Soluble expression of human glycoprotein Ibα in Escherichia coli through replacement of the N-terminal capping domain.

Glycoprotein Ibα (GpIbα), a family of LRR (leucine-rich repeat) proteins, is a membrane protein on the platelet, and plays an important role in atherothrombotic events. The complex formation of GpIbα with the von Willebrand Factor (vWF) has been revealed to lead to acute coronary syndrome (ACS) or stroke. A considerable attention has been paid to understand the biological functions of GpIbα and its regulation. However, difficulty with the soluble expression of human GpIbα in bacteria has hampered the relevant research. Herein, we present a soluble expression of GpIbα in Escherichiacoli by replacing the N-terminal capping domain of GpIbα with that of Internalin B using a computational approach. The resulting protein was expressed as a soluble form in E. coli, maintaining its structural feature and binding property for vWF. The present approach can be broadly used for the soluble expression of human LRR proteins in E. coli.

Platelets induce endothelial tissue factor expression in a mouse model of acid-induced lung injury.

Although the lung expresses procoagulant proteins under inflammatory conditions, underlying mechanisms remain unclear. Here, we addressed lung endothelial expression of tissue factor (TF), which initiates the coagulation cascade and expression of which signifies development of a procoagulant phenotype in the vasculature. To establish the model of acid-induced acute lung injury (ALI), we intranasally instilled anesthetized mice with saline or acid. Then 2 h later, we isolated pulmonary vascular cells for flow cytometry and confocal microscopy to detect the leukocyte antigen, CD45 and the endothelial markers VE-cadherin and von Willebrand factor (vWf). Acid increased both the number of vWf-expressing cells as well as TF and P-selectin expressions on these cells. All of these effects were markedly inhibited by treating mice with antiplatelet serum, suggesting the involvement of platelets. The increased expressions of TF, vWf, and P-selectin in response to acid also occurred in platelets. Moreover, the effects were replicated in endothelial cells derived from isolated, blood-perfused lungs. However, the effect was inhibited completely in lungs perfused with platelet-depleted and, to a lesser extent, with leukocyte-depleted blood. Acid injury increased endothelial expressions of the platelet proteins, CD41 and CD42b, providing evidence that platelet proteins were transferred to the vascular surface. Reactive oxygen species (ROS) were implicated in these responses, in that the endothelial and platelet protein expressions were inhibited. We conclude that acid-induced ALI causes NOX2-mediated ROS generation that activates platelets, which then generate a procoagulant endothelial surface.

Classification of Iranian patients with Glanzmann's Thrombasthenia using a flow cytometric method.

Glanzmann's Thrombasthenia (GT) is a rare inherited autosomal recessive platelet disorder caused by a deficiency or dysfunction of the GPIIb-IIIa receptor on platelets, which is characterized by a lack of platelet aggregation in response to multiple physiologic agonists and a life-long bleeding disorder. Flow cytometry is a rapid and highly sensitive method that can detect reduced levels of receptors, as well as absolute deficiency. The aim of this study was to classify Iranian GT patients by a flow cytometric method, and to correlate these findings with the severity of clinical bleeding. The expression of GPIIb-IIIa on the platelet surface was assessed in 123 GT patients using quantitative flow cytometry to determine the most common subtype among these patients. We used a panel of antibodies to detect the expression of glycoproteins GPIb, GPIIb, GPIIIa, as well as Integrin αv. Patients were also interviewed with regard to the severity and frequency of bleeding, according to history and gender, in order to evaluate the nature of their bleeding phenotype, and classify them as mild, moderate or severe bleeders, in accordance with the Glanzmann's Thrombasthenia Italian Team (GLATIT) protocol. In the detailed analysis of the results of our investigation, 95 out of 123 (77.5%) were classified as type I; 20 (16%) as type II with residual GPIIb-IIIa, and eight (6.5%) as GT variants. The variant type was diagnosed by the inability of GPIIb-IIIa to bind fibrinogen, as evidenced by the absence of platelet aggregation in response to physiologic agonists. There was no significant correlation between bleeding severity and different subtypes of GT. This study demonstrates that GT type I is the most common subtype among Iranian patients. There was no correlation between severity of symptoms and cytometric phenotype of the disease. The identification of families at risk may significantly decrease the incidence of the severe form of the disorder if genetic counseling is provided.

