Kidney graft outcome using an anti-Xa therapeutic strategy in an experimental model of severe ischaemia-reperfusion injury.

BACKGROUND: Deceased after cardiac death donors represent an important source of organs to reduce organ shortage in transplantation. However, these organs are subjected to more ischaemia-reperfusion injury (IRI). Reducing IRI by targeting coagulation is studied here in an experimental model. METHODS: The effect of an anti-Xa compound (fondaparinux) was evaluated using an autotransplanted kidney model in pigs. Kidneys were clamped for 60 min (warm ischaemia) and then preserved for 24 h at 4 °C in University of Wisconsin solution (UW). The anti-Xa compound was injected intravenously before warm ischaemia and used during cold storage, and its effects were compared with those of intravenous injection of unfractionated heparin (UFH) before warm ischaemia and use during cold storage, or use of UW alone during cold storage. RESULTS: At 3 months after transplantation, anti-Xa treatment improved recovery of renal function and chronic serum creatinine levels compared with UW and UFH (mean(s.e.m.) 89(4), 250(4) and 217(8) µmol/l respectively). The anti-Xa treatment also reduced fibrosis, and decreased tissue expression of markers of the epithelial-mesenchymal transition compared with UW and UFH. Cleaved protease-activated receptor 2 was overexpressed in the UW group compared with the anti-Xa and UFH groups. Leucocyte infiltrates were decreased in the anti-Xa group compared with the UW and UFH groups. Macrophage invasion was also decreased by anticoagulation treatment. CONCLUSION: Peritransplant anticoagulation therapy was beneficial to graft outcome, in both the acute and chronic phases. Moreover, specific inhibition of coagulation Xa protease further protected kidney grafts, with better recovery and decreased expression of chronic lesion markers. Surgical relevance The increasing use of marginal donors highlights the importance of organ quality in transplantation. Renal ischaemia-reperfusion injury (IRI), which includes a deleterious activation of coagulation, plays a central role in determining graft quality and outcome. Using an established porcine renal autotransplantation preclinical model with high clinical relevance, the benefits of anticoagulation therapy using an antifactor Xa molecule were evaluated. Peritransplantion anticoagulation treatment, specifically with an anti-Xa compound, protected marginal kidney grafts, improving functional recovery and reducing chronic lesions. This study demonstrates the benefits of anticoagulation therapy at the time of organ collection, particularly for marginal organs, encountered in cases of extended criteria and deceased after circulatory death donors. This anticoagulation strategy could be an important addition to current donor and organ management protocols in order to limit IRI and improve outcome.

Anti-thrombin therapy during warm ischemia and cold preservation prevents chronic kidney graft fibrosis in a DCD model.

Ischemia reperfusion injury (IRI) is pivotal for renal fibrosis development via peritubular capillaries injury. Coagulation represents a key mechanism involved in this process. Melagatran (M), a thrombin inhibitor, was evaluated in an autotransplanted kidney model, using Large White pigs. To mimic deceased after cardiac death donor conditions, kidneys underwent warm ischemia (WI) for 60 min before cold preservation for 24 h in University of Wisconsin solution. Treatment with M before WI and/or in the preservation solution drastically improved survival at 3 months, reduced renal dysfunction related to a critical reduction in interstitial fibrosis, measured by Sirius Red staining. Tissue analysis revealed reduced expression of transforming growth factor-beta (TGF-beta) and activation level of its effectors phospho-Smad3, Smad4 and connective tissue growth factor (CTGF) after M treatment. Fibrinolysis activation was also observed, evidenced by downregulation of PAI-1 protein and gene expression. In addition, M reduced S100A4 expression and vimentin staining, which are markers for epithelial mesenchymal transition, a major pathway to chronic kidney fibrosis. Finally, expression of oxidative stress markers Nox2 and iNOS was reduced. We conclude that inhibition of thrombin is an effective therapy against IRI that reduces chronic graft fibrosis, with a significantly positive effect on survival.

A new preservation solution (SCOT 15) Improves the islet isolation process from pancreata of non-heart-beating donors: a Murine model.

