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Background and Objective: Despite the fact that several prognostic systems for myelodysplastic syndromes (MDS) have been proposed, few studies have been designed to test their effectiveness in independent patient populations. The aim of this study was to compare the prognostic value of 8 previously described prognostic systems in a series of consecutive MDS patients observed at a single institution over a 10-year period., Design and Methods: One hundred and forty-three patients were diagnosed as having myelodysplastic syndrome (MDS) according to the French-American-British (FAB) criteria. They were studied retrospectively in order to assess the prognostic value of the FAB classification and 7 other prognostic systems., Results: On the basis of data at diagnosis, all investigated systems effectively stratified patients into groups with different life expectancies and identified a subset of patients with poor clinical outcome. However, the systems had different outcomes concerning median survival of patients classified as low-risk, ranging from less than 3 years for the Mufti scoring system to more than 8 years for the FAB classification modified according to Rosati et al. Moreover, patient distribution into different risk categories was quite different with the different prognostic systems., Interpretation and Conclusions: When applied to our case series, some of the prognostic systems had a much lower prognostic value than in the patient population from which they derived. This evidence suggests that testing of prognostic systems in independent case series is necessary before using the systems in clinical practice.

We describe a new variant of Bernard-Soulier syndrome characterized by almost normal amounts of GPIb and severely reduced GPIX and GPV. Despite surface expression, GPIbalpha failed to support ristocetin-induced platelet agglutination and to bind two conformation-dependent monoclonal antibodies, suggesting a qualitative defect. Sequence analysis of the gene coding for GPIX revealed a T-to-C substitution at base 1811, leading to a Leu40Pro conversion, whereas no defects were found in the coding region of the GPIbalpha gene. Allele-specific restriction enzyme analysis showed that the propositus and one of his sisters. both with severe bleeding diathesis. were homozygous for the GPIX mutation: the members of the family with mild bleeding diathesis and/or giant platelets in the peripheral blood were heterozygous, whereas the healthy ones were homozygous for the normal allele. Infusion of 1-desamino-8-D-arginine vasopressin normalized bleeding time in the two severely affected patients, although it did not modify ristocetin-induced platelet agglutination or membrane expression of GPIbalpha, GPIX, GPIIb-IIIa and GMP-140. Moreover, in one patient, normalization of bleeding time and rise of von Willebrand factor plasma concentration did not seem to be directly related.

Human erythrocyte band 3 becomes rapidly phosphorylated on tyrosine residues after exposure of erythrocytes to hypertonic conditions. The driving force for this phosphorylation reaction seems to be a decrease in cell volume, because (1) changes in band 3 phosphotyrosine content accurately track repeated changes in erythrocyte volume through several cycles of swelling and shrinking; (2) the level of band 3 phosphorylation is independent of the osmolyte employed but strongly sensitive to the magnitude of cell shrinkage; and (3) exposure of erythrocytes to hypertonic buffers under conditions in which intracellular osmolarity increases but volume does not change (nystatin-treated cells) does not promote an increase in tyrosine phosphorylation. We hypothesize that shrinkage-induced tyrosine phosphorylation results either from an excluded-volume effect, stemming from an increase in intracellular crowding, or from changes in membrane curvature that accompany the decrease in cell volume. Although the net phosphorylation state of band 3 is shown to be due to a delicate balance between a constitutively active tyrosine phosphatase and constitutively active tyrosine kinase, the increase in phosphorylation during cell shrinkage was demonstrated to derive specifically from an activation of the latter. Further, a peculiar inhibition pattern of the volume-sensitive erythrocyte tyrosine kinase that matched that of p72syk, a tyrosine kinase already known to associate with band 3 in vivo, suggested the involvement of this kinase in the volume-dependent response.

Background and Objective: Concerns about the risk of transfusion therapy are driving towards new strategies which are designed to minimize exposure to allogeneic blood products. We aimed to find out whether it is possible to support the phase of thrombocytopenia following high-dose chemotherapy (HDC) and circulating progenitor cells (CPC) transplantation by autologous platelet concentrates (PC)., Design and Methods: PC were collected from 32 patients undergoing HDC and CPC transplantation for stage II/III breast cancer. A single plateletpheresis was performed at rebound after high-dose cyclophosphamide, when platelet count exceeded 250 x 10(9)/L. PC were cryopreserved in 5% DMSO after controlled-rate freezing and stored in liquid nitrogen. In vitro studies of cryopreserved platelets (aggregation, ATP release and change of mean platelet volume induced by EDTA) were performed. When platelet counts dropped below 20 x 10(9)/L following HDC (thiotepa 600 mg/m2, L-PAM 160 mg/m2) and CPC transplant (CD34+ cells > 5 x 10(6)/kg), PC were thawed in a 37 degrees C water bath, centrifuged to remove DMSO, resuspended in autologous plasma and reinfused within one hour., Results: Large quantities of platelets were harvested in all patients (median 6.6 x 10(11), range 4.8-12.2). In vitro studies showed preserved platelet function as compared to both fresh platelets and standard PC. Twenty-eight out of 32 patients received autologous PC. At the time of transfusion most of the patients were febrile (> 38 degrees C) and had mucositis > G2. The median number of platelets reinfused was 3.8 x 10(11) (range 2.0-8.1) with a median loss during the freeze-thaw-wash procedure of 37%. Autotransfusion was able to maintain platelet count above 20 x 10(9)/L in most patients, with a corrected count increment > 7.5 in 20 cases. Four patients required one additional allogeneic transfusion, two because of a poor increment and two due to a late-occurring epistaxis. No side effects related to PC infusion were recorded. Sixteen control patients who received the same HDC and a similar number of CD34+ cells required a total of 17 allogeneic PC units (1 patient did not require platelet transfusion)., Interpretation and Conclusions: Our data demonstrate that large doses of autologous platelets can easily be collected and safely administered to support the period of thrombocytopenia in patients undergoing HDC and CPC transplantation. Autologous PC in these patients can abrogate the risks deriving from allogeneic platelet transfusion.

