Platelet Calcium Research Articles

The mechanisms underlying the stimulatory effects of thrombin on myometrial smooth muscle

Objective: The mechanisms underlying the stimulation of uterine contractions in the presence of intrauterine hemorrhage have not been well defined. Thrombin, a blood coagulation factor, activates membrane receptors to result in the stimulation of the phosphatidylinositol signaling pathway and the mobilization of cytosolic calcium in platelets. Our studies sought to determine whether thrombin stimulates similar events in myometrial smooth muscle. Study Design: Cytosolic calcium imaging and in vitro contraction studies were performed with rat myometrial tissue. Results: At a concentration range of 1 to 100 U/mL thrombin produced phasic myometrial contractions, which were comparable in intensity to those produced by oxytocin and prostaglandin F2α. Thrombin-induced cytosolic calcium concentration oscillations were similar to those produced by oxytocin. Contractions stimulated by thrombin were significantly suppressed in response to inhibitors of the phosphatidylinositol signaling pathway. These studies also confirmed that membrane receptor–Gq protein coupling events play a more important role than tyrosine kinase–mediated events during thrombin stimulation of myometrial smooth muscle. Conclusion: Thrombin is a potent uterotonic agonist, and its effects in myometrium are mediated by intracellular signaling events comparable to those activated by classic uterotonic agents. The physiologic importance of thrombin appears to be related to its potential role in the stimulation of uterine contractions in the presence of intrauterine hemorrhage. (Am J Obstet Gynecol 2000;183:674-81.)

Platelet activation is increased in cyclosporin A-induced hypertensive rats.

One of the most severe side effects of the immunosuppressive agent, cyclosporin A (CsA), is increased risk of thromboembolic complications and drug-related hypertension. Because platelets might be involved in these processes, we tested the possibility of CsA affecting platelet activation, which might contribute to these adverse drug reactions. The experiments were done using Wistar rats, treated or not (control) with CsA (Sandimmun Neoral), 5 and 30 mg/kg/day, for 7 weeks. Systolic, diastolic, and mean blood pressures, intracellular free calcium concentration ([Ca2+]i), platelet serotonin (5-HT) contents, and aggregation were determined, at weeks 0, 2, and 7 of treatment. Inositol phosphates (InsP) production, platelet thromboxane A2 (TXA2) generation, and morphology of platelets, through electron microscopy studies, also were compared. It was demonstrated that blood pressures increased in the CsA-treated groups, when compared with the control group, after 2 and 7 weeks of administration. CsA at both "attack" and "maintenance" doses increased basal, 5-HT, and thrombin-evoked [Ca2+]i after 2 and 7 weeks versus the control group. However, basal and evoked InsP production was stimulated by 5 mg/kg of CsA, but inhibited by 30 mg/kg, when compared with the control. Platelet 5-HT contents decreased significantly after 2 and 7 weeks in the CsA-treated groups, when compared with the control group. Collagen-induced whole blood platelet aggregation increased drastically in the "attack" CsA-treated group, whereas adenosine diphosphate (ADP)-induced platelet aggregation did not reach statistical significance. Finally, in vitro basal, collagen-, and ADP-evoked platelet TXA2 generation increased in both CsA concentrations, versus the control. In conclusion, our study demonstrates that both CsA doses alter platelet calcium homeostasis (even affecting the calcium fluxes differently), 5-HT and TXA2 contents and aggregation, which might contribute to the development and/or maintenance of high blood pressures and increased risk of thromboembolic complications.

Beta-Amyloid fragment 25-35 induces changes in cytosolic free calcium in human platelets.

beta-Amyloid fragment 25-35 induces changes in cytosolic free calcium in human platelets.

Structures of the platelet calcium‐ and integrin‐binding protein and the αIIb‐integrin cytoplasmic domain suggest a mechanism for calcium‐regulated recognition; homology modelling and NMR studies

Structures of the platelet calcium‐ and integrin‐binding protein and the αIIb‐integrin cytoplasmic domain suggest a mechanism for calcium‐regulated recognition; homology modelling and NMR studies

