Does three-dimensional intraglandular location predict malignancy in parotid tumors?

Tumors arising within the parotid encompass a heterogeneous mix of benign and malignant neoplasms and other tissue growths. The purpose of this study was to determine the association between the location of intraparotid masses and the risk of malignancy. A retrospective cohort study was performed of patients diagnosed with parotid tumors following open tumor excision. The primary predictor variable was the location of the epicenter of the tumor in three-dimensional space, as determined from preoperative imaging. Other variables were patient demographics and clinical parameters. The primary outcome variable was the final histopathologic diagnosis of a benign or malignant process. A χ2 analysis was performed to test for any significant associations between demographic, clinical, and radiographic factors in relation to the outcome, and backwards stepwise logistic regression analysis was used to control for variables. Both increasing age (P = 0.002) and the presence of local pain (P = 0.020) were associated with malignancy. Tumors located anterior to the posterior border of the retromandibular vein had 2.18 times higher odds of malignancy (95% confidence interval 1.13-4.21; P = 0.020). Multivariate regression analysis suggested that patient age, the presence of pain, and tumor location anterosuperiorly and superoinferiorly could all assist in determining the odds of malignancy.

Role of alloplastic reconstruction of the temporomandibular joint in the juvenile idiopathic arthritis population.

We present outcomes following total joint replacement of the temporomandibular joint (TMJ) in adolescent and young adult patients with juvenile idiopathic arthritis (JIA), and discuss a multidisciplinary treatment model. A retrospective review was performed of patients presenting to the University of North Carolina Oral and Maxillofacial Surgery Service (Chapel Hill, NC) from 2016- 2018 who underwent unilateral or bilateral total replacement of the TMJ for a diagnosis of end-stage joint disease secondary to JIA. Inclusion criteria included diagnosis by a rheumatologist, presentation to our department in adolescence (under 18 years of age), surgical intervention in adolescence or young adulthood (under 25 years of age), and documentation of preoperative and postoperative pain, maximum incisal opening (MIO), and quality of life measures. A database was created and data were then analysed both qualitatively and quantitatively. Five patients met the inclusion criteria. All achieved MIO of more than 35mm with a mean improvement of 24mm, and were able to tolerate a regular diet. All preoperative pain had essentially been eliminated. All patients reported a considerable improvement in quality of life. To our knowledge, this is the first report to document a series of paediatric and young adult patients with JIA who required total replacement of the joint for end-stage joint disease. To our knowledge, it is also the first to describe the use of a collaborative clinic of oral and maxillofacial surgeons, neuroradiologists, dental radiologists, orofacial pain specialists, paediatric rheumatologists, and paediatric nurse practitioners, to care for these patients.

Atherosclerotic plaque injury-mediated murine thrombosis models: advantages and limitations.

In spite of current treatment strategies, myocardial infarction and stroke are still major causes of death worldwide. These events are triggered by damage of an atherosclerotic plaque, resulting in occlusive thrombus formation. Mouse studies have significantly contributed to our understanding of the mechanisms of atherogenesis and of thrombosis following plaque injury, but the extent to which the mouse serves as an accurate model of human disease is open to discussion. In this review, we provide a detailed overview and comparison of the described mouse models for atherothrombosis including their (dis)advantages. Herein guidance is provided on how to select a suitable atherothrombosis model for research questions primarily relevant to the field of thrombosis.

Functional properties of human platelets derived in vitro from CD34+ cells.

The in vitro production of blood platelets for transfusion purposes is an important goal in the context of a sustained demand for controlled products free of infectious, immune and inflammatory risks. The aim of this study was to characterize human platelets derived from CD34+ progenitors and to evaluate their hemostatic properties. These cultured platelets exhibited a typical discoid morphology despite an enlarged size and expressed normal levels of the major surface glycoproteins. They aggregated in response to ADP and a thrombin receptor agonist peptide (TRAP). After infusion into NSG mice, cultured and native platelets circulated with a similar 24 h half-life. Notably, the level of circulating cultured platelets remained constant during the first two hours following infusion. During this period of time their size decreased to reach normal values, probably due to their remodeling in the pulmonary circulation, as evidenced by the presence of numerous twisted platelet elements in the lungs. Finally, cultured platelets were capable of limiting blood loss in a bleeding assay performed in thrombocytopenic mice. In conclusion, we show here that cultured platelets derived from human CD34+ cells display the properties required for use in transfusion, opening the way to clinical trials.

