Shear stress-induced von Willebrand factor binding to platelet glycoprotein Ib initiates calcium influx associated with aggregation.

Platelets subjected to elevated levels of fluid shear stress in the absence of exogenous agonists will aggregate. Shear stress-induced aggregation requires von Willebrand factor (vWF) multimers, extracellular calcium (Ca2+), adenosine diphosphate (ADP), and platelet membrane glycoprotein (GP)Ib and GPIIb-IIIa. The sequence of interaction of vWF multimers with platelet surface receptors and the effect of these interactions on platelet activation have not been determined. To elucidate the mechanism of shear stress-induced platelet aggregation, suspensions of washed platelets were subjected to different levels of uniform shear stress (15 to 120 dyne/cm2) in an optically modified cone and plate viscometer. Cytoplasmic ionized calcium ([Ca2+]i) and aggregation of platelets were monitored simultaneously during the application of shear stress; [Ca2+]i was measured using indo-1 loaded platelets and aggregation was measured as changes in light transmission. Basal [Ca2+]i was approximately 60 to 100 nmol/L. An increase of [Ca2+]i (up to greater than 1,000 nmol/L) was accompanied by synchronous aggregation, and both responses were dependent on the shear force and the presence of vWF multimers. EGTA chelation of extracellular Ca2+ completely inhibited vWF-mediated [Ca2+]i and aggregation responses to shear stress. Aurin tricarboxylic acid, which blocks the GPIb recognition site on the vWF monomer, and 6D1, a monoclonal antibody to GPIb, also completely inhibited platelet responses to shear stress. The tetrapeptide RGDS and the monoclonal antibody 10E5, which inhibit vWF binding to GPIIb-IIIa, partially inhibited shear stress-induced [Ca2+]i and aggregation responses. The combination of creatine phosphate/creatine phosphokinase, which converts ADP to adenosine triphosphate and blocks the effect of ADP released from stimulated platelets, inhibited shear stress-induced platelet aggregation without affecting the increase of [Ca2+]i. Neither the [Ca2+]i nor aggregation response to shear stress was inhibited by blocking platelet cyclooxygenase metabolism with acetylsalicylic acid. These results indicate that GPIb and extracellular Ca2+ are absolutely required for vWF-mediated [Ca2+]i and aggregation responses to imposed shear stress, and that the interaction of vWF multimers with GPIIb-IIIa potentiates these responses. Shear stress-induced elevation of platelet [Ca2+]i, but not aggregation, is independent of the effects of release ADP, and both responses occur independently of platelet cyclooxygenase metabolism. These results suggest that shear stress induces the binding of vWF multimers to platelet GPIb and this vWF-GPIb interaction causes an increase of [Ca2+]i and platelet aggregation, both of which are potentiated by vWF binding to the platelet GPIIb-IIIa complex.     

Increased basal and thrombin-induced free calcium in platelets of essential hypertensive patients

Intracellular free calcium, [Ca2+]i, was studied in platelets of essential hypertensive subjects and normotensive controls under basal conditions and after stimulation with epinephrine, norepinephrine, angiotensin II, ouabain, and thrombin, using the fluorescent calcium indicator quin 2. Basal [Ca2+]i was significantly higher in hypertensive subjects (n = 32) than in normotensive controls (n = 30; 167.4 +/- 5.0 vs 143.2 +/- 3.1 nmol/L; p less than 0.001). Epinephrine, norepinephrine, angiotensin II, and ouabain had no effect on platelet calcium, whereas thrombin induced a dose-dependent increase in [Ca2+]i in both the presence and absence of extracellular calcium. This [Ca2+]i increase in the presence of extracellular calcium, which depends mainly on calcium influx, was significantly higher (p less than 0.05) in platelets of hypertensive subjects at all thrombin concentrations (ranging from 0.025-0.1 U/ml), while the [Ca2+]i increase in the absence of extracellular calcium, which depends only on release from intracellular stores, was similar in hypertensive subjects and controls. These results suggest that, in essential hypertension, there is not only increased platelet resting [Ca2+]i but also an increase in agonist-mediated calcium influx, which appears to indicate a cell membrane abnormality in the platelets of subjects with essential hypertension.     