Stimulation of human platelets with thrombin caused a 42% inhibition of the ADP-ribosyl cyclase activity of membrane CD38. This effect was mediated by the activation of the platelet thrombin receptor rather than by proteolysis of CD38, and was not due to a different distribution of the synthesised nucleotide or to a reduced accessibility of CD38 to the substrate. The inhibitory effect of thrombin required actin polymerisation and was not observed when interaction of CD38 with the cytoskeleton was prevented by cytochalasin D. Finally, we analysed whether cADPR could play a role as a Ca2+-mobilising agent in human platelets. Using saponin-permeabilised cells, we found that unlike IP3, cADPR did not induce any release of Ca2+ from intracellular stores. These results indicate that the enzymatic activity of membrane CD38 can be modulated by platelet activation, and that the function of this glycoprotein is probably not related to Ca2+ mobilisation.

Objectives: The use of circulating progenitor cell support following high-dose chemotherapy for malignancies decreases but does not entirely abolish platelet transfusion requirement. We investigated the feasibility of supporting the posttransplant thrombocytopenic phase exclusively with autologous platelets collected by apheresis and cryopreserved., Methods: 25 patients underwent plateletpheresis during the platelet rebound occurring after high-dose cyclophosphamide. Autologous platelets were cryopreserved in 5% dimethylsulfoxide, thawed and transfused during the aplastic phase after the myeloablative regimen whenever clinically required., Results: A single plateletpheresis was carried out in all patients, allowing the harvest of a platelet concentrate with a mean value of 7.7 x 10(11) platelets. No significant procedure- or transfusion-related side effects were recorded. Mean platelet recovery after freezing and thawing was 63% and the mean number of platelet reinfused was 4.8 x 10(11); 23 of 25 patients were fully supported with autologous platelets., Conclusion: Plateletpheresis performed in our selected group of patients was found to be a safe and effective procedure to collect large amounts of autologous platelets; the numbers obtained proved to be sufficient for the transfusion demand of almost all patients.

Human c-mpl ligand or thrombopoietin (TPO) has been proved to be a critical cytokine in the physiological regulation of thrombopoiesis. Previous evidence suggested that TPO production is constitutive and TPO plasma levels are regulated by the platelet-megakaryocyte mass through c-mpl receptor-mediated uptake and metabolism. To evaluate whether this mechanism of TPO level regulation is also operative in subjects with an elevated platelet count, we evaluated serum TPO in 32 patients with thrombocytosis due to essential thrombocythaemia (ET) or polycythaemia vera (PV) and in 70 subjects with reactive thrombocytosis; 32 healthy subjects were also studied. TPO levels were significantly higher in the ET and PV groups (median 246.2 pg/ml, range 93.5-4596) and reactive thrombocytosis patients (median 287 pg/ml, range 82.7-1960.0) than in normal subjects (median 156.7 pg/ml, range 62.2-352.7). No significant difference was found between the two groups of patients, indicating that serum TPO levels cannot differentiate between primary and reactive thrombocytosis. No significant correlation was found between platelet count and TPO levels in either ET-PV or reactive thrombocytosis, whereas a trend toward a correlation between acute-phase reactants and TPO was observed in patients with reactive thrombocytosis. These results indicate that TPO clearance by platelets-megakaryocytes is not fully operative or is not the only mechanism of TPO level regulation in primitive and reactive thrombocytosis.

Tendon and corneal decorins are differently iduronated dermatan sulphate/proteoglycan (DS/PG) and the biochemical parameter that differentiates type I collagens is the hydroxylysine glycoside content. We have examined the effect of tendon and corneal decorins on the individual phases (tlag, dA/dt) of differently glycosylated type I collagens fibril formation, at molar ratios PG:collagen monomer ranging from 0.15:1 to 0.45:1. The results obtained indicate that decorins exert a different effect on the individual phases of fibril formation, correlated to the degree of glycosylation of collagen: at the same PG:collagen ratio the fibril formation of highly glycosylated corneal collagen is more efficiently inhibited than that of the poorly glycosylated one (tendon). Moreover tendon and corneal decorins exert a higher control on the fibrillogenesis of homologous collagen with respect to the heterologous one. These data suggest a possible tissue-specificity of the interaction decorin/type I collagen correlated to the structure of the PG and collagen present in extracellular matrices.