INTRODUCTION: Due to the organ shortage, there is increased use of organs harvested from non-heart-beating donors (NHBD). These organs have been subjected to a period of warm ischemia that is most deleterious to functional recovery. We have designed a new preservation solution, "Solution de Conservation des Organes et des Tissus" (SCOT 15; Macopharma, Tourcoing, France) which contains an extracellular ionic composition including PEG 20 kD (15 g/L) as a colloid. METHODS: Our objective was to compare SCOT 15 with University of Wisconsin (UW) solution or islet culture medium CMRL 1066 + 1% of Bovine Serum Albumin (BSA), as the working and preservation solution for islet isolation from pancreata subjected to warm ischemia using a murine model. RESULTS: Warm ischemia decreased the islet yield and cellular viability regardless of the preservation solution. Either when the pancreas was or was not subjected to warm ischemia, the best islet yield was obtained with SCOT 15 (P < .05 vs UW or CMRL 1066). The same results were observed for islet viability as assessed using the 3,(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) test; namely, better viability with SCOT 15 as compared with UW or CMRL 1066 (P < .01). CONCLUSION: In a murine model SCOT 15 was a better preservation solution for islet isolation than UW solution or culture medium (CMRL 1066).

Effect of raloxifene -- a selective oestrogen receptor modulator -- on kidney function in post-menopausal women with Type 2 diabetes: results from a randomized, placebo-controlled pilot trial.

AIMS: Epidemiological and experimental data suggest that activation of the oestrogen receptor pathway limits the incidence and the progression of diabetic nephropathy. We tested the hypothesis that raloxifene protects against increasing urinary albumin excretion in post-menopausal women with Type 2 diabetes in a randomized pilot clinical trial. METHODS: We included 39 post-menopausal women with Type 2 diabetes and micro- or macro-albuminuria in a 6-month, double-blind, placebo-controlled trial: 20 received placebo and 19 received 60 mg raloxifene per day. The albumin : creatinine ratio (ACR) in urine was determined on three consecutive days during the week before randomization and during the week before the final visit. RESULTS: One patient in each group dropped out in the first 3 weeks, leaving 37 patients for the analysis (19 on placebo and 18 on raloxifene). From randomization to the final visit, mean ACR was unchanged in the placebo group {277 microg/mg (67; 651) [median (interquartile range)] vs. 284 microg/mg (79; 1508)} but decreased slightly in the raloxifene group [376 microg/mg (67; 615) vs. 243 microg/mg (103; 549)]. This corresponds to a change of +24 (-37; +517) for the placebo group vs. -10 microg/mg (-36; +16) for the raloxifene group (P = 0.11). In multivariate analysis, raloxifene treatment (P(adjusted) = 0.013), baseline low-density lipoprotein (LDL) cholesterol (P(adjusted) = 0.023) and change in LDL cholesterol (P(adjusted) = 0.008) were related to the absolute change in ACR. Adverse effects were similar in the two groups. CONCLUSIONS: These results suggest that raloxifene may limit the progression of albuminuria in post-menopausal women with diabetes; further studies in a larger population are warranted.

Protective effect of PEG 35,000 Da on renal cells: paradoxical activation of JNK signaling pathway during cold storage.

Polyethylene glycol (PEG), a high-molecular weight colloid, is added to preservation solutions in order to decrease cold- and ischemia-induced injuries of the grafted organ. We evaluated on LLC-PK1, a porcine proximal tubular epithelial cell line (1) the efficiency of several commercial preservation solutions (University of Wisconsin, Euro-Collins, Celsior, SCOT, IGL-1), and (2) whether adding PEG (400-35,000 Da) in a simple extracellular-type buffer modified cell integrity and mitogen-activated protein kinase (MAPK) signaling pathways. SCOT was the most efficient commercial solution. Moreover, only PEG 35,000 Da totally preserved cell viability, induced a decrease on reactive oxygen species production and a decrease on p38-MAPK activation. Furthermore PEG 35,000 Da stimulated c-Jun N-terminal kinase (JNK). However, the inhibition of JNK pathway, with the specific SP600125 inhibitor, in the presence of PEG 35,000 Da did not affect cell survival. We also confirmed on whole pig kidney the protective effect of PEG 35,000 Da on cold-induced tubular injuries. This study confirms PEG antioxidative properties, but we demonstrate that its effect on JNK signaling pathway had also a paradoxical effect on cell death. This sheds a new light on PEG effects during cell preservation, independently from the classical immuno-camouflaging hypothesis.

