Inhibition of platelet aggregation and the release of P-selectin from platelets by cilostazol

To evaluate the in vitro effects of cilostazol, a phosphodiesterase III inhibitor, on platelet responses, we measured platelet aggregation and the levels of soluble P-selectin, a glycoprotein present on the -granule membrane in resting platelets, and cAMP. Platelet-rich plasma and washed platelets from healthy human volunteers were treated with cilostazol (5, 25 and 50 μM). Platelet-rich plasma was stimulated by ADP (1 and 5 μM) or collagen (5 μg/ml). Washed platelets were stimulated by thrombin (4 U/ml) in the presence or absence of 1 μM forskolin. In vehicle-treated samples, soluble P-selectin levels in response to 1 μM ADP-induced primary aggregation were similar to those of circulating levels of healthy volunteers but the levels in response to 5 μM ADP-induced secondary aggregation and collagen-induced aggregation increased markedly compared to those in response to primary aggregation. This result suggests that P-selectin is released from platelets according to the extent of platelet aggregation. Cilostazol inhibited platelet aggregation as well as P-selectin release in a concentration-dependent manner. Cilostazol inhibited completely thrombin-induced aggregation in the presence of 1 μM forskolin, when cAMP levels were two-fold higher than those in the absence of forskolin. Cilostazol, which increases intracellular cAMP in platelets, may be useful in the treatment of arterial occlusive diseases.     

The involvement of calcium in epinephrine or ADP potentiation of human platelet aggregation

The mechanism by which low doses of epinephrine or ADP potentiate primary platelet aggregation was investigated. Aspirin (lmg/ml)-treated human blood platelets were isolated by albumin density gradient centrifugation. Platelet ⁴⁵Ca uptake associated with epinephrine or ADP addition was determined over a 240 sec time course. Pretreatment of the platelets with ADP (0.5μM) significantly increased aggregation in response to epinephrine (0.1μM). This increased aggregation was associated with a substantially greater ⁴⁵Ca uptake than that which occurred in the presence of epinephrine (0.1μM) alone. The potentiated epinephrine response was inhibited by the Ca²⁺ antagonist verapamil (25μM). This inhibition could in turn be reduced by Ca²⁺ (1mM) addition. Pretreatment of platelets with epinephrine (0.1μM) also increased aggregation in response to ADP (0.5μM). Although this potentiated response was not associated with measurable ⁴⁵Ca uptake, it was nevertheless completely abolished by verapamil (25μM) treatment. These findings suggest that low doses of ADP promote the ability of epinephrine to stimulate an increase in membrane permeability to Ca²⁺.     

Platelet aggregation studies in whole human blood

Since the development of the photometric aggregometer, platelet aggregation studies are routinely performed on platelet-rich plasma (PRP). We have studied platelet aggregation in fresh citrated whole human blood using the recently developed Ultra Flo 100 Whole Blood Platelet Counter. Aggregation of platelets in whole blood was induced with adenosine 5′-diphosphate (ADP; 0.25–10μM), collagen (0.25–1.0μg/ml) and thrombin (0.05–0.2U/ml). Platelet aggregation induced by ADP and thrombin was maximum within 1 min, and that of collagen within 3 mins. Aggregation responses to low concentration of ADP and thrombin were rapidly reversible, whereas responses to collagen and high concentrations of ADP and thrombin were virtually irreversible. The aggregation of platelets was indicated by a fall in platelet count; confirmed by scanning electron micrographs which revealed the presence of large aggregates of platelets, and was prevented when blood was treated with EDTA as anticoagulant. The present technique appears to be rapid, sensitive and reliable; and allows direct measurement of platelet aggregation and disaggregation in whole blood in vitro and ex-vivo.     

