Effects of ovariectomy on aggregation,secretion, and metalloproteinases in porcine platelets

Differences in the aggregation and release of growth factors including matrix metalloproteinases (MMPs) after loss of ovarian hormones could contribute to an exaggerated response to injury in arteries of ovariectomized animals. Therefore, experiments were designed to compare aggregation, dense granular ATP release, expression of MMPs (MMP-2, MMP-9, and MMP-14) and tissue inhibitors of metalloproteinase (TIMP-1 and TIMP-2) in circulating platelets from sexually mature (7 mo old) gonadally intact and ovariectomized (4 wk) female pigs. Numbers of circulating platelets did not change after ovariectomy, but the percentage of reticulated platelets increased significantly. Platelet aggregation and dense granular ATP secretion also increased significantly with ovariectomy. In platelet lysates, active MMP-2 increased, whereas MMP-14 significantly decreased, after ovariectomy; the expression of TIMP-1, TIMP-2, and P-selectin did not change. These results suggest that platelet turnover, aggregation, and ATP secretion increase with ovariectomy. Also, ovarian hormones selectively regulate the expression and activity of MMPs in porcine platelets. Increased platelet aggregation and activity of MMP-2 would alter platelet-platelet and platelet-vessel wall interactions, contributing to an exaggerated response to injury with loss of ovarian hormones.     

In vivo effects of lipopolysaccharide and TLR4 on platelet production and activity: implications for thrombotic risk.

Gram-negative bacteria release LPS, which activates Toll-like-receptor-4 (TLR4) in the host, initiating an inflammatory response to infection. Infection increases risk for thrombosis. Platelets contribute to defense from infection and to thrombosis. Experiments were designed to determine whether LPS, through TLR4 signaling, affects platelet phenotype. Platelet responses in wild-type (WT) mice and mice that lack the TLR4 gene (dTLR4) were compared following a single nonlethal injection of LPS (0.2 mg/kg iv). Compared with WT mice, mice without TLR4 had fewer circulating platelets with lower RNA content and were less responsive to thrombin-activated expression of P-selectin but were equally sensitive to aggregation or ATP secretion. One week following the LPS injection, the time it takes for the circulating platelet pool to turnover, the number of circulating platelets, thrombin-induced expression of P-selectin, and collagen-activated aggregation were increased comparably in both groups of mice. Therefore, the change of the platelet pool to an activated phenotype 1 wk after a single exposure to LPS appears to arise from a process that is independent of TLR4. The persistence of the effect 1 wk after the injection suggests that the changes reflect an action of LPS on megakaryocytes and their platelet progeny rather than on circulating platelets, which would have been cleared.     

Effect of male sex and obesity on platelet arachidonic acid in spontaneous hypertensive heart failure rats

Sexual dimorphism is observed in the progression to congestive heart failure and, ultimately, in longevity in spontaneously hypertensive heart failure (SHHF) rats. As platelet activation may impact development of cardiovascular diseases, we studied the effects of obesity and sex on platelet polyunsaturated fatty acid (PUFA) profile and its relationship to platelet aggregation in 6-month-old SHHF rats. After a 24-hr fast, blood was obtained for measurement of platelet phospholipid omega-6 (n-6) and omega-3 (n-3) PUFA. Collagen-induced platelet aggregation was measured by whole-blood impedance aggregometry. Obese male (OM) SHHF had significantly more platelet arachidonic acid (AA) and total n-6 PUFA than lean males (LMs), lean females (LFs), or obese females (OFs). Platelet aggregation was enhanced in males compared to females, with OMs by 45% compared to OFs and with LMs by 28% compared to LFs. Though no difference was found between OFs and LFs, platelet aggregation was increased in OMs by 20% compared to LMs. Though not significantly different, lag time to initiate platelet aggregation tended to be shortest in OMs and then, in increasing duration, LMs, LFs, and OFs, suggesting that platelets from male rats were quicker to aggregate than those from females. Platelet aggregation was correlated with platelet AA and total n-6 PUFA content. There was no relationship between n-3 PUFA and platelet aggregation. In SHHF rats, elevated AA and n-6 PUFA levels in platelets are associated with enhanced platelet aggregation. This relationship is potentiated by obesity and male sex.     

