Phosphoinositide breakdown as an indirect link between stimulation and aggregation of rat platelets by thrombin and collagen

When washed rat platelets (1.5 x 10(9)/ml) were stimulated by a threshold concentration of thrombin (0.3 unit/ml) or collagen (10 micrograms/ml), a lag period of about 10 or 30 s, respectively, was seen before the start of aggregation. During the lag period, [32P]phosphatidylinositol 4,5-bisphosphate was degraded as the earliest event within 5-10 s of addition of the stimulus. However, though the extent of phosphatidylinositol 4,5-bisphosphate degradation within 10 s of addition of collagen was greater than that within 20 s of addition of thrombin (0.3 unit/ml), a lag of about 20 s remained before the initiation of aggregation by collagen. This casts doubt on the hypothesis that the stimulus-dependent phosphatidylinositol 4,5-bisphosphate breakdown induces the aggregation of platelets. Phosphatidylinositol labeled with 32Pi or [1-14C]arachidonic acid was scarcely degraded during the lag period. As aggregation proceeded, [14C-arachidonic acid]phosphatidylinositol was degraded with generation of diacylglycerol, phosphatidic acid, arachidonic acid and its metabolites. The maximum aggregation by collagen of rat platelets in which arachidonic acid of phospholipids was replaced in vivo with eicosapentaenoic acid was reduced, but that by thrombin was not, though reduction of thromboxane A2 generation was caused by both stimuli. Indomethacin also fully inhibited the aggregation induced by collagen, but not that induced by thrombin. Hence, thromboxane A2 is required for full aggregation by collagen, but not that by thrombin. These results indicate that thrombin-induced phosphoinositide metabolism may proceed independently of aggregation.     

N-ethylmaleimide inhibits Ca2+ influx induced by collagen or arachidonate on rabbit platelets

N-Ethylmaleimide dose dependently inhibited platelet aggregation induced by collagen or arachidonate but did not inhibit the aggregation by thrombin or ionophore A23187 within the concentrations tested. [3H]Arachidonate release from membrane phospholipids of the collagen-stimulated platelets was inhibited by N-ethylmaleimide in parallel with the inhibition of aggregation, but not in response to A23187. N-Ethylmaleimide prevented 45Ca2+ influx into platelet cells from outer medium induced by collagen, and also inhibited the increase in the concentration of cytoplasmic free Ca2+, which probably results from Ca2+ influx, as monitored by quin2 fluorescence, under stimulation with arachidonate. The concentration of N-ethylmaleimide giving a complete inhibition of Ca2+ influx was consistent with that required to inhibit collagen- or arachidonate-induced aggregation. Prostaglandin metabolism from arachidonate to thromboxane A2 was not disturbed by N-ethylmaleimide, while phosphatidate formation induced by arachidonate was slightly inhibited by it at concentrations at which aggregation was completely inhibited. These data suggest that N-ethylmaleimide preferentially suppresses increase in cytoplasmic free Ca2+ which is linked to thromboxane A2-receptor occupation in collagen- or arachidonate-stimulated platelets, probably due to blockage of Ca2+ influx through Ca2+-channel protein, thereby inhibiting aggregation induced by these agonists.     

Elevation of cytosolic free Ca2+ is directly evoked by thromboxane A2 in human platelets during activation with collagen

The thromboxane A2 antagonist, ONO-3708, completely inhibited the increase in cytosolic free Ca2+ in human platelets during activation with collagen. Half-maximal Ca2+ release and influx required about 3 and 4 nM STA2, a stable thromboxane A2 mimetic, respectively. However, half maximal activation of phospholipase C required about 18 nM STA2. This suggests that thromboxane A2 directly causes Ca2+ mobilization without further activation of phospholipase C during activation of human platelets with collagen.     

