bluuebunny/crossref_metadata_2025 huggingface dataset:
Peroral endoscopic pyloromyotomy is efficacious and safe for refractory gastroparesis: prospective trial with assessment of pyloric function
Authors: Jacques Jérémie , Pagnon Lauriane , Hure Florent , Legros Romain , Crepin Sabrina , Fauchais Anne-Laure , Palat Sylvain , Ducrotté Philippe , Marin Benoit , Fontaine Sebastien , Boubaddi Nour , Clement Marie-Pierre , Sautereau Denis , Loustaud-Ratti Veronique , Gourcerol Guillaume , Monteil Jacques
Published: August 2018

Background Gastroparesis is a functional disorder with a variety of symptoms that is characterized by delayed gastric emptying in the absence of mechanical obstruction. A recent series of retrospective studies has demonstrated that peroral endoscopic pyloromyotomy (G-POEM) is a promising endoscopic procedure for treating patients with refractory gastroparesis. The aim of this prospective study was to evaluate the feasibility, safety, and efficacy of G-POEM.

Methods 20 patients with refractory gastroparesis (10 diabetic and 10 nondiabetic) were prospectively included in the trial. Patients were treated by G-POEM after evaluation of pyloric function using an endoscopic functional luminal imaging probe. Clinical responses were evaluated using the Gastroparesis Cardinal Symptom Index (GCSI), and quality of life was assessed using the Patient Assessment of Upper Gastrointestinal Disorders – Quality of Life scale and the Gastrointestinal Quality of Life Index scores. Gastric emptying was measured using 4-hour scintigraphy before G-POEM and at 3 months.

Results Feasibility of the procedure was 100 %. Compared with baseline values, G-POEM significantly improved symptoms (GCSI: 1.3 vs. 3.5; P < 0.001), quality of life, and gastric emptying (T½: 100 vs. 345 minutes, P < 0.001; %H2: 56.0 % vs. 81.5 %, P < 0.001; %H4: 15.0 % vs. 57.5 %, P = 0.003) at 3 months. The clinical success of G-POEM using the functional imaging probe inflated to 50 mL had specificity of 100 % and sensitivity of 72.2 % (P = 0.04; 95 % confidence interval 0.51 – 0.94; area under the curve 0.72) at a distensibility threshold of 9.2 mm2/mmHg.

Conclusion G-POEM was efficacious and safe for treating refractory gastroparesis, especially in patients with low pyloric distensibility.
Endoscopic gastric reduction with an endoluminal suturing device: a multicenter prospective trial with 1-year follow-up
Authors: Huberty Vincent , Machytka Evzen , Boškoski Ivo , Barea Marie , Costamagna Guido , Deviere Jacques
Published: August 2018

Background Obesity is the pandemic disease of this century. Surgery is the only effective treatment but cannot be offered to every patient. Endoscopic sutured gastroplasty is a minimally invasive technique that may potentially fill the gap between surgery and behavioral therapy. In this study, we prospectively investigated the efficacy and safety of a novel suturing device.

Methods After a pre-bariatric multidisciplinary work-up, class 1 and 2 obese patients were included. Using a simple triangulation platform, transmural sutures with serosa-to-serosa apposition were performed in the gastric cavity. Patients were followed according to the same routines as those performed for bariatric procedures.

Results Between November 2015 and December 2016, 51 patients were included across three European Centers. Mean body mass index at baseline was 35.1 kg/m2 (SD 3.0). Excess weight loss and total body weight loss at 1 year were 29 % (SD 28) and 7.4 % (SD 7), respectively, for the whole cohort (45 patients). At follow-up gastroscopy, 88 % of sutures were still in place (30 patients). No severe adverse events were observed.

Conclusions Endoscopic sutured gastroplasty using this novel device is safe and achieved weight loss results in line with criteria expected for these endoluminal techniques. Further prospective studies vs. placebo or nutritional support are needed.
Quality of Life, Glycemic Control, Safety and Tolerability Associated
with Liraglutide or Insulin Initiation in Patients with Type 2 Diabetes in
Germany: Results from the Prospective, Non-interventional LIBERTY
Study
Authors: Lundershausen Rainer , Müller Sabrina , Hashim Mahmoud , Kienhöfer Joachim , Kipper Stefan , Wilke Thomas
Published: August 2018

Purpose To assess quality of life, glycemic control, and
safety/tolerability associated with liraglutide versus insulin
initiation in patients with type 2 diabetes in Germany.

Methods Liraglutide/insulin-naïve adults with type 2
diabetes and inadequate glycemic control despite using oral antidiabetic
medication were assigned to liraglutide (≤1.8 mg daily;
n=878) or any insulin (n=382) according to the treating
physician’s decision and followed for 52 weeks. The primary
objective was to evaluate Audit of Diabetes-Dependent Quality of Life
(ADDQoL) scores.

Results At baseline, the liraglutide group was younger and had
shorter type 2 diabetes duration, lower glycated hemoglobin
(HbA1c), higher body mass index, and a lower prevalence of
certain diabetes-related complications than the insulin group (all
p<0.05). ADDQoL average weighted impact scores improved numerically
in both groups from baseline to 52 weeks (mean difference [95%
confidence interval], liraglutide vs. insulin: 0.159 [−0.023;0.340];
not significant). Changes in general wellbeing and five ADDQoL domains
significantly favored liraglutide (remaining 14 domains, not significant).
HbA1c reductions were greater with insulin than liraglutide
(−2.0% vs. −1.2%; p<0.01); however,
mean HbA1c after 52 weeks was 7.2% in both groups.
Compared with insulin, liraglutide significantly decreased body mass index
(−1.54 kg/m2 vs.
+0.27 kg/m2; p<0.001),
systolic blood pressure (−5.03 mmHg vs.
−1.03 mmHg; p<0.01) and non-severe hypoglycemia
(0.85% vs. 4.55% at 52 weeks; p<0.01). Adverse drug
reactions were reported for<3% of patients in both
groups.