Effects of Welsh onion extracts on human platelet function in vitro

Welsh onion has been consumed for prevention of cardiovascular disorders. However, its underlying mechanisms are still unclear. This study investigated whether Welsh onion extracts can alter human platelet function (ie, platelet adhesion, aggregation, and thromboxane release). To clarify the underlying mechanisms, we also measured the intracellular calcium ([Ca 2+] i) and cyclic nucleotide levels in platelets. Our results showed that 1) boiled extracts directly induced platelet aggregation in a dose-dependent manner; 2) raw extracts inhibited platelet adhesion and ADP-evoked platelet aggregation, while boiled extracts enhanced them; 3) raw green extract suppressed ADP-stimulated platelet [Ca 2+] i elevation and thromboxane production, whereas boiled green extract enhanced them; 4) raw green extract elevated platelet cAMP level, whereas boiled green extract had no effect on cAMP level. Furthermore, the boiled green extract, but not the raw extract, induced pronounced platelet morphological changes. In conclusion, raw extracts of Welsh onion inhibit platelet function in vitro while boiled extracts activate platelets.

Structures of the platelet calcium- and integrin-binding protein and the alphaIIb-integrin cytoplasmic domain suggest a mechanism for calcium-regulated recognition; homology modelling and NMR studies.

Calcium- and integrin-binding protein (CIB) binds to the 20-residue alphaIIb cytoplasmic domain of platelet alphaIIbbeta3 integrin. Amino acid sequence similarities with calmodulin (CaM) and calcineurin B (CnB) allowed the construction of homology-based models of calcium-saturated CIB as well as apo-CIB. In addition, the solution structure of the alphaIIb cytoplasmic domain in 45% aqueous trifluoroethanol was solved by conventional two-dimensional NMR methods. The models indicate that the N-terminal domain of CIB possesses a number of positively charged residues in its binding site that could interact with the acidic carboxy-terminal LEEDDEEGE sequence of alphaIIb. The C-terminal domain of CIB seems well-suited to bind the sequence WKVGFFKR, which forms a well-structured alpha helix; this is analogous to calmodulin and calcineurin B, which also bind alpha helices. Similarities between the C-terminal domains of CIB and calmodulin suggest that binding of CIB to the cytoplasmic domain of alphaIIb may be affected by fluctuations in the intracellular calcium concentration.

Effects of thrombin on intracellular calcium and pH in human and murine platelets

The aim of this study was to compare the effect of thrombin (Thr) on cytosolic calcium [Ca 2 ] + i and intracellular pH [pH]i in human and murine platelets. Rich-platelet suspensions from both species were loaded with Fura-2 (2 w M) or BCECF (0.75 w M) by incubation with their respective acetoxymethyl esters to measure cytosolic calcium [Ca 2+ ] i or intracellular pH [pH]i, respectively. Suspensions were challenged with increasing concentrations of Thr, from 0.1 to 10 IU/ml. Basal [Ca 2+ ] i in human platelets was 98 - 6 and 99.1 - 9 nM in rat platelets ( n =20). Thr increased \\[Ca2+] i , EC 50 was 1.1 - 0.04 in human and 0.97 - 0.06 IU/ml in rat platelets ( n =7). Extracellular Mg 2+ (4 or 8 mM) abolished Thr response on [Ca 2+ ] i . [pH]i in human was 7.09 - 0.08 and 7.11 - 0.04 in rat platelets. Thr induced alkalinization of platelets in both species. Our results indicate that the potency of Thr to change [Ca 2+ ] i and [pH]i was similar in both species, allowing for comparisons between human and murine platelets and to extrapolate results from an animal model to human pathology.

Augmented platelet calcium uptake in response to serotonin stimulation in patients with major depression measured using Mn 2+ influx and 45Ca 2+ uptake

There is an augmented platelet intracellular calcium response to serotonin stimulation in major depression. The role that calcium influx has in this process is not known. The objective of this study was to determine platelet calcium influx in response to serotonin by two methods, Mn 2+ influx and 45Ca 2+ uptake, in order to observe if the uptake response to serotonin was augmented in major depression by comparing the response to normal controls. The use of the two methods of calcium influx showed that serotonin stimulates calcium uptake into platelets. Furthermore, patients with major depression have significantly augmented platelet calcium uptake in response to serotonin. The interesting finding was that calcium uptake into platelets is biphasic, occurring immediately and after five minutes. These results may support the two pool model for calcium oscillations within cells whereby extracellular calcium is needed for intracellular calcium release, and for replenishment of depleted stores once intracellular calcium is released.

