Edwin W. Salzman

Cyclic 3′,5′-adenosine monophosphate in human blood platelets: II. Effect of N6-2′-0-dibutyryl cyclic 3′,5′-adenosine monophosphate on platelet function

The relation of cyclic 3,5-adenosine monophosphate to platelet function has been studied by investigating the influence of this compound and of its N(6)-2-0-dibutyryl derivative on platelet aggregation and other aspects of platelet behavior after demonstration of adenyl cyclase activity in disrupted platelets. Dibutyryl cyclic AMP inhibited platelet aggregation induced by ADP, epinephrine, collagen, and thrombin. Cyclic AMP was also inhibitory but was less effective. The platelet release reaction was also inhibited; specifically, there was inhibition of the induction of platelet factor 3 activity and of the release of labeled 5-hydroxytryptamine. Platelet swelling produced by ADP was not inhibited. The action of dibutyryl cyclic AMP did not result from contamination with 5-AMP, nor was it attributable to production of 5-AMP by plasma enzymes. Dibutyryl cyclic AMP was degraded to 2-O-monobutyryl cyclic AMP and to cyclic AMP in plasma, but plasma exhibited no cyclic nucleotide phosphodiesterase activity, and the production of 5-AMP did not occur. The in vitro effects of dibutyryl cyclic AMP were associated with uptake of the compound by platelets. Adenyl cyclase activity of platelet homogenates was demonstrated with production of 9.27 x 10(-11) (+/-2.62 x 10(-11)) mole cyclic AMP per min per 10(10) platelets. The activity was increased by NaF and by prostaglandin PGE(1) and was decreased by epinephrine. The effect of epinephrine was blocked by phentolamine but not by propanolol. Adenyl cyclase activity was also inhibited by collagen, 5-hydroxytryptamine, and thrombin. ADP, dibutyryl cyclic AMP, and cyclic AMP did not alter adenyl cyclase activity. These observations are consistent with the hypothesis that platelet aggregation is favored by a decrease in platelet cyclic AMP and inhibited by an increase in cyclic AMP.

Thrombolytic Therapy in Thrombosis: A National Institutes of Health Consensus Development Conference

Excerpt For over three decades, the primary method of therapy used by almost all physicians for the management of acute deep-vein thrombosis and pulmonary embolism has been anticoagulation. This fo...

CYCLIC 3′,5′‐ADENOSINE MONOPHOSPHATE IN HUMAN BLOOD PLATELETS IV. REGULATORY ROLE OF CYCLIC AMP IN PLATELET FUNCTION*

n Evidence which bears on the hypothesis that agents that increase platelet cyclic adenosine 3,5 monophosphate (cAMP) tend to inhibit platelet aggregation, whereas agents whose action is to induce or augment platelet aggregation are associated with a decrease in platelet content of this cyclic nucleotide is reviewed. Using radiolabeled cAMP in incubation with various agents, it was found that caffeine, colagen, thrombin, prostaglandins E1 and E2, and phosphodiesterase inhibitors are all agents that increase platelet cAMP and inhibit platelet aggregation.n

SURFACE‐INDUCED PLATELET ADHESION, AGGREGATION, AND RELEASE*†

The secretion of intracellular constituents (“release reaction”) and aggregation are common responses to stimulation of platelets by a variety of agonists and together constitute the major aspects of what Holmsen has termed “the basic platelet reaction.” These processes comprise the ultimate expression of a complicated series of biochemical reactions that have been extensively studied in vitro and about which much is known. There is evidence that an early event is activation of an endogenous phospholipase leading to hydrolysis of phospholipids in the platelet membrane, thought principally to involve phosphatidyl choline, with liberation of arachidonic acid.”-‘ Enzymatic conversion of this essential fatty acid to labile endoperoxide precursors of the stable prostaglandins, PGE, and PGF,,, can be blocked by aspirin and other nonsteroidal anti-inflammatory agents, which are powerful inhibitors of the release reaction and platelet clumping.s--7 Samuelsson and associates have reported that prostaglandins are only a minor fraction of endoperoxide breakdown, quantitatively speaking, several orders of magnitude more of the endoperoxide molecules being converted to an unstable biologically active substance termed thromboxane This compound is postulated to be the principal effector in induction of the platelet release reaction, aggregation then resulting from the products of release. Prostaglandin E,, which does not itself initiate platelet activity, apparently has an important amplifying effect on processes induced by other stirnuli.l0~ The release reaction of platelets in response to most stimuli is accompanied by a fall in the platelet content of cyclic AMP (CAMP) . l z % Like prostaglandin synthesis, this effect is blocked by aspirin. There is much evidence to suggest that CAMP is important in the regulation of platelet activity, although the details of its role are disputed. For example, marked inhibition of platelet function results from an increase in platelet CAMP, which may be produced in plateletrich plasma by incubation with dibutyryl cAMP,I-’ by inhibition of platelet

Effect of common agonists on cytoplasmic ionized calcium concentration in platelets. Measurement with 2-methyl-6-methoxy 8-nitroquinoline (quin2) and aequorin.