Conclusions Liraglutide improved certain ADDQoL components and
reduced body mass index, systolic blood pressure, and non-severe
hypoglycemia versus insulin. Both treatments improved glycemic control.
Colon cleansing efficacy and safety with 1 L NER1006 versus 2 L polyethylene glycol + ascorbate: a randomized phase 3 trial
Authors: Bisschops Raf , Manning Jonathan , Clayton Lucy , Ng Kwet Shing Richard , Álvarez-González Marco ,   on behalf of the MORA Study Group
Published: August 2018

Background Polyethylene glycol (PEG)-based bowel preparations are effective cleansers but many require high-volume intake. This phase 3, randomized, blinded, multicenter, parallel-group, central reader-assessed study assessed the 1 L PEG NER1006 bowel preparation vs. standard 2 L PEG with ascorbate (2LPEG).

Methods Patients undergoing colonoscopy were randomized (1:1:1) to receive NER1006, as an evening/morning (N2D) or morning-only (N1D) regimen, or evening/morning 2LPEG. Cleansing was assessed using the Harefield Cleansing Scale (HCS) and the Boston Bowel Preparation Scale (BBPS). Primary end points were overall bowel cleansing success and high-quality cleansing in the right colon. Modified full analysis set (mFAS) and per protocol (PP) analyses were performed. Mean cleansing scores were analyzed post hoc.

Results Of 849 randomized patients, efficacy was analyzed in the following patient numbers (mFAS/PP): total n = 822/670; N2D n = 275/220; N1D n = 275/218; 2LPEG n = 272/232. mFAS established noninferiority. PP showed superiority for N2D on overall success (97.3 % vs. 92.2 %; P = 0.014), and for N2D and N1D on right colon high-quality cleansing (N2D 32.3 % vs. 15.9 %, P < 0.001; N1D 34.4 % vs. 15.9 %, P < 0.001) vs. 2LPEG. Using HCS, N2D and N1D attained superior segmental high-quality cleansing (P ≤ 0.003 per segment). N2D showed superior mean segmental HCS scores (P ≤ 0.007 per segment). Both N2D and N1D achieved superior mean overall (P < 0.001 and P = 0.006) and right colon BBPS scores (P < 0.001 and P = 0.013). N2D demonstrated superior right colon polyp detection (P = 0.024). Adherence, tolerability, and safety were comparable between treatments.

Conclusions NER1006 is the first low-volume preparation to demonstrate superior colon cleansing efficacy vs. standard 2LPEG with ascorbate, with comparable safety and tolerability.

European Clinical Trials Database (EudraCT)2014-002185-78TRIAL REGISTRATION: Multicenter, randomized, parallel group, phase 3 trial 2014-002185-78 at https://eudract.ema.europa.eu/
Colon cleansing efficacy and safety with 1 L NER1006 versus sodium picosulfate with magnesium citrate: a randomized phase 3 trial
Authors: Schreiber Stefan , Baumgart Daniel , Drenth Joost , Filip Rafał , Clayton Lucy , Hylands Kerry , Repici Alessandro , Hassan Cesare ,   on behalf of the DAYB Study Group
Published: August 2018

Background Polyethylene glycol (PEG) bowel preparations are widely used for precolonoscopy bowel cleansing. This phase 3 trial assessed the efficacy, safety, and tolerability of the novel 1 L PEG-based NER1006 vs. sodium picosulfate plus magnesium citrate (SP + MC) in day-before dosing.

Methods Patients requiring colonoscopy were randomized (1 : 1) to receive NER1006 or SP + MC. Cleansing was assessed on the Harefield Cleansing Scale (HCS) and Boston Bowel Preparation Scale (BBPS) using central readers. Two primary end points were assessed: overall colon cleansing success and high-quality cleansing of the right colon. Intention-to-treat (modified full analysis set [mFAS]) and per protocol (PP) analyses were performed.

Results Of 515 patients, efficacy was analyzed in 501 (NER1006, n = 250; SP + MC, n = 251) and 379 patients (NER1006, n = 172; SP + MC, n = 207) in the mFAS and PP analyses, respectively. Non-inferiority of NER1006 vs. SP + MC was established in the mFAS for both overall cleansing (62.0 % vs. 53.8 %; P = 0.04) and high-quality cleansing in the right colon (4.4 % vs. 1.2 %; P = 0.03). Superiority of NER1006 was demonstrated using HCS in the PP set for overall cleansing success (68.0 % vs. 57.5 %; P = 0.02) and right colon high-quality cleansing (5.2 % vs. 1.0 %; P = 0.02) and using BBPS in the mFAS for overall cleansing success (58.4 % vs. 45.8 %; P = 0.003) and right colon high-quality cleansing (4.0 % vs. 0.8 %; P = 0.02). Mean segmental scores for 4/5 segments were higher with NER1006 (P ≤ 0.04). Both treatments were well tolerated, with more mild adverse events for NER1006 (17.0 % vs. 10.0 %; P = 0.03).

Conclusions Colon cleansing with NER1006 vs. SP + MC was non-inferior (mFAS) and superior (PP), with acceptable safety.

