Phosphorylation of factor Va and factor VIIIa by activated platelets

Platelet activation leads to the incorporation of 32[PO4(2-)] into bovine coagulation factor Va and recombinant human factor VIII. In the presence of the soluble fraction from thrombin-activated platelets and (gamma-32P) adenosine triphosphate, radioactivity is incorporated exclusively into the M(r) = 94,000 heavy chain (H94) of factor Va and into the M(r) = 210,000 to 90,000 heavy chains as well into the M(r) = 80,000 light chain of factor VIII. Proteolysis of the purified phosphorylated M(r) = 94,000 factor Va heavy chain by activated protein C (APC) gave products of M(r) = 70,000, 24,000, and 20,000. Only the intermediate M(r) = 24,000 fragment contained radioactivity. Because the difference between the M(r) = 24,000 and M(r) = 20,000 fragments is located on the COOH-terminal end of the bovine heavy chain, phosphorylation of H94 must occur within the M(r) = 4,000 peptide derived from the carboxyl-terminal end of H94 (residues 663 through 713). Exposure of the radioactive factor VIII molecule to thrombin ultimately resulted in a nonradioactive light chain and an M(r) = 24,000 radioactive fragment that corresponds to the carboxyl-terminal segment of the A1 domain of factor VIII. Based on the known sequence of human factor VIII, phosphorylation of factor VIII by the platelet kinase probably occurs within the acidic regions 337 through 372 and 1649 through 1689 of the procofactor. These acidic regions are highly homologous to sequences known to be phosphorylated by casein kinase II. Results obtained using purified casein kinase II gave a maximum observed stoichiometry of 0.6 mol of 32[PO4(2-)]/mol of factor Va heavy chain and 0.35 mol of 32[PO4(2-)]/mol of factor VIII. Phosphoamino acid analysis of phosphorylated factor Va by casein kinase II or by the platelet kinase showed only the presence of phosphoserine while phosphoamino acid analysis of phosphorylated factor VIII by casein kinase II showed the presence of phosphothreonine as well as small amounts of phosphoserine. The platelet kinase responsible for the phosphorylation of the two cofactors was found to be inhibited by several synthetic protein kinase inhibitors. Finally, partially phosphorylated factor Va was found to be more sensitive to APC inactivation than its native counterpart. Our findings suggest that phosphorylation of factors Va and VIIIa by a platelet casein kinase II- like kinase may downregulate the activity of the two cofactors.     

Clinical and metabolic features of the randomised controlled Diabetes Remission Clinical Trial (DiRECT) cohort

Aims/hypothesis

Substantial weight loss in type 2 diabetes can achieve a return to non-diabetic biochemical status, without the need for medication. The Diabetes Remission Clinical Trial (DiRECT), a cluster-randomised controlled trial, is testing a structured intervention designed to achieve and sustain this over 2 years in a primary care setting to determine practicability for routine clinical practice. This paper reports the characteristics of the baseline cohort.

Methods

People with type 2 diabetes for <6 years with a BMI of 27–45 kg/m² were recruited in 49 UK primary care practices, randomised to either best-practice diabetes care alone or with an additional evidence-based weight management programme (Counterweight-Plus). The co-primary outcomes, at 12 months, are weight loss ≥15 kg and diabetes remission (HbA_1c <48 mmol/mol [6.5%]) without glucose-lowering therapy for at least 2 months. Outcome assessors are blinded to group assignment.

Results

Of 1510 people invited, 423 (28%) accepted; of whom, 306 (72%) were eligible at screening and gave informed consent. Seven participants were later found to have been randomised in error and one withdrew consent, leaving 298 (176 men, 122 women) who will form the intention to treat (ITT) population for analysis. Mean (SD) age was 54.4 (7.6) years, duration of diabetes 3.0 (1.7) years, BMI 34.6 (4.4) kg/m² for all participants (34.2 (4.2) kg/m² in men and 35.3 (4.6) kg/m² in women) and baseline HbA_1c (on treatment) 59.3 (12.7) mmol/mol (7.6% [1.2%]). The recruitment rate in the intervention and control groups, and comparisons between the subgroups recruited in Scotland and England, showed few differences.

Conclusions/interpretation

DiRECT has recruited a cohort of people with type 2 diabetes with characteristics similar to those seen in routine practice, indicating potential widespread applicability. Over 25% of the eligible population wished to participate in the study, including a high proportion of men, in line with the prevalence distribution of type 2 diabetes.