Clopidogrel response variability and its correlation with early recurrent cardiovascular events in chinese patients undergoing percutaneous coronary intervention.

BACKGROUND/AIMS: Numerous studies conducted on Caucasian patients have reported that individual responsiveness to clopidogrel varies widely, whereas there are only a few published studies on the antiplatelet effect of clopidogrel therapy in Chinese patients undergoing percutaneous coronary intervention. The present study aimed to evaluate clopidogrel antiplatelet effects and their correlation with early recurrent cardiovascular (CV) events. METHODS: Platelet aggregation (with 5 and 20 µmol/l ADP) and the expression of glycoprotein Ib and P-selectin were measured at baseline and 12 and 36 h after the clopidogrel loading dose in 111 consecutive patients. The primary outcome was a definite CV event. RESULTS: There was marked interindividual variability in the drug response, as measured by platelet aggregation and P-selectin expression. The proportions of nonresponders at 12 and 36 h were 32 and 19%, respectively, with 5 µmol/l ADP, 38 and 28% with 20 µmol/l ADP, and 27 and 17% according to P-selectin expression. The maximal aggregation rates stimulated by 5 µmol/l ADP in nonresponders were significantly higher than those of the responders at 12 h (57.53 ± 14.24% vs. 33.91 ± 10.79%; p < 0.0001) and at 36 h (48.65 ± 15.46% vs. 30.31 ± 16.04%; p < 0.0001). During the 3-month follow-up period, 11 patients (32.4%) among the nonresponders, 2 patients (7.1%) among the low responders and none of the responders suffered a recurrent CV event (p < 0.0001). CONCLUSIONS: The antiplatelet effectiveness of clopidogrel has a wide interindividual variation, and nonresponsiveness to clopidogrel is associated with an increased risk of early recurrent CV events.

Platelet apoptosis and activation in platelet concentrates stored for up to 12 days in plasma or additive solution.

BACKGROUND: Several studies suggest that apoptosis of platelets occurs during storage of platelet concentrates (PC). We sought to determine whether storage of PC in additive solution alters levels of apoptosis during storage beyond the current shelf life (5-7 days). STUDY DESIGN AND METHODS: Pooled buffy coat PC (n = 7) were prepared in either 100% plasma or 70% Composol and stored at 22 °C for 12 days. A third arm of the study stored PC in 100% plasma at 37 °C, which is thought to induce apoptosis. PC were tested for mitochrondrial membrane potential, annexin V binding, microparticles, caspase-3/7 activity and decoy cell death receptor 2, as well as standard platelet quality tests. RESULTS: Composol units remained ≥pH 6·88, with 36% lower lactate and higher pH vs plasma by day 12 (P < 0·001). Platelet function was better maintained, and activation and apoptotic markers tended to be lower in Composol units towards the end of storage. However, levels of all apoptosis markers assessed were not significantly different in units stored in Composol. Storage at 37 °C saw stronger correlation of apoptotic markers with standard quality tests compared to 22 °C, but loss of correlation of caspase-3/7 activity with other apoptosis markers. CONCLUSION: We conclude that storage of platelets in 70% Composol vs 100% plasma does not increase the rate of platelet apoptosis. Our data agree with other studies suggesting that platelet apoptosis is sequential to high levels of activation, but share a significant degree of overlap.

Juxtamembrane basic residues in glycoprotein Ibbeta cytoplasmic domain are required for assembly and surface expression of glycoprotein Ib-IX complex.

Platelet glycoprotein (GP) Ib-IX complex requires all its three subunits for efficient expression on the cell surface, but the underlying molecular basis is not fully clear. Using transfected Chinese hamster ovary cells as the model system, we demonstrate that juxtamembrane residues 149-154 in the cytoplasmic domain of the GPIbbeta subunit is required for assembly and surface expression of the GPIb-IX complex. The complex, or GPIbbeta by itself, lacking these residues is retained in the endoplasmic reticulum. Our results thus have illustrated an important role of the GPIbbeta cytoplasmic domain in biosynthesis of the GPIb-IX complex.

