Aggregation Of Bovine Platelets Research Articles

Feedback regulation of platelet function by 12 S-hydroxyeicosatetraenoic acid: Inhibition of arachidonic acid liberation from phospholipids

We have proposed a mechanism that platelet aggregation is regulated by its 12-lipoxygenase product, 12 S-hydroxyeicosatetraenoic acid (12-HETE) (Sekiya, F., Takagi, J. and Saito, Y. (1989) Thrombos. Res. 56, 407–415). Inhibition of endogenous 12-HETE production by 15-HETE, a specific inhibitor of 12-lipoxygenase, accelerated aggregation of bovine platelets in response to collagen and arachidonic acid liberation from phospholipids was enhanced. Exogenously added 12-HETE suppressed collagen-induced liberation of arachidonic acid and the aggregation was also inhibited. On the other hand, 12-HETE did not interfere with thromboxane synthesis from free arachidonic acid in a cell-free system. These observations suggest that 12-HETE exerts a negative feedback to prevent excess aggregation through interference with arachidonic acid liberation from membrane phospholipids.

Elucidation of a role of plasma albumin during collagen-induced aggregation of bovine platelets

Bovine platelets of which phospholipids were labeled with [ 14C] arachidonate were stimulated by collagen. Omitting albumin in suspending medium suppressed aggregation and arachidonate liberation from phospholipids. Analysis of [ 14C] arachidonate metabolites released into medium revealed that release of 12 S -hydroxyeicosatetraenoic acid (12-HETE) from stimulated platelets was dependent on the existence of albumin, while thromboxane B 2 release was not. Moreover, exogenously added 12-HETE inhibited collagen-induced aggregation. These observations suggest that albumin potentiates aggregation by preventing intracellular accumulation of 12-HETE, and strengthen our previous finding that albumin in plasma is very essential for collagen-induced aggregation of platelets (Sekiya, F., Takagi, J., Kasahara, K., Inada, Y. and Saito, Y. (1988) Thrombos. Res. 50, 837–846.).

A Comparison of the Antiplatelet Effect of Aspirin on Human and Bovine Platelets

The risk of thromboembolism in human patients or animal models with blood contacting prosthetic devices is well documented. Aspirin is used frequently as an antiplatelet agent to minimize this risk. Although the inhibitory effect of aspirin on human platelets has been clearly established, preliminary studies to examine this effect on bovine platelets revealed a minimal inhibition of platelet function in vivo as well as in vitro. Because a considerable amount of implant research is carried out in bovine models, it was considered important to evaluate carefully the antiplatelet effect of aspirin on bovine platelets. To evaluate the effect of aspirin, experiments were conducted on human and bovine blood in vitro as well as after the administration of aspirin up to 4,000 mg/day (p.o.) in calves and 2,500 mg/day in humans. Appropriate amounts of buffered aspirin were added to heparinized or citrated blood incubated for 25 min and centrifuged to obtain platelet rich plasma (PRP). An aliquot of PRP was then challenged by a predetermined concentration of ADP (an aggregating agent) or collagen to evaluate platelet aggregation and release reaction. After aspirin was administered in vivo, blood was drawn from the animal at predetermined intervals to evaluate platelet function. In human blood, the inhibitory effect of aspirin was discernable at 80 mg/L in vitro and 2,500 mg/day when ingested orally. Under identical experimental conditions, no inhibition of bovine platelet aggregation was observed using dosages of aspirin up to 1,000 mg/L in vitro or 4,000 mg/day in vivo. These observations suggest that although the antiplatelet effect of aspirin is quite pronounced in humans, bovine platelets do not show a similar response to this drug. This should be an important consideration when platelet inhibition is desired during postoperative management of calves.

Bovine platelet aggregation by Fusobacterium necrophorum.

Fusobacterium necrophorum aggregated bovine platelets. The aggregation was paralleled by the haemagglutinating ability of the organism. Treatment of the bacterial cells with antiserum to the homologous purified haemagglutinin reduced the degree of platelet aggregation. Scanning electronmicroscopy revealed that little lysis of the affected platelets occurred during the 1-h incubation period. Purified haemagglutinin became bound to the surfaces of the platelet cells as shown by immunofluorescence microscopy. These observations suggest that platelet aggregation is mediated by the haemagglutinin and may be related to the pathogenicity of the bacterium.

