Increased binding of fibrinogen to glycoprotein IIIa-proline33 (HPA-1b, PlA2, Zwb) positive platelets in patients with cardiovascular disease.

AIMS: The GPIIb-IIIa complex on the platelet membrane plays an important part in thrombosis as it is the receptor for fibrinogen. The gene for platelet membrane glyco-protein IIIa has multiple alleles one of which, the GPIIIa-Proline33 (HPA-1b, PlA2, Zwb) allele has been reported in some, but not all studies, to be associated with an increased risk of myocardial infarction. We investigated whether the presence of the Pro33 form of GPIIIa on the platelet membrane is associated with increased fibrinogen binding. METHODS AND RESULTS: Blood samples from 70 patients (54 male) with stable angina of whom 22 (18 male) had a history of previous myocardial infarction, were analysed for the GPIIIa-Leu-Pro33 polymorphism at the genomic level, and for whole blood flow cytometric measurement of platelet fibrinogen binding. The GPIIIa-Pro33 form was present in 20 (28.6%) patients (1 homozygous) representing an allele frequency of 0.85 and 0.15 (GPIIIa-Leu33:Pro33). The incidence of myocardial infarction was higher (40.0%) in patients positive for GPIIIa-Pro33 than in those without (32.0%) but this was not significant (P=0.58). Fibrinogen binding to ADP-stimulated platelets was significantly higher in the GPIIIa-Pro33 positive group at all ADP concentrations (<0.0001; two way ANOVA). There was no association between fibrinogen binding and the level of expression of the GPIIb-IIIa complex, platelet volume or platelet count. Fibrinogen binding in response to thrombin stimulation was not different between the groups (P>0.05). CONCLUSIONS: The increased tendency of platelets from patients with the Pro33 form of GPIIIa may predispose patients with this allele to a higher risk of acute thrombotic events, and argues for selective use of therapeutic agents that inhibit ADP-mediated platelet activation in occlusive vascular disease states.     

Use of an improved quantitative polymerase chain reaction assay to determine differences in human rhinovirus viral loads in different populations

Human rhinoviruses (HRV) frequently cause acute respiratory infections and chronic respiratory disease exacerbations. However, testing is not generally offered. We developed a modified HRV quantitative polymerase chain reaction (qPCR) assay to assess viral loads in the community and hospital patients. The assay had a lower limit of detection of 2 log₁₀ viral copies/mL and displayed linearity over 5 log₁₀ viral copies, with a lower limit of quantitation of 4 log₁₀ viral copies/mL. Mean viral loads (95% confidence interval) for hospitalized children, university students, and institutionalized elderly, were 7.08 log₁₀ viral copies/mL (6.7–7.5), 6.87 log₁₀ viral copies/mL (6.5–7.2), and 7.09 log₁₀ viral copies/mL (6.9–7.3), respectively (P = 0.67). Serial specimens of 14 university students showed a decrease of mean viral loads from 6.36 log₁₀ viral copies/mL on day 1 to 2.32 log₁₀ viral copies/mL 7 days past symptom onset (P < 0.001). Using an HRV qPCR, we showed that viral loads did not differ between the community and hospitalized populations and significantly decreased following symptoms onset in healthy individuals.     

Comparison of the release of microRNAs and extracellular vesicles from platelets in response to different agonists

On activation platelets release microRNAs and extracellular vesicles (EV) into circulation. The release of EV from platelets has been shown to be dependent on the agonist; in this study, we investigated whether the microRNA profile or EV released from platelets was also agonist specific.

Washed platelets from healthy subjects were maximally stimulated with agonists specific for the receptors for collagen (Glycoprotein VI (GPVI)), thrombin (PAR1/PAR4), or ADP (P2Y1/P2Y12) with/without inhibiting secondary mediators, using aspirin to block cyclooxygenase-1 and apyrase to remove ADP. The released microRNAs were profiled using TaqMan microRNA microarray cards. Platelet-derived EV (pdEV) were characterized by size (Nanoparticle Tracking Analysis, NTA), for procoagulant activity (Annexin-V binding and support of thrombin generation), and for the EV markers CD63 and HSP70.

