Human Blood Platelets Research Articles

Pro- and antioxidant activities of docosahexaenoic acid on human blood platelets

n - 3 polyunsaturated fatty acids may protect against vascular diseases, however, their high accumulation in membranes may increase lipid peroxidation and subsequently induce deleterious effects in patients suffering from oxidative stress. This led us to investigate in vitro the dose-dependent effect of docosahexaenoic acid (DHA) on the redox status of human platelets. We have compared the effect of different DHA concentrations (0.5, 5 and 50 micro mol L(-1)) corresponding to DHA/albumin ratios of 0.01, 0.1 and 1. At the highest concentration, DHA elicited a marked oxidative stress, as evidenced by high malondialdehyde and low vitamin E levels whereas the lowest DHA concentration significantly decreased the malondialdehyde formation, with no change in vitamin E. The proportion of DHA was only increased in plasmalogen phosphatidylethanolamine at low concentration to rise in all phosphatidyl-choline and -ethanolamine subclasses at high concentration. Thus, the results show a biphasic effect of DHA with antioxidant and prooxidant effects at low and high concentrations, respectively, with a possible relationship with the phospholipid subclass in which it accumulates.

Inhibition of FMLP-stimulated neutrophil chemiluminescence by blood platelets increased in the presence of the serotonin-liberating drug chloroquine

Introduction: Previously, we reported that human blood platelets significantly decreased the concentration of reactive oxygen species (chemiluminescence) produced by Ca 2+-ionophore-stimulated neutrophils and that the reduction was partially mediated by serotonin liberated from platelets during their activation. The aim of the present study was to investigate whether platelet inhibition can occur independently of serotonin liberation and whether it can be pharmacologically enhanced. Materials and methods: Chemiluminescence was measured after stimulation of human neutrophils with N-formyl- l-methionyl- l-leucyl- l-phenylalanine (FMLP) in the presence of luminophore luminol. Concentration of platelet serotonin was estimated fluorometrically. Results: Platelets, added to neutrophils in the physiological cell ratio 50:1, decreased neutrophil chemiluminescence by 47%. The inhibition was not accompanied with liberation of platelet serotonin and rose after addition of chloroquine to platelet–neutrophil samples. In the absence of platelets, this drug did not affect neutrophil chemiluminescence. Chloroquine actively liberated serotonin; amine concentrations found in platelet supernatants were sufficient to inhibit neutrophil chemiluminescence. Conclusions: The presented results indicate that unstimulated platelets decreased neutrophil chemiluminescence by a serotonin-independent mechanism, yet their inhibitory effect could be enhanced pharmacologically through chloroquine-induced serotonin liberation.

Stabilization of membranes in human platelets freeze-dried with trehalose

Human blood platelets are normally stored in blood banks for 3–5 days, after which they are discarded. We have launched an effort at developing means for preserving the platelets for long term storage. In previous studies we have shown that trehalose can be used to preserve biological membranes and proteins during drying and have provided evidence concerning the mechanism. A myth has grown up about special properties of trehalose, which we discuss here and clarify some of what is fact and what is misconception. We have found a simple way of introducing this sugar into the cytoplasm of platelets and have successfully freeze-dried the trehalose-loaded platelets, with very promising results. We present evidence that membrane microdomains are maintained intact in the platelets freeze-dried with trehalose. Finally, we propose a possible mechanism by which the microdomains are preserved.

Generation of nitric oxide by peripheral blood leukocytes and platelets in healthy people and in patient with thermal trauma

