Combined Inhibitory Effects of Ethanol and Adenosine on the Responses of Rabbit Platelets to Thrombin

We have previously shown that acutely administered ethanol, resulting in blood alcohol concentrations of 40-00 mM, inhibits experimentally induced arterial thrombosis in rabbits. This inhibition by ethanol in vivo is more pronounced than that observed on stimulated platelets in vitro, when a similar concentration of ethanol is added before an aggregating agent. It may be, then, that ethanol has combined effects in vivo with other inhibitors of platelet function. Adenosine has been found to be an important mediator of some of the in vivo effects of ethanol, and we investigated whether ethanol has combined inhibitory effects with adenosine on thrombin-stimulated platelet responses in vitro. Aggregation and secretion of [¹⁴C]serotonin from washed, prelabeled rabbit platelets, pretreated with aspirin, were studied. Maximal aggregation induced by 0.15 units thrombin/ml was slightly inhibited by 87 mM ethanol; secretion of serotonin was reduced from 24% to 12%. However, when thrombin-induced aggregation was significantly reduced by 1 μM adenosine, ethanol, at 44 and 87 mM, further inhibited aggregation. Secretion of [¹⁴C]serotonin was reduced to <3%, with the combination of adenosine and the higher concentration of ethanol. Ethanol did not increase platelet cyclic AMP (cAMP) above basal levels, nor did it affect the increase in cAMP caused by adenosine. The adenosine receptor antagonist, 8-phenyltheophylline, at 1 μM, blocked the inhibitory effects of adenosine on platelet responses and prevented the adenosine-induced increase in cAMP. Unexpectedly, however, 8-phenyltheophylline (1–2 μM) did not completely block the combined inhibitory effects of ethanol and adenosine; this incomplete reversal was not associated with increases in cAMP over basal levels. 8-Phenyltheophylline may by useful in vivo in determining whether ethanol has combined inhibitory effects with adenosine on experimentally induced arterial thrombosis.     

Reduced thiols and the effect of intravenous nitroglycerin on platelet aggregation

Nitroglycerin inhibits platelet aggregation in vitro and this effect may be important in its overall mechanism of action. In addition, its use has been associated with prolonged bleeding times and hemorrhagic complications. Despite these experimental and clinical observations, no significant antiplatelet effect of nitroglycerin has been observed ex vivo during intravenous nitroglycerin administration to patients. Because the in vitro antiplatelet effects of nitroglycerin have been shown by one of the investigators participating in this study to depend on the presence of sufficient stores of reduced intracellular thiol--which are readily depleted ex vivo by nitroglycerin in the formation of S-nitrosothiols--an attempt was made to unmask nitroglycerin-mediated inhibition of platelet aggregation by exposing platelets taken from patients treated with nitroglycerin to the reduced thiol N-acetylcysteine ex vivo. The obtained data demonstrate that platelets taken from patients treated with intravenous nitroglycerin manifest attenuated aggregation responses ex vivo when thiol stores are repleted. It is therefore proposed that the mechanism of action of nitroglycerin is mediated in part by its antiplatelet effect and that this effect depends on the adequacy of reduced intracellular thiol stores.     

Inhibitory action of gliclazide on platelet functions

Gliclazide (GC), an oral hypoglycemic agent, inhibits platelet functions, but its effective concentration is reported to be much higher in vitro than in vivo. To determine why, we compared its inhibitory effect measured by impedance aggregometry using citrated whole blood with that measured by turbidometry using platelet-rich plasma. In addition, to see how GC inhibits platelet functions, we examined its effects on arachidonic acid metabolism in platelets in an in vitro system. Impedance aggregometry was found to be more sensitive than turbidometry for detecting the inhibition of platelet aggregation, and revealed significant inhibition at 1 x 10(-4) M GC. GC reduced the amount of prostaglandin I2 (PGI2) needed to inhibit ADP-induced platelet aggregation and the adhesiveness of platelets to a rabbit vessel wall after their preincubation with 1 x 10(-3) M GC for 10 min. GC (1 x 10(-4) -1 x 10(-2) M) had no effect on platelet cyclo-oxygenase activity. GC inhibited thromboxane A2 (TXA2)-induced platelet aggregation, but had no effect on the aggregation triggered by addition of mixtures of arachidonic acid (AA) and inhibitors of key enzymes regulating various steps of AA metabolism in platelets. GC had no significant effect on PGI2-stimulated cyclic AMP (cAMP) production in platelets. These results show that the difference in the effective concentrations of GC reported to modulate platelet functions in vivo and in vitro is partly due to differences in the methods used to evaluate its effect: turbidometry evaluates platelet aggregability, but not other platelet functions modulated by GC in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biphasic roles for soluble guanylyl cyclase (sGC) in platelet activation