Platelet activation triggered the release of 57–79 different microRNAs, dependent upon agonist, with a core of 46 microRNAs observed with all agonists. There was a high level of correlation between agonists (r² > 0.98; p < 0.0001 for all), and with the microRNA content of the parent platelets (r² > 0.98; p < 0.0001). The 46 microRNAs seen in all samples are predicted to have significant effects on the translation of proteins involved in endocytosis, cell cycle control, and differentiation. MiR-223-3p was the most abundant in all samples and has previously been implicated in myeloid lineage development and demonstrated to have anti-inflammatory effects. Stimulation through GPVI produced a pdEV population with significantly more procoagulant activity than the other agonists. Apyrase significantly reduced microRNA and pdEV release, while aspirin had little effect.

These data suggest that all tested agonists trigger the release of a similar microRNA profile while the procoagulant activity of the pdEV was agonist dependent. ADP was shown to play an important role in the release of both microRNAs and pdEV.     

Enhancement by testosterone of dimethylnitrosamine carcinogenesis in lung, liver and kidney of inbred NZR/Gd female rats

The effect of androgenic treatments on single dose dimethyl-nitrosamine(DMN) carcinogenesis was studied in 60 female NZR/Gd inbredrats, 25 of which were intact and received DMN 20 mg/kg i.p.in one dose after 48 h starvation, while 35 received DMN combinedwith testosterone injections every 4 weeks beginning 15 daysbefore or after DMN, and in 29 rats combined also with ovariectomy.Sixteen female rats underwent ovariectomy and received testosteroneevery 4 weeks with no DMN. There were also 107 untreated intactcontemporaneous control female rats. A single dose of DMN inducedalveologenic lung (16%), hepatocellular (24%) and kidney epithelial(44%) tumours in intact rats, and the incidences of lethal tumoursat these sites were significantly enhanced by androgenic treatmentsfollowing the carcinogen, most markedly in the kidney. Androgenictreatment given before DMN injection had no significant effecton kidney or liver tumour incidences, but the carcinogenic responsein lung was still strongly enhanced.     

Heparin-Associated Thrombocytopenia: Case Report and Prospective Study

Summary: Heparin-associated thrombocytopenia: Case report and prospective study. A. S. Gallus, K. T. Goodall, W. Beswick and C. N. Chesterman, Aust. N.Z. J. Med ., 1980, 10 , pp. 25–31.
After observing a patient with heparin-induced thrombocytopenia we prospectively recorded the incidence of thrombocytopenia associated with heparin treatment by measuring the platelet count every second day in 166 patients given therapeutic heparin for various thrombo-embolic disorders, and 51 patients given low-dose heparin prophylaxis. A platelet count below 100×10^g/litre developed in nine patients (5.4%) during or soon after full-dose heparin therapy, and in one patient given low-dose heparin. Careful clinical review suggested that heparin was either the most likely cause or a contributing cause of thrombocytopenia in 51166 patients (3.0%) receiving therapeutic heparin and none of the patients who received prophylactic heparin. Associated laboratory studies suggest that heparin-initiated platelet aggregation in vivo is a useful marker for heparin-induced thrombocytopenia.     

Preparation of factor IX deficient human plasma by immunoaffinity chromatography using a monoclonal antibody

A murine hybridoma clone is described that grows continuously in culture and produces a monoclonal antibody we have called Royal Free Monoclonal Antibody to factor IX No. 1 (RFF-IX/1). This has high affinity for a coagulation site on factor IX. RFF-IX/1 immobilised on sepharose can be used to deplete factor IX from normal human plasma. This immunoaffinity depleted plasma is indistinguishable from severe Christmas disease plasma and can be used as the substrate in a one stage coagulation assay for factor IX. The affinity column has high capacity and can be regenerated so that large scale production from normal plasma of factor IX deficient plasma as a diagnostic reagent is now feasible.