bluuebunny/crossref_metadata_2025_split huggingface dataset:
Isolation And Purification Of Collagen And α1 Receptor From Platelet Membrane
Authors: Chiang T M , Kang A H 
Published: July 2018

We have previously demonstrated that chick skin type I collagen and the α1(I) chain mediate platelet aggregation. Aggregation is associated with specific binding of these substances by platelet membranes. We now describe the isolation and purification of the receptor. Platelet membranes were prepared as described previously and isolated membranes were solubilized in 0.5% Triton. The receptor was then purified by a combination of gel filtration, affinity chromatography on α1(I)-sepharose or type I collagen-sepharose and preparative polyacrylamide gel electrophoresis. The receptor activity was assayed either directly by a binding assay using (14C)-glycine-labeled α1(I) or indirectly by an adhesion inhibition assay on Sepharose 2B with (14C)-sero- tinin-labeled platelets.The results show that the α1(I) receptor can be purified to a single band on SDS-gel electrophoresis with a recovery of 2.5%. Its activity is destroyed by preincubation with trypsin or pronase indicating it is a protein. The apparent molecular weight as estimated by gel filtration and SDS-gel electrophoresis is 95,000 daltons. The binding of (14C)- labeled α1(I) is specifically displaced by unlabeled α1(I), and the bound radioactivity can be removed by treatment with purified bacterial collagenase. The binding of (14C)- labeled α1(I) by the purified α1(I) receptor can also be inhibited by the receptor isolated from collagen-sepharose affinity chromatography. These data suggest that the α1(I) binding site is identical to the collagen binding site.
Salicylate Accumulating During Repeated Aspirin Administration Prevents The Inhibitory Effect Of Aspirin On Vascular Prostacyclin
Authors: Dejana E , Cerletti C , de Castellarnau C , Galletti F , de Gaetano G 
Published: July 2018

In vitro studies have shown that salicylate (SA) effectively protects cyclooxygenase from aspirin (ASA) inhibition. The purpose of this study was to investigate in vivo this pharmacological interaction and to evaluate the possibility that SA accumulating during repeated ASA treatment interferes with the inhibitory effect of the unchanged drug on vascular prostacyclin(PGI2). Male CD-COBS rats were given i.v. saline or SA (25-100 mg/kg) followed after 15 min by ASA (2.5-10 mg/kg). The animals were killed 30 min after the last treatment. PGI2 activity was bioassayed on thoracic aorta rings and confirmed by RIA of 6-Keto-PGF1α. Blood SA levels were measured by fluorimetric assay. SA pretreatment showed dose-related competitive prevention of the inhibitory effect of ASA on vascular PGI2 which was almost complete at a dose ratio of 5:1. Further experiments were performed with single or repeated doses of ASA (200 mg/kg). This resulted in different blood SA levels after 24 hours when in all conditions vascular PGI2 recovered by more than 50% of basal value. At that moment further inhibition (60-90%) of PGI2 by a test dose of ASA (10 mg/kg) could only be obtained if blood SA levels were lower than 100 μg/ml. Above this level an inverse linear correlation (r = 0.87, p< 0.01) was found between blood SA levels and the inhibitory activity of ASA. In particular, almost no ASA activity could be detected at blood SA levels around 250 μg/ml.These results show that SA and ASA interact in vivo at vascular levels. This interaction could have clinical implications in view of the rapid hydrolysis of ASA to SA and slow elimination of SA from blood. These observations add a new piece of information to the current debate on the optimal ASA treatment schedule for thrombosis prevention.
Dipyridamole Reverses The Inhibitory Effect Of Aspirin On Prostacyclin Synthesis
Authors: Jackson C A , Preston F E , Greaves M 
Published: July 2018