Nitric oxide (NO) stimulates cGMP synthesis by activating its intracellular receptor, soluble guanylyl cyclase (sGC). It is a currently prevailing concept that No and cGMP inhibits platelet function. However, the data supporting the inhibitory role of NO/sGC/cGMP in platelets have been obtained either in vitro or using whole body gene deletion that affects vessel wall function. Here we have generated mice with sGC gene deleted only in megakaryocytes and platelets. Using the megakaryocyte- and platelet-specific sGC-deficient mice, we identify a stimulatory role of sGC in platelet activation and in thrombosis in vivo. Deletion of sGC in platelets abolished cGMP production induced by either NO donors or platelet agonists, caused a marked defect in aggregation and attenuated secretion in response to low doses of collagen or thrombin. Importantly, megakaryocyte- and platelet-specific sGC deficient mice showed prolonged tail-bleeding times and impaired FeCl?-induced carotid artery thrombosis in vivo. Interestingly, the inhibitory effect of the NO donor SNP on platelet activation was sGC-dependent only at micromolar concentrations, but sGC-independent at millimolar concentrations. Together, our data demonstrate important roles of sGC in stimulating platelet activation and in vivo thrombosis and hemostasis, and sGC-dependent and -independent inhibition of platelets by NO donors.     

Aurin tricarboxylic acid: a novel inhibitor of the association of von Willebrand factor and platelets

Shear stress activated platelets undergo aggregation in the presence of large or unusually large von Willebrand factor (vWF) multimers without the addition of ristocetin or any other exogenous chemical. This phenomenon may be analogous to the platelet aggregation that leads to thrombosis in the narrowed arteries and arterioles of patients with atherosclerosis or vasospasm. A triphenyl-methyl compound, aurin tricarboxylic acid (ATA), inhibits shear-induced, vWF-mediated platelet aggregation in platelet-rich plasma (PRP) in concentrations above 200 mumol/L and in buffer suspensions of washed platelets at a concentration of 0.1 mumol/L. In a concentration-dependent manner, ATA also inhibits ristocetin-induced, vWF-mediated platelet clumping in both fresh and formaldehyde-fixed platelet suspensions. This inhibition can be overcome by increasing the concentration of vWF, following the kinetics of first order competitive inhibition. ATA prevents the attachment to platelets of the largest vWF multimeric forms found in normal plasma and of the unusually large vWF multimers derived from endothelial cells. The rate of aggregation and degree of inhibition by ATA is not accounted for by the binding of ristocetin or calcium. Arachidonic acid- and adenosine diphosphate (ADP)-induced aggregation are not inhibited by ATA. Platelets incubated with ATA can be easily separated from the compound. However, ATA binds to large vWF multimeric forms and inhibits their ristocetin-induced interaction with platelet glycoprotein Ib. Because ATA also inhibits shear-induced, vWF-mediated platelet aggregation in vitro in the absence of ristocetin, it may be a useful prototype compound to impede the development of arterial thrombosis in vivo.     

In vivo aspirin supplementation inhibits nitric oxide consumption by human platelets

Antiplatelet therapies improve endothelial function in atherosclerosis, suggesting that platelets regulate vascular nitric oxide (NO) bioactivity in vivo. Herein, washed platelets consumed NO on activation in an aspirin-sensitive manner, and aspirin enhanced platelet NO responses in vitro. To examine whether in vivo aspirin can inhibit platelet NO consumption, a double-blind placebo-controlled study was conducted. After a 2-week nonsteroidal anti-inflammatory drug (NSAID)-free period, healthy men were randomly assigned and administered aspirin (75 mg/d orally) or identical placebo for 14 days, then crossed over to the opposite arm. Following in vivo aspirin, NO consumption by platelets was inhibited 91%. Rate of onset and recovery following aspirin withdrawal was consistent with cyclooxygenase 1 (COX-1) inhibition. In a small substudy, NO consumption by platelets from postmenopausal women was faster in hypercholesterolemics and less sensitive to aspirin (ie, 39% versus 76% inhibition for hypercholesterolemics or normocholesterolemics, respectively). However, 150 mg aspirin/day increased inhibition of NO consumption by platelets of hypercholesterolemics to 80%. Comparisons of platelet COX-1 or -2 expression and urinary 11-dehydro-thromboxane B2 excretion suggested that aspirin was less able to block platelet activation in vivo in hypercholesterolemia. In conclusion, aspirin inhibits NO consumption by platelets from healthy subjects, but its beneficial effects on NO bioactivity may be compromised in some hypercholesterolemic patients.     

