bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Disulfide Bond In Antithrombin Required For Interaction With Heparin
Authors: Finlay T H , Ferguson W S 
Published: July 2018

Mild reduction of antithrombin (AT) with dithiothreitol followed by alkylation with [14C]-iodoacetamide results in the incorporation of 1.7 mol of acetamide/mol of protein with cysteine the only amino acid modified. Tryptic digestion of the reduced and alkylated AT results in the formation of only two labeled peptides indicating that mild reduction cleaves only one of the 3 disulfide bridges.The 2nd order rate constant for the reaction of thrombin with both native and mildly reduced AT is 5.9-9.6 × 105 M-1 min-1. Addition of heparin accelerates the reaction between native AT and thrombin (k >107 M-1 min-1) but has no effect on the reaction between reduced AT and thrombin (k = 8.3-12.2 ×105 M-1 min-1). Alkylation of reduced AT destroys all remaining thrombin-neutralizing activity.Native AT elutes from a heparin-Sepharose column with 1.0 M NaCl whereas reduced AT elutes at 0.5 M NaCl. Reduced and alkylated AT is not retained by the immobilized heparin even in 0.1 M NaCl. The intrinsic tryptophan fluorescence enhancement observed following binding of heparin to native AT is not seen with reduced AT. These data indicate that reduction of a single disulfide bond in AT lowers the affinity of the protein for heparin and eliminates the heparin-mediated acceleration of thrombin neutralization.Mildly reduced and alkylated AT was digested with CNBr and the peptides were fractionated by HPLC. Preliminary analysis of the CNBr peptides suggests that the crucial disulfide bond is between CYS239 and CYS422 (using the numbering in Magnusson’s sequence data).Supported by grants from the USPHS. THF is a Research Career Development Awardee of the NIH (HL-00277).
A Study On Fibrinolysis Induced By Defibrase Infusion
Authors: Kato M , Fujimaki M , Fukutake K 
Published: July 2018

When Defibrase as a defibrination agent was infused intravenously, it was followed by fibrinolytic phenomenon with release of FDP in high concentration. In this paper it will be reported which in actual inducer among fibrino- peptide A, des-A-fibrinmonomer and Defibrase itself in this occasion.The activity of plasminogen tissue activator, plasminogen and α2 plasmin inhibitor were measured by chromogenic method using S-2322, S-2251 and S-2251 respectively and fibrinogen concentration was assayed by tyrosine method. FDP, plasminogen and α2 plasmin inhibitor were also measured by immunological methods.After the infusion of Defibrase into four normal adults fibrionlytic activities were detected on the plasminogen-free fibrin plate and followed by decrease of plasminogen and α2 plasmin inhibitor and increase of FDP with plasminogen activator. On the other hand, Defibrase did not show any activities of plasminogen activator and plasmin. Therefore, it might be imagined that one of three substances including fibrinopeptide A, des-A-fibrinmonomer and Defibrase might be actual inducer of plasminogen activator from the vessel wall. When fibrinopeptide A separated from dog plasma was infused intravenously to dogs, no effect on fibrinolytic phenomenon, was indicated and, when the test-samples from the reflux experiments with Defibrase by the ues of dog’s leg were applied on standard fibrin plate, weak fibrinolytic reaction were detected in a few cases. However, when des-A-fibrinmonomer was tested, increase of FDP, decrease of fibrinogen and α2 plasmin inhibitor, showing the mode of dose-response, were detected in all cases of dog.According to the results obtained above, it might be suggested that the actual inducer of fibrinolytic related with Defibrase infusion might be des-A-fibrinmonomer.
Plasminogen Activators In Human Malignant Melanoma
Authors: Markus G , Madeja J , Evers J L , Hobika G H , Camiolo S M , Ambrus J L 
Published: July 2018

