bluuebunny/crossref_metadata_2025_split huggingface dataset:
Fibrinogen Biosynthesis: In Vitro Translation. Glycosylation And Translocation Of Fibrinogen Peptide Chains
Authors: Fuller G M , Nickerson J M 
Published: July 2018

Fibrinogen is a hepatically derived plasma glycoprotein that is composed of three pairs of nonidentical chains linked together by complex sets of disulfide bridges. In an effort to understand the molecular and cellular processes of translating and assembling this important multichained protein we have utilized an in vitro translating system using mRNA’s for rat fibrinogen. Highly specific antibodies to fibrinogen and to each chain have been developed and used to immunoprecipitate the nascent Aα, Bβ, and γ polypeptides. We have also used a rat hepatoma cell line which synthesizes and secretes fibrinogen to prepare nonglycosylated but processed fibrinogen subunits. SDS/PAGE analysis of the translation products clearly show that each polypeptide has a “signal” peptide located at its amino terminal end. The size of the signal peptide is different for each chain. These results demonstrate that separate mRNA’s exist for each of the fibrinogen subunits. Temporal analysis of the glycosylation of the Bβ and γ chain reveal that the γ chain receives its Asn-linked carbohydrate as an early cotranslational event. The Bβ chain’s core carbohydrate moiety is near the end of the polypeptide and our evidence shows that the glycosylation event likely occurs posttranslationally. When microsomal membranes are added to an on-going translation system, all three of fibrinogen's polypeptides translocate into the cisternal space, with an apparent equal stiochiometry. Additional experiments suggest that fibrinogen assembly occurs as a cotranslational process.These studies have been supported in part by NIH HL - 16445 and HL 00162.
Consumption Of Viiirrag During Delayed Coagulation Process
Authors: Hada M , Ikematsu S , Fujimaki M , Fukutake K 
Published: July 2018

Coagulational functions of VIIIR:AG are still unknown, while its serological significance in laboratory medicine has been established. In this study it will be analyzed the mechanism of the consumption of VIIIR:AG during the process of prolonged blood coagulation.Quantitative assay of VIIIR:AG in plasma and serum is measured by Laurell’s method using 1% agarose with anti-F.VIII rabbit serum and qualitative assay of VIIIR:AG is performed by crossed-immunoelectrophoresis with anti F.VIII rabbit serum.In 28 patients with Hemophilia A a negative relationship between serum VIIIR:AG / plasma VIIIR:AG ratio and prolongation of PTT is estimated and also a positive relationship between serum VIIIR:AG / plasma VIIIR:AG ratio and serum VIII:vW / plasma VIII:vW ratio is obtained. However, serum VIIIR:AG shows the similar quantity to plasma VIIIR:AG in the cases within normal clotting time, which have been treated by the in vitro addition of thrombin or by transfusion of AHG in vivo. When heparin or synthetic antithrombin ( MCI-9038 ) is added into normal whole blood or F.XIII deficient whole blood, the case of normal whole blood shows delayed clotting time and decrease of serum VIIIR:AG, but the case of F.XIII deficient whole blood indicates no decrease of serum VIIIR:AG. Furthermore, in those conditions change of the concentration of CIg in serum, which has been pointed out with fibrin crosslinking, indicate the similar behavior as serum VIIIR:AG.The results obtained above might suggest that serum VIIIR:AG tend to decrease in the cases with prolonged clotting time, and the F.XIII activity might be involved in the consumption mechanism of serum VIIIR:AG in such an abnormal condition, as fibrin crosslinkage could not carry out properly.
Kallikrein-Like Activity Of Crotalase, A Snake Venom Enzyme Which Clots Fibrinogen
Authors: Markland F S , Shaw E , Kettner C , Schiffman S , Bajwa S S , Reddy K N N , Kirakossian H , Pirkle H 
Published: July 2018