The value of aspirin (ASA) as an antithrombotic agent is limited by the fact that it inhibits the production of prostacyclin (PGI2) by the vessel wall. However, there is evidence that the combination of ASA and dipyridamole (DP) has antithrombotic potential for patients with certain types of thrombovascular disease and there appears to be some synergism between the two drugs. To study the relationship between ASA and DP we have examined the effect of the two drugs, singly and in combination on thromboxane (TX) production and PGI2 synthesis by platelets and vessel walls respectively. Rings of venous tissue prepared from veins obtained during surgery were incubated in Ringers solution with ASA, DP or a combination of both. PGI2 synthesis was assessed by radioimmunoassay for 6-keto PGF1α, its stable metabolite on aliquots of supernatant from the incubation mixture during a thirty minute period. All control samples synthesized PGI2. 0.1mM ASA caused 4299 per cent inhibition of PGI2. DP at a concentration of 0.05mg/ml had no significant effect on PGI2 production compared with values obtained in controls. The combination of DP and ASA caused significantly less inhibition of PGI2 synthesis compared with almost complete inhibition observed with ASA alone.In similar studies on platelet rich plasma, TXA2 synthesis, determined by radioimmunoassay for TXB2, was abolished by ASA 0.15mM, whereas DP 0.05mg/ml had minimal inhibitory action. However, at lower concentrations of ASA the inhibition of both aggregation and TXA2 synthesis was markedly potentiated by DP 0.05mg/ml.These results provide an explanation for the reported synergism between ASA and DP.
Activation Of Extravascular Coagulation In The Inflamed Joints In Rheumatoid Arthritis (RA)
Authors: van Ginkel C J W , Oh J I H , van Mourik J A , van Aken W G , Vreeken J 
Published: July 2018

Deposition of fibrin-like material on the synovival membrane of inflamed joints is a prominent and persistent feature of RA. To evaluate the involvement of the classical coagulation cascade in the formation of this material, fibrinopeptide A (FPA) and coagulation factors were analyzed in synovial fluid from RA patients. FPA was 1100 ng/ml (range 300-3200; n=l6), compared to 200 ng/ml (60-300;n=13) in synovial fluid from patients with osteoarthritis. No significant change (p> 0.2; n=l4) in FPA immunoreactivity (using R2 anti-FPA serum from Dr Nossel beside the antiserum from our institute) was observed after thrombin treatment of the diffusate of dialyzed synovial fluids. This finding indicates that the high levels of FPA immunoreactivity in joint fluids of RA reflect FPA (Aα 1-16) instead of FPA-like fibrinogen fragments (which can be cleaved from fibrinogen by e.g. granulocytic proteases). In view of the finding that all coagulation factors including fibrinogen were sufficiently present (30-100%; n=l8) - except FV (<5%) - the accumulation of FPA suggests activation of extravascular coagulation in the inflamed joints in RA.To investigate the mechanisms of this activation, normal plasma was incubated with human articular cartilage and activation of FXII was determined by measuring the generation of bradykinin (radioimmunoassay). Cartilage activated FXII whereas its activation capacity was strongly enhanced by pre-exposition to granulocytic enzymes (compare in vivo!). Among its constituents collagen (type II) activated FXII, but proteoglycans were only weak activators. Both substances were isolated from human cartilage, obtained from a normal knee joint at necropsy. With respect to the contribution of the extrinsic coagulation pathway it was shown that synovial fluids from RA patients stimulated the thromboplastin generation by normal human monocytes. Taken together, these findings suggest that both intrinsic and extrinsic coagulation pathways, presumably triggered by the inflammatory process itself, are involved in articular fibrin deposition.
Antithrombin Assay - A Study Of Reaction Conditions, Reagents And Kinetics
Authors: Friberger P , Knös M , Andersson M 
Published: July 2018

Antithrombin (AT) in plasma diluted as much as 1:600 in the final incubation mixture reacts rapidly and essentially quantitatively with thrombin in the presence of 3 IU/ml of heparin using tris buffer pH 8.3 and I 0.15. Normal plasma levels of AT consume approximately 60% of human thrombin when 7 nmol/1 is added. The reaction is similar to a 2nd order reaction, k2=8.106 1/mol.sec and is almost complete after 3 min incubation. The excess amount of thrombin is then determined with the chromogenic substrate S-2238.Data collected prove: 1. The toal amount of the AT cofactor activity is titrated. 2. Other plasma inhibitors do not interfere. 3. The quality and/or source of thrombin and heparin is of definite importance. 4. The procedure for preparing the standard curve and the selection of standard is critical. 5. Reagents with good stability can be selected.The assay is performed in two steps but easy handling is obtained by the use of the same volume of each reagent and the selection of suitable incubation times - all in order to allow reproducible results also in a routine laboratory. This is important since there are small differences between normal and pathologic values.
Determination Of Antithrombin III Heparin-Binding Capacity By High Performance Liquid Chromatography
Authors: Nunez H A 
Published: July 2018

