic peptide substrate (Tos-Gly-Pro-Arg-pNA.HCl). formation of an antithrombin III--thrombocytin complex was demonstrated on NaDodSO4--polyacrylamide gel electrophoresis. Hirudin and alpha 1-antitrypsin did not inactivate thrombocytin. Thrombocytin had a low fibrinogen-clotting activity (less than 0.06% that of thrombin). Thrombocytin also caused progressive degradation of the alpha chain of human fibrinogen, and it cleaved prothrombin, releasing products similar to intermediate 1 and fragment 1 produced by thrombin. Thrombocytin activated factor XIII by limited proteolysis and increased the procoagulant activity of factor VIII in a manner analogous to that of thrombin.     

Effect of low doses of ethanol on platelet function in long-life abstainers and moderate-wine drinkers

In vitro, high concentrations of ethanol (EtOH) reduce platelet aggregation. Less is known about the effect of low EtOH doses on platelet function in a selected human population of long-life abstainers and low moderate-wine drinkers to avoid rebound effect of EtOH on platelet aggregation. Results of our experiments suggest that moderate-wine drinkers have higher levels of high density lipoprotein (HDL) than long-life abstainers while fibrinogen levels are unchanged. Furthermore, platelets obtained from these individuals do not differ in their response when stimulated by agonists such as AA and collagen. The effect of in vitro exposure of low doses of EtOH has been studied in PRP and in washed platelets. EtOH (0.1-10 mM) inhibits platelet aggregation induced by collagen at its ED50 while is ineffective when aggregation was triggered by U-46619 and by 1 microM adenosine diphosphate (ADP). 5-10 mM EtOH partially reduces the second wave of aggregation induced by 3 microM ADP. 0.1-10 mM EtOH dose-dependently lowers the aggregation induced by AA at its ED50 but it is less effective at ED75 of AA. The antiaggregating effect of EtOH on aggregation induced by AA is unchanged by inhibitor of nitric oxide synthase. In addition, 10 mM EtOH reduces thromboxane (Tx) formation. In washed platelets, 1-10 mM EtOH partially inhibits platelet aggregation induced by thrombin. In washed resting platelets, 10 mM EtOH does not change the resting [Ca++]i while significantly reduces the increase in [Ca++]i triggered by AA. The results of ex vivo experiments have demonstrated that wine increases the HDL. However, this observation may or may not influence the response of platelets to agonists. Results of our studies demonstrate that low doses of alcohol reduces platelet function.     

Modulation of platelet cyclic nucleotide content by PGE1 and the prostaglandin endoperoxide PGG2.

The effects of prostaglandin E1 and prostaglandin G2, the prostaglandin endoperoxide, on platelet cyclic nucleotide concentrations were measured in platelet rich plasma (PRP), and in washed intact platelets. PGE1 was found to be a potent stimulator of platelet cAMP levels in both PRP and washed cells, and to inhibit aggregation in both systems. PGE1 did not change platelet cGMP levels in either PRP or washed cells. PGG2 which is a potent inducer of platelet aggregation, did not affect either the basal cAMP or the basal cGMP concentration. However, PGG2 was found to antagonize the increases in cAMP content in response to PGE1 in both PRP and washed platelets. The addition to our system of a cyclic nucleotide phosphodiesterase inhbitor, theophylline, did not change our findings. It is suggested that PGG2 may induce platelet aggregation by inhibiting PGE1-stimulated cAMP accumulation.     

