Distinct karyotypic and mutational landscape in trisomy AML.

Trisomy karyotype occurs in 5%-10% of AML. Its mutational landscape and prognostic significance are not well defined. A cohort of 156 trisomy AML patients was analysed, with reference to 615 cytogenetically normal (CN) AML patients. Trisomy AML showed distinct mutational landscape with more prevalent SMC1A, N/KRAS, ASXL1 and BCOR but fewer CEBPAbZIP and NPM1 mutations in patients ≤60, and fewer NPM1 mutations in those >60. NRAS mutations were associated with poor outcome in trisomy AML, whereas DNMT3A and FLT3-ITD mutations had neutral effect. Trisomy AML appeared biologically distinct from CN-AML.

Platelet Ca(2+) responses coupled to glycoprotein VI and Toll-like receptors persist in the presence of endothelial-derived inhibitors: roles for secondary activation of P2X1 receptors and release from intracellular Ca(2+) stores.

Inhibition of Ca(2+) mobilization by cyclic nucleotides is central to the mechanism whereby endothelial-derived prostacyclin and nitric oxide limit platelet activation in the intact circulation. However, we show that ∼ 50% of the Ca(2+) response after stimulation of glycoprotein VI (GPVI) by collagen, or of Toll-like 2/1 receptors by Pam(3)Cys-Ser-(Lys)(4) (Pam(3)CSK(4)), is resistant to prostacyclin. At low agonist concentrations, the prostacyclin-resistant Ca(2+) response was predominantly because of P2X1 receptors activated by ATP release via a phospholipase-C-coupled secretory pathway requiring both protein kinase C and cytosolic Ca(2+) elevation. At higher agonist concentrations, an additional pathway was observed because of intracellular Ca(2+) release that also depended on activation of phospholipase C and, for TLR 2/1, PI3-kinase. Secondary activation of P2X1-dependent Ca(2+) influx also persisted in the presence of nitric oxide, delivered from spermine NONOate, or increased ectonucleotidase levels (apyrase). Surprisingly, apyrase was more effective than prostacyclin and NO at limiting secondary P2X1 activation. Dilution of platelets reduced the average extracellular ATP level without affecting the percentage contribution of P2X1 receptors to collagen-evoked Ca(2+) responses, indicating a highly efficient activation mechanism by local ATP. In conclusion, platelets possess inhibitor-resistant Ca(2+) mobilization pathways, including P2X1 receptors, that may be particularly important during early thrombotic or immune-dependent platelet activation.

Efficient gene transfer using the human JC virus-like particle that inhibits human colon adenocarcinoma growth in a nude mouse model.

The JC virus (JCV) may infect human oligodendrocytes and consequently cause progressive multifocal leukoencephalopathy (PML) in patients with immune deficiency. In addition, the virus has also been detected in other human tissues, including kidney, B lymphocytes, and gastrointestinal tissue. The recombinant major structural protein, VP1, of JCV is able to self-assemble to form a virus-like particle (VLP). It has been shown that the VLP is capable of packaging and delivering exogenous DNA into human cells for gene expression. However, gene transfer is not efficient when using in vitro DNA packaging methods with VLPs. In this study, a novel in vivo DNA packaging method using the JCV VLP was used to obtain high efficiency gene transfer. A reporter gene, the green fluorescence protein, and a suicide gene, the herpes simplex virus thymidine kinase (tk), were encapsidated into VLPs in Escherichia coli. The VLP was used to specifically target human colon carcinoma (COLO-320 HSR) cells in a nude mouse model. Intraperitoneal administration of ganciclovir in the tk-VLP-treated mice greatly reduced tumor volume. These findings suggest that it will be possible to develop the JCV VLP as a gene delivery vector for human colon cancer therapy in the future.

Primary and secondary agonists can use P2X(1) receptors as a major pathway to increase intracellular Ca(2+) in the human platelet.