European Clinical Trials Database (EudraCT)

2014-002186-30

TRIAL REGISTRATION: Multicenter, randomized, parallel group, phase 3 study 2014-002186-30 at https://eudract.ema.europa.eu/
Dietary Intervention with Oatmeal in Patients with uncontrolled Type 2 Diabetes Mellitus – A Crossover Study
Authors: Delgado Graciela , Kleber Marcus E. , Krämer Bernhard K , Morcos Michael , Humpert Per M. , Wiegand Kerstin , Mauldin Andy , Kusterer Klaus , Enghöfer Michael , März Winfried , Segiet Thomas , Lammert Alexander
Published: August 2018

Background In a pilot study, we evaluated the efficacy of two days of oatmeal on insulin resistance and glucose metabolism and found a marked decrease of insulin requirements. The most important shortcoming of that study was that the interventions were not isocaloric (diabetes adapted diet: 1500 kcal/d vs. oatmeal 1100 kcal/d). To address these drawbacks we designed the OatMeal And Insulin Resistance (OMA-IR) study.

Methods The study was a randomized, open label crossover dietary intervention study with consecutive inclusion of 15 patients with uncontrolled type 2 diabetes. The intervention comprised two days of oatmeal on days 3 and 4 of a 5 days hospital stay. During the control period, patients received a diabetes mellitus adapted diet only. The primary endpoint was the daily insulin requirement and glycemic control.

Results Upon oatmeal treatment, the required insulin dose could be significantly reduced on the third and fourth day as compared to the second day of inpatient stay (82.0±30.3 and 69.9±29.9IU versus 112±36.2IU;P<0.001). During control treatment, insulin requirement did not change. There were no significant differences in the changes of mean blood glucose or fasting glucose between both treatments. HbA1c was lower four weeks after the oatmeal intervention.

Conclusion In this crossover study, two days of oatmeal intervention allowed a highly significant reduction of required daily insulin doses while maintaining adequate metabolic control as compared to a diabetes adapted diet only. The beneficial effects of the intervention might last for several weeks as shown by the lower HbA1c four weeks after the intervention.
The Prognostic Importance of Impaired Fasting Glycemia in Chronic Coronary Heart Disease Patients
Authors: Slezák David , Mayer Otto , Bruthans Jan , Seidlerová Jitka , Rychecká Martina , Gelžinský Julius , Mateřánková Markéta , Karnosová Petra , Wohlfahrt Peter , Cífková Renata , Filipovský Jan
Published: August 2018

Objectives Impaired glucose metabolism represents one the most important cardiovascular risk factors, with steeply raising prevalence in overall population. We aimed to compare mortality risk of impaired fasting glycaemia (IFG) and overt diabetes mellitus (DM) in patients with coronary heart disease (CHD).

Study design prospective cohort study

Methods A total of 1685 patients, 6–24 months after myocardial infarction and/or coronary revascularization at baseline, were followed in a prospective cohort study. Overt DM was defined as fasting glucose ≥ 7 mmol/L and/or use of antidiabetic treatment, while IFG as fasting glucose 5.6–6.99 mmol/L, but no antidiabetic medication. The main outcomes were total and cardiovascular mortality during 5 years of follow-up.

Results During follow-up of 1826 days, 172 patients (10.2%) deceased, and of them 122 (7.2%) from a cardiovascular cause. Both exposures, overt DM (n=623, 37.0% of the whole sample) and IFG (n=436, 25.9%) were associated with an independent increase of 5-year total mortality, compared to normoglycemic subjects [fully adjusted hazard risk ratio (HRR) 1.63 (95%CI: 1.01–2.61)]; p=0.043 and 2.25 (95%CI: 1.45–3.50); p<0.0001, respectively]. In contrast, comparing both glucose disorders one with each other, no significant differences were found for total mortality [HRR 0.82 (0.53–1.28); p=0.33]. Taking 5-years cardiovascular mortality as outcome, similar pattern was observed [HRR 1.96 (95%CI: 1.06–3.63) and 3.84 (95%CI: 2.19–6.73) for overt DM and IFG, respectively, with HRR 0.63 (95%CI: 0.37–1.07) for comparison of both disorders].

Conclusions Impaired fasting glycaemia adversely increases mortality of CHD patients in the same extent as overt DM.
Advances in Sharing Multi-sourced Health Data on Decision Support Science 2016-2017
Authors: Shankar Prabhu , Anderson Nick
Published: August 2018

Introduction: Clinical decision support science is expanding to include integration from broader and more varied data sources, diverse platforms and delivery modalities, and is responding to emerging regulatory guidelines and increased interest from industry.

Objective: Evaluate key advances and challenges of accessing, sharing, and managing data from multiple sources for development and implementation of Clinical Decision Support (CDS) systems in 2016-2017.

Methods: Assessment of literature and scientific conference proceedings, current and pending policy development, and review of commercial applications nationally and internationally.

Results: CDS research is approaching multiple landmark points driven by commercialization interests, emerging regulatory policy, and increased public awareness. However, the availability of patient-related “Big Data” sources from genomics and mobile health, expanded privacy considerations, applications of service-based computational techniques and tools, the emergence of “app” ecosystems, and evolving patient-centric approaches reflect the distributed, complex, and uneven maturity of the CDS landscape. Nonetheless, the field of CDS is yet to mature. The lack of standards and CDS-specific policies from regulatory bodies that address the privacy and safety concerns of data and knowledge sharing to support CDS development may continue to slow down the broad CDS adoption within and across institutions.

Conclusion: Partnerships with Electronic Health Record and commercial CDS vendors, policy makers, standards development agencies, clinicians, and patients are needed to see CDS deployed in the evolving learning health system.
Findings from 2017 on Consumer Health Informatics and Education: Health Data Access and Sharing
Authors: Staccini Pascal , Lau Annie ,   Section Editors for the IMIA Yearbook Section on Consumer Health Informatics and Education
Published: August 2018

Objective: To summarize the state of the art during the year 2017 in consumer health informatics and education, with a special emphasis on sharing health data and accessing personal health information (PHI) from patients' and consumers' perspective.