Trial registration

www.controlled-trials.com/ISRCTN03267836; date of registration 20 December 2013

     

The effects produced by prostaglandin D2 on serotonin turnover and release and tryptophan uptake

In earlier studies, it was proposed that there was a serotonergic involvement in the ability of prostaglandin D2 (PGD2) to potentiate pentobarbital sleeping time. The actions of PGD2 on neuronal turnover and release of serotonin and uptake of tryptophan were examined in mice. The effect of PGD2 administration on serum tryptophan levels was also investigated. PGD2 (1 and 4 mg/kg) increased the concentrations in whole brain of endogenous tryptophan (TRYP) and of 3H-tryptophan (3H-TRYP) following an intravenous (IV) injection of 3H-tryptophan. Formation of 3H-5-hydroxyindoleacetic acid (3H-5HIAA) was doubled after PGD2 administration (1 and 4 mg/kg). Whole brain concentrations of endogenous serotonin (5HT) and 3H-serotonin (3H-5HT) were unchanged after the administration of the prostaglandin. PGD2 (10(-4) to 10(-10)M) in vitro had no effect on spontaneous or K+-evoked release of 3H-5HT from whole brain synaptosomes. Uptake of 3H-tryptophan in synaptosomes was neither stimulated nor depressed by (10(-4) to 10(-12)M) PGD2. There was also no change in serum tryptophan levels after administration of this prostaglandin. Thus, PGD2 administration does affect the serotonergic system but no direct neurochemical correlate of sedation can be shown.     

Involvement of the serotonergic system in the prolongation of pentobarbital sleeping time produced by prostaglandin D2

In the present study, the depressant and sedative actions of prostaglandin D2 (PGD2) were investigated. Intravenous (IV) administration of PGD2 produced a significant decrease in the spontaneous locomotor activity of mice from 1 to 15 minutes following injection. Prostaglandin D2 was also able to potentiate pentobarbital sleeping time at doses of 0.4 and 4.0 mg/kg when administered intravenously. Distribution studies with 3H-PGD2 (6 microCi, 4 mg/kg) showed that only 0.04% of the tritium administered could be found in brain at 5 min after the injection, and that only 50% of this was parent 3H-PGD2. The role of the serotonergic neurotransmitter system in the depressant action of PGD2 was investigated with drugs which modulate this system. The ability of PGD2 to potentiate pentobarbital sleeping time was diminished by pretreatment with agents that reduce brain level or synthesis rate of serotonin. Such agents include para-chlorophenylalanine (PCPA), a tryptophan hydroxylase inhibitor, 5,7-dihydroxytryptamine (5,7-DHT), a neurotoxin with selectivity for serotonergic neurons, and quipazine, a serotonergic autoreceptor stimulant. On the other hand, pretreatment with 5-hydroxytryptophan (5-HTP), the precursor of serotonin, further enhanced the potentiation of pentobarbital sleeping time by PGD2. These data suggest that the depressant actions of PGD2 are linked to the serotonergic neurotransmitter system.     

Study on the effect of oral administration of carbocisteine on ventilatory parameters in the SO2 inhalation model of bronchitis in the rat

In order to study the physiological correlates of the beneficial action of carbocisteine (S-carboxy-methyl-cysteine), we have measured the changes occurring in ventilatory parameters in rats made bronchitic by prolonged exposure (2 weeks) to air containing sulfur dioxide (SO2). In animals treated with distilled water (1 ml/100 g/day), statistically significant (P < 0.05) changes in respiratory frequency (-20%) and tidal volume (+31%) were found. As a result of these opposing changes, the ventilation/min was stable. Moreover, the compliance was decreased (33%, P < 0.05) and the resistance was greatly enhanced (+ 99%, P < 0.05). The concomitant administration of carbocisteine (500 mg/kg po/day) with SO2 inhalation significantly (P < 0.05) prevented the development of resistance without effecting significant changes in the other parameters except for a slight improvement in ventilation/min. In conclusion, this improved respiratory resistance in the bronchitic carbocisteine-treated animals tallies with a decrease in mucus retention associated with the return to normal of rheological characteristics of the secreted mucus.     

Audit of post-operative auto-transfusion of shed blood in hip arthroplasty.

Auto-transfusion of shed blood for joint arthroplasty has several theoretical advantages: availability, compatibility, avoidance of transmission of infection and cost. The clinical records of 92 primary hip arthroplasties were reviewed to investigate the effect of auto-transfusion on the use of the National Blood Transfusion Service both in terms of blood ordered and subsequently used. Overall, post-operative auto-transfusion had no significant effect on blood ordering or usage and was very expensive. The majority of patients were over transfused.     

Fetal intraluminal gastric masses after second trimester amniocentesis.

Eight instances of homogeneous, well-defined echoes within the fetal stomach were identified on routine second trimester detailed scan over a 7 month period, a prevalence of 1 in 287 or 0.35%. This finding was significantly more frequent in women who had cytogenetic amniocentesis than in those who had not had the procedure (4 in 266 [1.5%] versus 4 in 2031 [0.2%], respectively; P < 0.01). Indirect signs of intra-amniotic bleeding, such as particles in the amniotic fluid, chorioamniotic separation, or hyperechogenic bowel, were present in four cases. The association between echogenic material within the fetal stomach and cytogenetic material within the fetal stomach and cytogenetic amniocentesis is discussed.