Inhibitory effects of phenol derivatives on bovine platelet aggregation and their effects on Ca 2+ mobilization

The effects of phenol derivatives on aggregation of bovine platelets induced by ADP, thrombin, platelet activating factor, collagen and A23187 were investigated. The phenol derivatives inhibited all these induced aggregations except that by the calcium ionophore. The derivatives each inhibited the aggregations induced by ADP, thrombin, platelet activating factor and collagen, respectively, within a similar concentration range. A linear relation was found between the inhibitory potencies of the phenol derivatives and their partition coefficients between n-octanol and water ( P oct values), suggesting that their interaction with hydrophobic regions of the cell was important for inhibition. Fluorescence analyses with fura-2-loaded platelets showed that in the concentration ranges in which the phenol derivatives inhibited aggregation, they also inhibited agonist-induced increases in Ca 2+ both in the presence and absence of extracellular Ca 2+. Moreover, a high correlation was found between the inhibitory effects of the derivatives on aggregation and their effects on Ca 2+ mobilization. These results suggest that inhibition of platelet aggregation by phenol derivatives is mainly due to inhibition of the increase in cytoplasmic Ca 2+ by inhibition of both intracellular Ca 2+ mobilization and Ca 2+ uptake.

Characterization of the normal bovine platelet aggregation response

1. Bovine platelets are more sensitive to stimulation by platelet activating factor (PAF) than adenosine-di-phosphate (ADP) or thrombin. 2. While epinephrine, arachidonic acid and serotonin are ineffective by themselves as aggregatory stimulants of bovine platelets they enhance the aggregation response of other platelet agonists. 3. There is no correlation between thromboxane A 2 production and release and the extent of platelet aggregation in bovine platelets. 4. The dependence of bovine platelet aggregation on a phospholipid pathway and calcium mobilization is indicated.

Comparison of the inhibitory effect of T-2 toxin on bovine platelet function with that of other known platelet inhibitors

The inhibitory effects of T-2 toxin on bovine platelet function and thromboxane A 2 production were compared with those of the known inhibitors of human platelet function, acetylsalicylic acid, dipyridamole and verapamil. T-2 toxin (1 × 10 −3M) effectively inhibited bovine platelet aggregation (33.2–64.3%), whereas neither acetylsalicylic acid nor dipyridamole did so. T-2 toxin appeared to be a less effective inhibitor of platelet aggregation than the calcium channel blocker, verapamil. T-2 toxin (1 × 10 −3M) added to platelet suspensions together with verapamil, produced an additive inhibitory response. T-2 toxin (2.5 × 10 −4M) effectively inhibited the release of thromboxane A 2 from ADP-stimulated bovine platelets as did acetylsalicylic acid and verapamil but not dipyridamole. T-2 toxin appears to inhibit bovine platelets by a biochemical mechanism distinct from that of the other inhibitors.

Inhibitory effects of long-chain alkyltrimethylammonium ions on aggregation of bovine platelets and the relation of their effects to Ca 2+ mobilization

The inhibitory efefcts of alkyltrimethylammonium ions on ADP-and thrombin-induced aggregation of bovine platelets were investigated. The ammonium cations inhibited the two aggregation reactions to similar extents. The relationship between their inhibitory efects on ADP-induced aggregation and their alkyl chain lengths from C 8 to C 18 was investigated. Results showed that the inhibitory effects of ammonium cations increased with increase of their alkyl chain lengths up to C 16, and that the increase was linear with chain lengths of up to C 14. This linear relation and slope of the linear regression line suggested that the inhibitory effects of the ammonium cations depended on their partitioning into the membrane. However, unlike long-chain unsaturated fatty acids, they did not affect the membrane fluidity of the platelets, Fluorescence analysis of fura-2 loaded platelets revealed that, in the concentration range where the alkyltrimethylammonium ions inhibited aggregation, they inhibited agonist-induced increase in cytosolic Ca 2+ both in the presence and absence of extracellular Ca 2+. These results suggest that inhibition of platelet aggregation by alkyltrimethylammonium ionsis mainly due to their inhibition of increase in cytoplasmic Ca 2+ by inhibition of both intracellular Ca + mobilization and Ca 2+ uptake.

