First Phase Of Aggregation Research Articles

Amyloid Fibrils Formation of Concanavalin A at Basic pH

Mechanisms of partial unfolding and aggregation of proteins are of extreme interest in view of the fact that several human pathologies are characterized by the formation and deposition of protein-insoluble material, mainly composed of amyloid fibrils. Here we report on an experimental study on the heat-induced aggregation mechanisms, at basic pH, of concanavalin A (ConA), used as a model system. Thioflavin T (ThT) fluorescence and multiangle light scattering allowed us to detect different intertwined steps in the formation of ConA aggregates. In particular, the ThT fluorescence increase, observed in the first phase of aggregation, reveals the formation of intermolecular β-sheet structure which constitutes a rate-limiting step of the process. The intertwining between the formation of β-aggregate structures and the whole aggregation process is discussed as a function of protein concentration: a coagulation process produces the same kind of aggregates at the different concentrations studied. Multiangle light-scattering data highlight the onset of the condensation process which gives rise to formation of compact fractal aggregates. AFM microscopy supports this conclusion showing thin fibrils of ConA, formed in the early stage of aggregation, which further interact to form larger structures with a netlike spatial organization.

Mucilage microcosms

In the summers of 2000, 2001 and 2002, large amounts of sticky mucilaginous material aggregated to form masses of impressive dimensions over large areas of the Adriatic Sea, particularly in its northern part. Aggregates differing in size were sampled by SCUBA divers and submitted to chemical (nutrient and organic matter concentrations) and biological analysis (virus, bacteria and phytoplankton abundances and bacterial metabolism). Suspended and sinking mucilaginous aggregates were biota-rich environments where the abundance of planktonic organisms and the concentration of nutrients were orders of magnitude higher than in the surrounding seawater. The embedded phytoplankton was mostly composed of diatoms, but the dinoflagellate Gonyaulax fragilis, previously reported in association with the aggregates, was also present. A variety of processes occurred within the mucilaginous aggregates which resulted in the transformation of the organic matter composition and lability and contributed to a partial degradation of mucilage. For an efficient degradation of mucilage, several conditions are necessary: high bacterial abundance and activity and an efficient recycling of nutrients within the aggregates. Most of these conditions, appear to change depending on the type and age of the aggregate. During the first phase of aggregation (cobwebs and ribbons), bacterial activities addressed the degradation of organic matter, particularly that of the nitrogen fraction. The degradation products were rapidly taken up by bacteria, supporting an increase in their abundance and production. In aged mucilage (clouds), the degradation processes decreased and the bacterial metabolism suggested the presence of new organic labile compounds probably due to phytoplankton production. On the basis of our results, stringers, generally considered the first step of the aggregation process, seemed to be the result of a mechanical disruption of other types of aggregates.

Inhibition of aggregation of rabbit and human platelets induced by adrenaline and 5-hydroxytryptamine by KB-R7943, a Na(+)/Ca(2+) exchange inhibitor.

1. We investigated the effect of KB-R7943, a Na(+)/Ca(2+) exchange inhibitor, on the aggregation response induced by adrenaline and 5-hydroxytryptamine (5-HT), alone or in combination in human and rabbit platelets in the presence or absence of ouabain. 2. KB-R7943 inhibited aggregation induced by the combination of adrenaline and 5-HT in a concentration-dependent manner. The IC(50) values of KB-R7943 were 4.2+/-2.0 or 3.0+/-0.7 microM with washed rabbit platelets with or without ouabain pretreatment, respectively. 3. In platelet-rich human plasma, the aggregation was biphasic. The IC(50) value of KB-R7943 was 17.2+/-4.4 microM for the first phase aggregation. 4. KB-R7943 did not inhibit the first phase of aggregation induced by adrenaline alone, or the monophasic aggregation induced by 5-HT alone. 5. The aggregation of rabbit platelets depended on the presence of K(+) in the medium, and K(+)-dependent and K(+)-independent Ca(2+) influx were observed in resting platelets. Ouabain treatment increased only the K(+)-dependent but not the K(+)-independent Ca(2+) influx. 6. KB-R7943 inhibited K(+)-dependent Ca(2+) influx with or without ouabain pretreatment, but not K(+)-independent Ca(2+) influx. 7. From these results, we conclude that KB-R7943 inhibits the adrenaline plus 5-HT induced aggregation of rabbit and human platelets by inhibiting K(+)-dependent Na(+)/Ca(2+) exchange (NCKX). Our results suggest that NCKX plays an important role in platelet aggregation.