Na+/H+ exchange modulates Ca2+ mobilization in human platelets stimulated by ADP and the thromboxane mimetic U 46619

According to recent observations ADP stimulates platelets via activation of Na+/H+ exchange which increases cytosolic pH (pHi). This event initiates formation of thromboxane A2 (via phospholipase A2) and, thereafter, inositol 1,4,5-trisphosphate (via phospholipase C) which is known to mobilize Ca2+ from intracellular storage sites. We investigated changes in pHi and cytosolic free Ca2+, [Ca2+]i, activating platelets with ADP and the thromboxane mimetic U 46619. We found that ADP (5 microM) increased pHi from 7.15 +/- 0.08 to 7.35 +/- 0.04 (n = 8) in 2'-7'-bis-(carboxyethyl)-5,6-carboxyfluorescein-loaded platelets, whereas thromboxane A2 formation was inhibited by indomethacin. ADP also induced a dose-dependent Ca2+ mobilization in fura2-loaded platelets which again was not affected by indomethacin. [Ca2+]i increased by 54 +/- 10 nM (n = 8) at 1 microM and by 170 +/- 40 nM (n = 7) at 10 microM ADP above the resting value of 76 +/- 12 nM (n = 47). Inhibition of Na+/H+ exchange by ethylisopropylamiloride (EIPA) reduced ADP-induced Ca2+ mobilization by more than 65% in indomethacin-treated platelets. This inhibition could be completely overcome by artificially raising pHi using either NH4Cl or the Na+/H+ ionophore monensin. We found that U 46619 increased pHi by 0.18 +/- 0.05 at 0.1 microM and by 0.29 +/- 0.07 (n = 7) at 1.0 microM above the resting value via an EIPA-sensitive mechanism. In conflict with the proposed role of the Na+/H+ exchange we found that U 46619 raised [Ca2+]i via a mechanism that for more than 50% depended on intact Na+/H+ exchange. Again, artificially elevating pHi restored U 46619-induced Ca2+ mobilization despite the presence of EIPA. Thus, our data show that Na+/H+ exchange is a common step in platelet activation by prostaglandin endoperoxides/thromboxane A2 and ADP and enhances Ca2+ mobilization independently of phospholipase A2 activity.     

Measurement of thromboxane A2-induced elevation of ionized calcium in collagen-stimulated platelets with the photoprotein, aequorin

Using aequorin-loaded rat platelets stimulated with collagen, we found two phases of Ca2+ mobilization, one coinciding with a shape change and the other with aggregation, which have not yet been detected in quin2-loaded platelets. U46619, a stable analogue of prostaglandin H2, induced only a shape change and a concomitant rapid rise in the cytoplasmic ionized calcium concentration ([Cai2+]). However, upon addition of U46619 to platelets previously stimulated with collagen in the presence of indomethacin, a rapid increase in [Cai2+] and a shape change occurred, and, after about 1 min, second increase in [Cai2+] and aggregation occurred. The actions of U46619 were inhibited by an antagonist for the thromboxane A2 (TXA2) receptor. These results suggest that the collagen-induced shape change is initiated by TXA2-induced Ca2+ mobilization, and aggregation is induced by the secondary Ca2+ mobilization induced by TXA2 and the occupation of the receptor by collagen.     

Prostacyclin inhibits platelet aggregation induced by phorbol ester or Ca2+ ionophore at steps distal to activation of protein kinase C and Ca2+-dependent protein kinases.

Suspensions of aspirin-treated, 32P-prelabelled, washed platelets containing ADP scavengers in the buffer were activated with either phorbol 12,13-dibutyrate (PdBu) or the Ca2+ ionophore A23187. High concentrations of PdBu (greater than or equal to 50 nM) induced platelet aggregation and the protein kinase C (PKC)-dependent phosphorylation of proteins with molecular masses of 20 (myosin light chain), 38 and 47 kDa. No increase in cytosolic Ca2+ was observed. Preincubation of platelets with prostacyclin (PGI2) stimulated the phosphorylation of a 50 kDa protein [EC50 (concn. giving half-maximal effect) 0.6 ng of PGI2/ml] and completely abolished platelet aggregation [ID50 (concn. giving 50% inhibition) 0.5 ng of PGI2/ml] induced by PdBu, but had no effect on phosphorylation of the 20, 38 and 47 kDa proteins elicited by PdBu. The Ca2+ ionophore A23187 induced shape change, aggregation, mobilization of Ca2+, rapid phosphorylation of the 20 and 47 kDa proteins and the formation of phosphatidic acid. Preincubation of platelets with PGI2 (500 ng/ml) inhibited platelet aggregation, but not shape change, Ca2+ mobilization or the phosphorylation of the 20 and 47 kDa proteins induced by Ca2+ ionophore A23187. The results indicate that PGI2, through activation of cyclic AMP-dependent kinases, inhibits platelet aggregation at steps distal to protein phosphorylation evoked by protein kinase C and Ca2+-dependent protein kinases.     