[Value of systematic biological markers of inflammation for the prognosis at 12 months of patients undergoing programmed coronary angioplasty]. / Intérêt du dosage de marqueurs systémiques de l'inflammation dans l'évalualion pronostique à 12 mois de patients traités par angioplastie coronaire programmée.

Value of systematic dosage of biological markers of inflammation for the prognosis at 12 months of patients undergoing programmed coronary angioplasty Systematic dosage of proteins of inflammation has been suggested for assessing the prognosis of athero-thrombotic diseases. The authors undertook a study of plasma C-reactive protein (CRP) and interleukin 6 (IL-6) for evaluating the prognosis of patients undergoing programmed coronary angioplasty. A prospective monocentric study of 117 patients (65 +/- 8 years) was divided into a control group of 28 patients undergoing coronary angiography (Group 1) and 89 patients undergoing programmed coronary angioplasty (Group 2). Serum IL-6 and CRP levels were measured before arterial puncture and at H12 and H24 after coronary catheterisation. The follow-up period was 12 months. The angioplasty did not significantly increase CRP and IL-6 concentrations compared with coronary angiography. Twenty patients (Group 2) (22%) suffered a cardiovascular event in the 12 months' follow-up. These patients had significantly higher CRP levels at H0, H12 and H24 after coronary angioplasty than those who had uncomplicated outcomes. This was not observed for IL-6 concentrations because of the wide dispersion of the results obtained. Increased CRP concentrations between H0 and H24 was also a good predictive factor independently of high basal CRP levels potentially due to other causes than atheroma. Coronary angioplasty is associated with increased CRP at H0, H12 and H24. These values are correlated with the risk of future events at 6 and 12 months. This information is easily obtained and should help management of these patients.

Resistance to aspirin in vitro is associated with increased platelet sensitivity to adenosine diphosphate.

BACKGROUND: Platelet activation plays an important role in arterial thrombosis and the widespread use of aspirin has reduced major events by 25% in the secondary prevention of cardiovascular diseases. However, it appears that aspirin antiplatelet effect is not uniform and 8-45% of the population are, in vitro, aspirin resistant, and it is well recognized that platelets can be activated by pathways that are not blocked by aspirin, such as adenosine diphosphate (ADP). OBJECTIVES: To investigate whether aspirin-resistant patients have a modified sensitivity to ADP-induced platelet activation MATERIALS AND METHODS: Seventy-two patients were enrolled. Platelet function was measured by the PFA-100(R) analyser; platelet GP IIb-IIIa activation by ADP 10 micro M was assessed by flow cytometry using PAC-1 MoAb. RESULTS: Using a collagen/epinephrine coated cartridge on the PFA-100(R), the prevalence of aspirin resistance was 29.2% (n=21). For aspirin-resistant patients, the collagen/ADP coated cartridge showed a closure time significantly shorter (p=0.004) compared to the sensitive and control groups. Platelets from aspirin-resistant patients bound PAC-1 significantly more (p=0.03) than the aspirin-sensitive patients and controls when activated with 10 micro M ADP. CONCLUSIONS: Platelets from aspirin-resistant patients appear to be more sensitive and activable by ADP. This hypersensitivity could provide a possible explanation for the so-called aspirin resistance, and this could justify therapeutic improvement with alternative antiplatelet agents.

The platelet cytoskeleton regulates the aggregation-dependent synthesis of phosphatidylinositol 3,4-bisphosphate induced by thrombin.

