Adenosine diphosphate receptors on blood platelets: potential new targets for antiplatelet therapy

Platelets play a key role not only in physiological haemostasis, but also under pathological conditions such as thrombosis. Platelet activation may be initiated by a variety of agonists including thrombin, collagen, thromboxane or adenosine diphosphate (ADP). Although ADP is regarded as a weak agonist of blood platelets, it remains an important mediator of platelet activation evoked by other agonists, which induce massive ADP release from dense granules, where it occurs in molar concentrations. Thus, ADP action underlies a positive feedback that facilitates further platelet aggregation and leads to platelet plug formation. Additionally, ADP acts synergistically to other, even weak, agonists such as serotonin, adrenaline or chemokines. Blood platelets express two types of P2Y ADP receptors: P2Y(1) and P2Y(12). ADP-dependent platelet aggregation is initiated by the P2Y1 receptor, whereas P2Y(12) receptor augments the activating signal and promotes platelet release reaction. Stimulation of P2Y(12) is also essential for ADP-mediated complete activation of GPIIb-IIIa and GPIa-IIa, and further stabilization of platelet aggregates. The crucial role in blood platelet biology makes P2(Y12) an ideal candidate for pharmacological approaches for anti-platelet therapy.     

Increased immunoreactive neuropeptide Y in platelets of spontaneously hypertensive rats (SHR)

A high concentration of immunoreactive neuropeptide Y was observed in rat platelets using a specific and sensitive radioimmunoassay for neuropeptide Y. Three kinds of high performance liquid chromatography combined with radioimmunoassay for neuropeptide Y showed that immunoreactive neuropeptide Y in rat platelets is identical to rat authentic neuropeptide Y. To investigate the pathological role of platelet neuropeptide Y in genetic hypertensive rats, the platelet content and plasma concentration of neuropeptide Y were measured by a sensitive radioimmunoassay for rat neuropeptide Y in 5-, 10- and 15-wk old spontaneously hypertensive rat and age-matched Wistar Kyoto rat. Platelet content of immunoreactive neuropeptide Y in 5-, 10- and 15-wk old spontaneously hypertensive rat was higher than that in Wistar Kyoto rat at each age. No difference was observed in plasma concentration of immunoreactive neuropeptide Y between spontaneously hypertensive rat and Wistar Kyoto rat at each age.     

Pomolic acid, triterpenoid isolated from Licania pittieri, as competitive antagonist of ADP-induced aggregation of human platelets

Pomolic acid (PA), triterpenoid isolated from Licania pittieri, has previously shown a potent ability to inhibit adenosine diphosphate (ADP)- and epinephrine-induced human platelet aggregation. To investigate whether PA could be an antagonist of ADP-activated receptors of human platelets (P2Y(1) and P2Y(12)), pharmacological studies were conducted to examining its ability to modulate the platelet shape change induced by a selective P2Y(1) receptor agonist MRS2365 and also the nature of its possible interaction with ADP receptors by analyzing the characteristics of log concentration-response curves of ADP constructed in the absence and in the presence of fixed concentrations of PA, using in vitro platelet aggregation assays. PA did not interfere with the activation of P2Y(1) receptor by MRS2365 to induce platelet shape change and displayed a competitive antagonism of ADP-induced platelet aggregation, which most probably involves competition for a single binding site in platelets. The estimated equilibrium dissociate constant (K(b)) of PA as ADP receptor antagonist was 15.4±0.06nM. Together, these findings give indirect evidence for the idea that PA could be a potent competitive antagonist of P2Y(12) receptor, and open the possibility to consider it as new member of the non-nucleotide generation of antiplatelet drugs.     

Re-evaluation of the effects of neuropeptide Y on aggregation of human platelets.

