Glycophorin A-based exclusion of red blood cells for flow cytometric analysis of platelet glycoprotein expression in citrated whole blood.

Objectives Analysis of platelet glycoprotein (GP) expression by flow cytometry is applied for diagnostic confirmation of GP-associated thrombocytopathies. While platelet-rich plasma may be used for distinct identification of target events, this strategy is not feasible for small sample volumes or for patients showing low platelet counts and/or giant platelets. However, also the use of whole blood (WB) is hampered by the difficulty to discriminate platelets from red blood cells (RBC) in such patients. To circumvent these limitations, we evaluated the feasibility of a RBC gating-out strategy. Methods In addition to platelet GPIb, GPIIa/IIIa, as well as P-selectin (CD62P), citrated whole blood (CWB) samples were stained for RBC-specific glycophorin A (CD235a). CD235a-negative platelet events were further discriminated by forward-/side-scatter characteristics and platelet GP expressions analyzed relative to that of a healthy control sample processed in parallel. Results Established reference intervals allowed for clear identification of decreased GPIIb/IIIa- or GPIb expression pattern in samples of patients with confirmed Glanzmann thrombasthenia or Bernard-Soulier syndrome, respectively. It could be shown that the analysis of 2,500 platelet events is sufficient for reliable GP expression analysis, rendering the proposed method applicable to samples with low platelet counts. Conclusions This study demonstrates the feasibility of CD235a-based exclusion of RBC for platelet GP expression analysis in CWB. In contrast to direct staining of platelet-specific antigens for target identification, this indirect gating out approach is generally applicable independent of any underlying platelet GP expression deficiency.

Device-induced platelet dysfunction in mechanically assisted circulation increases the risks of thrombosis and bleeding.

Thrombotic and bleeding complications are the major obstacles for expanding mechanical circulatory support (MCS) beyond the current use. While providing the needed hemodynamic support, those devices can induce damage to blood, particularly to platelets. In this study, we investigated device-induced alteration of three major platelet surface receptors, von Willebrand factor (VWF) and associated hemostatic functions relevant to thrombosis and bleeding. Fresh human whole blood was circulated in an extracorporeal circuit with a clinical rotary blood pump (CentriMag, Abbott, Chicago, IL, USA) under the clinically relevant operating condition for 4 hours. Blood samples were examined every hour for glycoprotein (GP) IIb/IIIa activation and receptor loss of GPVI and GPIbα on the platelet surface with flow cytometry. Soluble P-selectin in hourly collected blood samples was measured by enzyme linked immunosorbent assay to characterize platelet activation. Adhesion of device-injured platelets to fibrinogen, collagen, and VWF was quantified with fluorescent microscopy. Device-induced damage to VWF was characterized with western blotting. The CentriMag blood pump induced progressive platelet activation with blood circulating time. Particularly, GPIIb/IIIa activation increased from 1.1% (Base) to 11% (4 hours) and soluble P-selectin concentration increased from 14.1 ng/mL (Base) to 26.5 ng/mL (4 hours). Those device-activated platelets exhibited increased adhesion capacity to fibrinogen. Concurrently, the CentriMag blood pump caused progressive platelet receptor loss (GPVI and GPIbα) with blood circulating time. Specifically, MFI of the GPVI and GPIbα receptors decreased by 17.2% and 16.1% for the 4-hours sample compared to the baseline samples, respectively. The device-injured platelets exhibited reduced adhesion capacities to collagen and VWF. The high molecular weight multimers (HMWM) of VWF in the blood disappeared within the first hour of the circulation. Thereafter the multimeric patterns of VWF were stable. The change in the VWF multimeric pattern was different from the progressive structural and functional changes of platelets with the circulation time. This study suggested that the CentriMag blood pump could induce two opposite effects on platelets and associated hemostatic functions under a clinically relevant operating condition. The device-altered hemostatic function may contribute to thrombosis and bleeding simultaneously as occurring in patients supported by a rotary blood pump. Device-induced damage of platelets may be an important cause for bleeding in patients supported with rotary blood pump MCS systems relative to device-induced loss of HMWM-VWF.

Increased soluble GPVI levels in cirrhosis: evidence for early in vivo platelet activation.

