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A diverse range of biochemical and cellular assays are used by medicinal chemists to guide compound optimization. The data collected from these assays influence decisions taken on structure-activity relationship (SAR) campaigns. Therefore, it is paramount that medicinal chemists have a solid understanding of the strengths and limitations of each assay being used to characterize synthesized analogs. For the successful execution of a medicinal chemistry campaign, it is our contention that an early partnership among assay biologists, informaticians, and medicinal chemists must exist. Their combined skill sets are necessary to not only design and develop robust assays but also implement an effective screening cascade in which multiple orthogonal and counter assays are selected to validate the activity and target(s) of the synthesized compounds. We review multiple cases of drug and chemical probe discovery from collaborative National Center for Advancing Translational Sciences/National Institutes of Health projects and published scientific literature in which the evaluation of compounds in secondary or orthogonal assays led to the discovery of unexpected activities, forcing a reconsideration of the original assay design that was used to discover the biological activity of the compound. Using these retrospective case studies, the goal of this Perspective is to hedge toward the development of physiologically relevant assays that are able to capture the true bioactivity of compounds being developed in a medicinal chemistry campaign.

Background: Human trafficking is a global problem and results in deleterious psychological, social, and physical effects on the lives of those who are trafficked; however, it is not clear how to best intervene with survivors. The purpose of this review was to synthesize the evidence of exit and postexit intervention programs for survivors of human trafficking to inform practice and research. Method: Systematic review methods were used to search, select, and extract data from published and unpublished experimental, quasi-experimental, and preexperimental studies that assessed the effects of any exit or postexit interventions for victims of human trafficking. The authors searched eight databases, reviewed bibliographies, and conducted forward citation searches from relevant reports and prior reviews to find studies authored between 2005 and 2015. Results: The search yielded six eligible studies that included 155 female and 6 male survivors from four countries. Interventions were diverse, with three using a trauma-informed approach. Authors measured a myriad of outcomes, including mental health, social network, community reintegration, and employment; however, the quality of most studies was poor. Discussion: Evidence of effects of exit and postexit interventions is sparse, and much of the research is poorly designed and executed; however, the needs of trafficking survivors are complex and effective interventions are desperately needed. Implications for practice and research are discussed.

Background: The purpose of this study was to review the outcomes associated with different surgical treatments in the management of suppurative flexor tenosynovitis (SFT). Methods: Patients treated by open drainage (OD) through a single incision and closed catheter irrigation (CCI) of the tendon sheath were evaluated from 2003 to 2009 at a single institution. Variables examined included culture results, time to surgery, duration of hospitalization and antibiotic therapy, pain, reoperation, and functional outcomes were recorded. Statistical analyses used included parametric and nonparametric t tests. Results: Twenty-four patients were treated for SFT with a mean follow-up of 3 ± 2 years. There were 13 (100%) males in the OD group compared to 8 (72%) males within the CCI group. Ninety-two percent of patients who underwent OD and 100% who underwent CCI reported none or mild pain. There were no differences between the 2 groups with regard to functional outcome scores. Factors leading to worse functional outcomes included prolonged time to receiving antibiotics and Staphylococcus aureus infection. Smokers required prolonged antibiotic treatment compared to nonsmokers. Accounting for planned returns to the operating room, there was no difference in reoperation rates between the OD or CCI groups. Conclusions: Surgical treatment of SFT with either OD or CCI resulted in similar outcomes for pain, function, and need for reoperation. Factors that lead to poor outcomes include S aureus culture, prolonged time to antibiotics, and smoking.

Background— It has been proposed that under hypoxic conditions, nitrite may release nitric oxide, which causes potent vasodilation. We hypothesized that nitrite would have a greater dilator effect in capacitance than in resistance vessels because of lower oxygen tension and that resistance-vessel dilation should become more pronounced during hypoxemia. The effect of intra-arterial infusion of nitrite on forearm blood flow and forearm venous volumes was assessed during normoxia and hypoxia. Methods and Results— Forty healthy volunteers were studied. After baseline infusion of 0.9% saline, sodium nitrite was infused at incremental doses from 40 nmol/min to 7.84 μmol/min. At each stage, forearm blood flow was measured by strain-gauge plethysmography. Forearm venous volume was assessed by radionuclide plethysmography. Changes in forearm blood flow and forearm venous volume in the infused arm were corrected for those in the control arm. The peak percentage of venodilation during normoxia was 35.8±3.4% (mean±SEM) at 7.84 μmol/min ( P P P Conclusions— Nitrite is a potent venodilator in normoxia and hypoxia. Arteries are modestly affected in normoxia but potently dilated in hypoxia, which suggests the important phenomenon of hypoxic augmentation of nitrite-mediated vasodilation in vivo. The use of nitrite as a selective arterial vasodilator in ischemic territories and as a potent venodilator in heart failure has therapeutic implications.