The association between platelet autoantibody specificity and response to intravenous immunoglobulin G in the treatment of patients with immune thrombocytopenia.

We retrospectively investigated the association between platelet autoantibody specificity and response to intravenous immunoglobulin G (IVIG) in 17 patients with immune thrombocytopenia (ITP). Platelet-associated antibodies against glycoprotein (GP) IIb/IIIa, GPIb/IX, and GPIa/IIa were detected in 13, 10, and 8 patients, respectively. A response occurred in 7 of 7 patients without anti-GPIb/IX, but in only 3 of 10 patients with anti-GPIb/IX (p<0.01). There was no difference in the response rates in patients with or without anti-GPIIb/IIIa or anti-GPIa/IIa. We conclude that ITP patients with anti-GPIb/IX may be less responsive to IVIG.

Properties of ets-1 binding to chromatin and its effect on platelet factor 4 gene expression.

Ets-1 is important for transcriptional regulation in several hematopoietic lineages, including megakaryocytes. Some transcription factors bind to naked DNA and chromatin with different affinities, while others do not. In the present study we used the megakaryocyte-specific promoters platelet factor 4 (PF4), and glycoprotein IIb (GPIIb) as model systems to explore the properties of Ets-1 binding to chromatin. Chromatin immunoprecipitation assays indicated that Ets-1 binds to proximal regions in the PF4 and GPIIb promoters in vivo. In vitro and in vivo experiments showed that Ets-1 binding to chromatin on lineage-specific promoters does not require lineage-specific factors. Moreover, this binding shows the same order of affinity as the binding to naked DNA and does not require ATP-dependent or Sarkosyl-sensitive factors. The effect of Ets-1 binding on promoter activity was examined using the PF4 promoter as a model. We identified a novel Ets-1 site (at -50), and a novel Sarkosyl-sensitive DNase I-hypersensitive site generated by Ets-1 binding to chromatin, which significantly affect PF4 promoter activity. Taken together, our results suggest a model by which Ets-1 binds to chromatin without the need for lineage-specific accessory factors, and Ets-1 binding induces changes in chromatin and affects transactivation, which are essential for PF4 promoter activation.

Comparison of platelet function and viscoelastic test results between healthy dogs and dogs with naturally occurring chronic kidney disease.

OBJECTIVE To compare platelet function and viscoelastic test results between healthy dogs and dogs with chronic kidney disease (CKD) to assess whether dogs with CKD have platelet dysfunction and altered blood coagulation. ANIMALS 10 healthy control dogs and 11 dogs with naturally occurring CKD. PROCEDURES Blood and urine were collected once from each dog for a CBC, serum biochemical analysis, urinalysis, and determination of the urine protein-to-creatinine ratio, prothrombin time, activated partial thromboplastin time, plasma fibrinogen concentration, and antithrombin activity. Closure time was determined by use of a platelet function analyzer and a collagen-ADP platelet agonist. Thromboelastography (TEG) variables (reaction time, clotting time, α angle, maximum amplitude, and global clot strength [G value]) were determined by use of recalcified nonactivated TEG. Platelet expression of glycoprotein Ib (GPIb; receptor for von Willebrand factor), integrin αIIbß3 (αIIbß3; receptor for fibrinogen), and P-selectin (marker for platelet activation) was assessed by flow cytometry. RESULTS Compared with healthy control dogs, the median closure time was prolonged, the median maximum amplitude and G value were increased, and the median clotting time was decreased for dogs with CKD. Platelet expression of both αIIbß3 and P-selectin was also significantly increased for dogs with CKD, compared with that for control dogs. Platelet expression of GPIb, αIIbß3, and P-selectin was not correlated with closure time or any TEG variable. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that dogs with CKD frequently had evidence of platelet dysfunction and hypercoagulability that were not totally attributable to alterations in platelet surface expression of GPIb, αIIbß3, and P-selectin.

Sensing developing biofilms: the bitter receptor T2R38 on myeloid cells.