Cirrhosis is a consequence of prolonged liver injury and is characterised by extensive tissue fibrosis: the deposition of collagen-rich extracellular matrix. The haemostatic balance is disordered in cirrhosis and coagulation activation appears to promote fibrosis. In spite of recent studies demonstrating a role for anticoagulant therapy in preventing cirrhosis progression, there has not been a change in clinical practice, suggesting that physicians are reluctant to anticoagulate patients with cirrhosis due to bleeding risks. Platelets play an important role in facilitating coagulation. Glycoprotein VI (GPVI) is a platelet-specific collagen receptor that is shed from the platelet surface in a metalloproteinase-dependent manner in response to GPVI ligation and coagulation activation. Our aim was to use soluble GPVI levels to determine whether there was evidence for collagen and coagulation-induced platelet activation in early, well-compensated cirrhosis. Plasma soluble GPVI levels were quantified in 46 patients with mixed aetiology cirrhosis and 55 healthy controls using an immunoassay. In the cirrhosis group, soluble GPVI levels were significantly increased (5.8 ± 4.4 ng/ml, n = 46) compared to healthy controls (3.3 ± 3.4 ng/ml, n = 55, p < 0.05). This increase in soluble GPVI levels was still evident when levels were adjusted for platelet count (Healthy controls; 0.015 ± 0.018 ng/106 platelets/ml vs. cirrhosis; 0.048 ± 0.04 ng/106 platelets/ml, p < 0.0001). This study provides evidence for early platelet activation in patients with well-compensated cirrhosis. This may have translational implications for prognosis, treatment, and risk stratification.

[The preparation and evaluation of the quality control materials for detection of platelet membrane glycoproteins by flow cytometry].

Objective: To prepare the quality control material for detection of platelet membrane glycoproteins by flowcytometry and evaluate the appearance traits, homogeneity and stability of it. Methods: Fresh platelets from the blood group O donors were fixed by the certain concentration of aldehyde solution and then washed by the imidazole buffer. After that, adding certain concentration of lyophilized protection solution into the preparations. The preparations were dispensed to be lyophilized and then were kept refrigerated in 2-8 ℃.According to the protocol of control of lyophilized biological products, the quality indicator for monitoring the prepared process, containing the appearance traits, the residual water, the platelet recovery and the rehydration quality were evaluated. The homogeneity and stability of these preparations were evaluated according to the CNAS-GL03 Guidance on evaluating the homogeneity and stability of samples used for proficiency testing and the ISO Guide 35 Reference material-general and statistical principles for certification. Results: The appearance traits and the rehydration quality of the quality control materials meeted the requirements, with the residual water distributed between 3.96% to 4.04% and the platelet recovery rate ranged from 68% to 72%.The homogeneity evaluation showed that there was no significant difference among the groups(P>0.05). The stability test indicated that the positive rate of platelet membrane glycoproteins CD42b, CD41 and CD62P of the quality control material was -0.14%, -0.14% and 0.74%, respectively, at 16 weeks after storage. There was no linear trend between the percentage of positive platelets with membrane glycoproteins and time(P>0.05). Conclusions: The quality control material for detection of platelet membrane glycoproteins by flow cytometry prepared by us meets the needs of the appearance traits, the residual water, the rehydration quality, the homogeneity and the longtime stability.It is hopeful to be used as internal quality control of the assay in clinic laboratory, the external quality assessment and proficiency evaluation.

Evaluation of soluble glycoprotein V as an in vitro quality marker for platelet concentrates: a correlation study between in vitro platelet quality markers and the effect of storage medium.

BACKGROUND AND OBJECTIVES: Extent of shape change (ESC), hypotonic shock response (HSR), the adhesive glycoprotein CD62P and lactate have been widely used as in vitro quality makers of platelet concentrates. Our aim was to evaluate soluble glycoprotein V (sGPV) as a platelet in vitro activation marker for platelet concentrates. Data were obtained from different validations during a twelve-year period. MATERIALS AND METHODS: Platelet concentrates stored in PAS B, PAS E with 20-35% plasma carry-over or in plasma were prepared either from buffy coats or collected by apheresis. The samples were analysed up to day seven using sGPV, ESC, HSR, CD62P and lactate in addition to normal quality control assays. RESULTS: sGPV correlated statistically significantly with ESC, HSR, CD62P and lactate in platelet concentrates stored in PASs. CONCLUSION: We propose that quantitative sGPV which is calculated per platelet number could be used as the first choice in vitro platelet activation marker in validations and monitoring platelet production processes. sGPV could also be a promising alternative to platelet activation assays used in interlaboratory comparisons of the quality of platelet concentrates. Quantitative lactate and lactate production rate can be used to evaluate the quality of platelets concentrates during storage. However, the formation of lactate depends on the platelet count and the amount of glucose in the product making the comparison more difficult.

