Addition Of Adenosine Diphosphate Research Articles

Centruroides sculpturatus venom and platelet reactivity: possible role in scorpion venominduced defibrination syndrome

The effect of Centruroides sculpturatus venom and some purified fractions on the aggregation behavior of dog platelets has been examined. The venom causes a sustained partial aggregation, which proceeds almost instantly to complete aggregation on addition of adenosine diphosphate (ADP): the maximum aggregation to ADP is unaffected. The effects of the venom are not mimicked by any of the currently isolated neurotoxins, but are by a fraction obtained from the primary gradient separation of the venom. The effect of the venom is heat labile and unaffected by prior treatment of the platelets with tetrodotoxin, tetraethylammonium or aspirin. Heat lability rules out effects from most inorganic salts, serotonin or phospholipase A 2, which are heat stable. A defibrination syndrome has been noted following envenomation by a member of the family Buthidae, and possibly occurs for C. sculpturatus venom as well: the effects of the venom on platelets may account in part for the occurrence of this syndrome.

Reversibility of the Association of Fibrinogen With Rabbit Platelets Exposed to ADP

Fibrinogen enhances the adenosine diphosphate (ADP) induced aggregation of washed rabbit platelets and has previously been shown to associate with platelets upon the addition of ADP. This association has been investigated further by measuring the binding of 126I-flbnnogen to platelets exposed to ADP under physiologic conditions of aggregation and deaggregation. To determine the extent of 126I-fibrinogen binding, the platelets were removed by rapid centrifugation and the radioactivity in the platelet pellet was measured; trivalent chromium-51 was used as a space marker. If fibrinogen is present before ADP is added, the fibrinogen binds maximally to the platelets as they change shape in response to ADP. When ADP is not removed from the platelet suspension, deaggregation is slow, and fibrinogen also binds when added shortly after the ADP, but the amount that binds is less. The ability of the platelets to bind fibrinogen upon exposure to ADP is lost rapidly if an enzyme that removes the ADP is present in the suspension. A large portion of the 125I-fibrinogen bound to platelets during ADP stimulation, can be displaced by unlabeled fibrinogen added shortly after the ADP. Bound fibrinogen dissociates from platelets when they deaggregate upon removal of the ADP stimulus, and characterization of this fibrinogen by SDS-PAGE did not show any change resulting from its reactions with platelets. If the platelets are allowed to recover and then are stimulated a second time with ADP, they aggregate and bind fibrinogen to the same extent as platelets not previously aggregated with ADP. Thus, fibrinogen binding sites become available when platelets are exposed to ADP, become unavailable within a short time, and, if conditions are such that the platelets can recover, fibrinogen binding sites may become available again upon subsequent addition of ADP. It appears that neither the fibrinogen nor the fibrinogen binding site is permanently altered during the processes of aggregation and deaggregation.

Interaction of Normal, Thrombasthenic and Bernard-Soulier Platelets With Immobilized Fibrinogen: Defective Platelet-Fibrinogen Interaction in Thrombasthenia

Fibrinogen is required for normal platelet adhesion to glass and aggregation by adenosine diphosphate (ADP), epinephrine, thrombin, and connective tissue, The abnormalities of platelet function in fibrinogen-free medium are similar to those found in thrombasthenia, raising the possibility that the platelets in this disorder do not interact with fibrinogen normally. To investigate the fibrinogen-platelet interaction, highly purified fibrinogen was covalently coupled to solid beads, which were then used to develop an assay in which platelets agglutinate the beads when they interact with the fibrinogen. Unstimulated normal platelets agglutinated the beads; the agglutination could be inhibited by EDTA, lidocaine, sodium azide, antifibrinogen Fab′2, and exogenous fibrinogen in solution. Aspirin caused no inhibition, while high doses of PGE1 and apyrase produced only partial inhibition. ADP and epinephrine dramatically enhanced the agglutination. Platelet-rich plasma from two patients with Bernard-Soulier syndrome also agglutinated the beads. In contrast, the platelet-rich plasma from two patients with thrombasthenia completely failed to agglutinate the beads, even with the addition of ADP and epinephrine. The inability of thrombasthenic platelets to agglutinate fibrinogen beads confirms the presence of an abnormality in interaction with fibrinogen, and since with normal platelets this interaction is independent of platelet aggregation, it suggests that the abnormal fibrinogen interaction may be the cause of the platelet aggregation defect in thrombasthenia.

