Secondary Platelet Aggregation Research Articles

Two platelet aggregation inhibitors in tsetse (Glossina) saliva with studies of roles of thrombin and citrate in in vitro platelet aggregation.

Two inhibitors of platelet aggregation have been identified in saline extracts of Glossina morsitans (tsetse) salivary glands. A protein fraction (MW greater than 30 000) inhibited primary and secondary aggregation to ADP, secondary aggregation to adrenalin, and aggregation to collagen. It also caused disaggregation of platelets stimulated by ADP and adrenalin. These properties could be explained by ADP hydrolytic activity. A previously identified antithrombin fraction (MW 11 000-13 000) abolished thrombin-induced aggregation. It did not affect platelet aggregation to ADP, adrenalin or collagen, as compared to aggregation in citrate, when used as sole anticoagulant for platelet rich plasma or when added to citrated platelet rich plasma. These results fail to support hypotheses (i) that thrombin plays an important role in platelet aggregation by other agonists and (ii) that secondary platelet aggregation is an artefact induced by citrate. It is proposed that these inhibitors may be important in maintaining mouthpart and crop patency during feeding. Their discovery suggests that other arthropods may also have antiplatelet agents in their saliva which might be entomologically important, provide useful tools for platelet studies, and be of potential therapeutic interest.

Effects of N-acetyl glucosamine on platelet aggregation

N-acetyl glucosamine (NAG) alone did not aggregate platelets but in low concentrations shortened the delay phase of the Staphylococcus aureus induced platelet aggregation in a concentration dependent manner, which suggests that NAG may be one of the components of the S. aureus cell wall responsible for platelet aggregation. NAG, on the other hand, inhibited collagen induced platelet aggregation and the secondary platelet aggregation induced by epinephrine and ADP. This inhibition appears to be due to impurities that occur at higher levels in some NAG lots than in others. NAG has also been shown to decrease the length of time before the release of ATP from platelets when S. aureus is the aggregating agent.

The mechanism of the inhibitory effect of protease inhibitors on platelet aggregation and cellular synthesis of prostaglandins. I. The effect on the release of arachidonic acid from phospholipids.

Abstract The inhibitory effect of proteinase inhibitors on platelet aggregation was investigated. The proteinase inhibitors tested were SBTI, leupeptin and FOY (a synthetic proteinase inhibitor). Also, synthetic substrates for serine proteinases (TLME, ATEE) were tested. They completely inhibited the secondary aggregation of platelets induced by ADP or epinephrine. They also completely inhibited the platelet aggregation induced by collagen or thrombin. The aggregation induced by arachidonic acid was completely inhibited by all the proteinase inhibitors and synthetic substrates. The aggregation induced by Ca ionophore A 23187 was completely inhibited by leupeptin, FOY, TLME or ATEE but not by SBTI. It is generally accepted that platelet prostaglandin metabolism plays an important role in platelet aggregation. As the first step to elucidate the possible mechanism of the inhibitory effect of the proteinase inhibitors, their effect on the release of arachidonic acid from platelet phospholipids was investigated. The release of arachidonic acid from 14 C-arachidonate incorporated gel filtered platelets ( 14 C-AA-GFP) by thrombin or A 23187 was directly measured in the presence or absence of a proteinase inhibitor or synthetic substrate, utilizing thin layer chromatography (TLC) and a scintillation counting. The release was almost completely blocked when the aggregation of 14 C-AA-GFP by thrombin or A 23187 was completely inhibited by a proteinase inhibitor or a synthetic substrate.

The mechanism of the inhibitory effect of protease inhibitors on platelet aggregation

The inhibitory effect of protease inhibitors on platelet aggregation was investigated. The inhibitors tested were SBTI, leupeptin, aprotinin and FOY (a synthetic protease inhibitor).They completely inhibited the secondary aggregation of platelets induced by ADP or epinephrine. They also completely inhibited the platelet aggregation induced by collagen, thrombin or sodium arachidonate. The mechanism of their inhibitory effect was investigated, utilizing radioactive arachidonic acid and prostaglandin H2. All the protease inhibitors tested blocked the release of arachidonic acid from platelet phospholipids. SBTI and aprotinin inhibited the cellular synthesis of thromboxane B2 from arachidonic acid but not from prostaglandin H2, suggesting their inhibitory effect on cyclooxygenase. Leupeptin did not possess any effect on thromboxane synthesis either from arachidonic acid or from prostaglandin H2. FOY exhibited selective inhibition on the formation of thromboxane B2 both from arachidonic acid and from prostaglandin H2, yielding higher amount of other stable end products of cyclooxygenase pathway. Therefore, FOY seemed to possess an inhibitory effect on thromboxane synthetase and this drug could be an ideal antiplatelet without suppressing the formation of a natural inhibitor of platelet aggregation; prostaglandin I2.