Background and Objective: It has been previously suggested that activation of coagulation and fibrinolysis may sometime occur in patients with unruptured aortic aneurysm. However, the incidence of this complication and the effect of surgical repair are unknown. The objective of our study was to gain further information on this topic., Methods: We investigated activation of the hemostatic process in 20 consecutive patients with unruptured abdominal aortic aneurysm. We then evaluated the effect of surgical repair of the vascular abnormalities., Results: Both before and in the first week after surgery, the large majority of patients showed clear signs of activation of coagulation (increased plasma levels of prothrombin fragment 1 + 2 and fibrin peptide A), and many had low levels of the natural anticoagulant antithrombin III. Platelets were activated in all cases (high levels of plasma beta-thromboglobulin), and signs of platelet consumption (thrombocytopenia and/or increased mean platelet volume) were present in most of them., Interpretation and Conclusions: Activation of the hemostatic process occurs in nearly all patients with abdominal aortic aneurysm and could play a role in the hemorrhagic and thrombotic events that can complicate the clinical development of these subjects.

Lysophosphatidic acid is a biologically active phospholipid able to induce cell proliferation and platelet aggregation. In this study we investigated the biochemical mechanisms of platelet activation by lysophosphatidic acid. We found that lysophosphatidic acid stimulated the binding of the photoreactive GTP-analog 4-azidoanilido-[alpha(32)P]GTP to a 40-kDa protein on platelet membranes. Moreover, lysophosphatidic acid induced the rapid decrease of the intracellular concentration of cAMP in intact platelets, indicating that this lipid activates platelets by binding to a membrane receptor coupled to the inhibitory GTP-binding protein Gi. In agreement with a receptor-mediated action, we found that platelet activation by lysophosphatidic acid underwent homologous desensitization. In the absence of extracellular CaCl(2), lysophosphatidic acid did not induce platelet aggregation, and did not stimulate phospholipase C. However, under the same conditions, lysophosphatidic acid produced the rapid tyrosine phosphorylation of several platelet proteins. This effect was not mediated by the formation of thromboxane A(2). Our results demonstrate that, in lysophosphatidic acid-stimulated platelets, activation of protein-tyrosine kinases occurs in the absence of phospholipase C activation and platelet aggregation, and may be directly related to the activation of the G-protein-coupled lysophosphatidic acid-receptor.

Human erythrocytes were induced to release membrane vesicles by treatment with Ca2+ and ionophore A23187. In addition to the biochemical changes already known to accompany loading of human erythrocytes with Ca2+, the present study reveals that tyrosine phosphorylation of the anion exchanger band 3 protein also occurs. The relationship between tyrosine phosphorylation of band 3 and membrane vesiculation was analysed using quinine (a non-specific inhibitor of the Ca(2+)-activated K+ channel, and the only known inhibitor of Ca(2+)-induced vesiculation) and charybdotoxin, a specific inhibitor of the apamin-insensitive K(+)-channel. Both inhibitors suppressed tyrosine phosphorylation of band 3. In the presence of quinine, membrane vesiculation was also suppressed. In contrast, at the concentration of charybdotoxin required to suppress tyrosine phosphorylation of band 3, membrane vesiculation was only mildly inhibited (16-23% inhibition), suggesting that tyrosine phosphorylation of band 3 is not necessary for membrane vesiculation. Phosphorylation of band 3 was in fact observed when erythrocytes were induced to shrink in a Ca(2+)-independent manner, e.g. by treatment with the K+ ionophore valinomycin or with hypertonic solutions. These observations suggest that band 3 tyrosine phosphorylation occurs when cell volume regulation is required.

Cytochalasin D was used to investigate the role of intracellular cytoskeleton in the stabilization of platelet aggregation induced by strong platelet agonists. Incubation of gel-filtered platelets with increasing concentrations of cytochalasin D resulted in a dose-dependent inhibition of actin polymerization and association of actin-binding proteins with the Triton X-100-insoluble material induced by the thromboxane analogue, U46619, and the thrombin receptor activating peptide, TRAP. The same concentrations of cytochalasin D did not significantly inhibit platelet aggregation promoted by the two agonists. The addition of the chelating agent EDTA to fully aggregated platelets, that had been treated with cytochalasin D, resulted in the rapid and almost complete disaggregation. EDTA did not cause disaggregation of control, solvent-treated, aggregated platelets. The degree of platelet disaggregation induced by EDTA was dependent on the dose of cytochalasin D used, and was correlated with the inhibition of the cytoskeletal reorganization. Aggregation of cytochalasin D-treated platelets stimulated with U46619 or TRAP was also reverted by the addition of the tetrapeptide RGDS or the fibrinogen gamma-chain dodecapeptide, which competitively interfere with fibrinogen binding to the glycoprotein IIb-IIIa complex. These results indicate that the intracellular cytoskeleton plays an essential role in the stabilization of the fibrinogen-platelet interaction, and is necessary for the irreversibility of platelet aggregation induced by strong agonists.