Paradoxical Effect of Nonphysiological Shear Stress on Platelets and von Willebrand Factor.

Blood can become hypercoagulable by shear-induced platelet activation and generation of microparticles. It has been reported that nonphysiological shear stress (NPSS) could induce shedding of platelet receptor glycoprotein (GP) Ibα, which may result in an opposite effect to hemostasis. The aim of this study was to investigate the influence of the NPSS on platelets and von Willebrand factor (vWF). Human blood was exposed to two levels of NPSS (25 Pa, 125 Pa) with an exposure time of 0.5 s, generated by using a novel blood-shearing device. Platelet activation (P-selectin expression, GPIIb/IIIa activation and generation of microparticles) and shedding of three platelet receptors (GPIbα, GPVI, GPIIb/IIIa) in sheared blood were quantified using flow cytometry. Aggregation capacity of sheared blood induced by ristocetin and collagen was evaluated using an aggregometer. Shear-induced vWF damage was characterized with Western blotting. Consistent with the published data, the NPSS caused significantly more platelets to become activated with increasing NPSS level. Meanwhile, the NPSS induced the shedding of platelet receptors. The loss of the platelet receptors increased with increasing NPSS level. The aggregation capacity of sheared blood induced by ristocetin and collagen decreased. There was a loss of high molecular weight multimers (HMWMs) of vWF in sheared blood. These results suggest that the NPSS induced a paradoxical effect. More activated platelets increase the risk of thrombosis, while the reduction in platelet receptors and the loss of HMWM-vWF increased the propensity of bleeding. The finding might provide a new perspective to understand thrombosis and acquired bleeding disorder in patients supported with blood contacting medical devices.

Platelet-activating factor (PAF) receptor expression is associated with histopathological stage and grade and patients' survival in gastric adenocarcinoma.

Platelet activating factor (PAF) has been considered as potent inflammatory lipid mediator that exerts its actions by binding to PAF receptor (PAFR). PAF/PAFR system has been implicated in several pathophysiological states, including tumor progression, angiogenesis and metastasis. The present study aimed to evaluate the clinical significance of PAFR expression in gastric adenocarcinoma. PAFR protein expression was assessed immunohistochemically on 54 gastric adenocarcinoma tissue samples and was analyzed in relation with clinicopathological parameters, tumor proliferative capacity and patients' survival. PAFR was abundantly expressed in all gastric adenocarcinoma cases examined. Increased PAFR expression was significantly more frequently observed in well/moderately compared to poorly differentiated gastric adenocarcinoma cases (p=0.011). PAFR expression was significantly increased in intestinal- compared to diffuse-type cases (p=0.020). Elevated PAFR expression was significantly associated with smaller tumor size, absence of lymph node and organ metastasis and low tumor histopathological stage (p=0.025, p<0.001, p=0.009 and p<0.001, respectively). Additionally, patients presenting elevated PAFR expression had significantly longer survival times compared to those with low PAFR expression (log-rank test, p<0.001). These results support an important potential role of PAFR signalling in gastric malignant disease progression and render further research in this field a necessity.

In macrophages, HIV-1 assembles into an intracellular plasma membrane domain containing the tetraspanins CD81, CD9, and CD53.

In macrophages, HIV-1 has been shown to bud into intracellular structures that contain the late endosome marker CD63. We show that these organelles are not endosomes, but an internally sequestered plasma membrane domain. Using immunofluorescence microscopy and immunoelectron microscopy, we find that HIV-1 buds into a compartment that contains the tetraspanins CD81, CD9, and CD53. On uninfected macrophages, these proteins are seen at the cell surface and in intracellular vacuole-like structures with a complex content of vesicles and interconnected membranes that lack endosome markers, including CD63. Significantly, these structures are accessible to small tracers (horseradish peroxidase or ruthenium red) applied to cells at 4 degrees C, indicating that they are connected to the cell surface. HIV assembles on, and accumulates within, these intracellular compartments. Furthermore, CD63 is recruited to the virus-containing structures and incorporated into virions. These results indicate that, in macrophages, HIV-1 exploits a previously undescribed intracellular plasma membrane domain to assemble infectious particles.