Activation of human platelets by N-substituted aminophospholipids

N-(7-nitro-2,1,3-benzoxadiazol-4-yl) phosphatidylserine (NBD-PS), a fluorescent phospholipid synthesized from phosphatidylserine by reaction with NBD-chloride, caused platelet shape change and aggregation when added at micromolar concentrations to suspensions of washed human platelets in the absence of added fibrinogen. Platelet aggregation by NBD-PS was accompanied by thromboxane synthesis and secretion of contents from dense, alpha-, and lysosomal granules in the absence of appreciable platelet damage. Indomethacin completely inhibited NBD-PS-induced thromboxane synthesis, but platelet aggregation and [14C]serotonin secretion were only slightly inhibited. Neither inhibition of the ADP-dependent pathway with creatine phosphate/creatine kinase plus ATP, alone or in combination with indomethacin, nor maximum elevation of cyclic AMP by treatment with prostaglandin I2 and theophylline completely inhibited NBD-PS-induced platelet aggregation or [14C]serotonin secretion. Platelet effects of NBD-PS were specific in that neither phosphatidylserine nor lyso-NBD-PS were similarly active. The activation of platelets by NBD-PS is not attributable to the NBD moiety exclusively since acylation of the amino group with 5-dimethylaminonaphthalene-1-sulfonyl-chloride yielded a similarly active derivative. Dansylated phosphatidylethanolamine was also active. The findings indicate that NBD-PS and other N-substituted aminophospholipids can activate a central pathway of platelet secretion and aggregation that is independent of released ADP and thromboxane formation and is only partially controlled by platelet cyclic AMP.     

Characterization of platelet abnormalities of Tester Moriyama (TM) rats with storage pool deficiency

Platelet abnormalities of Tester Moriyama (TM) rats, which have prolonged bleeding time with normal platelet count, were characterized by comparison with those of fawn-hooded (FH) rats with platelet storage pool deficiency (SPD). Morphologically, the dense granules were virtually lacking in platelets from TM and FH rats. Platelets from TM and FH rats aggregated in response to adenosine diphosphate (ADP), but failed to have secondary aggregation. In contrast, platelet aggregation was completely absent in response to 1 to 20 micrograms of collagen/ml, although partial aggregation was observed at the higher dosage of 50 micrograms/ml. Normal amounts of platelet membrane glycoproteins IIb/IIIa were expressed in TM and FH rats, but platelet adenosine triphosphate (ATP) and ADP contents were lower than those in platelets from control Wistar rats. Platelet ATP-to-ADP ratio of TM and FH rats was significantly higher than that of Wistar rats. Serotonin content in platelets from TM and FH rats was 20 to 25% that of Wistar rat platelets. These results suggested that platelet abnormalities of TM rats are a typical characteristic of platelet SPD and are similar to those of FH rats, which are genetically different from TM rats. Therefore, TM rats may serve as a useful animal model for the study of platelet SPD.     

Helicobacter pylori urease activates blood platelets through a lipoxygenase‐mediated pathway