Intracellular Ca2+ mobilization and not calcium influx promotes phorbol ester-stimulated thromboxane A2 synthesis in human platelets.

Phorbol esters, potent activators of protein kinase C (PKC), greatly enhance the release of arachidonic acid and its metabolites (TXA2, HETES, HHT) by Ca2+ ionophores in human platelets. In this paper, we report the relationship between intracellular Ca2+ mobilization and external calcium influx into platelets and the ability of PMA plus A23187 to promote thromboxane A2 (TXA2) synthesis. The enhanced levels of TXA2 due to the synergistic stimulation of the platelets with A23187 and phorbol esters are not affected significantly by the presence of external Ca2+ or the calcium-chelator EGTA. PKC inhibitors, staurosporine and sphingosine, abolished phorbol myristate acetate (PMA) potentiation of TXA2 production which strongly supports the role of PKC in the synergism. Platelet aggregation is more sensitive to PMA and external calcium than TXA2 formation. PMA increased TXA2 production as much as 4-fold at low ionophore concentrations. The A23187-induced rise in [Ca2+]i was reduced by pretreatment of human platelets with phorbol esters, both in the presence and absence of EGTA, and staurosporine reversed this inhibitory effect. These results indicate that the synergistic stimulation of TXA2 production by A23187 and phorbol esters is promoted by intracellular Ca2+ mobilization and not by external calcium influx. Our data also suggest that PKC is involved in the regulation of Ca2+ mobilization from some specific intracellular stores and that PKC may also stimulate the Ca(2+)-dependent phospholipase A2 at suboptimal Ca2+i concentrations.     

Antiplatelet effect of butylidenephthalide

Butylidenephthalide inhibited, in a dose-dependent manner, the aggregation and release reaction of washed rabbit platelets induced by collagen and arachidonic acid. Butylidenephthalide also inhibited slightly the platelet aggregation induced by PAF and ADP, but not that by thrombin or ionophore A23187. Thromboxane B2 formation caused by collagen, arachidonic acid, thrombin and ionophore A23187 was in each case markedly inhibited by butylidenephthalide. Butylidenephthalide inhibited the aggregation of ADP-refractory platelets, thrombin-degranulated platelets, chymotrypsin-treated platelets and platelets in the presence of creatine phosphate/creatine phosphokinase. Its inhibition of collagen-induced aggregation was more marked at lower Ca2+ concentrations in the medium. The aggregability of platelets inhibited by butylidenephthalide could be recovered after the washing of platelets. In human platelet-rich plasma, butylidenephthalide and indomethacin prevented the secondary aggregation and blocked ATP release from platelets induced by epinephrine. Prostaglandin E2 formed by the incubation of guinea-pig lung homogenate with arachidonic acid could be inhibited by butylidenephthalide, indomethacin and aspirin. It is concluded that the antiplatelet effect of butylidenephthalide is mainly due to an inhibitory effect on cyclo-oxygenase and may be due partly to interference with calcium mobilization.     

Effect of calcium concentration and inhibitors on the responses of platelets stimulated with collagen: contrast between human and rabbit platelets.

1. Variations in the concentration of Ca2+ [Ca2+] in the suspending medium have different effects on the responses of human and rabbit platelets to collagen. 2. When rabbit platelets are stimulated with a low concentration of collagen (0.5 micrograms/ml), aggregation, release of granule contents, and formation of thromboxane are maximal when the suspending medium contains [Ca2+] in the physiological range (0.5-2.0 mM), and very slight in a medium with no added Ca2+. 3. In contrast, human platelets respond most strongly when the suspending medium contains no added Ca2+ [( Ca2+] approx. 20 microM); this is attributable to the enhanced formation of thromboxane A2 (TXA2) upon close platelet-to-platelet contact in this medium. 4. When TXA2 formation is blocked by inhibition of cyclo-oxygenase with aspirin or indomethacin, rabbit platelet aggregation and release in response to 1.25-10 micrograms/ml collagen is also maximal at [Ca2+] of 0.5-2.0 mM and least at 20 microM; human platelets do not aggregate and the extent of release is relatively independent of [Ca2+]. 5. In 1 mM [Ca2+], use of apyrase and/or ketanserin with rabbit platelets in which TXA2 formation is blocked shows that released ADP and serotonin make large contributions to aggregation and release in response to high concentrations of collagen; human platelet aggregation is largely dependent on TXA2. 6. Use of fura-2-loaded platelets shows that the collagen-induced rise in cytosolic [Ca2+] is only slightly inhibited by aspirin or indomethacin in rabbit platelets, but almost completely inhibited in human platelets. 7. Responses of rabbit platelets to collagen are less dependent on TXA2 than those of human platelets. Released ADP and serotonin make major contributions to the responses of rabbit platelets to collagen.     