Platelet surface receptor glycoprotein VI-dimer is overexpressed in stroke: The Glycoprotein VI in Stroke (GYPSIE) study results.

OBJECTIVES: Platelet activation underpins thrombus formation in ischemic stroke. The active, dimeric form of platelet receptor glycoprotein (GP) VI plays key roles by binding platelet ligands collagen and fibrin, leading to platelet activation. We investigated whether patients presenting with stroke expressed more GPVI on their platelet surface and had more active circulating platelets as measured by platelet P-selectin exposure. METHODS: 129 ischemic or hemorrhagic stroke patients were recruited within 8h of symptom onset. Whole blood was analyzed for platelet-surface expression of total GPVI, GPVI-dimer, and P-selectin by flow cytometry at admission and day-90 post-stroke. Results were compared against a healthy control population (n = 301). RESULTS: The platelets of stroke patients expressed significantly higher total GPVI and GPVI-dimer (P<0.0001) as well as demonstrating higher resting P-selectin exposure (P<0.0001), a measure of platelet activity, compared to the control group, suggesting increased circulating platelet activation. GPVI-dimer expression was strongly correlated circulating platelet activation [r2 = 0.88, P<0.0001] in stroke patients. Furthermore, higher platelet surface GPVI expression was associated with increased stroke severity at admission. At day-90 post-stroke, GPVI-dimer expression and was further raised compared to the level at admission (P<0.0001) despite anti-thrombotic therapy. All ischemic stroke subtypes and hemorrhagic strokes expressed significantly higher GPVI-dimer compared to controls (P<0.0001). CONCLUSIONS: Stroke patients express more GPVI-dimer on their platelet surface at presentation, lasting at least until day-90 post-stroke. Small molecule GPVI-dimer inhibitors are currently in development and the results of this study validate that GPVI-dimer as an anti-thrombotic target in ischemic stroke.

Sandwich immunoassay for soluble glycoprotein VI in patients with symptomatic coronary artery disease.

BACKGROUND: Platelet glycoprotein VI (pGPVI) expression is increased in acute coronary syndrome (ACS), reflecting platelet activation. There is no reliable method available to measure pGPVI. Our aim was to develop a bead-based sandwich immunoassay to measure soluble GPVI (sGPVI). METHODS: Based on antibodies for sGPVI developed earlier, we established and validated a bead-based sandwich immunoassay in 2438 consecutive patients with stable angina pectoris (SAP; n = 1371), non-ST-elevation myocardial infarction (NSTEMI; n = 724), and ST-elevation MI (STEMI; n = 343). In a subgroup (n = 1011), we measured surface expression of pGPVI using flow cytometry. RESULTS: The assay revealed a working range of 8-500 ng/L. Intra- and interassay imprecision was <7% and <14%, respectively. Patients with NSTEMI and STEMI showed significantly lower mean sGPVI concentrations than patients with SAP [mean (SD), 8.4 (3.6) µg/L and 8.6 (4.1) µg/L vs 9.8 (4.8) µg/L; P = 0.002], whereas subgroup analysis revealed significantly enhanced pGPVI in NSTEMI (n = 276) and STEMI (n = 80) patients compared with SAP (n = 655) [mean fluorescence intensity (SD), 21.2 (8.1) and 19.8 (6.8) vs 18.5 (7.7); P = 0.002 and P = 0.018]. pGPVI and sGPVI were inversely correlated (r = -0.076; P = 0.023). Area under the ROC curve was 0.716, 95% CI 0.681-0.751, for sGPVI, distinguishing patients with SAP from those with ACS, and was superior (P = 0.044) to the curve of subgroup analysis for pGPVI (0.624, 95% CI 0.586-0.662). sGPVI (P = 0.023) and pGPVI (P = 0.028) had better association with the development of ACS than troponin I (P = 0.055) in the very early stage of disease, based on logistic regression analysis. CONCLUSIONS: This sandwich immunoassay reliably measures sGPVI and may help to identify patients with ACS earlier than other laboratory markers.