Necrotizing soft tissue infection following routine third molar extraction: report of two cases and review of the literature.

Necrotizing cellulitis, necrotizing fasciitis, and necrotizing myositis are a constellation of severe soft tissue infections characterized by rapid progression, dusky soft tissue changes, and edema and induration expanding beyond the clinical wound edges. The cases of two female patients with type II necrotizing soft tissue infections occurring after routine third molar extraction are reported here. The patients were treated for the infections at the University of North Carolina Hospitals in 2016. Both were previously healthy. Of particular interest, recent inoculation of group A Streptococcus appears to have contributed to the infection in both cases.

Ultrasound versus magnetic resonance imaging of the temporomandibular joint in juvenile idiopathic arthritis: a systematic review.

A systematic review of published articles on ultrasound (US) and magnetic resonance imaging (MRI) of the temporomandibular joint (TMJ) in juvenile idiopathic arthritis (JIA) was performed to answer the question "What is the sensitivity and specificity of US as compared to MRI in diagnosing acute and chronic joint changes in patients with JIA?" The most recent evidence was sought in published articles via a search of the PubMed, Ovid, and Embase databases. Article appraisal was performed by two reviewers. Nineteen articles reporting prospective or ambispective studies comparing US to MRI in TMJ imaging were found. Six of these articles were specific to JIA patients. The heterogeneity of these articles made comparison difficult. Of the acute and chronic changes assessed (disk displacement, joint effusion, bony deformity), only joint effusion was appropriately assessed by multiple authors, with US having a sensitivity of 0-72% and specificity of 70-83% as compared to MRI. There was a paucity of studies specific to JIA, with many studying adult, non-rheumatic patients. This systematic review found that dynamic imaging with high-resolution US improves sensitivity and specificity compared to static, low-resolution US. Additionally, there is evidence to suggest that US imaging following a baseline MRI can increase US sensitivity and specificity and may have a future role in disease surveillance.

Platelets are dispensable for antibody-mediated transfusion-related acute lung injury in the mouse.

UNLABELLED: Essentials Role of platelets in immunological transfusion-related acute lung injury (TRALI) is debated. Immunological TRALI was tested in mice exhibiting severe thrombocytopenia or platelet dysfunction. Platelets are required to prevent lung hemorrhage but not edema formation and respiratory distress. Platelets are dispensable for the initiation and development of TRALI. SUMMARY: Background Transfusion-related acute lung injury (TRALI) is a serious transfusion-related complication. Previous conflicting studies have indicated that platelets are either crucial or dispensable for TRALI. Objectives To evaluate the role of platelets in major histocompatibility complex (MHC) I-induced-TRALI. Methods Antibody-mediated TRALI was experimentally induced in mice by lipopolysaccharide priming followed by the administration of an anti-MHC I mAb. Results TRALI was tested in the context of severe thrombocytopenia provoked by the administration of diphtheria toxin (DT) in transgenic iDTR mice selectively expressing DT receptor in megakaryocytes. The pathologic responses occurring within the first 10 min following the injection of the anti-MHC I mAb, i.e. the severity of lung edema and the drop in aortic blood oxygenation, were similar in severely thrombocytopenic DT-iDTR and control mice. At later times, mortality was nevertheless increased in DT-iDTR mice, owing to lung hemorrhages. When less severe thrombocytopenia was induced with an antiplatelet mAb, TRALI started and developed similarly as in control mice, but hemorrhages were absent. Furthermore, when platelet functions were defective because of administration of aspirin or clopidogrel, or because of glycoprotein (GP)IIbIIIa deficiency, TRALI still developed but no lung hemorrhages were observed. In contrast, when GPVI was immunodepleted, TRALI still occurred, but was occasionally accompanied by hemorrhages. Conclusions Platelets are dispensable for the initiation and development of MHC I-induced TRALI. Although they do not protect against the disruption of the vascular endothelial cell barrier and the subsequent plasma leakage and edema formation, platelets are essential to prevent more serious damage resulting in hemorrhages in alveoli.

ATP-binding cassette transporter 1 (ABCA1) deficiency decreases platelet reactivity and reduces thromboxane A2 production independently of hematopoietic ABCA1.

