bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Effects Of Chymotrypsin And Of ADP On The Binding Site For Bovine Factor VIII On Human Platelets
Authors: Kirby Edward P , Mills David C B 
Published: July 2018

The aggregation of human platelets by bovine Factor VIII (Platelet Agglutinating Factor-PAF) is inhibited by exposure of the cells to ADP or Chymotrypsin. We have investigated the mechanism of these effects using washed platelets. The washing procedure was modified from the method of Mustard et al. (Brit. J. Haematol. 22:193, 1972), omitting heparin and using a protein-free Tyrode’s solution for the final resuspension. The washed platelets were stable and responded to ADP (0.1-1 μM) with a shape change and, if fibrinogen was added, with aggregation. Bovine Factor VIII was purified to >90% homogeneity and was labeled with 125I (approx. 1 atom/subunit) by the IodoGen procedure, with no loss of activity. Aggregation was measured in the aggregometer in the presence of 7 mM EDTA. Binding was measured after incubation of labeled Factor VIII with washed platelets in the presence of 7 mM EDTA for 5 min at 37° without stirring.Treatment of washed platelets with Chymotrypsin progressively destroyed their ability to bind Factor VIII and to be agglutinated by it. Responsiveness to Factor VIII disappeared before any alteration was detected in the ability of platelets to undergo ADP-induced shape change. Treatment of platelets with ADP, however, inhibited agglutination induced by Factor VIII without affecting the binding of Factor VIII to the platelets. Agglutination by wheat germ agglutinin or phytohemagglutinin was not inhibited by ADP treatment. We conclude that Chymotrypsin probably inhibits Factor VIII- induced agglutination by destroying the platelet binding site for Factor VIII, but that ADP must act at a point distal to Factor VIII binding. Agglutination of metabolically intact platelets by Factor VIII may not be a simple process, because ADP can specifically inhibit it without affecting Factor VIII binding or aggregation of the platelets by lectins.
Randomized Study Of Ticlopidine On Cerebral Vasospasm Following Ruptured Aneurysm
Authors: Mizukami M , Kikuchi H , Ono H , Taneda M 
Published: July 2018

Based on our hypothesis for the mechanisms of chronic cerebral vasospasm following subarachnoid hemorrhage (SAH), we have made a randomized study of Ticlopidine in preventing cerebral vasospasm in 71 patients with ruptured aneurysm.In a double blind cross-over set up-S. 200 mg T.I.D. vs. placebo - the induction of circulating platelet aggregates by venous occlusion was highly significantly inhibited.Materials and Methods: Ticlopidine is a potent inhibitor of platelet aggregation and adhesiveness. The inhibitory nature of this drug is distinct from that of Aspirin which not only is an inhibitor of platelet aggregation butalso suppresses PGI2 generation in the vascular wall. Fourty one patients were given Ticlopidine (400 mg/day) and 30 patients served as a control group. All patients underwent direct intracranial operation and 46 of them were operated upon 4 days after SAH. High density which indicates subarachnoid clot was observed on pre- and postoperative CT scans in the majority of both groups.Results: Mortality was 12% in Ticlopidine group and 17% in control group. As for morbidity, excellent results were obtained 57% in Ticlopidine group and 42% in control group in Grade 1-3. In order to know the effects of Ticlopidine on cerebral vasospasm and ischemic neurological deficit caused by vasospasm, 32 patients who did not show preoperative neurological deficit and underwent surgery within 4 days after SAH were analized in relation to the presence or absence of high density on postoperative CT. The development of postoperative vasospasm was almost same (71.4-76.5%) in two groups, but permanent ischemic neurological deficit was 28.6% in Ticlopidine group and 66.7% in control group when postoperative CT scan showed high density in subarachnoid space.This preliminary randomized study indicates that Ticlopidine may be effective on prevention of postoperative ischemic neurological deficits due to vasospasm.
Ultrastructural Study Of Experimental Venous Thrombosis
Authors: Thomas D P , Merton R E , Hiller K F , Hockley D 
Published: July 2018