Effects of Ethanol Administration on Platelet Function in the Rat

When ethanol is administered to rats chronically by inhalation for 5–7 days to produce a final ethanol concentration in plasma of about 50 TTIM platelet agggregation in vitro to collagen is markedly inhibited. Platelet aggregation to ADP is relatively unaffected and there is no evidence of thrombocytopenia or morphological change in platelets on scanning and transmission electron microscopy. The changed sensitivity to collagen is not simply due to the presence of ethanol in plasma during the in vitro tests because in vitro addition of a similar concentration of ethanol produces a much smaller inhibition of platelet aggregation to collagen in platelet-rich plasma from control animals. The acute or subacute administration of ethanol either by injection (3 g/kg^-1 intraperitoneally) or by inhalation (4–6 h) produced very variable results, platelets from some animals being inhibited with respect to collagen as much as those from animals chronically exposed to ethanol, whereas others showed no inhibition other than that attributable to the presence of ethanol in plasma. When ethanol was administered chronically by inhalation for 30 days, thrombocytopenia was apparent and platelets from these animals were unresponsive to all aggregating agents tested. It is concluded that even relatively short periods of ethanol intake may lead to significant inhibition of platelet function, specifically aggregation to collagen. This may be of relevance to the suggested protective effect of ethanol intake in cardiovascular disease.     

Platelet Antithrombins: Role of Thrombin Binding and the Release of Platelet Fibrinogen

The nature of platelet antithrombin was elucidated by comparison of thrombin binding and antithrombin activities of intact platelets and by purification of antithrombin from platelet lysates using glycerol osmotic lysis, ethanol precipitation and Sephadex gel filtration techniques. The major portion of the antithrombin and thrombin binding activity of intact platelets is lost after brief sonication. The antithrombin activity in destroyed platelets is found to be due to platelet fibrinogen. Treatment of platelets with PGE₁ (100 μg/ml) markedly inhibits (>80%) the release of platelet fibrinogen induced by thrombin. However, the PGE₁ treatment produced slight (<30%) but significant decrease of antithrombin activity of intact platelets, whereas the binding of thrombin to platelets was not affected by PGE₁ treatment. The amounts of thrombin bound to and inactivated by PGE₁-treated platelets at the same cell concentration are identical. The above results suggest that platelets contain at least two antithrombin activities. One, which accounts for the major portion of platelet antithrombin is mediated by thrombin binding to platelets. The other, which attributes to a lesser extent to platelet antithrombin activity, is due to the release of platelet fibrinogen. Also, antithrombin is readily demonstrated in a plasma medium indicating physiological significance of platelet antithrombin.     

Effects of Recombinant Human Megakaryocyte Growth and Development Factor (rHuMGDF) on Platelet Production,Platelet Aggregation,and Thrombosis

