Some properties of mouse platelets

Mouse platelets were aggregated by arachidonate, thrombin, collagen and ADP. In general they were, like rat platelets, more aggregable in heparinized PRP than in citrated (3.8%) PRP. Mouse platelets underwent the release reaction when aggregated by arachidonate, collagen and thrombin, but not when stimulated by ADP. The aggregation of the platelets to arachidonate was inhibited by cyclooxygenase inhibitors and by prostacyclin. Studies with tritiated arachidonate showed that mouse platelets possess the lipoxygenase and cyclooxygenase pathways found in other mammalian platelets and produce thromboxane and 12-HETE. The mouse provides a convenient model for the study of many conditions known to affect platelet aggregation. The similarity of mouse platelets to the platelets of other mammals together with the ability to study large numbers of animals at low cost, should encourage further use of mouse platelets.     

Influence of time, temperature and platelet concentration on dog platelet aggregation.

Previously encountered variability in platelet aggregation responses in laboratory and clinical investigations prompted this study of common physical factors that affect aggregability of platelets (specifically, canine platelets). Valid comparison of platelet responses under the various conditions of this study was made possible by calibrating the aggregometer-recorder system using the method described below. Using this technique a direct relationship was found between platelet concentration and the amplitude of the aggregation response. For the first 90 min after venepuncture, the magnitude of platelet aggregation was stable, but deterioration in platelet aggregability occurred after two hr in vitro. Choice of short-term (one hr) storage temperature affected the amplitude of aggregation induced by 30 uM epinephrine (EPI), but not by 21 uM adenosine diphosphate (ADP). EPI-induced aggregation was significantly greater after short term storage at room temperature (20°C) than after storage at freezing (0°C) or body (37°C and 40°C) temperatures. Comparing aggregating agents, the average amplitude of aggregation in response to ADP was significantly greater than to EPI even though, in administering equal weights of agents per volume of platelet rich plasma (PRP), the resulting molar concentration of EPI was stronger than that of ADP (30 uM EPI vs 21 uM ADP).     

Overnight food deprivation in normal and diabetic mice markedly enhances thromboxane production by arachidonate stimulated platelet rich plasma and markedly increases platelet phospholipase A2 activity

These studies report the effect of overnight food deprivation on thromboxane production by mouse platelets stimulated with arachidonic acid. Phospholipase activity was also measured. We used radioimmunoassay to measure thromboxane B₂ (TxB₂) in platelet rich plasma (PRP) stimulated with 0.5 mM sodium arachidonate. TxB₂ levels increased from 30 to 180 seconds after stimulation, and at 90 and 180 seconds were approximately twice as high in PRP from food deprived mice as compared with levels in PRP from fed animals (P<.01). The same period of food deprivation (17–21 hours) doubled the phospholipase A₂ (PLA₂) activity of the mouse platelets. No significant differences were observed between the TxB₂ levels in PRP from normal mice and the PRP from mice with streptozotocin diabetes, although the marked elevation of TxB₂ levels produced by food deprivation was somewhat greater in the PRP from diabetics. No differences were observed in PLA₂ activity in diabetic as compared with control platelets. The marked enhancement of PLA₂ activity produced by food deprivation, was seen in both control and diabetic platelets.     

Unexpected effects of aurin tricarboxylic acid on human platelets.

