Specific activation,signalling and secretion profiles of human platelets following PAR-1 and PAR-4 stimulation

Blood platelets play a central haemostatic function; however, they also play a role in inflammation and are capable of secreting various cytokines, chemokines and related products. The purpose of this study was to identify subtle variations in platelet physiology using proteomics. We compared the levels of membrane proteins (n?=?3), α and δ granule proteins (n?=?18), and signalling proteins (n?=?30) from unstimulated platelets with those of protease-activated receptor (PAR)-1- and PAR-4-stimulated platelets (n?=?10). The vast majority of these proteins responded similarly to PAR-1 or PAR-4 engagement. However, differences were observed within membrane CD40L expressed, and α granule GRO-α and MDC secreted proteins.     

Proteinase-activated receptors 1 and 4 counter-regulate endostatin and VEGF release from human platelets

The roles of proteinase-activated receptors (PARs) in platelet functions other than aggregation are not well understood. Among these is the release of factors that regulate the process of angiogenesis, such as endostatin and VEGF, which, respectively, inhibit and promote angiogenesis. PAR1 and PAR4 are expressed on the surface of human platelets and can be activated by thrombin. In the present study, we have attempted to determine the roles of PAR1 and PAR4 in regulating release of endostatin and VEGF from human platelets. Aggregation and endostatin release could be elicited by a specific PAR4 agonist (AYPGKF-NH(2)). The PAR4 agonist concentration dependently suppressed VEGF release. A selective PAR1 agonist (TFLLR-NH(2)) induced platelet aggregation and VEGF release but suppressed endostatin release. Thrombin did not affect endostatin or VEGF release. However, in the presence of a selective PAR1 antagonist (SCH79797), thrombin stimulated endostatin release and suppressed VEGF release. Conversely, in the presence of a selective PAR4 antagonist (transcinnamoyl-YPGKF-NH(2)), thrombin stimulated VEGF release. In vivo, treatment of rats with established gastric ulcers with a PAR1 antagonist each day for 1 wk resulted in a significant retardation of healing. We conclude that PAR1 and PAR4 counter-regulate the release of endostatin and VEGF from platelets. These protease-activated receptors could therefore play a crucial role in regulating angiogenesis and in turn could regulate the processes of wound healing and tumor growth.     

Actin polymerisation regulates thrombin-evoked Ca2+ signalling after activation of PAR-4 but not PAR-1 in human platelets

The role of actin polymerisation in regulating thrombin-evoked Ca²⁺ signalling was investigated in human platelets. We have previously reported that cytochalasin D (Cyt D) inhibits thapsigargin-evoked store-operated Ca²⁺ entry (SOCE), which is believed to contribute a major component of thrombin-evoked Ca²⁺ entry in platelets. In contrast, Cyt D increased thrombin-evoked Ca²⁺ entry to 147.5?±?9.2% and Sr²⁺ entry to 134.2?±?6.4% of control. Similar results were obtained with latrunculin A. This potentiation was not affected if protein kinase C was inhibited using Ro-31-8220, suggesting that it did not involve PKC-dependent non-capacitative Ca²⁺ entry. Ca²⁺ entry evoked by the PAR-4 agonist, AYPGKF, was increased to 133.7?±?12.8% of control by Cyt D, whereas Ca²⁺ signalling evoked by the PAR-1 agonist, SFLLRN, was unaffected. The PAR-4 antagonist, tcY-NH₂, abolished the effect of Cyt D on thrombin-evoked Ca²⁺ entry. Biotinylation of cell-surface proteins showed that PAR-4 was internalised after stimulation by thrombin. Cyt D reduced this internalisation. These data suggest that Cyt D prevents the internalisation of PAR-4, which may lead to prolonged signalling from this receptor. This may mask a direct effect of Cyt D on the activation of SOCE after the activation of PAR-4.     

