Quantitative live-cell imaging of human immunodeficiency virus (HIV-1) assembly

Advances in fluorescence methodologies make it possible to investigate biological systems in unprecedented detail. Over the last few years, quantitative live-cell imaging has increasingly been used to study the dynamic interactions of viruses with cells and is expected to become even more indispensable in the future. Here, we describe different fluorescence labeling strategies that have been used to label HIV-1 for live cell imaging and the fluorescence based methods used to visualize individual aspects of virus-cell interactions. This review presents an overview of experimental methods and recent experiments that have employed quantitative microscopy in order to elucidate the dynamics of late stages in the HIV-1 replication cycle. This includes cytosolic interactions of the main structural protein, Gag, with itself and the viral RNA genome, the recruitment of Gag and RNA to the plasma membrane, virion assembly at the membrane and the recruitment of cellular proteins involved in HIV-1 release to the nascent budding site.     

PGE(2) reverses G(s)-mediated inhibition of platelet aggregation by interaction with EP3 receptors, but adds to non-G(s)-mediated inhibition of platelet aggregation by interaction with EP4 receptors

Prostaglandin E(2) (PGE(2)) has intriguing effects on platelet function in the presence of agents that raise cyclic adenosine 3'5'-monophosphate (cAMP). PGE(2) reverses inhibition of platelet aggregation by agents that stimulate cAMP production via a G(s)-linked receptor, but adds to the inhibition of platelet function brought about by agents that raise cAMP through other mechanisms. Here, we used the EP receptor antagonists DG-041 (which acts at the EP3 receptor) and ONO-AE3-208 (which acts at the EP4 receptor) to investigate the role of these receptors in mediating these effects of PGE(2). Platelet aggregation was measured in platelet-rich plasma obtained from healthy volunteers in response to adenosine diphosphate (ADP) using single platelet counting. The effects of a range of concentrations of PGE(2) were determined in the presence of (1) the prostacyclin mimetic iloprost, which operates through G(s)-linked IP receptors, (2) the cAMP PDE inhibitor DN9693 and (3) the direct-acting adenylate cyclase stimulator forskolin. Vasodilator-stimulated phosphoprotein (VASP) phosphorylation was also determined as a measure of cAMP. PGE(2) reversed the inhibition of aggregation brought about by iloprost; this was prevented in the presence of the EP3 antagonist DG-041, indicating that this effect of PGE(2) is mediated via the EP3 receptor. In contrast, PGE(2) added to the inhibition of aggregation brought about by DN9693 or forskolin; this was reversed by the EP4 antagonist ONO-AE3-208, indicating that this effect of PGE(2) is mediated via the EP4 receptor. Effects on aggregation were accompanied by corresponding changes in VASP phosphorylation. The dominant role of EP3 receptors circumstances where cAMP is increased through a Gs-linked mechanism may be relevant to the situation in?vivo where platelets are maintained in an inactive state through constant exposure to prostacyclin, and thus the main effect of PGE(2) may be prothrombotic. If so, the results described here further support the potential use of an EP3 receptor antagonist in the control of atherothrombosis.     

Lipoprotein (a) reduces platelet aggregation via apo(a)-mediated decreases in thromboxane A(2)production

Agonist (collagen- or ADP-)-stimulated platelet aggregation and thromboxane B(2) (T X B(2) ) production was reduced in human whole blood (WB) and washed platelets (WP) that were co-incubated with lipoprotein (a)[Lp(a)] at levels of 25, 50 and 100 mg % but not at 5 mg % relative to a baseline concentration of 1 mg %. Significant decreases in agonist-stimulated aggregation and T X B(2) levels were seen with 5, 25, 50 and 100 mg %purified apo (a) that was co-incubated with WB and WP relative to a baseline concentration of 1 mg %. Purified Lp(a) that was free of apo(a) [Lp(a)(-)], at concentrations of 5, 25, 50 and 100 mg %, that were co-incubated with WB and WP, had no impact on agonist-induced platelet aggregation and T X B(2) production relative to a baseline level of 1 mg %. A monoclonal antibody (Mab) (3B1) against apo(a) blocked Lp(a)-mediated reduction in platelet aggregation and T X B(2) concentrations in WB and WP that were stimulated by either agonist. Various Mabs against apoB failed to affect an Lp(a)-induced reduction in WB and WP aggregation or T X B(2) levels in response to either agonist. These results strongly suggest that Lp(a)-induced decreases in collagen or ADP stimulated platelet aggregation and T X B(2) production are mediated by apo(a).     