Aurin tricarboxylic acid (ATA) is a potent inhibitor of ristocetin-mediated platelet agglutination and of shear-induced, von Willebrand factor (vWf)-mediated platelet aggregation, probably via inhibition of vWf interaction with glycoprotein Ib (GPIb). We examined the effects of ATA (both the sodium salt and a solution of ATA in ethanol) on platelet functions in citrated plasma (PRP) and in suspensions of washed platelets in Tyrode-albumin solution (contains 2 mM Ca2+). ATA (42-211 micrograms/ml) blocked aggregation and release of granule contents induced by thrombin (0.15 U/ml in PRP; 0.03 U/ml in platelet suspension). Responses to higher concentrations of thrombin were not inhibited. ATA also prolonged thrombin-induced clotting of fibrinogen. Since ATA had no effect on fibrinogen-induced responses of chymotrypsin-treated platelets, ATA probably acts on thrombin rather than on fibrinogen. In PRP and platelet suspensions, ATA (acid form 106 micrograms/ml; sodium salt 122 micrograms/ml) had little effect on ADP-induced platelet aggregation. The sodium salt of ATA (61-122 micrograms/ml) enhanced collagen-induced aggregation and release by platelets in citrated plasma and by washed platelets; the enhancement was extensively inhibited by aspirin. With platelet suspensions, ATA significantly enhanced aggregation and release caused by low concentrations of sodium arachidonate (15-50 microM); aggregation and release caused by higher concentrations of arachidonate were somewhat inhibited by ATA. Arachidonate-induced aggregation and release were also enhanced by ATA in PRP. ATA enhanced aggregation and release induced by the calcium ionophore A23187; aspirin had little effect on the enhancement.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence that the rat is not an appropriate model to study the role of prostaglandins in normal or abnormal platelet aggregation

Abnormal platelet aggregation seen in experimentally induced diabetic, hypercholesterolemic and spontaneously hypertensive rats (SHR) has been linked with increased prostaglandin synthesis. The present study was conducted to examine the role of prostaglandins in rat platelet activation using normal Wistar Kyoto (WKY) and SHR rats. Up to 30 microM ADP did not induce secondary phase of platelet aggregation in rat PRP and up to 30 microM epinephrine did not produce any response in rat PRP. In other experiments ADP (1.0 microM) and epinephrine (2.0 microM) induced typical biphasic aggregation responses in human PRP. Up to 20 microM U46619, a stable analog of prostaglandin H2, did not induce platelet aggregation in rat PRP or washed rat platelets. In contrast 2.0 microM U46619 caused maximal aggregation in human PRP and washed human platelets. Arachidonic acid (1.5-2.0 mM) induced aggregation in washed rat platelets. However, this was associated with excessive (67% and 94%) loss of cytoplasmic LDH. The low concentrations of thrombin (0.04 and 0.05 U/ml), induced two to three-fold increase in aggregation response in SHR platelets as compared to WKY platelets. Higher concentrations of thrombin (0.1 and 0.3 U/ml) induced similar aggregation responses in SHR and WKY platelets. Thrombin (0.04-0.3 U/ml) induced serotonin secretion in a concentration dependent manner. The extent of secretion was the same in SHR and WKY platelets at all concentrations. Thrombin-induced synthesis of thromboxane A2 (TXA2) in WKY and SHR platelets was quantified using a radioimmunoassay for TXB2. Thrombin (0.04-0.3 U/ml) produced TXB2 in WKY and SHR platelets in a concentration dependent manner. The SHR platelets produced significantly larger amounts of TXB2 as compared to WKY platelets. In other experiments aspirin (500 microM) inhibited thrombin (0.05 U/ml) induced TXB2 synthesis by 75% in both WKY and SHR platelets but failed to inhibit aggregation or secretion in either WKY or SHR platelets. Based on these data it is suggested that: (a) rat platelets inspite of their ability to synthesize TXA2 do not require TXA2 for aggregation; and (b) the rat may not be an appropriate model to study the role of prostaglandins in normal or abnormal platelet aggregation.     

Investigation on a selective non-prostanoic thromboxane antagonist, BM 13.177, in human platelets