Methods: We conducted a systematic search of articles published in PubMed using a predefined set of queries which identified 228 potential articles for review. The section editors then screened these articles according to topic relevance and selected 15 candidate best papers for full review and scoring by a panel of international experts. Based on the scores and the reviews, four papers received the highest score and were selected in a consensus meeting as the best papers on health data access and sharing from consumers' and patients' perspective.

Results: These four papers were categorised into the following topics: 1) data sharing for research and governance in privacy protection; 2) use of personal health information and individual privacy concerns; and 3) consumers' views and demographic characteristics regarding health data sharing and the use of digital health portals. Overall, it was surprising to see only a small number of papers reporting original research in this area.

Conclusions: Patients understand the need for sharing information to facilitate best care and to enrich biomedical knowledge. When confronted with the reality of accessing information systems for their own information, patients are concerned about usability as well as privacy. Overall, there is a need for more emphasis on: 1) considering privacy as a feature defined by design; 2) using specific consent approaches and data sharing mechanisms for recruiting clinical trial participants; 3) taking into account socio-demographic characteristics when promoting consumer access to personal health information; and 4) defining indicators of high-quality care to incorporate healthcare professionals' level of caution when accessing patients' medical information and fostering patient trust in data exchange. Ultimately, privacy mechanisms should be part of the design process and not only be implemented when security has been breached and violated.
PROTECTION BY α1-ANTITRYPSIN ALA-357 ARG-358 AGAINST ARTERIAL HYPOTENSION INDUCED BY FACTOR XII FRAGMENT
Authors: Schapira M , Waeber B , Brunner H R , Crystal R , Courtney M
Published: August 2018

The specificity of serine protease inhibitors belonging to the serpin superfamily depends on the nature of the reactive center amino acid residues. For example, Met→Arg mutation at the reactive center P1 residue (position 358) alters the specificity of α1-antitrypsin (AT) from the Met-specific enzyme neutrophil elastase to the Arg-specific proteases thrombin, plasma kallikrein (K) and activated Factor XII fragment (XIIf). To obtain an inhibitor species which would inhibit K and XIIf but not thrombin, we now have produced by site-directed mutagenesis of cloned AT cDNA an AT variant having Arg at P1 and Ala at P2. This modification at P2 was made because C1-inhibitor, the major inhibitor of K and XIIf, also has Ala at P2. In purified systems, AT Ala-357 Arg-358 inactivated thrombin, K and XIIf with 2nd-order rate constants of 1.1, 21.8 and 0.6 μM-1 min-1 whereas values of 8.5, 4.2 and 2.1 μM-1 min-1 were found with AT Arg-358. Thus, when compared to AT Arg-358, AT Ala-357 Arg-358 was 5.2 times more efficient for inhibiting K but 7.7 times less efficient for inhibiting thrombin. In vivo, AT Ala-357 Arg-358 (0.7 mg i.v.) did not modify the thrombin time of male Wistar rats while a 2-fold prolongation was seen with 0.7 mg AT Arg-358. However, AT Ala-357 Arg-358 (0.7 mg i.v.) partially prevented the kinin-mediated circulatory collapse induced by XIIf (0.1 μg i.v.) since rats (n=4) treated with this double mutant had a blood pressure fall of 14 ±3 (meaniSD) mmHg while control animals (n = 8) receiving saline or AT Val-358 (0.7 mg i.v.) had a decrease of 27 ± 3 mmHg (p<0.01 by t test). AT Ala-357 Arg-358 has therapeutic potential for disease states with activation of the plasma kinin-forming system such as angioedema attacks or septic shock.
PLATELET GLYCOPROTEIN IIb-IIIa-LIKE PROTEINS MEDIATE ENDOTHELIAL CELL ATTACHMENT TO ADHESIVE MATRIX PROTEINS AND ARE UP-REGULATED BY PHORBOL ESTERS
Authors: Charo I F , Fitzgerald L A , Meyer D , Bekeart L S , Phillips D R
Published: August 2018

Human endothelial cells (EC) express glycoproteins that are similar to the platelet glycoprotein IIb-IIIa complex (GP IIb-IIIa), the platelet receptor for adhesive proteins. Although GP IIb—IIIa is abundant in both platelets and EC, its only known function is to mediate platelet aggregation. The present study tests the hypotheses that EC attachment to adhesive proteins in the extracellular matrix is mediated by the GP IIb-IIIa-1ike proteins. Endothelial cells attached well to glass slides that were previously coated with adhesive proteins, but not albumin. To determine whether GP IIb-IIIa was involved, EC adherence was measured in the presence and absence of a GP IIb-IIIa monoclonal antibody (7E3) which inhibits fibrinogen (Fg) binding to platelets. The attachment of EC to Fg and von Willebrand factor (vWf), but not fibronectin (Fn) coated slides, was completely inhibited by 7E3. Attachment to vitronectin was partially inhibited. In contrast, EC attachment to Fn was specifically inhibited by a Fn-receptor antibody. Endothelial cell adherence to vWf was also inhibited by a monoclonal antibody (Mab9) against the GP IIb-IIIa binding domain of vWf, but not by antibodies agains.t other portions of vWf. We have further found that 7E3 disrupts monolayers of endothelial cells by detaching the cells from their extracellular matrix. EC incubated in phorbol myris-tate.acetate (PMA) increase in size and appear more tightly adherent to their extracellular matrix. To determine if PMA increases synthesis of cellular receptors for matrix proteins, we have used cDNA probes to measure the mRNA levels of the large subunit of the Fn-receptor (FnRα) and GP IIIa in EC. After a 4 hour incubation in the presence of PMA (10 nM), there was a 2-fold increase in the mRNA levels of both FnRα and GP IIIa, as well as increased cell spreading on the matrix. We conclude: i) the GP Ilb-IIIa complex in EC is a surface receptor for specific adhesive proteins, and is distinct from the FnR, and ii) both GP IIIa and FnRα synthesis are increased by PMA, which causes a concomittant change in cell morphology.
NORMAL SYNTHESIS AND EXPRESSION OF ENDOTHELIAL GP IIb/IIIa IN GLANZMANN'S THROMBASTENIA
Authors: Giltay J C , Leeksma O C , Breederveld C , van Mourik J A
Published: August 2018