Relationship of the effects of nigericin on the aggregation and cytoplasmic pH of bovine platelets in the presence of different cations

The effect of nigericin on aggregation of bovine platelets was investigated in media containing the chloride salts of various alkali metal cations of quarternary ammonium cations. In medium with K +, which has the highest permeability with the ionophore among the cations tested, nigericin slightly enhanced both ADP- and thrombin-induced aggregation. In medium with Na +, nigericin scarcely affected ADP-induced aggregation, and slightly inhibited thrombin-induced aggregation. In media with Cs +, choline and tetramethylammonium, it inhibited the aggregations induced by both ADP and thrombin. Measurement of the cytoplasmic pH with the fluorescent probe 2′,7′-bis(carboxyethyl)5,6-carboxyfluorescein showed that nigericin increased the intracellular pH in K + medium and caused its stable decrease (of about 0.6) in Cs +, choline and tetramethylammonium media, but caused only a small transient decrease in medium with Na +. These results suggest that the effects of nigericin on platelet aggregation are mainly due to its effects on the cytoplasmic pH. This conclusion is supported by the findings that the effects on platelet aggregation of other types of ionophore tested were also proportional to their effects on the cytoplasmic pH.

Effects of long-chain cis-unsaturated fatty acids and their alcohol analogs on aggregation of bovine platelets and their relation with membrane fluidity change

The effects of long-chain cis-unsaturated fatty acids with different alkyl chain lengths and different numbers of double bonds on aggregation of bovine platelets and membrane fluidity were investigated. All the cis-unsaturated fatty acids tested inhibited aggregation and at the same time increased membrane fluidity in accordance with their inhibitory effects. The saturated fatty acids and trans-unsaturated fatty acid tested for comparison had much lower or no effects on aggregation and membrane fluidity. The inhibitory effects of mono cis-unsaturated fatty acids increased with increase of their alkyl chain length. cis-Unsaturated fatty acids with two or more double bonds had more inhibitory effects than mono-unsaturated fatty acids. The position of the double bonds had less influence than the number of double bonds. We also examined the effects of cis-unsaturated fatty acids on membrane fluidity with diphenylhexatriene and anthroyloxy derivatives of fatty acids as probes and observed increased fluidity to be considerable in the membrane. The alcohol analogs of cis-unsaturated fatty acids also inhibited aggregation and increased membrane perturbation. These results suggest that the inhibition of platelet aggregation by cis-unsaturated compounds is due to perturbation of the lipid layer.

Effects of monovalent cations and anions on ADP-induced aggregation of bovine platelets, and mechanism thereof

The effects of monovalent cations - inorganic alakali metal cations and organic quanternary ammonium cations - and monovalent inorganic anions on ADP-induced aggregation of bovine platelets were investigated. In the presence of K +, Rb +, Cs +, choline or tetramethylammonium, aggeregation proceeded. However, aggregation was markedly restricted in media containing Li +, Na +, tetrabutylammonium or dimethyldibenzylammonium. With anions, aggregation proceeded in the order Cl − > Br − > I − > Clo 4 − > SCN −. The effects of cations significantly depended on Ca 2+ concentration, whereas those of the anions depended little of Ca 2+. Anions such as SCN − and ClO 4 − markedly decreased the fluorescence of the surface charge probe 2- p-tuluidinylnaphthalene-6-sulfonate, whereas cations had less pronouced effects. The relative effects of the anions on the fluorescence were consistent with their relative inhibitory effects on aggregation. These results suggest that inhibition of platelet aggregation by the anions is due to a change in the surface change of the platelet plasma membrane. On the other hand, kinetic analysis suggests that the effects of monovalent cations on platelet aggregation are due to their competition with Ca 2+ during the process of aggregation.