Pretreatment of intact platelets with cytochalasin D prevented actin polymerization and cytoskeleton reorganization induced by thrombin, but did not affect platelet aggregation. Under these conditions, synthesis of phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P2) stimulated by thrombin was strongly inhibited, while production of phosphatidic acid was unaffected. The inhibitory effect of cytochalasin D was not observed when platelet aggregation was prevented by the RGDS peptide. We also found that cytochalasin D did not affect PtdIns(3,4)P2 synthesis induced by concanavalin A (ConA), which is known to occur through an aggregation-independent mechanism. Moreover, thrombin, but not ConA, induced the translocation of phosphatidylinositol 3-kinase to the cytoskeleton. This process was equally inhibited by both the RGDS peptide and cytochalasin D. These results demonstrate that the cytoskeleton represents a functional link between thrombin-induced aggregation and synthesis of PtdIns(3,4)P2.

Head-down tilt bed rest and immune responses.

Head-down tilt bed rest (HDT) is used as a model for studying the physiological changes occurring in weightlessness during spaceflight. In the present study, eight volunteers were subjected to a strict HDT of -6 degrees for 42 days. Blood samples were obtained 37 and 13 days before, at days 13, 34, and 41 during, and 12, 33, and 47 days after HDT. FACScan analysis was used to determine cell subpopulations. Plasma was used to quantify various circulating hormone levels. Whole blood and reconstituted blood were stimulated with various activators such as phytohaemagglutinin-P (PHA), PHA combined with phorbol-12-myristate 13-acetate (PMA), anti-CD2, anti-CD3, and lipopolysaccharide. Supernatants were collected and analysed for the interleukins IL-1beta, IL-2, IL-6, and IL-10, interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha). The total number of T lymphocytes and monocytes did not change significantly, whereas the number of polymorphonuclear cells increased during HDT. The percentage of CD2+ and CD3+ cells was increased at day 35 of HDT. The percentage and total number of natural killer cells (CD2+/CD3-/CD56+) was increased 12 days before and 14 days after HDT. TNF-alpha secretion did not change significantly during HDT. IL-2, IL-10 and IFN-gamma were increased at day 34 of HDT. IL-1beta levels were increased before and during HDT compared to post-HDT measurements. No significant changes were observed in plasma immunoglobulin, complement factors and other factors of the inflammatory system. Prolactin levels increased slightly but significantly at day 35 of HDT, thyreotropin and growth hormone levels remained virtually unchanged. Cortisol decreased slightly but significantly over the entire duration of the study. The changes observed during HDT do not indicate that the immune system is blunted, and these changes do not seem to correlate with the duration of HDT. Taken together these results show that a HDT does not reproduce the changes in immune responses observed after spaceflight.

Lipid products of phosphoinositide 3-kinase interact with Rac1 GTPase and stimulate GDP dissociation.

A number of reports suggest that under different conditions leading to cytoskeleton reorganization the GTPase Rac1 and possibly RhoA are downstream targets of phosphoinositide 3-kinase (PI 3-kinase). In order to gain more insight into this particular signaling pathway, we have addressed the question of a possible direct interaction of PI 3-kinase products with the Rho family GTPases RhoA, Rac1, and Cdc42. Using recombinant proteins, we found that Rac1 and, to a lesser extent, RhoA but not Cdc42 were capable to selectively bind to phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) in a mixture of crude brain phosphoinositides. Nucleotide-depleted Rac1 was the most efficient, but the GDP- and GTP-bound forms retained significant PtdIns(3,4,5)P3 binding activity. This protein-lipid association involved electrostatic as well as hydrophobic interactions, since both phosphate groups located at specific positions of the inositol ring and fatty-acyl chains were absolutely required. Based on the sequence of Rac1, two potential binding sites were identified, one at the C terminus and one in the extra alpha-helical domain. Deletion of these two domains resulted in a complete loss of binding to PI 3-kinase products. Finally, PtdIns(3, 4,5)P3 strongly stimulated GDP dissociation from Rac1 in a dose-dependent manner. In agreement, data obtained in intact cells suggest that PtdIns(3,4,5)P3 might target Rac1 to peculiar membrane domains, allowing formation of specific clusters containing not only small GTPases but other partners bearing pleckstrin homology domains such as specific exchange factors required for Rac1 and RhoA activation.

