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Biomedical subjects

M L Lozano

Publications and source records attributed to M L Lozano.

At least 19 recordsLinked to original sources
PubMed2005

Flavonoids inhibit platelet function through binding to the thromboxane A2 receptor.

BACKGROUND: Dietary flavonoids are known for their antiplatelet activity resulting in cardiovascular protection, although the specific mechanisms by which this inhibition occurs has not been fully established. OBJECTIVE: The aim of this study was to investigate the interaction of nine flavonoids representative of various chemical classes, with platelet responses dependent on thromboxane A(2) (TxA(2)) generation and on receptor antagonism, and to analyze the structural requirements for such effects. METHODS: The effect of several types of flavonoids on platelet aggregation, serotonin release, and TxA(2) generation was investigated. Competitive radioligand binding assays were used to screen for affinity of these compounds to TxA(2) receptors. RESULTS: Flavones (apigenin and luteolin) and isoflavones (genistein) abrogated arachidonic acid and collagen-induced platelet responses, such as aggregation and secretion, with a less substantial effect on TxA(2) synthesis. These compounds were identified as specific ligands of the TxA(2) receptor in the micromol L(-1) range, this effect accounting for antiplatelet effects related to stimulation with those agonists. Tight binding of flavonoids to the human TxA(2) receptor relies on structural features such as the presence of the double bond in C2-C3, and a keto group in C4. CONCLUSIONS: The inhibition by specific flavonoids of in vitro platelet responses induced by collagen or arachidonic acid seems to be related, to a great extent, to their ability to compete for binding to the TxA(2) receptor. Therefore, antagonism of this TxA(2) receptor may represent an additional mechanism for the inhibitory effect of these compounds in platelet function.

Apigenin↗
PubMed2004

Evaluation of refrigerated platelet concentrates supplemented with low doses of second messenger effectors.

With the goal of producing haemostatically effective platelet concentrates (PCs) with a longer shelf-life, we aimed to identify a simple combination of platelet inhibitors, with a low pharmacological load, which could avoid the unacceptable loss of platelets stored under refrigerated conditions. PCs stored with different combinations of second messenger effectors were analysed at days 5, 10 and 15 of storage and compared with those supplemented with ThromboSol--a combination of six platelet inhibitors that protects cells from cold damage. The following parameters were analysed: platelet counts, biochemical parameters (glucose, pH, bicarbonate, lactate), cell lysis (lactic dehydrogenase, LDH), membrane glycoproteins (GPs), platelet aggregation, fibrinogen binding and hypotonic shock response. We characterized the combination of amiloride and sodium nitroprusside (at 1/2 the dose included in ThromboSol). This was found to be similar to ThromboSol and superior to nontreated units in the prevention of cold-induced platelet aggregation at day 15 of storage (maintenance of 78% and 80% of initial platelet counts, respectively), preservation of GPIbalpha (11% and 12% better maintenance of mean fluorescence intensity compared with control units, respectively), and reduced cell lysis (13% and 11% decrease in supernatant LDH, respectively). The reduced pharmacological load with the identified solution compared with ThromboSol is an argument in favour of the potential use of these agents when designing strategies to improve PC storage.

Blood Platelets↗
PubMed2003

Application of a new enzyme-linked immunosorbent assay for detection of total hepatitis C virus core antigen in blood donors.

Recent studies have shown that total hepatitis C virus (HCV) core antigen, both free and antibody bound, is an accurate indirect marker of viral replication that can be used in clinical practice. The aim of the present study was to evaluate the performance of a new total HCV core antigen enzyme-linked immunosorbent assay (ELISA) for detection and quantification of total core antigen in blood donors, testing positive for anti-HCV antibodies and for prospective low-risk population screening. A population comprising 257 samples, from blood donors detected reactive for anti-HCV antibodies [137 recombinant immunoblot assay (RIBA) positive and 120 RIBA indeterminate], were tested by using a new total HCV core antigen ELISA. HCV-RNA was quantified by using quantitative polymerase chain reaction (PCR) assays in all RIBA-positive samples and RIBA-indeterminate samples that were positive for the total core antigen. Specificity of the assay was studied in 1070 healthy blood donors negative for anti-HCV antibodies. Compared with quantitative PCR assays, the total HCV core antigen assay showed 97.37% sensitivity. The three HCV-RNA-positive samples, which tested negative for the total core antigen, had a low viral load (< 1.4 x 10(4) IU mL(-1)). All samples with more than 1.4 x 10(4) IU mL(-1) of viral RNA were positive for total core antigen, independent of the HCV genotype. Concentration of total core antigen correlated significantly with those of HCV-RNA (r = 0.614, P < 0.0001). Overall specificity in freshly collected blood donor specimens was 99.63%. Our data indicate that the total HCV core antigen ELISA has a sensitivity close to PCR assays in diagnosing HCV infection in blood donors with anti-HCV antibodies and shows an excellent specificity in volunteer donors. This assay, in combination with anti-HCV antibodies screening tests, could be an alternative to molecular assays for HCV infection screening in blood donors.