In the platelet, it is well established that many G-protein- and tyrosine kinase-coupled receptors stimulate phospholipase-C-dependent Ca(2+) mobilization; however, the extent to which secondary activation of adenosine 5'-triphosphate (ATP)-gated P2X(1) receptors contributes to intracellular Ca(2+) responses remains unclear. We now show that selective inhibition of P2X(1) receptors substantially reduces the [Ca(2+)](i) increase evoked by several important agonists in human platelets; for collagen, thromboxane A(2), thrombin, and adenosine 5'-diphoshate (ADP) the maximal effect was a reduction to 18%, 34%, 52%, and 69% of control, respectively. The direct contribution of P2X(1) to the secondary Ca(2+) response was far greater than that of either P2Y receptors activated by co-released ADP, or via synergistic P2X(1):P2Y interactions. The relative contribution of P2X(1) to the peak Ca(2+) increase varied with the strength of the initial stimulus, being greater at low compared to high levels of stimulation for both glycoprotein VI and PAR-1, whereas P2X(1) contributed equally at both low and high levels of stimulation of thromboxane A(2) receptors. In contrast, only strong stimulation of P2Y receptors resulted in significant P2X(1) receptor activation. ATP release was detected by soluble luciferin:luciferase in response to all agonists that stimulated secondary P2X(1) receptor activation. However, P2X(1) receptors were stimulated earlier and to a greater extent than predicted from the average ATP release, which can be accounted for by a predominantly autocrine mechanism of activation. Given the central role of [Ca(2+)](i) increases in platelet activation, these studies indicate that ATP should be considered alongside ADP and thromboxane A(2) as a significant secondary platelet agonist.

Multiple primary cancers in families with Li-Fraumeni syndrome.

BACKGROUND: Li-Fraumeni syndrome is a dominantly inherited disorder characterized by early-onset breast cancer, sarcomas, and other cancers in children and young adults. Members of families with this syndrome also develop multiple primary cancers, but the frequency is unknown. To approach this issue, we quantified the incidence of second and third primary cancers in individuals from 24 Li-Fraumeni kindreds originally diagnosed with cancer during the period from 1968 through 1986. METHODS: The relative risk (RR) of subsequent cancers and 95% confidence intervals (CIs) were calculated by use of population-based incidence data from the Connecticut Cancer Registry. Kaplan-Meier analysis was used to determine the cumulative probability (+/- standard error) of subsequent cancers. RESULTS: Among 200 Li-Fraumeni syndrome family members diagnosed with cancer, 30 (15%) developed a second cancer. Eight individuals (4%) had a third cancer, while four (2%) eventually developed a fourth cancer. Overall, the RR of occurrence of a second cancer was 5.3 (95% CI = 2.8-7.8), with a cumulative probability of second cancer occurrence of 57% (+/- 10%) at 30 years after diagnosis of a first cancer. RRs of second cancers occurring in families with this syndrome were 83.0 (95% CI = 36.9-187.6), 9.7 (95% CI = 4.9-19.2), and 1.5 (95% CI = 0.5-4.2) for individuals with a first cancer at ages 0-19 years, 20-44 years, and 45 years or more, respectively. Thirty (71%) of 42 subsequent cancers in this group were component cancers of Li-Fraumeni syndrome. CONCLUSIONS: Compared with the general population, members of Li-Fraumeni syndrome families have an exceptionally high risk of developing multiple primary cancers. The excess risk of additional primary cancers is mainly for cancers that are characteristic of Li-Fraumeni syndrome, with the highest risk observed for survivors of childhood cancers. Cancer survivors in these families should be closely monitored for early manifestations of new cancers.

Clinical stage I carcinoma of the testis: a review.

Significant advances in the management of disseminated carcinoma of the testis have had a tremendous influence on the management of all stages of disease, including both stage I and stage II. Because the disease is so uniformly curable in advanced forms, strategies have evolved for lower stages of disease to maximize curative potential with minimization of therapeutic intervention and potential long-term side effects. This review addresses issues of diagnosis and initial clinical staging of patients with stage I carcinoma of the testis. The usefulness of biological tumor markers and the accuracy, sensitivity, and specificity of radiographic imaging modalities are emphasized. The traditional treatment of stage I seminomas and nonseminomas includes the role of both radiation therapy and retroperitoneal lymph node dissection (RPLND). Treatment results and complications of each modality are also reviewed. The importance of orchiectomy followed by meticulous surveillance, as well as a critical analysis of prognostic features of subsequent locoregional or systemic relapse in patients with stage I testis cancer, focuses attention on changing concepts of this disease. The rationale underlying improved ability to stage patients meticulously and offer therapeutic intervention when necessary following orchiectomy, constitutes the focus of present and future clinical research. Such strategies are discussed in detail.

Stage I nonseminomatous germ cell testicular tumor: prediction of metastatic potential by primary histopathology.