CD38 is a cell surface molecule widely used as a marker for immature and activated lymphocytes. It has been recently shown that CD38 displays three enzymatic activities: hydrolysis of NAD+ to ADP-ribose, synthesis of cyclic ADP-ribose from NAD+, and hydrolysis of cyclic ADP-ribose to ADP-ribose. Thus, CD38 plays a key role in the synthesis of cyclic ADP-ribose, a calcium-mobilizing compound. We investigate here the expression and cellular localization of CD38 in human platelets using a specific monoclonal antibody. Results showed that CD38 is expressed by human platelet membranes. Moreover, we show that platelet CD38 possesses NAD glycohydrolase, ADP-ribose cyclase, and cyclic ADP-ribose hydrolase activities. This finding indicates that the calcium-mobilizing agent cyclic ADP-ribose can be synthetized by human platelets and raises the question about the possible role of CD38 expression and enzymatic activities in the signal transduction pathways leading to platelet activation.

The biochemical mechanisms of platelet activation by lysophosphatidic acid were investigated. Lysophosphatidic acid interacts with a membrane receptor coupled to the inhibitory GTP-binding protein Gi and produces a rapid decrease of the intracellular concentration of cAMP. Aggregation of gel-filtered platelets by lysophosphatidic acid requires the presence of extracellular CaCl2, as this phospholipid does not induce secretion of platelet dense granules. Platelet activation by lysophosphatidic acid in the absence of extracellular CaCl2 does not involve phospholipase C activation, as evaluated by measuring mobilization of Ca2+ from internal stores and pleckstrin phosphorylation, but causes the rapid tyrosine phosphorylation of several intracellular proteins. Our results indicate that activation of intracellular tyrosine kinases is not secondary to Ca2+ mobilization and protein kinase C activation in lysophosphatidic acid-stimulated platelets.

Background: Previous studies showed severe biochemical and functional damage to platelets in patients undergoing cardiopulmonary bypass for cardiac surgery, and suggested that this derived from the proteolytic action of plasmin on the platelet surface., Methods: A double-blind study was carried out to compare platelet function and composition in patients randomized to receive the protease inhibitor aprotinin or placebo during reoperation for valvular prosthesis replacement or coronary artery bypass grafting., Results: Flow cytometry with specific monoclonal antibodies and polyacrylamide gel electrophoresis did not show any significant proteolysis of platelet glycoprotein Ib and IIb-IIIa either in the placebo or the aprotinin group. Functional studies were consistent with these results, since ristocetin-induced platelet agglutination was unchanged and platelet aggregation and ATP release induced by collagen and ADP were only slightly reduced by cardiopulmonary bypass. These mild defects in platelet function were partially prevented by aprotinin infusion., Conclusions: On the basis of our data and those from literature, we suggest that platelets may be affected very little or severely damaged during cardiopulmonary bypass for cardiac surgery, probably depending on some aspects of the technical procedure which remain to be identified. Aprotinin infusion significantly protects platelets in the latter condition, while its role is obviously slight in the former.

We found that intracellular Ca2+ concentration ([Ca2+]i) increased during ristocetin-induced agglutination of aequorin loaded platelets resuspended in plasma. Chelation of extracellular Ca2+ had no effect on platelet clumping, but delayed and greatly reduced Ca2+ increase, indicating that it derived for the most part from Ca2+ influx. Nine monoclonal antibodies (MA) against glycoprotein (GP) Ib largely prevented ristocetin-induced platelet clumping and [Ca2+]i increase, while three anti-GPIb MA with no effect on platelet clumping did not interfere with Ca2+ movement. In unstirred samples platelet agglutination was greatly reduced and [Ca2+]i increase was abolished, suggesting that close platelet-to-platelet contact, in addition to von Willebrand factor (vWF) binding to GPIb, is necessary for Ca2+ transient. Nine MA against GPIIb/IIIa, the gly-arg-gly-asp-ser (GRGDS) peptide and GPIIb/IIIa complex dissociation had no effect on platelet agglutination, but significantly reduced Ca2+ increase. Our results suggest that platelet clumping induced by vWF binding to GPIb is responsible for GPIIb-IIIa dependent Ca2+ influx.

Treatment of human platelets with the lectin Concanavalin A (Con A) resulted in the tyrosine phosphorylation of several proteins with molecular masses 65, 80, 85, 95, 120, 135, and 150 kDa. These proteins were divided in two groups: the first group included the 65-, 85-, 95-, and 120-kDa bands, which were tyrosine phosphorylated also in thrombin-stimulated platelets; the second group (80-, 135-, and 150-kDa bands) included proteins whose tyrosine phosphorylation was exclusively promoted by Con A, but not by thrombin. Members of the second group were rapidly dephosphorylated when the lectin was displaced from the cell surface by methyl alpha-D-mannopyranoside. Pretreatment of intact platelets with the prostacyclin analog iloprost, inhibited Con A-induced tyrosine phosphorylation of the first group of proteins, but had no effect on the tyrosine phosphorylation of the proteins of the second group. Succinyl-Con A, a dimeric derivative of the lectin, which binds to the platelet surface but does not promote clustering of the receptor, did not induce tyrosine phosphorylation of the second group of proteins, although phosphorylation of some members of the first group was observed. Our results demonstrate the presence of two different mechanisms leading to protein-tyrosine phosphorylation in Con A-stimulated platelets, and identify a new signal transduction pathway, promoted by the clustering of membrane glycoproteins, that produces tyrosine phosphorylation of specific substrates. This new pathway may be activated by platelet interaction with multivalent ligands, such as adhesive proteins, during adhesion, spreading, and aggregation.

