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.     

Protective effect of three diallyl sulphides against glucose-induced erythrocyte and platelet oxidation,and ADP-induced platelet aggregation

Isolated human erythrocyte membrane and platelet were used to study the antioxidative and anti-aggregative effects of three diallyl sulphides (diallyl sulphide, DAS; diallyl disulphide, DADS; diallyl trisulphide, DAT) against glucose-induced oxidation and adenosine 5′-diphosphate (ADP)-induced platelet aggregation. Three sulphide agents showed dose-dependent antioxidative protection against glucose-induced erythrocyte membrane oxidation (p<.05), and these agents at 10 μM significantly increased the retention of -tocopherol in erythrocyte membrane (p<.05), in which DAT was the most effective agent (p<.05). Three sulphide agents significantly inhibited 30 and 50 mM of glucose-induced platelet oxidation (p<.05). The anti-aggregative activity of each sulphide agent was dose dependent (p<.05), in which DAT showed the greatest inhibitory effect on platelet aggregation than DADS, followed by DAS (p<.05). After ADP stimulation, the malondialdehyde (MDA) formation in platelets treated with sulphide agents was significantly less (p<.05), in which DADS and DAT showed similar antioxidative activities (p>.05). These results suggested that DADS and DAT could be considered as strong antioxidative and antithrombotic agents to prevent or control oxidative damage and platelet hyperactivity.     

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.     

The influence of glutathione and other thiols on human platelet aggregation

The platelet membrane contains sulfhydryl groups which are essential for normal platelet function. Reduced glutathione (GSH) and other thiols such as cysteine and 6-mercaptopurine were found to inhibit human platelet aggregation induced by adenosine diphosphate (ADP), collagen and arachidonic acid. The inhibition of ADP-induced aggregation by GSH (IC₅₀=0.61 ± 0.05 mM) was greater than that by cysteine (IC₅₀=13 ± 1 mM) or 6-mercaptopurine (IC₅₀=5.4 ± 0.2 mM). Two other thiols, dithiothreitol and beta-mercaptoethanol were found to cause platelet aggregation instead of inhibition. The interaction of GSH with the ADP receptor was noncompetitive in nature.     

Vitamin E inhibition on platelet procoagulant activity: involvement of aminophospholipid translocase activity

Background

Activated platelets provide an important procoagulant surface, because exposed negatively charged phosphatidylserine (PS) is an important cofactor of the coagulation cascade. Aminophospholipid translocase (APLT) can transport PS from the outer to the inner membrane leaflet. Although vitamin E has been investigated for its anti-aggregating effect on platelets, its effect on platelet procoagulant activity has not been reported.

Methods

Phorbol 12-myristate 13-acetate (PMA), a well-known PKC activator, and thrombin were used to induce PS exposure on platelet surface. The expression of PS was measured by annexin A5 binding with flow cytometry. Platelet procoagulant activity was measured by a prothrombinase assay. APLT activity was measured by flow cytometry by determining the percent of 1-palmitoyl-2-[6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]caproyl]-sn-glycero-3-phosphatidylserine (NBD-PS) translocated from the outer to the inner membrane leaflet. Inhibition effects of vitamin E on platelet aggregation were simultaneously measured by a Multiplate aggregometer, a Chrono-log aggregometer, and a PFA-100 system.

Results

Vitamin E significantly attenuated PMA-induced conformational change of glycoprotein IIb/IIIa and P-selectin expression. Vitamin E significantly inhibited PMA and thrombin-induced PS externalization and reduced prothrombinase activity on platelet surfaces both in vitro and ex vivo. APLT activity was increased by vitamin E in a dose-dependent manner, indicating that reduced procoagulant activity may be attributed, at least in part, to this increased APLT activity. Vitamin E inhibited platelet aggregation induced by combined chemokine SDF-1 and low-dose ADP as well as by usual doses of ADP or collagen when measured by the Multiplate and Chrono-log aggregometers but not when measured by PFA-100.

Conclusions

These in vitro and ex vivo results showed that vitamin E inhibited platelet PS exposure and procoagulant activity partly by increasing APLT activity. These actions of vitamin E on platelet function provide new insights into the anticoagulation properties of vitamin E.     