The bacterium Helicobacter pylori causes peptic ulcers and gastric cancer in human beings by mechanisms yet not fully understood. H. pylori produces urease which neutralizes the acidic medium permitting its survival in the stomach. We have previously shown that ureases from jackbean, soybean or Bacillus pasteurii induce blood platelet aggregation independently of their enzyme activity by a pathway requiring platelet secretion, activation of calcium channels and lipoxygenase‐derived eicosanoids. We investigated whether H. pylori urease displays platelet‐activating properties and defined biochemical pathways involved in this phenomenon. For that the effects of purified recombinant H. pylori urease (HPU) added to rabbit platelets were assessed turbidimetrically. ATP secretion and production of lipoxygenase metabolites by activated platelets were measured. Fluorescein‐labelled HPU bound to platelets but not to erythrocytes. HPU induced aggregation of rabbit platelets (ED₅₀ 0.28 μM) accompanied by ATP secretion. No correlation was found between platelet activation and ureolytic activity of HPU. Platelet aggregation was blocked by esculetin (12‐lipoxygenase inhibitor) and enhanced ～3‐fold by indomethacin (cyclooxygenase inhibitor). A metabolite of 12‐lipoxygenase was produced by platelets exposed to HPU. Platelet responses to HPU did not involve platelet‐activating factor, but required activation of verapamil‐inhibitable calcium channels. Our data show that purified H. pylori urease activates blood platelets at submicromolar concentrations. This property seems to be common to ureases regardless of their source (plant or bacteria) or quaternary structure (single, di‐ or tri‐chain proteins). These properties of HPU could play an important role in pathogenesis of gastrointestinal and associated cardiovascular diseases caused by H. pylori.     

Adenine nucleotide metabolism of blood platelets. X. Formaldehyde stops centrifugation-induced secretion after A23187-stimulation and causes breakdown of metabolic ATP.

A23187 induced shape change, aggregation and secretion of platelets in plasma. When rapid cooling was used to stop secretion and centrifugation to separate the cells from the medium, maximal amounts of storage ATP plus ADP and preadsorbed [14C]serotonin were found in the supernatant immediately (less than 5 s) after A23187 addition. These results suggested that A23187 could cause shape change and aggregation through secreted ADP and not directly. When secretion was stopped with chilling and formaldehyde treatment before centrifugation, the secreted substances appeared after a lag of 60-120 s, i.e. after shape change was terminated and aggregation was well on its way. These two platelet responses thus seemed to be independent of secretion and induced directly by A23187. The absence of a lag period when secretion was stopped by chilling alone was thought to be due to centrifugation-induced secretion of platelets conditioned by A23187. Formaldehyde completely inhibited centrifugation-induced secretion. At 37 degrees C, formaldehyde caused rapid breakdown of metabolic ATP in platelets with a pattern dependent on the formaldehyde concentration: Below 50 mM, ATP was converted to inosine plus hypoxanthine via ADP, AMP and IMP and the adenylate energy charge was preserved. Above 100 mM, AMP was the end product with a drastic reduction in the adenylate energy charge. These changes were not due to lysis of the platelets, but were apparently caused by an formaldehyde-induced increase in cellular ATP consumption. Platelet secretion is usually associated with a conversion of metabolic ATP to hypoxanthine. Formaldehyde had to be used to stop secretion and since it caused breakdown of ATP, additional smaples were taken out for nucleotide determination during stirring of platelet-rich plasma with A23187. It was found that metabolic ATP was converted to inosine plus hypoxanthine only during the secretion step.     

A conformation-dependent epitope of human platelet glycoprotein IIIa.

This study explores conformational states of human platelet glycoprotein IIIa (GP IIIa) and possible mechanisms of fibrinogen receptor exposure. D3GP3 is an IgG1, kappa monoclonal antibody generated against purified GP IIIa and found to be specific for GP IIIa by immunoprecipitation and Western blot analysis. The binding of D3GP3 to resting platelets caused fibrinogen binding (approximately 5,000 molecules/platelet) and platelet aggregation but not secretion. Platelets express 40,000-50,000 GP IIb-IIIa molecules in their surface membranes. However, resting platelets only bound approximately 5,000 D3GP3 molecules/platelet. D3GP3 binding to platelets could be increased 2-3-fold by dissociation of the GP IIb-IIIa complex with 5 mM EDTA or by occupying the fibrinogen receptor with either RGDS peptides or fibrinogen. Platelet stimulation with ADP in the absence of fibrinogen did not cause increased D3GP3 binding above control levels. These data suggest that 1) GP IIb-IIIa can exist in multiple conformations in the platelet membrane, 2) D3GP3 binding to GP IIIa can expose the fibrinogen receptor, 3) the binding of either RGDS peptides or fibrinogen causes exposure of the D3GP3 epitope, and 4) platelet activation in the absence of ligand does not induce the same conformational changes in GP IIb-IIIa as does receptor occupancy by RGDS peptides or fibrinogen.     