Haemostatic Platelet Plug Formation in the Isolated Rabbit Mesenteric Preparation - An Analysis of Red Blood Cell Participation

In a model using an isolated rabbit mesenteric preparation microvessels were transected and the time until haemostatic plugs formed was registered. Perfusion of platelet rich plasma gave no haemostasis whereas whole blood did. Addition of chlorpromazine or adenosine to the whole blood significantly prolonged the time for haemostasis, and addition of ADP to the platelet rich plasma significantly shortened it. It is concluded that red cells are necessary for a normal haemostasis in this model, probably by a combination of a haemodynamic and ADP releasing effect.

The Effect of Pretreatment of Human or Rabbit Platelets With Chymotrypsin on Their Responses to Human Fibrinogen and Aggregating Agents

Platelet function and the interaction of platelets with human fibrinogen have been studied following degradation of membrane glycoproteins by chymotrypsin. Washed human and rabbit platelets were treated with α-chymotrypsin (1-50 U/ml), which cleaved glycopeptides from membrane glycoproteins without causing aggregation or release of granule contents. Chymotrypsin removed a large percentage of the sialic acid and periodic acid Schiff (PAS) staining material from the platelet membrane, but glycoprotein IV (of human platelets) or III (of rabbit platelets) were not affected. Chymotrypsin-treated platelets were either less responsive or unresponsive to aggregation induced by adenosine diphosphate (ADP) and aggregation and release induced by thrombin or collagen, depending on the concentration of chymotrypsin with which the platelets had been incubated. Unlike untreated platelets, chymotrypsin-treated platelets aggregated in response to human fibrinogen. This was not an agglutination reaction, since it was prevented by EDTA or EGTA or (with rabbit platelets) by the omission of calcium from the suspending medium. ADP enhanced fibrinogen-induced aggregation of these platelets. Fibrinogen becomes associated with untreated human or rabbit platelets during ADP-induced aggregation. 125I-fibrinogen became associated with the chymotrypsin-treated platelets during fibrinogen-induced aggregation, and more 125I-fibrinogen associated with them upon the addition of ADP than became associated with untreated control platelets during ADP-induced aggregation. Normal, untreated, disc-shaped platelets do not seem to have available fibrinogen-binding sites. It may be that when they are induced to change shape by ADP, such sites are revealed so that fibrinogen can form bridges between adjacent platelets. It appears that treatment of platelets with chymotrypsin, which removes glycopeptides from membrane glycoproteins, also causes rearrangement of platelet membrane components so that fibrinogen-binding sites become available. These observations support the concept that fibrinogen has a major role in platelet aggregation.

血清リポ蛋白の血小板凝集能に及ぼす影響

The effects of plasma lipoproteins on platelet aggregation were examined after incubation of platelet rich plasma (PRP) and different concentrations of plasma lipoproteins for 30-60min at room temperature.Platelet aggregation was measured after addition of adenosine diphosphate (ADP) by Aggregation Meter of SIENCO company. Final concentration of ADP was 2μM.PRP were prepared from normal subjects and patients with cerebral thrombosis and plasma lipoproteins were concentrated and divided into three fraction of very low density (VLDL d<1.006), low density (LDL 1.006<d<1.63) and high density (HDL 1.063<d<1.21) lipoproteins by the ultracentrifugation of Havel's method.No significant changes of platelet aggregation were observed in both groups by the incubation of PRP with VLDL, LDL and HDL.We conclude that plasma lipoproteins have no effects on platelet aggregation in short time incubation.

Quantification of the morphological reaction of platelets to aggregating agents and of its reversal by aggregation inhibitors.