Platelet spontaneous aggregation in platelet-rich plasma is increased in habitual smokers

Cigarette smoking is a well-known risk factor for atherosclerotic disorders. Several authors have suggested that platelet aggregability is important in smoking-induced vascular injury. When platelet-rich plasma is stirred at 37°C in the absence of chemical stimulants, small aggregates of platelets may be formed, but it was difficult to detect small aggregates by conventional aggregometer using optical density. Recent technological advances have made it possible to detect small aggregates by using a newly developed assay system that employs laser light scattering. In the present study, we attempted to measure platelet aggregation by this method, using laser light scattering in 54 nonsmoking healthy males and 51 healthy male habitual smokers who were age matched. In smokers, blood was obtained after 10 hours of smoking abstinence. No significant difference in platelet aggregation was induced by 1 μM or 5 μM of ADP between smokers and nonsmokers. In smokers, plasma fibrinogen levels and the number of small aggregates formed in the absence of chemical stimulants was significantly higher than in nonsmokers. Small aggregates formed in the absence of stimulants correlated positively with the concentrations of von Willebrand factor (vWF) antigen (r=0.2654, p<0.01) and of fibrinogen (r=0.2834, p<0.01). The formation of these small aggregates was inhibited by monoclonal antibody against GPIIb/IIIa blocking fibrinogen binding to GPIIb/IIIa but not inhibited at all by monoclonal antibody against GPIb blocking vWF binding to GPIb. From these results, enhanced platelet aggregability in smokers was confirmed, and it was suggested that GPIIb/IIIa is concerned in platelet spontaneous aggregation, although vWF may not directly influence on the platelet spontaneous aggregation. Since the mechanism of spontaneous aggregation and the effect of increased spontaneous aggregability on the progression of atherosclerosis remains unclear, further study was considered necessary.     

The effect of red blood cells on the ADP-induced aggregation of human platelets in flow through tubes

The effect of red blood cells, rbc, and shear rate on the ADP-induced aggregation of platelets in whole blood, WB, flowing through polyethylene tubing was studied using a previously described technique (1). Effluent WB was collected into 0.5% glutaraldehyde and the red blood cells removed by centrifugation through Percoll. At 23 degrees C the rate of single platelet aggregation was upt to 9 x greater in WB than previously found in platelet-rich plasma (2) at mean tube shear rates G = 41.9, 335, and 1,920 s-1, and at both 0.2 and 1.0 microM ADP. At 0.2 microM ADP, the rate of aggregation was greatest at G = 41.9 s-1 over the first 1.7 s mean transit time through the flow tube, t, but decreased steadily with time. At G greater than or equal to 335 s-1 the rate of aggregation increased between t = 1.7 and 8.6 s; however, aggregate size decreased with increasing shear rate. At 1.0 microM ADP, the initial rate of single platelet aggregation was still highest at G = 41.9 s-1 where large aggregates up to several millimeters in diameter containing rbc formed by t = 43 s. At this ADP concentration, aggregate size was still limited at G greater than or equal to 335 s-1 but the rate of single platelet aggregation was markedly greater than at 0.2 microM ADP. By t = 43 s, no single platelets remained and rbc were not incorporated into aggregates. Although aggregate size increased slowly, large aggregates eventually formed. White blood cells were not significantly incorporated into aggregates at any shear rate or ADP concentration. Since the present technique did not induce platelet thromboxane A2 formation or cause cell lysis, these experiments provide evidence for a purely mechanical effect of rbc in augmenting platelet aggregation in WB.     

Modification of platelet functions by monobromobimane, a fluorescent thiol group label