Lipoprotein (a) reduces platelet aggregation apo(a)-mediated decreases in production via thromboxane A 2

Agonist (collagen- or ADP-)-stimulated platelet aggregation and thromboxane B reduced in human whole blood (WB) and washed platelets (W P) that were co-incubated with lipoprotein (a) \[Lp(a)] at levels of 25, 50 and 100 mg% but not at 5 mg% relative to a baseline concentration of 1 mg% . Significant decreases in agonist-stimulated aggregation and T X B purified apo (a) that was co-incubated with W B and WP relative to a baseline concentration of 1 mg%. Purified Lp(a) that was free of apo(a) \[Lp(a)-], at concentrations of 5, 25, 50 and 100 mg%, that were co-incubated with WB and W P, had no impact on agonist-induced platelet aggregation and T X B level of 1 mg% . A monoclonal antibody (Mab) (3B1) against apo(a) blocked Lp(a)-mediated reduction in platelet aggregation and T X B concentrations in WB and W P that were stimulated by either agonist. Various Mabs 2 against apoB failed to affect an Lp(a)-induced reduction in WB and WP aggregation or T X B to either agonist. These results strongly suggest that Lp(a)-induced decreases in collagen or ADP stimulated platelet aggregation and T X B production are mediated by apo(a). 2 (T X B ) production was 2 2 levels were seen with 5, 25, 50 and 100 mg% 2 production relative to a baseline 2 levels in response 2     

A whole blood assay for platelet HPA1 (PLA1) phenotyping applicable to large-scale screening

Any future programme of antenatal screening of pregnancies for risk of neonatal alloimmune thrombocytopenia will have as a major requirement the availability of cost-effective assays which can be applied to large numbers of samples. To address this, we developed a competitive ELISA to type whole blood samples for the platelet alloantigen HPA1a, based on the use of purified glycoprotein (GP) IIb/IIIa from donors of known HPA1 genotype along with well characterized anti-HPA1a antiserum. Microtitre plates were coated with purified GPIIb/IIIa from donors of genotype HPA1a1a/3a3a. Anticoagulated whole blood of unknown HPA1 type was added to each well followed by anti-HPA1a. Residual antiHPA1a antibody not bound to the platelets in the test blood sample, bound to the immobilized HPA1a on the plate and was quantitated by standard ELISA. 475 blood donors were typed by the assay and the results compared in a blinded comparison with typing in the Capture-P^tm assay. Concordance was 100% (468 HPA1a positive and seven HPA1a negative). The HPA1 type of control samples stored as whole blood could be discriminated by this assay for up to 23 d of storage at 4°C. This assay should be suitable for use in large-scale population screening programmes.     

A whole blood assay for platelet HPA1 (PLA1) phenotyping applicable to large-scale screening

Any future programme of antenatal screening of pregnancies for risk of neonatal alloimmune thrombocytopenia will have as a major requirement the availability of cost-effective assays which can be applied to large numbers of samples. To address this, we developed a competitive ELISA to type whole blood samples for the platelet alloantigen HPA1a, based on the use of purified glycoprotein (GP) IIb/IIIa from donors of known HPA1 genotype along with well characterized anti-HPA1a antiserum. Microtitre plates were coated with purified GPIIb/IIIa from donors of genotype HPA1a1a/3a3a. Anticoagulated whole blood of unknown HPA1 type was added to each well followed by anti-HPA1a. Residual antiHPA1a antibody not bound to the platelets in the test blood sample, bound to the immobilized HPA1a on the plate and was quantitated by standard ELISA. 475 blood donors were typed by the assay and the results compared in a blinded comparison with typing in the Capture-P^tm assay. Concordance was 100% (468 HPA1a positive and seven HPA1a negative). The HPA1 type of control samples stored as whole blood could be discriminated by this assay for up to 23 d of storage at 4°C. This assay should be suitable for use in large-scale population screening programmes.     