The mode of action of BM 13.177 (4-[2-(benzenesulfonamido)-ethyl] phenoxyacetic acid), a new anti-aggregating and anti-thrombotic agent, was studied in human washed platelets and citrated PRP. With ASA-treated platelets, BM 13.177 (0.1 - 100 microM) did not inhibit the shape change and the aggregation induced by ADP, serotonin, adrenaline, thrombin, or collagen. Therefore, BM 13.177 is neither an antagonist of ADP, serotonin, adrenaline, thrombin, or collagen nor a common pathway inhibitor like PGE1, or an inhibitor of the platelet interactions during aggregation. However, BM 13.177 (greater than or equal to 0.1 microM) produced a dose-dependent reduction of shape change, aggregation and release of [3H]serotonin induced by the stable PGH2 analogues U 46619 and U 44069 in ASA-treated platelets or ASA-treated citrated PRP. In untreated platelets, BM 13.177 inhibited platelet activation by U 46619 or U 44069 and by exogenous arachidonic acid or by endogenous arachidonic acid mobilized by hydrogen peroxide. Consequently, the ADP- and adrenaline-induced secondary aggregation and [3H]serotonin release in citrated PRP and the major effects of collagen were also inhibited. In washed platelets treated with 10 microM arachidonic acid or 100 microM hydrogen peroxide, the formation of TXB2 was not inhibited by 10 microM BM 13.177. However, the TXB2 formation after stimulation with 1,200 microM hydrogen peroxide was partially reduced by BM 13.177 to the same extent as by PGE1. This reduction may be due to the absence of a secondary release of arachidonic acid from phospholipids if the platelets were prevented from activation by BM 13.177 or PGE1. Arachidonic acid and hydrogen peroxide also induced the shape change, aggregation and release of washed platelets when thromboxane formation was inhibited by dazoxiben. Under these conditions, BM 13.177 was able to abolish the platelet response which was due to accumulating prostaglandin endoperoxides. These results show that BM 13.177 acts as a selective antagonist of TXA2 and prostaglandin endoperoxides. Its inhibitory effect on platelet function does not depend on an inhibition of either the primary release of arachidonic acid or the activities of cyclooxygenase or thromboxane synthetase.     

Extracellular-free Ca++ accounts for the sex difference in the aggregation of human platelets in citrated platelet-rich plasma

It has been shown in Poiseuille flow, that the ADP-induced aggregation of human platelets in citrated plasma from female donors is significantly greater than from male donors over a range of mean tube shear rate, G, from 41.9 s-1 to 1920 s-1 and mean transit time, t, from 0.2 to 86 s. The present work verifies the sex difference at G = 335 s-1 and t = 43 s and deals with the effect of free Ca2+ on it. An inverse correlation between the extent of single platelet aggregation and donor hematocrit, and between hematocrit and the plasma ionized calcium concentration, [Ca2+], as well as a positive correlation between the extent of single platelet aggregation and [Ca2+] was found. This indicated that the sex difference is due to hematocrit-dependent differences in the [Ca2+] that result when a fixed volume of the chelating agent citrate is used to anticoagulate blood. When the initial citrate concentration was adjusted to compensate for the variable volume dilution of citrate in plasma among donors and the [Ca2+] of males raised above that of females, the sex difference was reversed. Again, aggregation correlated with [Ca2+]. At the physiological [Ca2+] in both heparinized PRP and hirudinized PRP, the rate of aggregation and aggregate size were much greater than in citrated plasma but no sex difference was detected.     

Thromboxane A2 and the endoperoxides mediate canine platelet activation

With canine platelets, arachidonate consistently induces a shape change and potentiates the response to a submaximal ADP stimulus even though it does not generally induce aggregation. We have determined that TXA₂ and possibly the endoperoxides are responsible for these effects based on the following data: (i) indomethacin (14uM) blocks both actions of arachidonate, (ii) imidazole (5uM) partially blocks both, and (iii) the endoperoxide analog, U-46619, produces effects similar to arachidonate, whereas other stable metabolites of arachidonate (PGE₂, PGF₂, PGD₂, PGI₂, 6-keto-PGF₁ and TXB₂) do not. Specifically, PGI₂ and PGD₂ inhibit ADP-induced aggregation; low concentrations of PGE₂ and PGF₂ facilitate secondary aggregation; and the other metabolites are relatively inactive. With the exception of PGF₂ , these activities are qualitiatively identical to those observed with human platelets. A decreased sensitivity of canine platelets to TXA₂ and the endoperoxides is advanced as the explanation of their diminished responsiveness to arachidonate relative to other species.     