The heparin-binding capacity of purified human antithrombin III (AT) both native and after a variety of denaturat- ing conditions was measured using a high performance liquid chromatographic (HPLC) system.An exclusion chromatography, TSK-3000, 0.75 (ID) × 50 cm column equilibrated with 0.1M NaCl in 0.02M potassium phosphate buffer pH 7.35, at a flow rate of 1 ml/min was used. Under these conditions two essentially resolved peaks were obtained at 10.5 to 14 min. The peak corresponding to the unbound AT, or native AT in the absence of heparin (H), is sharp; that of the AT-H complex is broad due, probably, to the size heterogeneity of the H used in the tests. Without H, aggregates of AT are also resolved by this technique. The areas under the curve of the protein peaks monitored spectrophotometrically in the absence and in the presence of an excess of H are used to quantitate these species. A titration of AT with increasing amounts of H indicates the concentration of H sufficient to bind all the H-binding AT present in a given AT containing sample.The results of the HPLC method correlate well with those obtained by two-dimensional or crossed Immunoelectrophoresis (CIEP), a technique that can be used to estimate the H-binding capacity of AT. Thus, for example, in non- pasteurized, pasteurized and heated AT samples, for which the HPLC method indicated that 5, 25 and 80% of the AT had lost its H-binding capacity, the CIEP gave similar results.The results indicate that the HPLC method can adventage- ously be used to determine quantitatively the H-binding capacity of AT and, if present, AT aggregates, thus appearing more informative than CIEP. The analysis time is less than 30 minutes and uses inexpensive reagents.
Gyki 14,451, A Specific Inhibitor Of Thrombin:“In Vitro” And“In Vivo”Studies
Authors: Tremoli E , Maderna P , Colli S , Morazzoni G , Paoletti R 
Published: July 2018

The effects of a synthetic tripeptide, Gyki 14,451(Boc-D Phe-Pro-Arg-H)have been studied in vitro on human platelet aggregation and arachidonic acid (AA) metabolism and in vivo on experimentally induced venous thrombosis in the rat.1 μM Gyki 14,451 concentration selectively inhibits thrombin induced platelet aggregation as well as malondial- dehyde (MDA) and thromboxane B2 (TXB2) formation by platelet rich plasma (PRP) stimulated with thrombin. Far higher concentrations (400μM)of the peptide are required to exert an inhibitory effect when collagen, ADP and AA are used to stimulate platelets. No effect has been observed on the conversion of 14C AA to metabolites using unstimulated platelets. Kinetic studies of MDA production by platelets stimulated with thrombin and its inhibition by Gyki 14,451 (0.15,0.3,0.6 uM) suggest that the peptide interacts with thrombin by an apparently competitive mechanism.4 mg/Kg of Gyki 14,451, intravenously injected in the rat caudal vein, completely inhibited the occurrence of venous thrombosis, obtained by vena cava ligature.The oral administration of the peptide (50 mg/Kg by gastric intubation) failed to reduce the percentage of incidence of venous thrombosis (88% in controls versus 90% in treated rats) resulting only in a reduction of the thrombus weight. These data suggest that the anticoagulant peptide Gyki 14,451, given intravenously, possesses a consistent activity in the prevention of experimentally induced venous thrombosis.
Interaction Of Protamine Sulfate With Thrombin
Authors: Cobel-Geard R , Penner J A , Hassouna H I 
Published: July 2018

Protamine sulfate, a simple protein M.W. 4,600, is a known antiheparin agent which, in the absence of heparin, demonstrates an anticoagulant activity both in vitro and in vivo. Much work has been done to elucidate the interactions of heparin with thrombin and ATIII, however, little is known about the mechanism of protamine sulfate anticoagulant activity and its effect on thrombin.In binding studies, radiolabeled thrombin was reacted with protamine for 1 hour and ATIII was then used as a probe to test the availability of the thrombin active site. ATIII/*thrombin complex was separated from free *thrombin on a Sephacryl S200 molecular sieve column. As a result of preincubating thrombin with protamine, ATIII was unable to bind thrombin. Functional assays for thrombin activity, were carried out in a system where time taken for formation of a thrombin catalyzed fibrin clot was the measure used for thrombin activity. Effect of protamine on thrombin activity when tested in this system demonstrated that with increasing molar concentrations of protamine, there is a corresponding decrease in thrombin activity. Maximum inhibition of activity occurred at a protamine concentration of 200 μM. This inhibition was concentration dependent, partial and reversible. In contrast the amydolysis of the synthetic substrate H-D-Phe-Pip-Arg-p-nitroanilide by thrombin was unaffected by protamine.Results of these studies have confirmed the anticoagulant role of protamine sulfate and provided information on its interaction with purified thrombin. Since the drug did not totally inhibit functional activity of thrombin, we feel that it binds the enzyme, partially blocking the active site.
Sex-Related Differences In Aspirin Pharmacokinetics Following IV Injection In Rabbits
Authors: Buchanan M R , Rosenfeld J , Hirsh J 
Published: July 2018