Intracellular Ca2+ mobilization and its regulation in platelet activation]

It has been generally accepted that platelets play etiological roles for the development of atherosclerosis and arterial thrombosis. Platelet activation may be dependent upon the cytosolic free Ca2+ concentration ([Ca2+]i), and regulated by PGI2 and endothelium-derived relaxing factor (EDRF) released by vascular endothelium. We have studied here the effect of endothelial cells (EC) on platelet activation and intracellular Ca2+ mobilization. Effluent of non-stimulated EC column inhibited thrombin-induced platelet aggregation and intracellular Ca2+ mobilization. An addition of this effluent to platelet suspension leaded to increase in intraplatelet cyclic AMP (cAMP) which was inhibited by the treatment of indomethacin to EC, suggesting that this effect was involved in PGI2 released by EC. On the other hand, effluent of thimerosal-stimulated EC column inhibited platelet aggregation and increase in [Ca2+]i stimulated with thrombin, and leaded to increase in intraplatelet cyclic GMP (cGMP). But the treatment of indomethacin to EC had no effect of this inhibition. The effect of thimerosal-stimulated EC was inhibited by the addition of 1-NG-monomethylarginine (NMA), EDRF/NO inhibitor, suggesting that EDRF released by thimerosal-stimulated EC produced an increase in cGMP and inhibited platelet activation. Although forskolin-induced in cAMP caused a marked prevention of inositol 1, 4, 5-trisphosphate (IP3) production stimulated with thrombin, 8-bromo cGMP and EDRF-induced increase in cGMP had no effect of IP3 production. An increase in cAMP and cGMP was considered to inhibit intracellular Ca2+ mobilization by different mechanisms in platelets.     

Endothelium-derived relaxing factor inhibits thrombin-induced platelet aggregation by inhibiting platelet phospholipase C.

Endothelium-derived relaxing factor (EDRF) inhibits platelet function, but the mechanism underlying this inhibitory effect is not known. To examine this, cultured acetylsalicylic acid (ASA)-treated endothelial cells (EC) from bovine aorta (BAEC) or from human umbilical vein (HUVEC) were incubated with washed, ASA-treated human platelets. Incubation of platelets with either BAEC or HUVEC resulted in inhibition of thrombin-induced platelet aggregation that was dependent on the number of EC added. This effect was potentiated by superoxide dismutase and reversed by treating EC with NG-nitro-L-arginine or by treating platelets with methylene blue, indicating that the inhibition of platelet aggregation was due to the release of EDRF by EC. EC significantly blocked the thrombin stimulated breakdown of phosphatidylinositol-4,5-bisphosphate (PIP2) and the production of phosphatidic acid in [32P]orthophosphate-labeled platelets and of inositol trisphosphate in [3H]myoinositol-labeled platelets. In addition, the thrombin-mediated activation of protein kinase C (PKC) and phosphorylation of myosin light chain were inhibited in the presence of EC. Finally, thrombin stimulated an increase in cytosolic ionized calcium concentration ([Ca2+]i) in fura2-loaded platelets that was abolished by concentrations of EC which also blocked thrombin-induced aggregation. These data indicate that EDRF blocks thrombin-induced platelet aggregation by inhibiting the activation of PIP2-specific phospholipase C and thereby suppressing the consequent activation of PKC and the mobilization of [Ca2+]i.     

Platelet activation and subsequent inhibition by plasmin and recombinant tissue-type plasminogen activator.