The generation of nitric oxide (NO) by human peripheral blood leukocytes and platelets has been studied in healthy subjects and patients with burns (with the affected area varying from 10 to 45% of the body surface). Differential centrifugation was used to isolate leukocytes and platelets from the blood. The leukocyte suspension was diluted with a complete medium to a concentration of 1 × 107 cells/ml, and the platelet suspension, to 1 × 108 cells/ml; the suspensions were then cultured for 15 h (37°C). The concentration of nitrite, an NO metabolite, was determined using the Griss reaction. The relative production of NO by leukocytes of healthy subjects and patients was 0.75 ± 0.06 and 2.93 ± 0.16 μmol/l, respectively (p < 0.001), and its relative production by platelets of healthy subjects and patients was 2.15 ± 0.14 and 3.62 ± 0.13 μmol/l, respectively (p < 0.01). The absolute generation of NO by leukocytes of healthy subjects and patients is 0.47 ± 0.05 and 3.02 ± 0.28 μmol/l, respectively (p < 0.001), and its absolute generation by platelets of healthy subjects and patients was 7.70 ± 0.55 and 14.68 ± 0.84 μmol/l, respectively (p < 0.001). Thus, the absolute production of NO by platelets is 16 times higher than the absolute production of NO by leukocytes of healthy subjects. Stress increases the generation of NO by both leukocytes and platelets. The absolute generation of NO by platelets in thermal trauma is positively correlated with the plasma content of fibrinogen in the patients.

Temporal aspects of onion-induced antiplatelet activity.

Organosulfur compounds in onion extracts are formed following the lysis of the S-alk(en)yl-L-cysteine sulfoxides by alliinase. These compounds inhibit the aggregation of human blood platelets and offer the potential for positive cardiovascular health benefits. An experiment was designed to examine temporal and temperature effects on onion-induced antiplatelet activity. Platelet aggregation is induced by various agonists, including ADP, collagen, and thrombin. Unexpectedly, all freshly-juiced onion extracts (ca. 5 minutes post-juicing) appeared to exhibit both an agonist-free aggregation peak (AFP) and a platelet inhibitory peak (PIP) characteristic of inhibition of platelet aggregation. The AFP was minimal by 30 minutes and dissipated in all treatments by 120 minutes, while the PIP increased as onion extracts aged and did not change after 30 minutes at 25 degrees C. This finding confirms the observation that the in vitro platelet inhibitory activity of onion organosulfur compounds is time dependent. Freshly-prepared onion extracts were incubated with the ADP scavenger enzyme apyrase (E.C. 3.6.1.5). AFPs were abolished in apyrase-treated extracts, suggesting that this response may have been due to free ADP in onion extracts, although an amount of ADP required to generate such a response would be unexpected in onion extracts. In addition, platelet aggregates were not observed in the AFP, suggesting this response may be associated with changes in light transmission through platelet rich plasma that are not associated with platelet aggregation. Artifacts of analysis are, therefore, possible when assessing onion-induced antiplatelet activity with freshly-juiced extracts. Temporal formation of platelet-inhibiting organosulfur compounds should be taken into account during both in vitro and in vivo assessment of onion-induced antiplatelet activity.

Activation-dependent surface expression of gC1qR/p33 on human blood platelets

GC1qR/p33 (gC1qR) is expressed by a variety of somatic and cultured cells, including blood platelets. It interacts with several cellular, viral, bacterial, and plasma proteins, suggesting a potential role in thrombosis, inflammation, and infection. Considerable controversy has surrounded the surface membrane localization of gC1qR, however, since its cDNA sequence does not predict a traditional membrane-anchoring domain, and bears a typical mitochondrial targeting sequence. The present study examined gC1qR expression on resting and activated human blood platelets using flow cytometry and confocal microscopy with two monoclonal antibodies, 74.5.2 and 60.11, directed against gC1qR C-terminal amino acids 204-218, and N-terminal amino acids 76-93, respectively. Unstimulated platelets reacted minimally with either antibody. In contrast, platelet activation with TRAP, epinephrine, or ADP produced markedly increased gC1qR expression as reflected by 74.5.2 binding but not 60.11 binding. Platelet activation was verified using PAC-1 and anti CD 62 antibodies. Whereas PAC-1 binding to activated platelets could be reversed following platelet incubation with PGE1, 74.5.2 binding remained unchanged, suggesting the sustained expression of gC1qR following platelet stimulation. The data further demonstrate that detection of cell surface gC1qR may be dependent on antibody specificity. The ability of gC1qR to bind proteins involved in complement, coagulation, and kinin systems, as well as viral and bacterial pathogens including S. aureus protein A, supports the hypothesis that gC1qR expressed on activated platelets may contribute directly to thrombosis, inflammation, and endovascular infections.

