Inhibition of protease-activated receptor 4 impairs platelet procoagulant activity during thrombus formation in human blood.

UNLABELLED: Essentials The platelet thrombin receptor, PAR4, is an emerging anti-thrombotic drug target. We examined the anti-platelet & anti-thrombotic effects of PAR4 inhibition in human blood. PAR4 inhibition impaired platelet procoagulant activity in isolated cells and during thrombosis. Our study shows PAR4 is required for platelet procoagulant function & thrombosis in human blood. SUMMARY: Background Thrombin-induced platelet activation is important for arterial thrombosis. Thrombin activates human platelets predominantly via protease-activated receptor (PAR)1 and PAR4. PAR1 has higher affinity for thrombin, and the first PAR1 antagonist, vorapaxar, was recently approved for use as an antiplatelet agent. However, vorapaxar is contraindicated in a significant number of patients, owing to adverse bleeding events. Consequently, there is renewed interest in the role of platelet PAR4 in the setting of thrombus formation. Objectives To determine the specific antiplatelet effects of inhibiting PAR4 function during thrombus formation in human whole blood. Methods and Results We developed a rabbit polyclonal antibody against the thrombin cleavage site of PAR4, and showed it to be a highly specific inhibitor of PAR4-mediated platelet function. This function-blocking anti-PAR4 antibody was used to probe for PAR4-dependent platelet functions in human isolated platelets in the absence and presence of concomitant PAR1 inhibition. The anti-PAR4 antibody alone was sufficient to abolish the sustained elevation of cytosolic calcium level and consequent phosphatidylserine exposure induced by thrombin, but did not significantly inhibit integrin αII b ß3 activation, α-granule secretion, or aggregation. In accord with these in vitro experiments on isolated platelets, selective inhibition of PAR4, but not of PAR1, impaired thrombin activity (fluorescence resonance energy transfer-based thrombin sensor) and fibrin formation (anti-fibrin antibody) in an ex vivo whole blood flow thrombosis assay. Conclusions These findings demonstrate that PAR4 is required for platelet procoagulant function during thrombus formation in human blood, and suggest PAR4 inhibition as a potential target for the prevention of arterial thrombosis.

RNA polymerase II elongation factors Spt4p and Spt5p play roles in transcription elongation by RNA polymerase I and rRNA processing.

Previous investigations into the mechanisms that control RNA Polymerase (Pol) I transcription have primarily focused on the process of transcription initiation, thus little is known regarding postinitiation steps in the transcription cycle. Spt4p and Spt5p are conserved throughout eukaryotes, and they affect elongation by Pol II. We have found that these two proteins copurify with Pol I and associate with the rDNA in vivo. Disruption of the gene for Spt4p resulted in a modest decrease in growth and rRNA synthesis rates at the permissive temperature, 30 degrees C. Furthermore, biochemical and EM analyses showed clear defects in rRNA processing. These data suggest that Spt4p, Spt5p, and, potentially, other regulators of Pol I transcription elongation play important roles in coupling rRNA transcription to its processing and ribosome assembly.

Surgical removal of a radicular odontogenic cyst in a four-year-old Dalmatian dog.

A cystic structure was identified radiographically in a four-year-old dog during routine dental prophylaxis. Surgical removal of the cyst lining was achieved by exposure of the site through extraction of the right first to third maxillary incisor teeth (101, 102, 103). The cyst lining was removed en-bloc. The cavity was curetted and filled with decalcified freeze-dried bone. Histological examination revealed a radicular cyst. The proposed etiology is blunt trauma to tooth 103, pulpal necrosis, apical granuloma and resulting cyst formation. Fourteen months following surgery, there was no recurrence of the cyst.

Ultrastructural in situ hybridization to nascent transcripts of highly transcribed rRNA genes in chromatin spreads.

The amplified rRNA genes of amphibian oocytes were used as a model system for the development of an in situ hybridization technique to label nascent transcripts in dispersed chromatin. A biotinylated complementary RNA probe was hybridized to nascent transcripts from dispersed nucleoli, and detected by a two step antibody technique utilizing colloidal gold as an electron dense marker. A specific sequence on the rRNA nascent transcript was labeled in a pattern consistent with its location; however, gene morphology was difficult to analyze following in situ hybridization owing to low sample contrast. Proteins associated with the transcripts were apparently lost during the procedure, leading to decreased electron density of the transcripts. The technique was systematically modified in an attempt to identify conditions that preserved gene morphology adequately for ultrastructural analysis, while simultaneously maintaining sufficient levels of specific labeling.