Two waves of platelet secretion induced by thromboxane A2 receptor and a critical role for phosphoinositide 3-kinases

Thromboxane A2 (TXA2)-mediated platelet secretion and aggregation are important in thrombosis. Here, we present a novel finding that the stable TXA2 analogue, U46619, induces two waves of platelet secretion, each of which precedes a distinct wave of platelet aggregation. ADP released from platelets during the first wave of secretion played a major role in augmenting the first wave of platelet aggregation. The second wave of platelet secretion and aggregation required the first wave of both ADP secretion and aggregation and were blocked by either the integrin inhibitor RGDS or a P2Y12 receptor antagonist, indicating a requirement for both the integrin outside-in signal and ADP-activated Gi pathway. U46619 stimulated phosphoinositide 3-kinase (PI3K)-dependent phosphorylation of Akt, which was augmented by ADP but did not require integrin outside-in signaling. Platelets from PI3Kgamma knock-out mice or PI3K inhibitor-treated platelets showed an impaired second wave of platelet secretion and aggregation. However, the second wave of platelet aggregation was restored by addition of exogenous ADP to PI3Kgamma deficient or PI3K inhibitor-treated platelets. Thus, our data indicate that PI3K, together with the integrin outside-in signaling, play a central role in inducing the second wave of platelet secretion, which leads to the second wave of irreversible platelet aggregation.     

Changes in the platelet membrane glycoprotein IIb.IIIa complex during platelet activation

Platelet activation is accompanied by the appearance on the platelet surface of approximately 45,000 receptor sites for fibrinogen. The binding of fibrinogen to these receptors is required for platelet aggregation. Although it is established that the fibrinogen receptor is localized to a heterodimer complex of the membrane glycoproteins, IIb and IIIa, little is known about the changes in this complex during platelet activation that result in the expression of the receptor. In the present studies, we have developed and characterized a murine monoclonal anti-platelet antibody, designated PAC-1, that binds to activated platelets, but not to unstimulated platelets. PAC-1 is a pentameric IgM that binds to agonist-stimulated platelets with an apparent Kd of 5 nM. Binding to platelets is dependent on extracellular Ca2+ (KCa = 0.4 microM) but is not dependent on platelet secretion. Platelets stimulated with ADP or epinephrine bind 10,000-15,000 125I-PAC-1 molecules/platelet while platelets stimulated with thrombin bind 20,000-25,000 molecules/platelet. Several lines of evidence indicate that PAC-1 is specific for the glycoprotein IIb.IIIa complex. First, PAC-1 binds specifically to the IIb.IIIa complex on Western blots. Second, PAC-1 does not bind to thrombasthenic platelets or to platelets preincubated with ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid at 37 degrees C, both of which lack the intact IIb.IIIa complex. Third, PAC-1 competitively inhibits the binding of 125I-A2A9, and IgG monoclonal antibody that is specific for the IIb.IIIa complex. Fourth, the antibody inhibits fibrinogen-mediated platelet aggregation. These data demonstrate that PAC-1 recognizes an epitope on the IIb.IIIa complex that is located near the platelet fibrinogen receptor. Platelet activation appears to cause a Ca2+-dependent change involving the glycoprotein IIb.IIIa complex that exposes the fibrinogen receptor and, at the same time, the epitope for PAC-1.     

Decreased platelet aggregation, increased bleeding time and resistance to thromboembolism in P2Y1-deficient mice.