A new synthetic substrate for protein methionine sulfoxide reductase is proposed. We show that extracts from human polymorphonuclear leukocytes can reduce 4-dimethylaminoazobenzene-4'-sulfonyl-L-methionine-dl-sulfoxide [DABS-L-Met-dl-(O)] to the corresponding methionine derivative, in the presence of dithiothreitol or dithioerythritol. The product of the reaction (DABS-Met) was separated by reversed-phase HPLC and detected by reading the absorbance at 436 nm. Due to the chirality of the sulfur atom in the sulfoxide, two diastereomers of Met(O) exist, namely Met-l-sulfoxide and Met-d-sulfoxide. After separation of the two forms and preparation of the DABS-derivatives, we observed a preferential reduction of the l-sulfoxide by polymorphonuclear leukocytes extracts. We discuss the possibility that the observed stereospecificity might have physiological relevance in the field of the oxidative modifications of proteins.

The stimulation of human platelets with physiological agonists results in the incorporation of several proteins into the cytoskeleton, fibrinogen binding, and platelet aggregation. We recently demonstrated that the Ras-related low molecular weight GTP-binding protein Rap2B associates with the cytoskeleton in activated platelets and that this interaction requires platelet aggregation. In the present study we demonstrate that agonist-induced actin polymerization is necessary for the translocation of Rap2B to the cytoskeleton, suggesting that Rap2B interacts with the newly formed actin filaments. Moreover, the association of Rap2B with Triton X-100-insoluble material from platelets was totally blocked by treatment of intact platelets with monoclonal antibodies against the fibrinogen receptor glycoprotein IIb-IIIa. Platelets from patients affected by Glanzmann thrombastenia, a genetic disorder in which platelet plasma membranes lack glycoprotein IIb-IIIa but possess normal levels of Ras-related proteins, failed to incorporate Rap2B into the cytoskeleton upon activation by thrombin. Comparative immunoblotting revealed that the translocation of Rap2B to the cytoskeleton during platelet aggregation was accompanied by the simultaneous translocation of glycoprotein IIb-IIIa. Moreover, the cytoskeleton from aggregated platelets contained Rap2B and glycoprotein IIb-IIIa in comparable amounts. These results demonstrate the association of Rap2B and glycoprotein IIb-IIIa and their translocation to the cytoskeleton in aggregated human platelets.

Following activation of human platelets changes in cytoskeletal organization occur: some proteins, which are present in the cytosol or membrane-associated in resting platelets, are recovered in the Triton-insoluble residue in activated cells. Assembly and disassembly of complex effector units on the membrane and inside cells is under the control of low molecular weight GTP-binding proteins, particularly those in the ras family. We investigated the interaction of small GTP-binding proteins with the platelet cytoskeleton and the effect of high cAMP levels on these interactions. At least two GTP-binding proteins of 24 and 28 kDa were detected in the Triton-insoluble residue of resting platelets. Stimulation of platelets with thrombin or concanavalin A (Con A), under non-aggregating conditions, resulted in increased 24 kDa protein-bound GTP, which also contained a significant amount of rap1B. High cAMP levels differently affected this interaction depending on the type of agonist used. cAMP increased association of G-proteins with the cytoskeleton following Con A-activation, while it decreased G-proteins interaction after thrombin stimulation. The activation did not influence the cAMP-dependent phosphorylation of rap1B. No phosphoprotein corresponding to rap1B could be detected in the Triton-insoluble residues, however. These findings could be related to the different mechanisms of cytoskeletal protein recruitment in platelets activated with either thrombin or Con A.

A periodic fall of platelet number characterizes an acquired pathological condition named cyclic thrombocytopenia. We observed a patient in whom the episodes of thrombocytopenia (platelet number less than 50 x 10(9)/l) were followed regularly by thrombocytosis (700-2300 x 10(9) platelets/l). The period of platelet count fluctuation was about 40 d. Morphological examination of bone marrow showed the cyclic disappearance of mature and immature megakaryocytes; bone marrow cultures revealed a periodic severe defect of both multilineage and single-lineage progenitor cell growth. When platelet count was falling, a mild defect of platelet aggregation and ATP release was observed, while platelet function was normal when platelet count was rising. Prednisone, thymopentin, high-dose intravenous gamma-globulin and splenectomy were without effect. After 4 years of cyclic platelet and megakaryocyte fluctuations, stable amegakaryocytic thrombocytopenia developed and the patient died of haemorrhagic stroke.

