Mechanisms of platelet autoimmunity: The role of macrophages

Taken together, the above results suggest that, in AITP, activated macrophages are present that could potentially interact with platelets and induce platelet modifications; one of these modifications is the expression of macrophage-derived HLA-DR molecules normally restricted to the golgi, endosomal, and membrane compartments. These macrophage markers on platelets may have a role in CD4+ Th₁ cell regulation; a platelet or microparticle expressing a macrophage-derived class II MHC molecule (as found in AITP) may be able to interact with CD4+ T cells (particularly in the presence of inflammatory-like stimulating factors, i.e., help) and may either initiate autoimmune pathogenesis in AITP or aggravate/perpetuate it. Thus, there is compelling evidence to suggest that a) activated macrophages are present in patients with AITP, b) macrophages have enhanced interactions with platelets in AITP, and c) activated macrophages can induce platelet abnormalities such as platelet HLA-DR expression. Based on these data and the fact that macrophages are known to be potent APC stimulators of Th¹ cell activation, it could be hypothesized that macrophages may play a critical role in the development and maintenance of autoimmune Th₁ pathology in AITP that may lead to autoantibody production, i.e., that macrophages, in addition to their role at the “end” stage of platelet destruction in the RES, have an important role in the initiation of the process. In AITP, macrophages become activated (e.g., secondary to infection or other agonists) and process normal or modified platelets in an altered fashion that relates to platelet HLA-DR expression. These processing events, particularly platelet HLA-DR expression, may have an influence on Th¹ cell activation and ultimately autoantibody production.     

The Effect of Lipopolysaccharide from <Emphasis Type="Italic">Proteus mirabilis</Emphasis> on the Level of the Stable End Metabolic Products of Nitric Oxide in Blood Platelets

Nitric oxide (^•NO) plays an important role in a number of physiologic processes. Evidence exists that ^•NO, which stimulates soluble guanylate cyclase and enhances cyclic guanosine monophosphate (cGMP) levels, may inhibit platelet activation. In contrast, during platelet activation induced by different agonists, synthesis of ^•NO in platelets occurs. In these studies, production of the stable end-products of ^•NO-nitrite and nitrate (NO_x) in human platelets, stimulated by different doses of lipopolysaccharide from Proteus mirabilis (LPS; endotoxin), has been evaluated. LPS is a weak platelet agonist that may activate various steps of platelet activation with the generation of reactive oxygen species. The mechanism of platelet activation induced by the endotoxin is not known. The aim of the present study was to measure the level of nitrite and NO_x in blood platelets treated with LPS and to examine the level of nitrotyrosine in platelet proteins caused by LPS. Our results show that LPS at a low concentration (6.8 ng/ml) caused a decrease (approximately 80%) in the NO_x level, whereas at higher concentrations (13.6 and 25 ng/ml) it induced an increase in the NO_x level (approximately 210% and 260%, respectively). Our results indicate that LPS, like other agonists (thrombin, platelet-activating factor), can stimulate ^•NO production in platelets. After incubating platelets with LPS, we also observed a distinct increase in platelet protein nitration (3-nitrotyrosine).     

<Emphasis Type="SmallCaps">l</Emphasis>-carnitine modulates blood platelet oxidative stress

The oxidative stress induced by acute exertion may interfere with blood platelet activation. The beneficial effect of l-carnitine (γ-trimethylamino-β-hydroxybutyric acid) on oxidative stress in blood platelets has not been fully investigated; however, different studies indicate that this compound modulates platelet functions. The aim of our study was to assess the effects of l-carnitine on platelet activation and oxidative/nitrative protein damage (determined by the levels of protein carbonyl groups, thiol groups, and 3-nitrotyrosine residues) in resting blood platelets or platelets treated with peroxynitrite (ONOO⁻, a strong physiological oxidant) in vitro. We also investigated the effects of l-carnitine on the level of platelet glutathione and on the formation of superoxide anion radicals ( O₂ ^{- ·} ) \left( {{\hbox{O}}_2^{ - \bullet }} \right) , lipid peroxidation measured by thiobarbituric acid reactive substances (TBARS) in blood platelets stimulated by thrombin (a strong physiological agonist), and platelet aggregation induced by adenosine diphosphate (a strong physiological stimulator). We have observed that carnitine decreases platelet activation (measured by platelet aggregation, the generation of O₂ ^{- ·} {\hbox{O}}_2^{ - \bullet } , and TBARS production). Moreover, our results in vitro demonstrate that carnitine may protect against oxidation of thiol groups induced by ONOO⁻. Thus, carnitine may have some protectory effects against oxidative changes induced in blood platelets.     

