Effect of dipyridamole on prostaglandin generation by human platelets and vessel walls

These experiments were conducted to determine the effects of dipyridamole on human platelet aggregation, platelet thromboxane A2 (TXA2) and human vessel wall prostacyclin (PGI2) generation. Dipyridamole in varying concentrations (5 to 50 micrograms/ml) had no direct effect on ADP-induced platelet aggregation in vitro, but it potentiated PGI2-induced platelet aggregation inhibition at these concentrations. Dipyridamole also inhibited arachidonic acid-induced platelet TXA2 generation at these concentrations. In continuously perfused umbilical vein segments, dipyridamole treatment resulted in stimulation of PGI2 release determined by bioassay and by measurement of its stable metabolite 6-keto-PGF1 alpha. Minimum concentration of dipyridamole causing PGI2 release was 50 micrograms/ml. These in vitro studies suggest that anti-thrombotic effects of dipyridamole in man are mediated mainly by potentiation of PGI2 activity and to some extent by TXA2 suppression. Stimulation of PGI2 release by human vessels may not be seen in usual therapeutic concentrations.     

Effect of aspirin on prostaglandin synthesis by human platelets

When platelet rich plasma is exposed to N-ethylmaleimide, a ten fold increase in measurable prostaglandin E synthesis occurs. This effect is almost completely abolished within 2 hours of ingestion of 600 mg of aspirin by human volunteers. Recovery of this platelet function is slow for the first two days, returning sharply to normal over the next six days and plateauing approximately 8 days following initial removal from aspirin. It is suggested from these studies that platelet prostaglandin E production following NEM may be a useful test of platelet function.     

Lipoxin generation by permeabilized human platelets.

Human platelets convert leukocyte-derived leukotriene (LT) A4 to lipoxins during transcellular lipoxin biosynthesis. Here, we examined lipoxin generation in intact human platelets and compared it with that elicited from permeabilized platelets. Conversion of LTA4 to lipoxins by permeabilized cells exceeded (10-15 times) that to peptidoleukotrienes, while intact cells exposed to thrombin generated similar amounts of these two series (LT/LX). Permeabilized platelets also generated 3-5 times more lipoxins than intact cells. Lipoxin A4 (LXA4), lipoxin B4 (LXB4), and their respective all-trans isomers were identified by physical methods including HPLC and GC-MS. Chiral analysis of platelet-derived all-trans-containing LXs revealed that greater than 69.5 +/- 0.5% carried alcohol groups in the R configuration at carbons 6 and 14 (e.g., 11-trans-LXA4 and 8-trans-LXB4), respectively. More than 50% of these all-trans LX were formed by isomerization of native LXA4 and LXB4 during isolation. Lipoxin formation with permeabilized platelets gave an apparent Km of 8.9 microM and Vmax of 83.3 ng/(min-10(9) platelets) with maximal conversion in pH range 7-9. In addition, permeabilized platelets converted 14,15-LTA4 and LTA5, but not LTA3, to lipoxins. Consecutive exposure to LTA4 did not alter LXA4 generation but inhibited LXB4 by 40-50%, suggesting that LXB4 formation can be regulated by suicide inactivation. Unlike platelets, human endothelial cells did not convert LTA4 to lipoxins. These results indicate that lipoxin formation is a major route of LTA4 metabolism in thrombin-activated platelets and those that have undergone a loss of membrane barriers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modulation of TXA2 generation of platelets by human lipoproteins