Nitric Oxide Pathway, Ca2+, and Serotonin Content in Platelets from Patients Suffering from Chronic Daily Headache

An alteration in serotonin concentration has been found in patients with chronic headache caused by abuse of analgesic substances as well as an up-regulation of 5HT2 platelet receptors, which has been correlated with chronicization of the headache. In a previous study we demonstrated an increase in L-arginine/nitric oxide (NO) pathway activity in platelets from patients affected by migraine with or without aura, particularly during attacks. In the present research we assessed the variations in platelet L-arginine/NO pathway and cyclic guanosine monophosphate (cGMP) levels in 32 patients affected by chronic daily headache (CDH) (8 M, 24 F, age range 34-50 years) both during and between attacks. In these same patients, the platelet aggregation to different collagen concentrations (0.3, 1, 3 micrograms/ml) was determined as well as the intracellular platelet calcium concentration using fluorescence polarization spectrometry. These parameters were compared with those of an age- and sex-matched control group (n = 25; n = 10, n = 15, age range 35-51 years). A reduction found in platelet aggregation response to each collagen concentration used (p < 0.001) was coupled with an increased NO and cGMP production (NO: p < 0.0001; cGMP: p < 0.001). This was accompanied by a significant increase in intracytosolic Ca2+ (p < 0.0001) concentration and a reduced platelet serotonin content compared to those in control individuals (p < 0.0002). Changes in the above platelet parameters were accentuated more in patients with analgesic abuse than in CDH patients with no drug abuse. These findings suggest the occurrence of an activation of cGMP-Ca2+ mediated events in CDH patients with analgesic abuse. This physiologic compensatory mechanism, which intervenes in overcoming the increase in cytosolic Ca2+ levels, is not as efficient at limiting serotonin depletion by platelet dense bodies. A similar depletion in the central serotoninergic pathway can be assumed in the same patients.

Advantages of Calcium Green-1 over Other Fluorescent Dyes in Measuring Cytosolic Calcium in Platelets

Fluorescent indicators are widely used in the measurements of cytosolic calcium in many cell types for many purposes because they are relatively easy to use. Notwithstanding, they have some defects to prevent accurate measurements under certain conditions, such as significant dye leakage and UV-quenching effect. Menadione, a representative quinone derivative with antiaggregating effect, is also UV-absorbent. To investigate whether menadione can affect the change of cytosolic calcium in platelets by agonist, we measured the change of cytosolic calcium level using calcium green-1. Since this dye has not been used previously in platelets, we determined that the optimal loading of calcium green-1 to platelets was achieved using 3 μM dye incubated for 60 min at 37°C. Our study compared the use of calcium green-1 with fura-2 and fluo-3 (two widely used dyes) in measurements of cytosolic calcium. Fura-2 is UV-excited, so when menadione was treated in fura-2-loaded cells, it had a quenching effect. Fluo-3, the other visible fluorescent indicator, leaked from platelets very rapidly and required the use of anion channel blockers which are known to affect physiological response of platelets. Our study demonstrated that changes in cytosolic calcium levels can be accurately measured without these problems by using calcium green-1. We therefore were able to demonstrate that menadione inhibited calcium increase by thrombin in a dose-dependent manner similar to menadione's antiaggregating effect in platelets.

Dichotomous Regulation of Myosin Phosphorylation and Shape Change by Rho-Kinase and Calcium in Intact Human Platelets

AbstractBoth Rho-kinase and the Ca2+/calmodulin-dependent myosin light chain (MLC) kinase increase the phosphorylation of MLC. We show that upon thrombin receptor stimulation by low-dose thrombin or the peptide ligand YFLLRNP, or upon thromboxane receptor activation by U46619, shape change and MLC phosphorylation in human platelets proceed through a pathway that does not involve an increase in cytosolic Ca2+. Under these conditions, Y-27632, a specific Rho-kinase inhibitor, prevented shape change and reduced the stimulation of MLC-phosphorylation. In contrast, Y-27632 barely affected shape change and MLC-phosphorylation by adenosine diphosphate (ADP), collagen-related peptide, and ionomycin that were associated with an increase in cytosolic Ca2+ and inhibited by BAPTA-AM/EGTA treatment. Furthermore, C3 exoenzyme, which inactivates Rho, inhibited preferentially the shape change induced by YFLLRNP compared with ADP and ionomycin. The results indicate that the Rho/Rho-kinase pathway is pivotal in mediating the MLC phosphorylation and platelet shape change by low concentrations of certain G protein–coupled platelet receptors, independent of an increase in cytosolic Ca2+. Our study defines 2 alternate pathways, Rho/Rho-kinase and Ca2+/calmodulin-regulated MLC-kinase, that lead independently of each other through stimulation of MLC-phosphorylation to the same physiological response in human platelets (ie, shape change).