Recombinant human megakaryocyte growth and development factor (rHuMGDF) is a c-mpl ligand that promotes the differentiation of CD34+ precursor cells into megakaryocytes and then platelets. In experimental animals, injection of this and other c-mpl ligands leads to profound increases in the circulating platelet count in a matter of days. However, c-mpl ligands have also been shown to sensitize platelets to aggregating agents in vitro, raising the possibility that c-mpl ligands may have prothrombotic effects in vivo. Therefore, characterizing rHuMGDF in an in vivo model of thrombosis is a necessary and critical step in defining the in vivo pharmacology of this novel and important hematopoietic factor, a pegylated form of which is currently in clinical trials. To determine the biologically effective doses in the rabbit, daily subcutaneous injections of rHuMGDF at 0.1, 1.0, or 10 µg/kg were administered over 7 days. Daily injection of 10 µg/kg produced an approximate fourfold increase in platelet count and 1.0 µg/kg doubled platelet count over the injection period, both of which were statistically significant. The serum concentrations of rHuMGDF were determined 10 minutes following a single intravenous injection with 0.1, 1.0, and 10 µg/kg, and were 0.05 ± 0.02, 0.98 ± 0.07, and 21.32 ± 2.35 ng/ml. To determine whether rHuMGDF can sensitize platelets in vivo, platelet aggregometry was performed on platelets isolated from animals immediately before and 10 minutes after they had been injected intravenously with rHuMGDF (0.1, 1.0, and 10 µg/kg). Intravenous injection of 10 µg/kg produced measurable changes in platelet aggregometry ex vivo, as evidenced by an increased sensitivity of platelets to adenosine diphosphate (ADP). To assess the in vivo prothrombotic potential of rHuMGDF, a rabbit carotid artery model of cyclic flow reduction (CFR) was used to measure the effect of intravenous rHuMGDF administration on the rate of thrombus formation as assessed by CFR slope and frequency. Intravenous administration of rHuMGDF had no effect on CFR slope or frequency when administered in doses ranging from 0.1 to 10 µg/kg. Control experiments demonstrated that CFR slope and frequency can be enhanced by intravenous infusion of epinephrine and can be abolished by the combined administration of aspirin and ketanserin, indicating that potentially prothrombotic and antithrombotic agents can be identified in this model. We conclude that biologically active doses of rHuMGDF used in this study (1.0 and 10 µg/kg) produce measurable serum levels, induce a thrombopoietic effect, and sensitize platelets in vivo, as determined by ex vivo aggregometry, at 10 µg/kg. Despite the sensitization of platelets to aggregation induced by ADP, it is clear that rHuMGDF does not alter the pattern of CFRs observed in the rabbit carotid artery, whereas agents known to sensitize platelets (epinephrine) and to inhibit platelets (aspirin and ketanserin) readily affected the CFR pattern. These findings indicate that intravenous rHuMGDF administration, while capable of sensitizing platelets, does not enhance platelet-dependent thrombosis in vivo.     

Ethanol Exposure Results in a Transient Decrease in Human Platelet cAMP Levels: Evidence for a Protein Kinase C Mediated Process

At concentrations between 2 and 32 mM, ethanol is shown to depress human platelet cAMP levels. The effect is biphasic, maximal at 30 sec, with platelet concentrations of cAMP returning to baseline values at higher ethanol concentrations and at longer incubation times. The cAMP lowering effect of ethanol can be blocked by a phosphodiesterase (PPDE) inhibitor, 3-isobutyl-1-methyl-xanthine (IBMX), at a concentration of 2 mM, suggesting that an increase in PPDE activity may be responsible for this effect. Exposure of platelets to 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7), a protein kinase C (PKC) inhibitor, blocks the ethanol-induced decrease in platelet cAMP, suggesting ethanol may be acting through activation of PKC.     

Zinc-induced platelet aggregation is mediated by the fibrinogen receptor and is not accompanied by release or by thromboxane synthesis

We demonstrate that zinc (0.1 to 0.3 mmol/L) induces aggregation of washed platelet suspensions. Higher concentrations (1 to 3 mmol/L) of zinc were needed to aggregate platelets in platelet-rich plasma obtained from blood anticoagulated with low-molecular-weight heparin, probably due to the binding of zinc to the plasma proteins. Zinc- induced aggregation of normal washed platelets required added fibrinogen and no aggregation occurred with thrombasthenic platelets or with normal platelets pretreated with a monoclonal antibody (10E5) that blocks the platelet fibrinogen receptor. These data indicate that the platelet membrane fibrinogen receptor-glycoproteins IIb and IIIa mediate the effect of zinc. Zinc-induced aggregation was blocked by the agent TMB-8, which interferes with the internal calcium flux, and by prostacyclin, which elevates platelet cyclic adenosine monophosphate levels. Zinc-induced aggregation was not accompanied by thromboxane synthesis or by the secretion of dense-body serotonin and was not affected by preexposure of platelets to acetylsalicylic acid. Experiments with creatine phosphate/creatine phosphokinase showed that the zinc effect on platelets was independent of extracellular adenosine diphosphate (ADP). Zinc had an additive effect when platelet aggregation was stimulated with subthreshhold concentrations of collagen or ADP. Together with the known effects of nutritional zinc on in vivo bleeding, on platelet aggregation, and on lipid metabolism, the results suggest that zinc may have an important bearing on normal hemostasis, thrombosis, and atherosclerosis.     