Liberation of [3H]arachidonic acid and changes in cytosolic free calcium in fura-2-loaded human platelets stimulated by ionomycin and collagen.

Cytosolic Ca2+ levels and arachidonate liberation were investigated in platelets loaded with the fluorescent Ca2+ indicator dye fura-2, and labelled with [3H]arachidonate. Fura-2 was used in preference to quin2 because the latter interfered with [3H]arachidonate labelling of phospholipids. From a resting free Ca2+ level of around 100 nM, ionomycin (10-200 nM) evoked an instantaneous, concentration-dependent increase in cytosolic Ca2+ that only resulted in [3H]arachidonate liberation (up to 4-fold over control) at Ca2+ levels greater than 1 microM. Addition of collagen (10 micrograms/ml) evoked an elevation in Ca2+ up to 461 +/- 133 nM. These changes in Ca2+ were accompanied by a 2-4-fold elevation in [3H]arachidonate with depletion of [3H]phosphatidylcholine by 17 +/- 4% and [3H]phosphatidylinositol by 41 +/- 7%. Indomethacin (10 microM) reduced the elevation in Ca2+ by collagen to 115 +/- 18 nM but did not significantly inhibit the 2-4-fold increase in [3H]arachidonate. [3H]Phosphatidylcholine and [3H]phosphatidylinositol were decreased by 9 +/- 7% and 10 +/- 6%, respectively, with collagen in the presence of indomethacin. Stimulation of phosphoinositide turnover by collagen in the presence and absence of indomethacin was indicated by [32P]phosphatidate formation in cells prelabelled with [32P]Pi. This phosphatidate formation was decreased (75%) by the presence of indomethacin. In the presence of indomethacin, phorbol myristate acetate (20 nM) alone or in combination with ionomycin (30 nM) failed to stimulate arachidonate liberation despite a marked stimulation of aggregation. These results indicate that, whereas ionomycin requires Ca2+ in the microM range for arachidonate liberation, collagen, notably in the presence of indomethacin, does so at basal Ca2+ levels. The mechanisms underlying the regulation of arachidonate release by collagen are not clear, but do not appear to involve activation of protein kinase C, or an elevation of cytosolic free Ca2+.     

The formation of [3H]inositol phosphates in human platelets by palmitoyl lysophosphatidic acid is blocked by indomethacin

The intracellular Ca2+ thresholds for platelet shape change and aggregation by A23187 and palmitoyl lysophosphatidic acid were approximately 350 and 750 nM, respectively, as estimated using quin2. The similar thresholds for these two agonists imply they activate platelets through a similar mechanism. In the absence of cyclooxygenase inhibitors, both agents induce the formation of [3H]inositol phosphates, reflecting the activation of phospholipase C. This activation of phospholipase C is blocked by the cyclooxygenase inhibitor indomethacin. It is suggested that platelet activation by palmitoyl lysophosphatidic acid involves an initial mobilization of intracellular Ca2+ with subsequent activation of phospholipase A2; the arachidonic acid metabolites formed then stimulate phospholipase C.     

Signal transduction in collagen-stimulated rat platelets is composed of three stages

A stable thromboxane A2 (TXA2) mimetic, U46619, induced a shape change of rat platelets, but did not induce phosphoinositide breakdown, aggregation or secretion. However, when U46619 was added to platelets which had been previously stimulated with collagen in the presence of indomethacin, all biological responses were induced about 1 min after the occurrence of shape change. Furthermore, two phases of phosphorylation of myosin light chain (MLC) were observed under the same conditions, one coinciding with shape change and the other with aggregation. Similar two-phase Ca2+ mobilization has been observed using aequorin (Nakano, T., Terawaki, A., & Arita, H. (1986) J. Biochem. 99, 1285-1288). From these results, collagen-induced signal transduction is considered to be composed of three stages. The first stage is the initial TXA2 generation. The second stage involves inositol trisphosphate-independent first-phase Ca2+ mobilization and the first-phase MLC phosphorylation by the action of TXA2 alone, leading to the shape change of platelets. The third stage is initiated by an abrupt phosphoinositide breakdown via the synergistic action of TXA2 and occupation of the collagen receptor, and the resulting inositol trisphosphate may induce the second-phase Ca2+ mobilization to produce the second-phase MLC phosphorylation together with aggregation and secretion.     