In Glanzmann’s thrombastenia (GT), an autosomal recessive inherited hemorrhagic disease, the platelet membrane glycoprotein (GP) IIb/IIIa complex is absent or reduced. Recently we and others demonstrated that cultured human umbilical vein endothelial cells synthesize a membrane protein complex that is structurally closely related to GP IIb/IIIa. Endothelial cells of thrombasthénie patients could, therefore be deficient in GP IIb/IIIa. We had the opportunity to culture endothelial cells isolated from the umbilical cord of a newborn with GT and to examine the plasma membrane composition of these cells. Employing a variety of immunochemical techniques, including immunoprecipitation, immunofluorescence staining and crossed immunoelectrophoresis, we demonstrated that the endothelial cells of the patient were indistinguishable from normal endothelial cells in their ability to synthesize and express GP IIb/IIIa. Our results indicate that GT is not accompanied by and "endotheliopathy".

Zwa (P1A1) is an alloantigen, located on platelet GP IIIa, and, consequently, Zwa is absent on GT platelet. Data will he presented which show that not only normal endothelial IIIa carries the Zwa antigen, hut also GT endothelial cells normally express Zwa. This finding supports our view that GT endothelial GP IIb/IIIa is indistinguishable from normal endothelial GP IIb/IIIa. Moreover, this finding directly shows that the thrombastenic glycoprotein abnormality and the inheritance of Zwa antigen are controlled by different genes.
AT III BINDING SITE OF HEPARIN. DETERMINATION OF THE ROLE OF SULFATE GROUPS AT THE REDUCING-END DISACCHARIDE THROUGH NEW SYNTHETIC PENTASACCHARIDES
Authors: Petitou M , Lormeau J C , Choay J
Published: August 2018

A unique sequence is required in heparin for binding to antithrombin III (AT III) and eliciting anti-factor Xa activity. We have previously synthesized a pentasaccharide containing all the structural features of this sequence. It binds to AT III with the same affinity as high affinity heparin. The complex thus formed has a high anti-factor Xa activity. We have now synthesized a new series of α-methylated pentasaccharides which better mimic the naturally occurring configuration at C1 of the reducing-end glucosamine unit. The synthesis of the inactive pentasaccharide II allowed us to confirm in this series the essential role of the 3-sulfate group borne by glucosamine unit F. Wehave also particularly addressed the still unanswered question regarding the role of the 2-sulfate group borne by iduronic acid G and of the 6-sulfate group borne by glucosamine unit H. Pentasaccharides III and IV elicit the same anti-factor Xa activity which is substantially decreased as compared to I. A similar decrease has been recently reported for pentasaccharide V (T. Beetz & C.A.A. van Boeckel, Tetrahedron Lett., 1986, 27, 5889). We therefore conclude that the presence of both these sin fate groups is required for full expression of the antifactor Xa activity.
A FREQUENT TAQI RFLP AND A GENE LESION OR RARE TAQI RFLP IN THE VON WILLEBRAND FACTOR GENE
Authors: Marchetti G , Guerra S , Ballerini G , Patracchini P , Volinia S , Bernardi F
Published: August 2018

A cDNA for vonWi11ebrand factor (vWf) has been used to investigate lesions and RFLPs in the vWf gene.

In hybridizations with the 3'cDNA portion (a 2 Kb SacI fragment) a frequent polymorphism has been found with TaqI restriction enzyme. The alleles are 3.3 Kb and 2.6 Kb ; the frequencies of 0.51 and 0.49 respectively enable to investigate an appreciable portion of vW disease (vWd) families.

In a patient with type III vWd an abnormal TaqI pattern has been observed. A 4.5 Kb band is absent and an additional band of 2.3 Kb is present.

This pattern has been inherited from the consanguineous heterozygous parents and has been traced in several members of this large family. The presence of the abnormal gene pattern is related to total or partial vWf deficiency in the family and has not been found in several normal subjects. The BamHI and BglII restriction patterns are normal and suggest a small mutation originating a new TaqI site.

These findings are compatible with a gene lesion or a rare RFLP.

Work supported by Ricerca Sanitaria Finalizzata Regione Emilia Romagna.
THE PRACTICAL VALUE OF THE IN VITRO FILTER BLEEDING TIME
Authors: Salmon G P , O’Brien J R
Published: August 2018