Clinical and experimental studies suggest that aspirin (ASA) is a useful antithrombotic agent; however, its efficacy seems limited to males. This may be due to differences in i) the effects of ASA on platelets and/or vessel wall or ii) the pharmacokinetics of ASA, between males and females.We examined the possible sex-related differences in 1) enhanced arterial thrombosis 3 h after treatment with 0, 3, 10 or 100 mg/kg ASA and 2) the distribution (α) and clearance (β) of ASA between male and female rabbits. Enhanced arterial thrombosis was determined by measuring 51Cr-platelet accumulation onto injured carotid arteries. ASA-plasma levels were determined, using a GC/MS assay.
51Cr-platelet accumulation onto injured arteries was 0.67 ± 0.15 × 106 (males) and 0.52 ± 0.07 × 106/100 mm2 vessel wall (females; mean ± SEM, n=8). 3 mg/kg ASA had no effect. 10 and 100 mg/kg ASA significantly enhanced 5lcr- platelet accumulation to 1.94 ± 0.41 × 106 and 2.59 ± 0.93 × 106 in males, and to 1.65 ± 0.32 × 106 and 2.16 ± 0.73 × 106 in females (p<0.001). There were no sex-related differences.ASA-plasma levels were determined in serial samples collected from 10 rabbits injected with 10 mg/kg ASA. One week later, the study was repeated in the same animals. The α and β T½’s were 3.3 ± 0.2 and 29.2 ± 3.0 (min) respectively in males after the 1st treatment. Similar T½’s were found after the 2nd treatment (3.4 ± 0.1, 23.3 ± 3.5 respectively). In females, the initial α and β T½’s were 3.7 ± 0.2 and 26.1 ± 3.5 respectively. The α T½ was similar (3.6 ± 0.1) whereas the β T½ was significantly decreased (14.8 ± 0.3, p<0.007) after the 2nd ASA treatment. This decrease in females was associated with an increase in the apparent volume of distribution, p<0.02The significance of these findings is uncertain.
Sex-Associated Differences On The Effect Of Asa Or Flurbiprofen On Platelet Function Ex Vivo
Authors: Nishizawa E E , Molony B A , Meinzinger M M , Williams D J 
Published: July 2018

It has been reported that although ASA may have some beneficial effects in males, no differences from placebo was detected in females in clinical trials on venous thrombosis, TIA and stroke. Since ASA inhibits platelet function and because the end-points measured in the above clinical studies may be related to platelet function, we measured bleeding time and platelet aggregation induced by collagen or arachidonic acid following oral administration of ASA (650 mg) or flurbiprofen (100 mg) in normal male and female volunteers (10 per group, total of 40 subjects) between the age of 50 and 70.No sex-associated differences in response with either drug were observed in bleeding time or platelet function. Thus, we were unable to confirm that the observation of beneficial effect of ASA in males was due to its effect on platelets. However, more interestingly there was a statistically shorter bleeding time in males (p = 0.026) before administration of drug. It was also found that despite the normal response to collagen, PRP prepared from pre-drug blood samples from 6 individuals (15%) did not aggregate when stimulated with arachidonic acid at concentrations as high as 1 mM. These results suggest that, at least in some individuals, collagen-induced aggregation may proceed by a pathway independent of the arachidonic acid pathway.
DES-AA Fibrin Clearance In Healthy Human Subjects
Authors: Brosstad F , Harrbora T , Holm B , Godal H C , Kierulf P 
Published: July 2018