Lanthanum stimulates the accumulation of cyclic AMP and inhibits secretion and thromboxane B2 formation in human platelets

La³⁺ was found to inhibit the secretion of 5-hydroxytryptamine and the production of thromboxane B₂ by washed platelets exposed to collagen or thrombin. In addition, La³⁺ inhibited secretion in response to sodium arachidonate, although the conversion of arachidonate to thromboxane B₂ was not affected.La³⁺ was also found to enhance the accumulation of cyclic AMP under basal conditions and in response to prostaglandin E₁, in washed platelets. The inhibition of cyclic AMP accumulation by ADP was prevented by La³⁺, suggesting that the effect of ADP on cyclic AMP metabolism was dependent upon the presence or flux of calcium at the platelet membrane.La³⁺ inhibited the activity of adenylate cyclase in platelet lysates both in response to prostaglandin E₁ and to F^?, indicating a possible effect at the catalytic subunit of the enzyme. None of the observed effects of La³⁺ could be reversed by the addition of Ca²⁺ up to 10 mM. The stimulation of cyclic AMP production by La³⁺ may largely explain the inhibitory effect of La³⁺ upon platelet secretion and thromboxane B₂ production. These results also suggest that Ca²⁺ localised at the platelet plasma membrane may be important in the regulation of cyclic AMP metabolism.     

Synthesis of hydroxy fatty acids from linoleic acid by human blood platelets

The metabolism of linoleic acid by washed human platelets was investigated. 1.¹⁴C linoleic acid was converted to 1.¹⁴C hydroxy octadecadienoic acids (HODES) at about the same rate with which 1.¹⁴C 12-HETE was produced from 1.¹⁴C arachidonic acid. The total radioactivity in HODEs was distributed among two isomers: 13-HODE (85%) and 9-HODE (15%) as defined by GC-MS. The production of HODES by intact washed platelets was inhibited by indomethacin (IC50:5×10⁻⁷M) which suggest that hydroxy fatty acids were produced by PGH-synthase. By contrast, the production of HODEs by platelet cytosolic fractions was not modified under indomethacin treatment but completely abolished by NDGA (10⁻³M) and inhibited by the platelet lipoxygenase inhibitors 15-HETE (2.10⁻⁵M) and baicalein (10⁻⁵M). Platelets thus contain two different active systems which may convert linoleic acid to hydroxy fatty acids. Since these compounds remained essentially associated with the platelets, their presence may significantly participate in the mechanisms of platelet activation.     

Phosphatidylinositol-specific phospholipase-C of platelets: association with 1,2-diacyglycerol-kinase and inhibition by cyclic-AMP.

Horse platelets prelabeled with [¹⁴C]arachidonate (AA) rapidly degrade [¹⁴C]phosphatidylinositol (PI) to [¹⁴C]1,2-diacylglycerol (DG) upon treatment with deoxycholate (DOC). This phospholipase-C (PLC) activity is specific for PI since other phospholipids or neutral lipids are not affected. Although exogenous Ca²⁺ is not required for activity, EGTA or EDTA abolishes PI degradation. Addition of Mg²⁺ (1 mM) and ATP (1 mM) results in phosphorylation of the DG and production of phosphatidic acid (PA). Higher concentrations of DOC inhibit DG-kinase. These observations, together with the fact that different platelet agonists induce a rapid degradation of PI and production of PA, indicate that PLC and DG-kinase activities are intimately linked. Incubation of platelets with dibutyryl cyclic-AMP, cyclic AMP-phosphodiesterase inhibitors and pyridoxal-5′-phosphate, which prevent platelet aggregation, inhibits the DOC-dependent conversion of PI to DG. The activity of PLC may play a central role in mediating platelet function and aggregation.     

Characterization of the Rac guanine nucleotide exchange factor P-Rex1 in platelets

Background

Blood platelets undergo a carefully regulated change in shape to serve as the primary mediators of hemostasis and thrombosis. These processes manifest through platelet spreading and aggregation and are dependent on platelet actin cytoskeletal changes orchestrated by the Rho GTPase family member Rac1. To elucidate how Rac1 is regulated in platelets, we captured Rac1-interacting proteins from platelets and identified Rac1-associated proteins by mass spectrometry.