Any hypothesis for arterial thrombosis must be able to account for the following facts: (1) thrombi do not form in normal arteries; (2) thrombi form in atherosclerotic arteries; (3) arterial thrombi consist initially of aggregated platelets; (4) atherosclerosis increases slowly, whereas thrombosis occurs rapidly and is individually unpredictable. Therefore, atherosclerotic arteries must be subject to sudden, unpredictable events capable of initiating platelets aggregation; (5) most occlusive thrombi are associated with fissures in underlying atheromatous placques.

Reconstruction of the multiligament-injured knee often involves extended surgical and tourniquet use times and often is performed in patients who have sustained concomitant fractures as well as vascular injuries, all of which would appear to predispose the patient to the potentially serious complications of deep vein thrombosis and perhaps pulmonary embolism, yet little is known about the frequency of venous thromboembolic (VTE) events after multiligamentous knee reconstruction.The purposes of this study were to (1) determine the frequency of symptomatic VTE after multiligamentous knee reconstruction at a single institution; and (2) to determine associated risk factors for VTE in these patients.The records of 134 (63% of the 213 consented individuals in our longitudinal database) patients who underwent primary (129 [96%]) or revision (five [4%]) multiligamentous knee reconstruction at a single institution between 1992 and 2013 were retrospectively reviewed. With two patients undergoing procedures bilaterally, this resulted in a total of 136 multiligamentous knee reconstructions. VTE for which clinical symptoms were evident and confirmed by imaging within 3 months after the reconstructive procedure was noted. Pre- and perioperative data were collected with respect to demographics, associated injuries, medical history, smoking status, and surgical data. Standard rehabilitation and thromboprophylaxis protocols were used in all patients.Three cases of symptomatic VTE (three deep vein thromboses) occurred after the 136 reconstructions (2%; 95% confidence interval, 0.53%-7.3%). Two of the three patients were obese and the remaining patient smoked and abused alcohol. However, as a result of a low frequency of VTE, no risk factors could be identified in this series.Symptomatic VTE occurred in 2% of multiligamentous knee reconstructions at our institution while receiving thromboprophylaxis. This is similar to rates documented after anterior cruciate ligament reconstruction without thromboprophylaxis. Further multicenter research is required to identify the true frequency of and risk factors for developing VTE in patients undergoing multiligamentous knee reconstruction after knee dislocation.Level III, therapeutic study. See Guidelines for Authors for a complete description of levels of evidence.

Background —Antiplatelet agents are widely recognized for their efficacy in reducing the occurrence of vascular events in patients with atherothrombotic disease. Aspirin is currently considered to be the “reference standard” antiplatelet agent and is recommended by the American Heart Association for use in patients with a wide range of manifestations of cardiovascular disease on the basis of its high benefit-to-risk and benefit-to-cost ratios. Recently, clopidogrel (Plavix, Bristol-Myers Squibb Co), another antiplatelet agent, was approved by the Food and Drug Administration for many of the same indications as aspirin. Summary of Review —Because physicians will be faced with deciding whether to switch from the well-established practice of recommending aspirin for use in patients with atherothrombotic disease, both aspirin and clopidogrel are compared with respect to the primary factors that influence such decisions (ie, their relative efficacy, safety, cost, and convenience of use). Conclusions —Based on the available evidence, aspirin is preferred for the majority of stroke or myocardial infarction patients at risk of recurrent atherothrombotic events. Clopidogrel may, however, provide valuable therapeutic benefit over aspirin in patients with peripheral arterial disease and in stroke or myocardial infarction patients for whom aspirin treatment is contraindicated or for whom aspirin fails to achieve the desired therapeutic effect.

