The influence of lysophosphatidic acid on platelet protein phosphorylation

Lysophosphatidic acid (LPA) is a lysophospholipid that is produced during thrombin stimulation of platelets, which can promote platelet aggregation. The mechanism of the effect of LPA was explored in normal platelets and in platelets from a patient with a storage pool deficiency (SPD). A comparison with other lysophospholipids showed that only LPA exerted significant effects to cause or potentiate platelet aggregation. Aspirin, an inhibitor of prostaglandin endoperoxide synthetase, had little effect on LPA-induced aggregation, but completely blocked LPA-induced serotonin secretion. LPA also promoted phosphorylation of myosin light chain (MLC), a 47 kilodalton (kDa) protein, and actin-binding protein. Aspirin significantly inhibited the phosphorylation of the 47-kDa and actin-binding proteins at 3-8 min after the addition of LPA, but had no effect on protein phosphorylation within the 1st min and had no significant effect on MLC phosphorylation. In SPD platelets, aspirin partially inhibited both aggregation and phosphorylation of the 47-kDa protein (less than 30% inhibition) and MLC (less than 40% inhibition) at time points of 1 min or less. The addition of ADP to SPD platelets enhanced the LPA response in platelets either pretreated or not pretreated with aspirin. Studies with SPD platelets indicate that thromboxane and secreted ADP contribute to, but are not necessary for, LPA-induced aggregation and phosphorylation. A23187 (a calcium ionophore) and LPA showed some selectivity to promote MLC as opposed to the 47-kDa protein phosphorylation, particularly at low concentrations of agonists and at earlier time points. The protein phosphorylation changes seen are consistent with a role for MLC phosphorylation in the granule centralization promoted with LPA.     

Morphologic and hematologic characteristics of storage pool deficiency in beige rats (Chédiak-Higashi syndrome of rats)

Characterization of beige rats as having a platelet storage pool deficiency (SPD) was undertaken. Platelets from beige rats, an animal model of Chédiak-Higashi syndrome (CHS), completely lacked the ability to aggregate when stimulated with high dosages of collagen (50 micrograms/ml), and lacked secondary aggregation induced by adenosine diphosphate (ADP). Concentrations of ADP, ATP, and serotonin in the platelets of beige rats were significantly lower than those of control rats. However, platelet count remained within normal values. Electron microscopy revealed that platelets had fewer dense granules, whereas other organelles had normal structure. This morphologic and functional evidence confirms that platelets of beige rats have the typical characteristics of SPD.     

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.     

Irreversible platelet aggregation does not depend on lipoxygenase metabolites

Previous investigations in our laboratory demonstrated the existence of an intrinsic mechanism, termed membrane modulation, capable of restoring sensitivity to aspirin treated platelets, resulting in irreversible aggregation in response to arachidonic acid (AA). The mechanism underlying correction of aspirin induced inhibition of platelet function, however, was not clear. In the present study we have evaluated the role of lipoxygenase (LO) metabolites of AA in securing irreversible aggregation of drug induced cyclooxygenase (CO) deficient platelets. Platelets treated with aspirin or Ibuprofen did not convert radiolabeled AA to thromboxane, but generated significant quantities of hydroxy acids via the LO pathway. However, drug exposed platelets, when stirred with epinephrine first and then challenged with AA, aggregated irreversibly. Eicosatetraynoic acid (ETYA 1, U53119) inhibited AA conversion by the LO pathway, whereas 5,8,11,14-eicosatetraynoic acid (ETYA 2) inhibited AA conversion by both CO and LO enzymes. Yet, at the inhibitory concentration these fatty acids failed to prevent AA induced irreversible aggregation of CO deficient, alpha adrenergic receptor stimulated platelets. Results of four studies show that the generation of LO metabolites of AA are not essential for securing irreversible aggregation of platelets.     

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.     