UNLABELLED: ESSENTIALS: The role of ATP-binding cassette transporter 1 (ABCA1) in platelet functions is poorly characterized. We studied the impact of ABCA1 deficiency on platelet responses in a mouse model and two Tangier patients. ABCA1-deficient platelets exhibit reduced positive feedback loop mechanisms. This reduced reactivity is dependent on external environment and independent of hematopoietic ABCA1. BACKGROUND: The ATP-binding cassette transporter ABCA1 is required for the conversion of apolipoprotein A-1 to high-density lipoprotein (HDL), and its defect causes Tangier disease, a rare disorder characterized by an absence of HDL and accumulation of cholesterol in peripheral tissues. The role of ABCA1 in platelet functions remains poorly characterized. OBJECTIVE: To determine the role of ABCA1 in platelet functions and to clarify controversies concerning its implication in processes as fundamental as platelet phosphatidylserine exposure and control of platelet membrane lipid composition. METHODS AND RESULTS: We studied the impact of ABCA1 deficiency on platelet responses in a mouse model and in two Tangier patients. We show that platelets in ABCA1-deficient mice are slightly larger in size and exhibit aggregation and secretion defects in response to low concentrations of thrombin and collagen. These platelets have normal cholesterol and major phospholipid composition, granule morphology, or calcium-induced phosphatidylserine exposure. Interestingly, ABCA1-deficient platelets display a reduction in positive feedback loop mechanisms, particularly in thromboxane A2 (TXA2) production. Hematopoietic chimera mice demonstrated that defective eicosanoids production, particularly TXA2, was primarily dependent on external environment and not on the hematopoietic ABCA1. Decreased aggregation and production of TXA2 and eicosanoids were also observed in platelets from Tangier patients. CONCLUSIONS: Absence of ABCA1 and low HDL level induce reduction of platelet reactivity by decreasing positive feedback loops, particularly TXA2 production through a hematopoietic ABCA1-independent mechanism.

The future of glycoprotein VI as an antithrombotic target.

The treatment of acute coronary syndromes has been considerably improved in recent years with the introduction of highly efficient antiplatelet drugs. However, there are still significant limitations: the recurrence of adverse vascular events remains a problem, and the improvement in efficacy is counterbalanced by an increased risk of bleeding, which is of particular importance in patients at risk of stroke. One of the most attractive targets for the development of new molecules with potential antithrombotic activity is platelet glycoprotein (GP)VI, because its blockade appears to ideally combine efficacy and safety. This review summarizes current knowledge on GPVI regarding its structure, its function, and its role in physiologic hemostasis and thrombosis. Strategies for inhibiting GPVI are presented, and evidence of the antithrombotic efficacy and safety of GPVI antagonists is provided.

Mechanisms underlying FeCl3-induced arterial thrombosis.

BACKGROUND: The FeCl(3)-induced vascular injury model is widely used to study thrombogenesis in vivo, but the processes leading to vascular injury and thrombosis are poorly defined. OBJECTIVES: The aim of our study was to better characterize the mechanisms of FeCl(3)-induced vascular injury and thrombus formation, in order to evaluate the pathophysiological relevance of this model. METHODS: FeCl(3) was applied at different concentrations (from 7.5% to 20%) and for different time periods (up to 5 min) to mouse carotid or mesenteric arteries. RESULTS: Under all the conditions tested, ultrastructural analysis revealed that FeCl(3) diffused through the vessel wall, resulting in endothelial cell denudation without exposure of the inner layers. Hence, only the basement membrane components were exposed to circulating blood cells and might have contributed to thrombus formation. Shortly after FeCl(3) application, numerous ferric ion-filled spherical bodies appeared on the endothelial cells. Interestingly, platelets could adhere to these spheres and form aggregates. Immunogold labeling revealed important amounts of tissue factor at their surface, suggesting that these spheres may play a role in thrombin generation. In vitro experiments indicated that FeCl(3) altered the ability of adhesive proteins, including collagen, fibrinogen and von Willebrand factor, to support platelet adhesion. Finally, real-time intravital microscopy showed no protection against thrombosis in GPVI-immunodepleted and ß(1)(-/-) mice, suggesting that GPVI and ß(1) integrins, known to be involved in initial platelet adhesion and activation, do not play a critical role in FeCl(3)-induced thrombus formation. CONCLUSION: This model should be used cautiously, in particular to study the earliest stage of thrombus formation.

Arterial thrombosis: relevance of a model with two levels of severity assessed by histologic, ultrastructural and functional characterization.