Design and reshaping of an scFv directed against human platelet glycoprotein VI with diagnostic potential.

Blood platelets play a key role in physiological hemostasis and in thrombosis. As a consequence, platelet functional analysis is widely used in the diagnosis of hemorrhagic disorders as well as in the evaluation of thrombosis risks and of the efficacy of antithrombotics. Glycoprotein (GP) VI is a platelet-specific collagen-signaling receptor. Clinical studies suggest that increased GPVI expression is associated with a risk of arterial thrombosis. Conversely, GPVI deficiencies have been identified in patients with defective platelet responses to collagen. Currently, there is no standard test available for measuring GPVI expression, essentially because antibodies usually cross-link GPVI upon binding, leading to platelet activation and consecutive changes in GPVI expression. Here, we designed a recombinant monovalent antibody fragment (scFv) derived from an anti-GPVI monoclonal IgG, 3J24, with the characteristics required to analyze GPVI expression. Guided by in silico modeling and V-KAPPA chain analysis, a Protein L (PpL) recognition pattern was engineered in the scFv, making possible its purification and detection using PpL conjugates. The PpL affinity-purified scFv is functional. It retains GPVI-binding specificity and allows detection of platelet surface-expressed GPVI without inducing platelet activation. In conclusion, the reshaped scFv may be very useful in the development of diagnostic approaches.

Degranulation plays an essential part in regulating cell surface expression of Fas ligand in T cells and natural killer cells.

Fas ligand (FasL) triggers apoptosis during cytotoxicity mediated by cytotoxic T lymphocytes and during immune downregulation. The ability of T cells and natural killer cells to trigger apoptosis through this mechanism is controlled by the cell surface expression of FasL (ref. 2). Because FasL expression is up-regulated on activation, FasL was thought to be delivered directly to the cell surface. Here we show that newly synthesized FasL is stored in specialized secretory lysosomes in both CD4+ and CD8+ T cells and natural killer cells, and that polarized degranulation controls the delivery of FasL to the cell surface. In this way, FasL-mediated apoptosis is finely controlled by receptor-mediated target-cell recognition. The cytoplasmic tail of FasL contains signals that sort FasL to secretory lysosomes in hemopoietic cells. This pathway may provide a general mechanism for controlling the cell surface appearance of proteins involved in immune regulation.

Loading solution prevents activation damage of human platelets before lyophilization.

The current study aims to optimize the compositions of platelet activation-inhibitors for a loading solution of lyophilizing protectants and to establish a series of perfect pretreatment methods for platelet lyophilization. The optimal combination of six kinds of inhibitors and loading solutions of lyophilizing protectants, including prostaglandin E1 (PGE1), adenosine, L-arginine, phyticacid, bivalirudin, and cilostazol, was analyzed using the orthogonal experimental design. The values of the expression rates of p-selectin (CD62p) and platelet membrane glycoprotein (PAC-1), as well as of platelet and mean platelet volume (MPV), were selected as indices of platelet activation. The values of CD62p and Pac-1 induced by thrombin were determined as indices of platelet reactivity. The maximal aggregation and slide platelet aggregation test (SPAT) induced by the inducer were calculated as indices of the aggregation function of platelets. Level I of the loading condition factor had no adverse action on MPV, CD62p, PAC-1, SPAT, and the maximum platelet aggregation rate. Level II of factors PGE1, L-arginine, phycicacid sodium, and Bivalirudin could inhibit the activation of platelets and enable them to retain their function. The results show that the optimal solution compounding was the third group. The loading solution, which includes plasma, 1 µM prostaglandin E1, 5 mM L-arginine, 0.5 mM phyticacid, and 0.5 µM bivalirudin, could prevent the activation damage of platelets before lyophilization.

Search for a platelet-activating factor receptor in the Trypanosoma cruzi proteome: a potential target for Chagas disease chemotherapy.