Effect of the concentration of Ca2+ in the suspending medium on the responses of human and rabbit platelets to aggregating agents

The effect of the concentration of Ca2+ in the suspending medium of human and rabbit platelets on aggregation, release of 14C-serotonin, and TXB2 formation in response to ADP, thrombin, 1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine (PAF), collagen and arachidonic acid was studied in either platelet-rich plasma anticoagulated with D-phenylalanyl-prolyl-arginyl chloromethylketone (PPACK) or citrate, or suspensions of washed platelets in modified Tyrode-albumin solutions containing 1 mM Mg2+ and concentrations of added Ca2+ ranging from 0 to 5 mM. In response to ADP, thrombin, or PAF, human platelets were stimulated to form TXA2 by close platelet contact in a low-Ca2+ medium; at physiological concentrations of Ca2+, TXB2 formation was much less and declined progressively as the concentration of Ca2+ was raised. When the formation of TXA2 was blocked with aspirin or indomethacin, aggregation and release by human platelets were strongest at physiological concentrations of Ca2+. Rabbit platelet responses differed markedly from those of human platelets because close contact of rabbit platelets in a low-Ca2+ medium did not promote TXA2 formation. Rabbit platelet responses were more strongly inhibited by the lack of added Ca2+ in the medium than the responses of human platelets, possibly because rabbit platelets do not contain releasable Ca2+. In all studies of human platelets in media with low concentrations of Ca2+, the additional contribution to platelet responses of TXA2 formed because of close platelet contact should be considered because TXA2 formation is not usually stimulated in this way at physiological concentrations of Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanisms of vascular graft thrombosis: Role of altered canine platelet sensitivity to thromboxane

Using the standard turbidimetric method of platelet aggregation and quantitation of platelet secretion with ¹⁴C-Serotonin, we have examined the responsiveness of the platelets of mongrel dogs to arachidonic acid (AA), and the thromboxane agonist U46619 in the presence and absence of a subthreshold concentration of epinephrine. In response to stimulation with 750 μM AA, the platelets of 18 dogs produced irreversible aggregation (Group I), the platelets of 22 dogs showed, at most, reversible aggregation (Group II), while the platelets of 8 dogs demonstrated no aggregatory response (Group III). In the presence of AA and a subthreshold concentration of epinephrine (0.5 μM), the platelets of all three groups demonstrated enhanced aggregatory and secretory responses although the extent of ¹⁴C-Serotonin secretion differed significantly between all three groups. These differences in platelet aggregation correlate with the deposition of platelets onto synthetic vascular grafts and the maintenance of graft patency. When stimulated with 0.5 μM U46619 and a subthreshold concentration of epinephrine, the platelets of 97% Group I dogs and 75% of Group II dogs exhibited irreversible aggregation, while the platelets of all Group III dogs showed only reversible aggregation. In addition, significant differences in the extent of ¹⁴C-Serotonin secretion to this combination of agonists were observed between groups. Further examination of the specific effects of U46619 on canine platelets revealed that although the aggregatory and secretory responses to U46619 vary between the different canine platelet populations, the threshold concentration of U46619 required to produce platelet shape change is identical among all groups. Quantitation of the stable metabolite of AA produced via the cyclooxygenase pathway, thromboxane B₂(TxB₂), revealed no significant differences in the production of TxB₂ by the platelets of these different populations upon stimulation with AA. Our results suggest that the mechanisms underlying the differences in responsiveness of canine platelets to AA, are likely due to differences in sensitivity of canine platelets to TxA₂, and may be localized to the mechanism responsible for mediating platelet aggregation and secretion in response to TxA₂.     

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 B₂ formation caused by all aggregating agents, except epinephrine, was incompletely suppressed by 50μM trifluoperazine, and 300μM trifluoperazine only caused complete inhibition of thromboxane B₂ 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 A₂, and that higher concentrations inhibit platelet responses by interfering with protein kinase C.     

Inhibitory effects of sulphated flavonoids isolated from Flaveria bidentis on platelet aggregation

Flaveria bidentis is a plant species that has as major constituents sulphated flavonoids in the highest degree of sulphatation. Among them, quercetin 3,7,3′,4′-tetrasulphate (QTS) and quercetin 3-acetyl-7,3′,4′-trisulphate (ATS) are the most important constituents. Both showed anticoagulant properties. The objective of the present study was to evaluate the effects of these flavonoids on human platelet aggregation in comparison with the well-known inhibitor quercetin (Qc) by using several agonists. Platelet-rich plasma (PRP) or washed human platelets (WP) were incubated with different concentrations of the flavonoids to be tested (1 to 1000 μM, final concentration), and the platelet aggregation was induced by using adenosine 5′-diphosphate (ADP), epinephrine (EP), collagen, arachidonic acid (AA) and ristocetin as agonists. QTS (500 μM) and Qc (250 μM) markedly inhibited platelet aggregation with all the aggregant agents, except ristocetin, whereas ATS (1000 μM) showed only slight antiplatelet effects. In addition, QTS and Qc antagonized the aggregation of PRP or WP induced by U-46619, a mimetic thromboxane A2 (TxA₂) receptor agonist. Challenged with collagen or arachidonic acid, the thromboxane B₂ (TxB₂) formation was also inhibited by the flavonoids, mainly by QTS and Qc, in WP. These results demonstrate that QTS and in minor extension ATS induce a deleterious effect on the production of TxA₂, as judged by TxB₂ formation, in stimulated WP and a marked interference on the TxA₂ receptor according to the profile of inhibition of the agonist-induced platelet aggregation when using ADP, EP, AA and collagen and confirmed with U-46619.     