Purpose For patients with purulent flexor tenosynovitis, our purpose was to (1) calculate the diagnostic accuracy of white blood count (WBC), erythrocyte sedimentation rate (ESR), and C-reactive protein (CRP) for those who underwent surgical drainage, (2) to correlate these markers for those treated with antibiotics alone, and (3) to evaluate the accuracy of diagnosis for surgical patients. Methods A total of 82 consecutive patients (71 surgical and 11 nonsurgical) with flexor tenosynovitis were identified from orthopedic databases at 2 academic centers. We evaluated inflammatory markers (WBC, ESR, and CRP), radiographs, descriptions of surgical findings, and intraoperative cultures for all patients. For nonsurgical patients, we evaluated inflammatory markers for possible correlation with the presumed diagnosis of purulent flexor tenosynovitis. For surgical patients, sensitivity, specificity, positive predictive value, and negative predictive value were calculated individually for inflammatory markers. Results For nonsurgical patients, WBC, ESR, and CRP were elevated in 3 of 11 patients (27%), 6 of 8 patients (75%), and 6 of 7 patients (86%), respectively. For surgical patients, the intraoperative findings or cultures were consistent with infection in 69 of 71 cases (97%), whereas calcific tendinitis was diagnosed in 2 cases. Cultures were positive in 56 patients (79%). All 3 markers had a specificity and positive predictive value of 100%. For WBC, ESR, and CRP, respectively, the sensitivity was 39%, 41%, and 76% and the negative predictive value was 4%, 3%, and 13%. Conclusions Commonly used inflammatory blood markers (WBC, ESR, and CRP) may be helpful in diagnosing purulent flexor tenosynovitis. If the levels of any of these markers are elevated in patients suspected of having the diagnosis, the likelihood of infection is extremely high. However, with low negative predictive values, these markers cannot reliably rule out infection. Type of study/level of evidence Diagnostic III.

Reduced endothelial surface charge markedly increases the rate of LDL uptake into blood vessels. Previous work in the streptozotocin diabetic rat reported reduced endothelial surface charge. We compared endothelial surface charge density in internal mammary artery rings from patients with type 2 diabetes (n = 12) and from non diabetic patients undergoing coronary artery bypass grafting, and observed a substantial (52%) reduction in the former. This was associated with higher plasma sialic acid levels suggesting loss of sialic acid residues from the glycocalyx as a possible mechanism.

Skeletal muscle is rarely a site of malignant metastasis; the molecular and cellular basis for this rarity is not understood. We report that myogenic cells exert pronounced effects upon co-culture with metastatic melanoma (B16-F10) or carcinoma (LLC1) cells including conversion to the myogenic lineage in vitro and in vivo, as well as inhibition of melanin production in melanoma cells coupled with cytotoxic and cytostatic effects. No effect is seen with non-tumorigenic cells. Tumor suppression assays reveal that the muscle-mediated tumor suppressor effects do not generate resistant clones but function through the down-regulation of the transcription factor MiTF, a master regulator of melanocyte development and a melanoma oncogene. Our findings point to skeletal muscle as a source of therapeutic agents in the treatment of metastatic cancers.

Recently, fatigability and muscle oxygen consumption (mVO(2)) during sustained isometric contractions were found to be less at shorter (30 degrees knee angle; 0 degrees = full extension) compared to longer knee extensor muscle lengths (90 degrees ) and, at low torques, less in the rectus femoris (RF) muscle than in the vastus lateralis and medialis. In the present study we hypothesized that these findings could be accounted for by a knee angle- and a muscle-dependent activation respectively. On two experimental days rectified surface EMG (rsEMG) was obtained as a measure of muscle activation in nine healthy young males. In addition, on day 1 maximal torque capacity (MTC) was carefully determined using superimposed nerve stimulation on brief high intensity contractions (> 70%MVC) at 30, 60 and 90 degrees knee angles. On day 2, subjects performed longer lasting isometric contractions (10-70%MTC) while mVO(2) was measured using near-infrared spectroscopy (NIRS). At 30 degrees , maximal mVO(2) was reached significantly later (11.0 s +/- 6.5 s) and was 57.9 +/- 8.3% less (average +/- SD, across intensities and muscles) than mVO(2) at 60 and 90 degrees (p < 0.05). However, rsEMG was on average only 18.0 +/- 11.8% (p = 0.062) less at the start of the contraction at 30 degrees . At 10%MTC at all knee angles, maximal mVO(2) of the RF occurred significantly later (28.8 +/- 36.0 s) and showed a significantly smaller increase in rsEMG compared to both vasti. In conclusion, it is unlikely that the tendency for less intense muscle activation could fully account for the approximately 60% lower oxygen consumption at 30 degrees , but the later increase in RFmVO(2) seemed to be caused by a less strong activation of the RF.

An unusual fracture of the trapezium was found on computed tomography examination after plain radiographs failed to demonstrate any bony pathology. This coronal fracture has not been previously mentioned in the literature. Management included open reduction and internal fixation with 2 lag screws.