Blood Donors↗
PubMed2002

Platelet aggregation through prothrombinase activation induced by non-aggregant doses of platelet agonists.

Activation of the prothrombinase complex, which catalyzes the formation of thrombin from prothrombin, is crucial for the (patho)physiological processes of hemostasis and thrombosis. We here report that washed platelets supplemented with prothrombin can be irreversibly aggregated with otherwise non-aggregant doses of adenosine diphosphate (10 micromol/l), thrombin (0.06 U/ml), or collagen (1 microg/ml). Prothrombinase-catalyzed prothrombin to thrombin conversion most probably supports this aggregation response, since inhibitors of thrombin (hirudin or heparin) and an inhibitor of activated factor X (DX-9065a) impair the response. A certain degree of agonist-induced platelet activation seems to be required for this prothrombin-supported aggregation response, since prothrombin alone does not induce aggregation, and blockade of glycoprotein Ia/IIa with a specific antibody inhibits the platelet aggregation response to collagen and prothrombin. These results may suggest that activation of the prothrombinase complex could be a common step of the platelet response to distinct agonists, which may be achieved at low levels of platelet stimulation.

Adenosine Diphosphate↗
PubMed2001

Administration of post-autologous PBSCT rhG-CSF is associated with long-term low concentrations of bone marrow hematopoietic progenitor cells.

Recombinant human granulocyte colony-stimulating factor (rhG-CSF) has been widely used after autologous peripheral blood stem cell transplant (APBSCT) in an attempt to reduce the duration of neutropenia, but whether this treatment has any influence on long-term engraftment remains unknown. We have retrospectively analyzed data from breast cancer patients to compare post-APBSCT rhG-CSF administration in terms of the short-term benefit and myeloid marrow regeneration after 1 year. Group A included 10 patients not treated with post-APBSCT rhG-CSF, while groups B and C comprised 15 and 13 patients treated with this drug from days +1 and +6, respectively. No differences among the three groups were found in age, diagnosis, previous chemo-radiotherapy, CD34+/CD71- cell concentration in pre-transplant bone marrow (BM), mobilization schedule, CD34+ cell yield, conditioning regimen and post-transplant radiotherapy. Post-APBSCT rhG-CSF was shown to accelerate neutrophil recovery, but there were no significant differences in platelet recovery, transfusion requirements, days of fever, antibiotic administration or inhospital stay. With regard to BM hematopoietic precursors 1 year after APBSCT, significantly lower concentrations of total CD34+ cells, committed CD34+/CD33+ subsets, and more immature CD34+/CD71- cells were found in both groups B and C compared with patients not having received the cytokine (group A). Thus, post-APBSCT rhG-CSF administration does not appear to beneficially affect procedure outcome, and might even impair long-term marrow hematopoiesis.

Adult↗
PubMed2001

Polymorphisms of P-selectin glycoprotein ligand-1 are associated with neutrophil-platelet adhesion and with ischaemic cerebrovascular disease.