The plasminogen activator (PA) content of metastatic malignant melanoma was determined in Triton X-100 extracts of 11 surgical specimens and adjacent normal tissue, using azocaseinolysis with added plasminogen. The mean PA content of the tumors was 8.4 ± 10 (SD) CTA u/g tissue (6 times that of the surrounding tissue), lower than found earlier in lung, colon and breast tumors. Inhibition by goat IgG against purified human urokinase showed that the predominant activator was of the UK type, as was the case with the tumors examined earlier, except those of the prostate. This is in contrast with recent reports which showed that human melanoma-derived cells secrete into the culture fluid almost exclusively the “vascular type” PA (Wilson et al., Cancer Res. 40, 933, 1980; Roblin and Young, Ibid. 40, 2706, 1980). While this type of PA was present in all extracts here examined, in 3 of them only trace amounts (<1%) could be found. The vascular type PA, when present, could be inhibited by rabbit IgG against human melanoma cell culture-derived vascular PA (kind gift of Dr. E. Dowdle). SDS-gel electrophoresis in conjunction with fibrin-agar overlay zymography showed multiple activator bands ranging in mol. wt. from 100,000 to 30,000. All but the 70,000 mol. wt. vascular type PA band were inhibited by inclusion of anti-UK IgG into the fibrin-agar mixture.The discrepancy between the tumor extracts and the culture media may be due to a selective advantage of vascular type PA-secreting cells in culture, or to the turning off of the UK gene in the culture environment. It is also possible that production of vascular type PA is suppressed iii vivo.
New Synthetic Peptide Substrates Of -Val-pNA Type For An Insoluble Fibrinolytic Serine Enzyme Extracted From Human Spleen
Authors: Okamoto U , Nagamatsu Y , Horie N , Okada Y , Tsuda Y 
Published: July 2018

An insoluble spleen fibrinolytic serine proteinase(SFP) extracted from the human spleen with 2 M NaClO4 and partially purified by the authors was characterized with its enzymatic capability to split fibrin and fibrinogen at neutral pH. Its substrate specificity was much limited; any synthetic peptide substrates for factors of coagulation and fibrinolysis were not hydrolyzed. The present studies were therefore made to search for SFP peptide substrate which mimicked possible natural substrates, using the partially purified enzyme preparation showing single protein band possessing fibrinolytic activity on polyacrylamide gel disk electrophoresis.Despite of fact that various chromogenic peptide substrates having principal structure of -Arg-pNA and -Lys-pNA and various esters of Lys, Arg, Try, Phe and Tyr were not decomposed by SFP, a clue was however found that Suc-Ala-Ala-Ala-pNA (substrate for elastase) was decomposed by SFP but very slowly. Then, trial to mimic a part of insulin B chain (a substrate for elastase) led us to reach Suc-Tyr-Leu-Val-pNA which was well amidolyzed by SFP with Km of 0.17 mM and Kcat/Km(M) of 22,600. Further, Suc-Ala- Tyr-Leu-Val-pNA was found more appropriate substrate than the former. It was noteworthy that ratio of SFP susceptibility of Suc-Ala-Pro-Val-pNA, Suc-Ala-Pro-Ala-pNA and Suc-Ala-Pro-Leu-pNA was approximately 9:1:0.Results obtained sofar concluded that synthetic peptide substrates appropriate for SFP are dominantly characterized by the C-terminal-Val; other part amino acid sequences is also significant however.
Activation Of Human Factor VII By Activated Factors IX And X In Purified Systems
Authors: Masys D R , Bajaj S P , Rapaport S I 
Published: July 2018

Factor VII activity, as measured in a one-stage clotting assay, increases when whole blood is clotted in glass. Prior studies in this laboratory using factor-deficient plasmas suggested that this factor VII activation was due to activated factor IX (IXa). We therefore studied activation of VII by IXa and by activated factor X (Xa) in purified systems. Human factors II, VII, IX, and X were each purified to homogeneity as judged by SDS-polyacrylamide gel electrophoresis. Reaction mixtures of VII, IXa or Xa, and other cofactors and enzymes were made, and subsampled for VII activity. The activation state of VII was judged by comparison of one-stage clotting assay to a coupled amidolytic assay using a synthetic substrate. In the presence of phospholipid (PL) and calcium (Ca), both IXa and Xa activated VII 25 fold; however, Xa was roughly 800 times more efficient than IXa. In the absence of PL, Xa was roughly 20 times more efficient than IXa, in Ca-containing solutions. Only slight activation of VII by either enzyme occurred in the absence of Ca. The addition of prothrombin (II) markedly slowed activation of VII by both Xa and IXa; however, this effect did not occur if fully-decarboxylated II was used. The addition of anti thrombin III and thrombin-modified factor VIII at physiologic concentrations did not change rates of VII activation by IXa or Xa.These results confirm the ability of IXa and Xa to activate factor VII at physiologic concentrations in purified systems. The higher relative efficiency of Xa over IXa under all conditions studied contrasts strikingly with observations in whole plasma systems where the VII activation measurable after clotting is greater in X-deficient than in IX-deficient plasma. The activation of VII by Xa and IXa may serve as an amplification loop in the generation of clotting by either “intrinsic” or “extrinsic” cascades.
Antiplatelet Agents In Diabetic Lower Extremity Vascular Disease. A Va Cooperative Study
Authors: Colwell J 
Published: July 2018