Quorum-sensing molecules, also known as autoinducer, are essential for bacterial biofilm formation. Our focus is on N-(3-oxododecanoyl)-L-homoserine lactone (AHL-12), because it is also known as an 'interkingdom signalling molecule', which means that it also interacts with mammalian cells. AHL-12 activates defence-relevant functions of phagocytic cells, including enhancement of phagocytosis, increased expression of adhesion receptors and induction of chemotaxis. This leads to the hypothesis that early recognition of developing biofilms might be the key to a successful host defence against biofilm infection. In that context we studied activation of phagocytic cells by AHL-12, and found that phagocytes are activated via a rather specialized receptor that was not previously described on myeloid cells, the bitter taste receptor T2R38. Taste receptors are commonly associated with cells of the gustatory system. The extragustatory expression, however, suggests an additional role, namely the sensing of the onset of bacterial biofilm infection.

Sodium selenite and cancer related lymphedema: Biological and pharmacological effects.

A significant percentage of cancer patients develop secondary lymphedema after surgery or radiotherapy. The preferred treatment of secondary lymphedema is complex physical therapy. Pharmacotherapy, for example with diuretics, has received little attention, because they were not effective and only offered short-term solutions. Sodium selenite showed promise as a cost-effective, nontoxic anti-inflammatory agent. Treatment with sodium selenite lowers reactive oxygen species (ROS) production, causes a spontaneous reduction in lymphedema volume, increases the efficacy of physical therapy for lymphedema, and reduces the incidence of erysipelas infections in patients with chronic lymphedema. Besides biological effects in reducing excessive production of ROS, sodium selenite also displays various pharmacological effects. So far the exact mechanisms of these pharmacological effects are mostly unknown, but probably include inhibition of adhesion protein expression.

Platelet Reactivity in Patients With Stroke and Hyperlipidemia, GPIbα Assessment.

The aim of this study was to assess platelet reactivity in patients after ischemic stroke and to investigate the influence of hyperlipidemia (HL) on platelet activity markers. A total of 41 patients after ischemic stroke were divided into the following 2 groups: patients with HL and patients with normolipidemia. Expression of CD42b on resting, thrombin-activated blood platelets, and fibrinogen level was assessed. The CD42b-positive platelets were analyzed using the flow cytometer, anti-CD61, and anti-CD42b monoclonal antibodies. The results confirmed increased platelet reactivity to thrombin in all patients after ischemic stroke manifested by significantly lower CD42b expression and percentage of CD42b(+) platelets after activation by thrombin. The influence of HL on the expression of CD42b on resting and thrombin-activated platelets was not found. However, increased level of fibrinogen but no influence of HL on fibrinogen concentration was observed in patients after ischemic stroke. Increased susceptibility to platelet agonists was found in patients after ischemic stroke in the convalescent phase.

Cell-to-cell variability in the differentiation program of human megakaryocytes.

Differentiation of CD34(+) stem/progenitor cells into megakaryocytes is thought to be a uniform, unidirectional process, in which cells transform step by step from less differentiated precursor stages to more differentiated megakaryocytes. Here we propose the concept and present evidence based on single-cell analysis that differentiation occurs along multiple, partially asynchronous routes. In all CD34(+) cells cultured with thrombopoietin, surface appearance of glycoprotein IIIa (GPIIIa) preceded that of GPIb, indicating that the expression of these glycoproteins occurs in a timely ordered manner. Cellular F-actin content increased in parallel with GPIb expression. Only cells that expressed GPIb were polyploid, pointing to co-regulation of GPIb expression, actin cytoskeleton formation and polyploidization during megakaryocytopoiesis. On the other hand, most progenitor cells responded to thrombin but not to thromboxane A(2) analogue by rises in cytosolic [Ca(2+)](i). The appearance of thromboxane-induced responses during megakaryocytopoiesis was not strictly linked to glycoprotein expression, because cells showed responsiveness either before or after GPIb expression. The same non-strictly sequential pattern was observed for disappearance of the Ca(2+) response by prostacyclin mimetic; in some megakaryocytes it occurred before and in others after GPIb expression. Thus, megakaryocytic differentiation follows along independent routes that are either strictly sequential (GPIIIa and GPIb expression) or proceed at different velocities (Ca(2+) signal regulation).

Ex vivo expansion of megakaryocyte progenitor cells: cord blood versus mobilized peripheral blood.