Venous stasis is known to be a necessary but usually not a sufficient cause of venous thrombogenesis. The nature of the additional factor(s) required is uncertain, but the two most likely candidates are local generation of thrombin and vessel wall damage. Autopsy studies show that most venous thrombi develop in apparently normal vessels, although this evidence is based primarily on light microscopy. If thrombin generation is the additional factor, it is uncertain whether such thrombin acts solely on the blood or also damages the endothelium, leading to platelet deposition on exposed subendothelium. We studied the effect of locally injected thrombin on the jugular veins of rabbits, using transmission and scanning electronmicroscopy. One unit of thrombin was sufficient to clot blood contained in an isolated venous segment within a few minutes. The thrombus so formed was then embolised and autologous 11 indium-labelled platelets were injected. Blood flow was re-established for 30 minutes, following which the venous segment was fixed in situ and removed for study.We found no evidence of significant vessel wall damage, as judged by ultrastructural studies or the deposition of labelled platelets. At least 10 u. of injected thrombin were required before the radioactivity in the thrombosed segment exceeded that found in a control vein. Even then, the endothelial lining appeared intact by electronmicroscopy. Following administration of aspirin (10 mg/kg) there was a marked increase in radioactivity present in the thrombosed venous segment, suggesting that inhibition of vessel wall cyclo-oxygenase had led to increased platelet deposition.
We conclude that a fresh stasis thrombus resulting from the direct action of thrombin on platelets and fibrinogen does not damage the endothelium, which appears to be relatively resistant in vivo to even high local concentrations of thrombin.
Chromogenic Substrates For Granulocyte Elastase, Chymo-Trypsin And The Subcomponents Of Cl-Esterase
Authors: Simonsson R , Arielly S , Friberger P , Aurell L 
Published: July 2018

In order to improve and facilitate studies of various proteases and their possible interactions with the thromboembolic mechanisms, chromogenic substrates for granulocyte elastase, chymotrypsin, and Cls and Clr have been studied.Substrates of the type Suc-Ala-Ala-Ala-pNA or Ac-Ala-Pro- Ala-pNA (S-2483) are useful for pancreatic elastase but not particularly sensitive towards granulocyte elastase. By placing valine in the PI position, the specificity requirements of granulocyte elastase are better satisfied. Thus pGlu-Pro-Val-pNA (S-2484) was found to be a suitable substrate for this enzyme. In tris buffer pH 8.2 I 0.40 Km was 0.4 mmol/1 with S-2484 and the substrate was approximately 30 times more sensitive than Suc-Ala-Ala-Ala-pNA.The enzyme concentration of 4 nmol/1 gave a change in absorbance per minute of approximately 0.01.In our search for water soluble chromogenic substrates for chymotrypsin, tripeptide derivatives of p-nitroanilin were synthezised where the aliphatic amino acid residues in the P3 position present in most chymotrypsin substrates earlier described were substituted with arginine. In the PI position both tyrosine, phenylalanin and tryptophan were used. The two substrates Sue- and Suc(OMe)-Arg-Pro- Tyr-pNA (S-2558 and S-2586 respectively) were found to be the most potent ones. In tris buffer pH 7.8 I 0.40 and with 3 mmol/1 of Ca2+ Km was 0.05-0.1 mmol/1 and kcat 50-60 per sec. This type of substrates would allow chymotrypsin assays of biological samples without the presence of organic solvents.By studying purified preparations obtained from two different laboratories it was concluded that Cls and Clr have a similar specificity pattern towards pNA-derivatives of tripeptides. In all cases, the substrates split by these preparations have a C-terminal arginine residue. Some already known substrates seem to be sensitive enough to be utilized in biochemical studies of these enzymes.
FVIII:C Inhibitor Assays By Amidolytic Methods
Authors: Mackie I J , Seghatchian M J 
Published: July 2018