Platelet Function in Patients of Ischemic Cerebral Vascular Disease and Ménière's Disease

ADP-induced platelet aggregation was examined in patients with a history of ischemic cerebral vascular disease (CVD). When a threshold concentration of ADP for production of the secondary platelet aggregation was taken as a marker of platelet function, no remarkable difference was observed between CVD patients and control subjects. However, when thrombosis of perforating arteries diagnosed from computotomography findings was compared with that of main trunks of cerebral arteries, enhanced platelet aggregation was noted in the patients of perforating artery occulsion. The role of platelet aggregability in thrombogenesis thus seems to be manifested more readily in this type of vessel branching. As previously reported, platelet aggregation is remarkably enhanced in the presence of red blood cells. Our results also suggested that higher platelet aggregability might be compensated for by a lower hematocrit value.β-Thromboglobulin (βTG), a new indicator of intravascular platelet activation, was determined in plasma of CVD patients but no increase was observed in the chronic phase after the attack. In patients with Ménière's disease, the BTG level was elevated during the attack, but was within normal range in the intermittent periods.

A dominant role of thromboxane formation in secondary aggregation of platelets.

Aggregation of human platelets in vitro can occur in two phases—primary and secondary aggregation1. Primary aggregation is due to the direct interaction of the aggregating agent with its receptor2,3, whereas secondary aggregation is associated with both the secretion of dense granule constituents, including the aggregating agents, serotonin and ADP (ref. 4) and the activation of a pathway for the conversion of arachidonic acid to prostaglandins (PGs)5. Both secondary aggregation and the associated secretion of dense granule constituents are contingent on the formation of PGG2 and PGH2 which can, in part, be transformed into thromboxane (TX) A2 (refs 1,3,6,7). TXA2 is a potent aggregating agent and apparently the functionally active compound in the arachidonate pathway8–10. However, the precise role of TXA2 in mediating secondary aggregation is a matter of controversy as it is not clear whether secondary aggregation can occur independently of secretion. We report here that platelets from cats with the Chediak–Higashi (CH) syndrome11 aggregate with arachidonate in a cyclooxygenase-dependent manner and that biphasic aggregation produced by serotonin is accompanied by TXB2 formation without detectable ADP secretion. These results demonstrate that secondary platelet aggregation can be mediated through thromboxane formation without participation of secreted ADP.

Platelet coagulant activities in arterial occlusive disease of the eye.

Ischemic optic neuropathy and retinal arterial occlusion are 2 forms of arterial occlusive disease affecting the eye. Reports in the literature suggest platelet hyperactivity in acute arterial occlusive diseases affecting other organ systems. Therefore, 14 patients with ischemic optic neuropathy and 17 patients with central or branch retinal artery occlusion were studied to determine whether platelets have a role in the pathogenesis of these vascular occlusive disorders. The results of the following investigations were no different in these patients compared with those in 18 control patients with non-vascular eye diseases: prothrombin times, partial thromboplastin times, plasma fibrinogen, factor V, factor VIII, platelet counts and threshold concentrations of ADP, epinephrine and collagen resulting in secondary platelet aggregation and serotonin release. In contrast, platelet coagulant activities concerned with the early stages of intrinsic coagulation were significantly increased in patients with retinal artery occlusion without hypertension or type IV hyperlipoproteinemia, but generally normal in patients with ischemic optic neuropathy and in patients with retinal artery occlusion associated with hypertension, type IV hyperlipoproteinemia, diabetes mellitus and generalized atherosclerosis. These results are consistent with a platelet contribution to retinal arterial occlusive disease in patients without other known contributing factors such as hypertension, serum lipid abnormalities, diabetes mellitus and generalized atherosclerosis and may have implications regarding prophylaxis.