The success of plasminogen activators in recanalizing occluded coronary arteries may be influenced by their effect on blood platelets; however, some previous studies have shown platelet activation by plasmin and thrombolytic agents while others have shown an inhibitory effect. Moreover, it has not been determined whether these effects reflect an alteration of intracellular signal transduction, fibrinogenolysis, degradation of adhesive protein receptors, or a combination of these events. To distinguish among these possibilities, the increase of cytoplasmic [Ca2+] [( Ca2+]i), which is an intracellular marker of platelet activation that precedes fibrinogen binding to the surface of activated platelets, was measured along with aggregation and release of 5-hydroxytryptamine (5-HT) in washed human platelets incubated with plasmin or recombinant tissue-type plasminogen activator (rt-PA). Plasmin (0.1 to 1.0 CU/mL) induced a prompt, concentration-dependent [Ca2+]i increase when added to platelets, but subsequently inhibited the [Ca2+]i increase in response to thrombin or the endoperoxide analog U44069. Platelet aggregation accompanied the [Ca2+]i increase if the platelets were stirred, while the aggregation of platelets unstirred during plasmin incubation was inhibited upon agonist addition and resumption of stirring. The release of 5-HT paralleled the [Ca2+]i increase induced by plasmin and was also inhibited after the subsequent addition of a second agonist. The effects of rt-PA, added with plasminogen (100 micrograms/mL), were similar to those of plasmin, and could be accounted for by the concentration of plasmin generated. The ADP scavengers apyrase and CP/CK each prevented the [Ca2+]i increase, and aggregation caused by plasmin or rt-PA, and also prevented their inhibitory effects on thrombin-induced activation. Thus, plasmin and rt-PA initially activate platelets, inducing a [Ca2+]i increase, and, if the platelets are stirred, aggregation. Such activation is followed by subsequent inhibition of cellular activation by a second agonist; the inhibitory effect is in proportion to the degree of initial activation, and ADP is an important cofactor in both processes. These platelet effects occur at rt-PA concentrations achievable clinically, and may affect the success of therapy with thrombolytic and adjunctive agents.     

Abnormal Ca2+ handling and increased Mg2+ permeability in platelets of hypertensive rats.

In order to test the hypothesis that intracellular Na+ accumulation and cellular Mg2+ deficiency may be involved in the abnormalities in Ca2+ handling and reactivity in spontaneously hypertensive rats (SHR) platelets, the metabolism of Na+, Ca2+ and Mg2+ was determined in fluorescent dye loaded platelets from 15 SHR and 15 Wistar-Kyoto rats (WKY) at 12 weeks of age. Mg2+ leak was estimated as the Mg2+ influx induced by an increase in extracellular [Mg2+] (from 1 to 5 mmol/l) and Mg2+/Na+ exchange activity was estimated as the Mg2+ influx induced by a decrease in extracellular [Na+] (from 140 to 50 mmol/l). Cellular metabolism of the fluorescent dye was similar in the two groups. Mean platelet [Ca2+]i was significantly increased under basal and thrombin (0.1 U/ml)-stimulated conditions in SHR compared to WKY, both in the presence and absence of extracellular Ca2+. Mean Ca2+ discharge capacity was similar between the two groups. There was no difference in mean [Na+]i between the two groups. Basal [Mg2+]i was also increased in SHR platelets. Mg2+ leak was higher in SHR than in WKY, while Mg2+/Na+ exchange activity was similar in the two groups. There was no difference in serum Mg2+ concentration between SHR and WKY. These data suggest that abnormal Ca2+ handling is accompanied by elevation in [Mg2+]i via increased permeability of platelet cell membranes to Mg2+ in SHR without any alteration in [Na+]i, and do not support the Mg2+ deficiency hypothesis in genetically hypertensive rats.     