Endothelin-1 and human platelet activity

Endothelin-1, a peptide produced by endothelium, causes vascular smooth muscle contraction possibly by mobilizing intracellular calcium. Shifts in ionized calcium may also play a role in platelet activation. Accordingly, the effects of endothelin on platelet ionized calcium and aggregation were studied. The measurements were made in aequorin-loaded gel-filtered human platelets derived from healthy donors. Endothelin even in a final concentration of 10⁻⁶M did not cause a measurable change in platelet ionized calcium or aggregation. When tested in combination with collagen, thrombin and platelet activating factor, endothelin showed no synergistic effect. These observations raise the possibility that endothelin may not interact with platelets in a physiologically significant way.     

Investigation of possible mechanisms of pyridoxal 5′-phosphate inhibition of platelet reactions

Pyridoxal 5′-phosphate (PLP) inhibits ADP-induced platelet shape change and aggregation. PLP also causes rapid deaggregation of platelets aggregated by ADP. Effects on platelet aggregation and coagulation are demonstrable following administration to man. Several suggestions concerning the possible mechanism of inhibition have been investigated. PLP did not inhibit the nucleoside diphosphokinase activity of intact rabbit platelets nor of isolated platelet membranes. PLP had no measurable effect on rabbit platelet cyclic AMP either alone or in the presence of prostaglandin E₁ and caffeine. ADP-induced aggregation and ¹²⁵I-fibrinogen binding of rabbit platelets were inhibited concomitantly; however, fibrinogen-induced aggregation of chymotrypsin-treated platelets was only slightly inhibited by PLP. ¹²⁵I-fibrinogen binding to chymotrypsintreated platelets was partially inhibited by PLP; when PLP was added to these platelets, bound fibrinogen was displaced. With chymotrypsin-treated platelets, PLP inhibited the ADP component of the synergistic aggregating effect of the combination of fibrinogen and ADP. It seems unlikely that the inhibitory effects of PLP are due to interference with nucleoside diphosphokinase, increasing platelet cyclic AMP or inhibition of fibrinogen binding. The mechanism of inhibition remains to be established.     

A polyclonal antibody to protein disulfide isomerase induces platelet aggregation and secretion

Monoclonal mouse antiplatelet antibodies against a variety of platelet surface components can activate platelets, causing platelet aggregation and secretion. The mechanism involves binding of the Fab domain to a platelet surface antigen, and the activation occurs through an interaction of the Fc domain with the platelet FcγRII receptor. There is almost no information on FcγRII receptor-dependent activation of platelets by polyclonal rabbit antibodies. We presently report that a polyclonal rabbit antibody to a platelet surface antigen, protein disulfide isomerase, induces platelet aggregation and secretion. These effects are seen with concentrations of the antiprotein disulfide isomerase antibody as low as 25 to 40 μg/mL. Fab and F(ab′)₂ preparations of the rabbit antiprotein disulfide isomerase antibody do not cause aggregation. Fab made from the rabbit antiprotein disulfide isomerase antibody as well as a monoclonal antibody to the FcγRII (IV.3) receptor block the aggregation and secretion responses. Aggregation and secretion are inhibited by an antiglycoprotein IIbIIIa antibody, which blocks fibrinogen binding and wortmannin, an inhibitor of phosphoinositide 3-kinase. Aspirin, prostaglandin E₁, and Ethylenediaminetetraacetic acid (EDTA) also block the platelet responses. These data suggest that activation of platelets by polyclonal antibodies occurs by mechanisms similar to those found with activating monoclonal antibodies.     