Dichotomous Regulation of Myosin Phosphorylation and Shape Change by Rho-Kinase and Calcium in Intact Human Platelets

Both Rho-kinase and the Ca2+/calmodulin-dependent myosin light chain (MLC) kinase increase the phosphorylation of MLC. We show that upon thrombin receptor stimulation by low-dose thrombin or the peptide ligand YFLLRNP, or upon thromboxane receptor activation by U46619, shape change and MLC phosphorylation in human platelets proceed through a pathway that does not involve an increase in cytosolic Ca2+. Under these conditions, Y-27632, a specific Rho-kinase inhibitor, prevented shape change and reduced the stimulation of MLC-phosphorylation. In contrast, Y-27632 barely affected shape change and MLC-phosphorylation by adenosine diphosphate (ADP), collagen-related peptide, and ionomycin that were associated with an increase in cytosolic Ca2+ and inhibited by BAPTA-AM/EGTA treatment. Furthermore, C3 exoenzyme, which inactivates Rho, inhibited preferentially the shape change induced by YFLLRNP compared with ADP and ionomycin. The results indicate that the Rho/Rho-kinase pathway is pivotal in mediating the MLC phosphorylation and platelet shape change by low concentrations of certain G protein–coupled platelet receptors, independent of an increase in cytosolic Ca2+. Our study defines 2 alternate pathways, Rho/Rho-kinase and Ca2+/calmodulin-regulated MLC-kinase, that lead independently of each other through stimulation of MLC-phosphorylation to the same physiological response in human platelets (ie, shape change).

Effect of recombinant forms of urokinase plasminogen activator on platelet aggregation and intracellular calcium accumulation

Urokinase caused plasmin-dependent inhibition of platelet aggregation in platelet-rich plasma, while its proteolytically inactive form had no such effect. Both forms potentiated the increase in platelet calcium concentration induced by aggregation inductors and facilitated aggregation of washed platelets. In contrast to full-length urokinase molecule, its aminoterminal fragment inhibited platelet aggregation and the corresponding elevation of intracellular calcium. These data suggest that urokinase exerts a plasmin-independent effect on platelet activity. This effect depends on urokinase structure.

Elevated basal and thapsigargin-stimulated intracellular calcium of platelets and lymphocytes from bipolar affective disorder patients measured by a fluorometric microassay

Background: A number of investigators have reported finding elevated basal and stimulated intracellular calcium levels in the platelets or lymphocytes of bipolar disorder patients. Methods: Intracellular calcium was measured by a micro fura-2 fluorometric method in the platelets and lymphocytes of 30 affective disorder patients and 14 control subjects. Results: We observed significantly elevated basal calcium concentrations in bipolar patient platelets and lymphocytes compared to control subjects. Bipolar patient platelet calcium responses to thrombin, serotonin, and thapsigargin were also significantly greater than control subjects. The peak calcium levels of lymphocytes of bipolar patients were greater than control subjects only when stimulated by thapsigargin. There were significant differences between bipolar and unipolar patients in basal and thapsigargin-stimulated calcium measures but not between bipolar I and bipolar II patients. Unmedicated versus medicated calcium measures were not significantly different. We also found little correlation between calcium measures and the severity of mood rating. Conclusions: Using this method, we were able to confirm and extend the work of others, indicating altered intracellular calcium homeostasis in the blood cells of bipolar disorder patients. In addition, our data suggest that storage operated calcium channels may be the source of the elevated intracellular calcium in platelets and lymphocytes of bipolar patients.

Marked Temperature Dependence of the Platelet Calcium Signal Induced by Human von Willebrand Factor

Interaction of von Willebrand factor (vWF) with the platelet is essential to hemostasis when vascular injury occurs. This interaction elevates the intracellular free calcium concentration ([Ca2+]i) and promotes platelet activation. The present study investigated the temperature dependence of vWF-induced [Ca2+]i signaling in human platelets. The influence of temperature can provide invaluable insight into the underlying mechanism. Platelet [Ca2+]i was monitored with Fura-PE3. Ristocetin-mediated binding of vWF induced a transient platelet [Ca2+]i increase at 37 degrees C, but no response at lower temperatures (20 degrees C to 25 degrees C). This temperature dependence could not be attributed to a reduction in vWF binding, as ristocetin-mediated platelet aggregation and agglutination were essentially unaffected by temperature. Most other platelet agonists (U-46619, alpha-thrombin, and adenosine 5'-diphosphate [ADP]) induced a [Ca2+]i signal whose amplitude did not diminish at lower temperatures. The [Ca2+]i signal in response to arachidonic acid, however, showed similar temperature dependence to that seen with vWF. Assessment of thromboxane A2 production showed a strong temperature dependence for metabolism of arachidonic acid by the cyclo-oxygenase pathway. vWF induced thromboxane A2 production in the platelet. Aspirin treatment abolished the vWF-induced [Ca2+]i signal. These observations suggest that release of arachidonic acid and its conversion to thromboxane A2 play a central role in vWF-mediated [Ca2+]i signaling in the platelet at physiological temperatures.