Measurements of total collagen, of the ratio of collagen types III/(I+III) and of sulphated glycosaminoglycans (GAGs) were compared with mechanical strength for individual ulcerated and non-ulcerated human aortic plaque caps and with intima adjacent to the plaques. The distributions of the collagen type ratio were similar for both ulcerated and non-ulcerated plaque caps but different from that of the adjacent intima. The proportions of different collagen types were not related to fracture stress and are thus unlikely to affect the potential to ulcerate. The distributions of the sulphated GAGs showed lower amounts for the plaque caps compared with the nearby intima, with the centres of ulcerated plaque caps having the lowest values. Total collagen had higher values in the peripheries of plaque caps compared with the nearby intima, but was distinctly lower in the centres of ulcerated plaque caps. Plaque caps appeared to require a higher collagen content than adjacent intima to support a given level of mechanical strength, suggesting that while collagen production had occurred in the plaque caps it was not as efficiently organized to resist fracture as a similar amount of collagen in the adjacent intima. Ulcerated plaque caps are notable for much larger transverse (centre vs. periphery) gradients of connective tissue constituents than for non-ulcerated plaque caps. The development of these transverse gradients may be a critical aspect in determining the propensity of a plaque to ulcerate.

The aim of this study was to evaluate the effects of a preparation of low molecular weight heparan sulphate (LMW-HS) on the fibrinolytic system. Twenty-five healthy volunteers received LMW-HS by mouth in three separate experiments. In the first experiment, 10 volunteers received either 80 mg LMW-HS or placebo in a single-blind cross-over study; blood samples were taken before and 1, 2, 3 and 6 h after treatment. LMW-HS caused an increase in global fibrinolysis, the effect being greatest after 2-3 h and disappearing by 6 h. However, neither plasminogen activator activity nor tissue-type plasminogen activator (tPA) antigen levels were changed. In the second experiment, daily doses of 80 mg LMW-HS were administered to 10 volunteers for 7 days; this regimen did not produce a statistically significant increase in fibrinolytic activity for the whole group although some individuals did respond markedly. In the third experiment, 160 mg LMW-HS administered to five volunteers did not affect ADP- and collagen-induced platelet aggregation. In vitro, LMW-HS added to plasma at concentrations of 20 and 30 micrograms/ml, brought about a significant increase in apparent plasminogen activator activity. These results suggest that the increased fibrinolytic activity seen after LMW-HS is due to the recruitment of additional amounts of tPA in the ex vivo test system. LMW-HS had no effect on plasminogen activator inhibitor.

Twenty two children, under 15 years of age, from southern Chile's Tenth Region, with advanced stages of Hodgkin's disease (8 III-A, 6 III-B and 8 IV-B) were treated with chemotherapy (modified COPP protocol) and radiotherapy between 1976 and 1985 at Valdivia's Regional Hospital. Overall survival rate was 77.3% and disease-free survival rate was 64.5% after a follow-up period from 3 to 13 years (median 70 months). Five patients died during the first two years. One relapsed 10 years after beginning of therapy, but achieved a second complete remission. The survival rate in patients with lymphocytic depletion type (5 cases) was 20.0%, whereas that of the mixed cellularity type (14 cases) was 92.9% (p less than 0.01). No cases of second neoplasms related to chemotherapy and radiotherapy were seen in this series.

some observations were made also on smallarteries forcomparison. Thevenousvessels werecho-sen for quantification in preference to arteries becausethe comparative slowness ofbloodflow madeit easierto develop and control the new methods required.The results showed that the injection of hamsterplatelets, made fluorescent by labeling in vitro withfluorescein isothiocyanate, into thecirculationofotherhamsters makesit possible to quantifythe flowingandsticking ofplatelets in vivo. Theexperiments provideevidence that there is very little tendency for circulat-ing platelets to stick to the walls of small veins andarterioles, evenunderconditions in whichtheadhesionpropensities ofbothplatelets andvesselwallsmightbeexpectedtobeincreasedbypreparativemanipulations.In venules such