Vasodilator-stimulated phosphoprotein (VASP) phosphorylation assay for platelet response to cilostazol

Recent clinical trials have demonstrated that cilostazol, an antiplatelet drug competent to inhibit phosphodiesterase 3, is effective in secondary prevention of atherothrombosis including ischemic stroke, myocardial infarction, and peripheral arterial disease. However, there is no reliable assay for detection of the platelet response to cilostazol. We attempted to establish such an assay for clinical use. Phosphorylation of vasodilator-stimulated phosphoprotein (VASP) subsequent to the pharmacological action of cilostazol was measured by a platelet VASP assay kit that has been widely used for monitoring platelet response to ADP receptor antagonists in clinical settings. We modified the kit and found the optimal conditions for detection of cilostazol-dependent VASP phosphorylation. The assay could detect the in vitro and in vivo platelet responses to cilostazol effectively in the presence of 50 nM PGE1. ROC analysis showed that our assay had 97% sensitivity and 75% specificity when blood drawn between 3 and 9?h after cilostazol ingestion was subjected to the assay. The assay results were verified by immunoblotting specific for VASP phosphorylation. Standard light transmission platelet aggregometry could not detect significant inhibition of agonist-induced platelet aggregation by cilostazol except for the in vitro effect of high concentrations of cilostazol. These results demonstrate that the platelet VASP assay can detect the platelet response to cilostazol with high sensitivity and specificity.     

P2X(1)-mediated activation of extracellular signal-regulated kinase 2 contributes to platelet secretion and aggregation induced by collagen

Adenosine triphosphate (ATP) and its stable analog, alpha,beta-methylene ATP, activate the platelet P2X(1) ion channel, causing a rapid Ca(++) influx. Here, we show that, in washed apyrase-treated platelets, alpha,beta-methylene ATP elicits reversible extracellular signal-regulated kinase 2 (ERK2) phosphorylation through a Ca(++)- and protein kinase C-dependent pathway. In contrast, high-performance liquid chromatography-purified adenosine diphosphate (ADP) did not trigger ERK2 phosphorylation. alpha,beta-Methylene ATP also activated the ERK2 pathway in P2X(1)-transfected HEK293 cells but not in cells expressing mutated P2X(1)delL nonfunctional channels. Because ATP released from the dense granules during platelet activation contributes to platelet aggregation elicited by low doses of collagen, and because collagen causes ERK2 phosphorylation, we have investigated the role of P2X(1)-mediated ERK2 activation in these platelet responses. We found that the antagonism of P2X(1) with ADP or desensitization of this ion channel with alpha,beta-methylene ATP both resulted in impaired ERK2 phosphorylation, ATP secretion, and platelet aggregation induced by low concentrations of collagen (< or = 1 microg/mL) without affecting the minor early dense granule release. Selective MEK1/2 inhibition by U-0126 and Ca(++) chelation with EGTA (ethyleneglycoltetraacetic acid) behaved similarly, whereas the PKC inhibitor GF109203-X totally prevented collagen-induced secretion and ERK2 activation. In contrast, when elicited by high collagen concentrations (2 microg/mL), platelet aggregation and secretion no longer depended on P2X(1) or ERK2 activation, as shown by the lack of their inhibition by alpha,beta-methylene ATP or U-0126. We thus conclude that mild platelet stimulation with collagen rapidly releases ATP, which activates the P2X(1)-PKC-ERK2 pathway. This process enhances further degranulation of the collagen-primed granules allowing platelet aggregation to be completed.     

Detection of platelet antibodies using a micro-enzyme-linked immunosorbent assay (ELISA)

An enzyme-linked immunosorbent assay (ELISA) for the measurement of circulating platelet antibody and platelet-associated IgG (PAIgG) is described. The test is done in microtiter plates and rapidly provides quantitative and highly reproducible results. Alloantibodies from 28 of 30 multiple transfused patients and isoantibodies from 3 of 4 patients with immune thrombocytopenic purpura (ITP) were detected. PAIgG was elevated in all 4 patients with ITP, and HLA and platelet-specific antigens were reliably detected using HLA typing sera and anti-PIA1 antibody, respectively. Platelets preserved wither by dessication in the wells of the microtiter plates or in liquid suspension in saline at 4 degrees C gave results comparable to values using fresh platelets. Storage periods ranged from 30 days for dessicated platelets to more than 1 yr for platelets stored in suspension. The ability to utilize preserved platelets may allow relatively convenient screening of large numbers of potential platelet donors for alloimmunized patients.     