Studies of the mechanism of the human platelet release reaction induced by immunologic stimuli. III. Relationship between the binding of soluble IgG aggregates to the Fc receptor and cell response in the presence and absence of plasma

Aggregated IgG coupled covalently with bis-diazobenzidine (BDB-IgG) and labeled with 3H-diazobenzene (3H-BDB-IgG) has been used to study the binding of soluble IgG aggregates to human platelets in relationship to the release of the contents of intracellular granules (e.g., serotonin). In washed cell suspensions a minimum of 0.14 to 0.2 mug 3H-BDB-IgG per 5 X 10(8) platelets (40 to 70 aggregates per cell) was required for the triggering of the release reaction and cell aggregation. Binding was independent of divalent cations. The Arrhenius plot gave a straight line between 0 to 37 degrees C and a Q10 of 1.6. Neither inhibitors of the release reaction nor energy metabolism, nor formaldehyde fixation of the platelets affected binding. Bound 3H-BDB-IgG was not significantly eluted by IgG, bovine albumin (BSA), buffer, or plasma. Binding to washed platelets was more strongly inhibited by human IgG than by F(ab')2, bovine IgG, human albumin (HSA), or BSA. Plasma was an even more effective inhibitor of both binding and release. Plasma deficient in IgG or depleted of complement retained its inhibitory capacity. In the presence of plasma, at physiologic ratios of plasma and platelets, no release of serotonin was observed. Binding, although inhibited in rate, nevertheless occurred. It was enhanced by divalent cation chelation and had a Q10 of 2.5. The release reaction of washed platelets to which 3H-BDB-IgG had been bound in the presence of HSA or BSA was also inhibited by the subsequent addition of plasma or plasma proteins (human IgG being more effective than bovine IgG, F(ab')2, HSA, or BSA). 3H-BDB-IgG bound in the presence of either plasma or human IgG did not induce release when the platelets were subsequently suspended in media lacking these proteins. Thus, it appears that the platelet Fc receptor binds 3H-BDB-IgG by a process which is effectively inhibited by plasma, or by free IgG with an intact Fc, and to some extent by high concentrations of other proteins. The effects of bound IgG aggregates are dependent on the other proteins present both during binding and subsequently added. The mechanism of such receptor modulation and its implications in vivo are discussed.     

Ecological tannin assays

Summary Estimations of condensed tannin content are generally based on calibration standard curves from Quebracho condensed tannins. We generated calibration standard curves from eight Sonoran Desert species for comparison with estimates of tannin concentrations derived from the Quebracho standard curve. Estimates of leaf tannin concentrations of each of the eight species using each species standard curve differed significantly with the estimates given by the Quebracho standard curve. Standard curves constructed from tannins from different individuals of three of the species varied significantly between, but not within, species. The efficiency of precipitation of protein bovine serum albumin (BSA) by each different tannin varied up to a factor of fifty for tannins of different species. Ordering species from highest to lowest based on tannin concentrations or binding efficiencies gave two different ranks. We argue that concentration or efficiency alone do not describe adequately tannin ecological activity. Instead, we suggest combining tannin concentrations and binding efficiencies to measure the protein precipitating potential of a leaf. Leaf protein precipitating potential is a more ecologically realistic parameter, we feel, for between-species comparisons than tannin content or binding efficiencies alone.Operated for the U.S. Department of Energy by the University of California under Contract No. DE-AC03-76-SF00012. This article was supported by the Director of the Office of Energy Research, Office of Health and Environmental Research     

Comparison of the inhibitory effect of E-prostaglandins in human and rabbit platelet-rich plasma and washed platelets

1. The anti-aggregatory potency of a number of E-type PGs was compared in human and rabbit platelet-rich plasma (PRP) and washed platelets. The potency of 13,14-dihydro-PGE₁ and 5,6-dihydro-PGE₃ is significantly higher in human than in rabbit washed platelets, while the potency of 15-keto-13,14-dihydro-PGE₁ is higher in rabbit.2. The potency of PGEs in rabbit PRP is very similar to that of washed platelets, with the exception of 1a,1b-dihomo-PGE₂, which is of a significantly lower potency in PRR.3. In human, 5,6-trans-PGE₂, PGE₃, and 15-keto-13,14-dihydro-PGE₁ are more potent in PRP than in washed platelets.4. The results indicate that the potency of E-type PGs in human and rabbit platelets is different and plasma can essentially influence the anti-aggregatory effect of PGEs; plasma can either decrease or increase potency.     