P-selectin glycoprotein ligand (PSGL-1) shares common features with platelet glycoprotein Ibalpha. A recently described polymorphism in this receptor that results in a variable number of tandem repeats (VNTR) sequence present either 16, 15 or 14 times (alleles A, B or C) could, similar to GPIbalpha, be functionally relevant. The allelic frequency of this polymorphism was investigated in 469 individuals from the south of Spain, and was similar to that previously described in other Caucasian populations: 85% A, 14% B and 1% C alleles. We identified two new polymorphisms genetically linked to the C isoform, resulting in the Ser273Phe and Met274Val changes. To assess the functional consequence of the polymorphisms in the receptor, we performed flow cytometric analysis of P-selectin dependent neutrophil-platelet interaction. Neutrophils carrying the shortest C allele and the amino acid variations in residues 273 and 274 exhibited a significantly lower capacity to bind activated platelets than A/B and A/A samples (mean fluorescence intensity of CD42b+ neutrophils 262 versus 303 and 319 respectively, P < 0.05). The distribution of the VNTR was analysed in three case-control studies including 104 cerebrovascular (CVD), 101 coronary heart disease (CHD) and 150 deep venous thrombosis (DVT) patients. The results showed that smaller (B and C) alleles seem to be associated with a lower risk of developing CVD (P = 0.008) but not to be related to CHD or DVT. In conclusion, polymorphisms of the PSGL-1 receptor may influence the neutrophil-platelet binding, and represent a risk factor for CVD.

Adolescent↗
PubMed2001

Binding of recombinant apolipoprotein(a) to human platelets and effect on platelet aggregation.

The interaction of lipoprotein(a) [Lp(a)] with platelets is not well defined, particularly with regards to the individual contribution of the protein components of Lp(a), the apo B-100 and the apolipoprotein apo(a). This study investigated the binding of different recombinant apo(a) [r-apo(a)] isoforms, to human platelets and its effect on platelet aggregation. Scatchard analysis of saturation binding experiments demonstrated that human platelets display a single class of high affinity r-apo(a) binding sites (71 +/- 46 molec./platelet, Kd = 5.6 +/- 2.0 nmol/L). Platelet activation with strong agonists (thrombin, arachidonic acid) increased 2- to 10-fold the r-apo(a) binding, without affecting the affinity. Competition assays showed that the binding sites are highly specific for r-apo(a) and Lp(a). At high concentration t-PA could also bind to the r-apo(a) binding sites. By contrast, neither fibrinogen nor plasminogen inhibited to the r-apo(a) binding. The lysine analogue EACA inhibits the binding of r-apo(a) to platelets, thus suggesting the involvement of lysine residues in that interaction. Moreover, the r-apo(a) binding to platelets is unlikely mediated by GPIIb/IIIa-attached fibrin since it is not affected by platelet treatment with either LJ-CP8, a monoclonal antibody that specifically blocks fibrinogen binding to GPIIb/IIIa, nor GPRP, an inhibitor of fibrin polymerisation. Finally, we show that the distinct recombinant apo(a) proteins, as well as native Lp(a), promote an aggregation response of platelets to otherwise subaggregant doses of arachidonic acid. This proaggregant effect of r-apo(a) is dependent on its binding to platelets since it requires a minimum incubation time, and it is prevented by EACA at concentration inhibiting the r-apo(a)-platelet interaction. These results suggest that the prothrombotic action of Lp(a) may be in part mediated by modulating the platelet function through the interaction of its apo(a) subunit with a specific receptor at the platelet surface.

Adenosine Diphosphate↗
PubMed2001

Effect of cold-storage in the accumulation of bioreactive substances in platelet concentrates treated with second messenger effects.

BACKGROUND AND OBJECTIVES: The mandatory 5-day of shelf-life platelet concentrates (PCs) creates outdating and inventory control problems in blood banking. Moreover, storage of PCs at 22-24 degrees C has been associated with a time-dependent accumulation of pyrogenic cytokines, potentially harmful for recipients. Previous studies have shown that supplementation of PCs with ThromboSol, a mixture of second-messengers effectors, might allow storage of functionally active platelets at refrigerated temperature to be extended. This study further investigates this storage approach by comparing the accumulation of bioactive compounds in standard and refrigerated PCs. DESIGN AND METHODS: The PCs were supplemented with ThromboSol or a control solution and stored in parallel at 24 degrees C with continuous agitation or undisturbed at 4 degrees C. Samples were removed on days 1, 5, 9 of storage, and assayed for their content of interleukin (IL)-6, IL-8, tumour necrosis factor (TNF)-alpha, transforming growth factor (TGF)-beta1, and anaphylatoxins C3a and C4a. RESULTS: Throughout storage, refrigerated PCs, both ThromboSol-treated and untreated units, displayed a slightly lower level of IL-6 and significantly lower concentration of IL-8 than conventionally stored PCs. ThromboSol slightly reduced the level of these cytokines in PCs. Throughout storage at 22 degrees C, an accumulation of anaphylatoxins C3a and C4a was seen both in both control and ThromboSol-treated PCs. This accumulation was significantly reduced in control PCs stored at 4 degrees C, but not in refrigerated PCs supplemented with ThromboSol. Cold-storage, with or without ThromboSol, had a minor effect on the accumulation of TGF-beta1 in PCs. INTERPRETATION AND CONCLUSIONS: Our data confirm that release of bioactive compounds during in vitro storage of PCs is a temperature-sensitive process. The ThromboSol-refrigeration system could be a useful alternative for extending storage of PCs, without increasing the accumulation of cytokines (IL-6, IL-8), known to be involved in febrile reactions in recipients. Nevertheless, this storage system has no benefit on the level of other bioactive compounds (TGF-beta1, anaphylatoxins C3a and C4a) in PCs.