The in vivo bleeding time has many disadvantages, some of which are obviated by an in vitro filter bleeding time. Normal native or heparinized blood is forced at 40mmHg through a filter made of glass microfibres with tortuous capillary sized pores which retain particles over lOμ. It is found that with high shear platelet aggregation occurs and when about 200,000 platelets (or 70/O of platelets are retained the filter becomes blocked. The test takes about two minutes to perform. Three routine measurements are easily made. (1) The % platelets retained between 10-20 secs; (2) the rate of blocking, e.g. drops 10-20/drops 0-5; (3) the blocking time. The within sample coefficient of variation (c of v) was 9% ± 4, n = 6. The c of v within stable patients studied over six months was 20% ± 11, n = 20. The correlation between platelet retention and the skin bleeding time is good overall when von Willebrand (VW) patients are included (r = ™0.73, n = 52) but absent when both tests are within the normal range. This test is sensitive to R:ag values from 0-160 (r = 0.62,n = 52) but poor within the VW and within normal groups. It is also abnormal in some patients with low platelet counts. Atherosclero-tics and diabetics have a normal test. Treatment of VW patients with DDAVP or with cryoprecipitate usually normalises this test. In one VW patient neither substance corrected this test and he bled. The test is thus useful in monitoring in vitro and in vivo the efficiency of various factor VIII concentrates to stop bleeding in VW disease. In vitro it is abnormal in the presence of many “membrane-active” drugs. Different drugs also have different effects on the retention of white cells; so it could be useful as a pharmacological screen. Thus it has uses as a quick and easy clinical screen for many forms of platelet-dependent haemostatic defects. It could also be used by the pharmaceutical industry and by producers of factor VIII concentrates.
REGULATION OF PLASMINOGEN ACTIVATOR INHIBITOR-1 mRNA IN HUMAN ENDOTHELIAL CELLS
Authors: van den Berg E A , Sprengers E , Jaye M , Burgess W , van Hinsbergh V W M
Published: August 2018

Cultured human endothelial cells (HEC) increase their production of plasminogen activator inhibitor (PAI-1) upon stimulation with endotoxin and IL-1, agents that are known to cause an increase in PAI-1 levels in vivo. In order to study the regulation of PAI-1 synthesis at the mRNA level, we isolated a cDNA clone for the human PAI-1 gene from an endothelial expression cDNA library in λ gt 11 by screening with a PAI-1 specific antibody. Three positive cross-hybridizing clones were isolated. The longest insert (1500 bp) was partially sequenced (1000 bp). The sequence was identical to the PAI-1 sequence recently reported by others. The identity of the cDNA clone was further confirmed by comparison with part of the amino acid sequence of PAI-1. For that purpose t-PA-PAI-1 complex was purified from HEC conditioned medium by immunoadsorption to anti-t-PA IgG, and a suitable peptide was sequenced after comparison of the HPLC elution profiles of CNBr digests of t-PA and t-PA-PAI-1 complex. The amino acid sequence (M)FRQFQADFT completely matches the sequence predicted from the cDNA sequence.

By hybridization of the cDNA probe to Northern blots of total cellular RNA from human umbilical vein and artery EC (HUVEC, HUAEC), two transcripts of 2.3 and 3 kb were found. Primary HUAEC, incubated for 18 hours in growth medium, produced considerable although variable levels of PAI-1 activity and contained PAI-1 mRNA levels comparable to those found in subcultured HUAEC. When subcultured HUEC were incubated for 6 h with endotoxin, IL-1 or TNF, a 2-fold increase in PAI-1 mRNA was found with each of these mediators. Stimulation of the cells in the presence of cycloheximide resulted in a further increase of the 3 kb PAI-1 transcript. The 3’ end of this transcript contains a 75 bp AT-rich sequence. Similar 3’ AT-rich sequences have been found in mRNA’s for a number of inflammatory mediators and cellular oncogenes, and in some cases it has been shown that removal of the sequence increased mRNA stability. The influence of cyclohex-imid on the larger PAI-1 transcript might be explained by inhibition of synthesis of a specific nuclease that controls the level of mRNA’s harbouring such an AT rich sequence.
THROMBOTIC AND INFLAMMATORY CHANGES IN ENDOTHELIAL CELLS INCUBATED WITH LEUKOCYTES
Authors: Schaub R G , Dunn C J , Tracey D E , Fleming W E , Burdick M D
Published: August 2018

Adhesion of leukocytes (WBC's) to vascular endothelial cells (EC's) is a component of inflammation, thrombosis and atherosclerosis. The purpose of this study was to assess the effect of WBC adhesion on the EC contribution to these pathologic events. Human WBC's were isolated and co-incubated with cultured human umbilical cord EC's. Supernatants and cell lysates (4 wells × 2) were obtained at 0.5,1,2, and 4 hours of incubation. EC's and WBC's (5 × 105) were-incubated alone or in combination. Supernatants and cell lysates were assayed for leukotriene B4 (LTB4), the thromboxane metabolite thromboxane B2 (TXB2) and the prostacyclin metabolite 6-keto prostaglandin FT alpha (6 keto) by RIA. Cell lysates were analyzed for cell associated procoagulant activity (PCA) by an APTT procedure, for plasminogen activator inhibitor (PAI) by an amidolytic assay and for IL-1 by a T-cell co-stimulator assay. Cellular and supernatant LTB4 was unmeasurable for both WBC's and WBC/EC cultures. WBC TXB2 showed a time dependent elevation which was unaffected by EC's. IL-1 activity was measurable at 2 hours and reach 14 U/ml in WBC's and 6 U/ml in EC/WBC cultures. Co-incubation of WBC's with EC's induced a 200% increase in both supernatant and cell associated 6 keto concentrations compared to EC's incubated alone. EC's and WBC's produced no PCA when incubated alone. PCA activity of the EC/WBC co-cultures was measurable at 2 hours and was 300-1500 U/ml after 4 hours. Coincubated EC's had a 50% decrease in cell PAI, suggesting an increased release of inhibitor from the cells. The prostacyclin and PAI release -along with the delayed expression of PCA activity are responses similar to those expected after EC exposure to cytokines. A source of these cytokines appears to be the WBC's which secreted measurable amounts of IL-1. WBC released IL-1 was sufficient to induce biochemical changes in EC's which can stimulate coagulation (PCA synthesis), inhibit fibrinolysis (PAI release), and enhance inflammation (prostacyclin synthesis). These results suggest that the release of WBC IL-1 can be sufficient to produce pro-thrombotic and inflammatory changes in EC's which are similar to those observed with the addition of exogenous IL-1 to EC cultures.
CARBOHYDRATE PREVENTS LOSS OF LARGE VON WILLEBRAND FACTOR MULTIMERS BY PROTECTING AGAINST AMINO TERMINAL PROTEOLYTIC CLEAVAGE
Authors: Federici A B , Berkowitz S D
Published: August 2018