Findings

Here, we demonstrate that Rac1 captures the Rac guanine nucleotide exchange factor P-Rex1 from platelet lysates. Western blotting experiments confirmed that P-Rex1 is expressed in platelets and associated with Rac1. To investigate the functional role of platelet P-Rex1, platelets from P-Rex1 ^-/- -deficient mice were treated with platelet agonists or exposed to platelet activating surfaces of fibrinogen, collagen and thrombin. Platelets from P-Rex1 ^-/- mice responded to platelet agonists and activating surfaces similarly to wild type platelets.

Conclusions

These findings suggest that P-Rex1 is not required for Rac1-mediated platelet activation and that the GEF activities of P-Rex1 may be more specific to GPCR chemokine receptor mediated processes in immune cells and tumor cells.     

Hydrogen peroxide and peroxynitrite enhance Ca2+ mobilization and aggregation in platelets from type 2 diabetic patients

Cytosolic Ca²⁺ mobilization, especially Ca²⁺ entry, is enhanced in platelets from type 2 diabetic individuals, which might result in platelet hyperaggregability. In the present study, we report an increased oxidant production in resting and stimulated platelets from diabetic donors. Pretreatment of platelets with catalase or trolox, an analog of vitamin E, reversed the enhanced Ca²⁺ entry, evoked by thapsigargin plus ionomycin or thrombin, observed in platelets from diabetic subjects, so that in the presence of these scavengers Ca²⁺ entry was similar in platelets from healthy and diabetic subjects. In contrast, mannitol was without effect on Ca²⁺ mobilization. Catalase and trolox reduced thrombin-induced aggregation in platelets from type 2 diabetic subjects, while mannitol did not modify thrombin-induced platelet hyperaggregability. We conclude that H₂O₂ and ONOO⁻ are likely involved in the enhanced Ca²⁺ mobilization observed in platelets from type 2 diabetic patients, which might lead to platelet hyperactivity and hyperaggregability.     

Activated Platelets from Diabetic Rats Cause Endothelial Dysfunction by Decreasing Akt/Endothelial NO Synthase Signaling Pathway

Diabetes is associated with endothelial dysfunction and platelet activation, both of which may contribute to increased cardiovascular risk. The purpose of this study was to characterize circulating platelets in diabetes and clarify their effects on endothelial function. Male Wistar rats were injected with streptozotocin (STZ) to induce diabetes. Each experiment was performed by incubating carotid arterial rings with platelets (1.65×10⁷ cells/mL; 30 min) isolated from STZ or control rats. Thereafter, the vascular function was characterized in isolated carotid arterial rings in organ bath chambers, and each expression and activation of enzymes involved in nitric oxide and oxidative stress levels were analyzed. Endothelium-dependent relaxation induced by acetylcholine was significantly attenuated in carotid arteries treated with platelets isolated from STZ rats. Similarly, treatment with platelets isolated from STZ rats significantly reduced ACh-induced Akt/endothelial NO synthase signaling/NO production and enhanced TXB₂ (metabolite of TXA₂), while CD61 (platelet marker) and CD62P (activated platelet marker) were increased in carotid arteries treated with platelets isolated from STZ rats. Furthermore, the platelets isolated from STZ rats decreased total eNOS protein and eNOS dimerization, and increased oxidative stress. These data provide direct evidence that circulating platelets isolated from diabetic rats cause dysfunction of the endothelium by decreasing NO production (via Akt/endothelial NO synthase signaling pathway) and increasing TXA₂. Moreover, activated platelets disrupt the carotid artery by increasing oxidative stress.     

Platelet Content of Nitric Oxide Synthase 3 Phosphorylated At Serine1177 Is Associated with the Functional Response of Platelets to Aspirin

Objective

To analyse if platelet responsiveness to aspirin (ASA) may be associated with a different ability of platelets to generate nitric oxide (NO).

Patients/Methods

Platelets were obtained from 50 patients with stable coronary ischemia and were divided into ASA-sensitive (n = 26) and ASA-resistant (n = 24) using a platelet functionality test (PFA-100).