Dual effects of heparin on VEGF binding to VEGF receptor-1 and transduction of biological responses

Vascular endothelial growth factor (VEGF) regulates blood vessel formation by binding to the receptor tyrosine kinases VEGF receptor-1 (Flt-1) or VEGF receptor-2 (KDR) and to the structurally unrelated neuropilins. As exon 7-containing isoforms of VEGF bind to heparin, angiogenesis may be modulated by heparin/heparan sulfate. We analyzed the effect of heparin on VEGF₁₆₅-binding and activation of VEGF receptor-1 in porcine aortic endothelial cells, which lack expression of VEGF receptor-2 and neuropilins. Heparin decreased binding of ¹²⁵I-VEGF to 50% at 5 μg/ml and cross-linking of ¹²⁵I-VEGF to VEGF receptor-1 on intact cells was similarly decreased. Schatchard analyses showed that the affinity for binding of ¹²⁵I-VEGF to VEGF receptor-1 was decreased in the presence of heparin. In contrast, VEGF receptor-1 kinase activity was elevated when cells were treated simultaneously with VEGF and heparin. In accordance, VEGF-induced tyrosine phosphorylation of phospholipase Cγ (PLCγ) and DNA synthesis were augmented by heparin. However, basal PLCγ tyrosine phosphorylation and DNA synthesis levels were to some extent increased by incubation of cells with heparin alone. We conclude that although heparin decreases binding of VEGF to VEGF receptor-1, the remaining binding results in more efficient kinase activation. Taken together, there is no loss of VEGF/VEGF receptor-1 function in the presence of heparin. This revised version was published online in June 2006 with corrections to the Cover Date.     

Protease-activated receptor 4 activity promotes platelet granule release and platelet-leukocyte interactions

Human platelets express two protease-activated receptors (PARs), PAR1 (F2R) and PAR4 (F2RL3), which are activated by a number of serine proteases that are generated during pathological events and cause platelet activation. Recent interest has focused on PAR4 as a therapeutic target, given PAR4 seems to promote experimental thrombosis and procoagulant microparticle formation, without a broadly apparent role in hemostasis. However, it is not yet known whether PAR4 activity plays a role in platelet-leukocyte interactions, which are thought to contribute to both thrombosis and acute or chronic thrombo-inflammatory processes. We sought to determine whether PAR4 activity contributes to granule secretion from activated platelets and platelet-leukocyte interactions. We performed in vitro and ex vivo studies of platelet granule release and platelet-leukocyte interactions in the presence of PAR4 agonists including PAR4 activating peptide, thrombin, cathepsin G, and plasmin in combination with small-molecule PAR4 antagonists. Activation of human platelets with thrombin, cathepsin G, or plasmin potentiated platelet dense granule secretion that was specifically impaired by PAR4 inhibitors. Platelet-leukocyte interactions and platelet P-selectin exposure the following stimulation with PAR4 agonists were also impaired by activated PAR4 inhibition in either a purified system or in whole blood. These results indicate PAR4-specific promotion of platelet granule release and platelet-leukocyte aggregate formation and suggest that pharmacological control of PAR4 activity could potentially attenuate platelet granule release or platelet-leukocyte interaction-mediated pathological processes.     

Role of Galphaq and phospholipase C-beta2 in human platelets activation by thrombin receptors PAR1 and PAR4: studies in human platelets deficient in Galphaq and phospholipase C-beta2

Thrombin responses in human platelets are mediated by the protease-activated receptors (PAR), PAR1 and PAR4. The signalling pathways mediating PAR activation have not been fully delineated for human platelets. We assessed cytoplasmic Ca2+ mobilization in response to activation with thrombin and PAR1 (SFLLRN) and PAR4 (GYPGKF) peptides in two patients whose platelets were deficient in two major signalling proteins, Galphaq or phospholipase (PLC)-beta2. In normal platelets, thrombin induced a biphasic Ca2+ response with a rapid rise to a peak followed by a sustained elevation in Ca2+. The peak Ca2+ rise was impaired in both patients at lower thrombin concentrations. At higher concentrations, it was decreased in PLC-beta2-deficient platelets; the sustained Ca2+ elevation observed in normal and Galphaq-deficient platelets was reduced in PLC-beta2-deficient platelets. The response to SFLLRN was decreased in both patients at lower concentrations. The peak Ca2+ in response to GYPGKF was reduced in both patients; the sustained Ca2+ increase was markedly decreased in PLC-beta2-deficient platelets. These studies provide evidence that, in human platelets, both Galphaq and PLC-beta2 play a major role in responses to PAR1 and PAR4 activation, and that PLC-beta2 is required for the sustained Ca2+ rise upon thrombin activation.     