Thrombocytopenia is a problematic and potentially fatal occurrence after transplantation of cord blood stem cells. This problem may be alleviated by infusion of megakaryocyte progenitor cells. Here, we compared the ability of hematopoietic progenitor cells obtained from cord blood and expanded in culture to that of mobilized peripheral blood cells. The CD34(+) cells were plated for 10 days in presence of thrombopoietin (TPO) alone and combined with stem cell factor (SCF), Flt3-ligand (FL), interleukin-3 (IL-3), IL-6, and IL-11. Cells were analyzed for the CD41 and CD42b expression and for their ploidy status. Ex vivo produced platelets were enumerated. We show that (1) TPO alone was able to induce differentiation of CD34(+) cells into CD41(+) cells, with limited total leucocyte expansion; (2) the addition of SCF to TPO decreased significantly CD41(+) cell percentage in CB, but not in MPB; and (3) in CB, the addition of FL, IL-6, and IL-11 to TPO increased the leukocyte expansion with differentiation and terminal maturation into MK lineage. In these conditions, high numbers of immature CD34(+)CD41(+) MK progenitor cells were produced. Our results thereby demonstrate a different sensitivity of CB and MPB cells to SCF, with limited CB MK differentiation. This different sensitivity to SCF (produced constitutively by BM stromal cells) could explain the longer delay of platelet recovery after CB transplant. Nevertheless, in CB, the combination of TPO with FL, IL-6, and IL-11 allows generation of a suitable number of immature MK progenitor cells expressing both CD34 and CD41 antigens, which are supposed to be responsible for the platelet recovery after transplantation.

Do platelet apoptosis, activation, aggregation, lipid peroxidation and platelet-leukocyte aggregate formation occur simultaneously in hyperlipidemia?

OBJECTIVES: The circulating lipoproteins may cause some abnormalities in platelet composition and function in hypercholesterolemia. The aim of this study was to investigate whether platelet apoptosis, platelet activation, platelet aggregation, platelet-leukocyte aggregate (PLA) formation and lipid peroxidation occur simultaneously in hyperlipidemia. DESIGN AND METHODS: Expression of GpIIb/IIIa (CD41a), P-selectin (CD62-P), platelet-bound fibrinogen (antifibrinogen), platelet membrane phosphatidylserine (PS), platelet-monocyte aggregates (mono-PLA) and platelet-neutrophil aggregates (neut-PLA) was measured in eight hyperlipidemic and eight normal subjects using flow cytometry. ADP (10 microM) was used to activate platelets. Furthermore, ADP induced platelet aggregation responses, platelet malondialdehyde (MDA) and glutathione (GSH) levels were determined. RESULTS: Before platelet activation, platelet CD62-P, antifibrinogen, annexin-V, mono-PLA, neut-PLA and platelet MDA levels as well as platelet aggregation responses in the hyperlipidemics were significantly higher than those in the controls (P<0.01, P<0.01, P<0.01, P<0.001, P<0.001, P<0.01, P<0.001, respectively), whereas GpIIb/IIIa expression and GSH levels were not different significantly (P > 0.05). In the control group, CD62-P, antifibrinogen and annexin-V levels increased significantly after ADP activation (P<0.05, P<0.05, P<0.01, respectively). In hyperlipidemic subjects, annexin-V expression increased significantly after activation (P<0.01), whereas expression of GpIIb/IIIa, CD62-P and antifibrinogen remained unchanged (P>0.05). The levels of total cholesterol (T-CHO), low density lipoprotein cholesterol (LDL-C), serum fibrinogen (S-FGN) and high density lipoprotein cholesterol (HDL-C) in patients were found to be correlated with platelet CD62-P, antifibrinogen, annexin-V, mono-PLA and MDA. CONCLUSIONS: In conclusion, it seems that in hyperlipidemia, some platelets are in an activated state in circulation, and that increased lipid peroxidation, early apoptosis, platelet-leukocytes aggregate formation and platelet aggregation altogether accompany this process.

Signalling through the platelet glycoprotein Ib-V-IX complex.