Stimulation of human platelets with concanavalin A resulted in a significant increase in the concentration of cytoplasmic free Ca2+. This effect was due to two different processes: Ca2+ mobilization from internal stores and Ca2+ influx from the extracellular medium. Kinetic analysis revealed that the release of Ca2+ from internal storage sites occurred sooner than the opening of plasma membrane Ca2+ channels. The ability of concanavalin A to induce a sustained increase in cytoplasmic Ca2+ concentration was antagonized and reversed by methyl alpha-D-mannopyranoside, demonstrating that it was promoted by the interaction of the lectin with cell surface glycoproteins. Succinyl-concanavalin A, a dimeric derivative of the lectin, that does not promote patching/capping of the receptor, was able to bind to the platelet surface, and antagonized the effects of native concanavalin A. In addition, succinyl-concanavalin A, per se, was unable to induce Ca2+ mobilization in human platelets. Therefore, the action of the native concanavalin A was mediated by receptor clustering events. Concanavalin A mobilized Ca2+ from the same internal stores from which Ca2+ was mobilized in response to strong platelet agonists, such as thrombin and arachidonic acid. However, while thrombin was ineffective in inducing Ca2+ release after stimulation of platelets with ConA, ConA was able to cause a full discharge of Ca2+ from internal stores even in platelets previously stimulated with thrombin. These results demonstrate for the first time that the clustering of specific membrane glycoproteins can trigger platelet activation. The physiological implications during platelet aggregation are discussed.

The intracellular distribution of the low-molecular-weight GTP-binding protein rap2B was investigated in resting and agonist-activated human platelets. In both cases, platelets were lysed by Triton X-100, and cell fractions were obtained by differential centrifugations. Using a specific polyclonal antiserum, we found that rap2B in resting platelets was completely detergent-soluble. When platelets were aggregated with thrombin, the thromboxane analogue U46619, or the Ca(2+)-ATPase inhibitor thapsigargin, a significant amount of rap2B became associated with the cytoskeleton. This association was paralleled by a decrease of rap2B in the Triton X-100-soluble fraction. Translocation of rap2B to the cytoskeleton strictly depended on platelet aggregation, and maximal incorporation was found when approximately 50% aggregation was measured. Inhibition of fibrinogen binding to the glycoprotein IIb-IIIa complex completely prevented the interaction of rap2B with the cytoskeleton. These results clearly demonstrate that changes in the intracellular localization of rap2B occur during platelet activation and represent evidence that this low molecular weight GTP-binding protein may be involved in platelet function.

We investigated ex vivo spontaneous platelet aggregation (SPA) in platelet-rich plasma in 37 patients with acute myocardial infarction. It occurred in about 50% of subjects receiving heparin after streptokinase treatment, while it rarely took place in patients who did not receive either streptokinase or heparin and in those treated with streptokinase alone. The study of patients receiving heparin for deep vein thrombosis suggested that SPA may derived from adenosine diphosphate released from platelets during sample handling. We suggest that heparin infusion may facilitate ex vivo platelet activation and that this mechanism is operative in patients with acute myocardial infarction who have undergone thrombolytic therapy.

Background: The technique of freezing blood platelets could be very useful in the transfusion support of thrombocytopenic patients. The best method of platelet cryopreservation still remains an object of debate, though it has been suggested that dimethyl sulfoxide (DMSO) is more effective than glycerol-glucose as a cryopreservative. However, few studies have directly compared platelets cryopreserved with different methods., Methods: We compared "in vitro" function of platelets cryopreserved with 5% dimethyl sulfoxide (DMSO) or 3% glycerol-glucose at -140 degrees C. Platelet aggregation and release reaction were studied with a Lumi aggregometer, thromboxane B2 (TxB2) production by radioimmunoassay, and Ca++ movement by the Fura 2 method., Results: Cryopreservation with both of the methods dramatically reduced the ability of platelets to release ATP and to aggregate in response to single agonists. In contrast, cryopreserved platelets maintained their ability to aggregate after stimulation with paired agonists and to produce TxB2. Cytoplasmic Ca++ increase induced by thrombin was observed in the glycerol-preserved platelets, while it was nearly absent in the DMSO-preserved ones., Conclusions: We suggest that cryopreservation with glycerol-glucose or DMSO induces similar defects of platelet function. The damage is severe, but platelets are still able to respond to strong stimulation.

A young patient developed chronic idiopathic thrombocytopenic purpura. Prednisone therapy normalized platelet number, but bleeding symptoms did not disappear. Platelet function was severely impaired, since platelet aggregation, ATP release and adhesion to collagen and subendothelial matrix were significantly reduced. Plasma and purified immunoglobulins of the patient reproduced the functional defects in normal platelets. Immunoblotting revealed that patient's plasma contained an antibody reacting with a component of platelets with the same electrophoretic mobility of glycoproteins IIIa of normal platelets. Moreover, patient's plasma inhibited the binding of an anti-GPIIb/IIIa monoclonal antibody to platelet surface. Additional immunosuppressive therapy with prednisone and azathioprine normalized platelet function and induced the disappearance of bleeding symptoms.

In response to concanavalin A, cytoplasmic calcium movement was observed in human platelets, both in the presence of 1 mM Ca2+ or 1 mM EGTA in the medium. Concanavalin A also caused the activation of inositide turnover and the production of inositol phosphates, suggesting that activation of phospholipase C occurs. The mechanism by which concanavalin A stimulates phospholipase C does not depend on GTP-binding transducers, because it was not inhibited by GDP beta S, while experiments performed in the presence of cytochalasin B suggested a role for membrane glycoprotein IIb-IIIa-cytoskeleton interaction in this process. Ca(2+)-proteases and Na+/H+ antiport also seemed to be related to concanavalin A-induced phospholipase C activation, as suggested by experiments performed in the presence of leupeptin and amiloride.