Increase in platelet aggregation following a rise in plasma free fatty acids.

Platelet aggregation was studied following intravenous injection of heparin to nine healthy adults. Heparin is known to produce an increase in the concentration of free fatty acids (FFA) within 10 minutes of intravenous injection. A significant correlation was found between the intensity of primary platelet aggregation and changes in plasma FFA levels. No correlation was found between plasma FFA levels and the intensity of secondary platelet aggregation.

The Effects of “Anti-platelet” Drugs on Bleeding Time and Platelet Aggregation in Normal Human Subjects

The effects on hemostasis of several commonly used drugs previously described as inhibiting platelet function were evaluated in a randomized, double-blind study of 54 normal volunteers. The subjects were each given a single dose of aspirin, chlorpromazine, glyceryl guaiacolate, diphenhydramine, indomethacin or lactose placebo. A single dose of aspirin significantly prolonged the template bleeding time and inhibited secondary platelet aggregation two and 24 hours after ingestion. Single doses of indomethacin and chlorpromazine affected aggregation at two hours but had no effect on bleeding time, although multiple doses of indomethacin did prolong bleeding time. Glyceryl guaiacolate inhibited aggregation one hour after ingestion but had no effect on bleeding time. Diphenhydramine did not affect either. These findings suggest that standard doses of many commonly used "anti-platelet" drugs may have little clinical effect on the hemostatic mechanism in normal man. Results of in-vitro platelet-drug incubations may not be directly applicable to in-vivo hemostasis.

Platelet Aggregation in Portal Cirrhosis

Primary and secondary platelet aggregation in response to adenosine diphosphate was studied in 24 patients with portal (Laënnec) cirrhosis and compared with platelet aggregation in 14 normal subjects. In 12 patients with cirrhosis, platelet aggregation was diminished when compared to controls. Of the 12 patients with impaired aggregation, 6 had elevated levels of fibrinogen-fibrin degradation products (FDPs), 11 had thrombocytopenia, 10 had shortened euglobulin lysis times, 11 had prolonged bleeding times, 4 had hypofibrinogenemia, and all had prolonged thrombin clotting times. The data suggest that elevated levels of serum FDPs do not explain fully the impairment of platelet aggregation or the prolongation of the thrombin clotting time that was noted in patients with advanced liver disease. A possible explanation for the prolongation of the thrombin clotting time is the presence of "altered" plasma fibrinogen.

Nucleotide and Serotonin Metabolism in Platelets With Defective Secondary Aggregation

Abstract Ten patients with qualitative platelet defects have been investigated. All of the patients had impairment of secondary platelet aggregation induced by ADP, epinephrine, and collagen, and a defective release reaction. In seven patients from four families, the abnormality was consistent with the lack of a metabolically inert adenine nucleotide pool. Four of these patients, from two families, were albinos. Platelets from all of these patients had lower than normal amounts of adenine nucleotides and 5HT; the ability of these platelets to incorporate the amine was reduced and 5HT was metabolized at an abnormally rapid rate in platelet-rich plasma. It was not possible to distinguish the defect present in the albinos from that in the normally pigmented patients. Three other patients had normal amounts of platelet adenine nucleotides and 5HT; platelet aggregation and the release of adenine nucleotides induced by collagen were impaired. Metabolic ATP breakdown, during collagen aggregation, was also decreased. This defect is similar to that induced in normal platelets by aspirin. Studies on intracellular synthesis of cyclic 3'5' AMP in both groups of patients showed that the platelets were normally responsive to PGE1 and the antagonism of PGE1 by ADP and by epinephrine was also normal.

Effects of antimycin and 2-deoxyglucose on adenine nucleotides in human platelets. Role of metabolic adenosine triphosphate in primary aggregation, secondary aggregation and shape change of platetets.