The glycoprotein Ib-V-IX is one of the major adhesive receptors expressed on the surface of circulating platelets. It is composed of four different polypeptides-GPIbalpha, GPIbbeta, GPIX, and GPV-and represents a multifunctional receptor able to interact with a number of ligands, including the adhesive protein von Willebrand factor, the coagulation factors thrombin, factors XI and XII, and the membrane glycoproteins P-selectin and Mac-1. Interaction of GPIb-V-IX with the subendothelial von Willebrand factor is essential for primary haemostasis, as it initiates platelet adhesion to the subendothelial matrix at the sites of vascular injury even under high flow conditions. Upon interaction with von Willebrand factor, GPIb-V-IX initiates transmembrane signalling events for platelet activation, which eventually result in integrin alpha(IIb)beta(3) stimulation and platelet aggregation. The investigation of the biochemical mechanisms for platelet activation by GPIb-V-IX has attracted increasing attention during the last years. This review will describe and discuss recent findings that have provided new insights into the events underlying GPIb-V-IX transmembrane signalling. In particular, it will summarise basic concepts on the structure of this receptor, extracellular ligands, and intracellular interactors potentially involved in transmembrane signalling. The recently suggested role of membrane Fc receptors in GPIb-V-IX-initiated platelet activation will also be discussed, along with the involvement of lipid metabolising enzymes, tyrosine kinases, and the cytoskeleton in the crosstalk between GPIb-V-IX and integrin alpha(IIb)beta(3).

Characterization of prostaglandin endoperoxide H synthase-1 enzyme expression during differentiation of the megakaryocytic cell line MEG-01.

OBJECTIVE: Because the prostaglandin endoperoxide H synthase-1 (PGHS-1)-dependent formation of thromboxane A(2) is an important modulator of platelet function, this pathway represents a pharmacologic target for the inhibition of platelet function by aspirin. The objective of our research was to study how PGHS-1 expression is regulated in platelets. MATERIALS AND METHODS: Because platelets are anucleated, their protein content is a consequence of gene expression in precursor cells known as megakaryocytes. We used the immortalized human megakaryoblastic cell line MEG-01 as a model to study the expression of PGHS-1, because MEG-01 cells can be induced to differentiate into platelet-like structures by adding nanomolar concentrations of 12-0-tetradecanoylphorbol-13-acetate (TPA). We determined the expression profiles of PGHS-1 protein and mRNA in the cells comprising the three different populations of MEG-01 cultures: nucleated floating, nucleated attached, and platelet-like structures. RESULTS: We determined that PGHS-1 protein levels were higher in the nucleated adherent population than in the nucleated floating population. PGHS-1 protein levels were greatest in the anucleated platelet-like population. In contrast, we found that PGHS-1 mRNA levels were highest in the cells that comprised the nucleated adherent population. Addition of TPA induced the expression of PGHS-1 protein and mRNA in all three populations but did not change the relationship of the amount of PGHS-1 protein or mRNA expressed in a given population relative to the other two fractions. We measured the expression of PGHS-1 protein on a cell-by-cell basis in the nucleated MEG-01 populations. We found that the percentage of MEG-01 cells expressing PGHS-1 protein in the adherent population was greater than in the floating population. We measured a time-dependent increase in the percentage of cells that expressed PGHS-1 over a period of 8 days after singular addition of TPA (1.6x10(-8)M). Importantly, we observed that TPA treatment stimulated floating MEG-01 to adhere to the surface of the tissue culture vessel and that, after such treatment, only floating MEG-01 cells suffered a compromised viability. We found that a high percentage of control cells expressed glycoprotein IIb/IIIa and that TPA treatment did not significantly alter this percentage. We did not detect glycoprotein Ib in control cells but did measure a slight increase in the percentage of MEG-01 cells that expressed this antigen in the TPA-treated population. CONCLUSION: We established a correlation between the level of PGHS-1 expression and the overall level of differentiation of MEG-01 cells. PGHS-1 protein expression, which increases consistently over the full course of differentiation, now may be used as an additional and perhaps better index by which to survey megakaryocytes.

Case-control study of platelet glycoprotein receptor Ib and IIb/IIIa expression in patients with acute and chronic cerebrovascular disease.