Effect of sodium arachidonate on thrombin generation through platelet activation--inhibitory effect of aspirin

BACKGROUND: Sodium arachidonate was used in this study to determine its capacity to generate thrombin through platelet activation. Whether aspirin prevent this effect was also investigated. METHODS AND RESULTS: Seventeen healthy volunteers without and after 160 mg/day aspirin intake for 3-5 days were studied. Lag-time and TG at basal condition and after platelet stimulation by sodium arachidonate (AA) were measured in normal non-aspirinated as well as "in vivo" aspirinated platelet rich plasma. (PRP). The lag-time was statistically significant shorter in non-aspirinated PRP activated with AA compared with non-activated PRP. This effect was inhibited by aspirin. In non-aspirinated PRP, there was an increase of TG at 4 and 6 min. incubation when platelets were activated with AA but the difference disappeared after 8 min. incubation, (84 +/- 71; 148 +/- 58 and 142 +/- 92 nmol/L respectively) compared with non-aspirinated. non-activated platelets (16 +/- 23; 55 +/- 56 and 111 +/- 76 nmol/L at 4,6 and 8 min, p < 0.0001, p < 0.0001 and p = 0.292, respectively). The AUCo-->22 min were 520.6 +/- 545.5 in non-aspirinated, non-stimulated PRP and 808.9 +/- 617, in non-aspirinated PRP activated with sodium arachidonate (p = 0.014). Aspirin administered in vivo produced a decrease of TG in PRP activated with AA. CONCLUSION: Platelet activated by AA trigged TG. This effect was inhibited by aspirin and could be an additional beneficial effect of aspirin in the prevention of thrombosis.     

Neferine exerts its antithrombotic effect by inhibiting platelet aggregation and promoting dissociation of platelet aggregates

Introduction

Neferine, a kind of isoquinoline alkaloid, extracted from the seed embryo of Nelumbo nucifera Gaertn, has long been recognized in traditional medicine as a medicinal plant with various usages. Neferine has many biological activities, including anti-hypertensive, anti-arrhythmic, negative inotropic effect and relaxation on vascular smooth muscle. Although previous studies have reported its antithrombotic effect, the mechanisms by which it exerts antithrombotic effect have not been thoroughly studied.

Method

Washed mice platelets and mice platelet-rich-plasma (PRP) were used to investigate the effects of neferine on platelet aggregation, secretion induced by various agonists and dissociation of agonist-formed platelet aggregates. Bioflux plates coated with collagen were used to investigate the effect of neferine on platelet adhesion and thrombosis in vitro. With collagen-epinephrine-induced acute pulmonary thrombus formation mouse model, the effect of neferine on thrombosis in vivo was also examined.

Results

Neferine, significantly and dose-dependently, inhibited collagen-, thrombin-, U46619-induced platelet aggregation in mice washed platelets, or ADP-induced platelet aggregation in PRP. Neferine treatment decreased platelet dense granule secretion initiated by collagen, thrombin and U46619. Also, Neferine dramatically and dose-dependently promoted the dissociation of platelet aggregates pre-formed by various agonists including collagen, thrombin, U46619 or ADP. Neferine can significantly reduce the area of mice platelets adhesion to the collagen and inhibit thrombosis in vitro. In collagen-epinephrine-induced acute pulmonary thrombus mouse model, neferine, at 6 mg/kg, significantly attenuated thrombus formation.

Conclusions

Neferine remarkably prevents thrombus formation by inhibiting platelet activation, adhesion and aggregation, as well as promoting disassembly of pre-formed platelet aggregates. The inhibitory effects of neferine on platelet activation might be relevant in cases involving aberrant platelet activation where neferine could be used as an anti-platelet and antithrombotic agent.     