Whereas in vitro studies showed that plasmin may induce both inhibition and activation of platelets, in vivo and ex vivo investigations suggested that thrombolytic agents are responsible for platelet stimulation. To gain further information on this topic, ex vivo platelet function was studied in 24 subjects with acute myocardial infarction treated with streptokinase or recombinant tissue-type plasminogen activator (rt-PA). Ten patients with acute myocardial infarction who did not receive thrombolytic treatment were also investigated. The data shows that at the end of thrombolytic infusion, the maximal extent of platelet aggregation and adenosine triphosphate release was reduced in treated patients compared with that in untreated ones. In subjects treated with streptokinase, the defect in platelet aggregation derived from both cellular and plasmatic defects. Plasmatic beta-thromboglobulin concentration was significantly reduced after streptokinase, but unchanged after rt-PA. Three days after thrombolytic treatment, platelet aggregation of patients receiving streptokinase or rt-PA was not significantly different from that of untreated subjects. A similar defect in platelet function was obtained in vitro, incubating normal platelet-rich plasma with pharmacologic concentrations of streptokinase. Again, platelet function defect derived from both cellular and plasmatic damages. It cannot be excluded that platelet activation occurs in patients with acute myocardial infarction during the very early phases of thrombolytic treatment. However, it is suggested that a transient defect in platelet function follows both streptokinase and rt-PA infusion.

Calf cornea slices were incubated with [U-14C]glucose, in varying pO2 or lactate concentrations. Acid glycosaminoglycans were separated by ion-exchange chromatography after papain digestion. The percentage radioactivity incorporated into keratan sulphate increased markedly with decreased oxygen tension, whereas a concomitant relative decrease of the biosynthesis of glycosaminoglycuronans occurred. Similar results were obtained with increased lactate concentration. Our findings support the idea that keratan sulphate is a functional substitute for chondroitin sulphate in conditions of oxygen lack (Scott, J.E. and Haigh, M. (1988) J. Anat. 158, 95-108).

Background: Anagrelide is a quinazolin compound developed initially as an inhibitor of platelet aggregation. Since "in vivo" studies demonstrated that it was responsible for thrombocytopenia in humans, anagrelide has been used recently in a small number of patients with thrombocytosis and myeloproliferative disorders. Platelet count was well controlled in the large majority of patients, and only minimal side effects were observed., Patients: Eight patients (5 with essential thrombocythemia, 2 with chronic granulocytic leukemia, and 1 with idiopathic myelofibrosis) received anagrelide (induction dose 4 mg/die; mean maintenance dose 2 mg/die; mean observation time 26 weeks). Complete blood counts were determined 4 times during the first month, and subsequently every month. "In vivo" and "ex vivo" platelet function was studied before anagrelide and after 4 and 10 days of therapy., Results: Platelet count was reduced and maintained below 500 x 10(9)/L in 5 of 8 patients. Headache, palpitation/tachycardia, gastrointestinal symptoms and a decrease in hemoglobin were the side effects. Anagrelide did not modify the leukocyte count or "in vivo"/"ex vivo" platelet function., Conclusions: Anagrelide may control thrombocytosis in patients with myeloproliferative disorders, even when traditional drugs have failed. When required, anti-aggregating drugs may be associated with anagrelide, since it has no effect on platelet function.

We studied the effect of inhibition of platelet aggregation (obtained by omitting sample stirring and by the addition of ASA and GRGDS peptide) on ATP secretion and Ca(2+) movements. When collagen was the agonist, platelet aggregation stimulated release reaction and Ca(2+) movements in both the presence and absence of extracellular Ca(2+). When platelets were stimulated by thrombin, ATP release and Ca(2+) movements were largely independent of aggregation. Our data suggest that platelet aggregation stimulates Ca(2+) movement, and that this phenomenon of feedback amplification of platelet activation plays an important role in platelet function when collagen is the agonist, while it has little or no role when thrombin is the stimulus.

The state of oxidation of membrane proteins was analyzed in red cell subpopulations of different age by quantifying the oxidation of methionine to its sulfoxide and by determining the amount of thiol groups in ghost membrane preparations and the reactivity of thiols of individual membrane proteins in intact cells. The results obtained show that oxidation of methionine occurs early during red cell life in the circulation, and can be detected in middle-aged and senescent cells. Thiol content of ghost membranes is kept constant in all the red cell subpopulations analyzed, but reactivity of thiol groups to the thiol reagent N-(7-dimethyl-amino-4-methyl-coumarinyl) maleimide (DACM) in intact cells decreased 30% in alpha-spectrin, band 3 (B3), 4.1 and 4.2 proteins, probably as a consequence of conformational changes of these molecules. Since the role played by band 3 in the exposure of senescence antigen has been described by many authors, the functional activity of the anion transporter has been analyzed by measuring the 4-4'-diisothiocyano-stilbene-2-2-'-disulfonate (DIDS) binding capacity in different red cell subpopulations. The results obtained are in agreement with the possibility that during senescence band 3 undergoes conformational changes involving the anion channel subsite being more exposed to the extracellular space and responsible for binding of DIDS.