Hypertonic saline solutions attenuate agonist-induced changes of intracellular calcium

The proper fluid for resuscitation of hemorrhagic shock is still controversial. Hypertonic saline solutions would cause an impairment of platelet function, aggravating blood loss in case of uncontrolled hemorrhage. This work examines the in vitro effect of hypertonic NaCl solutions on the changes in [Ca2+]i induced by 100 microM ADP, 0.1 IU/ml thrombin or 0.5 microM ionomycin in human platelets. Furthermore, it was investigated if the addition of NaCl reduces the mobilization from intracellular stores or the calcium entry from extracellular media. In a solution containing 1 mM CaCl2, the increase of [Ca2+]i produced by thrombin was 93, 75 or 55% of the 300 mosM control when osmolarity of the solution was 350, 400, or 500 mosM, respectively. The calcium signal induced by ADP decreased more rapidly in hypertonic media than in isotonic solution. In a calcium-free solution, the mobilization of stored calcium produced by thrombin was reduced when osmolarity was increased from 300 mosM to 350, 400 or 500 mosM by 86, 75 or 45% of the control, respectively. The increase of [Ca2+]i produced by subsequent introduction of 1 mM extracellular calcium was also reduced. Similar effects were found when platelets were stimulated by ADP. Instead, the capacitative calcium entry induced by ionomycin was increased in 500 mosM media by a 138% of the isotonic control. The decrease in the Ca2+ signal produced by receptor agonists in hypertonic media may play a role in the reduction of platelet responses such as aggregation or shape change when hypertonic resuscitation fluids are utilized.     

Tyrosine phosphorylation of phospholipase C induced by membrane immunoglobulin in B lymphocytes.

Ligation of membrane IgM on B lymphocytes causes activation of a protein-tyrosine kinase(s) (PTK) and of phospholipase C (PLC). To determine whether these are elements of a common signal-transduction pathway, the effect of three PTK inhibitors on the rise in intracellular free Ca2+ concentration [( Ca2+]i) in human B-lymphoblastoid cell lines was assessed. Tyrphostin completely suppressed the increase in [Ca2+]i and the generation of inositol phosphates induced by ligation of membrane immunoglobulin (mIg) M. Herbimycin and genistein reduced by 30% and 50%, respectively, the rise in [Ca2+]i caused by optimal ligation of mIgM, and they abolished it in cells activated by suboptimal ligation of mIgM. Tyrphostin had no effect on the capacity of aluminum fluoride to increase [Ca2+]i. To determine whether a function of PTK is the phosphorylation of PLC, immunoprecipitates obtained with anti-phosphotyrosine from detergent lysates of B-lymphoblastoid cells were assayed for PLC activity. Ligation of mIgM increased immunoprecipitable PLC activity 2-fold by 90 sec and 4-fold by 30 min. Specific immunoprecipitation and Western blot analysis identified tyrosine phosphorylation of the gamma 1 isoform of PLC after 60 sec of stimulation. Activation of PLC in B cells by mIgM requires PTK function and is associated with tyrosine phosphorylation of PLC-gamma 1, suggesting a mechanism of PLC activation similar to that described for certain receptor PTKs.     

Activation of phospholipases A and C in human platelets exposed to epinephrine: role of glycoproteins IIb/IIIa and dual role of epinephrine.

Human platelets stimulated by epinephrine undergo enhanced turnover of phosphatidylinositol 4,5-bisphosphate, accumulate inositol trisphosphate, diacylglycerol, and phosphatidic acid, and phosphorylate a 47-kDa protein. All of these phenomena indicate stimulation of phospholipase C. These responses are blocked completely by inhibitors of alpha 2-adrenergic receptors (yohimbine), cyclooxygenase (aspirin or indomethacin), phospholipase A [2-(p-amylcinnamoyl)amino-4-chlorobenzoic acid (ONO-RS-082)], Na+/H+ exchange [ethylisopropylamiloride (EIPA)], fibrinogen binding to glycoprotein IIb/IIIa (antibody A2A9), Ca2+/Mg+ binding (EDTA), or removal of fibrinogen. Epinephrine evokes (i) an increased turnover of ester-linked arachidonic acid in aspirin treated platelets that is inhibited by ONO-RS-082, EDTA, yohimbine, or the absence of fibrinogen and (ii) a rapid cytoplasmic alkalinization that is inhibited partially by blockage of cyclooxygenase activity and completely by A2A9 or EIPA. In contrast, when incubated with subaggregatory concentrations of the prostaglandin H2/thromboxane A2 analogue [(15S)-hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic acid (U46619) and epinephrine, aspirin-treated platelets show a potentiation of phospholipase C activation that is unaffected by the above inhibitors. We propose that epinephrine, in promoting exposure of glycoprotein IIb/IIIa sites for fibrinogen binding, leads to a cytoplasmic alkalinization, which, in conjunction with local shifts in Ca2+, promotes low-level activation of phospholipase A. The resulting free arachidonic acid is converted to cyclooxygenase products, which, potentiated by epinephrine, activate phospholipase C. This further amplifies the initial stimulatory response.     