PLATELTE AGGREGATION AFTER VAUVULAR SURGERY EVALUATED BY A LAZER-LITHT SCATTERING METHOD - EFFCTS OF ANTICOAGULANTS

Objectives. Effects of anticoagulants in the early phase of platelet aggregation in patients with artificial heart valves are unclear. Newly developed particle-counting methods that employ light scattering (LS) can be used to quantify changes in the number of platelet aggregates of different sizes after application of an aggregating stimulus. Using the LS method, we studied the effects of coumarin and aspirin administration in patients with artificial heart valves. Materials and Methods. Forty-seven patients undergoing valvular surgery were divided into 2 group, 26 patients treated with coumarin alone (group 1), and 21 patients treated with aspirin (81mg/day) and coumarin (group 2). Blood samples were obtained before surgery for control and on day 21 after surgery. Platelet aggregation after stimulation by 2.0 μg/ml collagen was determined; small (9 to 25 μm), medium (25 to 50 μm), and large (50 to 70 μm) aggregates were counted. Results. In group 1, platelet aggregation was not inhibited in both conventional optical density (OD) and LS methods. In group 2, OD methods demonstrated that aggregation was significantly reduced. In LS method, generation of large aggregates after stimulation were significantly decreased in patients treated with coumarin and aspirin than in the control (P 0.034), while, the quantity of the small aggregates was increased (P 0.0005). Conclusion. 1) Coumarin does not suppress platelet function. 2) Although aspirin suppresses platelet function, it does not affect the first phase of aggregation. Platelet dysfunction caused by aspirin is mainly due to an inhibition in the development of small aggregates into larger aggregates.

Platelet aggregation during cardiopulmonary bypass evaluated by a laser light-scattering method

Background. In regard to postoperative bleeding, the most important consequence of cardiopulmonary bypass (CPB) is the loss of aggregability. However, the mechanism of platelet aggregation loss during CPB is unclear. Newly developed particle-counting methods that use light scattering can be used to quantify changes in the number of platelet aggregates of different sizes after application of an aggregating stimulus. Using a light-scattering method, we investigated changes in platelet aggregation during cardiac operation. Methods. Nineteen patients undergoing CPB were evaluated. Blood samples were obtained before the operation, 1 hour after initiation of CPB, at the end of CPB, at the end of the operation, and on day 1 after the operation. Platelet aggregation after stimulation by 2.5 μmol/L adenosine diphosphate and 2.0 μg/mL collagen was determined; small (9 to 25 μm), medium (25 to 50 μm), and large (50 to 70 μm) aggregates were counted. Results. Generation of medium and large aggregates after stimulation with adenosine diphosphate and collagen were significantly decreased with CPB, whereas, in spite of hemodilution, the quantity of the small aggregates was maintained at the elevated level. Conclusions. These results reflect the fact that CPB does not affect the first phase of aggregation. It suggests that platelet dysfunction associated with CPB is mainly caused by an inhibition in the development of small aggregates into larger aggregates.

Measurement of human platelet microaggregates by a new method: Ultrasonic interferometry

We have adapted the ultrasonic interferometry technique (Echo-Cell), which was initially designed to study red blood cell aggregation and agglutination, to the detection of human platelet microaggregates. The experimental parameter chosen was the slope of the signal over the first 5 minutes of sedimentation. We compared our new method with the conventional aggregometry for the measurement of aggregates after thrombin-, collagen-, and epinephrine-induced platelet activation. Under these conditions we demonstrated the particular sensibility of the present method in detecting small platelet aggregates induced in the first phase of aggregation and formed by low concentrations of agonists. Furthermore, as an illustration of this method, we showed an inhibition of the formation of thrombininduced platelet aggregates in a concentration-dependent manner by the well-known antagonist arginine-glycine-aspartic acid-serine with a median inhibitory concentration of 0.4 μmol/L, which is 30 times lower than the median inhibitory concentration found by aggregometry.