Numerous problems are associated with the present assays of FVIII inhibitors, which give discrepant results between and within laboratories. We have attempted to remove some of the problems by using a FVIII assay based on FXa generation using a chromogenic substrate for end point detection instead of measuring a clotting time.Nine plasmas from FVIII inhibitor patients were assayed several times by Bethesda and New Oxford methods, using both clotting and chromogenic FVIII assays. For the latter, the incubated samples were diluted in glyoxaline buffer and mixed with Diagen FVIII reagent and the synthetic substrate S2222. The reaction was left to proceed until suitable amounts of PNA were produced and then stopped with acetic acid. An azo dye technique was used to intensify the colour, which was measured at 540 nm, parallel line bioassays were then performed. The table summarises the inhibitor unitages (mean values).A good correlation was obtained between clotting and amidolytic methods, except that the latter gave overall, slightly lower results. A marked discrepancy existed between Bethesda and New Oxford methods whichever type of FVIII assay was used. The chromogenic method therefore seems to measure a similar entity to clotting methods, and has the advantages that it may be modified in terms of incubation time and substrate concentration to suit the demand, and most importantly it requires minimal technician time. Most of the steps can be automated.
Chromogenic Substrate Methods For The Determination Of FVIIIc, Endotoxin And Plasminogen Activator
Authors: Eriksson E , Rosén S , Knös M , Friberger P 
Published: July 2018

FVIII clotting activity in plasma and concentrates can be assayed with good reproducibility in the range 20-120% (C.V. approximately 3%) using purified FIXfl and FX in excess. By incubating the sample with above factors, Ca2+ and phospholipid, FX is activated in proportion to the amount of FVIIIc. Generated FXa is measured with the substrate S-2222. FVIIIc in the range below 20% can be determined by modifying the reaction conditions.Endotoxin in water solutions can be accurately determined down to 1 pg/ml by incubating the sample with an excess of Limulus lysate. The generated enzymatic activity which is linear in proportion to the amount of endotoxin, is then measured with the substrate S-2423 (Ac-Ile-Glu-Gly-Arg-pNA). Accurate endotoxin determinations in plasma and cerebrospinal fluid are also possible after destroying interfering inhibitors by heat treatment.Plasminogen activator activities can be measured via plasminogen and the plasmin substrate S-2251 or directly by substrate S-2288 (H-D-Ile-Pro-Arg-pNA). Interfering activities can be minimized by strategic selection of various protease inhibitors.In all the methods the assay conditions, reagents and procedure have been optimized.
Central Venous Catheters And Thrombotic Complications. A Comparison Of Three Different Catheter Materials
Authors: Lindblad B , Efsing H O , Mark J , Wolff T 
Published: July 2018

Different frequences of thrombosis due to central venous catheters (CVC) is reported. Only few studies have compared the thrombogenecity of different catheter materials. This study was undertaken to evaluate and compare the frequency of thrombotic complications with three catheter materials.MATERIAL: The patients were randomized into three groups receiving: teflon catheter, siliconized polyethylen. catheter and heparinized polyethylen catheter.METHODS: Percutanous insertion (subclavian or internal jugular vein) was made under strict aseptic conditions as well as the daily care of the CVC. The condition of the veins was assessed by either phlebography or post-mortem examination. Phlebography was usually made on the seventh day after insertion of the CVC by contrast injection in a cubital vein.RESULTS: Of 110 patients 99 were examined by phlebography and 20 by post-mortem.Repeat phlebography was made after 3 to 4 weeks use of CVC in 14 patients and showed no late thrombotic development. Two patients had pulmonary embolism, one with fatal outcome. No other source for embolism than the thrombosis around the CVC was found.CONCLUSION: The siliconized polyethylencatheter had a significantly higher frequency of thrombosis than teflon or heparinized polyethylen catheters had. CVC did not give any late thrombotic development. Two patients developed pulmonary embolism, one with fatal outcome.
Evaluation Of The Thrombogenicity Of Four Dacron Arterial Grafts In An Artificial Circulation
Authors: Deacon P , Donaldson D R , Crow M J , Rajah S M , Kester R C 
Published: July 2018