Several studies have shown that platelets are a major source of circulating neuropeptide Y (NPY) immunoreactivity in rats, but the effects of this vasoconstrictor peptide on platelets are not well-defined. Recently, it was reported that porcine NPY was an inhibitor of in vitro human platelet aggregation induced by epinephrine, an observation which would have important implications regarding platelet-vascular interactions during states involving platelet activation and thrombosis. Thus, we undertook the present studies, in an attempt to confirm the earlier report, and to extend those observations to human NPY. In contrast to the recent report, we found no inhibitory effect of either human or porcine NPY on epinephrine- or collagen-induced aggregation of human platelets from normal subjects. Likewise, specific NPY Y-1 and Y-2 agonists had no direct or indirect action on platelet aggregation. Finally, the effect of human NPY on intraplatelet cAMP was measured. The peptide had no effect on either basal or iloprost-stimulated cAMP levels. We hypothesize that the role of NPY in the platelet-vascular interaction is in promoting vasoconstriction associated with platelet aggregation, and does not include inhibition of further thrombosis.     

Reversed phase chromatographic analysis of Nostoc and Euglena isoleucyl-tRNAs aminoacylated in vitro in homologous and heterologous systems.

To investigate the mechanisms governing collagen interaction with blood platelets, the effects of side-chain modifications on collagen-induced platelet aggregation and release of serotonin were studied. Since many chemical modifications alter the ability of collagen to form fibers that, according to current theory, may complicate interpretation of data, we eliminated this possibility by using collagen stabilized in a native-type fibrillar structure by treatment with either glutaraldehyde or ultraviolet irradiation. Acetylation, methylation, succinylation, treatment with 2,4-dinitrofluorobenzene, 2,4,6-trinitrobenzene sulfonic acid or 1,2-cyclohexanedione, and deguanidination with hypobromite were used to modify collagen side-chain reactive groups: amino, carboxyl, hydroxyl and guanidino. Both unmodified monomeric dispersed and fibrillar collagen preparations initiated platelet aggregation and release, although the kinetics and magnitude of the response were different. Monomeric collagen which had been modified by deguanidination, methylation or succinylation, failed to polymerize in physiological conditions and did not induce platelet aggregation and release. However, none of the chemical modifications of stabilized native-type collagen fibers, except treatment with hypobromite or cyclohexanedione, had an effect on collagen-induced platelet aggregation and release. Both hypobromite and cyclohexanedione modified guanidino groups of arginyl residues. Results showed that the ability of a collagen sample to induce platelet aggregation and release of serotonin is dependent on the arginine content of fibrillar collagen. These data demonstrate that manipulation of amino, carboxyl and hydroxyl groups is unimportant as long as the native-type fibrillar structure is maintained, and that arginyl residues are directly involved in collagen-platelet interaction. Moreover, the data suggest that only the arginyl residues in the Y position of the tripeptide unit Gly-X-Y of collagen are responsible.     

Collagen-induced platelet aggregation and release. I effects of side-chain modifications and role of arginyl residues

To investigate the mechanisms governing collagen interaction with blood platelets, the effects of side-chain modifications on collagen-induced platelet aggregation and release of serotonin were studied. Since many chemical modifications alter the ability of collagen to form fibers that, according to current theory, may complicate interpretation of data, we eliminated this possibility by using collagen stabilized in a native-type fibrillar structure by treatment with either glutaraldehyde or ultraviolet irradiation. Acetylation, methylation, succinylation, treatment with 2,4-dinitrofluorobenzene, 2,4,6-trinitrobenzene sulfonic acid or 1,2-cyclohexanedione, and deguanidination with hypobromite were used to modify collagen side-chain reactive groups: amino, carboxyl, hydroxyl and guanidino. Both unmodified monomeric dispersed and fibrillar collagen preparations initiated platelet aggregation and release, although the kinetics and magnitude of the response were different. Monomeric collagen which had been modified by deguanidination, methylation or succinylation, failed to polymerize in physiological conditions and did not induce platelet aggregation and release. However, none of the chemical modifications of stabilized native-type collagen fibers, except treatment with hypobromite or cyclohexanedione, had an effect on collagen-induced platelet aggregation and release. Both hypobromite and cyclohexanedione modified guanidino groups of arginyl residues. Results showed that the ability of a collagen sample to induce platelet aggregation and release of serotonin is dependent on the arginine content of fibrillar collagen.These data demonstrate that manipulation of amino, carboxyl and hydroxyl groups is unimportant as long as the native-type fibrillar structure is maintained, and that arginyl residues are directly involved in collagen-platelet interaction. Moreover, the data suggest that only the arginyl residues in the Y position of the tripeptide unit Gly-X-Y of collagen are responsible.     