Effects of α-tocopherol (Vitamin E) on the Ultrastructure of Human Platelets In Vitro

Washed human platelets were incubated with increasing concentrations of α-tocopherol. Spontaneous aggregation was induced by tocopherol (0.5 mM or above). Aggregation was inhibited by ethylenediaminetetraacetate and platelet activation was reduced by prostaglandin E(1). Using electron microscopy, it was confirmed that tocopherol caused platelet disruption to some extent and the released components may have generated aggregation. These effects were not observed in platelet-rich plasma. Spontaneous activation was not observed when the concentration of tocopherol was 0.03 mM or lower. Concentrations of tocopherol between 0.075 mM and 0.0075 mM had inhibiting influences on activation of washed platelets by thrombin. Tocopherol (between 0.1 mM and 0.005 mM) changed activation of washed platelets by cationized ferritin in that it facilitated the first phase of aggregation but reduced the second phase in an indirect proportional manner. The results show that the effects of tocopherol in washed platelet preparations are not comparable to those observed in plasma and that the platelet membrane must be regarded as a crucial target for vitamin E.

Release of immunoreactive-neuropeptide Y by rat platelets

Neuropeptide Y, a potent vasoconstrictor and cardiac depressant, is re-leased from sympathetic nerve endings. Its presence in megakaryocytes suggests this peptide might be stored in platelet granules and released during aggregation. Immunoreactive-neuropeptide Y was measured in platelet rich and platelet poor plasma, and was substantially greater in the former. Addition of collagen to platelets resulted in release of neuropeptide Y which paralleled, in a concentration-dependent manner, the degree of platelet aggregation. Adenosine diphosphate, at concentrations which induce only the first phase of aggregation and not the release reaction, caused only a minor release of neuropeptide Y. These results suggest that platelet release could be a major source of circulating neuropeptide Y and could contribute to hemodynamics of pathophysiological states involving platelet activation.

Sphingosine inhibition of agonist-dependent secretion and activation of human platelets implies that protein kinase C is a necessary and common event of the signal transduction pathways.

Sphingosine is a potent inhibitor of [3H]phorbol dibutyrate binding and protein kinase C activity in vitro and in human platelets (Hannun, Y., Loomis, C., Merrill, A., and Bell, R. (1986) J. Biol. Chem. 261, 12604-12609). Preincubation of platelets with sphingosine resulted in the inhibition of platelet secretion and second phase aggregation in response to ADP, gamma-thrombin, collagen, arachidonic acid, and platelet activating factor. Sphingosine did not affect the initial shape change of platelets or the first phase of aggregation in response to these agonists. Ristocetin-induced platelet agglutination was not affected by sphingosine. Sphingosine inhibition of secondary aggregation (secretion and second phase aggregation) was overcome by phorbol dibutyrate and by the cell-permeable protein kinase C activator, dioctanoylglycerol. Furthermore, platelet secretion and irreversible aggregation were induced by protein kinase C activators in platelets that had been "primed" to undergo initial shape change and first phase aggregation by low concentrations of agonists. These results suggest that protein kinase C activation is a necessary component in the signal transducing pathways that lead to platelet activation. Higher concentrations of agonists, however, induced irreversible aggregation and partial secretion in the presence of sphingosine, suggesting the existence of protein kinase C-independent pathways for platelet activation. These results demonstrate the utility of sphingosine as a pharmacologic tool in probing the role of protein kinase C in signal transduction.

Association of vinculin to the platelet cytoskeleton during thrombin-induced aggregation

Vinculin is a protein generally believed to be involved in membrane-cytoskeleton interaction, and its presence in platelets has been verified earlier. Here we show that in resting bovine platelets, vinculin is not associated with the Triton-insoluble cytoskeletal fraction but becomes incorporated into it during the thrombin-induced activation process. The incorporation starts around the same time as the release reaction and only after the shape change and the first phase of aggregation have taken place. Its time course parallels the cytoskeletal association of actin and certain other contractile proteins. Vinculin is a minor component of platelet cytoskeleton and only about 10 % of the total platelet vinculin becomes incorporated into the Triton X-100 residue.