Phosphatidylinositol 3,4,5-trisphosphate-dependent stimulation of phospholipase C-gamma2 is an early key event in FcgammaRIIA-mediated activation of human platelets.

Platelets express a single class of Fcgamma receptor (FcgammaRIIA), which is involved in heparin-associated thrombocytopenia and possibly in inflammation. FcgammaRIIA cross-linking induces platelet secretion and aggregation, together with a number of cellular events such as tyrosine phosphorylation, activation of phospholipase C-gamma2 (PLC-gamma2), and calcium signaling. Here, we show that in response to FcgammaRIIA cross-linking, phosphatidylinositol (3,4, 5)-trisphosphate (PtdIns(3,4,5)P3) is rapidly produced, whereas phosphatidylinositol (3,4)-bisphosphate accumulates more slowly, demonstrating a marked activation of phosphoinositide 3-kinase (PI 3-kinase). Inhibition of PI 3-kinase by wortmannin or LY294002 abolished platelet secretion and aggregation, as well as phospholipase C (PLC) activation, indicating a role of this lipid kinase in the early phase of platelet activation. Inhibition of PLCgamma2 was not related to its tyrosine phosphorylation state, since wortmannin actually suppressed its dephosphorylation, which requires platelet aggregation and integrin alphaIIb/beta3 engagement. In contrast, the stable association of PLCgamma2 to the membrane/cytoskeleton interface observed at early stage of platelet activation was fully abolished upon inhibition of PI 3-kinase. In addition, PLCgamma2 was able to preferentially interact in vitro with PtdIns(3,4,5)P3. Finally, exogenous PtdIns(3,4,5)P3 restored PLC activation in permeabilized platelets treated with wortmannin. We propose that PI 3-kinase and its product PtdIns(3,4,5)P3 play a key role in the activation and adequate location of PLCgamma2 induced by FcgammaRIIA cross-linking.

Calpains are involved in phosphatidylinositol 3',4'-bisphosphate synthesis dependent on the alpha IIb beta 3 integrin engagement in thrombin-stimulated platelets.

In thrombin-stimulated platelets alpha IIb beta 3 integrin engagement triggers both phosphatidylinositol 3',4'-bisphosphate synthesis and calpain activation. We checked the possible involvement of calpains in phosphatidylinositol 3-kinase signalling pathway using a cell permeant specific inhibitor of calpains, calpeptin. In conditions where thrombin-induced platelet aggregation and secretion were not impaired, we found a dose-dependent inhibition of phosphatidylinositol 3,4-bisphosphate synthesis by calpeptin from 50 micrograms/ml. Moreover, pretreatment of platelets by both calpeptin and the peptide RGDS, an inhibitor of fibrinogen binding to activated alpha IIb beta 3 integrin, did not induce additive effects on phosphatidylinositol 3,4-bisphosphate inhibition. Finally, the p85 regulatory subunit of phosphatidylinositol 3-kinase was still translocated to the cytoskeleton in calpeptin-treated platelets. These data indicate that calpains are involved in the regulation of alpha IIb beta 3 integrin-dependent phosphatidylinositol 3-kinase signalling pathway.

Reversible translocation of phosphoinositide 3-kinase to the cytoskeleton of ADP-aggregated human platelets occurs independently of Rho A and without synthesis of phosphatidylinositol (3,4)-bisphosphate.