1. In rabbit citrated platelet-rich plasma, the changes in shape of the platelets produced by adenosine diphosphate (ADP) or 5-hydroxytryptamine (5-HT) were observed by photometric and volumetric techniques and by measurements of platelet images on electron micrographs either directly or with an image analysing computer. This permitted the indirect manifestations of the shape changes to be correlated with the morphological features responsible for them, i.e. transformation of disks to more spherical forms and extrusion of blebs and spikes. 2. Following the addition of ADP, an initial brief peak in the light scattering records was associated with marked but transient irregularities in the surface of the platelets. These effects were absent when the other shape changes were produced by 5-HT. 3. When the optical manifestations of the shape changes induced by ADP were reversed by the addition of antagonists adenosine triphosphate, 2-chloroadenosine or prostaglandin E1, the morphological changes were reversed by a diminution in the number of spikes and the conversion of spherical platelet bodies to a more discoid form. 4. The volume of extracellular plasma trapped between platelets sedimented by centrifugation was proportional to the number of spikes which they extruded. Under all conditions, the volume of the platelets themselves remained remarkably constant at approximately 5 X 10(-9) microliter./platelet. 5. Addition of a calcium chelating agent alone produced a rapid persistent alteration in the optical properties of platelet-rich plasma. The magnitude of this alteration was proportional to chelator concentration but greater in some plasmas than in others. Similar optical effects were produced when the calcium concentration of platelet-rich plasma was increased by adding calcium. The optical effects produced by calcium or its chelators were unusual in that the changes in transmitted and horizontally scattered light were in opposite directions (transmitted light decreased, scattered light increased) whereas, in all other circumstances so far examined, these changes were in the same direction. The chelators caused the formation of spikes on the platelets without appreciably altering their disk shape, which may explain the unusual nature of the optical effects.

A study of rapid mitochondrial structural changes in vitro by spray-freeze-etching.

The spray-freeze-etching technique has been used to study energy-linked mitochondrial structural changes in rat liver mitochondria incubated in vitro. The technique involves spraying the suspension of mitochondria into liquid propane at -190 degrees C, and does not require the use of cryoprotectants or chemical fixatives. The results confirmed that freshly isolated mitochondria have a condensed matrix and that this expands at the expense of the outer compartment to give the orthodox configuration when the mitochondria are incubated in a K+ medium in the presence of substrate and phosphate. Addition of adenosine diphosphate (ADP) caused a rapid shrinkage of the matrix compartment, and the time-course and extent of this shrinkage has been measured quantitatively by coupling a rapid sampling device to the spray-freezing apparatus. These data show that for orthodox mitochondria the onset of phosphorylation is accompanied by a reduction of 30% in the matrix volume in 20's, and there is no evidence that the decrease in matrical volume affects the phosphorylation efficiency. These results suggest that natural ionophores in the mitochondrial inner membrane make it permeable enough to permit a rapid readjustment of matrix volume after the addition of ADP, and that the associated ion movement does not cause uncoupling of oxidative phosphorylation.

Shape and cytoplasmic filaments in control and lidocaine-treated human platelets

In order to examine the internal structure of platelets we used a negative stain technique following rapid lysis of gel-filtered platelets on electron microscope grids. Platelets activated by the preparative procedure contained an internal network of thin (7-8 nm) filaments in the body of the cells, continuous with parallel filaments in the microspikes. The filaments bound HMM-S1 to form arrowhead structures. The tight bundles of thin filaments in the microspikes possessed a striking axial periodicity similar to the periodicity seen in actin paracrystals containing muscle regulatory proteins. Nearly complete microtubule circlets were sometimes present in or adjacent to lysing cells with formed microspikes; they appeared as continuous loops. Lidocaine (30 mM) reversibly suppressed and reversed microspike formation in 70% or more of platelet populations even after adenosine diphosphate addition. Treated platelets were spherical, not discoid, lacked the thin-filament network and microtubules and contained an amorphous or granular internal cytoplasm. Bundles of thick 0.3-μm long filaments, similar to myosin aggregates, formed in vitro from purified platelet myosin were seen only in treated stored cells, and not in controls. We conclude that lidocaine suppression of microspikes is correlated with disassembly of thin-filament bundles, that reformation of microspikes involves reassembly of filaments into bundles, and that microtubules are not an obligatory part of microspikes.