In vitro effects of ethanol on rabbit platelet aggregation, secretion of granule contents, and cyclic AMP levels in the presence of prostacyclin

Ethanol, at physiologically tolerable concentrations, inhibits platelet responses to low concentrations of collagen or thrombin, but does not inhibit responses of washed rabbit platelets stimulated with high concentrations of ADP, collagen, or thrombin. However, when platelet responses to high concentrations of collagen or thrombin had been partially inhibited by prostacyclin (PGI2), ethanol had additional inhibitory effects on aggregation and secretion. These effects were also observed with aspirin-treated platelets stimulated with thrombin. Ethanol had no further inhibitory effect on aggregation of platelets stimulated with ADP, or the combination of ADP and epinephrine. Thus, the inhibitory effects of ethanol on platelet responses in the presence of PGI2 were very similar to its inhibitory effects in the absence of PGI2, when platelets were stimulated with lower concentrations of collagen or thrombin. Ethanol did not appear to exert its inhibitory effects by increasing cyclic AMP above basal levels and the additional inhibitory effects of ethanol in the presence of PGI2 did not appear to be brought about by further increases in platelet cyclic AMP levels.     

Platelet cytoskeletal components involved in shape change and secretion

We investigated the role of cytoskeletal components in platelet activation under conditions in which changes in the shape of platelets, their secreting reaction, or both, would occur. When rabbit platelets were stimulated with 0.1 U/ml thrombin, they changed their shape, secreted serotonin, and incorporated actin-binding protein (ABP), myosin, and actin into their cytoskeletons. Platelets treated with 2 μM cytochalasin E and stimulated with 0.1 U/ml thrombin secreted serotonin and assembled myosin and actin into their cytoskeletons, but did not change in shape. Stimulation with a low dose of thrombin (0.001 U/ml) or ADP (10 μM) caused a change in shape and incorporation of ABP, myosin, and actin, but not serotonin secretion. These results suggest that the assembly of myosin and actin into platelet cytoskeletons is related to the secreting reaction, and that ABP, myosin, and actin are all involved in the changes in platelet shape.     

Reduced functions of intracellular Ca in aggregation, secretion and protein phosphorylation of permeabilized platelets from stroke-prone spontaneously hypertensive rats

Aggregation, secretion and 47kDa protein (P47) phosphorylation by various agonists such as thrombin, ADP and ionophore A23187 were markedly reduced in platelets from stroke-prone spontaneously hypertensive rats (SHRSP) compared with those of age-matched Wistar Kyoto rat (WKY) platelets, suggesting defective functions of intracellular Ca²⁺ in SHRSP platelets (Tomita et al. Hypertension 1989: 14: 304–315). To clarify the mechanism of the platelet hypofunctions, saponin permeabilized platelets were prepared to compare the responses of platelets from both rats in varying concentrations of extracellular Ca²⁺. The leakage of lactate dehydrogenase from saponin (15 μg/ml)-treated platelets was approx. 5 % of total activity; the degree of the leakage in both platelets did not differ. In saponin-treated platelets, extracellular Ca²⁺ alone did not induce either aggregation or secretion in both strains. However, in the presence of 1-oleoyl-2-acetylglycerol (10 μg/ml), Ca²⁺ dose dependently stimulated both aggregation and secretion. Under this condition, Ca²⁺ sensitivity of aggregation, secretion and P47 phosphorylation in SHRSP platelets were significantly reduced compared with those in WKY platelets. These results strongly suggest that intracellular Ca²⁺ functions are impaired in SHRSP platelets.     

Effect of heparin on platelet monolayer adhesion,aggregation and production of malondialdehyde

Heparin inhibited monolayer adhesion of washed human and rabbit platelets to collagen-coated glass at 2.5 and 20 units/ml concentration, in the absence of red cells. Adhesion of rabbit platelets to de-endothelialized rabbit aorta, under similar conditions, was less strongly inhibited but no inhibition was seen at 40% haematocrit. Addition of plasma reduced, rather than enhanced heparin activity and hirudin 0.5 units/ml had no significant effect. Heparin also inhibited platelet aggregation, release of (14C) 5-HT and production of malondialdehyde in response to collagen and thrombin. Inhibition of thrombin-induced activity was greater in the presence of plasma. However, heparin enhanced aggregation and release evoked by ADP and did not consistently inhibit MDA synthesis produced by arachidonate. The results indicate that in addition to the effects of heparin on platelet function mediated by anti-thrombin activity and the previously described augmentation of responses to ADP, heparin has weak inhibitory activity against platelet-collagen interactions. Binding of heparin to the platelet membrane (and to surfaces to which platelets adhere) could account for these findings by causing non-specific interference with agonist-receptor interactions.     