Platelet activation is characterized by shape change, induction of fibrinogen receptor expression and release of granular contents, leading to aggregation and plug formation. While this response is essential for hemostasis, it is also important in the pathogenesis of a broad spectrum of diseases, including myocardial infarction, stroke and unstable angina. Adenosine 5'-diphosphate (ADP) induces platelet aggregation, but the mechanism for this has not been established, and the relative contribution of ADP in hemostasis and the development of arterial thrombosis is poorly understood. We show here that the purinoceptor P2Y1 is required for platelet shape change in response to ADP and is also a principal receptor mediating ADP-induced platelet aggregation. Activation of P2Y1 resulted in increased intracellular calcium but no alteration in cyclic adenosine monophosphate (cAMP) levels. P2Y1-deficient platelets partially aggregated at higher ADP concentrations, and the lack of P2Y1 did not alter the ability of ADP to inhibit cAMP, indicating that platelets express at least one additional ADP receptor. In vivo, the lack of P2Y1 expression increased bleeding time and protected from collagen- and ADP-induced thromboembolism. These findings support the hypothesis that the ATP receptor P2Y1 is a principal receptor mediating both physiologic and pathological ADP-induced processes in platelets.     

Platelet cryopreservation using a combination of epinephrine and dimethyl sulfoxide as cryoprotectants

Current methods of platelet storage are unsatisfactory because of the short shelf life of platelets and the rapid loss of platelet viability. We have developed a cryopreservation method that results in less damage from freezing and higher recovered function of platelets. Platelets were cryopreserved using a combination of epinephrine (EPN) and dimethyl sulfoxide (Me(2)SO) as cryoprotectants. The response of platelets to agonists was studied by flow cytometry and aggregation tests. Cryopreserving platelets with Me(2)SO decreased platelet annexin V binding due to freezing. The combination of EPN with Me(2)SO enhanced Me(2)SO cryoprotection and decreased platelet microparticle generation, suggesting that cryopreserving platelets using this combination is associated with increased platelet integrity. Platelet cryopreservation with an Me(2)SO/EPN combination also increased platelet aggregability, which was demonstrated by decreasing the lag phase and increasing the aggregation density to 66.39% +/- 6.6 that of fresh platelet-rich plasmas. We conclude that adding EPN as a combined cryoprotectant improves the quality of Me(2)SO-frozen platelets. As a method of aggregation of cryopreserved platelets, this method is comparable to that of normal fresh platelets and may improve the conditions for platelet transfusion.     

Platelet aggregation induced by alpha 2-adrenoceptor and protein kinase C activation. A novel synergism.

Adrenaline or UK 14304 (a specific alpha 2-adrenoceptor agonist) and phorbol ester (phorbol 12,13-dibutyrate; PdBu) or bioactive diacylglycerols (sn-1,2-dioctanoylglycerol; DiC8) synergistically induced platelet aggregation and ATP secretion. The effect on aggregation was more pronounced than the effect on secretion, and it was observed in aspirinized, platelet-rich plasma or suspensions of washed aspirinized platelets containing ADP scavengers. No prior shape change was found. In the presence of adrenaline, DiC8 induced reversible aggregation and PdBu evoked irreversible aggregation that correlated with the different kinetics of DiC8- and PdBu-induced protein kinase C activation. Adrenaline and UK 14304 did not induce or enhance phosphorylation induced by DiC8 or PdBu of myosin light chain (20 kDa), the substrate of protein kinase C (47 kDa), or a 38 kDa protein. Immunoprecipitation studies using a Gcommon alpha antiserum or a Gi alpha antiserum showed that Gi alpha is not phosphorylated after exposure of platelets to PdBu or PdBu plus adrenaline. Adrenaline, PdBu or adrenaline plus PdBu did not cause stimulation of phospholipase C as reflected in production of [32P]phosphatidic acid. Adrenaline caused a small increase of Ca2+ in the platelet cytosol of platelets loaded with Indo-1; this effect was also observed in the absence of extracellular Ca2+. However, under conditions of maximal aggregation induced by adrenaline plus PdBu, no increase of cytosolic Ca2+ was observed. Platelet aggregation induced by PdBu plus adrenaline was not inhibited by a high intracellular concentration of the calcium chelator Quin-2. These experiments indicate that alpha 2-adrenoceptor agonists, known to interact with Gi, and protein kinase C activators synergistically induced platelet aggregation through a novel mechanism. The synergism occurs distally to Gi protein activation and protein kinase C-dependent protein phosphorylation and does not involve phospholipase C activation or Ca2+ mobilization.     