Collagen-induced platelet aggregation and release. II critical size and structural requirements of collagen

To investigate the mechanisms governing collagen interaction with blood platelets, the effects of side-chain modifications on collagen-induced platelet aggregation and release of serotonin were studied. Since many chemical modifications alter the ability of collagen to form fibers that, according to current theory, may complicate interpretation of data, we eliminated this possibility by using collagen stabilized in a native-type fibrillar structure by treatment with either glutaraldehyde or ultraviolet irradiation. Acetylation, methylation, succinylation, treatment with 2,4-dinitrofluorobenzene, 2,4,6-trinitrobenzene sulfonic acid or 1,2-cyclohexanedione, and deguanidination with hypobromite were used to modify collagen side-chain reactive groups: amino, carboxyl, hydroxyl and guanidino. Both unmodified monomeric dispersed and fibrillar collagen preparations initiated platelet aggregation and release, although the kinetics and magnitude of the response were different. Monomeric collagen which had been modified by deguanidination, methylation or succinylation, failed to polymerize in physiological conditions and did not induce platelet aggregation and release. However, none of the chemical modifications of stabilized native-type collagen fibers, except treatment with hypobromite or cyclohexanedione, had an effect on collagen-induced platelet aggregation and release. Both hypobromite and cyclohexanedione modified guanidino groups of arginyl residues. Results showed that the ability of a collagen sample to induce platelet aggregation and release of serotonin is dependent on the arginine content of fibrillar collagen.These data demonstrate that manipulation of amino, carboxyl and hydroxyl groups is unimportant as long as the native-type fibrillar structure is maintained, and that arginyl residues are directly involved in collagen-platelet interaction. Moreover, the data suggest that only the arginyl residues in the Y position of the tripeptide unit Gly-X-Y of collagen are responsible.     

Activation of platelets induced by mAb P256 specific for glycoprotein IIb-IIIa. Possible evidence for a role for IIb-IIIa in membrane signal transduction

Monoclonal antibody P256, which is specific for glycoprotein IIb-IIIa complex, was found to induce aggregation of normal platelets in plasma. The mechanism of platelet activation induced by this monoclonal antibody was thoroughly studied. The divalent binding to the IIb-IIIa molecule was necessary for triggering aggregation since Fab' fragments did not induce aggregation as did IgG and F(ab')2 fragments; however, F(ab')2 did not induce the release as did the whole IgG. P256-induced aggregation was accompanied by release of all three granule constituents, namely dense granules, alpha-granules and lysosomes, with parallel kinetics showing half-maximum release 50 s after addition of P256. Thromboxane synthesis was initiated at the same time. Using 32P-prelabeled platelets, no variation in level of [32P]phosphatidylinositol 4,5-bisphosphate could be detected in the first minute after P256 addition, indicating no activation of the calcium-independent phospholipase C specific for polyphosphoinositol phospholipid. P256 induced a calcium mobilization as measured by Indo-1 fluorescence of about the third of that measured in the presence of a thrombin concentration giving the same intensity of aggregation. P256 induced phosphorylation of the myosin light chain p20 and of the main substrate of protein kinase C, p43. Addition of aspirin inhibited almost totally calcium mobilization and partially aggregation, release and protein phosphorylations. By contrast, in the absence of external calcium, although no aggregation could occur, the release reaction was only partially reduced. In this activation, the glycoprotein IIb-IIIa complex thus appears to play a role in modulating platelet response, not only via calcium fluxes but also in activating protein kinase C responsible for p43 phosphorylation.     

Modulation of platelet activation by native DNA.