Shape and Cytoplasmic Filaments in Control and Lidocaine-treated Human Platelets

In order to examine the internal structure of platelets we used a negative stain technique following rapid lysis of gel-filtered platelets on electron microscope grids. Platelets activated by the preparative procedure contained an internal network of thin (7-8 nm) filaments in the body of the cells, continuous with parallel filaments in the microspikes. The filaments bound HMM-S1 to form arrowhead structures. The tight bundles of thin filaments in the microspikes possessed a striking axial periodicity similar to the periodicity seen in actin paracrystals containing muscle regulatory proteins. Nearly complete microtubule circlets were sometimes present in or adjacent to lysing cells with formed microspikes; they appeared as continuous loops. Lidocaine (30 mM) reversibly suppressed and reversed microspike formation in 70% or more of platelet populations even after adenosine diphosphate addition. Treated platelets were spherical, not discoid, lacked the thin-filament network and microtubules and contained an amorphous or granular internal cytoplasm. Bundles of thick 0.3-μm long filaments, similar to myosin aggregates, formed in vitro from purified platelet myosin were seen only in treated stored cells, and not in controls. We conclude that lidocaine suppression of microspikes is correlated with disassembly of thin-filament bundles, that reformation of microspikes involves reassembly of filaments into bundles, and that microtubules are not an obligatory part of microspikes.

SHAPE CHANGE AND AGGREGATION OF BLOOD PLATELETS: INTERACTION BETWEEN THE EFFECTS OF ADENOSINE DIPHOSPHATE, 5‐HYDROXYTRYPTAMINE AND ADRENALINE

1 The interaction of effects between 5-hydroxytryptamine (5-HT) and adenosine diphosphate (ADP) on human or rabbit platelets was investigated in vitro. The initial platelet shape change and their aggregation were measured in stirred, citrated platelet-rich plasma (PRP) at 37 degrees C by recording the rate and extent of changes in light scattering and light transmission. 2 Both the velocity and extent of aggregation and the velocity and extent of the rapid morphological change caused by ADP were enhanced by simultaneous addition of 5-HT. Methysergide but not imipramine inhibited the 5-HT effects. 3 Platelets were made refractory to the aggregating and shape changing effect of either ADP or 5-HT by repeated aggregation with the particular agent; platelets made refractory to ADP retained their responsiveness to 5-HT and platelets made refractory to 5-HT responded to ADP. Platelets pre-incubated for 3-10 min with 5-HT without aggregation showed greatly reduced aggregation on subsequent addition of ADP. Methysergide inhibited all the effects of 5-HT whilst imipramine was inactive. 4 When the shape change or aggregation of platelets induced by ADP was submaximal, addition of 5-HT increased it further. Pre-incubation of PRP with 5-HT before the addition of ADP resulted in failure of the secondary induction of aggregation or shape change by 5-HT. The secondary induction by 5-HT also did not occur in the presence of methysergide; imipramine had no inhibitory effect. Similar secondary induction of aggregation was shown by adrenaline injected during aggregation by ADP; the adrenaline effect was removed by phentolamine but not by propranolol. 5 Our results show that the initial change in shape of platelets and their aggregation can be induced by ADP or 5-HT in specific manner. The interaction of the effect of these substances on platelets can result in either increase in platelet sensitivity or, under certain conditions, decrease in platelet responsiveness. The increase or depression of platelet reactivity appears to be a highly specific effect and is probably mediated at specific receptors involved with platelet activation.

Platelet dysfunction as a result of inhibition of ADP release (aspirin-like defect) in two identical twins(author's transl)

An abnormal release of platelet adenosine diphosphate (ADP), as seen after intake of acetylsalicylic acid, was demonstrated to be the cause of a clotting disorder in two identical female twins. The signs of bleeding occurred at the age of 26 and consisted of an increased frequency of haematomas, hypermenorrhoea not explained on gynaecological grounds, and prolonged bleeding after minor injuries. Increased bleeding time, abnormal aggregation after stimulation with collagen and absence of the second aggregation phase after addition of ADP were the abnormal findings of this clearly hereditary form of platelet dysfunction. The platelets were slightly larger than normal ones and there were changes in ultrastructure.

Essential thrombocythaemia and peripheral gangrene.

Six patients are described in whom gangrene of one or more toes occurred as the presenting feature of essential thrombocythaemia. Spontaneous platelet aggregation was observed in platelet-rich plasma from four patients and platelet aggregation after the addition of adenosine diphosphate and collagen was highly abnormal in samples from all six. All of the patients described dramatic relief of pain within six hours of ingestion of aspirin and this coincided with disappearance of the spontaneous platelet aggregation and collagen-induced platelet aggregation. Treatment with phosphorus-32 corrected the platelet count and there were no further recurrences of peripheral vascular disease. Platelet function tests performed at the time all gave normal results. It is concluded that essential thrombocythaemia is an important and treatable cause of peripheral vascular disease.