We have previously shown that carbohydrate (CHO) protects von Willebrand factor (vWF) from proteolytic degradation. We have now shown that removal of CHO from the vWF subunit exposes additional cleavage sites in the amino terminal region and that cleavages in this region are associated with loss of large multimers. We examined and compared the extent of large multimer loss with sites of subunit cleavage of native and GHO-modified vWF after treatment with plasmin, chymotrypsin, and trypsin. Highly purified vWF was treated with neuraminidase and β-galactosidase in the presence of proteinase inhibitors to remove 90-95% of the sialic acid and 45-50% of the D-galactose without loss of large multimers or diminution of the ristocetin cofactor activity. The extent and approximate location of subunit cleavage was determined by immunoblotting and monoclonal antibody epitope mapping. Multimeric analysis revealed an increasingly greater loss of large multimers when native vWF was digested with plasmin, chymotrypsin, and trypsin, respectively. Large multimer loss was more extensive with each enzyme after CHO-modification of vWF. On subunit analysis, plasmin, chymotrypsin, and trypsin were shown to produce both amino and carboxy terminal fragments. The number, location, and relative quantities of carboxy terminal fragments produced by these enzymes were unchanged after CHO modification. However, digestion of the amino terminal region was considerably more extensive as judged by a marked decrease or absence of the larger fragments seen when native vWF was digested, and by the appearance of new smaller molecular weight species. Thus, enzymatic digestion of vWF after removal of carbohydrate produced new cleavages in the amino terminal region but did not alter the location or extent of carboxy terminal cleavages. Therefore, the greater loss of large multimers that occurs after CHO modification is likely to be the result of cleavages in the amino terminal region of the molecule. It appears that by protecting the vWF subunit against amino terminal cleavage, carbohydrate inhibits the loss of large multimers.
AUTOACTIVATION OF HUMAN PLASMA PREKALLIKREIN
Authors: Tans G , Rosing J , Berrettini M , Lammle B , Griffin J H
Published: August 2018

Incubation of purified human plasma prekallikrein with sulfatides or dextran sulfate resulted in spontaneous activation of prekallikrein as judged by the appearance of amidolytic activity towards the chromogenic substrate H-D-pro-phe-arg-p-nitroanilide (S 2302). The time course of generation of amidolytic activity was sigmoidal with an apparent lag phase followed by a rapid activation until finally a plateau was reached. Soybean trypsin inhibitor completely blocked prekallikrein activation whereas corn, limabean and ovomucoid trypsin inhibitor did not. The Ki of the reversible inhibitor, benzamidine, for autoactivation (240 uM) was identical to the Ki of benzamidine for kallikrein. Thus, spontaneous prekallikrein activation and kallikrein showed the same specificity for a number of serine protease inhibitors, indicating that prekallikrein is activated by its own enzymatically active form, kallikrein. Immunoblotting analysis showed that, concomitant with the appearance of amidolytic activity, prekallikrein was cleaved. However, prekallikrein was not quantitatively converted into two-chain kallikrein since other polypeptide products were visible on the gels. This accounts for the observation that in amidolytic assays not all prekallikrein present in the reaction mixture was measured as active kallikrein. Kinetic analysis showed that prekallikrein activation can be described by a second-order reaction mechanism in which prekallikrein is activated kallikrein. The apparent second order rate constant was 27000 M-ls-1 (pH 7.2, 50 uM sulfatides, ionic strength 1=0.06, at 37°C). Autocatalytic prekallikrein activation was strongly dependent on the ionic strength, since there was a considerable decrease in the rate of the reaction at high salt concentrations. Our data support a prekallikrein autoactivation mechanism in which surface-bound kallikrein activates surface-bound prekallikrein. The rate constant of autoactivation is considerably lower than the rate constants reported for Factor Xlla dependent prekallikrein formation. Autocatalytic prekallikrein activation may, however, contribute to kallikrein formation during the initiating phase of contact activation.
DETECTION BY MONOCLONAL ANTIBODIES OF CONFORMATIONAL CHANGE IN FIBRINOGEN ADSORBED ON ARTIFICIAL SURFACES
Authors: Shiba E , Lindon J N , Kushner L , Kudryk B , Salzman E W
Published: August 2018

Adsorption of fibrinogen (Fg) appears to be an important precusor of platelet reactivity with artificial surfaces. We have reported that, contrary to the prevailing view, platelet reactivity is less well correlated with the total concentration of adsorbed Fg on the surface than with the concentration of adsorbed Fg detectable by labelled anti-Fg polyclonal antibodies ("native" Fg), and therefore presumably recognizable by the platelet's Fg receptors. To clarify the nature of the conformational change in Fg implied by these findings, we measured Fg adsorbed on a series of polyalkyl methacrylates, using a solid phase RIA in methacrylate-coated microtiter wells with monoclonal antibodies (MoAb) directed against a variety of Fg epitopes. Total Fg adsorption (125I-Fg) was the same on polymethyl methacrylate (PMMA, single carbon side chain) as on polybutyl methacrylate (PBMA, 4 carbon side chain), but platelet reactivity in a bead column was greater on PBMA than on PMMA. Adsorbed "native" Fg increased with the length of the alkyl side chain when assessed by MoAb against Fg fragment D or E, whole Fg, or γ/γγ chain at the saturated concentration of adsorbed Fg (0.3 ug/cm2) and was significantly greater on PBMA than on PMMA. Adsorbed "native" Fg decreased with length of the alkyl side chain at a lower concentration of Fg (<0.08 ug/cm2) measured with these MoAb. Platelet retention in a polymer bead column was blocked by Fab fragments of these MoAb. These results indicate that Fg molecules adsorbed to some artificial surfaces assume a specfic orientation and conformation that may be important in subsequent interaction of the surface with platelets.
MOLECULAR CLONING OF HUMAN PLATELET GLYCOPROTEIN Ib
Authors: Lopez J A , Chung D W , Fujikawa K , Hagen F S , Papavannopoulou T , Roth G J
Published: August 2018