Effect of endotoxin on arachidonic acid release and thromboxane B2 production by human platelets

Plasma thromboxane A₂, which is elevated during endotoxemia, has previously been shown to be a major factor contributing to the mortality and morbidity that occurs in endotoxin shock in the experimental animal. Using a minimal dose of Escherichia coli endotoxin (1 μg/ml), we have demonstrated that the preincubation of human platelets with endotoxin induces changes in platelet arachidonic acid release and the subsequent conversion of the released arachidonic acid to thromboxane B₂, the stable end product of thromboxane A₂. In paired experiments, in the presence of endotoxin, the addition of the aggregating agent thrombin (0.5 U/ml) caused human platelets to release 29.1 ± 3.4% of ¹⁴C-arachidonic acid from prelabeled platelet phospholipids. This value was significantly elevated (P < 0.02) when compared with the release of ¹⁴C-arachidonic acid from platelets in the absence of endotoxin (21.9 ± 3.6%). Similarly, comparison of the results of the conversion of the released arachidonic acid to platelet thromboxane B₂ (TxB₂) revealed that TxB₂ production was significantly increased (P < 0.01) when human platelets were preincubated with endotoxin prior to the addition of thrombin (6.1 ± 0.6%) when compared with TxB₂ formation observed in the absence of endotoxin (3.4 ± 0.5%). The absolute amount of released arachidonic acid that was converted to TxB₂ in the presence of endotoxin (1.8 ± 0.3%) was also significantly higher (P < 0.01) than the value observed in its absence (0.8 ± 0.2%). This study suggests that one of the tissue sources of the proaggregatory vasoconstrictor thromboxane A₂ during endotoxemia is the platelet.     

Heme oxygenase-1 promotes neovascularization in ischemic heart by coinduction of VEGF and SDF-1

Heme oxygenase-1 (HO-1) is a stress-inducible enzyme with multiple protective functions in cardiovascular systems. Studies have shown that the timely cardiac HO-1 overexpression at acute phase of ischemic infarction (MI) provides protection via its anti-apoptotic and anti-inflammatory effects. Here we demonstrate that a delayed HO-1 transduction mediated by a recombinant adeno-associated virus in ischemic hearts of mice with permanent coronary artery ligation significantly attenuated left ventricular fibrosis and cardiac dysfunctions examined at 4 weeks post MI. HO-1-mediated protection was correlated with enhanced vascularization in the ischemic myocardium. HO-1 gene transfer resulted in a notable increase in the number of c-kit⁺- stem cells recruited to the infarcted area at 10 days after ligation. HO-1-mediated stem cell recruitment was also demonstrated in the heart of non-ischemic mice receiving intravenous infusion of green fluorescent protein-bearing bone marrow stem cells. Additional experiments revealed that vascular endothelial growth factor (VEGF) and stromal cell-derived factor-1 (SDF-1) were highly induced in HO-1 transduced myocardium. Mononuclear cell infiltration was evident and colocalized with angiogenic factors in the same region. Flow cytometry analysis of the mononuclear cells isolated from HO-1-transduced left ventricles revealed that over 50% of cells expressed CD34, a marker of hematopoietic stem cells and endothelial progenitor cells. VEGF and SDF-1 blockade by neutralizing antibodies significantly attenuated HO-1-mediated neovascularization and protection in infarcted mice. These data suggest that cardiac HO-1 gene transfer post MI provides protection at least in part by promoting neovascularization through inducing angiogenic factors and the recruitment of circulating progenitor/stem cells.     

Molecular requirements involving the human platelet protease-activated receptor-4 mechanism of activation by peptide analogues of its tethered-ligand

Thrombin is the most potent agonist of human platelets and its effects are primarily mediated through the protease-activated receptors (PARs)-1 and -4. Although PAR-1 has higher affinity for thrombin than PAR-4, both receptors contribute to thrombin-mediated actions on platelets. Recently, a potent and selective PAR-1 antagonist (vorapaxar) was approved for clinical use in selected patients. In contrast, despite the fact that several PAR-4 antagonists have been developed, few of them have been tested in clinical trials.

The aim of the present study was to elucidate the molecular requirements involving the PAR-4 mechanism of activation by peptide analogues of its tethered-ligand.

Eight synthetic PAR-4 tethered-ligand peptide analogues were synthesized and studied for their agonistic/antagonistic potency and selectivity toward human washed platelet aggregation, using light transmittance aggregometry. In addition, in silico studies were conducted to describe the receptor–peptide interactions that are developed following PAR-4 exposure to the above analogues. To provide a first structure-activity relationship rationale on the bioactivity profiles recorded for the studied analogues, molecular docking was applied in a homology model of PAR-4, derived using the crystal structure of PAR-1.