The role of protein kinase C in alpha-thrombin-mediated endothelial cell activation.

Thrombin, the key regulatory protein of hemostasis, has been implicated in a variety of important endothelial cell processes closely linked to endothelial signal transduction mechanisms. An initial event, following receptor binding by catalytically active alpha-thrombin, appears to be the activation of a G-protein-coupled, PI-specific PLC, with resultant generation of IP3 and DAG, with increases in [Ca2+]i, and activation and translocation of PKC (Fig. 9). PKC activation results in down-regulation of PLC, as demonstrated by inhibition of agonist-induced increases in [Ca2+]i, whereas PLA2 activity is up-regulated, with a resultant increase in endothelial PGI2 synthesis. Recently, we have demonstrated that activity of membrane-bound, endothelial PLD, is also up-regulated by PKC activation. In addition to its modulatory role in endothelial cell phospholipase activities, PKC activation appears to play a critical role in thrombin-mediated endothelial barrier dysfunction, likely via specific cytoskeletal protein phosphorylation. A temporal relationship between alpha-thrombin-mediated signal transduction and specific cellular responses, such as PGI2 synthesis and barrier dysfunction, can be established (Fig. 2). Further investigations are ongoing to identify more clearly the precise biochemical intermediates involved in the endothelial cell response to thrombin, as well as the role of differential phosphorylation by various protein kinase systems in thrombin-mediated signal transduction in vascular endothelium.     

Cytosolic calcium in platelets of spontaneously hypertensive rats

To test the hypothesis that an abnormality of the intracellular concentration of ionized calcium, [Ca2+]i, is associated with high blood pressure, we measured [Ca2+]i in the platelets of spontaneously hypertensive (SHR) and Wistar-Kyoto control (WKY) rats using the Quin 2 technique after separation of the platelets in calcium-free medium, during calcium repletion, and upon exposure to agonists which increase platelet [Ca2+]i (thrombin, adenosine diphosphate, serotonin and ionomycin). Despite clear-cut changes in [Ca2+]i during these manipulations, there were no differences between the SHR and WKY rats in baseline levels of [Ca2+]i or in the kinetics of changes in [Ca2+]i. These results do not support the hypothesis that high levels of [Ca2+]i at rest or abnormal kinetics of changes in [Ca2+]i play a pathophysiological role in the hypertension of SHR.     

Subsecond calcium dynamics in ADP- and thrombin-stimulated platelets: a continuous-flow approach using indo-1

The regulation and kinetics (less than 5 seconds) of cytosolic calcium changes ([Ca2+]i) in stimulated blood platelets have been investigated under physiological blood flow conditions. Using a newly-developed continuous-flow approach with indo-1-loaded human platelets, adenosine diphosphate (ADP, 10 mumol/L) and thrombin (5 U/mL) were equally effective in significantly increasing [Ca2+]i by 0.5 seconds. ADP induced a transient [Ca2+]i peak of 1 to 2 mumol/L near 2 seconds, whereas thrombin caused a sustained and larger response. The first phase (less than 2 seconds) was not influenced by a lack of extracellular Ca2+, in contrast to the subsequent [Ca2+]i increase that only reached about 0.7 mumol/L for either ADP or thrombin. The shear rates used in our continuous-flow apparatus were physiological (less than 1,258 sec-1) and only slightly increased the basal [Ca2+]i of 0.1 mumol/L. Platelet aggregation (less than 5 seconds), assessed by single- particle counting, was not altered in platelets loaded with indo-1/AM (2.5 mumol/L).     