Quantitative parameterization of the light transmission properties of citrated, platelet-rich plasma as a function of platelet and adenosine diphosphate concentrations and temperature.

Abstract Seven parameters were simultaneously analyzed from light transmission records obtained with an aggregometer for "stable" adenosine diphosphate (ADP)-induced platelet aggregation of human and rabbit citrated platelet-rich plasma (PRP). We found for any given PRP sample that the initial per cent transmission (per cent T) preceding ADP addition (expressed as T i = 100 per cent − per cent T); and maximal rate of per cent T increase (S max ) along with the total per cent T increase (T max ) associated with ADP-induced platelet aggregation, all varied linearly with platelet concentration over the range of 380,000 to 185,000 per microliter for human and 680,000 to 110,000 per microliter for rabbit donors. In contrast, the averaged oscillation amplitudes of the untreated PRP (OA), the maximal per cent T decrease (T s ) following ADP addition, the latent time between this addition and "onset" of aggregation (LT), and the half-time for the "stable" aggregation reaction (t12), were independent of platelet concentration over the above ranges. T s and LT were studied only for human donors. Samples were prepared and kept at 37 °C. and studied between 1 to 2 hours after bleeding, corresponding to minimal "decay" in all of these parameters. Studies with PRP incubated at 4 °C. gave mainly decreases in S max and the shape-associated parameters, OA and T s , while studies with decreasing ADP concentrations used to induce aggregation gave closely parallel decreases in both S max and T max , with the other parameters unchanged. The results are discussed in terms of choice and setting of aggregometers; the usefulness of standardized slopes (Δ c ) of concentration-dependent parameters (T i , S max , and T max ) for estimating the "apparent" per cent of unreacted platelets and for evaluating the "effective" per cent PRP in control samples challenged with various stimuli; and the relationship of these parameters to platelet structure and function and their possible application to the mechanistic studies of thrombosis/hemostasis and the characterization of clinical disorders.

Aggregation patterns and release of adenosine diphosphate from guinea pig platelets

Guinea pig platelets aggregate biphasically following addition of adenosine diphosphate (ADP) to platelet-rich plasma, and second phase aggregation is associated with release of platelet ADP. Nucleotide release is dependent upon first phase aggregation, and is not caused by exogenous ADP itself. Desmethylimipramine and aspirin, which block second phase aggregation, also block tendon extract-induced aggregation. Results indicate a common factor in mechanism of ADP release caused by platelet-tissue adhesion, and platelet-platelet adhesion.

Second Phase Platelet Aggregation Induced by Adenosine Diphosphate in Patients with Cerebral Vascular Disease and in Control Subjects

SummaryAdenosine diphosphate-induced second phase platelet aggregation was studied in 70 patients with established cerebral vascular disease, 27 patients with chronic cerebral vascular disease taking dipyridamole, and 53 control subjects. The appearances of aggregation graphs relating changes in optical density in platelet-rich plasma to time after addition of adenosine diphosphate are described. An intermediate rate change in optical density was observed between first and second phase aggregation.Induction of second phase aggregation depends on the concentration of adenosine diphosphate and the platelet count of the plasma. Individual rseponses vary greatly, and there is appreciable variability in repeated estimations, but in the same plasma responses to adenosine diphosphate are accurately reproducible.In both patients and controls the mean logarithm of the adenosine diphosphate concentration that just induces second phase aggregation is inversely proportional to the mean platelet count of platelet-rich plasma. At all platelet concentrations studied, the smallest concentration of adenosine diphosphate that brought about second phase aggregation was significantly less in the patients than in controls. There was no significant correlation between the means of the two variables in patients taking dipyridamole. The findings in this group of patients accord with the assumption that dipyridamole favourably affects the abnormal reaction of platelets to adenosine diphosphate in some of the patients with cerebral vascular disease.

Heterogeneity of human platelets. II. Functional evidence suggestive of young and old platelets.