Chagas disease (CD) causes the highest burden of parasitic diseases in the Western Hemisphere and is therefore a priority for drug research and development. Platelet-activating factor (PAF) causes the CD parasite Trypanosoma cruzi to differentiate, which suggests that the parasite may express PAF receptors. Here, we explored the T. cruzi proteome for PAF receptor-like proteins. From a total of 23,000 protein sequences, we identified 29 hypothetical proteins that are predicted to have seven transmembrane domains (TMDs), which is the main characteristic of the G protein-coupled receptors (GPCRs), including the PAF receptor. The TMDs of these sequences were independently aligned with domains from 25 animal PAF receptors and the sequences were analysed for conserved residues. The conservation score mean values for the TMDs of the hypothetical proteins ranged from 31.7-44.1%, which suggests that if the putative T. cruzi PAF receptor is among the sequences identified, the TMDs are not highly conserved. These results suggest that T. cruzi contains several GPCR-like proteins and that one of these GPCRs may be a PAF receptor. Future studies may further validate the PAF receptor as a target for CD chemotherapy.

Molecular mapping of platelet hyperreactivity in diabetes: the stress proteins complex HSPA8/Hsp90/CSK2α and platelet aggregation in diabetic and normal platelets.

The molecular understanding of the pathophysiological changes elicited by diabetes in platelets may help in further elucidating the involvement of this pseudo-cell in the increased risk of developing cardiovascular disease and thrombosis in diabetic subjects. We aimed to investigate the differential characteristics of platelets from diabetic patients and nondiabetic controls to unveil the molecular mechanisms behind the increased platelet reactivity in diabetes. We compared platelets from diabetic and control subjects by 2 dimensional-electrophoresis followed by mass spectrometry. Changes in selected differential proteins were validated by immunoprecipitation assays and western blot. Platelet aggregation was measured by light transmittance aggregometry induced by collagen and ADP, and dynamic coagulation analysis of whole blood was measured by thromboelastometry. We observed significant differences in proteins related to platelet aggregation, cell migration, and cell homeostasis. Subjects with diabetes showed higher platelet aggregation and thrombogenicity and higher contents of the stress-related protein complex HSPA8/Hsp90/CSK2α than nondiabetic subjects. Changes in the chaperones HSPA8 and Hsp90, and in CSK2α protein contents correlated with changes in platelet aggregation and blood coagulation activity. In conclusion, the complex HSPA8/Hsp90/CSK2α is involved in diabetes-related platelet hyperreactivity. The role of the HSPA8/Hsp90/CSK2α complex may become a molecular target for the development of future preventive and therapeutic strategies for platelet dysfunction associated with diabetes and its complications.

P-selectin and vascular cell adhesion molecule 1 mediate rolling and arrest, respectively, of CD4+ T lymphocytes on tumor necrosis factor alpha-activated vascular endothelium under flow.

This report examines the adhesive interactions of human CD4+ T lymphocytes with tumor necrosis factor alpha-activated human endothelial cell monolayers in an in vitro model that mimics microcirculatory flow conditions. Resting CD4+ T cell interactions with activated endothelium consisted of initial attachment followed by rolling, stable arrest, and then spreading and transendothelial migration. P-selectin, but not E-, or L-selectin, mediated most of this initial contact and rolling, whereas beta 1-integrins (alpha 4 beta 1), interacting with endothelial-expressed vascular cell adhesion molecule 1, participated in rolling and mediated stable arrest. In contrast, beta 2-integrins were primarily involved in spreading and transmigration. These findings highlight an important role for P-selectin and suggest discrete functions for beta 1- and beta 2-integrins during lymphocyte recruitment to sites of immune-mediated inflammation.

A cytologic assay for diagnosis of food hypersensitivity in patients with irritable bowel syndrome.

BACKGROUND & AIMS: A percentage of patients with symptoms of irritable bowel syndrome (IBS) suffer from food hypersensitivity (FH) and improve on a food-elimination diet. No assays have satisfactory levels of sensitivity for identifying patients with FH. We evaluated the efficacy of an in vitro basophil activation assay in the diagnosis of FH in IBS-like patients. METHODS: Blood samples were collected from 120 consecutive patients diagnosed with IBS according to Rome II criteria. We analyzed in vitro activation of basophils by food allergens (based on levels of CD63 expression), as well as total and food-specific immunoglobulin (Ig)E levels in serum. Effects of elimination diets and double-blind food challenges were used as standards for FH diagnosis. RESULTS: Twenty-four of the patients (20%) had FH (cow's milk and/or wheat hypersensitivity); their symptom scores improved significantly when they were placed on an elimination diet. Patients with FH differed from other IBS patients in that they had a longer duration of clinical history, a history of FH as children, and an increased frequency of self-reported FH; they also had hypersensitivities to other antigens (eg, egg or soy). The basophil activation assay diagnosed FH with 86% sensitivity, 88% specificity, and 87% accuracy; this level of sensitivity was significantly higher than that of serum total IgE or food-specific IgE assays. CONCLUSIONS: A cytometric assay that quantifies basophils after stimulation with food antigens based on cell-surface expression of CD63 had high levels of sensitivity, specificity, and accuracy in diagnosing FH. This assay might be used to diagnose FH in patients with IBS-like symptoms.