Native DNA (dsDNA) was found to induce the aggregation of isolated human platelets and the release of platelet 5HT; this activation was inhibited by both theophylline and TYA, suggesting a role for cAMP and metabolic products formed from arachidonate. By contrast, nonaggregating amounts of dsDNA inhibited platelet activation induced by collagen or thrombin. This inhibition, which could be overcome by use of greater amounts of the stimulatory agents, was not associated with the loss of platelet viability. Activation of platelets by dsDNA was not observed in plasma or in isolated platelet systems to which small amounts of cell-free plasma were added. However, dsDNA maintained in plasma its ability to inhibit platelet aggregation induced by collagen and thrombin. RNA and single-stranded DNA failed to induce platelet aggregation or release of 5HT and to block the platelet activation stimulated by dsDNA. Further, dsDNA did not significantly inhibit platelet aggregation in platelet-rich plasma stimulated by ADP or epinephrine. These data implicate dsDNA as a selective and potentially important activator and modulator of platelet responsiveness.     

ATP augments von Willebrand factor-dependent shear-induced platelet aggregation through Ca2+-calmodulin and myosin light chain kinase activation

Shear stress triggers von Willebrand factor (VWF) binding to platelet glycoprotein Ibalpha and subsequent integrin alpha(IIb)beta(3)-dependent platelet aggregation. Concomitantly, nucleotides are released from plateletdense granules, and ADP is known to contribute to shear-induced platelet aggregation (SIPA). We found that the impaired SIPA of platelets from a Hermansky-Pudlak patient lacking dense granules was restored by exogenous l-beta,gamma-methylene ATP, a stable P2X(1) agonist, as well as by ADP, confirming that in addition to ADP (via P2Y(1) and P2Y(12)), ATP (via P2X(1)) also contributes to SIPA. Likewise, SIPA of apyrase-treated platelets was restored upon P2X(1) activation with l-beta,gamma-methylene ATP, which promoted granule centralization within platelets and stimulated P-selectin expression, which is a marker of alpha-granule release. In addition, during SIPA, platelet degranulation required both extracellular Ca(2+) and VWF-glycoprotein Ibalpha interactions without involving alpha(IIb)beta(3). Neither platelet release nor SIPA was affected by protein kinase C inactivation, even though protein kinase C blockade inhibits platelet responses to collagen and thrombin in stirring conditions. In contrast, inhibiting myosin light chain (MLC) kinase with ML-7 reduced platelet release and SIPA by 30%. Accordingly, the potentiating effect of P2X(1) stimulation on the aggregation of apyrase-treated platelets coincided with intensified phosphorylation of MLC and was abrogated by ML-7. SIPA-induced MLC phosphorylation occurred exclusively through released nucleotides and selective antagonism of P2X(1) with MRS2159-reduced SIPA, ATP release, and potently inhibited MLC phosphorylation. We conclude that the P2X(1) ion channel induces MLC-mediated cytoskeletal rearrangements, thus contributing to SIPA and degranulation during VWF-triggered platelet activation.     

Platelet aggregation may not be a prerequisite for collagen-stimulated platelet generation of nitric oxide

By determining the sum of the supernatant concentrations of nitrite and nitrate the stimulated generation of nitric oxide (NO) by human washed platelets induced by a range of fibrillar collagen concentrations (0.0156-25 microg ml(-1)) was investigated. Platelet serotonin (5-hydroxytryptamine, 5-HT) efflux and platelet aggregation were also measured. Under resting conditions (0 microg ml(-1) collagen) platelet NO release was equivalent to 1.06+/-0.17 nmol per 10(8) platelets. Maximal NO release, equivalent to 2.1+/-0. 37 nmol per 10(8) platelets, was observed with only 0.0625 microg ml(-1) collagen (P<0.02, stimulated vs. resting release), higher collagen concentrations producing no further increases in platelet NO output. By contrast, maximal platelet aggregation and 5-HT efflux did not occur until collagen concentrations of 2.5 microg ml(-1) and 10-25 microg ml-1), respectively, had been achieved. L-NAME (1 mmol l(-1)) and L-NMMA (1 mmol l(-1)) inhibited stimulated platelet NO generation by 78+/-6% and 72%, respectively. Contrasting with fibrillar collagen, fibrillar beta-amyloid protein had no effect on platelet NO generation, or on 5-HT efflux or aggregation. These data perhaps indicate that NO generation by human platelets is stimulated by concentrations of fibrillar collagen insufficient to elicit an aggregatory response. Such a mechanism could operate in vivo to inhibit platelet aggregation which might otherwise be induced by low concentrations of circulating agonists.     