The lipoprotein (LP) fractions VLDL, LDL, HDL2 and HDL3 were prepared by ultracentrifugation of plasma from healthy volunteers and from patients with coronary heart disease (CHD). We investigated the capacity of platelets from healthy volunteers and patients with atherosclerosis to generate thromboxane A2 (TXA2) during spontaneous clotting of whole blood under the influence of the lipoprotein fractions. In our experiments the serum concentration of TXB2, reflecting the capacity of platelets to generate TXA2 during clotting, depends on several factors: the type of LP fraction used, the blood used for generation of TXA2, and for the same LP fraction whether it was taken from plasma of healthy volunteers or patients with CHD. VLDL prepared from plasma of healthy volunteers inhibited but VLDL prepared from plasma of patients with CHD enhanced the TXA2 formation of platelets from healthy volunteers (p less than 0.05, resp.). LDL from CHD patients inhibited the TXA2 formation of platelets from atherosclerotic patients (p less than 0.01). The HDL subfractions HDL2 and HDL3 from healthy volunteers inhibited TXA2 formation by platelets from healthy volunteers as well as those from atherosclerotic patients (p less than 0.05; p less than 0.01, respectively). HDL2 from patients with CHD inhibited only the TXA2 formation of platelets from healthy volunteers (p less than 0.01), whereas HDL3 from CHD patients inhibited only the TXA2 formation of platelets from atherosclerotic patients (p less than 0.01).     

Inhibition by ADP of prostaglandin induced accumulation of cyclic AMP in intact human platelets

The influence of extracellular ADP on cyclic AMP accumulation within intact human platelets was studied. ADP inhibited the increase in cyclic AMP which occurs when platelets are exposed to prostaglandin E1 or I2. The degree of inhibition varied in the range 70-95% , and was half maximal at ADP concentrations of between 0.3 and 2 microM. Other naturally occurring diphosphates, i.e. GDP, IDP and UDP, were at least 100 fold less effective than ADP, and UDP at 1mM partially reversed the effect of ADP. The effect by ADP was completely reversed by ATP, but only attenuated to a minor degree of 10 mM EDTA. Increasing concentrations of ADP caused a progressive degree of inhibition of cyclic AMP accumulation, and the kinetics of this inhibition were compatible with a simple saturable process with no cooperativity. ADP added 10 seconds after prostaglandin E1 blocked cyclic AMP accumulation within 1-2 seconds, and addition of ATP after ADP and prostaglandin I2 relieved the inhibition due to ADP within 2-3 seconds. The action of ADP was blocked by sulphydryl reagents including N-substituted maleimides, cytochalasin A, NBD chloride and p-mercuribenzene sulphonate. The data were considered to be consistent with mediation of the ADP effect through a sulphydryl-bearing specific extracellular receptor coupled to the adenylate cyclase.     

Effect of prolonged prostaglandin exposure on prostaglandin synthesis by human lung fibroblasts

Pretreatment of human lung fibroblasts with PGE₂ but not PGF_2α enhanced synthesis of prostaglandins (PGs). The effect of the pretreatment on PG synthesis was related to the concentration of PGE₂ that was added to the culture medium. Pretreatment with PGE₂ at 5 × 10⁻¹²M did not enhance PG synthesis whereas pretreatment with PGE₂ at 5 × 10⁻⁶M induced a maximal effect. Production of PGs was increased following 1 day of pretreatment with PGE₂ and was increased further following 3 days of pretreatment. The PGE₂ treated cells showed only a slight increase in the bradykinin-induced release of radioactivity from cells prelabeled with [³H]arachidonic acid but showed a dramatic increase in the bradykinin-induced synthesis of radio-labeled PGs. The conversion of free arachidonate to PGs in both intact cells and in a cell-free preparation was increased by PGE₂ pretreatment. The presence of cyclohexamide during the pretreatment did not inhibit the PGE₂-induced activation of PG synthesis. Taken together, the results indicate that pretreatment of cells with PGE₂ increased PG synthesis by augmenting the conversion of arachidonate to PGs.     

Stimulation of prostaglandin D2 receptors on human platelets by analogs of prostacyclin.