Effects of trifluoperazine on platelet activation

Previous reports of the inhibitory effects of trifluoperazine on platelet responses to different aggregating agents have been conflicting, and the mechanism of action remains unclear. We have found that aggregation by minimum concentrations of collagen and arachidonic acid, and second phase aggregation by minimum concentrations of ADP, thrombin, epinephrine and the calcium ionophore A23187 were inhibited by 40–60μM trifluoperazine. The first phase of aggregation by a minimum concentration of epinephrine was completely inhibited by 100μM trifluoperazine, and the first phase of aggregation induced by ADP, thrombin or A23187 was decreased by 300μM trifluoperazine. The platelet shape change caused by collagen, but by no other aggregating agent examined, was inhibited by 300μM trifluoperazine. Secretion of 3H-5 hydroxytryptamine by minimum concentrations of ADP, collagen, epinephrine and arachidonic acid was completely suppressed by 50μM trifluoperazine. Secretion by thrombin and A23187 was incompletely inhibited by 300μM trifluoperazine. Thromboxane B 2 formation caused by all aggregating agents, except epinephrine, was incompletely suppressed by 50μM trifluoperazine, and 300μM trifluoperazine only caused complete inhibition of thromboxane B 2 formation by ADP, collagen and epinephrine. The phorbol ester, TPA, which mimics diacylglycerol by activating protein kinase C, caused aggregation and secretion. Aggregation, but not secretion, by low concentrations of TPA was inhibited by concentrations of trifluoperazine as low as 50μM. However, aggregation by a combination of TPA and A23187 was only inhibited by concentrations of trifluoperazine in excess of 100 μM. Secretion by TPA was inhibited by concentrations of trifluoperazine in excess of 200μM. Our findings suggest that low concentrations of trifluoperazine inhibit platelet activation by inhibiting phospholipase A 2, and that higher concentrations inhibit platelet responses by interfering with protein kinase C.

制癌剤ことにAnthracycline系制癌剤およびCoenzymeQ<SUB>10</SUB>のヒト血小板におよぼす影響について

Among the recent advances in cancer therapy, the development of chemotherapy is most remarkable. On the other hand, hemorrhagic diathesis is often caused by antineoplastic drugs, especially Anthracyclines, which are widely used in the treatment of various kinds of cancer.Reported here are the results of serial experiments on the influence of antineoplastic drugs on human platelets in vitro. The effects of Coenzyme Q10 (CoQ10), known to alleviate such adverse actions as cardiotoxicity and to increase the antineoplastic efficacy of Anthracyclines, were also investigated. The resu lts were as follows:(1) Anthracyclines, including Adrimycin (ADM), Daunomycin (DM), and Aclacinomycin A (ACR), inhbited platelet aggregation and release reaction in dose and time-dependen t manner.5-Fluorouracil (5-FU), a non-Anthracycline, did not show su c h inhibitory effects.(2) Differences were found in the intensity of the action between Anthracyclines, of which DM was the most intensive regardless of the aggregating agent. ADM inhibited pl a t elet aggregation more effectively than ACR when ADP, collagen, epinephrine, and A 23187were employed as agonists. ACR was more effective than ADM in the case of thro m bin. Anthracyclines had almost no inhibitory effect on arachidonic acid and STA2 induced platelet aggregation.(3) CoQ10 also had an inhibitory effect on platelet aggregation, which was more remarkable in the first phase of aggregation. Furthermore CoQ10 was shown to enhance the a n tiplatelet effect of ADM.(4) Neither Anthracyclines nor CoQ10 caused any significant change in cyclooxygenase metabolites or cyclic nucleotide levels, which might account for their inhibitory effec t s on platelet function.(5) The liberation of radioactivity from 14C-arachidonic acid-labeled platelets in response to collagen was reduced by Anthracyclines. It was suggested that the impairment of platele t function by Anthracyclines was partially attributable to the decrease of phospho l ipase activity.