Efficient assay for evaluating human thrombopoiesis using NOD/SCID mice transplanted with cord blood CD34+ cells

A suitable model for the preclinical study of human platelet production in vivo has not been available. NOD/SCID mice were characterized as representing an efficient engraftment model for human hematopoietic stem cells, which resulted in the production of human platelets. Here, we evaluated in vivo human thrombopoiesis and ex vivo human platelet functions in NOD/SCID mice transplanted with human cord blood (CB) CD34(+) cells. Human platelets and human CD45(+) cells appeared in peripheral blood of NOD/SCID mice from 4 wk after transplantation. Human platelets produced in these mice showed CD62P expression and the activation of GPIIb/IIIa on human platelets on stimulation with an agonist. PEG-rHuMGDF (0, 0.5 and 5 microg/kg/d s.c.) was injected for 14 d into mice that had been confirmed to produce human platelets stably. The number of human platelets increased about twofold at 0.5 microg/kg/d and about fivefold at 5 microg/kg/d after 14 d. Withdrawal of PEG-rHuMGDF administration caused the human platelet count to return to the pretreatment level. Further, re-administration of PEG-rHuMGDF induced a similar human thrombopoietic response as it did on initial administration. These results suggest that NOD/SCID mice engrafted with human CB CD34(+) cells will be useful for the study of human platelet production in vivo.     

Thrombospondin promotes platelet aggregation

Thrombospondin (TSP), isolated from human platelets, promotes aggregation of both nonstimulated platelets and platelets stimulated with thrombin or ADP. The TSP-promoted aggregation is specific since a monoclonal antibody against TSP inhibits the effect of exogenously added TSP and inhibits thrombin-induced platelet aggregation in the absence of added TSP. Several lines of evidence suggest that TSP mediates its effect on aggregation of nonstimulated and stimulated platelets through different platelet-surface receptor systems. The TSP- promoted aggregation of nonstimulated platelets was inhibited by a monoclonal antibody to platelet glycoprotein IV (GPIV), but not by a monoclonal antibody to the fibrinogen receptor, GPIIb-IIIa. In contrast, the antibody to GPIIb-IIIa totally inhibited the TSP- potentiated aggregation of thrombin-stimulated platelets, whereas the antibody to GPIV has no effect. Thus, these studies suggest that TSP promotes platelet aggregation by at least two mechanisms--one dependent on and one independent of the platelet fibrinogen receptor system.     

Recombinant fragment of von Willebrand factor AR545C inhibits platelet binding to thrombin and platelet adhesion to thrombin-treated endothelial cells

Activated, but not resting, platelets are capable of adhering to intact endothelial cells (ECs). We evaluated the effect of a recombinant von Willebrand factor (VWF) fragment AR545C, which inhibits glycoprotein Ib (GPIb)/VWF binding, on platelet adhesion to human ECs under static or flow conditions. Incubation of resting platelets with intact endothelium under flow conditions (350/s) resulted in minimal platelet adhesion. The adhesion was enhanced two- to threefold after either platelet activation by thrombin receptor agonist peptide (TRAP) or EC pretreatment with thrombin. The enhancing effect of thrombin was abolished by addition of either hirudin (10 u/ml) or PGE1 (1 microg/ml). Preincubation of resting platelets with increasing concentrations of AR545C under static or flow conditions resulted in a dose-dependent inhibition of thrombin-induced enhanced adhesion to ECs. AR545C (0.3 microM) completely abolished the effect of thrombin, reducing platelet adhesion to the control level observed with non-treated ECs. In contrast, the same concentration of AR545C had no effect on the adhesion of TRAP-activated platelets to ECs. AR545C also inhibited thrombin-induced platelet aggregation and binding in a dose-dependent manner. In addition, 0.3 microM of AR545C reduced thrombin-induced serotonin release by 57%, whereas monoclonal antibody AN51, which inhibits ristocetin-induced platelet aggregation, had no effect on either thrombin-induced platelet aggregation or binding or on serotonin release. Similarly, AR545C had no effect on TRAP-induced serotonin release. These findings suggest that (i) AR545C inhibits platelet activation mediated by thrombin and this inhibition occurs through blocking the high-affinity thrombin binding sites on the GPIb/IX complex and (ii) AR545C has no effect on the moderate affinity thrombin receptor (seven transmembrane domain thrombin receptor; STDR).     