Aggregation of bovine platelets by acetyl glyceryl ether phosphorylcholine (platelet activating factor)

Platelet activating factor is a multifaceted mediator of inflammation capable of stimulating platelet aggregation as well as anaphylaxis, neutropenia and numerous other in vitro and in vivo cellular changes. This lipid mediator, or autocoid, is released by a wide variety of inflammatory cells following an equally diverse group of cellular stimuli including phagocytosis or antigenic stimulation. The synthesized form of PAF is acetyl glyceryl ether phosphorylcholine (AGEPC). In this study AGEPC aggregated bovine platelets in a dose dependent manner. Maximal, irreversible aggregation occurred at 3.6 × 10 −11 M AGEPC with unwashed platelets and at 8.8 × 10 −12 M AGEPC with washed platelets. Aggregation failed to occur when platelets were tested with the biologically inactive structural analog of AGEPC. The possible contribution by platelet cyclooxygenase products was eliminated by showing lack of platelet aggregation to arachidonic acid and also by pretreating platelets with aspirin.

Inhibition of bovine platelet function by T-2 toxin, HT-2 toxin, diacetoxyscirpenol and deoxynivalenol

The aggregation of bovine platelets suspended in homologous plasma is inhibited in the presence of T-2 toxin, HT-2 toxin, diacetoxyscirpenol (DAS) or deoxynivalenol (DON) when either collagen or ADP is used as the stimulatory agent for aggregation. For each of the mycotoxins the degree of inhibition is dependent on the amount of trichothecene present in the platelet suspension but is not dependent on the time exposure of the platelets to the toxin. For both ADP- and collagen-stimulated platelets, the order of potency of inhibition is T-2 toxin > HT-2 toxin > DAS > DON. A significant ( P < 0.01) dose-dependent decrease was also observed in the amount of the thromboxane B 2 released from collagen-stimulated platelets in the presence of each of the mycotoxins.

Inhibition of ADP-induced aggregation of bovine platelets by saturated fatty acids and its relation with the change of membrane surface charge

The inhibitory effects of saturated fatty acids with 4 to 18 carbon atoms on ADP-induced aggregation of bovine platelets were investigated. The inhibitory effects of the acids increased with increase of their alkyl chain length up to C 14. On the other hand, from C 16 the inhibitory effects tended to decrease with increase of chain length, and stearic acid (C 18 was not inhibitory. There was a linear relationship between the inhibitory effects and alkyl chain lengths up to C 12. This linear relation and the slope of the linear regression line suggested that the inhibitory effects of the acids depended on their partition into the membrane. The fatty acids decreased the fluorescence of the surface charge probe 2-p- toluidinylnaphthalene-6-sulfonate , indicating that they increased the negative charge on the membrane surface. The relative effects of the acids on the fluorescence were consistent with their relative inhibitory effects on aggregation. These results suggest that the inhibition of platelet aggregation by saturated fatty acids is due to a change in the membrane surface charge of the platelet plasma membrane.

Effects of alcohols on ADP-induced aggregation and membrane fluidity of gel-filtered bovine blood platelets.

The effects of four alcohols--n-propyl, n-butyl, n-amyl and n-hexyl alcohol--on the ADP-induced aggregation of gel-filtered bovine platelets were examined. All four alcohols inhibited the aggregation, the order of their effects being n-propyl less than n-butyl less than n-amyl less than n-hexyl. Comparison of the inhibitory effects of the alcohols with their physico-chemical properties showed that their degrees of inhibition depended on their hydrophobicities. Moreover, it was suggested that their interaction with the lipid layer of the membrane was important for the inhibition. Studies on the effects of alcohols on the fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene-labeled platelets showed that the membrane fluidity of the platelets increased in the same concentration range in which aggregation inhibition was observed. Since the alcohols inhibited aggregation without affecting Ca2+ mobilization in the platelets, as revealed in this study, it was concluded that inhibition of platelet aggregation was due to perturbation of membrane lipids by the alcohols. This hypothesis is supported by several recent studies on the effects of cholesterol and cations, which suggest that a relatively rigid membrane favors platelet aggregation.