Why single daily dose of aspirin may not prevent platelet aggregation

The effect of different doses of aspirin on the synergistic activity of sodium arachidonate plus platelet activating factor (paf) ADP or collagen in platelet aggregation was studied in human volunteers. Aggregation studies in platelet rich plasma (PRP) showed that aspirinated platelets, unresponsive to arachidonate, when stirred with threshold concentrations of paf, ADP or collagen, reacted differently according to the dose of aspirin and the time elapsed since ingestion. After a single or daily 50 mg dose for 7-10 days independent of elapsed time until blood withdrawal, a complete synergistic activity was obtained. In PRP samples obtained 24 hours after the last aspirin intake, a complete synergistic aggregation was achieved after a single dose or after 7-10 days of 500 mg aspirin ingestion; synergistic effect did not appear when blood was drawn 2.5 hours after intake. The thromboxane B2 concentrations were very low in all samples after PRP stimulation with sodium arachidonate or paf or both. As rationale is that platelet activation in vivo occurs in response to several stimuli, the therapeutic implications of our results is that aspirin may not prevent the agonist potentiation effect when low dose or daily high dose (500mg) are administrated. This may explain the erratic results of most aspirin trials in which this drug was used to suppress platelet function.     

Arachidonate-induced platelet aggregation in the dog.

The platelet rich plasma (PRP) from most dogs aggregates in response to ADP, collagen and thrombin. Dog PRP generally does not aggregate in response to epinephrine, and previous studies found that dog PRP uniformly failed to aggregate when exposed to arachidonic acid prepared in an ethanol-sodium carbonate medium. Our studies demonstrate that 30% of randomly selected mongrel dogs have PRP which aggregates when exposed to sodium arachidonate dissolved in modified Tyrode's buffer, PRP from these dogs also aggregates with lower concentrations of ADP and collagen than PRP which is unresponsive to arachidonate. Pre-incubation of PRP with epinephrine uniformly transforms PRP which does not aggregate on exposure to arachidonate alone into arachidonate-aggregating PRP. Dog PRP which aggregates with arachidonate release ¹⁴C-serotonin, while non-aggregating PRP does not. However, arachidonate stimulates malondialdehyde production in both aggregating and non-aggregating PRP.The results of this study indicate that dogs are heterogeneous in regard to their aggregation response to arachidonate. The mechanism of this heterogeneity is unknown: however, since prostaglandin metabolism is intact and the platelets of some dogs respond to arachidonate alone, it appears to be the result of variable sensitivity to endoperoxides and thromboxane A₂.     

Synergistic actions of paf-acether and sodium arachidonate in human platelet aggregation. 2. Unexpected results after aspirin intake

The effect of sodium arachidonate and paf-acether on the activation of human platelet rich plasma from volunteers 2.30 to 36 hours after 500 mg of aspirin intake was studied. Concentrations of paf-acether which induce a reversible aggregation in platelet rich plasma (PRP) (0.29-0.029 microM) and concentrations of sodium arachidonate (AA) which don't produce aggregation (0.75-1mM) on the PRP from these volunteers, induced full aggregation when added together. But no cooperation activity was achieved in the 2.30 hours sample. Contrarily to the in vitro studies performed in human normal PRP, ASA (200 micrograms/ml) or indomethacin(12 microM) added to the PRP were unable to suppress the cooperative aggregation effect; neither did apyrase (12U/ml), esculetin (10 microM) or nordihydroguaiaretic acid (0.1 microM) have any action on the activated platelets but the synergistic action is completely suppressed by BW 755C (0.1 mM). TXB2 formation is very low in all these activated samples and insufficient to cause platelet aggregation. These results suggest 2 behaviors of platelets: synergistic activity of paf-acether and exogenous AA in vitro on normal human PRP is mediated mainly through active metabolites of AA formed via cyclooxygenase, as was previously published. When cyclooxygenase is inhibited in vivo by administration of 500 mg ASA, the cooperative effect of agonists is still present but the active aggregating product(s) is probably, formed through a pathway different of that of the cyclooxygenase or lypoxygenase.     