Formation by phospholipase A2 of prostaglandins and thromboxane A2-like activity in the platelets of normal and essential fatty acid deficient rats. Comparison with effect on human and rabbit platelets

When rat platelets are incubated with phospholipase A2, thromboxane A2-like activity and prostaglandins are formed. The amounts are approximately similar, whether aggregation is induced after the incubation or not. No aggregation is observed when the platelets are incubated with phospholipase A2. In the platelets of essential fatty acid deficient rats, only small amounts of thromboxane A2-like acitivity and prostaglandins are formed. No formation of these substances occurs when human and rabbit platelets are incubated with phospholipase A2. The results indicate that formation of thromboxane A2-like activity enhances aggregation in rat platelets, but that aggregation is not induced.     

Human platelet activation by an alkylacetyl analogue of phosphatidylcholine

The present study has evaluated the influence of semi-synthetic platelet-aggregating factor, (PAF) i.e., alkylacetylglycerophosphocholine, on human platelet morphology, biochemistry and function in order to determine if PAF serves as the corrective factor restoring sensitivity to refractory platelets after treatment with epinephrine. Threshold concentrations of PAF caused irreversible platelet aggregation which could be blocked by agents elevating endogenous levels or cyclic AMP or inhibited by antagonists of platelet prostaglandin synthesis and secretion. PAF did not stimulate platelets through α-adrenergic receptors or receptors for arachidonate, endoperoxides or thromboxanes. 24 h after aspirin ingestion, platelets could be aggregated irreversibly by high concentrations, but not by threshold amounts of PAF, even though they were still insensitive to arachidonate. Another less potent PAF derivative, alkenylacetylglycerophosphocholine, blocked aggregation of 24-h aspirin platelets by PAF, but did not inhibit restoration of arachidonate sensitivity and irreversible aggregation when the samples were treated first with epinephrine. Our findings indicate that threshold amounts of PAF activate human platelets in a physiologic manner and cause irreversible aggregation which is dependent on prostaglandin synthesis and the release reaction. The results do not support the concept that PAF is the mediator of the mechanism of membrane modulation through which epinephrine induces correction of the refractory state in prostaglandin I₂-treated or dissociated platelets, or cells obtained from individuals following aspirin ingestion. Thus, the mechanism of platelet membrane modulation is capable of securing irreversible aggregation of secretion, prostaglandin synthesis or PAF formation.     

Sandy: a new mouse model for platelet storage pool deficiency

Sandy (sdy) is a mouse mutant with diluted pigmentation which recently arose in the DBA/2J strain. Genetic tests indicate it is caused by an autosomal recessive mutation on mouse Chromosome 13 near the cr and Xt genetic loci. This mutation is different genetically and hematologically from previously described mouse pigment mutations with storage pool deficiency (SPD). The sandy mutant has diluted pigmentation in both eyes and fur, is fully viable and has prolonged bleeding times. Platelet serotonin levels are extremely low although ATP dependent acidification activity of platelet organelles appears normal. Also, platelet dense granules are extremely reduced in number when analysed by electron microscopy of unfixed platelets. Platelets have abnormal uptake and flashing of the fluorescent dye mepacrine. Secretion of lysosomal enzymes from kidney and from thrombin-stimulated platelets is depressed 2- and 3-fold, and ceroid pigment is present in kidney. Sandy platelets have a reduced rate of aggregation induced by collagen. The sandy mutant has an unusually severe dense granule defect and thus may be an appropriate model for cases of human Hermansky-Pudlak syndrome with similarly extreme types of SPD. It represents the tenth example of a mouse mutant with simultaneous defects in melanosomes, lysosomes and/or platelet dense granules.     

Formation by phospholipase A2 of prostaglandins and thromboxane A2-like activity in the platelets of normal and essential fatty acid deficient rats. Comparison with effect on human and rabbit platelets

When rat platelets are incubated with phospholipase A₂, thromboxane A₂-like activity and prostaglandins are formed. The amounts are approximately similar, whether aggregation is induced after the incubation or not. No aggregation is observed when the platelets are incubated with phospholinase A₂. In the platelets of essential fatty acid deficient rats, only small amounts of thromboxane A₂-like activity and prostaglandins are formed. No formation of these substances occurs when human and rabbit platelets are incubated with phospholipase A₂.The results indicate that formation of thromboxane A₂-like activity enhances aggregation in rat platelets, but that aggregation is not induced.     