Monobromobimane (mBBr, bimane), a compound that penetrates cells and forms a fluorescent adduct with thiol groups, was used to asses the significance of thiols in platelet function. Exposure of washed platelets for 1 min to 100 microM mBBr abolished ADP-induced aggregation; shape change was not inhibited by 500 microM mBBr. The nonpenetrating compound monobromotrimethylammoniobimane was ineffective. Established ADP-induced aggregation was reversed by bimane, and fibrinogen binding to ADP-stimulated platelets was inhibited, an effect mainly due to decreased number of binding sites. Aggregation stimulated by A23187 and arachidonate was less effectively inhibited whereas epinephrine- and collagen-induced aggregation were abolished by 50 microM mBBr. Similar effects on aggregation and secretion were observed in platelet-rich plasma except that higher mBBr concentrations were usually necessary. Aggregation and 14C-serotonin secretion stimulated by 0.1 U/ml thrombin were partially inhibited by pretreatment with bimane. With lower thrombin concentrations, they were often enhanced, as was 3H-arachidonate release. Bimane inhibited epinephrine-induced arachidonate release in gel-filtered platelets, possibly because it abolished the primary aggregation necessary for this release. mBBr did not elevate cyclic AMP but enhanced the increase induced by PGE1 and prevented the subsequent decrease typically caused by ADP. Examination of SDS polyacrylamide gels with ultraviolet light showed that mBBr reacted with many platelet proteins but not with GP IIb or IIIa. This observation, and the fact that bimane did not inhibit the fibrinogen-induced aggregation of DTT- or chymotrypsin-treated platelets suggest that it reacts with thiol group(s) that are involved in "exposing" the fibrinogen receptor.     

Inhibitory effect of sodium salicylate on ADP-induced platelet aggregation and on 45Ca2+ uptake into platelets

The effects of sodium salicylate (SS) on ADP-induced platelet aggregation and on a metabolism of calcium in platelets were studied, using gel-filtrated platelets (GFP). SS inhibited dose-responsively ADP-induced aggregation in the presence of fibrinogen and Ca2+. It was found that extracellular 45Ca2+ was rapidly taken up into platelets after stimulation by ADP, while SS significantly inhibited this activity. On the other hand, SS had no effect on platelet aggregation induced by 0.11-1.0 microM ionophore A23187. Therefore, it was found that the inhibitory effect of SS on ADP-induced platelet aggregation may be due to the inhibition of the active influx of extracellular Ca2+ into platelets during aggregation.     

Kinetic parameters of platelet aggregation as an expression of platelet responsiveness

The kinetics of platelet aggregation induced by collagen and by ADP were studied. The maximum aggregation (deltaLTmax) and the ADP and collagen concentrations required to produce half-maximum aggregation (Kd) were determined using platelets obtained from normal individuals, individuals who ingested aspirin and individuals whose platelet-rich plasma (PRP) demonstrated spontaneous aggregation. The Kd and deltaLTmax for ADP-induced platelet aggregation were variable and markedly affected by the citrate concentration. Conversely, kinetic parameters of collagen-induced aggregation were more reproducible and less affected by citrate. The Kd for collagen in platelet aggregation was increased following aspirin ingestion and decreased in samples of PRP that demonstrated spontaneous aggregation. These results suggest that kinetic parameters of platelet aggregation may be useful to express the responsiveness of platelets.     

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.     

A whole blood flow cytometric determination of platelet activation by unfractionated and low molecular weight heparin in vitro

The influence of unfractionated (Heparin–Natrium) and low-molecular heparin (Fragmin®) on platelet activation in whole blood was investigated by FACS analysis in vitro using antibodies against glycoprotein (gp) IIb/IIIa (CD 41), GMP 140 (CD 62P), gp 53 (CD 63) and fibrinogen. Samples were also labeled with anti-gp Ib (CD 42b). Neither unfractionated heparin (UFH) nor low molecular weight heparin (LMWH) led to significant (i.e., p<0.05) changes in fluorescence intensities of platelets labeled with anti-gp IIb/IIIa or anti-gp 53. Significant platelet activation due to unfractionated heparin could be observed by labeling with anti-GMP 140 (UFH: p=0.009; LMWH: p=0.16). The proportion of platelets with surface-bound fibrinogen was significantly increased (UFH: p=0.00006; LMWH: p=0.008). After incubation with heparins, activation ability of platelets by adenosine diphosphate (ADP) was significantly increased. The potentiating action of unfractionated heparin was larger. Therefore, flow cytometric results of platelet activation in patients receiving heparin should be interpreted carefully.     