The intravascular adhesion and aggregation of platelets initiate hemostasis and arterial thrombosis. In vitro, platelet aggregation is induced by many different agents; which of these is responsible for aggregation in vivo is now under investigation. For this purpose, we have developed novel techniques for the reproducible determination of bleeding times in mesenteric arterioles of rats and rabbits. Local infusions of enzyme systems which remove adenosine diphosphate (ADP) greatly increase the bleeding time at arterial injuries distal to the site of infusion. These observations establish the involvement of ADP in the activation of platelets for primary hemostasis. Direct measurement of free adenosine triphosphate (ATP), as an indicator of ADP, at such injury sites indicates that enough ADP for activating platelets is released very rapidly from damaged vessel walls and much later from the platelets themselves. This bleeding-time technique has also shown that hemostatic platelet aggregation is delayed less by the inhibition of the production of thromboxane A2 than by that of ADP. These observations provide an explanation for the ineffectiveness of any simple platelet-inhibiting drug (including aspirin) by itself, whenever arterial (e.g., coronary or cerebral) thrombosis is initiated by hemorrhages into atheromatous plaques. On the other hand, aspirin is significantly effective when myocardial infarction follows unstable angina and when strokes follow transient episodes of cerebral ischemia. This partial effectiveness can be explained by an action of aspirin on platelets, assuming that, in such cases, their thromboembolic aggregation is initiated by hemodynamic effects of atheromatous lesions.(ABSTRACT TRUNCATED AT 250 WORDS)

Atherosclerosis is accelerated in hyperlipidaemias but, apart from the concentration of low-density lipoprotein (LDL) in the blood, very little is known about other influences on the disease process. We now provide evidence that in anaesthetized rabbits the atherogenic uptake of LDL by arterial walls is accelerated by noradrenaline at its physiological concen­trations in rabbit and human blood. The principle of the experiments was to compare the uptake of intra­venously injected, radioactively labelled LDL, methylated to prevent removal by high-affinity receptors, in the two carotid arteries of anaes­thetized rabbits after infusing low concentrations of noradrenaline into one carotid and saline as control into the other, the volume rates of infusion being about 1% of the carotid blood flows. Human LDL, which behaves sufficiently like rabbit LDL for these purposes, was prepared, methylated and radio-iodinated by standard methods. At the end of the infusions, the arteries were excised and their radio activities determined. Noradrenaline infused for 2 h to produce local blood concentrations of nominally 1, 10, 50 and 100 nM significantly increased the LDL radio­ activities of the walls of the noradrenaline-infused carotids. Concen­trations of nominally 100 nM also increased the LDL radioactivities of the walls of the saline-infused carotids; this was associated with significant increases in their blood noradrenaline concentrations. These results may contribute towards an explanation for the acceler­ated atherosclerosis and the increased incidence of its clinical manifes­tations in conditions associated with elevated blood noradrenaline concentrations, including the episodic increases associated with stress and cigarette smoking as well as the more persistent increases caused by phaeochromocytoma.

Adenosine inhibits the aggregation of human but not of rat platelets whereas both are inhibited by prostaglandin E1 or by the pyrimido-pyrimidine compound RA233. In human platelets all three agents increase adenosine-3'-5'-cyclic monophosphate (cAMP). If the inhibition of aggregation depended on this increase, adenosine might be expected not to increase cAMP in rat platelets. Under conditions in which adenosine inhibited aggregation and increased cAMP in human platelets, adenosine caused a similar increase in cAMP in rat platelets without inhibiting their aggregation. The aggregation of rat platelets was inhibited as effectively as that of human platelets by PGE1 or RA233 at concentrations which caused greater increases in cAMP than did the highest concentrations (2.8 X 10(-4) M) of adenosine it was possible to use. When the increase of cAMP in rat platelets by PGE1 was limited to that produced by adenosine, PGE1 like adenosine failed to inhibit aggregation. Therefore, the difference in the inhibitory effectiveness of adenosine on rat and human platelets was quantitative rather than qualitative and apparently depended on the inability of adenosine to increase cAMP sufficiently in rat platelets. When cAMP had been increased by adenosine, PGE1 or RA233, the addition of ADP caused cAMP to decrease rapidly in both human and rat platelets to between +22 and -18% of control values, except that the decrease in rat platelets was to +40% after RA233 had been present for 0.5 min before ADP. The increase in cAMP produced in rat platelets by adenosine at 5 X 10(-6) to 2.8 X 10(-4) M for 3 min was associated with a small increase in aggregation velocity. It is suggested that the comparative ineffectiveness of adenosine as an inhibitor of platelet aggregation, particularly with rat but less so also with human platelets, is because, unlike PGE1 or RA233, adenosine has two opposing actions on aggregation; one being inhibition by activating adenylate cyclase and increasing cAMP, and the other being potentiation by uptake. This hypothesis accounts for the present results as well as for the earlier observation that dipyridamole which prevents the uptake of adenosine potentiates its inhibitory effect on the aggregation of human platelets.