A platelet viability assay (PVA) for the diagnosis of heparin-induced thrombocytopenia

Diagnosing heparin-induced thrombocytopenia (HIT) requires functional assays measuring platelet activation as they are highly specific and sensitive. A useful functional test for diagnosing HIT is the serotonin release assay (SRA), but this assay is technically demanding and requires a radioactive marker. We describe an alternate functional HIT assay, the platelet viability assay (PVA), that overcomes the need for a radioactive marker by using a viability dye endpoint to measure platelet activation. We compared the performance characteristics of the PVA to the SRA. Serum samples from 76 patients with suspected HIT were tested in both the PVA and the SRA. The PVA uses calcein-AM as a marker of platelet viability, with decreases in fluorescence and cell size as surrogate markers for platelet activation. A significant linear correlation (Spearman correlation, r = ?0.78, P < 0.0001) was observed between the PVA and SRA. Calcein-AM fluorescence decreased in a negative linear relationship with platelet activation as measured by ¹⁴C-serotonin release. The PVA detected all positive SRA samples, with an overall sensitivity of 100% and a specificity of 97% in comparison to the SRA. The measurement of platelet viability using the PVA provided similar results to the SRA when testing suspected HIT patient samples.     

Bleeding disorder due to platelet prostaglandin H synthase-1 (PGHS-1) deficiency

Defective platelet prostaglandin H synthase (PGHS) activity has been recognized as a cause of bleeding disorders, but the defect has not been characterized. We evaluated three female patients aged 37, 48 and 55 who presented with a mild bleeding disorder due to platelet dysfunction. None of the patients had underlying diseases or reported use of aspirin or other nonsteroidal anti-inflammatory drugs. Coagulation screening tests and platelet count were normal in each patient. Platelet aggregation in response to adenosine diphosphate (ADP), collagen and epinephrine were subnormal, characterized by an abnormal second-wave aggregation and propensity for disaggregation. Arachidonate-induced platelet aggregation was defective, whereas PGH₂-induced aggregation was normal. Platelet thromboxane A₂ (TXA₂) production in response to arachidonic acid was reduced in all three patients, i.e. 11.7, 4.6 and 4.4 ng TXB₂/3 10⁸ plt respectively (normal range was 49–81 ng/3 10 ⁸ plt), whereas they were normal in response to exogenous PGH₂, i.e. 71.4, 56.6 and 48.9 ng/3 10⁸ plts, respectively (normal range 49–85 ng/3 10⁸ plt). These results are consistent with a deficiency of platelet PGHS activity. The level of the constitutive platelet PGHS-1 and TXA₂ synthase (TXAS) proteins were determined on platelet microsomal fractions by Western blot analysis using affinity-purified polyclonal antibodies highly specific for human PGHS-1 and TXAS, respectively. In two patients the 70 kD PGHS-1 protein was undetectable, whereas it was normal in the third patient. The 60 kD TXAS band was normal in all three patients. These findings indicate that human platelet PGHS-1 deficiency is due to two types of enzyme defects: type 1 defect is manifested by an undetectable PGHS-1 protein in platelets whereas the type 2 defect is manifested by a normal quantity of PGHS-1 protein which has an impaired catalytic activity.     

Genetic typing of human platelet antigen 1 (HPA-1) by oligonucleotide ligation assay in a specific and reliable semi-automated system