A study of 60 patients with clinical stage I nonseminomatous germ cell testicular tumor (NSGCT) was conducted to identify prognostic factors that may predict the likelihood of metastasis. Clinical features and histopathologic features of the primary testicular tumor were examined and analyzed for correlations with the presence of retroperitoneal nodal metastasis documented by surgery (N+) and with development of relapse (R+). Pathologic tumor stage greater than or equal to 2, with tumor extension into the tunica albuginea, rete testis, epididymis, or spermatic cord, was correlated with an increased rate of N+ compared with pathologic tumor stage I (P = .001). Vascular invasion was correlated with a higher rate of N+ (P = .05) and had a similar association with R+ (P = .08). Tumors containing less than 50% teratoma were found to have a higher rate of N+ than tumors with greater than or equal to 50% teratoma (P = .02). Based on the identified prognostic factors, a model for predicting the probability of retroperitoneal nodal metastasis in clinical stage I patients is proposed. The risk factors for nodal metastasis are: pathologic tumor stage greater than or equal to 2, vascular invasion, and less than 50% teratoma. Patients with none of the risk factors are considered at low risk and may be offered orchiectomy alone with surveillance for initial treatment. Patients with all three risk factors are at high risk and should be treated with a retroperitoneal lymph node dissection (RPLND) or possibly chemotherapy. Patients with one or two risk factors are at intermediate risk; it is recommended that they undergo RPLND. This risk model facilitates a rational approach to the management of clinical stage I NSGCT.

Postoperative radiation therapy for high-risk colon carcinoma.

PURPOSE: This study examines the experience of patients treated with postoperative radiation therapy after resection of high-risk colon carcinoma in an effort to assess the potential role of this modality in combination with current systemic therapies. PATIENTS AND METHODS: From 1976 to 1989, 203 patients received postoperative radiation therapy with and without concurrent fluorouracil (5-FU) chemotherapy following resection of modified Astler-Coller B2, B3, C2, and C3 colon tumors. Of the 203 patients, 30 (15%) were identified as having residual local tumor after subtotal resection, whereas 173 (85%) had no known residual disease. The 173 patients treated with adjuvant radiation therapy were compared with a historical control group of 395 patients undergoing surgery only. RESULTS: Three groups of patients who appeared to benefit from postoperative radiation were identified. Improved local control and recurrence-free survival rates were seen for patients with stage B3 and C3 colon carcinoma treated with postoperative radiation therapy compared with a similarly staged group of patients undergoing surgery only. Irradiated patients whose tumors had an associated abscess or fistula formation had improved local control and recurrence-free survival rates compared with a similar group of patients undergoing surgery only. There appears to be a subset of patients with residual local disease after subtotal resection that may be salvaged by high-dose postoperative radiation therapy. CONCLUSION: Selected groups of patients with colon carcinoma may benefit from postoperative radiation in addition to current systemic therapies. Integration of 5-FU and levamisole with postoperative radiation therapy should be considered for patients with (1) stage B3 and C3 lesions, (2) tumors associated with abscess or fistula formation, and (3) residual local disease after subtotal resection.

Prognostic factors in invasive bladder carcinoma in a prospective trial of preoperative adjuvant chemotherapy and radiotherapy.

Clinical and pathologic factors were analyzed in 40 patients with localized muscle-invasive bladder carcinoma treated in a prospective bladder-preserving program consisting of transurethral tumor resection, neoadjuvant chemotherapy (methotrexate, cisplatin, and vinblastine [MCV]), and 4,000 cGy radiotherapy with concurrent cisplatin. Patients with biopsy-proven complete response after chemotherapy and 4,000 cGy radiation received full-dose radiotherapy (6,480 cGy) with cisplatin. Cystectomy was recommended to patients with residual disease. Distant metastasis rate was associated with tumor stage and size: 0% in T2 patients, 39% in T3-4 patients (P = .035), 6% for tumors less than 5 cm, and 59% for tumors greater than or equal to 5 cm (P = .002). Risk of bladder tumor recurrence was higher in patients with tumor-associated carcinoma in situ (CIS; 40%) than those without CIS (6%; P = .075). Papillary tumors and solid tumors both had similar treatment outcomes. By multivariate analysis, tumor stage T2 (P = .04) and absence of CIS (P = .03) were significant predictors of complete response; CIS was predictive of local bladder recurrence (P = .07); and tumor size (P = .03), response after chemoradiotherapy (P = .02), and vascular invasion (P = .08) were associated with distant metastasis. Six of eight local bladder tumor recurrences were superficial tumors. The low actuarial distant metastasis rate of T2 patients (0% at 3 years), the 3-year actuarial overall survival rates for T2 (89%) and T3-4 (50%) patients, and the similar treatment outcomes for papillary versus solid tumors are encouraging when compared with published historical controls. These results provide preliminary evidence (median follow-up, 30 months) that the current chemoradiotherapy regimen may have beneficial effects in the treatment of muscle-invasive bladder carcinoma. The true efficacy of neoadjuvant chemotherapy remains to be proven by ongoing randomized trials.

