Human platelet ribonuclease

Human platelets contain an RNase which has a pH optimum at 5.0. It hydrolyzes the secondary phosphate esters of uridine 3′-phosphates. It slowly converts uridine 2′:3′-and cytidine 2′:3′-cyclic phosphates to their corresponding nucleoside 3′-phosphates. Poly (A), poly (G) and poly (C) are not only refractory to the action of this enzyme, but also inhibit its action on poly (U). It differs from human granulocyte RNase, human serum RNase and bovine pancreatic RNase. Because of its unique property, this enzyme could serve as a biochemical marker in disorders involving the platelet destruction.     

Human platelet arginase

Summary We report here, for the first time the presence of arginase in human platelets. This enzyme has been partially purified and some of its properties studied. Its biological significance and its involvement in polyamine biosynthesis are considered.     

Use of mean platelet volume improves detection of platelet disorders

Classification of platelet disorders has been based on the platelet count. Addition of a second variable, mean platelet volume (MPV), to the routine blood count allows classification of patients into 9 categories: high, low, or normal MPV, and high, low or normal platelet count. We studied 1,244 adult inpatients. 1,134 had both platelet values normal. 11 patients had high MPV and low platelet count: all had hyperdestructive causes. 15 patients had high MPV and normal platelet count: 12 had heterozygous thalassemia, and three had iron deficiency. Seven patients had high MPV and high platelet count: causes included myeloproliferative disorders, inflammation, iron deficiency, and splenectomy, 25 patients had high platelet counts and normal MPV: the causes were inflammation, infection, sickle cell anemia, iron deficiency, or chronic myelogenous leukemia. 52 patients had an MPV that was inappropriately low for the platelet count (high, normal, or low). All had sepsis, splenomegaly, aplastic anemia, chronic renal failure, or a disease being treated with myelosuppressive drugs. High MPV thus appears correlated with myeloproliferative disease or thalassemia; and low MPV, with cytotoxic drugs or marrow hypoplasia. Addition of MPV to the platelet count allows subtler disorders to be detected (when the platelet count is normal), and allows distinction of the cause of thrombocytopenia.     

Finite platelet size could be responsible for platelet margination effect

Blood flows through vessels as a segregated suspension. Erythrocytes distribute closer to the vessel axis, whereas platelets accumulate near vessel walls. Directed platelet migration to the vessel walls promotes their hemostatic function. The mechanisms underlying this migration remain poorly understood, although various hypotheses have been proposed to explain this phenomenon (e.g., the available volume model and the drift-flux model). To study this issue, we constructed a mathematical model that predicts the platelet distribution profile across the flow in the presence of erythrocytes. This model considers platelet and erythrocyte dimensions and assumes an even platelet distribution between erythrocytes. The model predictions agree with available experimental data for near-wall layer margination using platelets and platelet-modeling particles and the lateral migration rate for these particles. Our analysis shows that the strong expulsion of the platelets from the core to the periphery of the blood vessel may mainly arise from the finite size of the platelets, which impedes their positioning in between the densely packed erythrocytes in the core. This result provides what we believe is a new insight into the rheological control of platelet hemostasis by erythrocytes.     

Thymoquinone-induced platelet apoptosis

Thymoquinone (TQ) is a nutrient with anticarcinogenic activity that stimulates suicidal death of tumor cells. Moreover, TQ triggers suicidal death of erythrocytes or eryptosis, an effect at least partially due to increase in cytosolic Ca²⁺ activity and ceramide formation. The present experiments explored whether TQ influences apoptosis of blood platelets. Cell membrane scrambling was determined utilizing Annexin V binding to phosphatidylserine exposing platelets, cytosolic Ca²⁺ activity utilizing Fluo 3‐AM fluorescence, caspase activity utilizing immunofluorescence and Western blotting of active caspase‐3 and inactive procaspase‐3, mitochondrial potential utilizing DiOC₆ fluorescence and ceramide by FACS analysis of ceramide‐binding antibodies. A 30 min exposure to TQ (≥5 µM) was followed by Annexin V binding, paralleled by caspase activation, increase of cytosolic Ca²⁺ activity, mitochondrial depolarization, and ceramide formation. P‐selectin exposure and integrin α_IIbβ₃ activation did not increase in response to TQ. Nominal absence of extracellular Ca²⁺ blunted but did not fully abolish the TQ‐induced activation of caspase‐3. The effects of TQ on platelets are significantly abolished with phosphoinositide‐3 kinase (PI3K) inhibitor wortmannin and G‐protein coupled receptor (GPCR) inhibitor pertussis toxin treatment prior to TQ stimulation. In conclusion, TQ triggers suicidal death of blood platelets in a PI3K‐dependent manner, possibly through a GPCR family receptor; an effect paralleled by increase of cytosolic Ca²⁺ activity, ceramide formation, mitochondrial depolarization, and caspase‐3 activation. J. Cell. Biochem. 112: 3112–3121, 2011. © 2011 Wiley Periodicals, Inc.     