Peripheral vascular disease is a devastating complication of diabetes mellitus. Post-operative vascular death rates are 10% or more following amputation for gangrene in diabetic patients, and three year mortality approaches 30%. An additional 20-30% of patients require subsequent amputation for diabetic gangrene. In view of these statistics, and because of the postulated role of the blood platelet in diabetic vascular disease, a collaborative study on antiplatelet agents was begun in 10 VA medical centers in 1977. In a double-blinded study, aspirin (325 mg tid) plus dipyridamole (75 mg tid) or placebos are given to adult diabetic males who had suffered a recent amputation for diabetic lower extremity vascular disease. End points are major vascular events after at least 3 years of follow-up.Recruitment of 231 subjects was completed by May, 1980. Baseline characteristics are well matched in both groups. Mean age is 59.6 years, duration of diabetes 12.7 years, smoking history 33.3 pack years, and treatment with insulin 68%. Previous myocardial infarction, congestive heart failure, and/or cerebrovascular disease is present in 17-18% and retinopathy is present in 40% at entry. About 43% have more than one vascular complication at baseline.Mean duration of follow-up is now 18 months. Major vascular events in separate patients are: 40 amputations, 31 deaths, 5 myocardial infarctions. Numerous less serious vascular events have also occurred, as have multiple events in single patients. Therapy has been stopped in 17% of patients, but rarely due to drug side effects. Compliance with therapy has been good.We conclude that this study will provide important new information on the natural history of lower extremity vascular disease in diabetes. It will also provide definitive data about the efficacy of antiplatelet agents in diabetic vascular disease.
Prevention Of Fatal Postoperative Pulmonary Embolism With Heparin-Dhe Or Dextran
Authors: Gruber U , Gorgerat J , Torhorst J 
Published: July 2018

Previous trials studying prevention of postoperative fatal pulmonary embolism (p.e.) concern elective general surgery. We have terminated a prospective, randomized multicentre trial comparing the effectiveness of s.c. heparindihydroergotamin (DHE) versus i.v. dextran in surgery of the musculoskeletal system including both emergency and elective cases. 14 Swiss hospitals participated. All adults operated upon were included and followed up for 6 weeks. 5000 IU heparin together with 0.5 mg DHE were given preoperatively and 12-hourly thereafter for at least one week. Patients in the dextran group received 6% dextran 70 in saline, a total of 3 × 500 ml. 8001 patients were admitted to the trial and 7413 fulfilled the criteria of the study. 5963 patients had been operated electively, 1450 as an emergency. 4368 patients had general anesthesia, 2259 were operated upon under epidural and 797 had other anesthetic procedures. 55 patients died during the first 6 postoperative weeks, the autopsy rate being 69%. 9 patients in the dextran group died of p.e. In 6 cases the pathologist decided the pulmonary embolus as being the only cause of death, in 3 the embolus was considered a contributory cause of death. In the heparin-DHE group 6 patients died of p.e. In 4 cases the embolus was the sole cause of death, in 2 it was contributory. These differences in the incidence of p.e. are statistically not significant. There were however significantly more bleeding and allergic complications in the dextran than in the heparin-DHE group.Conclusion: Both s.c. heparin-DHE and i.v. dextran are equally effective for the prevention of fatal pulmonary embolism. Heparin-DHE causes less bleeding complications.
Thrombin-Induced Responses In Human Platelets Differ In Their Requirement For Receptor Occupancy; Evidence For Tight Coupling Between Occupancy And Formation Of Phos- Phatidic Acid
Authors: Holmsen Holm , Dangelmaier Carol A , Holmsen Holm-Kjetil 
Published: July 2018