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.     

Sphingosine inhibition of agonist-dependent secretion and activation of human platelets implies that protein kinase C is a necessary and common event of the signal transduction pathways

Sphingosine is a potent inhibitor of [3H]phorbol dibutyrate binding and protein kinase C activity in vitro and in human platelets (Hannun, Y., Loomis, C., Merrill, A., and Bell, R. (1986) J. Biol. Chem. 261, 12604-12609). Preincubation of platelets with sphingosine resulted in the inhibition of platelet secretion and second phase aggregation in response to ADP, gamma-thrombin, collagen, arachidonic acid, and platelet activating factor. Sphingosine did not affect the initial shape change of platelets or the first phase of aggregation in response to these agonists. Ristocetin-induced platelet agglutination was not affected by sphingosine. Sphingosine inhibition of secondary aggregation (secretion and second phase aggregation) was overcome by phorbol dibutyrate and by the cell-permeable protein kinase C activator, dioctanoylglycerol. Furthermore, platelet secretion and irreversible aggregation were induced by protein kinase C activators in platelets that had been "primed" to undergo initial shape change and first phase aggregation by low concentrations of agonists. These results suggest that protein kinase C activation is a necessary component in the signal transducing pathways that lead to platelet activation. Higher concentrations of agonists, however, induced irreversible aggregation and partial secretion in the presence of sphingosine, suggesting the existence of protein kinase C-independent pathways for platelet activation. These results demonstrate the utility of sphingosine as a pharmacologic tool in probing the role of protein kinase C in signal transduction.     

Signalling events underlying platelet aggregation induced by the glycoprotein VI agonist convulxin.

We have investigated the role of secretion and intracellular signalling events in aggregation induced by the glycoprotein (GP)VI-selective snake venom toxin convulxin and by collagen. We demonstrate that aggregation induced by threshold concentrations of convulxin undergoes synergy with ADP acting via the P2Y12 receptor whereas there is no synergy via the P2Y1 receptor or with thromboxanes. On the other hand, apyrase, the P2Y12 receptor antagonist, AR-C67085, and indomethacin only marginally inhibit aggregation induced by convulxin. In comparison, these inhibitors severely attenuate the response to collagen. In order to investigate whether the weak inhibitory action against convulxin is due to release of agonists other than ADP from dense granules, experiments were performed on murine platelets deficient in this organelle (pearl mice platelets). A slightly greater reduction in aggregation induced by convulxin was observed in pearl platelets than in the presence of inhibitors of ADP, but a maximal response was still attained. Importantly, inhibition of protein kinase C further reduced the response to convulxin in pearl platelets demonstrating a direct role for the kinase in aggregation. Chelation of intracellular Ca2+ with 1,2-bis(2-aminophenoxy)ethane-N,N,N,N',N'-tetraacetic acid (acetoxymethyl)ester (BAPTA-AM) abolished aggregation induced by convulxin under all conditions. Activation of phospholipase C by convulxin was potentiated by ADP acting through the P2Y12 receptor. In conclusion, we show that Ca2+ and protein kinase C, but not release of the secondary agonists ADP and thromboxane A2, are required for full aggregation induced by convulxin, whereas the response induced by collagen shows a much greater dependence on secretion of secondary agonists.     