Glycoprotein Ib (GPIb) mediates von Willebrand factor-dependent platelet adhesion and participates in the resulting platelet activation process. In the present investigation, the primary structure of human platelet GPIb was studied. GPIb and its proteolytic fragment glycocalicin were purified to near homogeneity from human platelets by affinity chromatography using wheat germ agglutinin and anti-GPIb monoclonal antibody (D. Nugent, University of Washington) coupled to Sepharose. GPIba chain, β chain, and glycocalicin were isolated, reduced and carboxymethylated, and then fragmented by trypsin and S. aureus V8 protease. Peptides were isolated by HPLC and subjected to amino acid sequence analysis. Approximately 200 amino acid residues were identified. Affinity purified rabbit antibodies directed against the a chain, the ft chain, and glycocalicin were prepared and shown to be monospecific by Western blot analysis. Total RNA was prepared from human erythroleukemia cells grown in the presence of phorbol acetate. Poly(A)+ RNA was selected and used to prepare a cDNA library in λgt11. The library was screened with [125]I-labeled polyclonal antibody to glycocalicin. The clone with the largest cDNA insert was sequenced and shown to code for amino acid sequences corresponding to those determined by Edman degradation of glycocalicin. The predicted amino acid sequence contains at least six tandem repeats of 24 amino acids that are highly homologous with 13 repeats present in leucine rich α2 glycoprotein of human plasma. Another region in the protein contains a second repeat rich in threonine and serine, which shows some homology to a 9 amino acid repeat in the connecting region of human factor V. This region is probably the major site of attachment of clusters of O-linked carbohydrate in GPIbα. These results indicate that human platelet glycoprotein Ibα has a multi-domain structure composed of a number of repetitive sequences. Supported in part by grants from the American Heart Association, Robert Wood Johnson Foundation, Veterans Administration, and National Institutes of Health.
ON THE IDENTITY OF PLATELET FIBRINOGEN WITH PLASMA FIBRINOGEN
Authors: Kaudewitz H , Henschen A , Zimmermann R E
Published: August 2018

It is a well established fact that fibrinogen occurs not only in blood plasma but also in the α-granules of the platelets. Recently, it has been shown that fibrinogen is synthesised in the megakaryocytes as well as in the liver. Plasma fibrinogen is derived from the liver, but platelet fibrinogen either exclusively or partially from the megakaryocytes. Conclusive, proteinche-mical evidence for the identity of the fibrinogens from the two biosynthetic sources has previously not been produced. However, the two fibrinogen preparations have been shown to have the same overall peptide chain composition, to be thrombin-clottable and release fibrinopeptides of A- and B-type, and to react with antibodies against plasma fibrinogen. The two preparations differ in the way that platelet fibrinogen lacks the higher-molecular-mass γ-chain variant.

The aim of the present investigation was to conduct a detailed proteinchemical comparison between human plasma and platelet fibrinogen. For this purpose, fibrin(ogen)s from the two sources were mercaptolysed, alkylated and the three peptide chains isolated by reversed-phase high-performance liquid chromatography (HPLC). The peptide chains were then analysed directly for amino-terminal sequence and for carboxyterminal sequence by isolation of a terminal fragment. The HPLC-fingerprint patterns of the cyanogen bromide-cleaved chains were compared. The native fibrinogens were also treated with thrombin and the fibrinopeptide type distribution determined by reversed-phase HPLC. The carbohydrate side chain compositions were established by ion-exchange-chromatographic methods after acid hydrolysis. So far no previously unrecognized differences have been observed.
HEREDITARY THROMBOPHILIA CAUSED BY MISSENSE MUTATION IN PROTEIN C GENE
Authors: Hassan H J , Cianetti L , Mannucci P M , Vicente V , Cortese R , Peschle C
Published: August 2018

The structure of the gene for protein C was analyzed in 13 protein C deficient unrelated patients (11 heterozygous, 2 homozygous), who showed an equivalent reduction of this serine protease at both enzymatic and antigen level. No deletion(s) or rearrangement(s) was demonstrated by Southern blot after hybridization to a cDNA probe. One patient showed a variant restriction pattern after Bam HI digestion, characterized by an abnormal 9.6 kb band in addition to the 8.3 and 1.3 normal ones. Extensive family studies, including 7 heterozygotes with the same clinical phenotype, showed the same abnormal pattern in all and only these heterozygotes. Protein C gene from the propositus was cloned in EMBL3 lambda vector. A 411 bp PstI - SacI fragment from exon 9 encompassing the mutation in the Bam HI site was subcloned in M13mpl8. Its sequence showed a single transversion in the Bam HI palyndrome (GGATCC -> GCATCC) : this causes a substitution of the 402
thryptophan residue with a cystein. The 402 thryptophan residue is constantly conserved in a biochemical domain present in all eukaryotic serine proteases: substitution of the large thryptophan aromatic ring with the small cysteine hydrophilic side-chain conceivably leads to destabilization of the tertiary structure of protein C in these heterozygotes. Thus, the point mutation reported here is sufficient to explain the protein C deficiency in these subjects, and is apparently responsible for their clinical phenotype.