Because of controversy regarding the relationship of cytoplasmic ionized calcium concentration ([Cai2+]) to platelet activation, we studied the correlation of platelet aggregation and ATP secretion with [Cai2+] as determined by 2-methyl-6-methoxy 8-nitroquinoline (quin2) and aequorin in response to ADP, epinephrine, collagen, the Ca2+ ionophore A23187, and thrombin. Both indicators showed a concentration-dependent increase in [Cai2+] in response to all agonists except epinephrine when gel-filtered platelets were suspended in media containing 1 mM Ca2+. With epinephrine, a rise in [Cai2+] was indicated by aequorin, but not by quin2; [Cai2+] signals, aggregation, and secretion were suppressed by EGTA. ADP [0.5 microM] produced a rise in [Cai2+] that was registered by both aequorin and quin2 in platelets in Ca2+-containing media; addition of EGTA to the medium raised the threshold concentration of ADP to 5.0 microM for both indicators. Collagen produced progressive concentration-related increases in [Cai2+] and aggregation in aspirin-treated aequorin-loaded platelets. Quin2 failed to indicate a rise in [Cai2+]at lower collagen concentrations with EGTA or aspirin. [Cai2+] response to A23187 and thrombin was reduced by addition of EGTA to platelets loaded with either aequorin or quin2. With all five agonists in all conditions tested, aequorin [Cai2+] signals occurred at the same agonist concentration as that or lower than that which produced platelet shape change, aggregation, or secretion. Platelet activation was better correlated with changes in [Cai2+] indicated by aequorin than with the response of quin2, possibly because aequorin is more sensitive to local zones of [Cai2+] elevation.

Synergism of platelet-aggregating agents. Role of elevation of cytoplasmic calcium.

When a pair of platelet agonists, each in subthreshold concentration, is added together or in sequence to a platelet suspension, the platelet response is enhanced. Addition of two agonists to platelets loaded with aequorin also enhanced the observed rise in cytoplasmic ionized calcium ([Ca2+]i) in response to the second agonist if the agonists were added within 20 s of each other. Enhancement of aggregation and secretion required that an increase in [Ca2+]i (as indicated by aequorin but not necessarily indo-1) followed the first agonist, but not that the [Ca2+]i remain elevated until addition of the second agonist. Enhancement was not prevented by aspirin, ADP scavengers, or chelators of extracellular Ca2+. We conclude that a rise in [Ca2+]i induced by a first agonist primes platelets for an augmented functional response to a second agonist, which is not, however, determined by the [Ca2+]i at the time of addition of the second agonist.

Collagen banded fibril structure and the collagen-platelet reaction

Bovine hide collagen dispersions were swollen in the pH range 1.6-7.0, treated with glutaraldehyde, and dialyzed to neutral pH. The intensity with which these collagens reacted with human platelets in plasma was studied by aggregometry and scanning electron microscopy. Collagen swollen at a pH below 4.25 +/- 0.30 and treated with glutaraldehyde exhibited greatly reduced platelet aggregating ability after restoration of neutral pH. In addition, the state of supramolecular order in these collagens was investigated by transmission electron microscopy and infrared spectroscopy. Native, insoluble collagen fibrils were found to lose their banded structure, as observed by transmission electron microscopy, reversibly when exposed to low ionic strength aqueous solutions below pH 4.25 +/- 0.30. During the disorder transition, which occurred by time dependent swelling of fibrils, but without their disaggregation, the packing order in the fibrils was largely abolished while the triple helical structure of individual collagen molecules was retained. Chemical modification of collagen by glutaraldehyde treatment was found to prevent recrystallization of collagen during dialysis to neutral pH but did not otherwise affect the collagen-platelet reaction. The results of altering collagen mass dose (concentration) demonstrated the critical importance of traces of banded fibrils which resisted disordering below pH 4.25. The data suggest that collagen preparations which are free of significant traces of banded fibrils, but which are made up of collagen molecules possessing triple helical structure do not induce platelet aggregation, irrespective of dose.

Concurrent measurement of platelet ionized calcium concentration and aggregation: Studies with the Lumiaggregometer

Platelet aggregation and aequorin-indicated cytoplasmic ionized calcium concentration ([Cai2+]) were measured simultaneously using a modification of a commercially available instrument, the Chronolog Whole Blood Lumiaggregometer. The [Cai2+] rise in response to arachidonic acid, the Ca2+-ionophore A23187, collagen, and epinephrine occurred either before or simultaneously with platelet shape change or aggregation. PGD2 caused concentration-dependent inhibition of both the [Cai2+] rise and aggregation. However, suppression of the [Cai2+] rise alone did not fully account for the inhibition of aggregation due to the elevated cAMP produced by PGD2. Agonist-induced elevation of [Cai2+] did not depend on platelet aggregation, but was enhanced by close platelet-to-platelet contact. Concurrent measurement of platelet aggregation and [Cai2+] may further clarify pathways to platelet activation.

In vitro assessment of interaction of blood with model surfaces: Acrylates and methacrylates

An in vitro system for the assessment of blood compatibility of artificial surfaces is described. Anticoagulated human whole blood was pumped through columns of beads and then analyzed for evidence of platelet retention and the release of platelet constitutents. Platelets were retained on the columns by adhesion to the beads and by aggregation. Experiments were conducted with PVAc, PC, PMA and PS beads to provide data on the reproducibility of the system and the effects of anticoagulants and RBC concentration on platelet adhesion and release. The system is versatile, inexpensive, reproducibile, and also suitable for studies of the effects of pharmacologic agents and other aspects of platelet-artificial surface interaction.

Effect of platelet activity of inhibition of adenylate cyclase

Abstract SQ22536 (9-[tetrahydro-2-furyl]-adenine), an inhibitor of adenylate cyclase, blocks the inhibition of platelet function by substances that stimulate this enzyme, including PGE 1 , PGI 2 , PGD 2 , and adenosine. SQ22536 enhances platelet aggregation and serotonin release induced by prostaglandin endoperoxides and their analogs but does not consistently affect the platelet response to ADP or other agonists. SQ22536 reduced platelet cyclic AMP and inhibits the effect on this nucleotide of PGE 1 and adenosine. The elevation of cyclic GMP induced by ADP is prevented by SQ22536.