Increase of platelet thromboxane A2 formation and of its plasmatic half-life in diabetes mellitus

Platelet-rich plasmas (PRP) of diabetic subjects exhibited a hyperaggregability to either collagen or sodium arachidonate. These platelets, isolated from their plasma, only showed a normal response to sodium arachidonate. Moreover, the biosynthesis of prostaglandins and related compounds from endogenous arachidonate (thrombin as inducer) was enhanced in diabetic platelets whereas the biosynthesis from exogenous arachidonate was normal. On the other hand, the half-life of thromboxane A₂ was longer in diabetic platelet-poor plasma than in the control. It is concluded that a part of the hyperaggregability of diabetic PRP to sodium arachidonate could be due to the increase of the half-life of thromboxane A₂. Otherwise, hypersensitivity of isolated diabetic platelets to either collagen or thrombin shows a greater availability of phospholipidic arachidonate to the prostaglandin synthetase system.     

Characterization of U46619 binding in unactivated, intact human platelets and determination of binding site affinities of four TXA2/PGH2 receptor antagonists (13-APA, BM 13.177, ONO 3708 and SQ 29,548)

The binding of U46619 and the inhibition of this binding by four TXA2/PGH2 receptor antagonists (13-APA, BM 13.177, ONO 3708 and SQ 29,548) were studied in unactivated, intact human platelets. Washed platelets were equilibrated with [3H]-U46619 (5 nM) and the time course of binding determined. The receptor-specific binding reached equilibrium within 2-4 minutes, and could be displaced by addition of excess unlabelled ligand. Saturation of this binding was achieved at 750 nM. Scatchard transformation of the saturation binding curve yielded a single class of binding site with a Kd of 108 nM and Bmax of 360 fmole/10(8) platelets. When [3H]-U46619 (4 nM) was incubated with platelets in the presence of increasing concentrations of the antagonists, binding of U46619 was inhibited in a dose dependent manner. The potency series for inhibition of U46619 binding was: SQ 29,548 (IC50 = 7.9 nM) greater than ONO 3708 (IC50 = 38 nM) greater than BM 13.177 (IC50 = 0.91 microM) greater than 13-APA (IC50 = 6.2 microM). These findings are consistent with the notion that these compounds all act as competitive antagonists at the level of the platelet TXA2/PGH2 receptor.     

Effects of any epoxymethano stable analogue of prostaglandin endoperoxides (U-46619) on human platelets

U-46619 is a stable epoxymethano analogue of cyclic endoperoxide PGH2. We studied platelet aggregation, 14C-5HT release, LDH extrusion and prostaglandin and thromboxane production induced by this compound in platelet-rich plasma samples from 15 healthy volunteers. Each subject was tested both before and 90 min after aspirin (500 mg) ingestion. The threshold aggregating concentration (TAC) of U-44619 ranged between 0.18 and 0.90 micro M. Aggregation was maximal between 40 and 60 min after venipuncture and was concentration-dependent. At concentrations below the TAC, U-44619 induced primary reversible aggregation with minimal 14C-5HT release. At TAC or higher concentrations aggregation and release proceeded as parallel events. Neither prostaglandin or thromboxane production nor LDH loss could be detected in any of the situations tested. Aspirin ingestion did not modify the pattern of platelet responses. In unstirred, not aggregated platelet samples 14C-5HT release by U-46619 occurred to a similar extent as in stirred, aggregated platelet samples. Addition to citrated PRP of 0.3 mM Na2 EDTA blocked both aggregation and release induced by U-46619. This compound, however, aggregated washed platelets resuspended in Ca++-free-tyrode-albumin containing fibrinogen. The mechanism by which U-46619 activates platelets differs from that of all other common aggregating agents.     