Novel P2Y12 adenosine diphosphate receptor antagonists for inhibition of platelet aggregation (I): in vitro effects on platelets

ADP plays a key role in platelet aggregation which has led to the development of antiplatelet drugs that target the P2Y(12) receptor. The aim of this study was to characterize the effects of two novel P2Y(12) receptor antagonists, BX 667 and its active metabolite BX 048, on platelets. BX 667 and BX 048 block the binding of 2MeSADP to platelets and antagonize ADP-induced platelet aggregation in human, dog and rat washed platelets. Both compounds were shown to be reversible inhibitors of platelet aggregation. BX 048 prevents the decrease in cAMP induced by treatment of platelets with ADP. The specificity of BX 667 and BX 048 was demonstrated against cell lines expressing P2Y(1) and P2Y(6) as well as against a panel of receptors and enzymes. Taken all together these data show that both BX 048 and BX 667 are potent P2Y(12) antagonists with high specificity which, in the accompanying paper are demonstrated to behave predictably in vivo.     

The in vivo effect in humans of pyridoxal-5′-phosphate on platelet function and blood coagulation

Vitamin B₆ has an antithrombotic effect. This, based on the results of in vitro studies, has been attributed to an antiplatelet effect. We assessed the in vivo effect of vitamin B₆ by measuring the effect of long-term administration of vitamin B₆ on platelet function and blood coagulation. Vitamin B₆ (pyridoxine hydrochloride), 100mg twice daily p.o. for fifteen days, was administered to 10 healthy volunteers. The bleeding time was measured before the first dose and 15 days after. A baseline value, the acute effect, chronic effect, and the acute-on-chronic effect of vitamin B₆ was estimated by measuring platelet function. The following tests were performed: platelet aggregation induced by collagen, ADP and epinephrine; thromboxane A₂ (TxA₂)-production and prostacyclin inhibition of ADP-induced aggregation. The effects on the coagulation system were monitored by measuring: the prothrombin time, activated partial thromboplastin time and levels of coagulation factor. Vitamin B₆ significantly prolonged the bleeding time from 4.1 ± 1.1 minutes to 6.8 ± 1.0 minutes (p = 0.0063). Aggregation of platelets with collagen was slightly but not significantly inhibited. Platelet aggregation induced with the agonists ADP or epinephrine was significantly inhibited by vitamin B₆, and the platelets tended to aggregate at a slightly decreased rate. The mean TxA₂-production was slightly, but not significantly, decreased. Vitamin B₆ had no effect on the sensitivity of platelets to prostacyclin, or on the coagulation system. Our results indicate that the antithrombotic effects of vitamin B₆ is limited to inhibition of platelet function; there was no measurable influence on coagulation. The results of this in vivo study are however such that clinical trials are warranted to further assess the efficacy of vitamin B₆ as an antiplatelet drug.     

Effect of fibrinogen on ADP-induced platelet aggregation

The effect of fibrinogen on ADP-induced platelet aggregation was studied. In absence of ADP no binding of fibrinogen to platelets was observed. On addition of ADP to platelets, fibrinogen induced a dose-related aggregation and release reaction. Binding of approximately 1,000 fibrinogen molecules per platelet was observed. Since fragments of fibrinogen could inhibit this reaction it is suggested that ADP exposes structures on the platelet membrane which bind to fibrinogen. It is suggested that this binding of fibrinogen is of importance for the aggregation of platelets in presence of ADP.

Addition of ADP at high concentrations to platelets induces release of intracellullar fibrinogen which may explain the aggregation occurring at high concentration of ADP in the absence of added fibrinogen.     

Impairment of ADP-induced platelet aggregation by hashish components.

ADP-induced aggregation of washed human platelets is inhibited by the hashish components delta1-tetrahydrocannabinol (THC) and cannabidiol (CBD). The inhibition is counteracted by added ADP. When the cannabinoids are present at concentrations higher than 10(-5)M, the platelets aggregate non-reversibly, independently of an added inducer, apparently due to lysis and release of endogenous inducers. THC is clearly more potent than CBD in exhibiting the biphasic effect. Collagen- and thrombin-induced aggregation of washed platelets are hardly affected by the cannabinoids. THC and CBD also curtail ADP-induced reversible aggregation in platelet-rich plasma, while serotonin release and irreversible aggregation, caused by either ADP, collagen or thrombin, are not affected by the cannabinoids in platelet-rich plasma. The data point to associated sites for ADP and the cannabinoids on the platelet membrane.     