Metabolic and functional consequences of introducing inositol 1,4,5-trisphosphate into saponin-permeabilized human platelets.

In an earlier study we reported the effect of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] in releasing Ca2+ from highly purified human platelet intracellular membrane vesicles. [Authi & Crawford (1985) Biochem. J. 230, 247-253]. We have now investigated the metabolic and functional consequences of introducing Ins(1,4,5)P3 into saponin-permeabilized platelets. Washed human platelets when resuspended in a suitable medium were permeabilized with saponin (10-14 micrograms/ml) to allow entry of low-Mr water-soluble molecules without significant release of the cytoplasmic marker enzyme protein lactate dehydrogenase. Saponin-permeabilized platelets show identical platelet responses (shape change, aggregation and release of 5-hydroxy[14C]tryptamine) to both collagen (5 micrograms/ml) and thrombin (0.1 unit/ml) as obtained with intact cells, indicating that there is minimal disturbance to the surface membrane receptor topography for these two agonists. Ins(1,4,5)P3 (1-10 microM) added to saponin-treated platelets (but not to intact platelets) induced dose-related shape change, aggregation and release of 5-hydroxy[14C]tryptamine which at maximal doses was comparable with responses obtained with thrombin or collagen. The cyclo-oxygenase inhibitors indomethacin and aspirin, if added prior to saponization and Ins(1,4,5)P3 addition, completely inhibited both aggregation and release of 5-hydroxy[14C]tryptamine (EC50 for indomethacin, 50 nM; for aspirin, 30 microM). We believe that Ins(1,4,5)P3 induces the release of Ca2+ from intracellular storages sites which stimulates the Ca2+-dependent phospholipase A2 releasing arachidonic acid from membrane phospholipids. Arachidonic acid is then converted to the aggregatory prostanoids (prostaglandin H2 and thromboxane A2) resulting in the observed responses. This concept is supported by the use of the thromboxane receptor antagonists EPO 45 and EPO 92, both of which also completely inhibit Ins(1,4,5)P3-induced responses in saponin-permeabilized platelets. Electron microscopy of the platelet preparations revealed that thrombin- and collagen-induced platelet aggregates of intact and saponized cells were identical, showing extensive pseudopod formation and dense granule release. The Ins(1,4,5)P3-induced aggregates also showed similar dense granule release but an almost total absence of pseudopod formation. These results are discussed in the light of the second messenger role of Ins(1,4,5)P3 in stimulus-response coupling in platelets.     

Inhibitory effect of ginkgolide B on platelet aggregation in a cAMP- and cGMP-dependent manner by activated MMP-9

Extracts from the leaves of the Ginkgo biloba are becoming increasingly popular as a treatment that is claimed to reduce atherosclerosis, coronary artery disease, and thrombosis. In this study, the effect of ginkgolide B (GB) from Ginkgo biloba leaves in collagen (10 microg/ml)- stimulated platelet aggregation was investigated. It has been known that human platelets release matrix metalloproteinase- 9 (MMP-9), and that it significantly inhibited platelet aggregation stimulated by collagen. Zymographic analysis confirmed that pro-MMP-9 (92-kDa) was activated by GB to form an MMP-9 (86-kDa) on gelatinolytic activities. And then, activated MMP-9 by GB dose-dependently inhibited platelet aggregation, intracellular Ca2+ mobilization, and thromboxane A2 (TXA2) formation in collagen-stimulated platelets. Activated MMP-9 by GB directly affects down-regulations of cyclooxygenase-1 (COX-1) or TXA2 synthase in a cell free system. In addition, activated MMP-9 significantly increased the formation of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), which have the anti-platelet function in resting and collagen-stimulated platelets. Therefore, we suggest that activated MMP-9 by GB may increase the intracellular cAMP and cGMP production, inhibit the intracellular Ca2+ mobilization and TXA2 production, thereby leading to inhibition of platelet aggregation. These results strongly indicate that activated MMP-9 is a potent inhibitor of collagen-stimulated platelet aggregation. It may act a crucial role as a negative regulator during platelet activation.     