A major consideration in the choice of vascular prosthesis is the thrombogenic potential of its luminal surface. As platelets play a significant role in arterial thrombosis we studied graft-platelet interaction ‘in vitro' with an artificial circuit as a means of comparing the thrombogenicity of grafts. The types of grafts evaluated were, a plain knitted (Meadox ‘Cooley'), the pyrolitic carbon coated knitted (Meadox ‘Biolite', a double velour (Meadox ‘Microvel’ and a filamentous double velour (USCI ‘Sauvage’ The artificial circulation comprised twin loops of silicone tubing, one a test circuit containing a 15 cm × 8 mm diameter graft, the other a control circuit. Both circuits were perfused simultaneously with fresh heparinised blood from the same donor, for 60 mins at 37°C, with a pressure 120/80 mm Hg and a flow 140 mls/min. Six samples of each graft were tested. Thrombogenicity of the graft was assessed by the change in platelet count, adhesion and aggregation measured at 0, 15, 30 and 60 minutes of perfusion. The luminal surface was examined at the end of perfusion, by Scanning Electron Microscopy (SEM), and counts of adherent platelets made. There was a fall in platelet count, adhesion and aggregation in the test circuits during perfusion, this fall being similar for each type of graft, except the filamentous double velour where the fall in platelet count and adhesion was marginally greater. Serial counts of platelets adherent to the luminal surface (SEM) showed three grafts to be very similar (Biolite 84 ± 24, Cooley 2 111 ± 15 and Microvel 126 ± 37 respectively). The Sauvage graft had a very significantly higher number of adherent platelets (p<0.001) with a count of 490 ± 80.The results of this study indicate that the filamentous double velour has a more thrombogenic surface than the other grafts, when evaluated in our experimental model.
Endothelial Cell-Lined Vascular Prostheses
Authors: Burkel W 
Published: July 2018

While considerable effort has been expended to develop blood compatible artificial devices, the only truly non- thrombogenic surface known is that of vascular endothelium. Early prosthetic studies emphasized graft material from the surgical viewpoint. Later the emphasis changed to the physical configuration of the graft (woven, knitted, velour, porous, nonporous grafts, etc.) which gave the best tissue ingrowth, stable flow surface and hopefully an endothelial lining. Since vascular grafts seldom become lined with endothelium in man as a natural consequence of the healing process, methods to enhance the development of an endothelial surface would be of considerable value in reducing thrombosis, infection and the destruction of blood elements. To accomplish this objective, a wide variety of cells and tissue fragments have been added to grafts in order to produce pseudointimas.In our experiments endothelial cells have been isolated from the external jugular veins of 20-30kg foxhounds using trypsin and collagenase. The harvested cells were either cultivated in vitro for 14 days and then seeded into Dacron and PTFE vascular grafts during preclotting, or seeded into grafts immediately after derivation. Grafts were implanted as thoracoabdominal bypasses (25-30 cm in length) and incorporation of the graft and the formation of an endothelial lining followed from 1 day to 1 year. Grafts treated in an identical manner, but preclotted without endothelial cells served as controls.Endothelial cells are first visible in seeded grafts at 4 days, approach 70% coverage of the graft surface by 2 weeks and 90% at 1 month. Four month seeded grafts are completely lined with endothelium, while unseeded grafts still have 20-30% of their surface covered with a fibrin coagulum at 6 months. Preliminary results indicate that blood parameters are greatly perturbed initially, but return to normal levels with the evolution of an endothelial lining in seeded grafts.
Epidemiology Of Myocardial Infarction: Magnitude Of The Problem, Cause And Risk Factors
Authors: Kannel W B 
Published: July 2018

Coronary heart disease is a common, highly lethal, disease which frequently attacks without warning and too often presents with sudden death as the first symptom. Chances of an American male developing CHD before age 60 are one in five.Most angina, infarctions and sudden deaths represent medical failures which should have been forecasted and prevented. About 30% of first MI's will shortly develop angina and experience a per annum death rate, half of which will be sudden deaths. Reinfarctions will occur at 6% per year and half the recurrences will be fatal.No major innovations are needed to identify coronary candidates or to establish their risk from the joint effect of known risk factors. However, all have much to learn about motivating changes in behavior required to control the major risk factors such as cigarette smoking, faulty diet, overweight, sedentary living, abnormal lipids, hypertension and impaired glucose tolerance.Low density lipoprotein cholesterol promotes atherogenesis whereas HDL-cholesterol is protective, and the net effect is judged by their ratio. Hypertension, systolic or diastolic, labile or fixed, at any age in either sex is a powerful contributor to CHD. The impact of diabetes is greater for women, diminishes with age and varies depending on coexisting risk factors.Optimal risk evaluation requires quantitative combination of risk factors so as to include persons with multiple marginal risk factor abnormalities who are at high risk.
The Effect Of Thrombin On The Electrophoretic Mobility And Functional Activity Of Various Factor VIII Molecular Forms
Authors: Seghatchian M J , Mackie I I 
Published: July 2018