The following peptide analogues were synthesized: AYPGKF-NH₂ (1), GYPGKF-NH₂ (2), Ac-AYPGKF-NH₂ (3), trans-cinnamoyl-AYPGKF-NH₂ (4), YPGKF-NH₂ (5), Ac-YPGKF-NH₂ (6), trans-cinnamoyl-YPGKF-NH₂ (7), and caffeoyl-YPGKF-NH₂ (8). Peptide (1) is a selective PAR-4 agonist inducing platelet aggregation with an IC₅₀ value of 26.2 μM. Substitution of Ala-1 with Gly-1 resulted in peptide (2), which significantly reduces the agonistic potency of peptide (1) by 25-fold. Importantly, substitution of Ala-1 with trans-cinnamoyl-1 resulted in peptide (7), which completely abolishes the agonistic activity of peptide (1) and renders it with a potent antagonistic activity toward peptide (1)-induced platelet aggregation. All other peptides tested were inactive. Tyr-2, residue, along with its neighboring environment was a key determinant in the PAR-4 recognition mode. When the neighboring residues to Tyr-2 provided an optimum spatial ability for the ligand to enter into the binding site of the transmembrane receptor, a biological response was propagated. These results were compared with the predicted binding poses of small molecule antagonists of PAR-4, denoted as YD-3, ML-354, and BMS-986120. π–π stacking interaction with Tyr-183 appears to be critical and common for both small molecules antagonists and the peptide trans-cinnamoyl-YPGKF-NH₂.

Conclusively, the lipophilicity, size, and aromatic nature of the residue preceding Tyr-2 are determining factors on whether a human platelet PAR-4 tethered-ligand peptide analogue will exert an agonistic or antagonistic activity.     

RNA interference-directed silencing of VPAC1 receptor inhibits VIP effects on both EGFR and HER2 transactivation and VEGF secretion in human breast cancer cells

We used small-interference RNA (siRNA) to explore the mechanisms of some vasoactive intestinal peptide (VIP) actions on human breast cancer cells. Transfection of estrogen-dependent (T47D) and estrogen-independent (MDA-MB-468) breast cancer cells with VPAC₁-receptor siRNA completely abolished VIP stimulatory effect on secretion of the main angiogenic factor, vascular endothelial growth factor (VEGF), and transactivation of epidermal growth factor receptor (EGFR or HER1) and HER2, two members of HER family of tyrosine-kinase receptors. The silencing procedure suggested the involvement of EGFR and HER2 transactivation in VIP-stimulated VEGF secretion. It was further supported by blocking tyrosine kinase activity by the selective HER inhibitors AG-1478 (EGFR) and AG-825 (HER2). Results give value to the specific signaling of VIP through VPAC₁ receptor in human breast cancer cells and support the potential use of VPAC₁-receptor antagonists in combined targeted therapies for breast cancer. Molecular therapies involving RNA interference of VPAC₁-receptor expression could also be considered.     

Thrombin-induced lysosomal exocytosis in human platelets is dependent on secondary activation by ADP and regulated by endothelial-derived substances

Exocytosis of lysosomal contents from platelets has been speculated to participate in clearance of thrombi and vessel wall remodelling. The mechanisms that regulate lysosomal exocytosis in platelets are, however, still unclear. The aim of this study was to identify the pathways underlying platelet lysosomal secretion and elucidate how this process is controlled by platelet inhibitors. We found that high concentrations of thrombin induced partial lysosomal exocytosis as assessed by analysis of the activity of released N-acetyl-β-glucosaminidase (NAG) and by identifying the fraction of platelets exposing the lysosomal-associated membrane protein (LAMP)-1 on the cell surface by flow cytometry. Stimulation of thrombin receptors PAR1 or PAR4 with specific peptides was equally effective in inducing LAMP-1 surface expression. Notably, lysosomal exocytosis in response to thrombin was significantly reduced if the secondary activation by ADP was inhibited by the P2Y₁₂ antagonist cangrelor, while inhibition of thromboxane A₂ formation by treatment with acetylsalicylic acid was of minor importance in this regard. Moreover, the NO-releasing drug S-nitroso-N-acetyl penicillamine (SNAP) or the cyclic AMP-elevating eicosanoid prostaglandin I₂ (PGI₂) significantly suppressed lysosomal exocytosis. We conclude that platelet inhibitors that mimic functional endothelium such as PGI₂ or NO efficiently counteract lysosomal exocytosis. Furthermore, we suggest that secondary release of ADP and concomitant signaling via PAR1/4- and P2Y₁₂ receptors is important for efficient platelet lysosomal exocytosis by thrombin.     