In the previous communication, suggestive evidence was presented for large-heavy platelets being "young" platelets and light-small platelets being "old" platelets. Large-heavy, light-small, and total human platelet populations were compared with respect to their platelet function. After addition of adenosine diphosphate (ADP), thrombin, or epinephrine, platelet aggregation time was 3.0-, 4.5-, and 3.3-fold shorter with large-heavy platelets compared with light-small platelets, and large-heavy platelets released 3.7-, 7.6-, and 8.1-fold greater adenosine triphosphate (ATP) into the medium, respectively, than did light-small platelets. After platelet aggregation by thrombin or epinephrine, large-heavy platelets released 6.0- and 3.8-fold more ADP into the medium than did light-small platelets. After platelet aggregation by ADP, light-small platelets consumed 5.9-fold greater added extracellular ADP than did large-heavy platelets.Large-heavy platelets aggregated by ADP, thrombin, or epinephrine released 9.1-, 8.5-, and 12.7-fold greater platelet factor 4 than light-small platelets similarly treated.

Biochemical energetics of simulated platelet plug formation. Effect of thrombin, adenosine diphosphate, and epinephrine on intra- and extracellular adenine nucleotide kinetics.

Washed human platelets were incubated in a modified Ringer's solution, pH 7.1, at 37 degrees C for 1 hr. Intracellular basal levels for glycogen, adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), and orthophosphate were 31.1, 2.52, 1.39, 0.36, and 1.2 mumoles/ml of platelets, respectively. Extracellular ATP, ADP, and AMP remained fairly constant and represented 4, 2, and 4% of total adenine nucleotide content. Total adenine nucleotide content remained unchanged during the period of control incubation. Glycogen depletion was 17.8 mumoles/ml at the end of 1 hr; lactate production was 20.7 mumoles/ml per hr. In the presence of glucose, lactate production increased 100%, and glycogen depletion was spared 13%. Approximately 55% of glucose or glycogen fuel was converted to lactate. The agglutinating agents, thrombin, ADP, and epinephrine, resulted in increased glycogen depletion and lactate production both in the presence and absence of glucose. The effect of thrombin was greater than epinephrine. The effect of epinephrine was greater than ADP. All three agglutinating agents resulted in loss of high energy phosphates (net decline in adenine nucleotides) with release of adenine nucleotides into the extracellular environment. The effect of thrombin was greater than ADP. The effect of ADP was greater than epinephrine. In experiments with ADP addition, significant quantities of ADP were converted to AMP extracellularly. In experiments with thrombin and epinephrine appreciable quantities of extracellular orthophosphate were taken up by plateletes and could not be accounted for by changes in intracellular orthophosphate or adenine nucleotide. Sufficient ADP was released during exposure to thrombin and epinephrine to account for platelet agglutination. Changes in intracellular adenine nucleotides and orthophosphate could be correlated with the activation of regulator glycogenolytic and glycolytic enzymes.

Role of Enzyme-Enzyme Interactions in the Regulation of Glycolysis: Inactivation of D-Fructose 1,6-Diphosphatase by Kidney Cortex Mitochondria

Abstract Particulate preparations obtained from a number of tissues, including liver, kidney, and muscle inactivated renal d-fructose 1,6-diphosphatase when they were incubated with the enzyme in the presence of adenosine triphosphate, magnesium ion, and cysteine. Purified swine kidney d-fructose 1,6-diphosphatase and rat kidney uridine diphosphate-d-glucose-glycogen glucosyltransferase were inactivated by washed rat kidney cortex mitochondria. The initial rate of the reaction was dependent on the concentration of mitochondria, d-fructose 1,6-diphosphatase, and adenosine nucleotides, and magnesium ion and cysteine were required for maximum activity. The rate of inactivation of the enzyme by mitochondria was stimulated by the addition of adenosine diphosphate and adenosine monophosphate and to a lesser extent by adenosine triphosphate. The addition of malate and succinate to the incubation mixture decreased the rate of inactivation. Under the experimental conditions used in this study the rate of inactivation was approximately 1.1 x 10-3 µmole of d-fructose 1,6-diphosphatase per min per g of tissue.

Adenosine diphosphate and the measurement of platelet adhesiveness.

Platelet adhesiveness to glass beads was measured in citrated blood at intervals up to two hours after the addition of adenosine diphosphate (A.D.P.). Adhesiveness increased for 15 minutes, fell towards the original level, and then steadily increased. The initial changes were associated with the formation and subsequent dispersal of platelet clumps. The sequence of events resembled the reported platelet aggregation, the disaggregation and secondary aggregation effect of A.D.P. and emphasizes the importance of a standard technique in the measurement of A.D.P.-induced platelet adhesiveness.