Vitamin E reduces platelet adhesion to human endothelial cells in vitro

Although it has been reported that vitamin E (alpha-tocopherol) can reduce platelet adhesiveness and aggregation in vivo, the mechanism is still unknown. Therefore, the aim of the present study was to determine whether incubations of platelet-rich plasma (PRP) with vitamin E influence platelet adhesion to cultured endothelial cells. To exclude blood plasma involvement, also washed platelets were pretreated with alpha-tocopherol. Vitamin E (0.5-1.0 mM) was added to PRP or washed platelets. Endothelial cells in monolayer were incubated with thrombin-activated platelets (1 or 2 U/ml). After 1 hr of incubation, non-adhered platelets were removed and counted. Treating of PRP with alpha-tocopherol inhibited platelet adhesion to endothelial cell monolayer. This effect was dose dependent on concentrations of alpha-tocopherol and thrombin. In our experiments PRP was treated with alpha-tocopherol and endothelial cell monolayer was used as test surface. These findings agree with previous observations on the adhesivity of platelets to synthetic surfaces after dietary vitamin E in healthy volunteers. When washed platelets were incubated with alpha-tocopherol, no significant reduction of adhesion was detectable. As preincubation of washed platelets with alpha-tocopherol does not inhibit platelet adhesion, it may be supposed that the effect of vitamin E does not occur in a directly cellular mechanism. The data suggest that alpha-tocopherol may reduce platelet adhesiveness probably after incorporation by plasma lipoproteins.     

Effect of Ethanol on Platelet Function

Epidemiological studies have linked an inhibition of platelet function by ethanol, among other factors, to the cardioprotective effects of moderate ethanol consumption. Platelet defects have been noted in alcoholics and in human experimental studies. Importantly, in in vivo experimental settings, ethanol diminishes thrombus formation on damaged arterial walls. Ethanol inhibits platelet activation in vitro in response to diverse agonists. Phospholipase A2 is a major site for ethanol inhibition, corresponding to a reduction in the formation of stimulatory arachidonate metabolites. Additional signal transduction pathways are likely targets for ethanol including phosphoinositide-specific phospholipase C and cyclic AMP. The role of additional cofactors in the inhibition of platelet responses by ethanol is discussed.     

Platelet endothelial cell adhesion molecule-1 signaling inhibits the activation of human platelets.

Platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) is a 130-kd transmembrane glycoprotein and a member of the growing family of receptors with immunoreceptor tyrosine-based inhibitory motifs (ITIMs). PECAM-1 is expressed on platelets, certain T cells, monocytes, neutrophils, and vascular endothelial cells and is involved in a range of cellular processes, though the role of PECAM-1 in platelets is unclear. Cross-linking of PECAM-1 results in phosphorylation of the ITIM allowing the recruitment of signaling proteins that bind by way of Src-homology domain 2 interactions. Proteins that have been implicated in the negative regulation of cellular activation by ITIM-bearing receptors include the tyrosine phosphatases SHP-1 and SHP-2. Tyrosine phosphorylation of immunoreceptor tyrosine-based activatory motif (ITAM)-bearing receptors such as the collagen receptor GPVI-Fc receptor gamma-chain complex on platelets leads to activation. Increasing evidence suggests that ITIM- and ITAM-containing receptors may act antagonistically when expressed on the same cell. In this study it is demonstrated that cross-linking PECAM-1 inhibits the aggregation and secretion of platelets in response to collagen and the GPVI-selective agonist convulxin. In these experiments thrombin-mediated platelet aggregation and secretion were also reduced, albeit to a lesser degree than for collagen, suggesting that PECAM-1 function may not be restricted to the inhibition of ITAM-containing receptor pathways. PECAM-1 activation also inhibited platelet protein tyrosine phosphorylation stimulated by convulxin and thrombin; this was accompanied by inhibition of the mobilization of calcium from intracellular stores. These data suggest that PECAM-1 may play a role in the regulation of platelet function in vivo.     