Anti-tumoral effect of active immunotherapy in C57BL/6 mice using a recombinant human VEGF protein as antigen and three chemically unrelated adjuvants

Following the clinical success of Bevacizumab, a humanized monoclonal antibody that affects the interaction between vascular endothelial growth factor (VEGF) and its receptors, blocking tumor-induced angiogenesis has become one of the most important targets for the development of new cancer therapeutic drugs and procedures. Among the latter, therapeutic vaccination using VEGF as antigen presents itself as very attractive, with the potential of generating not only a growth factor blocking antibody response but also a cellular response against tumor cells and stromal elements, which appear to be a major source of tumor VEGF. In this paper, we report the development of a protein vaccine candidate, based on a human modified VEGF antigen that is expressed at high levels in E. coli. With respect to controls, immunization experiments in C57BL/6 mice using weekly doses of this antigen and three adjuvants of different chemical natures show that time for tumor development after subcutaneous injection of Melanoma B16-F10 cells increases, tumors that develop grow slower, and overall animal survival is higher. Immunization also prevents tumor development in some mice, making them resistant to second tumor challenges. Vaccination of mice with the human modified VEGF recombinant antigen produces antibodies against the human antigen and the homologous mouse VEGF molecule. We also show that sera from immunized mice block human VEGF-induced HUVEC proliferation. Finally, a possible contribution of T cell cytotoxicity to the overall anti-tumor effect is suggested from the results of vaccination experiments where CD8+ lymphocytes were impaired using neutralizing rat antibodies.     

重组人血管内皮抑制素联合顺铂、IL-2胸腔内注入治疗癌性胸水的临床观察

目的探讨胸腔内注入抗肿瘤新药重组人血管内皮抑制素（恩度）联合顺铂、IL-2治疗癌性胸水的有效性和安全性。方法经病理学和细胞学检查确诊癌性胸水患者73例,随机分成治疗组（40例）和对照组（33例）。其中肺腺癌25例,肺鳞癌18例,肺小细胞癌13例,乳腺癌10例,食道癌7例。两组间各项基线指标无显著差异。治疗组给予重组人血管内皮抑制素60mg（4支）,顺铂40mg,IL-2100万U胸腔内注入。对照组给予顺铂40mg,IL-2100万U胸腔内注入。两组胸腔治疗均每周2次,治疗组注药次数最多4次,最少2次。对照组注药次数最多8次,最少6次。按照WHO胸水评价标准评价近期疗效,参照Karnofsky评分（KPS）变化评价生活质量（QOL）。按照NCICTC3．0版标准评价毒性反应。治疗前后检测胸水VEGF的水平。结果治疗组40例患者,接受恩度治疗的周期数2—4,中位周期数为3,共完成的周期数103。获得CR15例,PR13例,SD5例,客观有效率70％,QOL改善率85％（33／40）,对照组33例患者,接受治疗的周期数6～8,中位周期数7,共完成的周期数208。获得CR例5例,PR13例,SD4例,客观有效率54．5％,QOL改善率66．7％,均较治疗组低。治疗组患者胸水VEGF基线水平40例患者有4例正常（10％）,36例（90％）患者胸水VEGF水平在治疗前有不同程度的升高,治疗后均有下降。而对照组33例胸水VEGF水平升高患者无明显下降。3～4级药物不良反应主要伴随化疗药物,包括白细胞减少、恶心、呕吐、皮疹、腹泻、没有心血管的反应。结论胸腔内注入重组人血管内皮抑制素、顺铂、IL-2治疗癌性胸水有一定的疗效,且毒副作用少,对恶性肿瘤晚期患者是一种较好的治疗选择,可以改善生存质量,延长生存期。值得临床进一步推广应用。     

Toll-like receptor 4 ligand can differentially modulate the release of cytokines by human platelets