BACKGROUND: We previously described a model of laser-induced thrombosis in mesenteric arterioles with superficial and deep levels of injury producing a transient thrombus resolving within 2 min and a larger almost occlusive thrombus, respectively. Both types of lesion were sensitive to platelet GPIIb-IIIa and P2Y(12) inhibition, whereas only deep injuries were sensitive to thrombin blockade. OBJECTIVE: The aim of the present study was to use histologic methods and electron and intravital microscopy to characterize the lesions and thrombi and to extend our knowledge of the sensitivity of this model to genetic and pharmacologic inhibition. RESULTS: A superficial injury was found to detach the endothelial cells and expose a collagen III- and IV-rich subendothelium where platelets could adhere. Tissue factor and fibrin were not detected. Deeper penetration of the external elastic lamina occurred in deep injuries, with exposure of collagen I, III and IV. Here the thrombus was composed of platelets exhibiting a decreasing gradient of degranulation from the deepest lesion area to the surface. Fibrin was found close to the most activated platelets. Consistently, glycoprotein VI (GPVI)-collagen and GPIb-von Willebrand factor (VWF) interactions were found to be critical in superficial injuries. After deep lesion, thrombus formation was modestly reduced in GPVI-immunodepleted mice and still strongly inhibited in VWF(-/-) mice. Combined hirudin infusion and GPVI depletion further inhibited thrombosis after deep injury. CONCLUSIONS: This study confirms the feasibility of inducing arterial thrombosis with distinct levels of severity and establishes the central roles of collagen and VWF in thrombus formation after superficial injury. Collagen, VWF and thrombin all appear to contribute to thrombosis after deep arterial lesion.

Ex vivo inhibition of thrombus formation by an anti-glycoprotein VI Fab fragment in non-human primates without modification of glycoprotein VI expression.

OBJECTIVES: Glycoprotein (GP)VI is an attractive target for the development of new antithrombotic drugs. Its deficiency protects animals in several models of thrombosis, arterial stenosis and ischemia--reperfusion while inducing no major bleeding tendency. The Fab fragment of one anti-GPVI monoclonal antibody (9O12.2) inhibits all GPVI functions in vitro. The aim of this study was to determine the ex vivo effects of 9O12.2 Fab on hemostasis, coagulation and thrombosis in non-human primates. METHODS AND RESULTS: Blood samples were collected from cynomolgus monkeys before and after (30, 90 and 150 min, 1 and 7 days) a bolus injection of 9O12.2 Fab (4 mg kg(-1)) or vehicle. Platelet counts and coagulation tests (prothrombin time, activated partial thromboplastin time) were not modified following Fab injection. The PFA-100 closure time increased during the first hours and returned to initial values on day + 1. Platelet-bound Fab was detected from 30 min to 24 h after Fab injection without GPVI depletion at any time. Collagen-induced platelet aggregation was selectively and fully inhibited at 30 min. Thrombus formation on collagen in flowing whole blood (1500 s(-1)) was delayed and decreased, and collagen-induced or tissue factor-induced thrombin generation in platelet-rich plasma was profoundly inhibited. CONCLUSION: The anti-GPVI 9O12.2 Fab inhibits thrombus formation ex vivo in non-human primates with a composite effect on platelet activation and thrombin generation in the absence of GPVI depletion.

A differential role of the platelet ADP receptors P2Y1 and P2Y12 in Rac activation.

The dynamics of the actin cytoskeleton, largely controlled by the Rho family of small GTPases (Rho, Rac and Cdc42), is critical for the regulation of platelet responses such as shape change, adhesion, spreading and aggregation. Here, we investigated the role of adenosine diphosphate (ADP), a major co-activator of platelets, on the activation of Rac. ADP rapidly activated Rac in a dose-dependent manner and independently of GPIIb/IIIa and phosphoinositide 3-kinase. ADP alone, used as a primary agonist, activated Rac and its effector PAK via its P2Y1 receptor, through a G(q)-dependent pathway and independently of P2Y12. The P2Y12 receptor appeared unable to activate the GTPase per se as also observed for the adenosine triphosphate receptor P2X1. Conversely, secreted ADP strongly potentiated Rac activation induced by FcgammaRIIa clustering or TRAP via its P2Y12 receptor, the target of antithrombotic thienopyridines. Stimulation of the alpha(2A)-adrenergic receptor/G(z) pathway by epinephrine was able to replace the P2Y12/G(i)-mediated pathway to amplify Rac activation by FcgammaRIIa or by the thrombin receptor PAR-1. This co-activation appeared necessary to reach a full stimulation of Rac as well as PAK activation and actin polymerization and was blocked by a G-protein betagamma subunits scavenger peptide.