Blood Platelets↗
PubMed2000

Platelet GP Ib/IX/V complex: physiological role.

Platelets play an essential role in primary hemostasis and in thrombotic events, particularly in arterial vessels, as rheological conditions originate closer interactions between platelets and endothelium than lower shear rates. In response to vascular injury, platelets adhere to the subendothelial matrix by membrane receptors potentiating the generation of thrombin, become activated, and a series of biochemical processes induce platelet aggregation and liberation of intracellular metabolic products to the extracelular medium. Among platelet receptors, glycoprotein (GP) Ib/IX/V complex is peculiar, as it binds adhesive proteins, mainly von Willebrand factor (vWF), and thrombin, the main platelet agonist. Platelet adhesion and subsequent aggregation under conditions of high shear flow, essentially relies upon this receptor's capacity of binding to the subendothelial matrix, initiating signal transduction. Two proteins associated to GP Ib/IX/V, actin-binding protein (ABP) 280 and 14-3-3zeta, are potential mediators of signal transduction by the complex, but their specific contribution in this process is not yet fully understood. Additionally, two proteins implicated in signal transduction by immune stimuli, FcgammaRIIA and FcR gamma-chain, associate with GPIb/IX/V complex, and increasing data indicate a potential role in GPIbalpha mediated signal transduction.

14-3-3 Proteins↗
PubMed2000

Mobilization of hematopoietic progenitor cells with paclitaxel (taxol) as a single chemotheraupetic agent, associated with rhG-CSF.

We assessed the mobilization capacity of taxol with rhG-CSF, both as a single chemotherapeutic agent and in the presence of cyclophosphamide (CY), and compared the effect with yields achieved when mobilization was performed solely with rhG-CSF. Fifteen patients with breast cancer received taxol 170 mg/m2 (continuous infusion, day 1) and rhG-CSF (8 microg/kg/day, from day 2 until the end of apheresis) (T-G group), while seven breast cancer patients were additionally treated with CY (4 g/m2) on day 2, followed by rhG-CSF starting at similar doses on day 3 (T-CY-G group). The PBSC collections after taxol with/without CY were compared with those of 30 breast cancer patients who had received rhG-CSF (8 microg/kg/day) for mobilization. No differences were found in the characteristics of patients included in any of the three mobilization groups. The median yield of CD34+ cells from all patients included in taxol containing schedules was 9 x 106/kg (range 2-26) collected with a median of one apheresis procedure (range 1-4). Leukaphereses began earlier in the T-G group (median day 8, range 7-10) than in the T-CY-G group (median day 13, range 11-17). In most patients (20 out of 22) who received taxol containing regimens, more than 2.5 x 106 CD34+ cells/kg, a threshold considered to be sufficient for hematopoietic reconstitution, were collected with a single apheresis. Those patients in the T-G group experienced less neutropenic and thrombocytopenic days, with all neutropenic fever episodes developing in patients treated with the T-CY-G schedule (43%). When considering priming with rhG-CSF alone in our historical cohort of 30 breast cancer patients, a significant detrimental effect was observed in comparison with taxol mobilizing schedules, in the number of aphereses performed, in the total yield CD34+cells and in the number of patients who achieved the target dose of 2.5 x 106/kg CD34+ cells within the first collection procedure. We conclude that taxol containing schedules are effective in mobilizing PBSC and facilitate the collection of high yields of CD34+ cells (usually more than 5 x 106/kg recipient body weight) with a reduced number of apheresis procedures. Taxol, as a single agent with rhG-CSF, exhibits less hematological toxicity than the combination chemotherapy mobilization regimen including CY. Bone Marrow Transplantation (2000) 25, 231-235.