Phase II study of fludarabine phosphate for gastric adenocarcinoma. An Illinois Cancer Center trial.

In an Illinois Cancer Center phase II trial, fludarabine phosphate was administered to a total of 14 patients (9 men, 5 women) with advanced, measurable, gastric adenocarcinoma. Fludarabine phosphate was given as a rapid intravenous (IV) bolus at a starting dose of 20 mg/m2/d for the first 5 days of a 28-day cycle. For subsequent cycles, the dose was escalated in increments of 2 mg/m2/d, provided that no toxicities greater than grade 1 were noted. In cases of grade 3 toxicity, dose reductions of 2 mg/m2/d were required, and patients who experienced grade 4 toxicities were removed from study. Receiving one complete 5-day course of fludarabine phosphate and surviving for 4 weeks on study were required for a patient to be evaluable for response. None of the patients responded to treatment. Although fludarabine phosphate was ineffective against gastric adenocarcinoma in this study, toxicity was acceptable at the 20 mg/m2/d times 5 every 28 days dose and schedule.

Phase II study of amonafide in gastric adenocarcinoma. An Illinois Cancer Center trial.

Twelve patients with recurrent, metastatic, or inoperable gastric adenocarcinoma were enrolled in an Illinois Cancer Center phase II trial of amonafide (nafidimide), a novel compound that acts as a DNA intercalator. Treatment consisted of a 60-minute infusion of amonafide which was administered daily for 5 consecutive days every 3 weeks at a starting dose of 300 mg/m2/d. Doses were modified according to the grade of toxicity experienced and eight patients underwent dose escalations. All 12 patients were evaluable for response and toxicities were predominantly hematologic. Stabilization of disease for at least 28 days was observed in seven patients and disease progression was noted in five. The median survival was 7.4 months. Doses were sufficient to produce severe bone marrow toxicity in one-third of the patients treated. None of the patients responded to therapy, implying a true response rate less than .221. Based on the results of this study, amonafide showed no activity against gastric adenocarcinoma; however toxicity appeared acceptable at the 300 mg/m2/d x 5 consecutive days every 3 weeks dose and schedule.

Development of physical fitness standards for Canadian Armed Forces younger personnel.

The purpose of this study was to develop minimum physical fitness standards based on common task criteria for personnel younger than 35 years of age in the Canadian Armed Forces. A random sample of 66 men and 144 women performed the Exercise Prescription (EXPRES) test and five physically demanding tasks that simulated common military tasks. Common cutting scores were selected as the point at which 75% of the total weighted sample passed each task. Since there were significant differences between the sexes in task performance and technique execution, the groups were analyzed separately. Results indicated a range in variance of 14 to 48% between military task performance and physical fitness test score, thus suggesting that fitness measures are a poor predictor of task performance. Because of the low variance observed the passing group of each task was treated as a representative sample of subjects whose fitness profiles were indicative of those able to meet task criteria. The 5th percentile fitness scores of the passing group were proposed as the minimum fitness standard. These conditions resulted in fitness standards that were physically more demanding for women than for men.

Confounding as a problem in relating life events to health status in elderly individuals.

Explored two types of confounding relevant to research relating major life events to physical illness in elderly individuals: (a) contamination of life events lists by health-related and subjective items; and (b) failure to control for illness existing prior to the life event assessment period. Community-residing elderly individuals (M = 70.4 years) completed two measures of health status in each of two waves of testing. During the second wave, participants also completed a life events list. Independent judges categorized the life events as health-related or not and further categorized non-health-related events as subjective or objective. Results indicated that life events-illness correlations were influenced by the presence of confounded items and that when illness present prior to the life event assessment period was controlled the relationship between confounded life events and illness decreased. These results underscore the importance of assessing possible sources of confounding when conducting life event illness research with elderly individuals.

rRNA transcription rate in Escherichia coli.