Decreased platelet vitamin C in diabetes mellitus: Possible role in hyperaggregation

To determine whether the handling of Vitamin C in the diabetic might be altered and might relate to the increased platelet sensitivity, we have investigated levels of platelet Vitamin C in the diabetic and determined the effects of Vitamin C or on platelet aggregation.

Levels of ascorbic acid, as tested by a lingual method, were significantly lower in diabetics than in normals (p < . 01). Ascorbic acid levels in washed platelets from diabetics were significantly lower than from normals (45. 2±3 μg/10¹⁰ platelets vs. 25. 5±2 μg/10¹⁰ platelets, p < . 001). The effects of ascorbic acid on platelet aggregation were studied by adding ascorbic acid in buffered solution (pH 7. 35) prior to aggregating agents. Ascorbic acid (1000 μg/ml) in platelet-rich plasma consistently inhibited platelet aggregation with threshold concentrations of ADP, epinephrine, and collagen, but enhanced aggregation with arachidonic acid. With washed platelets, ascorbic acid inhibited arachidonic acid-induced aggregation. To rule out an interaction of ascorbi acid and arachidonic acid in the medium, platelets were incubated at 37°C for 10 minutes with varying concentrations of ascorbic acid, rewashed, and aggregation with arachidonic acid tested. Aggregation was inhibited in a linear dose-dependent fashion. Oral ingestion of ascorbic acid (2 gm/day) for seven days by normal non-smoking males produced a marked inhibition of aggregation. In a similar study, platelets from an insulin-dependent diabetic showed no change in aggregation. These results suggest that platelet levels of ascorbic acid may relate to the hyper-aggregation of platelets from diabetics.     

Expression of fibrinogen on the surface of ADP-stimulated platelets: comparison of human and rabbit platelets

Fibrinogen is a cofactor in the aggregation of human platelets and must be added to suspensions of washed human platelets for extensive aggregation to occur in response to ADP. Although the patterns of fibrinogen binding and dissociation during platelet aggregation and deaggregation are similar for human and rabbit platelets, washed rabbit platelets aggregate extensively when stimulated with ADP even in the absence of added fibrinogen. To determine whether secreted fibrinogen was present on the surface of ADP-stimulated platelets and available to support aggregation, the binding of 125I-F(ab')2 fragments of anti-fibrinogen antibodies to formaldehyde-fixed platelets was measured. Although no fibrinogen was detected on human platelets either before or after treatment with ADP, fibrinogen was expressed on the surface of rabbit platelets after stimulation with ADP. However, this secreted fibrinogen did not dissociate during deaggregation of the platelets. Thus, the aggregation of rabbit platelets in the absence of exogenous fibrinogen may be supported by secreted fibrinogen, but a fibrinogen-independent component has not been completely ruled out.     

A comparison of the effects of three positive inotropic agents (amrinone, milrinone and medorinone) on platelet aggregation in human whole blood

Amrinone, milrinone and medorinone inhibit platelet aggregation in human whole blood. They are particularly potent inhibitors of arachidonic acid induced aggregation, inhibiting by 50% (IC₅₀) at concentrations of 1.5μM (milrinone), 7.5μM (medorinone) and 48μM (amrinone). Each drug was less potent at inhibiting ADP and collagen-induced aggregation. The rank order for inhibition of arachidonic acid - induced aggregation correlated well with the rank order of cyclic AMP phosphodiesterase inhibition for these drugs when coupared to the response of a reference cAMP phosphodiesterase inhibitor (CI-930) and a reference cAMP phosphodiesterase inhibitor (M & B 22948). Since inhibition of platelet aggregation occurred at clinically relevant concentrations, it is evident that these agents have potentially beneficial antithrombotic properties.     