PAR4, but not PAR1, signals human platelet aggregation via Ca2+ mobilization and synergistic P2Y12 receptor activation

Regulation of platelet activation plays a central role in hemostasis and pathophysiological processes such as coronary artery disease. Thrombin is the most potent activator of platelets. Human platelets express two thrombin receptors, PAR1 and PAR4, both of which signal platelet activation. Evidence is lacking on the mechanism by which PAR1 and PAR4 may differentially signal platelet aggregation. Here we show that at the relatively high concentration of agonist most likely found at the site of a local thrombus, dual inhibition of the P2Y12 receptor and calcium mobilization result in a complete inhibition of PAR4-induced aggregation, while having no effect on either thrombin or PAR1-mediated platelet aggregation. Both PAR1- and PAR4mediated aggregation are independent of calcium mobilization. Furthermore, we show that P2Y12 receptor activation is not required for protease-activated receptor-mediated aggregation at higher agonist concentrations and is only partially required for Rap1 as well as GPIIbIIIa activation. P2Y12 receptor inhibitors clinically in use such as clopidogrel are postulated to decrease platelet aggregation through partial inhibition of PAR1 signaling. Our data, however, indicate that at high local concentrations of thrombin, it is the signaling through PAR4 rather than PAR1 that may be regulated through purinergic feedback. Thus, our data identify an intra-platelet mechanism that may function as a future site for therapeutic intervention.     

Biphasic effect on platelet aggregation by phospholipase a purified from Vipera russellii snake venom

A basic phospholipase A was isolated from Vipera russellii snake venom. It induced a biphasic effect on washed rabbit platelets suspended in Tyrode's solution. The first phase was a reversible aggregation which was dependent on stirring and extracellular calcium. The second phase was an inhibitory effect on platelet aggregation, occurring 5 min after the addition of the venom phospholipase A without stirring or after a recovery from the reversible aggregation. The aggregating phase could be inhibited by indomethacin, tetracaine, papaverine, creatine phosphate/creatine phosphokinase, mepacrine, verapamil, sodium nitroprusside, prostaglandin E1 or bovine serum albumin. The venom phospholipase A released free fatty acids from synthetic phosphatidylcholine and intact platelets. p-Bromophenacyl bromide-modified venom phospholipase A lost its phospholipase A enzymatic and platelet-aggregating activities, but protected platelets from the aggregation induced by the native enzyme. The second phase of the venom phospholipase A action showed a different degree of inhibition on platelet aggregation induced by some activators in following order: arachidonic acid greater than collagen greater than thrombin greater than ionophore A23187. The longer the incubation time or the higher the concentration of the venom phospholipase A, the more pronounced was the inhibitory effect. The venom phospholipase A did not affect the thrombin-induced release reaction which was caused by intracellular Ca2+ mobilization in the presence of EDTA, but inhibited collagen-induced release reaction which was caused by Ca2+ influx from extracellular medium. The inhibitory effect of the venom phospholipase A and also lysophosphatidylcholine or arachidonic acid could be antagonized or reversed by bovine serum albumin. It was concluded that the first stimulatory phase of the venom phospholipase A action might be due to arachidonate liberation from platelet membrane. The second phase of inhibition of platelet aggregation and the release of ATP might be due to the inhibitory action of the split products produced by this venom phospholipase A.     