Results

ASA-sensitive platelets tended to release more NO (determined as nitrite + nitrate) than ASA-resistant platelets but it did not reach statistical significance. Protein expression of nitric oxide synthase 3 (NOS3) was higher in ASA-sensitive than in ASA-resistant platelets but there were no differences in the platelet expression of nitric oxide synthase 2 (NOS2) isoform. The highest NOS3 expression in ASA-sensitive platelets was independent of the presence of T-to-C mutation at nucleotide position −786 (T⁻⁷⁸⁶→C) in the NOS3-coding gene. However, platelet content of phosphorylated NOS3 at Serine (Ser)¹¹⁷⁷, an active form of NOS3, was higher in ASA-sensitive than in ASA-resistant platelets. The level of platelet NOS3 Ser¹¹⁷⁷ phosphorylation was positively associated with the closure time in the PFA-100 test. In vitro, collagen failed to stimulate the aggregation of ASA-sensitive platelets, determined by lumiaggregometry, and it was associated with a significant increase (p = 0.018) of NOS3 phosphorylation at Ser¹¹⁷⁷. On the contrary, collagen stimulated the aggregation of ASA-resistant platelets but did not significantly modify the platelet content of phosphorylated NOS3 Ser¹¹⁷⁷. During collagen stimulation the release of NO from ASA-sensitive platelets was significantly enhanced but it was not modified in ASA-resistant platelets.

Conclusions

Functional platelet responsiveness to ASA was associated with the platelet content of phosphorylated NOS3 at Ser¹¹⁷⁷.     

Group VIB Calcium-Independent Phospholipase A2 (iPLA2γ) Regulates Platelet Activation,Hemostasis and Thrombosis in Mice

In platelets, group IVA cytosolic phospholipase A₂ (cPLA₂α) has been implicated as a key regulator in the hydrolysis of platelet membrane phospholipids, leading to pro-thrombotic thromboxane A₂ and anti-thrombotic 12-(S)-hydroxyeicosatetranoic acid production. However, studies using cPLA₂α-deficient mice have indicated that other PLA₂(s) may also be involved in the hydrolysis of platelet glycerophospholipids. In this study, we found that group VIB Ca²⁺-independent PLA₂ (iPLA₂γ)-deficient platelets showed decreases in adenosine diphosphate (ADP)-dependent aggregation and ADP- or collagen-dependent thromboxane A₂ production. Electrospray ionization mass spectrometry analysis of platelet phospholipids revealed that fatty acyl compositions of ethanolamine plasmalogen and phosphatidylglycerol were altered in platelets from iPLA₂γ-null mice. Furthermore, mice lacking iPLA₂γ displayed prolonged bleeding times and were protected against pulmonary thromboembolism. These results suggest that iPLA₂γ is an additional, long-sought-after PLA₂ that hydrolyzes platelet membranes and facilitates platelet aggregation in response to ADP.     

TLR4-dependent upregulation of the platelet NLRP3 inflammasome promotes platelet aggregation in a murine model of hindlimb ischemia

Platelets play a critical role in the pathophysiology of peripheral arterial disease (PAD). The mechanisms by which muscle ischemia regulates aggregation of platelets are poorly understood. We have recently identified the Nod-like receptor nucleotide-binding domain leucine rich repeat containing protein 3 (NLRP3) expressed by platelets as a critical regulator of platelet activation and aggregation, which may be triggered by activation of toll-like receptor 4 (TLR4). In this study, we performed femoral artery ligation (FAL) in transgenic mice with platelet-specific ablation of TLR4 (TLR4 PF4) and in NLRP3 knockout (NLRP3^?/?) mice. NLRP3 inflammasome activity of circulating platelets, as monitored by activation of caspase-1 and cleavage of interleukin-1β (IL-1β), was upregulated in mice subjected to FAL. Genetic ablation of TLR4 in platelets led to decreased platelet caspase 1 activation and platelet aggregation, which was reversed by the NLRP3 activator Nigericin. Two weeks after the induction of FAL, ischemic limb perfusion was increased in TLR4 PF4 and NLRP3^?/? mice as compared to control mice. Hence, activation of platelet TLR4/NLRP3 signaling plays a critical role in upregulating platelet aggregation and interfering with perfusion recovery in muscle ischemia and may represent a therapeutic target to improve limb salvage.     

Use of[75Se]-methionine as a tracer of thrombocytopoiesis: II-kinetics in normal rats and in platelet disorders in man: a new approach

We have shown in the rat and in man that it is possible to determine the life span of autologous platelets and to quantify the production of these circulating cells by injecting[⁷⁵Se]-methionine and analysing the overall platelet radioactivity curves obtained: platelets and their precursors are labelled $\text{in vivo}$ by adsorption of circulating plasma proteins onto their membrane, and in the marrow, principally during synthesis of thrombosthenin. This last phenomenon takes place 2 to 3 days before platelet release into the rat blood circulation and 5 to 6 days in the case of man. When there is a greatly increased production, the maturation time is slightly decreased.     