Normal lytic granule secretion by cytotoxic T lymphocytes deficient in BLOC-1, -2 and -3 and myosins Va, VIIa and XV

Melanocytes and cells of the immune system share an unusual secretory mechanism which uses the lysosome as a regulated secretory organelle. Recently, a number of the proteins required for these 'secretory lysosomes' to undergo exocytosis have been identified. These include Rab27a, Lyst, Rab geranyl geranyl transferase and the adapter protein complex AP-3. Patients lacking any of these proteins are characterized by the rare combination of albinism and immunodeficiency, revealing roles for these proteins in both melanocyte and immune cell secretion. In order to ask how far the link between albinism and immunodeficiency extends we have examined cytotoxic T-lymphocyte (CTL) secretion from two BLOC-3-deficient patients and seven different mouse models of Hermansky-Pudlak syndrome, all of which display defects in pigmentation and platelet function. We find that CTL function is normal in HPS patients and pale-ear mice deficient in BLOC-3, pallid, muted and sandy mice deficient in BLOC-1, ruby-eye mice deficient in BLOC-2 and buff mice deficient in Vps33a. Similarly, the unconventional myosins, Va, VIIa and XV, which can act as effectors for Rab27a in some cell types, are not required in CTL. These results reveal differences in the protein machinery required for biogenesis and/or secretion of lysosome-related organelles in CTL and melanocytes.     

Interrelation of platelet aggregation, release reaction and thromboxane A2 production

Aggregation of platelets, stimulated by different agonists, was inhibited by omitting sample stirring or by preincubation of platelets with a monoclonal antibody against glycoproteins IIb-IIIa or with a pentapeptide containing the sequence Arg-Gly-Asp-Ser. In platelets stimulated by collagen, ADP and epinephrine, the inhibition of aggregation paralleled a reduction of both release reaction and thromboxane A2 formation. When thrombin was the stimulus, ATP release and thromboxane A2 production were unaffected (or only slightly modified) by the inhibition of platelet aggregation. These data add further evidence to the hypothesis that aggregation supports the activation of platelets stimulated by weak agonists.     

Effect of dietary lipid on collagen- and adenosine diphosphate-induced platelet aggregation and thromboxane A2 synthesis in the rat

Dietary lipids containing different proportions of long-chain polyunsaturated fatty acids can affect platelet thromboxane A(2) formation and aggregation. In the present work, the effects of dietary lipid, from animal and vegetable sources, on collagen- and adenosine diphosphate (ADP)-induced thromboxane A(2) (measured as thromboxane B(2)) production and aggregation in washed rat platelets were studied. In addition, plasma thromboxane B(2) levels in rats fed different dietary lipids were measured. Animals were fed 10% fat by weight as lard (LRD), corn oil, soy bean oil, canola oil (CAN), or cod liver oil (CLO) for a period of 7 weeks. Circulating thromboxane B(2) levels detected in platelet-poor plasma of the CLO-fed animals were significantly lower than those of rats fed all other dietary lipids. The platelets of CLO-fed animals synthesized significantly less thromboxane A(2) compared with those from other dietary groups following ex vivo stimulation of platelets with agonists such as collagen and ADP, with the exception of platelets from the LRD-fed animals. Ex vivo stimulation of platelets obtained from this group with collagen resulted in the synthesis of significantly greater levels of thromboxane A(2) compared with all other groups. However, aggregation responses to collagen and ADP were not significantly affected by dietary treatment, although relatively the lowest responses to these agonists were apparent in the CLO-fed and CAN-fed groups, respectively.     

The fibrinogen-derived peptide (RGDS) prevents proteolytic degradation of protein kinase C in platelets by inhibiting platelet aggregation

The effects of the fibrinogen-derived tetrapeptide, Arg-Gly-Asp-Ser (RGDS), on platelet activation processes was studied. At concentrations of 100-300 microM, RGDS completely prevented platelet aggregation induced by all the common platelet agonists, 'weak' and 'strong'. In agreement with earlier views on the aggregation-dependency of weak agonist-induced thromboxane synthesis and 5-hydroxytryptamine (5HT) secretion, RGDS (100-300 microM) inhibited these events induced by ADP, adrenaline and low concentrations of thrombin and collagen but not that induced by high concentrations of thrombin and collagen. 5HT secretion induced by the protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA), was also not affected by RGDS, but proteolytic degradation of the translocated membrane-bound enzyme in PMA-treated platelets, due to the actions of the Ca2+-dependent protease (Ca-DP), was completely prevented such that in the presence of RGDS, sustained increases in membrane-bound PKC activity were observed. PMA alone caused only transient increases in membrane-bound PKC. This effect of RGDS was similar to the effect of E64-d, a recently described inhibitor of Ca-DP in platelets, or the effects seen with PMA in unstirred non-aggregating platelets. It is concluded that RGDS inhibits the actions of Ca-DP in platelets via inhibition of aggregation.     