Effects of polyoxyethylene detergent (Brij 58) on platelet aggregation,release and clotting activity

Low concentrations of a polyoxyethylene detergent, Brij 58, inhibited the secondary phase of platelet aggregation induced by ADP in human citrated platelet-rich but had no effect on primary aggregation. Thrombin-induced aggregation of washed human platelets suspended in Tyrode's buffer was inhibited after incubation of cells with 4 · 10^?6 M detergent. Efflux of [¹⁴C]serotonin, ⁴⁵Ca²⁺ and labile aorta contracting substance (thromboxane A₂) and development of prothrombin-converting activity (platelet factor 3) were abolished concomitantly. Aggregation of washed platelets either by sodium arachidonate or by collagen was also inhibited by the same concentration of Brij 58 which inhibited thrombin aggregation. This concentration did not itself produce any release of a cytoplasmic marker, lactate dehydrogenase, from platelets. Higher concentrations of Brij 58, exceeding 4 · 10^?5 M, lysed the cells liberating lactate dehydrogenase, serotonin and Ca²⁺. When albumin was included as a platelet stabilizer in the suspending medium the concentration of detergent required for the inhibitory effects was increased ten-fold. This could be attributed to competitive binding of the detergent to albumin, demonstrated with [¹⁴C]acetylated Brij 58. A variety of other polyoxyethylene detergents, at concentrations from 8 · 10^?4 to 5 · 10^?3 M, also inhibited platelet aggregation induced by thrombin. It is concluded that low concentrations of Brij 58 stabilize the platelets against the action of aggregation agents, while higher concentrations produce membrane destabilization and cell lysis.     

Neomycin inhibits inositol phosphate formation in human platelets stimulated by thrombin but not other agonists

Neomycin (0.1-1 mM) added to human platelet-rich plasma or washed platelets prelabeled with [3H]inositol inhibits aggregation, ATP secretion (ID50 0.2 mM) and formation of [3H]inositol mono-, bis- and trisphosphate (ID50 0.6-0.8 mM) in response to thrombin (0.25 U/ml). The production of inositol phosphates in response to other platelet agonists (vasopressin, platelet activating factor, prostaglandin endoperoxide analogs and collagen) is not inhibited by neomycin, even at a concentration of 2 mM. At this concentration neomycin reduces the secretion of ATP stimulated by these agents (by up to 50%). The results indicate that neomycin has multiple effects on platelets that are unrelated to a specific inhibition of inositol phospholipid degradation by phospholipase C. Low concentrations (0.1-1 mM) of neomycin might selectively inhibit the interaction of thrombin with the platelet surface, and high concentrations (greater than 2 mM) might unspecifically reduce platelet secretion in response to various platelet agonists.     

Encrustation and Atherosclerosis: The Analogy Between Early in Vivo Lesions and Deposits Which Occur in Extracorporeal Circulations

A study was made of the relation between the pattern and topography of thrombus formation in models of various vessel configurations coupled into extracorporeal shunts in swine and the development of atherosclerosis at corresponding sites on swine aortas. The pattern and distribution of deposits formed in the models were strikingly similar to the pattern and distribution of incipient atherosclerosis at comparable sites in the vascular tree. The earliest and only consistent component of the flow chamber deposits was the blood platelet. The platelet deposits would frequently stain with oil red O. The cholesterol level of washed human platelets was found to show a good correlation with that in the plasma. This evidence suggests that deposition of particulate matter (chiefly platelets), largely determined by the hydraulic factors, may be an important factor in the early, as well as later, stages of atherosclerosis.     