Inhibition of PAR4 signaling mediates ethanol-induced attenuation of platelet function in vitro

BACKGROUND: Reduction in coronary heart disease morbidity in response to moderate consumption of alcoholic beverages may be partly mediated by ethanol-induced inhibition of platelet function. However, the precise mechanisms by which ethanol modulates platelet activation induced by thrombin, which plays a central role in hemostasis, remain unclear. The goal of this study was to investigate ethanol-induced changes in platelet function and clarify the underlying mechanisms including PAR1 and PAR4 activity and [Ca2+]i dynamics in vitro. METHODS: Platelet aggregation, increase in intracellular calcium ([Ca2+]i), and release of platelet factor 4 and beta-thromboglobulin induced by alpha-thrombin, PAR1-agonist peptide (AP), or PAR4-AP were assessed in the presence or absence of ethanol. RESULTS: Ethanol exposure inhibited low-dose thrombin (0.5 nM)-induced aggregation but not an increase in [Ca2+]i. In contrast, ethanol had no effect on high-dose thrombin (10 nM)-induced aggregation or the [Ca2+]i increase. Ethanol did not significantly inhibit thrombin-induced release of platelet factor 4 and beta-thromboglobulin. Ethanol reduced PAR1-AP-induced aggregation, but did not affect the spike form of [Ca2+]i increase. In contrast, ethanol inhibited the increase in [Ca2+]i as well as the aggregation in response to PAR4-AP and resulted in delayed [Ca2+]i peak time. Furthermore, ethanol inhibited both PAR1-AP- and PAR4-AP-induced platelet factor 4 and beta-thromboglobulin release. CONCLUSIONS: These data suggest that ethanol inhibits platelet aggregation via inhibition of PAR4 signaling and subsequent inhibition of Ca2+ influx and granule release. This phenomenon may contribute to the reduction in coronary heart disease morbidity in response to consumption of alcoholic beverages.     

Defective signal transduction induced by thromboxane A2 in a patient with a mild bleeding disorder: impaired phospholipase C activation despite normal phospholipase A2 activation

A patient with a mild bleeding disorder whose platelets responded defectively to thromboxane A2 (TXA2) was identified, and the mechanism of this dysfunction was analyzed. The platelets were defective in shape change, aggregation, and release reaction in response to synthetic TXA2 mimetic (STA2). When the platelet TXA2 receptor was examined with both a 125I-labeled derivative of a TXA2 receptor antagonist ([125I]-PTAOH) and [3H]-labeled TXA2 agonist ([3H]U-46619), the equilibrium dissociation rate constants (kd) and the maximal concentrations of binding sites (Bmax) of the platelets to both ligands were within normal ranges, suggesting that the binding capacity of their TXA2 receptor was normal. STA2 could not induce IP3 formation and intracellular Ca2+ mobilization, whereas these responses to thrombin were within normal ranges. GTPase activity was also decreased when the patient's platelet membrane was challenged with STA2. On the other hand, lysophosphatidylinositol formation, which is a direct indicator of phospholipase A2 (PLA2) activation, was found to be normal when the [3H]-inositol-labeled platelets were challenged with STA2. Thromboxane B2 (TXB2) was also produced in response to STA2. These results suggested that the abnormality in these platelets was impaired coupling between TXA2 receptor and phospholipase C (PLC) activation. Furthermore, it is also suggested that the activation of PLA2 and PLC are separable events in thromboxane-induced platelet activation.     