Involvement of platelet glycoprotein Ib in platelet microparticle mediated neutrophil activation

Summary Platelet microparticles (MPs) are membrane vesicles shed by platelets after activation, and carry antigens characteristic of intact platelets, such as glycoprotein (GP) IIb/IIIa, GPIb and P-selectin. Elevated platelet MPs have been observed in many disorders in which platelet activation is documented. Recently, platelet GPIb has been implicated in the mediation of platelet–leukocyte interaction via binding to its ligand Mac-1 on leukocyte. The role of GPIb for mediating adhesion-activation interactions between platelet MPs and leukocytes has not been clarified. In this study we investigate the role of GPIb in the interplay between platelet MPs and neutrophils. Platelet MPs were obtained from collagen-stimulated platelet-rich plasma (PRP). In a study model of neutrophil aggregation, platelet MPs can serve a bridge to support neutrophil aggregation under venous level shear stress, suggesting that platelet MPs may enhance leukocyte aggregation, which would bear clinical relevance in diseases where the platelet MPs are elevated. The level of aggregation can be reduced by GPIb blocking antibodies, AP1 and SZ2, but not by anti-CD18 mAb. The GPIb blocking antibodies also decreased platelet MP-mediated neutrophil activation, including β2 integrin expression, adherence-dependent superoxide release and platelet MP-mediated neutrophil adherence to immobilized fibrinogen. Our data provide the evidence for the involvement of GPIb–Mac-1 interaction in the cross-talk between platelet MPs and neutrophils.     

Association of age-related decrease in platelet membrane fluidity with platelet lipid peroxide

The influence of age on platelet lipid peroxide (LPO), platelet membrane fluidity and the composition of fatty acid was investigated in female Wistar rats widely ranging in age from 14 to 720 days old. LPO levels were significantly higher (p<0.05) in the platelets of upper age groups than in those of lower age groups, showing a significantly positive correlation with age (r=0.84, p<0.0001). Membrane fluidity, assessed by 1,6-diphenyl-1,3,5-hexatriene (DPH) fluorescence polarization, was significantly reduced with age. The composition of fatty acid demonstrated an age-related elevation (p<0.05) in the unsaturation index. The rises in the LPO levels revealed a significantly positive correlation with DPH-polarization (r=0.73, p<0.0001). Thus our results suggested that the age-related deterioration of platelet membrane fluidity, despite a significant elevation in the unsaturation index, was due to the age-related higher basal levels of LPO in platelets.     

Photoaffinity labeling of platelet activating factor binding sites in rabbit platelet membranes

A photoreactive, radioiodinated derivative of platelet activating factor (PAF), 1-O-(4-azido-2-hydroxy-3-iodobenzamido)undecyl-2-O-acetyl-sn- glycero-3-phosphocholine ([125I]AAGP), was synthesized and used as a photoaffinity probe to study the PAF binding sites in rabbit platelet membranes. The nonradioactive analog, IAAGP, induced rabbit platelet aggregation with an EC50 value of 3.2 +/- 1.9 nM as compared to 0.40 +/- 0.25 nM for PAF. Specific binding of [125I]AAGP to rabbit platelet membranes was saturable with a dissociation constant (Kd) of 2.4 +/- 0.7 nM and a receptor density (Bmax) of 1.1 +/- 0.2 pmol/mg protein. Photoaffinity labeling of platelet membranes with [125I]AAGP revealed several 125I-labeled components by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A protein species with apparent molecular weight of 52,000 was consistently observed and inhibited significantly by unlabeled PAF at nanomolar concentrations. The labeling was specific since the PAF antagonists, SRI-63,675 and L-652,731, at 1 uM also blocked the appearance of this band; whereas lysoPAF was not effective at the same concentration. These results suggest that the binding sites of PAF receptor in rabbit platelets reside in the polypeptide of Mr = 52,000.     

Cryopreservation of buffy-coat-derived platelet concentrates in dimethyl sulfoxide and platelet additive solution

Platelets prepared in plasma can be frozen in 6% dimethyl sulfoxide (Me₂SO) and stored for extended periods at −80 °C. The aim of this study was to reduce the plasma present in the cryopreserved product, by substituting plasma with platelet additive solution (PAS; SSP+), whilst maintaining in vitro platelet quality. Buffy coat-derived pooled leukoreduced platelet concentrates were frozen in a mixture of SSP+, plasma and 6% Me₂SO. The platelets were concentrated, to avoid post-thaw washing, and frozen at −80 °C. The cryopreserved platelet units (n = 9) were rapidly thawed at 37 °C, reconstituted in 50% SSP+/plasma and stored at 22 °C. Platelet recovery and quality were examined 1 and 24 h post-thaw and compared to the pre-freeze samples. Upon thawing, platelet recovery ranged from 60% to 80%. However, there were differences between frozen and liquid-stored platelets, including a reduction in aggregation in response to ADP and collagen; increased CD62P expression; decreased viability; increased apoptosis and some loss of mitochondrial membrane integrity. Some recovery of these parameters was detected at 24 h post-thaw, indicating an extended shelf-life may be possible. The data suggests that freezing platelets in 6% Me₂SO and additive solution produces acceptable in vitro platelet quality.