Adult↗
PubMed2000

Re-mobilization of peripheral blood progenitor cells within a short time interval fails to achieve effective progenitor cell yields.

We report the case of a healthy donor who was mobilized for the purpose of performing an unrelated donor transplantation with subcutaneous injections of rhG-CSF. Because of accidental loss of progenitors, 3 days after completing the first collection, the donor was mobilized again with rhG-CSF, and progenitors were collected. While a similar increase in the pre-apheresis leukocyte count was observed in both procedures, fewer mononuclear cells were mobilized during the second rhG-CSF course, resulting in a poor CD34+ yield. These data suggest that an 8-day interval between commencement of rhG-CSF mobilizations is insufficient to predict an efficient collection of hematopoietic progenitors to ensure engraftment after bone marrow transplantation.

Female↗
PubMed2000

Evaluation of pooled platelet concentrates using prestorage versus poststorage WBC reduction: impact of filtration timing.

BACKGROUND: Concern for the undesirable consequences of transfusing passenger WBCs is leading to the general use of WBC-reduced platelet concentrates (PCs). However, the impact of prestorage versus poststorage WBC reduction on the quality of platelet products has not been clearly defined. STUDY DESIGN AND METHODS: Pooled PCs were WBC reduced before or after 5-day storage, by use of a WBC filter (PXL-8, Pall Corp.). Samples from pools were taken on days 1 and 5, before and after filtration, and on Day 9 of storage and assessed for cell counts, biochemical values, expression of platelet glycoproteins, thrombin generation, and content of IL-6, IL-8, TNFalpha, transforming growth factor beta1 (TGFbeta1), and anaphylatoxins C3a and C4a. RESULTS: Filtration of fresh and 5-day-stored pooled PCs via a PXL-8 filter was similarly efficient, rendering pools with low WBC counts (<1 x 10(6) cells) and high platelet recovery (>95%). No major changes were found in the metabolic behavior or the expression of platelet GPIb, GPIIb/IIIa, CD62, and CD63 in PCs filtered before or after storage. Filtration, either before or after storage, increased by less than 5 percent the proportion of CD62+ platelets. Moreover, no changes were found in the concentration of prothrombin fragments 1 and 2 and thrombin-antithrombin complexes in the pooled PCs derived from the time of filtration. Finally, prestorage WBC reduction abrogated the accumulation of IL-6 and IL-8, but it did not prevent that of anaphylatoxins C3a and C4a nor of TGFbeta1. However, filtration through a PXL-8 filter significantly reduced (40-90%) the amount of IL-8, C3a, and C4a in the filtrate. CONCLUSIONS: The timing of PXL-8 filtration of PCs has little impact on the efficiency of WBC reduction and on platelet recovery, and it does not seem to affect the quality of platelets or the generation of thrombin in the PCs. As regards the goal of reducing the amount of bioactive products in PCs, it remains uncertain as to whether prestorage WBC reduction fully eliminates the need for poststorage filtration. Prestorage filtration leads to low levels of IL-6 and IL-8 in PCs, but it does not impair the poststorage content of TGFbeta1 or anaphyla-toxins. By contrast, poststorage PXL-8 filtration removes significant amounts of C3a and C4a, and thus it might provide clinical benefits beyond those of prestorage WBC reduction.

Blood Preservation↗
PubMed2000

A common polymorphism flanking the ATG initiator codon of GPIb alpha does not affect expression and is not a major risk factor for arterial thrombosis.