1. Human platelet-rich plasma prelabelled with [(3)H]adenine was incubated at 37 degrees C with antimycin A and 2-deoxy-d-glucose. Variations in the amounts of ATP, ADP and P(i), and in the radioactivity of ATP, ADP, AMP, IMP, hypoxanthine+inosine and adenine were determined during incubation. Adrenaline- and ADP-induced platelet aggregation and the ADP-induced shape change of the platelets were determined concurrently. 2. 2-Deoxyglucose caused conversion of [(3)H]ATP to [(3)H]hypoxanthine+inosine. The rate of this conversion increased with increasing 2-deoxyglucose concentration and was markedly stimulated by addition of antimycin, which had no effect alone. At maximal ATP-hypoxanthine conversion rates, the IMP radioactivity remained at values tenfold higher than control, whereas [(3)H]ADP and [(3)H]AMP radioactivity gave variations typical for product/substrates in consecutive reactions. The specific radioactivityof ethanol-soluble platelet ATP decreased during incubation to less than one-tenth of its original value. The amounts and radioactivity of ethanol-insoluble ADP did not vary during incubation with the metabolic inhibitors. 3. The rate of ADP- and adrenaline-induced primary aggregation decreased as the amount of radioactive ATP declined, and complete inhibition of aggregation was obtained at a certain ATP concentration (metabolic ATP threshold). This threshold decreased with increasing concentration of inducer ADP. 4. Secondary platelet aggregation (release reaction) had a metabolic ATP threshold markedly higher than that of primary aggregation. 5. Shape change was gradually inhibited as the ATP radioactivity decreased, and had a metabolic ATP threshold distinctly lower than that of primary aggregation, and which decreased with increasing concentration of ADP. 6. A small but distinct fraction of [(3)H]ATP disappeared rapidly during the combined shape change-aggregation process induced by ADP in platelets incubated with metabolic inhibitors, whereas no ATP disappearance occurred during aggregation in their absence.

Influence of lysolecithin on platelet aggregation initiated by 5-hydroxytryptamine.

PREVIOUS studies from this laboratory1,2 have shown that irreversible platelet aggregation initiated in vitro by adenosine diphosphate (ADP), adrenaline or collagen was inhibited by lysolecithin, whilst reversible ADP-induced aggregation was unaffected. This indicated that lysolecithin blocked the platelet release reaction which precedes secondary or irreversible platelet aggregation. Born3 has reported that exposure of human platelets to 5-hydroxytryptamine (5-HT) does not result in a release reaction and that platelet aggregation initiated by 5-HT is small, rapidly reversed and never enters the second phase which can be induced in human platelets by ADP or adrenaline. However, our results failed to confirm this and 5-HT was found on occasions to initiate biphasic aggregation resembling that induced by ADP. Table 1 shows the frequency of biphasic platelet aggregation, initiated by 5-HT, ADP and adrenaline, recorded during platelet studies of a group of normal male subjects and a group of male patients suffering from chronic peripheral arterial disease, none of whom was known to be taking drugs affecting platelet behaviour. The frequency of biphasic responses within both groups did not differ significantly and the response for individuals was usually the same when repeated at a later date. From the individual data summarized in Table 1 it was found that biphasic 5-HT induced aggregation was always accompanied by biphasic responses in parallel samples treated with ADP and adrenaline. Biphasic platelet aggregation curves initiated by 5-HT were all similar to that shown in Fig. 1 (control), in which secondary aggregation was preceded by partial disaggregation.

The effect of epinephrine on platelet aggregation in normal and atopic subjects

The effect of epinephrine on platelet aggregation was determined in 25 patients with allergic rhinitis, 26 patients with asthma, and 23 normal subjects. Three doses of epinephrine (0.42, 0.83, 3.3 μg) were evaluated in each preparation. Although 7 (28 per cent) of the patients with allergic rhinitis exhibited abnormalities in the pattern of platelet aggregation after addition of 0.83 μg of epinephrine, the mean results in this patient group were not significant compared to results in 23 normal subjects. At a lower epinephrine concentration (0.42 μg), 9 (36 per cent) of these patients showed abnormal responses that were significant. In asthmatic patients the mean aggregation-disaggregation curve differed significantly from the normal mean curve at all concentrations of epinephrine, and these abnormalities appeared 4 to 7 minutes after stimulation of platelets by epinephrine. It was demonstrated that humoral factors were involved in these reactions by incubating platelet-poor plasma (PPP) from 3 asthmatic patients with normal platelets prior to epinephrine stimulation. Normal platelet aggregation was subsequently impaired by this procedure. It was also shown that abnormal platelet aggregation in asthmatic patients could be partially reversed by prior incubation of their platelets with normal plasma. These results suggest that asthmatic patients have a humoral factor that specifically alters the receptor control mechanism of secondary platelet aggregation. However, since normal plasma partially restores a normal aggregation profile to asthmatic platelets, the platelet receptor in these patients is not permanently altered.