Temperature dependence of plasmin-induced activation or inhibition of human platelets.

It is known that at 37 degrees C plasmin may have two opposite effects on platelets: at high concentrations (greater than 1.5 caseinolytic units [CU]/mL), plasmin activates platelets; at lower concentrations (0.1 to 1.0 CU/mL) it inhibits platelet activation induced by thrombin, collagen, or calcium ionophore A23187. In this study, we report that when lowering the incubation temperature to 22 degrees C, plasmin at low concentrations (0.1 to 0.5 CU/mL) fully activated platelets. When platelets were treated with 0.2 CU/mL of plasmin, lowering the incubation temperature from 37 degrees C to 22 degrees C resulted in an increase in the expression of fibrinogen receptors, in platelet release and aggregation. Thromboxane A2 was not generated by plasmin treatment at either temperature. Ultrastructural studies showed that platelets responded to low-dose plasmin at 37 degrees C by forming pseudopods, centralizing granules without fibrinogen release, whereas at 22 degrees C the same dose of plasmin caused platelet degranulation with the appearance of alpha-granule fibrinogen within the lumen of the surface connected canalicular system. In addition, at 22 degrees C plasmin at doses insufficient to induce platelet aggregation potentiated platelet response to thrombin. Thus, we suggest that plasmin may initiate both activating and inhibitory processes within platelets and that the change of temperature could influence this balance. These results may be of clinical relevance, because the fibrinolytic system was found activated during cardiopulmonary bypass in which the temperature of patient's blood circulation was reduced. This temperature-dependent behavior is also an interesting model for a further study on platelet response to serine proteinases.     

Modulation of platelet function by extracellular adenosine triphosphate

A potential physiologic role of extracellular adenosine triphosphate (ATP) on platelet function is proposed in this report. It is widely accepted that ATP competitively inhibits adenosine diphosphate (ADP)- induced platelet aggregation. Our observations of platelet aggregation with the agonists, collagen, epinephrine, and ADP in the presence of 180 mumol/L ATP could support this competitive nature of ATP. However, the disaggregation of maximally aggregated platelets induced by ATP, theophylline, or ATP plus theophylline indicates that additional mechanisms of ATP action may be present. Extracellular gamma-32P-ATP (7 pmol) labels surface-membrane proteins in intact platelets as demonstrated by several criteria. The reaction is Ca++-dependent. Stimulation by calcium occurs in the physiologic range of 1 to 5 mmol/L. Significant levels of phosphorylation occur within one minute with near maximal levels reached by five minutes. Platelet cyclic AMP (cAMP) levels were elevated in a dose-dependent fashion in cells incubated for four minutes with increasing amounts of extracellular ATP (18 to 540 nmol). The addition of ATP plus theophylline resulted in a synergistic stimulation of cAMP levels. ATP was not being hydrolyzed to adenosine by plasma nucleotidases, as demonstrated by the lack of effect of ten U of adenosine deaminase. The phosphorylation of surface proteins by extracellular ATP released from activated platelets may modulate platelet responsiveness to agonists at distances removed from the site of vascular injury. Phosphorylation may also play a role in signal transduction to regulate the levels of intracellular cAMP, which further inhibits platelet activation.     

Effects of Ethanol on Human Platelets Stimulated with Platelet-Activating Factor, a Biologically Active Ether Phospholipid

The interaction between ethanol and 1-0-alkyl-2-acetyl-sn-glycerol-3-phosphocholine (platelet activating factor, PAF) was addressed using platelets obtained from normal nonalcoholic volunteers. Ethanol at concentrations of 20 to 100 mM inhibited PAF activation of human platelets. Ethanol inhibited prominently the second or arachidonic acid metabolite dependent wave of platelet aggregation, which occurs with human platelets in citrated plasma. It also inhibited serotonin release and thromboxane A2 formation associated with this secondary phase of aggregation. Ethanol did not readily inhibit the primary wave of PAF-induced aggregation. The incorporation of PAF into platelets or metabolism of PAF was not influenced by up to 100 mM ethanol. Since ethanol inhibited only the secondary response, a direct interaction between PAF, ethanol, and a platelet PAF receptor is unlikely. The effect of ethanol on PAF-induced platelet aggregation shows a selectivity similar to that demonstrated by other investigators for epinephrine and adenosine diphosphate.