Participation of Human Blood Platelets in Antimicrobial Host Defense

Background: Besides their classical functions in hemostasis, blood platelets actively contribute to inflammatory and defense reactions. As the first cellular elements that accumulate at any site of injury to the vascular wall, platelets start the innate host defense against any invading pathogens by means of internalization of bacteria and secretion of microbicidal peptides. Our own observations suggest that there are further platelet defense capabilities. Material and Methods: Gram-positive (Bacillus subtilis; B.s.), Gram-negative (Klebsiella pneumoniae; K.p. ) and intracellular (Chlamydia pneumoniae; C.p. ) bacteria were included in our study. Morphological aspects of bactericidal effects were investigated by transmission electron microscopy, and bacterial killing rates were determined by fluorescence double labeling of bacteria with LIVE/DEAD BacLight™. Bacteria were co-incubated with stimulated platelets or with stimulated, previously degranulated platelets, or were exposed to supernatants from stimulated platelets. Results: When co-incubated with stimulated platelets, bacteria from all three strains displayed swelling, loss of internal structures, and rupture of the cell wall. At binding sites of B.s. , the platelet membrane often showed a ruffled appearance. Exposure to supernatants from stimulated platelets yielded moderate killing rates (36/52/68% for B.s./K.p./C.p. ), whereas killing rates strongly increased if stimulated platelets were co-incubated instead (77/79/81% by B.s./K.p./C.p. ). Even degranulated platelets exerted bactericidal effects on B.s. (41%) if stimulated with thrombin. Conclusions: Besides the release of microbicidal peptides, human platelets also have a contact-dependent mechanism to kill invading bacteria. Since ruffled membranes are indicative for release of reactive oxygen species, platelets seem to contribute to antimicrobial host defense by a local release of free radicals.

Interaction of lithium with 5-HT 1B receptors in depressed unipolar patients treated with clomipramine and lithium versus clomipramine and placebo: preliminary results

Lithium is commonly used in combination with antidepressant drugs as a treatment for refractory depression; less often, it is used in non-resistant depression. The aim of this study was to examine the interaction of lithium with 5-HT 1B receptors in 10 non-resistant unipolar depressed patients treated with clomipramine+lithium (C+L) vs. clomipramine+placebo (C+P). A mediation of the serotonergic system has been proposed in the literature to explain the clinical effect of lithium. Indeed, in a previous study of healthy human blood platelets, we demonstrated the interaction of lithium with adenylate cyclase activity coupled to 5-HT 1B receptors. The functional activity of these receptors was measured by studying the inhibitory effect of L694,247, a 5-HT 1B receptor agonist, on the adenylate cyclase activity determined by the production of cAMP. Using the same technique in the present study, we found that lithium significantly reduced the inhibition of adenylate cyclase activity induced by 5-HT 1B receptor activation. This result confirms the specific interaction of lithium with 5-HT 1B receptors. Moreover, a correlation between the percentage of 5-HT 1B receptor-dependent adenylate cyclase inhibition and the clinical benefit of lithium was established, suggesting 5-HT 1B receptors may be a target for the therapeutic effect of lithium

Interactions between Eph kinases and ephrins provide a mechanism to support platelet aggregation once cell-to-cell contact has occurred.

Eph kinases are receptor tyrosine kinases whose ligands, the ephrins, are also expressed on the surface of cells. Interactions between Eph kinases and ephrins on adjacent cells play a central role in neuronal patterning and vasculogenesis. Here we examine the expression of ephrins and Eph kinases on human blood platelets and explore their role in the formation of the hemostatic plug. The results show that human platelets express EphA4 and EphB1, and the ligand, ephrinB1. Forced clustering of EphA4 or ephrinB1 led to cytoskeletal reorganization, adhesion to fibrinogen, and alpha-granule secretion. Clustering of ephrinB1 also caused activation of the Ras family member, Rap1B. In platelets that had been activated by ADP and allowed to aggregate, EphA4 formed complexes with two tyrosine kinases, Fyn and Lyn, and the cell adhesion molecule, L1. Blockade of Eph/ephrin interactions prevented the formation of these complexes and caused platelet aggregation at low ADP concentrations to become more readily reversible. We propose that when sustained contacts between platelets have occurred in response to agonists such as collagen, ADP, and thrombin, the binding of ephrins to Eph kinases on adjacent platelets provides a mechanism to perpetuate signaling and promote stable platelet aggregation.