Pathologic evidence indicates that thrombosis in coronary arteries is most frequently initiated by fissures in atheromatous plaques and that the associated hemorrhage induces platelet aggregation. Less frequently, thrombosis may be initiated by arterial spasm or by pathologic abnormalities affecting the platelets or the mechanisms of plasma coagulation. For the rational development of antithrombotic drugs on the basis of aggregation inhibitors, the cause (or causes) of plaque fissure and of the ensuing platelet aggregation need therefore to be elucidated. Our current research is based on the working hypothesis that fissuring occurs when plaques have acquired a particular composition that can be disrupted by the cumulative effect of continuously varying hemodynamic forces (reminiscent of fatigue failure in artificial materials), and that fissure-associated hemorrhage, like hemorrhage anywhere else, initiates platelet aggregation via a concurrence of hemodynamic and biochemical mechanisms. Detailed studies are currently being directed toward establishing the sequence of events that determine the contributions of adenosine diphosphate, thromboxane A2, and other endogenous agents in promoting hemostatic platelet aggregation in real life and, by implication, arterial thrombosis. Important recent evidence has demonstrated repeated thrombosis in unstable angina patients. In such patients, aspirin diminishes by about half the incidence of myocardial infarction and death. Presumably, it prevents the formation of platelet thrombi, which would tend to be produced in the turbulent blood flowing through arterial segments severely narrowed by hemorrhage plaques or in spasm. Several other platelet-active drugs are also under investigation.

A technique of, and the results obtained with, the intraoperative and postoperative intrapericardial instillation of microfibrillar collagen and fresh frozen plasma and platelet mixture are presented. Intraoperatively, the mixture was instilled into the exposed mediastinum in the course of open-heart operations in 31 patients in whom diffuse oozing of blood persisted following the termination of cardiopulmonary bypass and the neutralization of circulating heparin. Immediate clot formation and decrease of blood loss were observed in all instances. This method was also found effective in 9 of 20 patients who had continuing blood loss during the hours following open-heart surgery and in whom the mixture was instilled through the mediastinal drainage tubes.

1. Human erythrocytes incubated at 37° C in Krebs bicarbonate solution at pH 7·5 took up 5-hydroxytryptamine, histamine and (-)-noradrenaline at slow rates which were proportional to the concentration of the amines in the medium at least up to 16 μg/ml., in accordance with a simple diffusion process. 2. The initial velocity of uptake was greatest for 5-hydroxytryptamine, about 4 times less for histamine and about 20 times less for (-)-noradrenaline; (+)-noradrenaline was not taken up. 3. Comparison of uptake measurements by bio-assay and by radioactivity indicated that, after 1 hr, about 10% of each amine taken up had been inactivated. 4. At 2° C the uptake of all three amines was greatly diminished. The presence of the antagonists cocaine, chlorpromazine, lysergic acid diethylamide, mepyramine and pronethalol (at 10−6 or 10−5 g/ml.) did not affect uptake of the amines. 5. In the steady state the ratios of concentration of the amines between cells and medium were greater than could be accounted for by a Donnan distribution. 6. Cells which had taken up amines lost most but not all of them into amine-free medium, at rates which were slower than the corresponding rates of uptake. It was concluded that the amines are partially bound in the cells, noradrenaline most and histamine least. 7. The slow diffusion of amines into, and the small amounts retained by, erythrocytes indicate that they do not contribute significantly to the rapid termination of the pharmacological actions of these amines in vivo.

1. Measurements were made of the uptake, metabolism and release of [3H]-adrenaline by human platelets in citrated plasma or in an artificial medium. 2. Radioactive adrenaline was not taken up at 0-2° C. At 37° C there was a slow uptake which continued for at least 5 hours. 3. About half of the radioactivity in the platelets was intact adrenaline. The other half was an acidic metabolite from which adrenaline was released by acid hydrolysis. 4. The immediate uptake of adrenaline was proportional to its concentration in the plasma up to at least 1 × 10-5M. Uptake measured after 1 h also increased linearly with concentration up to about 1 × 10-4M but less with higher concentrations. The highest concentration ratio was about 12. 5. The concentration of metabolite in the platelets increased with the concentration of added adrenaline only up to about 2 × 10-4M. 6. The immediate uptake of adrenaline was partially inhibited by phentolamine and dihydroergotamine. Measurement of uptake both immediately and after 1 h showed that the inhibition produced was not increased beyond about 50% by these drugs or by (±)-propranolol, chlorpromazine or amitriptyline up to 1 × 10-4M. 7. Formation of the metabolite was inhibited by pyrogallol, 8-hydroxyquinoline, or tropolone. This inhibition was associated with a corresponding increase in the adrenaline accumulated intact. Formation of the metabolite was not inhibited by monoamine oxidase inhibitors. 8. Reserpine caused a small decrease in the uptake of adrenaline radioactivity in 1 h and a great increase in the proportion recovered as metabolite. 9. Thrombin caused the release from platelets of intact adrenaline but not of the metabolite. 10. Platelets of albino patients with spontaneous haemorrhages accumulated adrenaline radioactivity at the normal rate but this radioactivity was wholly accounted for by metabolite and not released by thrombin. 11. After taking up adrenaline, platelets resuspended in artificial medium at 37° C slowly released both adrenaline and its metabolite. At the same time, the intracellular adrenaline was slowly metabolized. 12. The Result suggest that human platelets take up adrenaline by two processes, one of which is inhibited by both α- and β-adrenoceptor blocking agents ad well as by phenothiazines; and that in the platelets adrenaline is partly stored in organelles from which, like 5-hydroxytryptamine, it can be specifically released.