Magnesium deficiency in drinking water modulates blood pressure, Ca and Mg distribution in tissues, and compartmentalization of membrane-bound calcium in platelets of normotensive WKY rats

Magnesium deficiency in drinking water increases calcium-accumulating capacity of the aortic and myocardial walls in normotensive WKY rats, induces Ca resorption from bones, and impairs compartmentalization of membrane-bound Ca in platelets, resulting in accumulation of Ca in the external plasma membrane without changing blood pressure. Increased systolic blood pressure was characteristic of rats in Ca-deficient groups.

Effect of Regular Exercise on Platelet Cytoplasmic Calcium during Treatmill Exercise in Healthy Young Males

Background and Objectives：Regular aerobic exercise plays a role in the prevention of cardiovascular diseases, but the risk of primary cardiac arrest transiently increases during vigorous exercise, particularly in sedentary men. There has been a controversy regarding the effect of exercise on platelet functional behavior, which is related to the pathogenesis of coronary ischemia. We evaluated the cytoplasmic free calcium concentration of platelets in healthy men after treadmill exercise. Materials and Method：Five physically active (group I；age, 24.2± 2.3 years and five sedentary men (group II；age, 22.4±1.4 yearswere included in this study. Platelet calcium was measured with fluorescent dye, quin2 before and after treadmill exercise. Results：Platelet calcium levels were increased from 86.8±11.8 nM to 128.8±15.0 nM in group I (p<0.05and from 102.6±14.4 nM to 162.4 ±26.5 nM in group II (p<0.05immediately after exercise. Thereafter, platelet calcium levels were decreased in group I, but continuously increased in group II. At fifteen minutes after exercise, platelet calcium concentrations were significantly higher than baseline values in group II (205.8±53.9 nM vs 102.6±14.4 nM；p<0.05, but not in group I (115.2±15.7 nM vs 86.8±11.8 nM. Conclusion：Cytoplasmic free calcium concentration in platelets were continuosly increased after treadmill exercise in sedentary men but not in physically active men. (Korean Circulation J 1999;29(10 :1112-1120

Effects of copper-aspirin complex on plasma 6-keto-prostaglandin F1α level and platelet cytosolic calcium in rabbits

Effects of copper-aspirin complex on washed platelet aggregation, thromboxane B2 formation and 6-ketoprostaglandin F1α level were monitored by Born's and Terashita's methods, respectively. The influence of copper-aspirin complex on cytosolic free calcium was examined using the fluorescent indicator, Fura 2-AM. Copper-aspirin complex significantly inhibited arachidonic acid-induced aggregation in washed platelets. The IC50 value was 9.6 μmolL-1. Copper-aspirin complex significantly decreased arachidonic acid-induced thromboxane B2 formation by 87.1% in washed platelets. Ten mg kg-1 of copper-aspirin complex given intragastrically markedly increased the plasma level of 6-keto-prostaglandin F1α . Aspirin, however, reduced both thromboxane B2 formation and 6-keto-prostaglandin F1α level. In the presence of CaCl2 1 mmolL-1, copper-aspirin complex (20, 40 and 80 mu mol L-1) markedly lowered arachidonic acid-induced increase in platelet calcium from the resting level (270±36 nmolL-1) to 213±14, 170±20 and 135±17 nmolL-1, respectively. In the presence of ethylene glycol-bis (β-aminoethyl ether) N,N,N',N'-tetraacetic acid 1 mmolL-1, copper-aspirin complex (20, 40 and 80 mu molL-1) significantly suppressed the release of intracellular calcium induced by arachidonic acid from 127±23 nmolL-1 to 108±17, 93±12 and 70±13 nmolL-1.