Clinically relevant HOCl concentrations reduce clot retraction rate via the inhibition of energy production in platelet mitochondria

Abstract

Using porcine blood, we examined the impact of hypochlorite, product of activated inflammatory cells, on clot retraction (CR), an important step of hemostasis. We found that, in vitro, HOCl is able to reduce CR rate and enlarge final clot size in whole blood (t.c. 100 μM), platelet-rich plasma (PRP) threshold concentration (t.c. 50 μM), and an artificial system (washed platelets and fibrinogen) (t.c. 25 nM). Combination of low HOCl and peroxynitrite concentrations resulted in synergistic inhibition of CR by these stressors. Concentrations of HOCl completely inhibiting CR failed to affect the kinetics of coagulation measured in PRP and in platelet-free plasma. Concentrations of HOCl reducing CR rate in PRP augmented production of lactate, inhibited consumption of oxygen by platelets, and decreased total adenosine triphosphate (ATP) content in PRP-derived clots. In an artificial system, concentrations of HOCl resulting in inhibition of CR (25–100 nM) reduced mitochondrial transmembrane potential and did not affect actin polymerization in thrombin-stimulated platelets. These concentrations of HOCl failed to affect the adhesion of washed platelets to fibrinogen and to evoke sustained calcium signal, thus excluding stressor action on glycoprotein IIb/IIIa receptors. Exogenously added Mg-ATP almost completely recovered HOCl-mediated retardation of CR. Concentrations of HOCl higher than those affecting CR reduced thromboelastometric variables (maximum clot firmness and α angle). We conclude that low clinically relevant HOCl concentrations may evoke the inhibition of CR via the reduction of platelet contractility resulted from malfunction of platelet mitochondria. At the inflammatory conditions, CR may be the predominant HOCl target.     

THE TUMOR-PROMOTER PHORBOL ESTER (12-0-TETRADECANOYL-PHORBOL-13-ACETATE), A POTENT AGGREGATING AGENT FOR BLOOD PLATELETS

The phorbol ester 12-0-tetradecanoyl-phorbol-13-acetate, a potent tumor-promoting agent, caused irreversible platelet aggregation when more than 0.02 µM was stirred with human citrated or heparinized platelet-rich plasma (PRP). With washed platelets, 1 nM was effective. The alcohol phorbol, which has little tumor-promoting activity, failed to cause platelet aggregation. With all but low concentrations of phorbol ester, aggregation was succeeded by a rapid phase. The latter was prevented or reduced by enzymes which destroy ADP and by aspirin, was associated with a change in platelet shape, and was presumably due to released ADP. At higher concentrations, only a rapid phase was seen, and these inhibitors were not effective. Low concentrations did not aggregate platelets in PRP containing sufficient EDTA or EGTA to chelate ionized calcium or in PRP from thrombasthenic patients; higher concentrations caused slight aggregation. Both the primary, non-ADP-dependent aggregation and the rapid ADP-dependent aggregation were markedly inhibited by substances which increase cyclic AMP, metabolic inhibitors, and the sulfhydryl inhibitor N-ethylmaleimide. Phorbol ester reduced platelet cyclic AMP only when it had been previously elevated by prostaglandin E₁. 1 µM did not release β-glucuronidase, lactic dehydrogenase, or inflammatory material from platelets in 4–5 min despite marked aggregation, but liberated all three in 30 min. The possibility is discussed that low phorbol ester concentrations cause primary aggregation by a direct action on platelet actomyosin.     

Desensitization of prostaglandin-activated platelet adenylate cyclase.

Prostaglandin D2 (PGD2) is one of several prostaglandins that can inhibit platelet aggregation and activate adenylate cyclase. Platelets were exposed to varying concentrations of PGD2 washed, and the adenylate cyclase response to prostaglandins, epinephrine, and sodium fluoride determined. Incubating platelets with 5 x 10(-5) M PGD2 for 2 hr resulted in a 45% decrease in PGD2 activation of adenylate cyclase and a 25% decrease in stimulation by PGE1. Fluoride activation (7-fold) epinephrine inhibition (30%) and basal enzyme activity were unchanged by exposure of the platelets to PGD2. Desensitization was concentration dependent, with loss of enzyme activity first noted when platelets were incubated with 10(-7) M PGD2. Enzyme sensitivity could be partially restored when desensitized platelets were washed free of PGD2 and incubated in buffer for 2 hr; complete resensitization required incubation for 24 hr in plasma. Regulation of prostaglandin sensitive platelet adenylate cyclase could be of importance in mediating the response of platelets to aggregating agents.