1. The specificity of platelet receptor sites for 5-HT uptake and for the rapid morphological change and aggregation was investigated with 5-hydroxy-tryptamine (5-HT) and seventeen analogues as well as with some antagonists of 5-HT.2. The analogues, with the exception of 5-hydroxy-N'N'-dibutyltryptamine, caused the rapid morphological change in platelets. In concentrations below those needed to produce the agonistic action (viz. 0.05-2.0 muM), these analogues themselves inhibited competitively the shape change caused by 5-HT.3. The velocity of change in shape caused by 5-HT was reduced in low Na media.4. Ten analogues produced platelet aggregation; three of these, viz. 5-methoxy-alpha-methyltryptamine, 5-hydroxy-alpha-methyltryptamine and 5-hydroxy-N'N'-diisopropyltryptamine), were approximately equipotent with 5-HT. Six analogues did not induce platelet aggregation.5. All the analogues which prevented the initial change in shape of platelets caused by 5-HT also inhibited its aggregating effect, apparently competitively with low K(i) values (0.02-1.6 muM).6. As with the inhibition of shape change, the inhibition of aggregation shows relatively low structural specificity of the receptor site.7. Methysergide was a potent inhibitor of shape change and aggregation (K(i) approximately 0.03 muM); imipramine was much less inhibitory (K(i) approximately 5-10 muM).8. Only one analogue (5-hydroxy-alpha-methyltryptamine) was taken up like 5-HT by platelets. All the other analogues inhibited the uptake of 5-HT by platelets (K(i)=0.2-2.7 muM).9. Methysergide was a weak inhibitor of 5-HT uptake (K(i) approximately 125 muM) whereas imipramine was very effective (K(i) approximately 0.3 muM).10. Our results show that the initial change in shape of platelets is required for and precedes aggregation. The structural specificity of the platelet receptor concerned with shape change and aggregation caused by 5-HT appears low whereas the uptake mechanism is a highly specific one. The uptake probably proceeds through more than one step, the relationship between the steps is not yet clear.

Human or rabbit platelets were activated by ADP or 5-hydroxytrypatamine and rapidly fixed with glutaraldehyde. The shape change associated with activation gave rise to an increase in sialic acids removable by neuraminidase. This increase, like the shape change, was prevented by adenosine or methysergide added before ADP or 5-hydroxytryptamine respectively. The results indicate the exposure of additional glycoprotein(s) on the platelet surface.

We discuss our study on the effects of discontinuation of the usage of bone wax as a hemostatic agent in sternotomy incisions. In 1976, we abandoned use of bone wax because it was suspected of causing several cases of Mycobacterium fortuitum sternal osteomyelitis. In a retrospective study involving 400 patients, we found that this step did not increase postoperative bleeding as measured by drainage from the chest tube and by the need to return patients to the operating room because of bleeding. It also was observed that there was an appreciable simultaneous drop in pulmonary complications. To test our theory that the wax pressed into the bone marrow can embolize to the lung, radioactively tagged bone wax was pressed into the cut sternum in animal models, and a search was made for radioactive deposits in the peripheral lung tissue. Shortly after the application of the wax, there was evidence of large radioactive deposits in the lungs. It is probable that this embolization occurs also under clinical conditions and may play a role in pulmonary complications following open-heart operations.

ADP released from erythrocytes causes adhesion and aggregation of platelets1. This can presumably happen in vivo when circulating red cells undergo haemolysis in pathological conditions2,3, even though ADP is hydrolysed by enzymes present in blood4–7. More important pathologically, as a factor in thrombogenesis, is the possibility that ADP is also released from red cells which are structurally and metabolically normal. ADP might leak from circulating cells undergoing reversible deformations8 which are much increased by abnormal conditions of flow, for example in vessels narrowed by atherosclerosis.