The effects of some salicylate analogues on human blood platelets. 2. The role of platelet acetylation in the inhibition of platelet aggregation

Aspirin, 2,3-diacetoxybenzoic acid, 2,6-diacetoxybenzoic acid and 2-propoxybenzoic acid were incubated in human platelet-rich plasma at 37°C for 5 and 10 min and the effects upon collagen induced platelet aggregation and the uptake by platelets of radioactive acetate and propionate groups from 14C-labelled analogues were studied to determine if a correlation existed between acylation of the platelet and inhibition of aggregation. Inhibition of aggregation and the uptake of radioactive label were both concentration-dependent and both increased with the time of incubation. Potency re inhibitors of aggregation was, in decreasing order, aspirin, 2,propoxybenzoic acid, 2,3-diacetoxybenzoic acid and 2,6-diacetoxybenzoic acid. Uptake of radioactive label however, was greatest with aspirin, intermediate with 2,3- and 2,6-diacetoxybenzoic acid, and lowest with 2-propoxybenzoic acid. Platelets exposed to a metabolic inhibitor (oligomycin, 10^?5M for 15 min) showed reduced uptake of labelled acetate and propionate and the degree of uptake did not correlate with the degree of inhibitory activity of the analogues on platelet aggregation. Platelet fragments produced by sonification did not take up radioactive label and chloroform: methanol extraction removed about 50% of the label from intact platelets. The results do not support the hypothesis that acetylation of platelets by aspirin is solely responsible for its inhibitory effects on aggregation but do not conflict with the suggestion that acetylation of platelets may be responsible for the persistent $\text{in}$$\text{vivo}$ effects of aspirin.     

Different effects of two thromboxane A2/prostaglandin H2 receptor ligands, U46619 and S-145, on rabbit platelets

Stimulation of rabbit platelets with U46619 induced platelet shape change, aggregation and secretion of ATP. However, S-145, which specifically binds to the thromboxane A2/prostaglandin H2 receptor like U46619, induced only shape change. Both compounds rapidly elevated cytoplasmic Ca2+ concentration although only U46619 evoked the formation of inositol phosphates. Chelating external Ca2+ with EGTA did not affect the S-145-induced platelet shape change while intracellular Ca2+ movement was severely reduced. These results suggest an essential role of phospholipase C in the induction of platelet aggregation and secretion and that some factor other than Ca2+ and phospholipase C participates in platelet shape change.     

In vitro binding of an IgE protein to human platelets

Bronchoconstriction in extrinsic asthma is initiated by mediators released from IgE-sensitized leukocytes after contact with polyvalent antigen. Because platelets also contain soluble mediators that can cause bronchoconstriction, platelet activation and release of the contents of platelet granules may play a role in IgE-mediated responses under some circumstances. We therefore sought to determine if platelets are capable of binding IgE and if cross-linking this cell-bound IgE initiates secretion of platelet granule contents. Platelets from 10 normal donors were studied by using automated fluorescence analysis and fluorescence microscopy. We detected binding of a purified myeloma IgE protein to 24.1 +/- 9.6% (mean +/- 2 SD) of the gel-filtered platelets from these normal individuals. Although we could detect the binding of IgE and anti-IgE to a minority of cells, every normal individual had a population of platelets that bound IgE. The amount of IgE that bound to normal platelets appeared to be distributed heterogeneously among the IgE-positive platelet population. Platelets from two individuals with type II Glanzmann's thrombasthenia bound normal amounts of heat-aggregated IgG, but less than 3% of the platelets bound detectable IgE. Moreover, a combination of monoclonal antibodies to glycoproteins IIb and IIIa inhibited the binding of the IgE protein to normal platelets but did not affect the binding of aggregated IgG. Thus, the binding of IgE to human platelets appeared to require the presence of the glycoprotein IIb-IIIa complex. Binding of monomeric IgE to platelets, by itself, did not initiate either platelet aggregation or release of 14C-serotonin. However, both aggregation and secretion of serotonin followed the addition of anti-IgE to IgE-sensitized platelets. These studies indicate that human platelets can bind an IgE myeloma protein in vitro and that cross-linking of surface-bound IgE with anti-IgE initiates aggregation and secretion. If platelets have a similar capacity to bind normal IgE in vivo, it is possible that platelets may participate directly in several atopic or inflammatory disorders in man mediated by this class of antibody.     