The aim of our study was to evaluate the effect of ADP and the role of cytoskeleton reorganization during reversible and irreversible platelet aggregation induced by ADP and thrombin, respectively, on the heterodimeric (p85alpha-p110) phosphoinositide 3-kinase translocation to the cytoskeleton and its activation. Reversible ADP-induced aggregation was accompanied by a reversible reorganization of the cytoskeleton and an increase in levels of the regulatory subunit p85alpha in this cytoskeleton similar to the increase observed in thrombin-activated platelets. This translocation followed a course parallel to the amplitude of aggregation. No increase in levels of both phosphatidylinositol (3, 4)-bisphosphate (PtdIns(3,4)P2) and phosphatidylinositol-(3,4,5)P3 could, however, be detected even at the maximum aggregation and PI 3-kinase alpha translocation. Moreover, in contrast to the situation for thrombin stimulation, the GTP-binding protein RhoA was hardly translocated to the cytoskeleton when platelets were stimulated with ADP, whereas translocation of pp60(c-)src and focal adhesion kinase did occur. These results suggest (i) translocation of signaling enzymes does not necessarily imply their activation, (ii) the reversibility of ADP-induced platelet aggregation may be the cause or the result of a lack of PI 3-kinase activation and hence of PtdIns(3,4)P2 production, and (iii) RhoA does not seem to be involved in the ADP activation pathway of platelets. Whether PtdIns(3,4)P2 or RhoA may contribute to the stabilization of platelet aggregates remains to be established.

Total inhibition of phospholipase C and phosphatidylinositol 3-kinase by okadaic acid in thrombin-stimulated platelets.

The strong inhibition of thrombin-induced platelet functions induced by okadaic acid is not correlated with the partial modification of pleckstrin phosphorylation, which remains still phosphorylated two min after stimulation, indicating that protein kinase C is not affected by okadaic acid. We then investigated the effect of okadaic acid on platelet lipid metabolism. Our data indicate that inhibition indeed strongly affects phosphatidic acid as well as phosphatidylinositol 3,4-bisphosphate synthesis at low concentrations of okadaic acid, and phosphatidylinositol 4,5-bisphosphate at higher concentrations. Since thrombin-induced tyrosine phosphorylations were completely inhibited in the presence of okadaic acid, as a consequence, phosphatidylinositol 3-kinase was no longer detected in antiphosphotyrosine immunoprecipitates, thus explaining the absence of phosphatidylinositol, 3,4-bisphosphate synthesis. Finally, okadaic acid inhibited thrombin-induced fibrinogen binding, indicating that serine/threonine phosphatases may affect the inside-out signalling which regulates the alpha 11bb3 integrin, downstream protein kinase C activation.

Afaâcytin, an alpha beta-fibrinogenase from Cerastes cerastes (horned viper) venom, activates purified factor X and induces serotonin release from human blood platelets.

Afaâcytin, a proteinase with caseinolytic, arginine-esterase and amidase activities, was purified from the venom of Cerastes cerastes (horned viper) in two steps by gel filtration through Sephadex G75, then HPLC on carboxymethyl-cellulose. Afaâcytin has an isoelectric point of 6.25, and consists of two subunits, alpha and beta, which have the same apparent molecular mass (40,000) and are indistinguishable in the absence of reduction or/and deglycosylation. Subunit beta is constituted of two disulfide-linked polypeptidic chains, beta and beta'. The respective apparent molecular mass of the chains are 43,000 (alpha), 35,500 (beta) and 10,200 (beta') as determined by SDS/PAGE under reducing conditions. Both chains alpha and beta are N-glycosylated. The two chains have the same N-terminal sequence (20 residues) which is similar to those of other proteinases from snake venom. Susceptibility of afaâcytin to diisopropyl fluorophosphate and benzamidine indicates the presence of a serine and an aspartic (or glutamic) acid residues in the catalytic site. Ca2+ appears to be required for structural cohesion of the afaâcytin molecule. Afaâcytin exhibits alpha beta-fibrinogenase and alpha-fibrinase properties. It replaces missing factors VIII and IX in deficient plasmas, and activates purified human factor X into factor Xa. It releases serotonin from platelets and directly aggregates human (but not rabbit) blood platelets. Despite its thrombin-like characteristics, however, afaâcytin is not inhibited by plasmatic thrombin inhibitors. The procoagulant properties of afaâcytin therefore have potential clinical applications.