The platelet membrane glycoprotein (GP) Ib alpha plays a key role in the initial formation of thrombi. Polymorphisms (VNTR and HPA-2) in this receptor are associated with increased risk of coronary heart disease (CHD) and cerebral vascular disease (CVD). We investigated whether a recently described polymorphism (S/R), due to a single base change (T-->C) five nucleotides upstream the initiator codon of GPIb alpha, might influence the expression of the protein, and be implicated in the development of arterial thrombosis. One hundred and thirty nine healthy individuals provided blood samples for DNA analysis of platelet GPIb alpha polymorphisms, and for flow cytometric analysis of the surface expression of the receptor. A group of 20 S/R normal individuals and an identical number of S/S participants, age and sex matched, was investigated for the analysis of the density of various platelet receptors. The distribution of the S/R polymorphism was also analyzed in two case/control studies including 104 CVD patients, 101 CHD patients, and one control age, sex, and environmental risk factors matched for each case patient. Surface density of GPIb alpha showed no wide variations between individuals, was not influenced by the presence of S or R alleles, nor associated with the VNTR or HPA-2 polymorphisms. The prevalence of the S/R genotype among CVD and CHD patients was not distinct from that in the control groups. We conclude that the S/R polymorphism of GPIb alpha, flanking the initiator codon of the receptor, does not seem to be associated with surface levels of the protein, and is not an independent risk factor for arterial thrombosis.

Adult↗
PubMed1999

[The education of type-2 diabetics: why not in groups?].

OBJECTIVE: To evaluate the efficacy of a programme of group education for diabetics, which succeeded in reducing base glucaemia, body mass index and glycosylated haemoglobin, stimulating insulin-treated patients' self-monitoring of glucaemia, improving their understanding of the disease, and bringing into group education 75% of the target population. DESIGN: A controlled clinical trial with paired samples, lasting two years. SETTING: "Las Fuentes Norte" Health Centre, Zaragoza. PATIENTS: 243 type-2 diabetics, distributed at random, with 120 in the intervention group and 123 in the control group. All patients with no history of group education in the previous two years and with no restrictions on their attending talks and monitoring were included. No-one gave up in the first year; 9 did in the second year. INTERVENTION: Group health education workshop for two days in the first and second years. MEASUREMENTS AND MAIN RESULTS: In the intervention group, at the end of the study, success in the survey increased by 4.2; base glucaemia dropped by 17.1 mg/dl, glycate haemoglobin went down by 0.5, and self-analysis went up by 14.2%, all with significant differences (p < 0.005 and CI 95%). The body mass index went down by 0.3 without significant differences. Knowledge was lost between the first and second year, with worse metabolic control. In the control group there were no significant differences in any variable. CONCLUSIONS: A stable programme of group education may be effective, especially because of the increase in knowledge of the disease, fostering excellent conditions for a change to a healthier life-style.

Adult↗
PubMed1999

Platelet cryopreservation using a reduced dimethyl sulfoxide concentration and second-messenger effectors as cryopreserving solution.

Cryopreservation of platelets is of great interest since it could extend to years the shelf life of therapeutic platelet concentrates (PCs) and facilitate stockpiling and inventory control in blood banking. We have compared the cryopreservation of PCs by the standard method using 6% Me(2)SO as cryoprotectant with the method of freezing employing low concentrations of Me(2)SO (2%) plus ThromboSol, a mixture of second-messenger effectors that protect platelets from cold damage. PC pools were treated either with 6% Me(2)SO or with ThromboSol and 2% Me(2)SO and then placed directly in a -80 degrees C freezer or in the vapor phase of a liquid nitrogen freezer (-120 degrees C). After storage for 1 week or for 3 months, samples were removed, thawed, and analyzed. Measurements included cell recovery, biochemical parameters, membrane glycoproteins (GPs), platelet aggregation, and binding of radiolabeled von Willebrand factor (vWF) and fibrinogen. PCs cryopreserved with ThromboSol and 2% Me(2)SO displayed a platelet recovery (90%) equivalent to those frozen with 6% Me(2)SO. Following either cryopreservation procedure, platelets showed increased surface expression of P-selectin and moderate loss of GP Ibalpha in comparison to fresh platelets. The aggregatory response to ristocetin and the binding of vWF were similar in platelets frozen by either procedure. Finally, both methods promoted comparable impairment of the reactivity of platelets to thrombin, aggregation and binding of fibrinogen and vWF, compared to that of fresh platelets. In summary, cryopreservation of PCs using reduced Me(2)SO concentration and ThromboSol yields platelets with in vitro functional characteristics equivalent to those of cells frozen with the conventional method using 6% Me(2)SO.

Blood Platelets↗