During amino acid sequence determination of crotalase, a thrombin-like enzyme from Crotalus adamanteus (eastern diamondback rattlesnake) venom, we found that in addition to the expected structural homology with bovine thrombin, there was even greater homology with porcine pancreatic kallikrein. In exploring further the similarity between crotalase and kallikrein, several completely unexpected and interesting observations were made. First, crotalase was rapidly and specifically inhibited by the tripeptide, affinity-labelling chloromethyl ketone inhibitor Pro-Phe-Arg-CH2Cl, which is known to be a specific inhibitor of urinary kallikrein. Further, crotalase exhibits significant activity not only with the thrombin chromogenic substrate S-2238 (H-D-Phe- pipecolyl-Arg-p-nitroanilide) and the general serine protease substrate S-2160 (N-benzoyl-Fhe-Val-Arg-p-nitroanilide) but also with the plasma kallikrein substrate S-2302 (H-D- Pro-Phe-Arg-p-nitroanilide) and the glandular kallikrein substrate S-2266 (H-D-Val-Leu-Arg-p-nitroanilide). Additionally, SDS polyacrylamide gel electrophoresis reveals that crotalase cleaves the plasma kallikrein susceptible bonds in human high molecular weight kiniogen (HMWK) producing intermediates with procoagulant activity. Analyses for bradykinin release from HMWK are presently in progress. Interestingly, one of the normal activities of plasma kallikrein, the activation of human plasminogen, was not one of the activities possessed by crotalase.In summary, it would appear that crotalase has significant kallikrein-like activity. Whether this will prove to be of importance in the ongoing clinical application of fibrinogen clotting snake venom enzymes, such as ancrod and batroxobin, remains to be shown.
UK-37,248, A Novel, Selective Thromboxane Synthetase Inhibitor With Anti-Aggregatory And Anti-Thrombotic Activity
Authors: Randall M J , Parry M J , Hawkeswood E E , Cross P E , Dickinson R P 
Published: July 2018

UK-37,248, 4-(2-(lH-imidazol-l-yl)ethoxy)benzoic acid hydrochloride, potently inhibited human blood platelet microsomal thronfooxane (Tx) synthetase, IC50=3xl0-9M. TxB2 was quantitated by a specific radioimmunoassay (RIA). In contrast (PG) endoperoxide synthesis by ram seminal vesicle microsomes and prostacylclin (PGI2) synthesis by pig aortic microsomes/were minimally affected by concentrations of UK-37,248 up to 1x10-4M. In the rabbit isolated perfused lung arachidonic acid (AA) metabolites were quantitated by differential bioassay. Concentrations of UK-37,248 from 10-7-10-6M selectively reduced TxA2 production from AA but increased the release of PGI2 and other PGs.The effect of UK-37,248 in the whole animal was studied by estimating TxB2 levels in blood samples removed before and after dosing. The samples were allowed to clot and the serum TxB2 levels were assessed by RIA. In anaesthetised rabbits, 15 minutes after injection of 0.3mg/kg i.v. UK-37,248, serum TxB2 levels were reduced by 75%. In dogs the compound was similarly effective, lmg/kg p.o. inhibiting TxB2 production by 79% two hours after dosing. Aggregation of human platelet-rich plasma in vitro, initiated by threshold collagen, was inhibited by UK-37,248 (IC50=4.8x10-6M). In rabbits, UK-37,248 at 2mg/kg i.v. prevented the mortality due to pulmonary embolism and reduced the associated thrombocytopenia and elevation of plasma TxB2 caused by i.v. AA. In conclusion, UK-37,248 is a selective inhibitor of platelet Tx-synthetase with antiaggregatory and anti-thrombotic activity.
Effect Of An Inhibitor Of Thromboxane Synthesis (UK-37, 248-01) On Thrombin Induced Pulmonary Insufficiency In The Rat
Authors: Sandler H , Saldeen T 
Published: July 2018

Severe trauma in man may be followed by an acute and/or delayed respiratory insufficiency due to microembolism in the lungs, the early and delayed microembolism syndrome, respectively. These conditions can be simulated in the rat by injection of thrombin and a fibrinolysis inhibitor. We have investigated the effect of a new inhibitor of thromboxane synthesis in this model.Intravenous injection of 500 IE thrombin/kg body weight in rats pretreated with trans-4-aminomethyl-cyclohexane-carboxylic acid 200 mg/kg b.w. resulted in a rapid, strong increase in the pulmonary arterial pressure (PAP) and a decrease in the mean arterial pressure (MAP). These reactions were followed by a progressive, slow increase in PAP, a slow decrease MAP and a heavy increase in the weight of the lungs due to interstitial and alveolar oedema.In rats pretreated with UK-37, 248-01 (Pfizer, England) 4 mg/kg b.w. the immediate increase in PAP and decrease in MAP was strongly inhibited. No effect on the later changes in PAP, MAP or lung weight was seen.The results speak for the release of thromboxane A2 being of importance in the early microembolism syndrome but not in the delayed syndrome. Thromboxane may be synthesized in platelets or leucocytes trapped in the lungs. Rats with depletion of the polymorphonuclear cells due to injection of a leucocyte antiserum reacted to the thrombin injection in the same way as rats pretreated with UK-37, 248-01 indicating a relationship between thromboxane A2 and the leucocytes trapped in the lungs.
Differential Uptake Of 111-Indium By Platelets And Granulocytes
Authors: Hawker R J , Hall Carolyn E 
Published: July 2018