Negative regulation of Gq-mediated pathways in platelets by G(12/13) pathways through Fyn kinase

Platelets contain high levels of Src family kinases (SFKs), but their functional role downstream of G protein pathways has not been completely understood. We found that platelet shape change induced by selective G(12/13) stimulation was potentiated by SFK inhibitors, which was abolished by intracellular calcium chelation. Platelet aggregation, secretion, and intracellular Ca(2+) mobilization mediated by low concentrations of SFLLRN or YFLLRNP were potentiated by SFK inhibitors. However, 2-methylthio-ADP-induced intracellular Ca(2+) mobilization and platelet aggregation were not affected by PP2, suggesting the contribution of SFKs downstream of G(12/13), but not G(q)/G(i), as a negative regulator to platelet activation. Moreover, PP2 potentiated YFLLRNP- and AYPGKF-induced PKC activation, indicating that SFKs downstream of G(12/13) regulate platelet responses through the negative regulation of PKC activation as well as calcium response. SFK inhibitors failed to potentiate platelet responses in the presence of G(q)-selective inhibitor YM254890 or in G(q)-deficient platelets, indicating that SFKs negatively regulate platelet responses through modulation of G(q) pathways. Importantly, AYPGKF-induced platelet aggregation and PKC activation were potentiated in Fyn-deficient but not in Lyn-deficient mice compared with wild-type littermates. We conclude that SFKs, especially Fyn, activated downstream of G(12/13) negatively regulate platelet responses by inhibiting intracellular calcium mobilization and PKC activation through G(q) pathways.     

Mechanisms involved in agonist-induced hyperaggregability of platelets from normal pregnancy

There is substantial evidence of increased platelet reactivity in vivo and in vitro during pregnancy. Platelet activation occurs in pregnancy with a risk of the development of preeclampsia. In this study, platelet behavior was studied during 28-40 weeks of gestation in a group of women who remained normotensive and a group of nonpregnant female controls. Platelet aggregation and ATP release stimulated by agonists (i.e. collagen and adenosine 5'-diphosphate) were markedly enhanced in washed platelets from pregnant subjects. Furthermore, the collagen-evoked increase in intracellular Ca(2+) ([Ca(2+)](i)) mobilization in fura-2-AM-loaded platelets was also enhanced in pregnant subjects. Moreover, the binding activity of fluorescein isothiocyanate-triflavin toward the platelet glycoprotein IIb/IIIa complex did not significantly differ between the nonpregnant and pregnant groups. In addition, the amount of thromboxane A(2) (TxA(2)) formation from pregnant subjects was significantly greater than that from nonpregnant subjects in both resting and collagen-activated platelets. On the other hand, prostaglandin E(2) formation in the presence of imidazole in either resting or arachidonic acid (100 microM)-treated platelets did not significantly differ between these two groups. The levels of cyclic AMP formation in both resting and prostaglandin E(1) (10 microM)-treated platelets from pregnant subjects were significantly lower than those in nonpregnant subjects. Nitric oxide production was measured by a chemiluminescence detection method in this study. The extent of nitrate production in either resting or collagen-stimulated platelets from pregnant subjects did not significantly differ from that of platelets from the nonpregnant group. We conclude that the agonist-induced hyperaggregability of platelets from normal pregnancy may be due, at least partly, to an increase in TxA(2) formation and a lowering of the level of cyclic AMP formation, which leads to increased [Ca(2+)](i) mobilization and finally to enhanced platelet aggregation and ATP release.