A major role for P2X1 receptors in the early collagen-evoked intracellular Ca2+ responses of human platelets.

In the platelet, ATP-gated P2X1 receptors have been reported to amplify functional responses to collagen, however the relative importance of early CCa2+ mobilisation events is unknown. We now report that selective desensitisation of P2X1 receptor activity leads to a major reduction in the initial intracellular Ca2+ responses to a wide range of collagen concentrations (0.25-2 microg ml(-1)). Peak [Ca2+](i) increases were reduced to 8.5 and 55% of control, and the maximum rate of rise was reduced to 12 and 33% of control, at low and high collagen concentrations, respectively. This P2X1-dependent acceleration and enhancement of collagen-stimulated Ca2+ responses was not observed in the absence of extracellular Ca2+. These results demonstrate a major role for ATP-gated Ca2+ influx in the early collagen-evoked Ca2+ signals and can at least partly explain the important contribution of P2X1 receptors to arterial thrombosis.

Time-dose considerations in the treatment of anal cancer.

PURPOSE: To analyze the impact of patient and treatment parameters in concurrent chemoradiation treatment for anal carcinoma. METHODS AND MATERIALS: Retrospective review of 50 MO anal cancer patients treated from 1984-1994. Most patients received concurrent 5-FU, mitomycin, and radiation. Local control and disease-free/overall survival were determined and analyzed according to patient and treatment parameters. RESULTS: With 43 month median follow-up, projected overall survival is 66% at 5 and 8 years. Disease-free survival is 67% at 5 years and 59% at 8 years. Local control is 70% at 5 and 8 years. Doses of > or =54 Gy are associated with improved 5-year survival (84 vs. 47%, p = 0.02), disease-free survival (74 v. 56%, p = 0.09), and local control (77 vs. 61%, p = 0.04). Although local control, disease-free survival, and overall survival were improved in patients whose overall treatment time was <40 days, this was not statistically significant. Outcome in the four patients with pretreatment hemoglobin (Hgb) <10 appeared worse with 3-year overall survival 50 vs. 68% (p = 0.07), disease-free survival 0 vs. 67% (p = 0.11), and local control 0 vs. 74% (p = 0.05). Projected 5-year overall survival, relapse-free survival, and local control in 4 HIV(+) patients is 0, 75, and 75%. Multivariate analysis reveals that dose (p = 0.02) and Hgb (p = 0.05) independently affect local control, dose (p = 0.02) affects disease-free survival, and dose (p = 0.01), Hgb (p = 0.03), T-stage (p = 0.03), and HIV-status (0.07) independently influence overall survival. CONCLUSION: Radiation doses of > or =54 Gy are associated with significantly improved survival and local control in anal cancer patients treated with chemoradiation. Overall treatment times of less than 40 days are associated with a trend towards improved outcome, but this is not significant. Pretreatment hemoglobin <10 is associated with worse treatment outcome. Survival of HIV (+) patient is poor, but the majority of such patients in this series died of intercurrent disease with their anal carcinomas controlled by chemoradiation.

Distinct mathematical behavior of apoptotic versus non-apoptotic tumor cell death.

PURPOSE: The presence or absence of a p53-dependent apoptosis response has previously been shown to greatly influence radiosensitivity in tumor cells. Here, we examine clonogenic survival curves for two genetically related oncogene transformed cell lines differing in the presence or absence of p53 and apoptosis. Solid tumor radiosensitivity patterns have been previously described for these lines. MATERIALS AND METHODS: Oncogene-transformed fibroblasts derived from E1A + Ras transfection of p53-wild-type or p53-null mouse embryonic fibroblasts were plated as single cells and irradiated at increasing radiation doses in single fractions from 1.5 to 11 Gy. Clonogenic cell survival assays were obtained. Survival data are fit to a linear-quadratic relationship: S = e(-alphaD-betaD2). Apoptosis was assessed and quantitated morphologically by staining with the fluorescent nuclear dye DAPI, by TUNEL assay for DNA fragmentation, and by measurement of apoptotic cysteine protease cleavage activity in cytosolic extracts. RESULTS: Whereas radiation triggers massive apoptosis in the presence of p53, it produces no measurable DNA fragmentation, apoptotic cysteine protease cleavage activity, or morphological changes of apoptosis in the cells lacking p53. These contrasting mechanisms of death display dramatically different quantitative behavior: log-survival of apoptotic cells is linearly proportional to dose (S = e(-alphaD)), whereas survival of non-apoptotic (p53 null) is linear-quadratic with a significant quadratic contribution. The surviving fraction at 2 Gy (SF-2) for p53-null cells was 70% verses 12% for p53-intact cells. CONCLUSIONS: In this system, apoptosis appears to exhibit a dominance of single-event which produces a very high alpha/beta ratio, and no significant shoulder; whereas non-apoptotic death in this system exhibits a comparatively small linear component, a low alpha/beta ratio, and a larger shoulder.