BACKGROUND: Animal models have been instrumental in defining thrombus formation, including the role of platelet surface glycoprotein (GP) receptors, in acute ischemic stroke (AIS). However, the involvement of GP receptors in human ischemic stroke pathophysiology and their utility as biomarkers for ischemic stroke risk and severity requires elucidation. AIMS: To determine whether platelet GPIb and GPIIb/IIIa receptors are differentially expressed in patients with AIS and chronic cerebrovascular disease (CCD) compared with healthy volunteers (HV) and to identify predictors of GPIb and GPIIb/IIIa expression. METHODS: This was a case-control study of 116 patients with AIS or transient ischemic attack (TIA), 117 patients with CCD, and 104 HV who were enrolled at our University hospital from 2010 to 2013. Blood sampling was performed once in the CCD and HV groups, and at several time points in patients with AIS or TIA. Linear regression and analysis of variance were used to analyze correlations between platelet GPIb and GPIIb/IIIa receptor numbers and demographic and clinical parameters. RESULTS: GPIb and GPIIb/IIIa receptor numbers did not significantly differ between the AIS, CCD, and HV groups. GPIb receptor expression level correlated significantly with the magnitude of GPIIb/IIIa receptor expression and the neutrophil count. In contrast, GPIIb/IIIa receptor numbers were not associated with peripheral immune-cell sub-population counts. C-reactive protein was an independent predictor of GPIIb/IIIa (not GPIb) receptor numbers. CONCLUSIONS: Platelet GPIb and GPIIb/IIIa receptor numbers did not distinguish between patient or control groups in this study, negating their potential use as a biomarker for predicting stroke risk.

Phytosphingosine promotes megakaryocytic differentiation of myeloid leukemia cells.

We report that phytosphingosine, a sphingolipid found in many organisms and implicated in cellular signaling, promotes megakaryocytic differentiation of myeloid leukemia cells. Specifically, phytosphingosine induced several hallmark changes associated with megakaryopoiesis from K562 and HEL cells including cell cycle arrest, cell size increase and polyploidization. We also confirmed that cell type specific markers of megakaryocytes, CD41a and CD42b are induced by phytosphingosine. Phospholipids with highly similar structures were unable to induce similar changes, indicating that the activity of phytosphingosine is highly specific. Although phytosphingosine is known to activate p38 MAPK-mediated apoptosis, the signaling mechanisms involved in megakaryopoiesis appear to be distinct. In sum, we present another model for dissecting molecular details of megakaryocytic differentiation which in large part remains obscure.

Redistribution of glycoprotein Ib within platelets in response to protease-activated receptors 1 and 4: roles of cytoskeleton and calcium.

Thrombin activates human platelets by hydrolyzing the protease-activated receptors PAR-1 and PAR-4, exposing new N-terminal sequences which act as tethered ligands, and binding to glycoprotein (GP) Ib, whose surface accessibility transiently decreases when platelets are stimulated by the enzyme. In an attempt to better understand this latter process, we used the peptides SFLLRNPNDKYEPF (PAR-1-AP or TRAP) and AYPGKF (PAR-4-AP) to study whether hydrolysis of both PAR receptors leads to GPIb redistribution. Both peptides induced surface clearance of GPIb with a maximum at 2 min and 5 min for PAR-1-AP and PAR-4-AP, respectively, followed by a slow return to the surface with levels normalizing between 30 and 60 min. Translocation was associated with the formation of clusters of GPIb as revealed by fluorescence microscopy. This transient redistribution of GPIb was blocked by cytochalasin D and in large part by the membrane permeable Ca2+ chelator, BAPTA. The inhibitor of phosphatidylinositol 3-kinase and myosin light chain kinase, wortmannin, did not significantly modify internalization of GPIb, although its return to the surface was delayed for PAR-1-AP. PAR receptor-mediated association of GPIb to the insoluble cytoskeleton was blocked by cytochalasin D, while BAPTA alone increased and stabilized the presence of GPIb. Globally, immunoprecipitation experiments and analysis of the cytoskeleton confirmed that GPIb translocation is powered by a contractile mechanism involving Ca2+ mobilization, actin polymerization, and myosin incorporation into the cytoskeleton and that both PAR-1 and PAR-4 can activate this process.