RS-93427, a novel analog of prostacyclin, increased adenylate cyclase activity in human platelet membranes (EC50 = 42 nM) to approximately the same maximum level as that produced by prostacyclin (EC50 = 87 nM). The concentration-response curve for RS-93427 appeared to be monophasic. However, a selective prostaglandin D2 antagonist (BW A868C) significantly reduced the stimulation of adenylate cyclase produced by low concentrations of RS-93427 (3.2 to 32 nM). RS-93520, a stereoisomer of RS-93427, also stimulated adenylate cyclase activity but in a biphasic pattern. BW A868C reduced the activation produced by low concentrations of RS-93520 with a 100-fold shift in the response curve. Maximum stimulation by RS-93520 (4.5-fold) was less than that obtained with prostaglandin D2 (7.3-fold). Thus, the stimulation of adenylate cyclase activity by low concentrations of RS-93520 is due to an interaction with prostaglandin D2 receptors while the activation by RS-93427 is mediated by both prostacyclin and prostaglandin D2 receptors. Additional data in support of these conclusions was obtained when these prostaglandins were tested as inhibitors of ADP-induced platelet aggregation in the presence or absence of BW A868C. The potent stimulation of prostaglandin receptors with chimeric molecules provides some insight into the structural features required for receptor activation.     

Synthesis of tritium-labelled prostaglandin E1 and its binding to human platelets

A method has been developed that makes it possible to obtain [5,6-3H2]PGE1 with a yield of 35% and a molar radioactivity of 1.7-1.8 TBq/mmol. The binding of [5,6-3H2]PGE1 to native platelets proved to be specific, saturating and reversible. It is characterized by low values (approximately 10(-9) M) of dissociation constants for high-affinity sites, correlates with the inhibition of ADP-induced aggregation of platelets and can be considered as receptor binding. Specific binding of 10 +/- 2 molecules of PGE1 with one platelet was found to cause 50% inhibition of the ADP-induced aggregation.     

Inhibition of prostaglandin synthesis in mouse 3T3 fibroblasts and human platelets by substituted phenols

Several substituted phenols with antioxidant properties were potent reversible inhibitors of prostaglandin synthesis in 3T3 cell cultures. The ID₅₀'s for prostaglandin (PG) E₂ synthesis in these cells were 0.1 μM for 2,6-xylenol, 5 μM for tricresol, 6 μM for -cresol, 7 μM for -cresol, 15 μM for 3,5-xylenol, 30 μM for -cresol and 100 μM for phenol. The corresponding values for aspirin and indomethacin were 4 μM and 0.02 μM, respectively.The substituted phenols also inhibited serotonin release, aggregation and prostaglandin synthesis in human platelets induced by arachidonic acid but not by PGG₂.     

Mechanisms of sphingosine and sphingosine 1-phosphate generation in human platelets

The bioactive molecule sphingosine 1-phosphate (S1P) is abundantly stored in platelets and can be released extracellularly. However, although they have high sphingosine (Sph) kinase activity, platelets lack the de novo sphingolipid biosynthesis necessary to provide the substrates. Here, we reveal a generation pathway for Sph, the precursor of S1P, in human platelets. Platelets incorporated extracellular 3H-labeled Sph much faster than human megakaryoblastic cells and rapidly converted it to S1P. Furthermore, Sph formed from plasma sphingomyelin (SM) by bacterial sphingomyelinase (SMase) and neutral ceramidase (CDase) was rapidly incorporated into platelets and converted to S1P, suggesting that platelets use extracellular Sph as a source of S1P. Platelets abundantly express SM, possibly supplied from plasma lipoproteins, at the cell surface. Treating platelets with bacterial SMase resulted in Sph generation at the cell surface, conceivably by the action of membrane-bound neutral CDase. Simultaneously, a time-dependent increase in S1P levels was observed. Finally, we demonstrated that secretory acid SMase also induces S1P increases in platelets. In conclusion, our results suggest that in platelets, Sph is supplied from at least two sources: generation in the plasma followed by incorporation, and generation at the outer leaflet of the plasma membrane, initiated by cell surface SM degradation.     