Evidence for a glycoprotein IIb-IIIa- and aggregation-independent mechanism of phosphatidylinositol 3',4'-bisphosphate synthesis in human platelets.

The synthesis of phosphatidylinositol 3',4'-bisphosphate (PtdIns(3,4)P2) in 32P-labeled human platelets induced by the tetrameric lectin concanavalin A and the physiological agonist thrombin were compared. Like thrombin, concanavalin A stimulated a time-dependent accumulation of PtdIns(3,4)P2, which reached maximal levels after 5 min of stimulation. However, while synthesis of PtdIns(3,4)P2 induced by thrombin was dependent on platelet aggregation, the production of PtdIns(3,4)P2 induced by concanavalin A was unchanged when aggregation was prevented by the omission of stirring or when fibrinogen binding to platelets was inhibited by the tetrapeptide RGDS. Accumulation of PtdIns(3,4)P2 was not observed in platelets stimulated with succinyl-concanavalin A, a dimeric derivative of the lectin that binds to the same receptors on the platelet surface but does not promote clustering of membrane glycoproteins. The synthesis of PtdIns(3,4)P2 induced by concanavalin A was also independent of the membrane glycoprotein IIb-IIIa, as normal accumulation of this lipid was observed in platelets from two patients affected by Glanzmann thrombasthenia. In contrast, thrombin showed a strongly reduced ability to stimulate PtdIns(3,4)P2 production in thrombasthenic platelets. Although concanavalin A was able to induce association of the regulatory subunit of the phosphatidylinositol 3-kinase with tyrosine-phosphorylated proteins, the tyrosine kinase inhibitor tyrphostin AG-213 did not inhibit the lectin-induced synthesis of PtdIns(3,4)P2. These results demonstrate the existence of a novel mechanism of PtdIns(3,4)P2 synthesis in human platelets, which is independent of glycoprotein IIb-IIIa and aggregation, but requires clustering of membrane glycoproteins. As clustering events occur during platelet aggregation promoted by physiological agonists, this new mechanism may also be involved in the aggregation-dependent production of PtdIns(3,4)P2 in thrombin-stimulated platelets.

Integrin-dependent translocation of phosphoinositide 3-kinase to the cytoskeleton of thrombin-activated platelets involves specific interactions of p85 alpha with actin filaments and focal adhesion kinase.

Thrombin-induced accumulation of phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P2) but not of PtdIns(3,4,5,)P3 is strongly correlated with the relocation to the cytoskeleton of 29% of the p85 alpha regulatory subunit of phosphoinositide 3-kinase (PtdIns 3-kinase) and is accompanied by a significant increase in PtdIns 3-kinase activity in this subcellular fraction. Actually, PtdIns(3,4)P2 accumulation and PtdIns 3-kinase, pp60c-src, and p125FAK translocations as well as aggregation were concomitant events occurring with a distinct lag after actin polymerization. The accumulation of PtdIns(3,4)P2 and the relocalization of PtdIns 3-kinase to the cytoskeleton were both dependent on tyrosine phosphorylation, integrin signaling, and aggregation. Furthermore, although p85 alpha was detected in anti-phosphotyrosine immunoprecipitates obtained from the cytoskeleton of thrombin-activated platelets, we failed to demonstrate tyrosine phosphorylation of cytoskeletal p85 alpha. Tyrphostin treatment clearly reduced its presence in this subcellular fraction, suggesting a physical interaction of p85 alpha with a phosphotyrosyl protein. These data led us to investigate the proteins that are able to interact with PtdIns 3-kinase in the cytoskeleton. We found an association of this enzyme with actin filaments: this interaction was spontaneously restored after one cycle of actin depolymerization-repolymerization in vitro. This association with F-actin appeared to be at least partly indirect, since we demonstrated a thrombin-dependent interaction of p85 alpha with a proline-rich sequence of the tyrosine-phosphorylated cytoskeletal focal adhesion kinase, p125FAK. In addition, we show that PtdIns 3-kinase is significantly activated by the p125FAK proline-rich sequence binding to the src homology 3 domain of p85 alpha subunit. This interaction may represent a new mechanism for PtdIns 3-kinase activation at very specific areas of the cell and indicates that the focal contact-like areas linked to the actin filaments play a critical role in signaling events that occur upon ligand engagement of alpha IIb/beta 3 integrin and platelet aggregation evoked by thrombin.