Thrombin causes secretion of dense granule constituents from platelets. The time course and extent of this response is unaltered if thrombin is rapidly neutralized after secretion has started, indicating that a sustained occupancy of the thrombin receptors is not required.Using a 50-fold excess of hirudin to neutralize thrombin, we show here that the shape change and aggregation responses also are independent on sustained receptor occupancy. In contrast, the secretion of acid hydrolases, liberation of [3H-]arachidonate (from phospholipids in platelets prelabeled with [3H]arachidonate) and formation of [32P]phosphatidic acid (in platelets prelabeled with 32P-orthophosphate) induced by thrombin were immedately stopped by hirudin. However, the incorporation of 32P into phosphatidylinositol and breakdown of [3H]phospha- tidyl inositol continued unaffected after thrombin removal. Thrombin-induced platelet responses can thus be subdivided into two classes according to their requirement for receptor occupancy: The first class (shape change, aggregation, dense granule secretion phosphatidylinositol breakdown and phosphatidylinositol synthesis) requires a short, initial occupancy, while the second class (acid hydrolase secretion, arachidonate liberation and phospha- tidic acid synthesis) requires an occupancy that is sustained as long as the response is executed. The increased turnover of phosphoinositides is a cyclic process, and our results show that only one step - the formation of phosphatidic acid from diacylglyceride and ATP - is tightly coupled to receptor occupancy by the agonist.
Reduced Invaginated Platelet Membrane In Idiopathic Thrombocytopenic Purpura
Authors: Nouvel C , El-Khoury M , Liverato C , Sié A , Robert A , Boneu B 
Published: July 2018

An indirect approach of the amount of invaginated platelet membrane has been provided by size measurements of platelets submitted to an hypotonic shock. We investigated this new morphological parameter in 15 normal subjects and in 11 ITP.Platelet volume was measured by the electronic method (Coulter counter, channelyser C 1000) on EDTA-PRP and on EDTA-PRP diluted with distilled water. One min. after dilution, paraformaldehyde (final concentration 0.08%) was added to prevent any return to the initial volume by an active process. Platelet lysis was quantified by LDH assay. Isoton was diluted in the same proportion. Arithmetic mean platelet volume (MPV) was calculated ; graphic analysis of the data allowed to determine the mode and the standard deviation (SD) of the log volume distribution.An inverse linear relationship between MPV and the log of osmolarity was observed. The maximum MPV increase without any detectable platelet lysis was obtained at 120 m Osm. For lower osmolarities a progressive lysis occured without further volume increase.Macrothrombocytosis and increased SD of the log volume distribution were found in undiluted ITP-PRP (12.2 fl and 0.31 respectively versus 9.2 fl and 0.27 in controls). After hypotonic treatment, the MPV increased in ITP and controls (respectively 17.2 fl and 15.9 fl) but the relative increase was lower in ITP than in controls (42% and 63% respectively) ; the SD of the log volume distribution was unmodi fied.These results suggest that large platelets occuring in ITP possess a reduced amount of invaginated plasma membrane.
The Duration Of Thrombotic Activity In Maturing Prosthetic Vascular Grafts In Man
Authors: Goldman M , Simpson D , Hawker R J , Norcott H , Drolc Z , McCollum C N 
Published: July 2018

Prosthetic graft occlusion is most frequent in the early postoperative period when the luminal surface is highly thrombogenic. It is generally believed that graft maturation ultimately results in a non-thrombogenic surface. The accumulation of 111-Indium labelled autologous platelets in Dacron aorto-femoral grafts has been measured one week following surgery and at intervals of 3 months to 1 year.Platelets from 9 patients were labelled with 111-In-oxine and reinjected. Isotope emissions over the graft and a reference site (aortic arch) were measured daily for 7 days and gamma camera images taken on alternate days. Graft thrombo-genicity was calculated as the daily rise in the ratio of emissions graft/reference.All grafts, regardless of age, accumulated platelets and were imaged by gamma camera. Mean thrombogenicity (± SEM) one week after surgery was 0.207 ± 0.037 compared with 0.08 ± 0.025 at follow-up (p <0.01). The platelet survival during the early study was reduced at 6.805 ± 0.61 days but had recovered to a near normal value of 8.57 ± 0.78 days (p < 0.001) at follow up.Although at a reduced rate, platelet accumulation still occurs on Dacron grafts 1 year following surgery despite the presence of normal platelet survival times.
Comparative Thrombogenicity And Healing Of Vascular Prostheses
Authors: Clagett G P , Graeber G M , Hufnagel H , Carter R , Gregory W , Robinowitz M , Langloss J M , Maddox Y , Ramwell P 
Published: July 2018