Management of patients with clinical stage I or II nonseminomatous germ cell tumors of the testis. Evolving therapeutic options.

Eighty patients with clinical stage I or II nonseminomatous germ cell tumors of the testis were managed with modified protocols, including modified nerve-sparing retroperitoneal lymph node dissection for patients with stage I cancer, retroperitoneal lymph node dissection for patients with low-volume stage II cancer, and initial chemotherapy with or without subsequent retroperitoneal lymphadenectomy for patients with high-volume stage II cancer. Patients with low-stage disease (clinical stage I) were treated successfully with modified retroperitoneal lymph node dissection (relapse rate, three of 40 patients). Clinical understaging was evidenced in 14 of 48 patients with clinical stage I disease who were found to have pathologic involvement of the retroperitoneal lymph nodes, including six patients with extensive retroperitoneal nodal involvement (pathologic stage B2). Of nine patients with retroperitoneal tumors less than 3 cm in diameter, four patients were satisfactorily treated with retroperitoneal lymph node dissection alone while five patients required chemotherapy after retroperitoneal lymph node dissection. Of 26 patients with retroperitoneal tumors 3 to 5 cm in diameter, 17 patients were treated with chemotherapy alone. All patients remain free of disease after the completion of definitive therapy. We conclude that therapeutic options should be modified based on histologic factors in the primary tumor, extent of retroperitoneal disease as indicated on a computed tomographic scan, and presence or absence of elevated tumor markers. By consideration these factors, optimum therapy can be selected to achieve the highest long-term survival rate with the least morbidity.

A simple method for detecting human polyomavirus DNA in urine by the polymerase chain reaction.

In order to develop a simple and sensitive method for detecting human polyomavirus DNA in the urine of patients by the polymerase chain reaction (PCR), it was found that the viral DNA could be released from urine by proteinase K and then amplified by PCR directly, without additional treatment such as ultracentrifugation or DNA extraction. Direct PCR amplification of viral DNA from urine was volume limited and 5 microliters of urine appeared to be the optimum amount for direct PCR amplification. When the urine volume was greater than 10 microliters, the results of PCR were inconsistent. However, the urine volume could be increased after dialysis to remove possible inhibitor(s) which may interfere with PCR. Direct PCR amplification of patient urine is convenient and eliminates several steps which can cause loss of DNA from the sample.

Production of the antigen and the antibody of the JC virus major capsid protein VP1.

The DNA of the major capsid protein VP1 of the human polyomavirus JC virus (JCV), Taiwan-3 strain, was generated from the urine of an autoimmune disease patient by polymerase chain reaction (PRC). The VP1 DNA was cloned into a prokaryotic expression vector, pGEX-4T-1, for expression in E. coli. The nucleotide sequences and the deduced amino acid sequences were determined and compared with the JC virus prototype, Mad-1. Thirty nucleotides were different between these two strains. Six of the altered nucleotides affected amino acid coding and ten of them caused changes in endonuclease recognition sites. The recombinant VPI protein was purified and used to raise monospecific antiserum in rabbit. Recombinant JCV VP1 protein and its monospecific antiserum are important clinical reagents and could possibly be developed as a subunit vaccine and as a serological diagnostic antigen in the future. In addition, the region between amino acid residues 40 and 80 of JCV VP1 is predicted to be an antigenic epitope on the basis of its hydropathy plot and comparison with the VP1 sequences of SV40 and BK virus.

Human anti-JC virus serum reacts with native but not denatured JC virus major capsid protein VP1.