Phosphoinositide 3-phosphatase segregates from phosphatidylinositol 3-kinase in EGF-stimulated A431 cells and fails to in vitro hydrolyse phosphatidylinositol(3,4,5)trisphosphate.

Beside 4- and 5-phosphatases playing a role in the interconversion between the D-3 phosphorylated polyphosphoinositides, the only enzyme described so far to be responsible for a phosphomonesterasic activity on the D-3 position of inositol lipids is a specific 3-phosphatase that hydrolyzes PtdIns(3)P in NIH 3T3 cells. We report here the presence of a potent 3-phosphatase activity in different cell types. This activity is detected both in cytosol and membranes of A431 cells and is inhibited by VO4(-3) and Zn2+. Interestingly, the cytosolic activity from A431 cells selectively hydrolyzes in vitro PtdIns(3)P and PtdIns(3,4)P2, whereas PtdIns(3,4,5)P3 remains a very poor substrate under the same conditions. Finally, assays of phosphatidylinositol 3-kinase and 3-phosphatase activities in the pool of phosphotyrosine-containing proteins isolated from EGF-stimulated A431 cells suggest a compartmentation of these two antagonistic activities during cell activation.

Rapid and transient thrombin stimulation of phosphatidylinositol 4,5-bisphosphate synthesis but not of phosphatidylinositol 3,4-bisphosphate independent of phospholipase C activation in platelets.

When platelets are stimulated by thrombin they immediately undergo inositol lipid hydrolysis via phospholipase C activation. However, subsequently an increased production of phosphatidylinositol 4,5-bisphosphate is observed. Phospholipases C were inhibited by lowering the cytoplasmic free calcium concentration by preincubation with Quin-2-tetra(acetoxymethyl) ester. Aggregation and secretion were also totally suppressed. Under these conditions we observed an increased labeling of phosphatidylinositol 4,5-bisphosphate, indicating a stimulation of inositol lipid kinases, independent of lipid hydrolysis by phospholipase C. Conversely the production of phosphatidylinositol 3,4-bisphosphate was totally abolished. These results suggest a different regulation of the kinases/phosphatases responsible for the production of phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4-bisphosphate.

Tyrosine kinases and phosphoinositide metabolism in thrombin-stimulated human platelets.

In this study we have examined the implication of tyrosine kinase activities in aggregation, 5-hydroxytryptamine secretion and mainly phosphoinositide metabolism in response to human platelet stimulation by thrombin. Using the potent tyrosine kinase inhibitor tyrphostin AG-213, we have observed a significant inhibition of aggregation and 5-hydroxytryptamine release; however, this percentage inhibition was lower at high thrombin concentrations. On the other hand, tyrphostin treatment of metabolically 32P-labelled platelets significantly inhibited the thrombin-dependent accumulation of PtdIns(3,4)P2, which involves at least a PtdIns 3-kinase and/or a PtdIns3P 4-kinase, whereas the synthesis of phosphatidic acid (PtdOH), a good reflection of the phospholipase C (PLC) activation in platelets, was partially blocked. Inositol phosphate production was also inhibited by about 40% when tyrphostin-treated platelets were stimulated with thrombin. In addition, we show by Western-blot analysis that PLC gamma 1, as well as the regulatory subunit (p85) of the PtdIns 3-kinase, were present in the anti-phosphotyrosine immunoprecipitate isolated from thrombin-stimulated platelets. Furthermore, tyrphostin treatment clearly decreased the PLC gamma 1 and p85 contents in such an anti-phosphotyrosine immunoprecipitate. Our results provide the first evidence for a direct or indirect regulation of PtdIns(3,4)P2 accumulation and PLC gamma 1 activity by tyrosine phosphorylation during thrombin stimulation of human platelets.