Human platelet calcium measurements. Methodological considerations and comparisons with calcium mobilization in vascular smooth muscle cells

Platelets are used as models for vascular smooth muscle cells (VSMC) in evaluating intracellular calcium ([Ca2+]i) metabolism in humans. This study was designed to determine if agonist-induced increases in [Ca2+]i in platelets occur via release from intracellular stores as previously demonstrated for VSMC. Incubation of purified platelets loaded with fura-2-AM in media containing 1.5 mmol/L Ca2+ resulted in higher basal [Ca2+]i than platelets incubated in Ca(2+)-free media. In addition, vasopressin-induced platelet [Ca2+]i transients were almost completely blocked by Ca2+ channel blockers. Thus, in contrast to VSMC, the transmembranous flux of extracellular Ca2+ is the major mechanism in vasopressin-induced increases in platelet [Ca2+]i, while mobilization of intracellular Ca2+ stores is only minimally involved.     

Induction of platelet Ca2+ influx and mobilization by a monoclonal antibody to CD9 antigen

We found that a monoclonal antibody (MoAb) to CD9 antigen, PMA2, induced a rise in cytosolic free calcium concentration ([Ca2+]i) in fura-2-loaded platelets, and we examined whether this response was due to direct action of PMA2 on CD9 antigen. The rise in [Ca2+]i was dependent on the PMA2 concentration, irrespective of the presence or absence of extracellular Ca2+. The role of secreted adenosine diphosphate (ADP) and thromboxane in the [Ca2+]i response to PMA2 was studied using creatine phosphate/creatine phosphokinase (CP/CPK) and aspirin. Combined treatment with CP/CPK and aspirin abolished the rise in [Ca2+]i, although either CP/CPK or aspirin alone produced only partial inhibition. Inhibition of adenosine triphosphate (ATP) secretion and thromboxane B2 synthesis by an MoAb to the glycoprotein IIb-IIIa complex, PMA1, resulted in little [Ca2+]i response to PMA2. In contrast, thrombasthenic platelets, in which ATP secretion and thromboxane B2 synthesis were normal, showed a normal [Ca2+]i response. When PMA2 was added to CD9+ mononuclear cells, no rise in [Ca2+]i was observed. Thus, we conclude that binding of monoclonal immunoglobulin G molecules to the CD9 antigen raises [Ca2+]i through the effect of secreted ADP and thromboxane on platelets, and that CD9 antigen is not directly involved in induction of Ca2+ influx and mobilization.     

Anti-GPIIb/IIIa (CD41) monoclonal antibody-induced platelet activation requires Fc receptor-dependent cell-cell interaction

We studied platelet activation by UR1, a murine IgG1 anti-CD41 mAb. Like thrombin and crosslinked anti-Fc gamma RII mAb IV3, UR1 initiates prompt aggregation and Ca2+ mobilization. UR1 F(ab')2 fragments failed to activate, yet inhibited UR1 IgG-mediated activation. UR1-induced activation was blocked by anti-Fc gamma RII mAb. High viscosity (15% dextran or Ficoll), which impedes cell-cell interaction, inhibited activation by UR1. Cell-cell interaction was confirmed by cell-mixing studies. UR1 binding to platelets of one pool was blocked with UR1 F(ab')2 allowing UR1 binding only to Fc gamma RII. IV3 Fab fragments blocked ligand binding to Fc gamma RII on platelets of a second pool; thus, UR1 could bind only its epitope. UR1 initiated an immediate [Ca2+]i increase in the intermixed pools at low ionic strength. These studies indicate that UR1 IgG binds CD41 on one platelet to form immune complexes which then crosslink and stimulate Fc gamma RII on nearby platelets. Two other anti-CD41 mAb, 6C9 and C17, and two anti-CD9 mAb, AG1 and mAb7, activated platelets in a UR1-like manner. We propose that platelet Fc gamma RII crosslinking that follows the interaction of IgG-opsonized platelets may be a common mechanism by which anti-platelet antibodies activate platelets.     

Lack of effect of parathyroid hormone on calcium homeostasis in rat platelets.