Costimulation of Gi- and G12/G13-mediated signaling pathways induces integrin alpha IIbbeta 3 activation in platelets

Platelet activation is a complex process induced by a variety of stimuli, which act in concert to ensure the rapid formation of a platelet plug at places of vascular injury. We show here that fibrillar collagen, which initiates platelet activation at the damaged vessel wall, activates only a small fraction of platelets in suspension directly, whereas the majority of platelets becomes activated by mediators released from collagen-activated platelets. In Galpha(q)-deficient platelets that do not respond with activation of integrin alpha(IIb)beta(3) to a variety of mediators like thromboxane A2 (TXA2), thrombin, or ADP, collagen at high concentrations was able to induce aggregation, an effect that could be blocked by antagonists of the TXA2 or P2Y12 receptors. The activation of TXA2 or P2Y12 receptors alone, which in Galpha(q)-deficient platelets couple to G12/G13 and Gi, respectively, did not induce platelet integrin activation or aggregation. However, concomitant activation of both receptors resulted in irreversible integrin alpha(IIb)beta3-mediated aggregation of Galpha(q)-deficient platelets. Thus, the activation of G12/G13- and Gi-mediated signaling pathways is sufficient to induce integrin alpha(IIb)beta3 activation. Although G(q)-mediated signaling plays an important role in platelet activation, it is not strictly required for the activation of integrin alpha(IIb)beta3. This indicates that the efficient induction of platelet aggregation through G-protein-coupled receptors is an integrated response mediated by various converging G-protein-mediated signaling pathways involving G(q) and G(i) as well as G12/G13.     

The platelet protein phosphorylation induced by a monoclonal antibody against human platelets (TP82)

In human platelets, a monoclonal anti-human platelet antibody (TP82) induced platelet aggregation and release of granules (i.e., serotonin, platelet factor 4, N-acetyl-beta-D-glucosaminidase). The release reaction occurred even in the absence of aggregation and was preceded by not only the protein phosphorylation, but the transient formation of endogenous diacylglycerol (DG). These results suggest that polyphosphoinositide breakdown plays an essential role in antibody-induced release of platelet granules.     

Calcitonin gene-related peptide-mediated antihypertensive and anti-platelet effects by rutaecarpine in spontaneously hypertensive rats

We have previously reported that Chinese traditional medicine rutaecarpine (Rut) produced a sustained hypotensive effect in phenol-induced and two-kidney, one-clip hypertensive rats. The aims of this study are to determine whether Rut could exert antihypertensive and anti-platelet effects in spontaneously hypertensive rats (SHR) and the underlying mechanisms. In vivo, SHR were given Rut and the blood pressure was monitored. Blood was collected for the measurements of calcitonin gene-related peptide (CGRP), tissue factor (TF) concentration and activity, and platelet aggregation, and the dorsal root ganglia were saved for examining CGRP expression. In vitro, the effects of Rut and CGRP on platelet aggregation were measured, and the effect of CGRP on platelet-derived TF release was also determined. Rut exerted a sustained hypotensive effect in SHR concomitantly with the increased synthesis and release of CGRP. The treatment of Rut also showed an inhibitory effect on platelet aggregation concomitantly with the decreased TF activity and TF antigen level in plasma. Study in vitro showed an inhibitory effect of Rut on platelet aggregation in the presence of thoracic aorta, which was abolished by capsazepine or CGRP(8-37), an antagonist of vanilloid receptor or CGRP receptor. Exogenous CGRP was able to inhibit both platelet aggregation and the release of platelet-derived TF, which were abolished by CGRP(8-37). The results suggest that Rut exerts both antihypertensive and anti-platelet effects through stimulating the synthesis and release of CGRP in SHR, and CGRP-mediated anti-platelet effect is related to inhibiting the release of platelet-derived TF.     

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.     

Cathepsin G is a strong platelet agonist released by neutrophils.