Plasma clearance of I125 des-AA fibrin mcncmers was studied in healthy subjects (N=7). Labelled mcnaners (fm), made fran labelled fibrinogen (F) in 2,5 M urea, pH 7,4 exposed to insoluble reptilase (Brosstad et al., Thrarto .Res. 13, 1978) vere solubilized upon addition to test-subject plasma, and subsequently injected. At intervals, citrated plasma samples ware tested for: I. Total radioactivity (Tr). II. Radioprecipitability (Rp) with trichloroacetic acid. III. Radioclottability (Rc) with plasmin-free thrcmbin (pH 6,3 1h). IV. Radiodistribution after ultrasentrifuga-ticn (Airfug, Beckmann) and V. Radiodistributicn in SDS-PAGE gels after electrophoresis of harvested EDTA-plasma clots, solubilized in urea-SDS.RESULTS: Prior to injection, plasma demonstrated: Rp = 100 %, Rc = 94 %, single peak ultracentrifuge pattern and 95 % of total radioactivity moving as F on SDS-gels. Almost identical results were obtained in all subjects. After 30 min., Tr had dropped to 50 % of Tr value at 5 min., being 10 % and 2 % after 420 min. and 48 h., correspondingly.
A parallell drop in Rp and Rc scarcely noticeable at 10 min., reached a mininun of 30 % at 360 min; the Airfug and SDS-gel patterns substantiating these findings. Due to non-linearity on semilog. plots, a definite half-life of des-AA fibrin cannot be stated from decay parameters (Tr) alone, but metabolic data as outlined above (Rp, Rc, Airfug, SDS-elpho) suggest a half-life of 30-60 min .. In five subjects, I131-des-AA fibrin and I125-fibrinogen was injected simultaneously. Clearance rates characteristic of that of the individual molecular species were obtained. The extremely rapid elimination of fibrin ccnpared to that of fibrinogen suggests specific receptors on RES- cells to be involved.
Plasma Fibronectin Levels: Effect Of Obtaining Blood For Assay VIA Indwelling Plastic Catheters
Authors: Snyder E , Barash P , Mosher D 
Published: July 2018

Plasma fibronectin (PFN) concentrations are reported to be decreased in patients with sepsis and disseminated intravascular coagulation. In such critically ill patients vascular access is often limited, and the indwelling plastic catheters inserted for clinical monitoring are often used for obtaining blood samples. Despite slow continuous infusion of heparinized saline, these catheters often become coated with fibrin. Since PFN is known to be precipi table by heparin and to be bound to fibrin(ogen), levels of PFN obtained from blood drawn through such indwelling catheters may be artifactually depressed. To provide data, levels of PFN were determined from blood samples drawn simultaneously through the distal port of Swan-Ganz (110 cm length), plastic arterial (3.2 cm length) or central venous pressure(CVP 20.3 cm length) catheters, and compared with those levels obtained after a percutaneous venipuncture. All catheters were in situ for 24-48 hours prior to blood sampling. The arterial and Swan-Ganz lines were flushed with heparinized saline continuously (2 units/ml;3 ml/hour). Prior to sample collection, 5 ml of blood was aspirated through the catheter and discarded. Plasma was frozen at -80C until assay. PFN concentration was determined using an electroimmunoassay.We conclude that PFN levels determined from blood drawn through any of the plastic catheters studied were equivalent to those levels obtained from a percutaneous venipuncture. In critically ill patients in whom vascular access is limited, PFN concentration may be reliably obtained from blood samples collected through indwelling catheters.
Increased Platelet Aggregation and Decreased Sensitivity of Platelets to Synthesized Prostacyclin in Alloxan-Diabetic Rabbits
Authors: Yamada K , Yoda T , Goto Y , Hirata T , Serizawa K 
Published: July 2018

Platelet aggregation and sensitivity of platelets to prostacyclin were examined with a view to clarify platelet function in alloxan-diabetic rabbits. Diabetic rabbits were obtained by intravenous injection of alloxan (100mg/ kg body weight) and range of plasma glucose in these diabetic rabbits was 350-500mg/100ml. Blood was collected from the central artery of the ear of normal and alloxan- diabetic rabbits. Platelet aggregation was measured turbidometrically as rate of light transmission at maximal aggregation to light transmission of platelet poor plasma by a NKK aggregometer (Tokyo, Japan). Platelet aggregation was induced by ADP with each final concentration of 0.5, 1.0, 2.0 and 6.0 μM. Sensitivity of platelets to synthesized prostacyclin (Ono Pharma. Co., Tokyo, Japan) was represented as prostacyclin concentration of fifty percent inhibition of ADP-induced platelet aggregation.Platelet aggregation rate in normal and alloxan-diabetic rabbits was as follows; 8.4±1.0, 22.8±2.6%(0.5 μM), 22.3±1.5, 35.2±3.5%(1.0 μM), 37.1±1.7,50.0±2.6%(2.0 μM), 51.0±2.2, 66.7±7.0%(6.0 μM ADP)(Mean±S.E., P< 0.005). Prostacyclin concentration of fifty percent inhibition of ADP-induced platelet aggregation in normal and alloxan- diabetic rabbits was as follows; 42.37±1.68, 48.50±0.48 ng/ml (Mean±S.E., P< 0.01).In conclusion, platelets of alloxan-diabetic rabbits presented a significant decrease in sensitivity to synthesized prostacyclin as compared to platelets from normal rabbits, with a significant increase in ADP-induced platelet aggregation.