The Platelet High Affinity Binding Site for Thrombin Mimics Hirudin, Modulates Thrombin-induced Platelet Activation, and Is Distinct from the Glycoprotein Ib-IX-V Complex

The platelet high affinity binding site for thrombin appears to be described by a classical receptor-ligand interaction that is distinct from the platelet thrombin receptor/substrate, PAR-1. However, the identification and function of the high affinity binding site with respect to its physiological importance have continued to elude investigators. Prior studies using two mutant thrombins suggested that thrombin interaction with the platelet high affinity binding site is mediated through an extensive portion of the thrombin molecule involving residues within the substrate binding pocket and the anion binding exosite (Leong, L., Henriksen, R. A., Kermode, J. C., Rittenhouse, S. E., and Tracy, P. B. (1992) Biochemistry 31, 2567-2575) and may mimic a thrombin-hirudin interaction. To test this hypothesis, an anti-hirudin peptide antibody (anti-hirpeptide Ab) was raised against a peptide mimicking the COOH terminus of hirudin. The Ab recognized adherent platelets and those in suspension as determined by enzyme-linked immunosorbent assay and immunofluorescence microscopy, respectively. 125I-Thrombin binding to platelets was inhibited in the presence of the anti-hirpeptide Ab in a dose-dependent manner with maximal inhibition >90%. Analyses of data from binding studies of 125I-thrombin to platelets at a fixed Ab concentration indicated that the anti-hirpeptide Ab inhibited the high affinity binding interaction exclusively. In addition, thrombin-induced increases in platelet [Ca2+]i were enhanced by blocking the high affinity binding site with the Ab due to redistribution of the agonist to PAR-1. Thrombin Quick I-induced platelet calcium mobilization was unaffected by the presence of the Ab, consistent with the inability of thrombin Quick I to bind to the high affinity site. Even though glycoprotein (GP) Ib contains a hirudin-like region within the alpha subunit, the postulated high affinity binding site, direct binding of 125I-thrombin could not be demonstrated to transfected Chinese hamster ovary and L cells expressing the GP Ib-IX-V complex. Furthermore, an anti-GP Ib Ab, raised to the peptide region proposed as the thrombin high affinity site, did not enhance thrombin-induced platelet calcium mobilization. The anti-hirpeptide Ab recognized a population of platelet membrane proteins distinct from PAR-1 and GP Ib by three-color immunofluorescence using confocal microscopy. These combined studies demonstrate that the high affinity binding site for thrombin is a unique platelet protein distinct from GP Ib which modulates the effective thrombin concentration localized at the human platelet surface.

Modulation Of Calcium Fluxes In Human Platelets By Human Milk in vitro † 21

The effect of adding human milk (HM) to washed fura-2 loaded human platelets was investigated in vitro. The effect of thrombin (0.01 U/ml) to increase intracellular free calcium ([Ca2+]i), when calcium was present in the medium, was inhibited approx. 60% by a brief (10 second) treatment with HM (6% produced the maximum inhibition). Similar inhibition of the ability of vasopressin to increase ([Ca2+]i) was also observed. The effect of thrombin to mobilize intracellular calcium in platelets (5 mM EGTA in the medium) was inhibited approx. 70% by HM (4% produced the maximum inhibition). This result suggests the HM contains a factor or factors that prevent thrombin from increasing([Ca2+]i) in platelets, perhaps by either stimulating calcium efflux from the platelet, inhibiting intracellular calcium release or stimulating calcium uptake into the intracellular stores. When calcium influx into platelets was measured using barium ions as a calcium surrogate, HM blocked the influx by approx. 70% (4% produced the maximum inhibition). This result also implies that HM also contains a factor that inhibits calcium influx across the plasma membrane. Significant effects could be observed with 1% HM on all three parameters. The inhibitory activity did not pass through a 10 KDa molecular weight cut off filter. The inhibitory effect of HM was substantially reduced by heating the milk for 15 min. at 100 C. Treating HM with 0.3 M HCl or 0.5M NaOH at room temperature for 10 min. had no effect on activity. It is concluded that HM contains a factors or factors that reduce the ability of thrombin to increase [Ca2+]i in platelets by both blocking calcium influx and preventing mobilization from internal calcium stores, alternatively the plasma membrane calcium ATPase pump may be activated. The factor is stable to mild acid and base treatment is heat labile and has a molecular weight in excess of 10 KDa. Because platelets participate in propagation of the acute inflammatory response, human milk factors that modify intracellular signalling in platelets might contribute to the anti-inflammatory character of human milk.