The formation of platelet aggregates in plasma affects its optical density. The relationship between the optical density and the numbers and sizes of aggregates has been explored.

Comparative Effectiveness of Adenosine Analogues as Inhibitors of Blood-Platelet Aggregation and as Vasodilators in Man

Twelve apparently healthy male volunteers were maintained for 6 weeks on a fish diet, the haemostatic effects of which were determined as standardized skin bleeding times; thromboxane A2 formation in vivo in the bleeding time blood and in vitro in clotting venous blood; platelet aggregability; and platelet phospholipids. Within 1 week the fish diet changed the fatty acid composition of platelet membrane phospholipids, with increases in omega-3 and decreases in omega-6 fatty acids; described platelet aggregability by ADP and collagen; but the bleeding time was unchanged. Not until 6 weeks on the diet did the bleeding time increase (by 37%: P less than 0.01) and it was still increased 3 weeks after the end of the diet, when the fatty acid composition of the platelets had returned to normal. The effect of the diet on decreased aggregability of platelets also failed to follow the changes in their fatty acid composition and in the bleeding time. Aggregation by ADP and by the highest dose of collagen was decreased throughout the diet and for several weeks thereafter, i.e. even when the fatty acid composition had reverted to the pre-dietary pattern and long after the normalization of the bleeding time. The diet caused no change in the in vivo appearance of thromboxane A2 in the bleeding time blood, contrasting with its effect in decreasing the in vitro formation of thromboxane A2 in clotting venous blood. These observations suggest that such fish diets do not delay haemostasis by diminishing the formation of thromboxane A2 locally nor directly by decreasing the aggregability of platelets or directly by the induced changes in their omega-3 fatty acids.

The 'Simplate' technique for measuring skin bleeding time was adapted to quantify thromboxane A2 in the emerging blood as the stable degradation product thromboxane B2 in twelve Swedish and ten English volunteers. During the bleeding time thromboxane B2 concentrations increased, but as the rate of blood loss fell the rate of production of thromboxane A2 was constant. The English subjects had shorter bleeding times and produced more thromboxane A2 than the Swedish subjects. When the Swedish subjects were grouped according to bleeding times those with the shortest had more thromboxane A2 than those with longer bleeding times. Clotting venous blood in vitro produced much more thromboxane A2 than bleeding-time blood and there was no correlation with bleeding time. Determination of the capacity of clotting blood to form thromboxane A2 is therefore irrelevant to in-vivo haemostasis. Acetylsalicylic acid greatly diminished the appearance of thromboxane A2 in the bleeding time and prevented the increase of thromboxane A2 concentration with time.

Since December 1984 to July 1987, 18 patients with NEC were studied. Vásquez-Estévez's protocol, was applied in order to define the surgical indication, taking in to account ten different clinical and laboratory parameters, each one scored 0 to 3 points. Only patients with score 15 or more should be operated and this was the case in 30% of our NEC. In all surgically treated patients intestinal necrosis was a constant finding, two of them had also bowel perforations. No patients died in the conservative management group (score under 15). Lethality among operated subjects was 16%.

We practised 17 cases of oocyte retrieval between January and June 1988 in our programme of IVF using a vaginal sound and local analgesia in order to retrieve oocytes using a transvaginal ultrasonic guide. The method is described. The recent literature has been reviewed and our results are given: 7.9 oocytes retrieved on an average from each patient, 4.4 oocytes fertilized per patient and 18% of clinical pregnancies. In view of the results that have been obtained and the lessening of risk by avoiding general anaesthesia and the lower cost for each case, as well as the fact that the patients tolerate the method very well, we are now recommending its application routinely and reserve laparoscopy for rare cases.

Summary. Human red cells were incubated at 37°C in physiological saline at pH 7.4 with 8-3H-adenine to label the adenine nucleotides. After washing, the cells were incubated in similar salines which were buffered to different values of pH. The loss of radioactivity from cells into medium was very slow below pH 7.4 and accelerated greatly with increasing pH up to about 7.8. At that pH the loss increased for at least 3 h when it represented more than 30% of the initial radioactivity of the cells. The loss could not be accounted for by haemolysis which remained minimal. The extracellular radioactivity was present mostly in hypoxanthine and some inosine. The loss of purine did not depend on oxygenation of the cells. These results, together with the high-affinity uptake system for adenosine, could provide a partial explanation of the turnover of adenine nucleotide purines of red cells which is many times faster than that of the cells themselves.