The in vivo antithrombotic effect of wine consumption on human blood platelets and hemostatic factors.

We compared the in vivo effect of red vs. white wine consumption on platelet aggregation, responsiveness and membrane viscosity, plasma total antioxidant status, thromboxane B(2) levels, and fibrinolysis. Diet and red wine had a synergistic effect in decreasing platelet aggregation. Red wine did not have a significantly more favorable effect on the fibrinolytic factors than white wine. The reduction in platelet membrane viscosity after red wine, which could contribute to the protective antithrombotic role of red wine, needs further explanation.

Thymosin beta4 is released from human blood platelets and attached by factor XIIIa (transglutaminase) to fibrin and collagen.

The beta-thymosins constitute a family of highly conserved and extremely water-soluble 5 kDa polypeptides. Thymosin beta4 is the most abundant member; it is expressed in most cell types and is regarded as the main intracellular G-actin sequestering peptide. There is increasing evidence for extracellular functions of thymosin beta4. For example, thymosin beta4 increases the rate of attachment and spreading of endothelial cells on matrix components and stimulates the migration of human umbilical vein endothelial cells. Here we show that thymosin beta4 can be cross-linked to proteins such as fibrin and collagen by tissue transglutaminase. Thymosin beta4 is not cross-linked to many other proteins and its cross-linking to fibrin is competed by another family member, thymosin beta10. After activation of human platelets with thrombin, thymosin beta4 is released and cross-linked to fibrin in a time- and calcium-dependent manner. We suggest that thymosin beta4 cross-linking is mediated by factor XIIIa, a transglutaminase that is coreleased from stimulated platelets. This provides a mechanism to increase the local concentration of thymosin beta4 near sites of clots and tissue damage, where it may contribute to wound healing, angiogenesis and inflammatory responses.

Determination of serum aluminum, platelet aggregation and lipid peroxidation in hemodialyzed patients.

Aluminum (Al3+) overload is frequently associated with lipid peroxidation and neurological disorders. Aluminum accumulation is also reported to be related to renal impairment, anemia and other clinical complications in hemodialysis patients. The aim of the present study was to determine the degree of lipid peroxidation, platelet aggregation and serum aluminum in patients receiving regular hemodialytic treatment. The level of plasma lipid peroxidation was evaluated on the basis of thiobarbituric acid reactive substances (TBARS). Mean platelet peroxidation in patients undergoing hemodialysis was significantly higher than in normal controls (2.7 +/- 0.03 vs. 1.8 +/- 0.06 nmol/l, P<0.05). Platelet aggregation and serum aluminum levels were determined by a turbidimetric method and atomic absorption spectrophotometry, respectively. Serum aluminum was significantly higher in patients than in normal controls (44.5 +/- 29 vs. 10.8 +/- 2.5 microg/l, P<0.05). Human blood platelets were stimulated with collagen (2.2 microg/ml), adenosine diphosphate (6 microM) and epinephrine (6 microM) and showed reduced function with the three agonists utilized. No correlation between aluminum levels and platelet aggregation or between aluminum and peroxidation was observed in hemodialyzed patients.