Several distinct populations of factor VIII procoagulant activity (VIIIC) were observed in some clinical concentrates after agarose gel electrophoresis. It is not yet known whether these different forms are the products of proteolytic digestion, or subtle conformational changes of FVIII structure, which may occur during concentrate preparation. Low thrombin concentrations activate FVIII in a time/concentration dependent manner, and the 2 proteins have high affinities for one another. We have studied the properties of the various FVIII forms before and after incubation of concentrate with highly purified α-thrombin. Agarose gel electrophoresis was used to monitor: a) quantitative changes in functional activity (by 2 stage clotting (VIII:C) and amidolytic (VIII: CAm) assays); b) the binding of 125I- anti VIII:C (*IgG) to FVIII: C; c) the crossed immunoelec- trophoretic (CIE) pattern of FVIII: RAg.In the absence of thrombin, FVIII concentrate showed 2 main populations of FVIII: C and VIII: CAm. The radioactivity (VIII: CAg) pattern correlated with the slower moving of these populations. Low thrombin concentrations (0.0001-0.01 u/nl) caused no detectable change in FVIII: C level in the slow peak but additional peaks of activity appeared at the sample well, and between the well and slow peak. This was accompanied by a decrease in the fast moving peak, and the appearance of a third new peak with slightly less mobility. Higher thrombin concentrations (0.5u/ml) caused a marked decrease in the slow moving peak, with a concomitant increase in the well peak, by all methods. Increasing thrombin concentration progressively reduced *IgG binding, and a pattern similar to serum was obtained. Thrombin caused a slightly faster migration of FVIII: RAg, a well peak was sometimes seen. Thrombin therefore appeared to act preferentially on the lower molecular weight FVIII activity, and the high molecular weight peaks that occurred may represent complexes of FVIII with thrombin and/or other contaminant proteins present in concentrate such as fibrinogen or fibronectin.
The Functional Heterogeneity Of Clinical FVIII Concentrates
Authors: Mackie I J , Seghatchian M J 
Published: July 2018

There is an increasing tendency to use more highly purified FVIII preparations, and more sophisticated fractionation methods. In some cases new populations of FVIII:RAg have been produced that have a greater or lesser physiological activity. The characterisation of clinical concentrates on the basis of their biological activity is therefore extremely important in the search for more effective therapy.We have investigated the activity of several high purity concentrates (Hemofil, Koate, Actif-VIII) as well as low purity pooled cryoprecipitate, with and without added 125I- anti-VIII:C(*IgG or *FAB), after electrophoresis in 1% agarose gels.In concentrates with a high FVIII:RAg concentration(25-30Iu/ml), 2 or more FVIII:RAg peaks were seen, with partial or complete identity. The main one migrated in the α 2 region, but a more anodal peak was frequently seen. The relative concentration of fast moving VIII:RAg in cryoprecipitate was too low to allow detection by immunoelectrophoresis. In the presence of *IgG or *FAB, only one main peak of radiolabelled VIII:C was noted, which corresponded with the larger molecular forms of VIII:RAg. In contrast, bioassays based on FXa generation (VIII:C and VIII:CAm), revealed 2 main peaks of activity in concentrates, cryoprecipitate and plasma. The slower moving peak correlated well with the radiolabelled VIII:C pattern. The fast moving peak in concentrate corresponded with the trailing edge of the fast moving VIII:RAg peak. This activity was not neutralised by increasing the *IgG level, or addition of anti-IXc IgG to the sample. No enzymic activity was detected in this region using a wide range of chromogenic substrates. However, Al(OH)3 adsorption did remove the activity. These forms of FVIII may represent a native subpopulation or proteolytic breakdown product, which has lost some antigenic determinants, but retained biological activity. This material, which has a lower affinity for anti VIII:C and RAg, could be present in plasma and be selectively concentrated during fractionation procedures.