Genotyping of platelet alloantigens with the possibility of using any type of cellular material as a source of DNA has become a preferred procedure, particularly in thrombocytopenic patients when platelet counts are too low for phenotyping. Recently human platelet antigen 1 (HPA-1) has been identified as an inherited risk factor for coronary thrombosis. The different detection methods currently used have disadvantages for large-scale DNA diagnosis, including the need for electrophoresis (allele-specific restriction enzyme analysis, amplification with sequence-specific primers) or the potential risk of reduced specificity (allele-specific oligonucleotide hybridization). In this report we describe the adaptation of an automated oligonucleotide ligation assay to genotype HPA-1 in polymerase chain reaction (PCR)-amplified DNA samples. HPA-1a and HPA-1b phenotypes corresponded to the results of the different genotyping assays. The genotypes determined with the ELISA-based PCR-oligonucleotide ligation assay were in 100% concordance with the results obtained by conventional allele-specific restriction enzyme site analysis and PCR amplification with sequence-specific primers. The automated oligonucleotide ligation assay provides a rapid, reliable, nonisotopic method to genotype human platelet antigens that can rapidly be applied to large population screening.     

Induction of transforming growth factor-beta 1 (TGF-beta 1), receptor expression and TGF-beta 1 protein production in retinoic acid-treated HL-60 cells: possible TGF-beta 1-mediated autocrine inhibition.

Treatment of HL-60 cells, a human promyelocytic leukemia cell line, with the vitamin A derivative retinoic acid (RA) for 7 days resulted in a dose-dependent decrease in proliferation and increase in granulocytic differentiation. The role of transforming growth factor-beta 1 (TGF-beta 1), a protein with pleiotropic effects on the proliferation and differentiation of various cell types, was examined during RA-induced differentiation of HL-60 cells. Although TGF-beta 1 alone had little effect on proliferation or differentiation of HL-60 cells, addition of TGF-beta 1 to HL-60 cells treated with a suboptimum concentration of RA (1.0 nmol/L) resulted in a marked decrease in proliferation with no effect on granulocytic differentiation. Studies of the mechanism of RA-induced TGF-beta sensitivity showed that although untreated HL-60 cells expressed low levels of TGF-beta 1 binding proteins on the cell surface, the levels were increased in a dose-dependent manner after RA treatment. Maximum induction was achieved after treatment with 10 nmol/L RA and consisted predominantly of the 65-Kd TGF-beta 1 receptor type. Moreover, RA treatment also resulted in a dose-dependent increase in both TGF-beta 1 steady-state mRNA expression and production of active TGF-beta with maximum induction at 10 nmol/LRA. RA treatment of HL-60 cells had no effect on TGF-beta 2 and TGF-beta 3 mRNA expression. These data suggest that the effects of RA may be mediated by a TGF-beta 1-mediated autocrine antiproliferative loop during differentiation of HL-60 cells.     

Hypothermia-induced platelet aggregation: no effect of aprotinin (trasylol) but inhibition by eptifibatide (integrilin)

OBJECTIVES: The serine-protease inhibitor aprotinin protects platelet function during cardiopulmonary bypass. However, its safety and efficacy during deep hypothermic circulatory arrest (DHCA) is controversial, and aprotinin is suspected to cause thrombosis especially during hypothermia. The platelet GP IIb/IIIa inhibitor eptifibatide has been assumed to preserve platelet function during cardiopulmonary bypass without increasing bleeding complications. The aim of this study was to compare the effect of aprotinin and eptifibatide on platelet function under conditions of DHCA. METHODS: Heparinized blood from healthy volunteers (n = 10) was incubated in stasis for 30 minutes at 18 degrees C to simulate DHCA and compared to samples incubated at 37 degrees C. The effect of eptifibatide (2.5 microg/ml) and aprotinin (300 KIU/ml) on platelets under these conditions was analyzed by flow cytometry. Platelet aggregates were identified using CD41-antibody binding and size. GPIIb/IIIa function was evaluated with the activation-specific antibody PAC-1 after stimulation with 10 microM ADP. Aggregate numbers and antibody mean-fluorescence are reported as mean +/- standard deviation. RESULTS: Hypothermia induced a 2.5-fold increase of aggregates ( p < 0.001) and a 2.6-fold increase of GPIIb/IIIa activation ( p < 0.001). This effect was not influenced by aprotinin but almost completely inhibited by eptifibatide ( p < 0.001). CONCLUSIONS: Aprotinin has no procoagulatory effect on platelet function during hypothermia but is not protective either. Eptifibatide inhibits hypothermia-induced platelet aggregation in vitro and may prevent aggregate sequestration in the microvasculature and consecutive ischemic organ damage in vivo.