Interactions of human blood and tissue cell types with 95-nm-high nanotopography

Two of the major concerns for tissue engineering materials are inflammatory responses from blood cells and fibrous encapsulation by the body in order to shield the implant from blood reaction. A further hurdle is that of vascularization. In order to develop new tissues, or to repair parts of the vascular system, nutrients need to be carried to the basal cell layers. If a material promotes tissue formation, but not vascularization, necrosis will be observed as multilayered cells develop. In this paper, polymer demixed island topography with a 95-nm Z axis was tested using human mononuclear blood cells, platelets, fibroblasts, and endothelial cells. The results showed no difference in blood response between the islands and the flat controls, suggesting that in vivo there would be negligible immunological difference. Fibroblasts reacted by changing morphology into a rounded shape with thick processes and poorly developed cytoskeleton. Retardation of fibroblast growth may be an advantageous, as it is this cell type that forms the fibrous capsule, preventing growth of the required tissue type. Finally, endothelial cells were seen to form arcuate, or curved, morphologies in response to the islands. This is the normal, in vivo, morphology for vascular endothelium. This result suggests that the nano-features are promoting a more phenotypically correct morphology.

Correlation between serotonin uptake in human blood platelets with the 44-bp polymorphism and the 17-bp variable number of tandem repeat of the serotonin transporter.

Dysfunctions of the central serotonin (5-hydroxytryptamine, 5-HT) system seem to be associated with psychiatric disorders such as schizophrenia or depression. Previous studies suggested that a 44-bp insertion/deletion polymorphism of the 5-HT transporter (5-HTT) promoter region might influence the transcriptional activity of the 5-HTT gene, and the insertion variant resulted in increased 5-HTT expression and 5-HT uptake. Moreover, a 17-bp variable number of tandem repeat (VNTR) polymorphism of the second intron may act as a transcriptional regulator with allele dependent differential enhancer-like properties. Since the 5-HTT of human platelets shares many properties with the transporter of neural tissue, platelets are widely used as a surrogate tissue source, possibly reflecting central 5-HT metabolism. Therefore, we investigated the impact of the 44-bp polymorphism and the 17-bp VNTR for 5-HT uptake in platelets of 50 male subjects. We found no significant effect of the 44-bp polymorphism and of the 17-bp VNTR on maximum rate (Vmax) of 5-HT uptake. However, individuals homozygous for the 5-HTT intron 2 allele with 12 repeats (STin2.12) of the 17-bp VNTR appeared to have lower affinity of 5-HT uptake than individuals heterozygous for the STin2.10/STin2.9 allele. This was also observed for the combined analysis of both polymorphisms. In conclusion, we found no association between the different genotypes of the 44-bp polymorphism and the 17-bp VNTR and maximum rate of 5-HT uptake into platelets.

Actin dynamics in platelets

The human blood platelet circulates in the blood as a non-adherent disk. Upon receiving signals of blood vessel damage, the platelet reorganizes its actin cytoskeleton which transforms it into a spiky dynamic adherent glue. This transformation involves a temporal sequence of four morphologically distinct steps which is reproducible in vitro. The actin dynamics underlying these shape changes depend on a large number of actin-binding proteins. Maintenance of the discoid shape requires actin-binding proteins that inhibit these reorganizations, whereas transformation involves other proteins, some to disassemble old filaments and others to polymerize new ones. F-Actin-affinity chromatography identified a large set of actin-binding proteins including VASP, Arp2 and 2E4/kaptin. Recent discoveries show that VASP inhibits filament disassembly and Arp2/3 is required to polymerize new filaments. Morphological analysis of the distribution of these actin-binding proteins in spread platelets together with biochemical measurements of their interactions with actin lead to a model of interactions with actin that mediate shape change.

Dietary components and human platelet activity

Platelet hyperactivity is one of the most important factors responsible for the incidence of cardiovascular disease. There are many nutritive and non-nutritive compounds present in the diet which may affect platelet function in various ways. Recent discovery of anti-platelet factors in plants, vegetables and fruits provides a new dietary means for a long-term strategy to favorably modify human blood platelet activity. This review summarises the effects of these dietary components on human platelet function both in vitro and in vivo .