The rate of in vivo transcription elongation for Escherichia coli rRNA operons was determined by electron microscopy following addition of rifampin to log-phase cultures. Direct observation of RNA polymerase positions along rRNA operons 30, 40, and 70 s after inhibition of transcription initiation yielded a transcription elongation rate of 42 nucleotides per s.

Dynamic analysis of isoinertial lifting technique.

The objectives of the study were threefold: (1) to quantify dynamic measures (displacement, velocity, force/acceleration, and power) of a 1·83 m isoinertial lift on an incremental lifting machine (ILM); (2)to identify any gender differences in ILM lifting technique; and (3) to assess the implications of these data for the use of the ILM as a screening device. One hundred and thirty-two military personnel (33 females and 99 males) completed a maximal isoinertial lifting test from a starting height of 0·34 m to a target height of 1·83 m on the ILM. A force transducer attached to the back of the armature provided continuous velocity and displacement data from which the displacement, velocity, acceleration/force, and power profiles were determined. These data were summarized into 37 lift parameters; 33 representing the dynamic components of the lift, and four representing averages taken across the entire lift. The results revealed that the 1·83 m isoinertial lift could be described in three phases: (1) a powerful pulling phase, which incorporated measures of maximal acceleration/force, velocity, and power; 2) a wrist changeover manoeuvre, wherein momentum was required to compensate for minimal force and acceleration values; and (3) a pushing phase, during which second maximal force and acceleration measures were attained. Statistically significant differences were found between genders on various parameters of the technique profiles, suggesting that the testing protocol may have placed different demands on males and females. Females spent a greater proportion of the total lift time in the pushing phase, and had less opportunity to generate power during the pulling phase. The resulting TLM scores may have underestimated the lifting capacity of females. It was recommended that females and males be given independent consideration in the design of ILM lifting protocols.

Four factor model to describe an isoinertial lift.

The objectives of the study were threefold: (1) to develop an empirical description of dynamic factors involved in a test of lifting performance on an Incremental Lifting Machine (TLM) through application of principal components analysis; (2) to conduct gender analyses of the factor structures; and (3) to determine the stability of the structures with repeated sampling. An initial sample of 175 participants (79 females and 96 males) completed a maximal isoinertial lifting test from a starting height of 0·34 m to a target height of 1·83 m. A confirmatory sample of 132 participants (33 females and 99 males) completed the same 1LM test under identical conditions. A force transducer attached to the back of the armature provided continuous displacement data from which displacement, velocity, acceleration, force, and power profiles were determined. These data were summarized into 32 lift parameters, and then subjected to principal components analyses. For the dynamic parameters recorded for the initial sample, a four factor solution accounting for 78·9% of the variance was found to be optimal. Factor one (named Mid-Body Coordination and accounting for 24·7% of the variance) related to the timing and displacement of maximum velocity and power. Factor two (named Maximum Strength) contained maximum force and power measurements and accounted for 22·5% of the variance. Factor three (named Minimum Strength) related to minimum measures of force and power and accounted for 17·2% of the variance. Finally, factor four (named Lower Body Co-ordination and accounting for 14·4% of the variance) related to the timing and displacement of maximum force. Descriptions of the factors were developed in terms of their underlying biomechanical relationships. The four factor solution was found to be stable across genders, and it was replicated for the confirmatory sample. It was concluded that these dynamic factors possessed considerable scientific utility for future research.

Transcription mapping of the Escherichia coli chromosome by electron microscopy.

The distinctive double Christmas tree morphology of rRNA operons as visualized by electron microscopy makes them easy to recognize in chromatin spreads from Escherichia coli. On the basis of the pattern of nascent transcripts on nearby transcription units and the relative distances of the operons from one another and the replication origin, we are now able to specifically identify five of the seven rRNA operons in E. coli. The use of rRNA operons as markers of both position and distance has resulted in the morphological mapping of a significant portion of the E. coli chromosome; over 600 kilobase pairs in the 84- to 90-min and 72-min regions can now be recognized. Since individual rRNA operons could be identified, direct comparisons could be made of their transcriptional activities. As judged by the densities of RNA polymerases along the operons, rrnA, rrnB, rrnC, rrnD, and rrnE were all transcribed at similar levels, with one RNA polymerase every 85 base pairs. The ability to recognize individual operons and specific regions of the chromosome allows direct comparisons of various genetic parameters.