Effect of cAMP phosphodiesterase inhibitors on ADP-induced shape change, cAMP and nucleoside diphosphokinase activity of rabbit platelets

The effects of cAMP phosphodiesterase inhibitors on ADP-induced shape change and cAMP concentrations have been studied. Caffeine (10 mM), theophylline (8 mM), dipyridamole (0.2 mM), or papaverine (0,05 mM) prevented the shape change of washed rabbit platelets induced by 0.4 microM ADP. At these concentrations, none of these cAMP phosphodiesterase inhibitors increased 14C-cAMP in platelets in which the cytoplasmic adenine nucleotides had been labelled with 14C-adenine. By a protein binding assay, only papaverine by itself increased platelet cAMP above its basal level. These results indicate that two pools of cAMP may exist in platelets. Both methods showed that stimulation of platelet adenylate cyclase with PGE1 (1 microM) resulted in an increase in platelet cAMP and all these cAMP phosphodiesterase inhibitors potentiated this increase caused by PGE1. By themselves, some of these compounds may act through mechanisms that do not involve platelet cAMP. The effects of these cAMP phosphodiesterase inhibitors on platelet nucleoside diphosphokinase (NDK) activity were also investigated. At concentrations that prevented ADP-induced shape change, papaverine and dipyridamole had no effect on the formation of 14C-ATP from 14C-ADP by washed rabbit platelets. The methylxanthines partially inhibited NDK activity of washed rabbit platelets and of isolated platelet membranes, probably due to the structural similarity between the adenine ring of ADP and these substances. However, adenine (8 mM) inhibited ADP-induced shape change and platelet NDK activity but was a less effective inhibitor of ADP-induced platelet aggregation. Thus it seems unlikely that interference with platelet NDK or the ADP receptor is the major mechanism by which the methylxanthines inhibit platelet functions.     

Ticlopidine and clopidogrel (SR 25990C) selectively neutralize ADP inhibition of PGE1-activated platelet adenylate cyclase in rats and rabbits

Ticlopidine and its potent analogue, clopidogrel, are powerful inhibitors of ADP-induced platelet aggregation. In order to improve the understanding of this ADP-selectivity, we studied the effect of these compounds on PGE1-stimulated adenylate cyclase and on the inhibition of this enzyme by ADP, epinephrine and thrombin. Neither drug changed the basal cAMP levels nor the kinetics of cAMP accumulation upon PGE1-stimulation in rat or rabbit platelets, which excludes any direct effect on adenylate cyclase or on cyclic nucleotide phosphodiesterase. However, the drop in cAMP levels observed after addition of ADP to PGE1-stimulated control platelets was inhibited in platelets from treated animals. In contrast, the drop in cAMP levels produced by epinephrine was not prevented by either drug in rabbit platelets. In rat platelets, thrombin inhibited the PGE1-induced cAMP elevation but this effects seems to be entirely mediated by the released ADP. Under these conditions, it was not surprising to find that clopidogrel also potently inhibited that effect of thrombin on platelet adenylate cyclase. In conclusion, ticlopidine and clopidogrel selectively neutralize the ADP inhibition of PGE1-activated platelet adenylate cyclase in rats and rabbits.     

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.     

Effect of amino sugars on platelet aggregation and on fibrinogen binding

The amino sugars glucosamine, galactosamine and mannosamine (30 mM) inhibited aggregation of human or rabbit platelets induced by ADP, collagen, thrombin, PAF or high concentrations of sodium arachidonate. 125I-fibrinogen binding during ADP-induced aggregation, and release of amine storage granule contents were also inhibited. Increasing the calcium concentration of the suspending medium to 5 mM did not overcome the inhibitory effect on the release reaction. The amino sugars deaggregated rabbit platelets that had been aggregated by ADP, collagen or thrombin, but deaggregated human platelets readily only when ADP was used as the aggregating agent. Fibrinogen-induced aggregation of chymotrypsin-treated platelets was blocked by the amino sugars. They did not inhibit platelet adherence to a collagen-coated glass surface, nor affect release of granule contents from the adherent platelets. Aggregation and release induced by low concentrations of sodium arachidonate or the divalent cation ionophore A23187 were potentiated, indicating that the effects of the amino sugars on platelets are more complex than simple inhibition of the lectin-like activity that becomes available on the surface of platelets that have undergone the release reaction. One of the effects of the amino sugars, however, is interference with the binding of fibrinogen to platelets. The effects of the amino sugars are shared by other primary amines.