Aggregation Mechanism of Blood Platelets Studied by the Time-Resolved Light Scattering Method

A laser light-scattering aggregometer was applied to study epinephrine-induced aggregation of human blood platelets. We improved the analytical software for the aggregometer; thus, it has become feasible to use a time-resolved light scattering method (TRLSM) based on Mie's theory, allowing one to monitor changes in both size and particle concentration as a function of time. By combination of TRLSM with microscopic analysis, it was found that there is a critical condition at which almost all the single platelets aggregate with one another to result in small size aggregates (AGS), at 2 min after addition of epinephrine (1.5 ± 0.5 μM). The resulting AGS was spherical in shape, 15 μm in diameter (as a number-averaged value), and it was composed of about 1900 single platelets closely packed. After the formation of AGS particles went essentially to completion, they aggregated with one another to result in large particles (AGL), with an accompanying decrease in the AGS concentration. The AGL formed is not spheri...

Characterisation of Rac activation in thrombin- and collagen-stimulated human blood platelets

In this study, we characterised the mechanisms of Rac GTPase activation in human platelets stimulated by two physiological agonists, either thrombin, acting through membrane receptors coupled to heterotrimeric G-proteins, or collagen which is known to mobilise a tyrosine kinase-dependent pathway. Both agonists induced a rapid activation of Rac that was not significantly affected by the inhibition of integrin α IIbβ 3 engagement. Using pharmacological inhibitors, we found that phospholipase C activation and calcium mobilisation were essential for platelet Rac activation by either thrombin or collagen whereas protein kinase C inhibition was without effect. In contrast to Rac, Cdc42 activation was independent of phospholipase C activation, indicating that the two GTPases are differently regulated. We also found that phosphoinositide 3-kinase was not required for Rac activation in response to thrombin but was involved in its activation by collagen.

Subcellular localization of thrombopoietin in human blood platelets and its release upon thrombin stimulation.

Thrombopoietin (TPO) is a major regulator of platelet production. The concentration of circulating TPO seems to be determined by its binding and internalization by megakaryocytes and platelets. To elucidate the platelet compartments involved in TPO metabolism, we investigated intraplatelet TPO by post-embedding immunoelectron microscopy, incubated platelets with recombinant human (rh)TPO coupled to colloidal gold and visualized the TPO uptake using electron microscopy. TPO concentrations were measured in 12 platelet concentrates (PC) before and after stimulation with thrombin and after disruption of platelets by freezing-thawing. In resting platelets, immunogold labelling revealed a prevailing cytoplasmic localization of TPO antigen and minor labelling within the surface-connected canalicular system (SCCS); storage granules were devoid of labelling. In tracer experiments, TPO-gold was observed on the plasma and SCCS membranes and within the cytoplasm. Upon thrombin stimulation, endogenous TPO was still detected within the cytoplasm by immunolabelling, and tracer experiments revealed TPO-gold within the cytoplasm and on fibrin fibres. After thrombin stimulation of PC, the plasma TPO levels increased to an average of 535%, and after platelet lysis to an average of 1625% compared with plasma values in unstimulated PC. We conclude that platelets contain releasable immunoreactive TPO within the SCCS and within their cytoplasm, but not within granular compartments. Stored immunoreactive TPO is released upon thrombin stimulation, but only to a minor degree.

Neutrophils promote the release of α-6-fucosyltransferase from blood platelets through the action of cathepsin G and elastase

Human blood platelets release α-6-fucosyltransferase during coagulation of blood or after stimulation with thrombin or other agonists that cause platelet activation (Antoniewicz et al., FEBS Lett. 244 (1989) 388-390). However, in the absence of neutrophils the thrombin-stimulated platelets release only a small fraction of α-6-fucosyltransferase activity (Kościelak et al., Acta Biochim. Polon. 42 (1995) 35-40). We show that the effect of neutrophils is reproduced by cathepsin G or (less efficiently) by elastase, the two enzymes that are released by neutrophils during coagulation of blood. We have also localized α-6-fucosyltransferase to membrane and α-granule fractions of platelets that had been disrupted by nitrogen cavitation. It is concluded that thrombin-activated neutrophils release cathepsin G and elastase that promote degranulation of platelets and hence the secretion of α-6-fucosyltransferase.