Effects of Ticlopidine, a new platelet function suppressing drug in patients with prosthetic heart valves

The effect of ticlopidine (fig. 1), a new platelet aggregation inhibitor, was studied in 15 patients with Björk-Shiley prosthetic heart valves (6 aortic, 6 mitral, 3 aortic and mitral). The patients were 9 men and 6 women, aged 22 to 55 years old, who had had valve prostheses placed 2 to 43 months (mean 23.7 months) prior to entry into the study. They received 200mg of ticlopidine, divided into two oral doses, daily, and were followed up for more than 3 months. Platelet adhesiveness and aggregation were examined 1, 2, and 4 weeks, and 3 months after the treatment, and the values were compared to those obtained before the treatment. Platelet adhesiveness was determined by a modified technique of Salzman. A Sienco dual sample aggregation meter was used to measure changes of platelet aggregation through light transmission of citrated platelet-rich plasma to which adenosine diphosphate (ADP) had been added. The platelet count of the plasma sample was adjusted to a range of 250-350, 000/mm3. Student's paired t-test was used for statistical significance.Platelet adhesiveness decreased significantly at 1 week, 2 weeks, and 3 months after the treatment, but no significant change was found at 4 weeks (fig. 2).In every patient, platelet aggregation induced by 4μM ADP (final concentration) showed an obvious reduction one week after the treatment, and this effect continued for more than 3 months (fig. 3). A similar reduction in the aggregation caused by 20μM ADP (final concentration) was observed. In many cases, the aggregation caused by 20μM ADP, which showed the second phase of aggregation, changed after the treatment to show only the first phase of aggregation (fig. 4). Ticlopidine inhibited both the first and the second phase of ADP-induced aggregation. This dosage seemed to cause a reduction in platelet aggregation about 3 days after the start of the treatment.We did not observe any interaction between ticlopidine and warfarin in this study.None of the patients had clinical symptoms or side effects due to ticlopidine and no episodes of thromboembolism occurred during the examination period. Bleeding time increased slightly at 4 weeks after the treatment. No abnormal alteration was observed in the white blood cell counts, coagulation time, fibrinogen level, fasting blood sugar, serum electrolites, total protein, hepatic and renal function tests at 4 weeks and 3 months after the treatment.Ticlopidine seems to be a useful platelet function suppressing drug for patients with prosthetic heart valves.

Inhibition of aggregation of human platelets by 8,15-dihydroperoxides of 5,9,11,13-eicosatetraenoic and 9,11,13-eicosatrienoic acids

The dihydroperoxy derivative of arachidonic acid, 8,15-dihydroperoxy-5,9,11,13-eicosatetraenoic acid, at 1×10 5 M , is an effective inhibitor of the aggregation of human blood platelets, induced by ADP, epinephrine and collagen. The nature of the effect is time dependent. If ADP is added 60 sec after the inhibitor, the first phase of aggregation occurs but the second phase is suppressed. When the time is prolonged to 240 sec, the first phase of aggregation is almost completely eliminated. The corresponding dihydroxy derivative is without effect on aggregation. The corresponding dihydroperoxy derivative obtained from 8,11,14-eicosatrienoic acid is also an effective inhibitor.

Influence of humoral shock factors on in vitro aggregation of dog platelets

We studied the effect of humoral factors present during shock on platelet aggregation. At plasma concentrations present in shock, prostaglandin E 1 significantly inhibited ADP induced aggregation of dog platelets. Prostaglandins E 2, F 2α, and D 2 at shock concentrations exerted no significant effect on ADP induced aggregation. PGE 2 inhibited only the first phase of aggregation at a concentration which was much higher than that present in shock. Although arachidonic acid is a potent aggregation inducing agent in certain species, its effect on dog platelets is only very slight. Purified preparations of cathepsin B1 and C and a myocardial depressant factor (MDF) did not induce dog platelet aggregation nor did MDF or cathepsin B1 modify ADP induced aggregation. Although cathepsin C inhibited ADP induced aggregation, this effect was independent of its proteolytic activity. Thus, humoral factors released in shock do not appear to accentuate tissue hypoperfusion by inducing or modifying 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.