Biosynthesis of prostaglandin D2 1. Formation of prostaglandin D2 by human platelets

Formation of prostaglandin D₂ (PGD₂) during the aggregation of platelets was determined, employing a specific bioassay. PGD₂ was synthesized in human platelet rich plasma (PRP) in response to thrombin, collagen and epinephrine. Indomethacin pretreatment abolished the biosynthesis of PGD₂. When thrombin treated PRP was incubated for different periods of time and denatured in the presence of SnCl₂ to prevent the formation of PGD₂ from endoperoxides during the extraction procedure, PGD₂ formation was noted within the first minute of incubation and reached a peak level after 4 minutes. PGD₂ from thrombin stimulated PRP was conclusively identified by gas chromatography-mass spectrometry.The formation of PGD₂ during platelet aggregation could represent a mechanism of feedback inhibition of aggregation.     

Inhibition of prostaglandin synthesis in mouse 3T3 fibroblasts and human platelets by substituted phenols.

Several substituted phenols with antioxidant properties were potent reversible inhibitors of prostaglandin synthesis in 3T3 cell cultures. The ID50's for prostaglandin (PG) E2 synthesis in these cells were 0.1 muM for 2,6-xylenol, 5 muM for tricresol, 6 muM for p-cresol, 7 muM for o-cresol, 15 muM for 3,5-xylenol, 30 muM for m-cresol and 100 muM for phenol. The corresponding values for aspirin and indomethacin were 4 muM and 0.02 muM, respectively. The substituted phenols also inhibited serotinin release, aggregation and prostaglandin synthesis in human platelets induced by arachidonic acid but not by PGG2.     

Specific binding sites for prostaglandin D2 on human platelets.

³H-PGD₂ was biosynthesized from ³H-arachidonate and used to study the binding of PGD₂ to intact human platelets. The binding of ³H-PGD₂ to platelets was rapid, being essentially complete within two min. Bound ³H-PGD₂ PGD₂. Scatchard analysis of concentration-dependent binding indicated a single class of binding sites with a dissociation constant (K_D) of 4.12 × 10^?7M and a capacity of 760 sites per platelet. The relative ability of PGD₂, PGE₂, PGE₁ and PGI₂ to displace ³H-PGD₂ bound to these sites was 100:2:2<1. We conclude therefore, that these PGD₂ binding sites are specific for PGD₂ and independent of those previously demonstrated to recognize ³H-PGI₂ and ³H-PGE₁.     

Purification and properties of prostaglandin E1/prostacyclin receptor of human blood platelets

Activation of platelet adenylate cyclase by prostaglandin E1 or prostacyclin is initiated through the interaction of the agonists with the same receptors on membrane. Prostaglandin E1/prostacyclin receptors of human platelets were solubilized in buffer, containing 0.05% Triton X-100 and protease inhibitors. The soluble membrane protein was chromatographed on a DEAE-cellulose column and assayed by a microfiber filter by equilibrium binding technique. The active fractions eluted at 0.7 M KCl were pooled, and the receptors were purified to homogeneity by Sephadex G-200 gel filtration with an overall recovery of 30%. The isolated receptor was 2,200-fold purified over the starting platelets. As evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the receptor showed a molecular mass of 190,000 daltons and is composed of two nonidentical subunits with molecular masses of 85,000 and 95,000 daltons. The interaction of prostaglandin E1 with the purified receptor was rapid, saturable, reversible, and highly specific. Among all prostaglandins tested, only prostacyclin was capable of displacing [3H]prostaglandin E1 bound to the receptor. Scatchard analysis of [3H]prostaglandin E1 binding to the purified receptor suggested the presence of a single class of high affinity binding sites (Kd = 9.8 nM) and a second population of low affinity binding sites (Kd = 0.7 microM) in the same protein molecule. Incubation of the purified receptor with platelets stripped of the receptor by washing with low concentrations of Triton X-100 efficiently restored the ability of prostaglandin E1 and prostacyclin to activate adenylate cyclase in these cells.     