Abciximab treatment in vitro after aspirin treatment in vivo has additive effects on platelet aggregation, ATP release, and P-selectin expression

To prevent arterial thrombosis, abciximab is administered together with aspirin. However, whether or not there are benefits to combine abciximab with aspirin is not yet well defined. Healthy volunteers were studied for the effect of aspirin+abciximab using sodium arachidonate and adenosine diphosphate (ADP) alone or in combination to induce platelet activation/aggregation. Abciximab produced complete inhibition of platelet aggregation induced with ADP but only 40% inhibition of aggregation induced by 0.75-mmol/l sodium arachidonate. Abciximab added in vitro to platelet-rich plasma (PRP) from platelets from aspirin-treated donors produced an almost complete inhibition of platelet aggregation. Aspirin, and abciximab alone, did not inhibit adenosine triphosphate (ATP) release as thoroughly as aspirin+abciximab did. Abciximab (3–5 μg/ml) produced inhibition of P-selectin expression induced with 5 (from 46.2±6.0% to 27.4±7.0%, P=.002) and 20-μmol/l ADP (from 53.1±8.1% to 35.1±11.0%, P=.019), but no effect was observed when 0.75-mmol/l sodium arachidonate was used (P=.721). Aspirin diminished P-selectin expression in sodium arachidonate-stimulated platelets (from 77.7±11.8% to 40.2±3.6%, P<.0001) in non-aspirinated and platelets from aspirin-treated donors, respectively. Abciximab (3, 4, and 5 μg/ml) added to platelets from aspirin-treated donors decreased P-selectin expression in platelets stimulated with sodium arachidonate from 40.2±8.6% to 25.6±11.5% (P=.027), to 20.5±3.5% (P<.0001), and to 22.5±1.8% (P<.0001). We concluded that the antiplatelet effect of abciximab is greatly increased by aspirin.     

Pharmacological profile of G619, a new platelet aggregation inhibitor.

G619, a 4-OH-isophthalic acid derivative, was studied for its capacity to inhibit platelet aggregation. G619 dose-dependently inhibited U46619, collagen, ADP, PAF, thrombin and epinephrine-induced platelet aggregation in vitro. The IC50 values for inhibition of U46619-induced human and rabbit platelet aggregation were 39 and 43 microM, respectively. G619, at 100 microM, inhibited high concentration collagen (10 micrograms/ml)-induced aggregation of rabbit platelets pretreated with indomethacin and increased the level of cAMP in washed rabbit platelets by 30% (p less than 0.01 vs basal). However, G619, did not inhibit fibrinogen binding to GPIIb/IIIa receptor, phosphodiesterase, U46619-induced contractile responses on canine saphenous vein or rabbit aorta, calcium-induced vasoconstriction and thrombin or PAF-induced elevation of [Ca++]i in platelets in vitro. In vivo, the U46619-induced maximal thrombocytopenia in rats was reduced from 40% (vehicle) to 22% and 18% by 10 and 30 mg/kg of G619 i.v., respectively. G619 (30 mg/kg) had no effect on the U46619-induced vasopressor response or sudden death in rats, and had no effect on TxB2 formation. Our results indicate that G619 is a broad-spectrum platelet aggregation inhibitor and may have its effect on a common mechanism for platelet aggregation besides an effect on the thromboxane A2 receptor.     

Effects of bupranolol, a new beta-blocker, on platelet functions of rabbit and human in vitro.

The effects of bupranolol, a new beta-blocker, on platelet functions were investigated in vitro in rabbits and humans as compared with propranolol, a well-known beta-blocker. At first, the effect of adrenaline on ADP-induced rabbit platelet aggregation was studied because adrenaline alone induces little or no aggregation of rabbit platelets. Enhancement of ADP-induced rabbit platelet aggregation by adrenaline was confirmed, as previously reported by Sinakos and Caen (1967). In addition the degree of the enhancement was proved to be markedly affected by the concentration of ADP and to increase with decreasing concentration of ADP, although the maximum aggregation (percent) was decreased. Bupranolol and propranolol inhibited the (adrenaline-ADP-)induced aggregation of rabbit platelets, bupranolol being approximately 2.4-3.2 times as effective as propranolol. Bupranolol stimulated the disaggregation of platelet aggregates induced by a combination of adrenaline and ADP, but propranolol did not. Platelet adhesion in rabbit was also inhibited by the beta-blockers and bupranolol was more active than propranolol. With human platelets, aggregation induced by adrenaline was inhibited by bupranolol about 2.8-3.3 times as effectively as propranolol. From these findings. We would suggest that bupranolol might be useful for prevention or treatment of thrombosis.