Synergistic stimulation of thromboxane biosynthesis by calcium ionophore and phorbol ester or thrombin in human platelets

Phorbol ester PMA and low concentrations of calcium ionophore A-23187, which given separately have minimal effect in stimulating thromboxane synthesis in human platelets, showed marked synergism when given simultaneously. A similar synergism can be also demonstrated between thrombin or collagen and low concentrations of A-23187 but not of PMA. Simultaneous addition of thrombin and PMA results in less synthesis of thromboxane than that of thrombin alone. These studies suggest that protein kinase C activation by agonists may not only induce but also regulate thromboxane synthesis in human platelets.     

Thromboxane synthetase inhibitors as pharmacological tools: differential biochemical and biological effects on platelet suspensions

The comparative effects of three so called "thromboxane-synthetase-inhibitors" (imidazole, N-0164, and U-51605) on arachidonate metabolism and on platelet aggregation were studied. All three compounds blocked platelet microsomal thromboxane synthesis from prostaglandin endoperoxides without affecting platelet adenyl cyclase. Imidazole, blocked thromboxane synthesis in intact platelets either from arachidonic acid or PGH2, without affecting aggregation. U-51605 simultaneously inhibited thromboxane synthesis and platelet suspension aggregation. N-0164 inhibited aggregation probably at extracellular sites, at concentrations that did not alter arachidonate or PGH2 metabolism. High concentrations of N-0164 simultaneously inhibited PG cyclo-oxygenase and thromboxane synthetase. The lack of specificity of these compounds requires that other actions of these compound must be considered when they are used as pharmacological tools to inhibit thromboxane synthetase.     

rim2 (recombination-induced mutation 2) is a new allele of pearl and a mouse model of human Hermansky-Pudlak Syndrome (HPS): genetic and physical mapping

A mouse mutation, rim2, is one of a series of spontaneous mutations that arose from the intra-MHC recombinants between Japanese wild mouse-derived wm7 and laboratory MHC haplotypes. This mutation is single recessive and characterized by diluted coat color and hypo-pigmentation of the eyes. We mapped the rim2 gene close to an old coat color mutation, pearl (pe), on Chromosome (Chr) 13 by the high-density linkage analysis. The pearl mutant is known to have abnormalities similar to Hermansky-Pudlak syndrome (HPS), a human hemorrhagic disorder, characterized by albinism and storage pool deficiency (SPD) of dense granules in platelets. A mating cross of C57BL10/Slc-rim2/rim2 and C57BL/6J-pe/pe showed no complementation of coat color. Additionally, characteristics similar to SPD were also observed in rim2. Thus, rim2 appeared to be a new allele of the pe locus and serves as a mouse model for human HPS. We have made a YAC contig covering the rim2/pe locus toward positional cloning of the causative gene. Received: 23 July 1997 / Accepted: 26 August 1997     

A congenital defect in platelet prostaglandin production associated with impaired hemostasis in storage pool disease

Prostaglandin (PG) production was found to be impaired in platelets from patients with 'storage pool disease', a well characterized hemostatic disorder. This finding adds to the growing body of evidence which implicates prostaglandin synthesis or a similar arachidonate-consuming process in second phase aggregation and the accompanying platelet release reaction. Prostaglandin production is not impaired in platelets from patients with thrombasthenia. It is thus unlikely that aggregation 'per se' is a stimulus for PG production, because thrombasthenic platelets are virtually incapable of aggregation.