Technetium-99m-labeled platelets: Comparison of labeling with a new lipid-soluble Sn(II)-mercaptopyridine-N-oxide and 99mTc-HMPAO

Platelets pretinned with a neutral Sn(II)-2-mercaptopyridme-N-oxide (SN-MPO) were labeled with ^99mTc and compared to those labeled with ^99mTc-HMPAO. The conditions of labeling platelets, e.g. concentrations of platelets and Sn(II)-MPO, ^99mTc in ACD-saline or ACD-plasma media, pH and incubation time, were optimized using canine platelets. Moderate labeling efficiency was obtained with 20 μg of tin(II) chloride and 30 min incubation with Sn-MPO and pertechnetate. The viability of labeled platelets was determined by platelet recovery and platelet survival times in Beagle dogs. The labeling efficiency with platelets from 43 mL of blood was 62.8 ± 7.6%. The platelet recovery was 35.7 ± 5.0% and exponential survival time was 34.6 ± 3.1 h compared to 43.3 ± 12.0% and 29.5 ± 3.3 h for ^99mTc-HMPAO-labeled platelets. These values were significantly (P < 0.01) lower than ¹¹¹In-labeled platelets. Biodistribution in dogs indicates lower retention in blood, spleen and liver after some initial ^99mTc excretion in urine. The platelet deposition with ^99mTc platelets (Sn-MPO method) on polyurethane angio-catheters was similar to ^99mTc-HMPAO-labeled platelets. This study indicates that the platelets could be successfully labeled with pertechnetate in a cost-effective manner for the evaluation of thromboembolic complications.     

Response of Blood Platelets to Proteus mirabilis Lipopolysaccharide

The effects of the lipopolysaccharide (LPS) of Proteus mirabilis on the production of thiobarbituric acid reactive substances (TBARS) and the generation of superoxide radicals (O₂^?) by pig blood platelets were studied in vitro. The effect of LPS on TBARS formation in platelets was dependent on the concentration of endotoxin. LPS at concentrations above 0.1 μg/10⁸ platelets caused the production of TBARS concomitant with the generation of superoxide radicals. The responses of platelets to LPS suggest that endotoxin, like thrombin (a strong platelets agonist), stimulates an enzymatic cascade of platelet arachidonate via cyclooxygenase and produces thromboxane A₂ (TXA₂) concomitant with malonyldialdehyde (MDA).     

Platelet phosphoinositide turnover in streptozotocin-induced diabetes

Increased platelet aggregation and secretion in response to various agonists has been described in both diabetic humans and animals. Alterations in the platelet membrane fatty acid composition of phospholipids and changes in the prostacyclin and thromboxane formation could only partly explain the altered platelet function in diabetes. In the present study, we have examined the role of phosphoinositide turnover in the diabetic platelet function. We report alterations in 2-[³H] myo-inositol uptake, phosphoinositide turnover, inositol phosphate and diacylglycerol (DAG) formation, phosphoinositide mass, and phospholipase C activity in platelets obtained from streptozotocin (STZ)-induced diabetic rats. There was a significant increase in the 2-[³H) myo-inositol uptake in washed platelets from diabetic rats. Basal incorporation of 2-[³H] myo-inositol into phosphatidylinositol 4,5-bisphosphate (PIP₂), phosphatidylinositol 4-phosphate (PIP) or phosphatidylinositol (PI) in platelets obtained from diabetic rats was, however, not affected. Thrombin stimulation of platelets from diabetic rats induced an increase in the hydrolysis of [³²P]PIP₂ but indicated no change in the hydrolysis of [³²P]PIP and [³²P]PI as compared to their basal levels. Thrombin-induced formation of [³H]inositol phosphates was significantly increased in both diabetic as well as in control platelets as compared to their basal levels. This formation of [³H]inositol phosphates in diabetic platelets was greater than controls at all time intervals studied. Similarly, there was an increase in the release of DAG after thrombin stimulation in the diabetic platelets. Based on these results, we conclude that there is an increase in the transport of myoinositol across the diabetic platelet membrane and this feature, along with alterations in the hydrolysis of PIP₂, inositol phosphates and DAG in the diabetic platelets, may play a role in increased phosphoinositide turnover which could explain the altered platelet function in STZ-induced diabetes.     