Blood platelets link the processes of haemostasis and inflammation. This study examined the immunomodulatory factors released by platelets after Toll-Like Receptor 4 (TLR4) engagement on their surfaces. Monoclonal anti-human FcγRII Ab (IV.3)-treated human platelets were cultured with TLR4 ligands in the presence or absence of blocking monoclonal antibody to human TLR4. The release of sCD62p, epidermal growth factor (EGF), transforming growth factor β (TGFβ), interleukin (IL)-8, platelet activating factor 4 (PAF4), platelet-derived growth factor, α, β polypeptide (PDGF-AB), Angiogenin, RANTES (regulated upon activation, normal T-cell expressed, and presumably secreted) and sCD40L were measured by specific enzyme-linked immunosorbent assay. TLR4 ligand [ Escherichia coli lipopolysaccharide (LPS)] bound platelet TLR4, which differentially modulates the release of cytokines by platelets. It was noted that (i) sCD62p, IL-8, EGF and TGFβ release were each independent of platelet activation after TLR4 engagement; (ii) RANTES, Angiogenin and PDGF-AB concentration were weaker in platelet supernatant after TLR4 engagement; (iii) sCD40L and PAF4 are present in large concentration in the releaseate of platelets stimulated by TLR4 ligand. The effects of LPS from E. coli on the modulation of secretory factors were attenuated by preincubation of platelets with an anti-TLR4 monoclonal antibody, consistent with the immunomodulation being specifically mediated by the TLR4 receptor. We propose that platelets adapt the subsequent responses, with polarized cytokine secretion, after TLR4 involvement.     

Storage of platelets in additive solutions: the effects of magnesium and potassium on the release of RANTES, beta-thromboglobulin, platelet factor 4 and interleukin-7, during storage

BACKGROUND AND OBJECTIVES: Several studies have suggested that the accumulation of cytokines during storage of platelet concentrates may mediate non-haemolytic transfusion reactions. Prestorage leucodepletion can prevent the release of cytokines from white blood cells during storage, but not the release of platelet-derived cytokines. Therefore, we investigated whether the addition of magnesium and potassium to platelets stored in a platelet additive solution (PAS) would affect the generation of cytokines during platelet storage. MATERIALS AND METHODS: Platelets were prepared from buffy coats using different suspension media: plasma; 70% PAS-III + 30% plasma; 70% PAS-III supplemented with magnesium and potassium +30% plasma; and 80% PAS-III supplemented with magnesium and potassium +20% plasma. The levels of certain cytokines--regulated on activation, normal, T-cell expressed, and secreted (RANTES), beta-thromboglobulin (beta-TG), platelet factor 4 (PF4) and interleukin-7 (IL-7)--were measured by enzyme-linked immunosorbent assay (ELISA) on days 1, 5 and 7. RESULTS: The concentrations of RANTES, beta-TG, PF4 and IL-7 increased, during storage, in all units. The increase was significantly greater in units stored in 70% PAS-III +30% plasma than in the other three suspension media. The storage of platelets in 70% PAS-III supplemented with magnesium and potassium +30% plasma significantly reduced the concentrations of platelet derived-cytokines during storage, as compared to platelets stored in 70% PAS-III + 30% plasma alone. CONCLUSIONS: The concentrations of platelet-derived cytokines increased, to a significantly greater extent, when platelets were stored in PAS-III than in plasma. However, when magnesium and potassium were added to PAS-III, the concentrations of platelet-derived cytokines obtained during storage were about the same as those produced by platelets stored in plasma.     

Loss of phosphotyrosine phosphatase activity and changes in the tyrosine phosphorylation state of proteins after storage of sheep platelets in plasma or Seto solution at 4 degrees C

BACKGROUND AND OBJECTIVES: During platelet storage an array of deleterious changes occur, through mechanisms not fully understood, which impair platelet haemostasis. Transfused platelets should maintain the integrated networks of signalling pathways that regulate platelet activation and functionality. We hypothesized that protein phosphorylation and dephosphorylation, which play a fundamental role in these pathways, might be affected by platelet storage. We therefore investigated whether the activity of phosphotyrosine phosphatase (PTP), which belongs to an oxidant-susceptible group of enzymes involved in the platelet signal-transduction pathways that ensure platelet functionality, is affected by platelet storage. MATERIALS AND METHODS: Using sheep platelet species as a model system, we conducted serial studies on the membranes of platelets and microparticles shed during platelet storage, in their own plasma or in a synthetic medium called Seto, for up to 5 days at 4 degrees C. RESULTS: A progressive decrease in both total and specific membrane-associated PTP activities from whole platelets (but not from microparticles) located within each platelet storage bag was observed from day 1 onwards in both types of storage media. These decreases could be partly avoided by the addition of vitamin E. Additionally, the observed decrease in PTP activity was accompanied with increases in the tyrosine phosphorylation of proteins from whole platelets or crude platelet membranes, the tyrosine phosphorylation state of proteins from microparticles remaining basically unchanged. CONCLUSION: Our findings suggest that alterations of at least the tyrosine phosphorylation balance might be one of the reasons for the decrease in the haemostatic function of stored platelets.