Sodium arachidonate induced platelet aggregation is independent of secreted adenosine diphosphate

Human gel-filtered platelets or platelet-rich plasma were stimulated by sodium arachidonate or by ADP in the presence of two compounds known to inhibit ADP mediated aggregation and secretion - ATP and N-ethylmaleimide. Using gel-filtered platelets and the lowest concentration of agonist necessary to elicit maximum aggregation, fifty percent inhibition of ADP-mediated aggregation required 9 μM N-ethylmaleimide or 23 μM ATP. Sodium arachidonate-mediated aggregation was significantly less sensitive; equivalent inhibition required 30 uM N-ethylmaleimide or >500 μM ATP. Concentrations of both inhibitors were determined that would completely inhibit ADP-induced aggregation yet would not completely prevent sodium arachidonate-induced aggregation. Furthermore, this concentration of N-ethylmaleimide could not be overcome by up to 500 uM ADP, demonstrating that the observed arachidonate-induced aggregation was not due to the effects of a small amount of secreted ADP acting at the platelet surface. Therefore, aggregation of human platelets induced by arachidonic acid can occur by a mechanism that is independent of secreted ADP.     

Characteristics of thrombin-degranulated human platelets: development of a method that does not use proteolytic enzymes for deaggregation

A method has been developed for preparing suspensions of washed human platelets that have lost as much as 90% of their dense granule and alpha granule contents as a result of stimulation by thrombin (0.9 U/ml for 3 min at 37 degrees C), and recovering the platelets without using a proteolytic enzyme. Glycyl-L-prolyl-L-arginyl-L-proline (GPRP) was used to prevent polymerization of released fibrinogen and arginyl-glycyl-aspartyl-serine (RGDS) to block the interaction of released fibrinogen, vWf or fibronectin with the glycoprotein IIb/IIIa complex. The thrombin used to degranulate the platelets was neutralized with D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone (FPRCH2Cl) and prostaglandin E1 was added to return the platelets towards a disc shape. The degranulated platelets aggregated in response to ADP, platelet activating factor, arachidonate and the thromboxane A2 mimetic, U46619 in the presence of added fibrinogen; the platelets changed shape but did not aggregate in response to collagen. Thrombin and the calcium ionophore, A23187, caused aggregation without added fibrinogen. Synergism between pairs of aggregating agents at low concentrations was observed. Little TXB2 was formed when the platelets were reaggregated by thrombin. RGDS and F(ab')2 fragments of an antibody to fibrinogen inhibited reaggregation induced by thrombin and A23187 indicating that small amounts of fibrinogen at the platelet surface may support aggregation by strong agonists. Adherence of thrombin-degranulated platelets to a collagen-coated surface was less than for controls, but spreading was more extensive. Electron-microscopic immunogold cytochemistry with anti-human fibrinogen IgG showed numerous gold particles in platelet vacuoles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of protein kinase C in U46619-induced platelet shape change, aggregation and secretion

Stimulation of rat platelets with U46619 induced Ca2+ mobilization and platelet shape change, but aggregation and secretion were induced when platelets were stimulated with U46619 plus phorbol 12-myristate 13-acetate (PMA). However, stimulation of rabbit platelets with U46619 induced all the three platelet responses. Aggregation and secretion of rabbit platelets were enhanced by simultaneous addition of PMA and inhibited by staurosporine, but platelet shape change was not affected by them. These results suggest that protein kinase C is important for aggregation and secretion, but not for platelet shape change. On the other hand, pretreatment of platelets with PMA inhibited platelet shape change as well as Ca2+ mobilization and inositol phosphate formation, indicating that the shape change was mediated by PMA-sensitive mechanism which was not clarified.     

Two phasic generation of thromboxane A2 by the action of collagen on rat platelets

Two-phase thromboxane A2 (TXA2) generation was observed in washed rat platelets stimulated by collagen. One coincided with shape change and the other with aggregation, and both reactions were inhibited by indomethacin. The collagen-induced first phase was not affected by treatment of the platelets with TXA2 receptor antagonist BM13177, while the shape change, the second phase TXA2 generation and aggregation were completely suppressed. U46619, a stable TXA2 mimetic, induced only platelet shape change and not aggregation or endogenous icosanoid mobilization. However, upon addition of U46619 to platelets previously stimulated by collagen in the presence of indomethacin, icosanoids formation was induced together with aggregation. These results suggest that both the collagen-induced initial TXA2 and the occupation of the receptor by collagen might be a trigger for the second-phase TXA2 formation in concert with platelet aggregation.