NADP-linked 15-hydroxyprostaglandin dehydrogenase for prostaglandin D2 in human blood platelets

Two forms of NADP-linked 15-hydroxyprostaglandin dehydrogenase for prostaglandin D₂ were found in the cytosol fraction of human blood platelets. These enzymes were purified by ammonium sulfate fractionation, Blue Sepharose, and Sephadex G-100 column chromatography. The two enzymes differed in molecular weights (65,000 for peak I enzyme and 31,000 for peak II as estimated by gel filtration) and their substrate specificities. The relative rates for reaction with peak I enzyme were: prostaglandin D₂, 100(%); E₂, 14; F_2α, 2; I₂, 29; and B₂, 0; whereas for peak II enzyme, D₂, 100; E₂, 23; F_2α, 61; I₂, 29; and B₂, 131. Prostaglandin D₂ was converted to 15-ketoprostaglandin D₂ and then 13,14-dihydro-15-ketoprostaglandin D₂, which were identified by spectrophotometry and gas chromatography/mass spectrometry, respectively. These metabolites were three orders of magnitude less potent in inhibiting human platelet aggregation than prostaglandin D₂. The results indicated that NADP-linked dehydrogenases participated in the metabolic inactivation of prostaglandin D₂ in the platelets. Furthermore, the dehydrogenase activity for prostaglandin D₂ was high in monkey (0.128 nmol/min · mg at 24 °C) and human platelets (0.066), but was not detectable (less than 0.007) in the rabbit, rat, and chicken. Because prostaglandin D₂, which was demonstrated by several authors to be synthesized in platelet-rich plasma during platelet aggregation, exhibited significant antiaggregatory activity only in human and monkey platelets, these prostaglandin dehydrogenases appear to play a physiological role in the circulatory system.     

The inactivation of prostaglandin E2 is decreased by dipyridamole and sulfinpyrazone in isolated rat lungs

The inactivation of prostaglandin E₂ (PGE₂) was decreased in the pulmonary circulation of isolated rat lungs, when either dipyridamole or sulfinpyrazone was infused into the pulmonary artery at the concentration of 20 μM. After pulmonary injection of 7.1 nmoles of ¹⁴C-PGE₂ the amount of 15-oxo-metabolites of PGE₂ in the effluent was 3.91 ± 0.19 nmoles from control lungs and 2.05 ± 0.19 nmoles (2P < 0.001) in that from 20 μM dipyridamole treated lungs. The corresponding values for control and 20 μM sulfinpyrazone lungs were 4.11 ± 0.25 and 3.03 ± 0.14 nmoles (2P < 0.01), respectively. The amounts of unmetabolized PGE₂ were correspondingly increased in the effluents from dipyridamole and sulfinpyrazone (20 μM) lungs. Neither dipyridamole nor sulfinpyrazone had at concentration of 2 μM any significant effect on the amount of 15-oxo-metabolites in the effluent, although the amount of unmetabolized PGE₂ was slightly increased in 2 μM sulfinpyrazone experiments.     

Some unusual considerations about vessel walls and wall stresses

The "zero-stress state" of blood vessels is usually defined with respect to the atmospheric pressure p(a) ( approximately 750 mmHg). As a consequence, circumferential and axial wall stresses due to a positive transmural pressure can only be positive and thus, by definition, only tensile. If the zero-stress state were defined with respect to vacuum pressure (0 mmHg), the compressive stress -p(a) generated by p(a) everywhere in the wall would, however, be included so that negative (=compressive) wall stresses would formally become possible. In order to examine the consequences this alternative definition would have for arteries, we have compared radial, circumferential, and axial stresses calculated "conventionally" to the values they take when the zero-stress state is defined "correctly" by reference to the vacuum pressure. It turns out that, under normal physiologic conditions, axial stress and perhaps also circumferential stress might well be compressive in many elastic and conductance arteries, contrary to the intuitive conviction of many people. Since the type of stresses a vessel wall is submitted to may be highly relevant for its structure and mechanical properties, this unconventional way of considering wall stresses may reveal unsuspected relationships between wall stresses on one side, and wall structure, vessel growth, adaptation and repair processes, atherosclerosis, angioplasty or stenting on the other side. Similar considerations might also prove useful with regard to cardiac hypertrophy.