Inhibitory effects of 4,4′-diisothiocyanostilbene-2,2′-disulfonate (DIDS) on the ADP-stimulated aggregation of gel-filtered bovine blood platelets

The effect of DIDS, a specific inhibitor of anion transport in the erythrocyte membrane, on the ADP-stimulated aggregation of gel-filtered bovine blood platelets was examined. Marked inhibition of aggregation was observed at concentrations of more than 5 x 10(-5)M DIDS. On preincubation with platelets for 30 min, DIDS was more potent and significant inhibition was observed at concentrations of over 2 x 10(-7)M. Since ADP-stimulated aggregation of bovine gel-filtered platelets precedes the release reaction, these results suggest that an anion transport system in the plasma membrane is involved in platelet aggregation.

Effects of anions on ADP-stimulated aggregation of bovine blood platelets

ADP-stimulated aggregation of bovine blood platelets was observed in media containing isotonic potassium salts of various monovalent anions. The aggregation depended on the anion in the medium, the order of aggregation being Cl −, Br −>I −>SCN −, ClO 4 − . After 30-min incubation, the extent of aggregation of platelets in Cl − or Br − medium was little changed, whereas, that in SCN − or ClO 4 − medium was remarkably decreased. This anion dependency of aggregation may be due to change in the membrane potential.

The binding of bovine platelet aggregating factor to human platelets

Bovine platelet-aggregating factor was labeled with 125I by the solid phase lactoperoxidase method with full retention of biological activity. The unlabeled platelet-aggregating factor was shown to compete with labeled platelet-aggregating factor in binding to fresh or formalin-fixed platelets. Scatchard saturation analysis revealed two classes of binding sites with dissociation constants of 0.4 and 6.5 nM and estimated 3,000 high affinity and 12,000 low affinity sites. Ristocetin (1.25 mg/ml) enhanced the binding of PAF to both washed and formalinized platelets by increasing the apparent number of both high and low affinity binding sites, while stimulating the rate of aggregation induced by platelet-aggregating factor on both washed and fixed platelets. Binding of platelet-aggregating factor to platelets was rapid with maximal binding at ten minutes. Rabbit antibody to platelet-aggregating factor formed a rapid (one minute) complex with platelet-aggregating factor which sedimented following ten minutes' centrifugtion at 8,700 g. This complex was soluble in antibody excess.

The influence of 2-mercaptoethanol on von Willebrand factor and bovine platelet aggregating factor.

<h2>Abstract</h2> The influence of a reducing agent, 2-mercaptoethanol (2-ME), on the activity of von Willebrand factor (vWF) and bovine platelet aggregating factor (PAF) was studied. 2-ME was found to inhibit the human platelet agglutination induced by vWF plus ristocetin or by PAF alone. However, binding ability of vWF in the presence of ristocetin or PAF to the platelets was not affected by the addition of 2-ME at a high concentration. Both vWF and PAF were found to be dissociated into subunits in the presence of 2-ME at concentrations inducing the inhibition of platelet agglutination. These findings indicate that although each subunit of vWF or PAF is capable of binding to platelets, a macromolecular constitution of vWF or PAF subunits, linked with disulfide bonds, is required for inducing the platelet agglutination.

Identical binding site on human platelets for von Willebrand factor and bovine platelet aggregating factor

Identity of the binding site on human platelets for von Willebrand factor (vWF) and bovine platelet aggregating factor (PAF) was investigated. Although the 125I labelled bovine PAF alone bound to human platelets fixed with paraformaldehyde, the binding of 1251 labelled vWF was dependent on the presence of ristocetin. The binding of 125I labelled bovine PAF was not inhibited by the presence of human plasma with ristocetin (at a final concentration of 1.5 mg/ml) or vWF alone, but was inhibited by vWF with ristocetin (at a final concentration of 4 mg/ml). Moreover, the binding of 125I labelled vWF with ristocetin was inhibited by the presence of bovine PAF. These results suggest that the binding sites on human platelets for vWF and bovine PAF are identical.