Proteolysis of platelet receptors in humans and other species.

In the past 5 years, metalloproteinase-mediated ectodomain shedding of platelet receptors has emerged as a new mechanism for modulating platelet function. By regulating surface expression of the platelet-specific receptors, glycoprotein (GP)VI that binds collagen, and GPIbalpha (the major ligand-binding subunit of the GPIb-IX-V complex) that binds von Willebrand factor (VWF) and other procoagulant and proinflammatory ligands, shedding not only irreversibly downregulates GPVI/GPIbalpha function, but generates proteolytic fragments that might be unique biomarkers or modulators in plasma. This is potentially significant because GPVI and GPIbalpha are involved in initiating thrombotic diseases such as heart attack and stroke, as well as autoimmune diseases where anti-platelet antibodies result in thrombocytopenia. Altered expression levels of GPIbalpha/GPVI are associated with both thrombotic propensity and platelet aging, suggesting an additional role in platelet clearance. Although emerging data are elucidating molecular mechanisms underlying GPIbalpha/GPVI shedding, evidence for the functional consequences of shedding in vivo, either clinically or in animal models, is far more limited. Here we consider recent published evidence for GPVI or GPIbalpha shedding in humans, nonhuman primates and mice, and whether conservation of sheddase cleavage sites across species points to a functional role for metalloproteolytic shedding in vivo.

Serum total tryptase levels are increased in patients with active chronic urticaria.

BACKGROUND: We have demonstrated previously mast cell histamine release upon incubation with chronic urticaria (CU) sera, presumably by degranulation. OBJECTIVE: To explore total and mature tryptase in order to assess whether any increase in total tryptase levels is due in part to mast cell degranulation or to mast cell burden. We also wanted to explore differences between the autoimmune groups called idiopathic (serum unable to activate basophils), and to correlate total and mature tryptase levels with different urticaria features. METHODS: We measured total and mature tryptase serum levels in 81 CU patients, 16 atopic donors and 21 healthy control sera. We assessed autoimmunity by measuring the CD63 expression in normal basophil donors upon incubation with CU sera. RESULTS: We found significantly higher levels of total tryptase in the sera of CU patients (6.6 ±4.1 µg/L) than in sera from healthy non-atopic subjects (4.4 ±2.8 µg/L) and from atopic subjects (4.5 ±1.7 µg/L). Mature tryptase levels were undetectable (<1 ng/mL). Total tryptase levels in the autoimmune urticaria group were significantly higher (9.8 ±5.4 µg/L) than the idiopathic urticaria group (4.4 ±2.2 µg/L). A significant difference in total tryptase was found between symptomatic patients (7.3 ±4.1 µg/L) compared with asymptomatic ones (5.7 ±4.1 µg/L) at the time of venesection. No difference was found in mature tryptase levels either. CONCLUSION: Total elevated tryptase levels are not accompanied by an elevated mature tryptase levels, as might be expected if the serum levels reflected mast cell degranulation.

Diagnostic and therapeutic potentials of platelet glycoprotein VI.

Platelets respond immediately to vascular injury by adhesion, aggregation, and thrombus formation. Disruption of the endothelial cell layer exposes extracellular matrix to the bloodstream. Collagen binding to platelet glycoprotein VI (GPVI) mediates the initial adhesion of the rolling platelet to the vascular wound. Signaling by GPVI leads to the onset of the platelet activation cascade that is finally crowned by a firm and shear-resistant integrin-based adhesive clot. Blockade of collagen binding to GPVI would prevent initial adhesion and further activation of the platelet and would have an enormous impact in antithrombotic therapy. Besides the therapeutical implication, radiolabeled GPVI gives us a valuable diagnostic means that may be used clinically for plaque imaging in the near future.