Adenine triphosphate nucleotides are antagonists at the P2Y receptor.

The aim of the present study was to characterize the pharmacological profile of the P2Y(12) receptor for several adenine triphosphate nucleotides in view of their possible roles as partial agonists or true antagonists. Two distinct cellular systems were used: P2Y(1) receptor deficient mouse platelets ( platelets) previously shown to express a native and functional P2Y(12) receptor and 1321 N1 astrocytoma cells stably expressing the human P2Y(12) receptor (1321 N1 P2Y(12)). ADP and its structural analogues inhibited cAMP accumulation in a dose-dependent manner in both platelets and 1321 N1 P2Y(12) cells with a similar rank order of potency, 2 methylthio-ADP (2MeSADP) >>ADP - Adenosine 5'-(betathio) diphosphate (AlphaDPbetaS). Commercial ATP, 2 chloro; ATP (2ClATP) and 2 methylthio-ATP (2MeSATP) also inhibited cAMP accumulation in both cell systems. In contrast, after creatine phosphate (CP)/creatine phosphokinase (CPK) regeneration, adenine triphosphate nucleotides lost their agonistic effect on platelets and behaved as antagonists of ADP (0.5 microm)-induced adenylyl cyclase inhibition with IC(50) of 13.5 +/- 4.8, 838 +/- 610, 1280 +/- 1246 microm for 2MeSATP, ATP and 2ClATP, respectively. In 1321 N1 P2Y(12) cells, CP/CPK regenerated ATP and 2ClATP lost their agonistic effect only when CP/CPK was maintained during the cAMP assay. The stable ATP analogue ATPgammaS antagonized ADPbetaS-induced inhibition of cAMP accumulation in both platelets and 1321 N1 P2Y(12) cells. Thus, ATP and its triphosphate analogues are not agonists but rather antagonists at the P2Y(12) receptor expressed in platelets or transfected cells, provided care is taken to remove diphosphate contaminants and to prevent the generation of diphosphate nucleotide derivatives by cell ectonucleotidases.

Lineage-specific overexpression of the P2Y1 receptor induces platelet hyper-reactivity in transgenic mice.

In order to investigate the role of the platelet P2Y1 receptor in several aspects of platelet activation and thrombosis, transgenic (TG) mice overexpressing this receptor specifically in the megakaryocytic/platelet lineage were generated using the promoter of the tissue-specific platelet factor 4 gene. Studies of the saturation binding of [33P]2MeSADP in the presence or absence of the selective P2Y1 antagonist MRS2179 indicated that wild-type (WT) mouse platelets bore 150 +/- 31 P2Y1 receptors and TG platelets 276 +/- 34, representing an 84% increase in P2Y1 receptor density. This led to a well defined phenotype of platelet hyper-reactivity in vitro, as shown by increased aggregations in response to adenosine 5'-diphosphate (ADP) and low concentration of collagen in TG as compared with WT platelets. Moreover, overexpression of the P2Y1 receptor enabled ADP to induce granule secretion, unlike in WT platelets, which suggests that the level of P2Y1 expression is critical for this event. Our results further suggest that the weak responses of normal platelets to ADP are due to a limited number of P2Y1 receptors rather than to activation of a specific transduction pathway. TG mice displayed a shortened bleeding time and an increased sensitivity to in vivo platelet aggregation induced by infusion of a mixture of collagen and epinephrine. Overall, these findings emphasize the importance of the P2Y1 receptor in hemostasis and thrombosis and suggest that variable expression levels of this receptor on platelets might play a role in thrombotic states in human, which remains to be assessed.

Mpl ligand increases P2Y1 receptor gene expression in megakaryocytes with no concomitant change in platelet response to ADP.