Thrombogenicity and healing characteristics were evaluated for 3 commonly used vascular prostheses: expanded poly-tetrafluorethylene (PTFE), knitted Dacron (KD), and velour knitted Dacron (VKD). Adult mongrel dogs were randomly allocated to receive thoracoabdominal aortic prostheses (6 animals for each type prosthesis) according to a randomized block design. Prostheses were 28-32 cm in length and 10 mm in diameter. Platelet survival (PS) studies were performed pre-op and every 6-8 weeks post-op for 1 year. PS data were subjected to analysis of variance and are shown {X ± SEM} below.PS, normal in each group pre-op, was shortened by all prostheses post-op; however, VKD reduced PS significantly more than KD and PTFE. At the end of 1 year, PS in animals with KD returned to normal but remained shortened in those with VKD and PTFE. Animals with VKD had the shortest PS throughout. Prostheses were then removed to assess coverage of luminal surface with prostacyclin-producing pseudointima. KD demonstrated the most complete coverage (90.6±5.5% of surface area) followed by VKD (56.3±9.6%) and PTFE (34.0±5.3%), p<0.02. These studies demonstrate that important differences exist among vascular prosthetic materials in initial reactivity with platelets following implantation and in subsequent rate and degree of healing.
Structure Abnormality Of Fibrinogen Metz And Its Relationship To The Clotting Defect
Authors: Henschen A , Southan C , Soria J , Soria C , Samama M 
Published: July 2018

Fibrinogen Metz was discovered in a young woman and was associated with a mild bleeding disorder and repeated abortions. Her parents are cousins and both have an abnormal fibrin formation but less intense than in the propositus.Fibrinogen Metz is therefore an homozygous case characterized by an unclottability of citrated sample, even in the presence of calcium and large quantities of thrombin. In addition, the clotting time is prolonged when the blood is collected in the absence of sodium citrate and the clot is extremely weak.By immunoenzymological assay, no release of fibrinopeptide A was noted after thrombin addition to purified fibrinogen. Furthermore, by disc electrophoresis in polyacrylamide gel in absence of SDS, an abnormal migration of the A α chain was observed.These results led us to analyze the sequence of the N terminal part of the A α chain by the Edman procedure. Arginine in position 16 was replaced by cysteine in fibrinogen Metz. This mutation localized on the site cleaved by thrombin explains the absence of fibrinopeptide A release, and the abnormal clottability of fibrinogen Metz.
Structure Of Fibrin
Authors: Freyssinet J M , Hudry-Clergeon G , Torbet J 
Published: July 2018

Highly oriented fibrin gels are formed when polymerization takes place into a strong magnetic field-up to 200,000 G. Neutron low-angle diffraction show that the protofibrils pack into a three-dimensional crystalline lattice.Kinetics parameters of the polymerization process can be estimated by recording the magnetically induced birefringence and the degree of orientation can be determined by measuring the final birefringence. To allow almost 100 % orientation, protein concentration has to be kept below 20 mg/ml (2 % w/v). Under these conditions X-ray low-angle diffraction patterns only show the familiar 225 Å-spaced meridional reflections. Neutron low-angle diffraction patterns are much more elaborated. In addition to the 223 Å (± 5 Å)-spaced meridional reflections, there are equatorial and other Bragg reflections which indicate that the fibrin monomers are arranged in a three-dimensional lattice. Meridional reflections due to a fibre axial periodicity are sharp, thus indicating that there is a high degree of crystallinity along the fibre axis. The equatorial diffraction arises from the regular, lateral arrangement of the fibrin monomers. In the pattern there is a discrete equatorial peak at 184 ± 5 Å and an extended band with a maximum at about 184/51/2 Å favouring a unit cell with a square base of side about 184 Å. Within each quadrant between the equator and the meridian, there are two maxima. The stronger of these is situated at the intersection of the first layer and row lines. The second is not so well resolved and has a meridional position between the second and third layer lines. So the true axial repeat might be 446 Å which agrees with the prevalent view that the approximately 450 Å-long molecules form a half-staggered arrangement along the fibre axis. Therefore this analysis, which is partly qualified, shows that the diffraction pattern can be indexed on a tetragonal unit cell (with a = 2 × 184 A and c =446 Å) that contains 8 molecules. Space groups P42 and P4222 satisfy our experimental conditions.