The immunoreactivity of human anti-JC virus (JCV) serum against the major capsid protein VP1 of JCV was analyzed by Western blot, dot blot, and hemagglutination inhibition (HAI) assays. JCV-positive human serum reacted with native but not denatured JCV major capsid protein VP1, as demonstrated by dot blot and Western blot. Rabbit antiserum raised against native JCV capsid had immunoreactivities similar to those of human anti-JCV serum. These results indicate that the antigenecity of native and denatured JCV VP1 is different. In addition, both JCV-positive human serum and rabbit antiserum raised against native JCV capsid protein inhibited the hemagglutination activity of JCV capsid particles. In contrast, rabbit antiserum raised against denatured JCV VP1 did not inhibit hemagglutination. These findings reveal that denaturation may alter the antigenic epitopes of JCV VP1. Therefore, keeping the JCV capsid protein native appears to be essential for serological or other immunological analyses of the virus.

Genomic cloning and sequence analysis of Taiwan-3 human polyomavirus JC virus.

Four different-strains of human polyomavirus JC virus (JCV), CY, Taiwan-1, Taiwan-2, and Taiwan-3, have been found in pregnant women and autoimmune disease patients in Taiwan. In this study, we report the cloning and sequencing of the Taiwan-3 JCV, virus isolated from the urine of an immunosuppressed patient with rheumatoid arthritis. The viral genome was amplified by polymerase chain reaction and then cloned into a prokaryotic replicative plasmid, pGEM-7Zf(-). The genomic DNA was sequenced and found to comprise 5,111 base pairs. The enhancer-promoter region of the viral genome lacks a copy of pentanucleotide-A (GGGAA) and pentanucleotide-B (AAAGC) compared to the CY archetypal JCV. There are 108 nucleotides altered in the total genome, excluding the variable part of the enhancer-promoter region, between Mad-1 (the prototype JC virus) and Taiwan-3. The enhancer-promoter region has approximately 25% of the altered nucleotides, resulting in amino acid changes in the open reading frames for I.T. capsid proteins (VP1, VP2, and VP3), and the agno protein. The cloned Taiwan-3, genome will provide an source for physiologic and pathologic investigation of the JCV virus in the future.

P2X(1) receptor inhibition and soluble CD39 administration as novel approaches to widen the cardiovascular therapeutic window.

Thrombus formation at sites of disrupted atherosclerotic plaques is a leading cause of death and disability worldwide. Although the platelet is now recognized to be a central regulator of thrombus formation, development of antiplatelet reagents that selectively target thrombosis over hemostasis represents a challenge. Existing prophylactic antiplatelet therapies are centered on the use of aspirin, an irreversible cyclooxygenase inhibitor, and a thienopyridine such as clopidogrel, which inactivates the adenosine diphosphate-stimulated P2Y(12) receptor. Although these compounds are widely used and have beneficial effects for patients, their antithrombotic benefit is complicated by an elevated bleeding risk and substantial or partial "resistance." Moreover, combination therapy with these two drugs increases the hemorrhagic risk even further. This review explores the possibility of inhibiting the platelet-surface ionotropic P2X(1) receptor and/or elevating CD39/NTPDase1 activity as new therapeutic approaches to reduce overall platelet reactivity and recruitment of surrounding platelets at prothrombotic locations. Because both proteins affect platelet activation at an early stage in the events leading to thrombosis but are less crucial in hemostasis, they provide new strategies to widen the cardiovascular therapeutic window without compromising safety.

Differential sensitivity of human platelet P2X1 and P2Y1 receptors to disruption of lipid rafts.

ATP-stimulated P2X1 and ADP-stimulated P2Y1 receptors play important roles in platelet activation. An increase in intracellular Ca2+ represents a key signalling event coupled to both of these receptors, mediated via direct gating of Ca2+-permeable channels in the case of P2X1 and phospholipase-C-dependent Ca2+ mobilisation for P2Y1. We show that disruption of cholesterol-rich membrane lipid rafts reduces P2X1 receptor-mediated calcium increases by approximately 80%, while P2Y1 receptor-dependent Ca2+ release is unaffected. In contrast to artery, vas deferens, bladder smooth muscle, and recombinant expression in cell lines, where P2X1 receptors show almost exclusive association with lipid rafts, only approximately 20% of platelet P2X1 receptors are co-expressed with the lipid raft marker flotillin-2. We conclude that lipid rafts play a significant role in the regulation of P2X1 but not P2Y1 receptors in human platelets and that a reserve of non-functional P2X1 receptors may exist.