The P2Y(1) receptor is responsible for the initiation of platelet aggregation in response to ADP and plays a key role in thrombosis. Although this receptor is expressed early in the platelet lineage, the regulation of its expression during megakaryocyte differentiation is unknown. In the mouse megakaryocytic cell line Y10/L8057, we detected P2Y(1) mRNA of three sizes (2.5, 4.4, and 7.4 kb). These cells have previously been shown to respond to Mpl ligand, the pivotal regulator of megakaryocytopoiesis, by increasing their expression of differentiation markers. Mpl ligand enhanced levels of P2Y(1) mRNAs in Y10/L8057 cells and this effect was selective: the same cytokine did not increase levels of A2a adenosine receptor mRNA. Although Mpl ligand did not affect the short half-lives of the P2Y(1) mRNAs, it enhanced transcription of the P2Y(1) gene. It also increased cell size and the number of cell surface P2Y(1) receptors, but not P2Y(1) receptor density. Injection of Mpl ligand into mice up-regulated P2Y(1) receptor mRNAs in megakaryocytes, as shown by in situ hybridization. However, platelets isolated from these mice did not exhibit a higher P2Y(1) receptor density or increased reactivity to ADP. This correlates with the finding that Mpl ligand increases GPIIb mRNA in megakaryocytes but not the density of the protein per platelet. Thus, the enhancement of P2Y(1) receptor expression induced by Mpl ligand in megakaryocytes may be an integral feature of their differentiation, whereas clinical use of this compound might not be associated with platelet hyper-reactivity to ADP.

Differential involvement of the P2Y1 and P2YT receptors in the morphological changes of platelet aggregation.

The relative contributions of the P2Y1 and P2YT receptors to the morphological changes induced in platelets by ADP or ADP-releasing agonists were assessed using two P2 antagonists, A2P5P and AR-C67085, selective for P2Y1 and P2YT, respectively. The P2Y1 receptor was found to be involved in i) the centralization of secretory granules elicited by ADP, ii) the formation of filopodia induced by released ADP in weakly activated platelets and iii) actin polymerization and the cytoskeletal translocation of cdc42, rac1 and rhoA, in an integrin alphaIIbbeta3 dependent manner, in ADP-stimulated platelets. In contrast, the P2YT receptor was shown i) to be essential for the formation of stable macroaggregates, ii) to enhance actin polymerization and the cytoskeletal translocation of small GTPases, probably through amplification of platelet aggregation, and iii) not to be involved in the early steps of platelet activation since its blockade did not affect the cytoskeletal translocation of rhoA.

Desensitization of the platelet aggregation response to ADP: differential down-regulation of the P2Y1 and P2cyc receptors.

Platelets activated by ADP become refractory to restimulation, but the mechanism of this process is not well understood. A normal platelet response to ADP requires coactivation of the P2Y(1) receptor responsible for shape change and the P2cyc receptor, responsible for completion and amplification of the response. The aim of the present study was to characterize the desensitization of platelets to ADP and to determine whether or not these two receptors are desensitized simultaneously through identical pathways when platelets become refractory to ADP. It was found that full inhibition of platelet aggregation in response to restimulation by ADP required the presence of ADP in the medium or use of a high concentration (1 mM) of its non-hydrolysable analogue ADPbetaS. Platelets incubated for 1 h at 37 degrees C with 1 mM ADPbetaS and resuspended in Tyrode's buffer containing apyrase displayed a stable refractory state characterized by the inability to aggregate or change shape in response to ADP. ADPbetaS treated platelets loaded with fura-2/AM showed complete blockade of the calcium signal in response to ADP, whereas the capacity of ADP to inhibit PGE1 stimulated cAMP accumulation in these platelets was only diminished. Consequently, serotonin was able to promote ADP induced aggregation through activation of the Gq coupled 5HT(2A) receptor while adrenaline had no such effect. These results suggested that the refractory state of ADPbetaS treated platelets was entirely due to desensitization of the P2Y(1) receptor, the P2cyc receptor remaining functional. Binding studies were performed to determine whether the P2Y(1) and/or P2cyc binding sites were modified in refractory platelets. Using selective P2Y(1) and P2cyc antagonists (A3P5P and AR-C66096 respectively), we could demonstrate that the decrease in [33P]2MeSADP binding sites on refractory platelets corresponded to disappearance of the P2Y(1) sites with no change in the number of P2cyc sites, suggesting internalization of the P2Y(1) receptor. This was confirmed by flow cytometric analysis of Jurkat cells expressing an epitope-tagged P2Y(1) receptor, where ADPbetaS treatment resulted in complete loss of the receptor from the cell surface. We conclude that the P2Y(1) and P2cyc receptors are differently regulated during platelet activation.