Primary and secondary platelet aggregation in uraemia.

ADP‐induced aggregation (primary and secondary) of platelet‐rich plasma from uraemic subjects did not differ significantly from that of normal individuals. Biphasic aggregation occurred in 13 of 14 uraemic patients and in 11 of 14 normal subjects. The optimal concentration of ADP‐required to demonstrate secondary aggregation was 1 μmolar.It is postulated that the normal response to ADP of platelets from uraemic subjects observed in this study resulted from the addition of optimal amounts of calcium to the platelet‐rich plasma.

Effect of prostaglandin E 2 and aspirin on the secondary aggregation of human platelets.

THE lack of cellular specificity1 of the prostaglandins creates potential hazards in their use as therapeutic agents: we describe one associated with PGE2 and the reversal of the effect with aspirin.

Depression of plasma lipid fractions and inhibition of platelet aggregation by action of glucagon

Venous blood was taken from 20 normal people following an overnight fast, and glucagon (1 mg) dissolved in 1.0 ml diluent was given i.v. Blood was taken at 1, 2, and 3 hr. Control studies were done in seven of the subjects at another time by administering the diluent only. Glucagon caused significant depression of plasma total lipid, and triglyceride at 1 and 2 hr, while plasma total cholesterol was significantly depressed at 2 hr. Epinephrine-induced platelet aggregation was studied in the hourly specimens of platelet-rich plasma (PRP) by measuring change in optical density (OD). Epinephrine caused both primary and secondary platelet aggregation in the 0-hr PRP of ten subjects and primary aggregation only in the other ten. Intravenous glucagon caused significant inhibition of secondary platelet aggregation at 2 and 3 hr. Secondary platelet aggregation was not inhibited when there was no depression of plasma lipids following glucagon (two of ten subjects). Glucagon (1 mg) dissolved in 1.0 ml diluent was given subcutaneously to five of the subjects at another time, and the studies were repeated. Depression of plasma total lipid, triglyceride, and cholesterol was greater than when glucagon was given i.v. Magnitude and duration of the depression were both increased. Secondary platelet aggregation induced by epinephrine was also inhibited. It was concluded that depression of plasma lipids by transfer of the lipid to platelets by action of glucagon is associated with inhibition of epinephrine-induced platelet aggregation.

Secondary platelet aggregation: a quantitative study.

Summary.The lowest concentration of ADP and adrenaline which resulted in a secondary wave of platelet aggregation at 37°C was determined in 60 normal subjects. The range of such ‘threshold’ concentrations was 0.2–1.4μM for ADP and 0.1–1.0μM for adrenaline. The mean threshold concentration of each reagent was unrelated to age but was higher for men than for women; this difference disappeared when the results were corrected for the effect of differences in PCV on the citrate concentration in platelet‐rich plasma. The variation between separate determinations of threshold concentration in the same individual was of the same order as that between individuals; no evidence was found that the threshold concentration of either reagent was related to the phase of the menstrual cycle in four young women tested repeatedly. There was a highly significant correlation between the threshold concentrations of the two reagents in individual subjects.No correlation was found between the threshold concentration of either reagent and the total platelet ATP, ADP or ATP: ADP ratio. Concentrations of adrenaline at or slightly above the threshold caused the release of up to about 50% of the platelet ADP and 25% of the ATP during the second phase of aggregation, while lower concentrations produced no significant release.The ultrastructural changes during the first phase of aggregation were similar with all concentrations of ADP tested; threshold concentrations and above caused dense packing of platelets during the second phase, with the formation of giant pseudopodia and no tendency to disaggregation during the period of observation, while at lower concentrations disaggregation was accompanied by a retraction of pseudopodia. There was no clear evidence of platelet degranulation during either first or second phase aggregation.