The present studies were undertaken to characterize a serine protease released by N-formyl-L-Met-L-Leu-L-Phe (fMet-Leu-Phe)-stimulated neutrophils that rapidly induces platelet calcium mobilization, secretion and aggregation. The biological activity associated with this protease was unaffected by leupeptin, was only weakly diminished by N-p-tosyl-L-Lys-chloromethane, but was strongly inhibited by alpha 1-antitrypsin, soyabean trypsin inhibitor, N-tosyl-L-Phe-chloromethane and benzoyloxycarbonyl-Gly-Leu-Phe-chloromethane (Z-Gly-Leu-PheCH2Cl). These observations indicated that the biological activity of neutrophil supernatants could be attributed to a chymotrypsin-like enzyme such as cathepsin G. Furthermore, platelet aggregation and 5-hydroxytryptamine release induced by cell-free supernatants from fMet-Leu-Phe-stimulated neutrophils were found to be blocked by antiserum to cathepsin G in a concentration-dependent manner but were unaffected by antiserum to elastase. The biological activity present in neutrophil supernatants co-purified with enzymic activity for cathepsin G during sequential Aprotinin-Sepharose affinity chromatography and carboxymethyl-Sephadex chromatography. SDS/polyacrylamide-gel electrophoresis of the reduced, purified protein, demonstrated three polypeptides with apparent Mr values of 31,500, 29,000 and 28,000 and four polypeptides were resolved on acid-gel electrophoresis. Purified cathepsin G from neutrophils cross-reacted with anti-(cathepsin G) serum in a double immunodiffusion assay and elicited platelet calcium mobilization, 5-hydroxytryptamine secretion and aggregation. Calcium mobilization and secretion induced by low concentrations of cathepsin G were partially dependent on arachidonic acid metabolites and ADP, while stimulation by higher enzyme concentrations was independent of amplification pathways, indicating that cathepsin G is a strong platelet agonist. These results suggest that pathological processes which stimulate neutrophils and release cathepsin G can in turn result in the recruitment and activation of platelets.     

血小板膜P2Y12受体基因T744C多态性对外伤性脑梗死(PTCI)患者血小板聚合率及疗效的影响

目的:外伤性脑梗死(posttraumatic cerebral infarction,PTCI)是颅脑损伤的常见并发症之一,P2Y12受体介导血小板聚集是血栓形成的重要通路,与血小板聚集形成密切相关。本研究探讨外伤性脑梗死发生发展与血小板膜P2Y12受体基因T744C基因多态性的关系。方法:用聚合酶链反应(PCR)和限制性酶切片段长度多态性(RFLP)技术对186例外伤性脑梗死患者P2Y12受体基因T744C多态性进行分析。分别在治疗前和治疗后对所有颅脑外伤患者的伤情GCS评分,并按基因型分组对照分析结果。结果:血小板膜T744C血小板膜T744C基因型基因频率分别为TT基因型59.14%、TC型32.26%、CC型8.60%,T等位基因75.27%、C等位基因24.73%;其中TT基因型对奥扎格雷反应较敏感,GCS评分预后好;而CC型对奥扎格雷反应性低,预后差。结论:T744C基因多态性中CC基因型可能导致外伤性脑梗死临床及预后存在明显的个体差异,与其对抗血小板药物抵抗有关。T744C的C等位基因可能是脑梗死的遗传危险因素,开展相关遗传学风险研究,对于进一步缓解脑梗症状、改善预后具有重要意义。     

The effects of short-term training on platelet functions and total antioxidant capacity in rats

The purpose of the present study was to investigate the effect of short-term endurance training on plasma total antioxidant status (TAS) and on in vitro platelet aggregation and ATP release. Blood samples were collected from the abdominal aorta of rats following short-term treadmill exercise (25 m/min, 0 % grade, 30 min) for three consecutive days, as well as in non-exercised control group. Platelet aggregation and platelet ATP release were evaluated by impedance and bioluminescence techniques, respectively. Plasma TAS was measured spectrophotometrically. Plasma TAS was higher and ADP-induced platelet ATP release was lower in the short-term training group with respect to the control group (p<0.001). Significant negative correlation (r = -0.56, p<0.05) was found between plasma TAS and ADP-induced platelet ATP release. Neither ADP- and collagen-induced maximum aggregation rate nor collagen-induced platelet ATP release were significantly different between the groups. According to these results, short-term training caused an alteration in platelet functions limited to the secretion response, which may be related to the oxidant/antioxidant balance changes favoring the antioxidants. The improved plasma total antioxidant capacity was possibly sufficient to prevent exercise-induced oxidative damage, and the adaptive response of platelets might be associated with enhanced antioxidant status.