Platelet membrane glycoprotein IIb-IIIa has been widely studied in the last years because of its role as an activation-dependent, adhesive protein receptor. Recently we demonstrated that occupancy of glycoprotein IIb-IIIa-receptor sites by specific ligands exerts an inhibitory effect on platelet responses induced by mild stimulation, leading us to suppose that this event may interact with activation pathways. Although the mechanisms of signal transduction in human platelets are not completely elucidated, the hypothesis that GTP-binding proteins are involved is generally accepted. Our results demonstrate that platelet ConA receptors, known to be located mainly on GP IIb-IIIa, are able to bind [35S]GTP gamma S; the GTP-binding activity is specific and is due to the association with the receptors of two G-proteins, with apparent molecular masses of 25 and 21 kDa, respectively. After the purification of GP IIb-IIIa, a glycoprotein complex electrophoretically pure was obtained that was still associated with a GTP-binding activity, migrating in SDS-polyacrylamide gel electrophoresis as a narrow band of about 21 kDa.

Among 761 consecutive patients with chronic myeloproliferative disorders (CMD), it was found that 18 (nine men and nine women) did not fulfill at presentation the established diagnostic criteria for the typical forms. In seven patients, the diagnosis of CMD was made on the basis of an intense and persistent thrombocytosis that complicated splenectomy. The other 11 patients had various combinations of the following signs suggesting CMD: splenomegaly, bone marrow myeloid hyperplasia and/or slight myelofibrosis, mild thrombocytosis and/or leukocytosis, and rare immature myeloid cells in the peripheral blood. All patients were younger than 46 years of age (median age, 31.5 years; range, 20 to 45 years). A major thrombotic event was the most frequent presenting feature (eight of 18 cases), and thrombotic complications supervened in seven of the eight splenectomized patients (six in the portal system), raising the overall rate of patients with thrombotic events in their history to 11 of 18. At a median follow-up of 50 months (range, 24 to 241 months), three patients had died of thrombotic complications (two after splenectomy). The 15 surviving patients had stable disease, and 12 of them were not receiving cytoreductive therapy. Spontaneous growth of circulating burst-forming units erythroid was demonstrated in one patient, and erythroid responsiveness to erythropoietin appeared higher than in the normal controls in four. Spontaneous in vitro platelet aggregation in whole blood and/or platelet-rich plasma was seen in five of seven patients. It was concluded that a difficult to identify, slowly progressive form of CMD occurs in young people, that it carries a high risk of thrombosis, and that splenectomy is a high risk procedure in these cases.

A young man with bleeding symptoms, mild thrombocytopenia and abundant marrow megakaryocytes was classified as having idiopathic thrombocytopenic purpura. Neither prednisone therapy nor splenectomy modified the clinical picture. Subsequently, a severe defect of platelet aggregation and release reaction was demonstrated. Fifteen months after the onset of bleeding symptoms, fever and hepatomegaly appeared and the diagnosis of T cell non Hodgkin lymphoma was made on the basis of a histologic review of paraffin sections of the spleen. Chemotherapy induced remission of the lymphoma, disappearance of bleeding symptoms and normalization of the platelet count and function.

Oxidative lesions to membrane proteins were studied in human erythrocytes of different age and were evaluated on ghost membrane preparations by assaying thiol and methionine sulphoxide groups, and in situ on intact cells, after treating erythrocytes with the fluorochrome N-(7-dimethyl-amino-4-methyl-coumarinyl) maleimide (DACM). DACM reacts with thiol groups and the amount of this reagent bound by membrane proteins was quantified after SDS-PAGE separation. Results obtained show that during aging of normal cells the oxidative state of membrane proteins increases: this was better shown by the assay of methionine sulphoxide residues rather than by the thiol titration, when studies were carried out on ghost membranes. After separation of individual membrane proteins by SDS-PAGE, decreased accessibility of DACM to thiol groups of band 3 and of the main proteins of the membrane skeleton was evident in senescent erythrocytes. These results show that during aging, band 3 and membrane skeleton proteins undergo conformational changes and/or oxidation. Similar results were obtained when thiol distribution was studied in membrane proteins separated by SDS-PAGE in both reducing and non-reducing conditions.

In vitro platelet aggregation in platelet-rich plasma (PRP) and in whole blood (WB) was assessed in 31 patients with idiopathic myelofibrosis, 32 with essential thrombocytosis, 23 with polycythemia vera, and 34 with chronic myelogenous leukemia. In PRP most subjects showed normal or reduced platelet aggregation, whereas in WB the majority of patients showed increased platelet function. Spontaneous platelet aggregation (SPA) was observed frequently in WB, whereas it was seldom observed in PRP. SPA in WB was inhibited by in vitro addition of aspirin and apyrase, and SPA was only partially dependent on high platelet count because it also occurred in samples with normal platelet content (at variance with 13 subjects with reactive thrombocytosis, in which SPA was observed only in samples with high platelet concentration). Platelets from patients with idiopathic myelofibrosis had the highest tendency to undergo SPA.