Inhibitory effects of phenol derivatives on bovine platelet aggregation and their effects on Ca2+ mobilization

The effects of phenol derivatives on aggregation of bovine platelets induced by ADP, thrombin, platelet activating factor, collagen and A23187 were investigated. The phenol derivatives inhibited all these induced aggregations except that by the calcium ionophore. The derivatives each inhibited the aggregations induced by ADP, thrombin, platelet activating factor and collagen, respectively, within a similar concentration range. A linear relation was found between the inhibitory potencies of the phenol derivatives and their partition coefficients between n-octanol and water (Poct values), suggesting that their interaction with hydrophobic regions of the cell was important for inhibition. Fluorescence analyses with fura-2-loaded platelets showed that in the concentration ranges in which the phenol derivatives inhibited aggregation, they also inhibited agonist-induced increases in Ca2+ both in the presence and absence of extracellular Ca2+. Moreover, a high correlation was found between the inhibitory effects of the derivatives on aggregation and their effects on Ca2+ mobilization. These results suggest that inhibition of platelet aggregation by phenol derivatives is mainly due to inhibition of the increase in cytoplasmic Ca2+ by inhibition of both intracellular Ca2+ mobilization and Ca2+ uptake.     

Collagen stimulates [3H]inositol trisphosphate formation in indomethacin-treated human platelets.

The present study investigates the pathway of metabolism of inositol phospholipids in human platelets exposed to collagen. Platelet activation by collagen was preceded by a lag phase usually lasting 10-20 s. Formation of [3H]inositol trisphosphate (IP3) was not observed during this period, but occurred in parallel with the onset of aggregation, release of ATP and phosphorylation of a 20 000 Da and a 40 000 Da protein. Indomethacin treatment partially inhibited all of these responses. Aggregation and ATP release, but not IP3 formation, were further inhibited in indomethacin-treated platelets loaded with the fluorescent Ca2+ indicator, quin2. Under these conditions there was no detectable mobilization of Ca2+. These results demonstrate that activation of platelets by collagen is associated with rapid hydrolysis of polyphosphoinositides by phospholipase C, thereby producing IP3. This observation is discussed in relation to IP3 as a possible Ca2+-mobilizing agent.     

Extract of a spice--omum (Trachyspermum ammi)-shows antiaggregatory effects and alters arachidonic acid metabolism in human platelets

An ethereal extract of omum (Trachyspermum ammi; Hindustani: ajwan)--a frequently consumed spice--was found to inhibit platelet aggregation induced by arachidonic acid (AA), epinephrine and collagen; in this respect it was most effective against AA-induced aggregation. Inhibition of aggregation by omum could be explained by its effect on platelet thromboxane production as suggested by the following experimental observation. (i) Omum reduced TxB2 formation in intact platelet preparations from added arachidonate, and (ii) it reduced the formation of TxB2 from AA-labelled platelets after stimulation with Ca2+-ionophore A23187 by a direct action on cyclooxygenase as it did not affect the release of AA from labelled platelets. An increased formation of lipoxygenase-derived products from exogenous AA in omum-treated platelets was apparently due to redirection of AA from cyclooxygenase to the lipoxygenase pathway.     

Inhibitory mechanisms of metallothionein on platelet aggregation in in vitro and platelet plug formation in in vivo experiments