The in vitro effects of parathyroid hormone on Ca2+ handling by fura-2 loaded rat platelets were studied. The incubation of these platelets with rat parathyroid hormone (1-34) for 10 min had no effect on intracellular fura-2 metabolism or [Ca2+]i in the resting state. The [Ca2+]i response to 0.1 U/mL thrombin was unaffected by preincubation with parathyroid hormone in the presence or absence of extracellular Ca2+. Furthermore, the recovery rate of [Ca2+]i after the thrombin-induced peak in Ca(2+)-depleted media was not altered with parathyroid hormone. These data indicate that parathyroid hormone may not have a significant effect on Ca2+ homeostasis in rat platelets in unstimulated and stimulated conditions.     

Effect of strenuous, acute exercise on alpha2-adrenergic agonist-potentiated platelet activation.

Vigorous exercise transiently increases the risk of primary cardiac arrest. Strenuous, acute exercise can also increase the release of plasma epinephrine. Previous investigations have indicated that epinephrine can potentiate platelet activation by activating platelet alpha2-adrenoceptors. This study investigated how strenuous, acute exercise affects alpha2-adrenergic agonist-potentiated platelet activation by closely examining 15 sedentary men who exercised strenuously on a bicycle ergometer. Before and immediately after exercise, platelet adhesiveness on fibrinogen-coated surfaces, [Ca2+]i in platelets, the number and affinity of alpha2-adrenergic sites on the platelet surface, and plasma catecholamine levels were determined. The results of this study can be summarized as follows: (1) The affinity of alpha2-adrenergic receptors on platelets decreases while the maximal binding number significantly increases after strenuous exercise, thereby correlating with the rise in plasma catecholamine levels. (2) Basal, clonidine-treated, ADP-treated, and clonidine plus ADP-treated adhesiveness and [Ca2+]i in platelets increased after strenuous exercise. (3) Strenuous exercise is associated with higher percentages of ADP- and clonidine plus ADP-enhanced platelet adhesiveness and [Ca2+]i than at rest. (4) The synergistic effects of clonidine on ADP-enhanced platelet adhesiveness and [Ca2+]i after strenuous exercise are much greater than those at rest. Therefore, we conclude that strenuous, acute exercise enhances platelet activation, possibly by altering the performance of platelet alpha2-adrenergic receptors, facilitating the ability of ADP-activated fibrinogen receptors, and enhancing fibrinogen binding to platelet fibrinogen receptors.     

Effect of extracellular magnesium on platelet activation and intracellular calcium mobilization.

A dose-dependent effect of magnesium on the inhibition of platelet aggregation and release of ATP from dense granules was observed in human platelets (in whole blood, platelet-rich plasma, or washed platelets) against various aggregation agents (ADP, U46619, collagen, or thrombin). The synthesis and release of the proaggregatory cyclooxygenase (CO) and lipoxygenase (LO) products, thromboxane A2 (TXA2) and 12-hydroxyeicosatetraenoic acid (12-HETE), respectively, in platelets were also inhibited by Mg in a dose-dependent manner (IC50 4 to 6 mmol/L). These Mg-mediated activities were further enhanced when platelets were preincubated with insulin (100 microU/mL). The effect of extracellular Mg on the change of intracellular calcium concentration ([Ca2+]i) was assessed using Fura-2/AM loaded cells in the presence or absence of extracellular Ca. Thrombin-stimulated influx of Ca ions decreased from 194 +/- 30 nmol/L to 156 +/- 21 nmol/L in the presence of 5 mmol/L Mg and to 111 +/- 16 nmol/L in 10 mmol/L Mg. However, the intracellular Ca release (as determined in the presence of 5 mmol/L EGTA) was not affected by Mg. The intracellular Ca-dependent protein kinase C and myosin light chain kinase activities on the phosphorylation of endogenous p47 and p20 proteins studied after 2 min of thrombin addition decreased only 10 to 25% in the presence of 5 to 10 mmol/L Mg. Similar results were obtained when EGTA was added prior to the initiation of protein phosphorylation. We conclude that Mg can dose dependently inhibit a wide variety of agonists on platelet aggregation. Furthermore, insulin can potentiate the inhibitory effects of Mg on platelet activation.(ABSTRACT TRUNCATED AT 250 WORDS)