The perturbation of thrombin binding to human platelets by trypsin and neuraminidase

The initial step in the interaction of thrombin with human platelets in binding of the enzyme to the platelet surface. The effects of digestion of isolated platelets with trypsin and neuraminidase on aggregation, release of serotonin and binding of thrombin have been examined.Trypsin is a powerful inducer of platelet aggregation as well as the release reaction. The aggregation effect of trypsin may be blocked with disodium ehtylenediaminetatraacetate (EDTA). Further, in the presence of EDTA, trypsin-induced release of [¹⁴C]serotonin is 15–20% lower compared to controls and the initial lag period is prolonged. Conditions were developed under which trypsin did neither aggregate nor release serotonin from platelets. Even under these conditions, trypsin caused a profound loss in the thrombin binding capacity of platelets. Thus, the trypsin-induced fall in the thrombin binding capacity and the platelet response are dissociated. This loss in the thrombin binding by trypsin is due to a lower number of binding sites available on the platelet surface and is not due to an altered affinity.Neuraminidase did not induce platelet aggregation or the release reaction. The ability of platelets to bind thrombin was also unimpaired by prior digestion with neuraminidase. Thus, the sialic acid at the platelet surface is not essential in the function of thrombin recognition by the receptor. This moiety may nontheless be a constituent of a glycoprotein which might act as the thrombin receptor.     

Role of lipoxygenase metabolism in platelet function: Effect of aspirin and salicylate

Aspirin inhibits thromboxane A₂ (TxA₂) production whereas its salicylate moiety inhibits 12-hydroxy-eiosatetraenoic acid (12-HETE) production in the platelet. The significance of the latter effect on platelet function is unclear. We examined the effects of aspirin and salicylate on (i) platelet/ collagen adhesion using ³H-adenine-labelled human platelets and collagen- coated discs, (ii) platelet aggregation induced by thrombin, collagen, ADP and arachidonic acid, and (iii) platelet TxA₂ and 12-HETE synthesis as measured by radioimmunoassay and high pressure liquid chromatography respectively. Aspirin (50 μM) decreased platelet aggregation and increased platelet adhesion. The decrease in aggregation was associated with inhibition of TxA₂ production and the increase in adhesion was associated with enhanced 12-HETE production. Salicylate had the opposite effects. Platelet aggregation was increased and platelet adhesion decreased. The increased aggregation was associated with enhanced TxA₂ production and the decrease in aggregation was associated with inhibition of 12-HETE production. These observations suggest that 12-HETE facilitates platelet adhesion which can be altered by salicylate treatment.     

Involvement of the antiplatelet activity of magnesium sulfate in suppression of protein kinase C and the Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> exchanger

Magnesium sulfate is widely used to prevent seizures in pregnant women with hypertension. The aim of this study was to examine the inhibitory mechanisms of magnesium sulfate in platelet aggregation in vitro. In this study, magnesium sulfate concentration-dependently (0.6–3.0 mM) inhibited platelet aggregation in human platelets stimulated by agonists. Magnesium sulfate (1.5 and 3.0 mM) also concentration-dependently inhibited phosphoinositide breakdown and intracellular Ca⁺² mobilization in human platelets stimulated by thrombin. Rapid phosphorylation of a platelet protein of M_r 47,000 (P47), a marker of protein kinase C activation, was triggered by phorbol-12-13-dibutyrate (PDBu, 50 nM). This phosphorylation was markedly inhibited by magnesium sulfate (3.0 mM). Magnesium sulfate (1.5 and 3.0 mM) further inhibited PDBu-stimulated platelet aggregation in human platelets. The thrombin-evoked increase in pHi was markedly inhibited in the presence of magnesium sulfate (3.0 mM). In conclusion, these results indicate that the antiplatelet activity of magnesium sulfate may be involved in the following two pathways: (1) Magnesium sulfate may inhibit the activation of protein kinase C, followed by inhibition of phosphoinositide breakdown and intracellular Ca⁺² mobilization, thereby leading to inhibition of the phosphorylation of P47. (2) On the other hand, magnesium sulfate inhibits the Na⁺/H⁺ exchanger, leading to reduced intracellular Ca⁺² mobilization, and ultimately to inhibition of platelet aggregation and the ATP-release reaction.