Metallothionein (MT) is a low-molecular-weight, cysteine-rich protein that contains heavy metals such as cadmium and zinc. The biological function of MT in platelets is not yet understood. Therefore, the aim of this study was to systematically examine the inhibitory mechanisms of metallothionein in platelet aggregation. In this study, metallothionein concentration-dependently (1-8 microM) inhibited platelet aggregation in human platelets stimulated by agonists. Metallothionein (4 and 8 microM) inhibited phosphoinositide breakdown in [3H]-inositol-labeled platelets, intracellular Ca+2 mobilization in Fura-2 AM-loaded platelets, and thromboxane A2 formation stimulated by collagen. In addition, metallothionein (4 and 8 microM) significantly increased the formation of cyclic GMP but not cyclic AMP in human platelets. Rapid phosphorylation of a protein of Mr 47,000 (P47), a marker of protein kinase C activation, was triggered by PDBu (100 nM). This phosphorylation was markedly inhibited by metallothionein (4 and 8 microM) in phosphorus-32-labeled platelets. In an in vivo thrombotic study, platelet thrombus formation was induced by irradiation of mesenteric venules in mice pretreated with fluorescein sodium. Metallothionein (6 microg/g) significantly prolonged the latency period for inducing platelet plug formation in mesenteric venules. These results indicate that the antiplatelet activity of metallothionein may involve the following pathways: (1) metallothionein may inhibit the activation of phospholipase C, followed by inhibition of phosphoinositide breakdown and thromboxane A2 formation, thereby leading to inhibition of intracellular Ca+2 mobilization; (ii) Metallothionein also activated the formation of cyclic GMP in human platelets, resulting in inhibition of platelet aggregation. The results strongly indicate that metallothionein provides protection against thromboembolism.     

Characteristics of collagen-induced Ca2+ mobilization in bovine platelets

We characterized the collagen-induced increase in cytosolic Ca2+ ([Ca2+]i) of bovine platelets loaded with the Ca2+ indicator Fura-PE3/AM. Collagen (10 micrograms/ml)-induced increase in [Ca2+]i was only partially inhibited by aspirin, a cyclooxygenase inhibitor, or adenosine 3'-phosphate 5'-phosphosulfate (A3P5PS, a P2Y1 receptor antagonist), while in human platelets it was almost completely suppressed by aspirin. Collagen-induced increase in [Ca2+]i of bovine platelets was inhibited by U73122 (0.3-5 microM), a phospholipase C inhibitor. Collagen (10 micrograms/ml) increased production of inositol 1,4,5-trisphosphate, which was prevented by pretreatment with U73122 (5 microM). Collagen (10 micrograms/ml) accelerated Mn2+ entry, since the rate of Fura-PE3 quenching by Mn2+ was enhanced by 13-fold following stimulation with collagen. U73122 inhibited the acceleration of Mn2+ entry induced by collagen. PGE1 (2.5 microM) partially inhibited the collagen (50 micrograms/ml)-induced increase in [Ca2+]i in bovine platelets but not in human platelets. The data suggest that collagen-induced Ca2+ mobilization in bovine platelets is mediated by phospholipase C. The Ca2+ mobilization in bovine platelets is different from that in human ones as to the dependency on arachidonic acid metabolites and sensitivity to PGE1.     

Characterization of a potent platelet aggregation inhibitor from Agkistrodon rhodostoma snake venom

By means of CM-Sephadex C-50 column chromatography and gel filtration on Sephadex G-75 and G-50 columns, a potent platelet aggregation inhibitor was purified and characterized. It was a glycoprotein with a molecular weight of 31,000. It was devoid of phospholipase A, ADPase, esterase and fibrino(geno)lytic activities. It inhibited dose-dependently the aggregation of washed platelets induced by collagen, thrombin, sodium arachidonate, platelet activating factor and ionophore A23187 with a similar IC50 (5-10 micrograms/ml). It was also active in platelet-rich plasma, with an IC50 of 10-15 micrograms/ml. The venom inhibitor reduced the elasticity of whole blood clot and inhibited the thrombin-induced clot retraction of platelet-rich plasma. These activities were related to its inhibitory activity on platelet aggregation rather than blood coagulation. The venom inhibitor had various effects on [14C]serotonin release stimulated by aggregation agonists. It had no effect on thromboxane B2 formation of platelets stimulated by sodium arachidonate, collagen and ionophore A23187. The presence of this venom inhibitor prior to the initiation of aggregation was a prerequisite for the maintenance of its maximal activity. It showed a similar inhibitory effect on collagen or thrombin-induced aggregation even when it was added after the platelets had undergone the shape change. High fibrinogen levels partially antagonized its activity. The venom inhibitor completely inhibited the fibrinogen-induced aggregation of alpha-chymotrypsin-treated platelets. It is concluded that this venom inhibitor interferes with the interaction of fibrinogen with fibrinogen receptors, leading to inhibition of aggregation.