Mixed cell,leucocyte or platelet preparations labelled with 111-Indium oxine have been used to visualise septic foci, renal rejection and venous and arterial thrombosis. Pure polymorphonuclear leucocyte preparations are difficult to prepare without modification of cellular activity and are generally contaminated with platelets. Up to 27% of the Indium label is associated with platelets when a granulocyte preparation contains as little as 2% of the original platelet population.Under conditions of limiting Indium-oxine,uptake by platelets is 16.8 and 17.5 times more effective than granulocytes or erythrocytes per unit area of membrane; the accumulation by these latter cell types is related to their surface area with a ratio of 4.7:1. This data is further evidence for a facilitated mechanism for the uptake of Indium by platelets.Labelled platelets have been used for visualisation of sterile and septic abscesses in animals, and in man minor inflammatory responses can be detected. 99.8% pure platelet preparations are simple to prepare and label. These cells which have a more efficient uptake of Indium and long biological survival (up to 9 days) are capable of detecting a wide range of lesions. The use of labelled granulocytes with a short biological survival (about 4 hours) is restricted to haematological investigations and diagnosis of acute bacterial infections or necrosis.
Platelet Activating Factor (PAF)-Induced Aggregation And Release Responses In Vitro In Three Species
Authors: Cargill D I , Cohen D S , Van Valen R , Lee M L , Saunders R N 
Published: July 2018

PAF-induced aggregation in human, rabbit and guinea pig platelets was studied, along with concomitant dense granule (ATP) release. Platelet rich plasma (PRP) was prepared from citrated whole blood (9:1) and adjusted to 5 × 10° plt/ ml. Aggregation and release assays were performed in a Chronolog Lumi-Aggregometer. PAF was dissolved in Tris- Tyrode-Albumin (0.25%) buffer, pH 7.4. ATP standards were run prior to each assay.ATP release was most sensitive in the guinea pig (range 10-10 (max) to 10-11(min) M PAF), less in the rabbit (10-8 to 10-9M PAF) and least sensitive to PAF in human platelets. In human platelets PAF elicited a biphasic release of ATP, the first immediately following addition of PAF and the second at_4-5 min. Release was induced in human platelets by 2 ß l0-7 to 6 × 10-7M PAF.Throughout the range of PAF concentrations which caused a dose-related release of ATP in guinea pig platelets, platelet aggregatory response remained constant (85% or more). Similar findings were observed in rabbit platelets.Human PRP exhibited biphasic release and irreversible aggregation at 10-6M PAF. At 10-7M the initial phase of ATP release disappeared, while a biphasic aggregatory response remained, accompanied by a secondary release pattern. Aspirin (ASA 200 μM final concentration) inhibited both the secondary phases of release and aggregation.PAF-induced aggregation in rabbit and guinea pig platelets appears to be independent of the release reaction. In human platelets, PAF gives a biphasic release pattern, the second phase being inhibited by ASA. The significance of the novel first phase of release is being studied.
Pathways Of Phospholipid Bound Arachidonic Acid To Prostaglandin-Like Metabolites In Rat Platelets After Aggregation
Authors: Zijlstra F J , van Vliet H H D M , Vincent J E 
Published: July 2018

The distribution of [l-14c] arachidonic acid (AA) over phospholipids (PL) and neutral lipids and the formation of labelled thromboxane B2 (TxB2), 12L-hydroxy-5,8,10 hepta- decatrienoic acid (HHTJand 12L-hydroxy-5,8,10,14 eicosa- tetraenoic acid (HETE) was measured in rat platelets at different times after the induction of aggregation by collagen. PL, neutral lipids and prostag1andin-(PG) like substances were separated by several systems of TLC and the radioactivity determined in the individual compounds.Platelet PL were labelled with [l-14c] AA by 10 min incubation and the platelets washed to remove free AA.After aggregation, there is rapid decrease in the amount of labelled phosphatidy1-inositol (PI) in the first 5 min and a slower decrease in that of phosphatidylcholine (PC) lasting about 20 min. There are no consistent changes in the amount of labelled phosphatidyl-ethanol amine. In the neutral lipids, only a decrease in the amount of labelled diglycerides occurs during the first 5 min. The amounts of TxB2 and HHT formed augment, rapidly after the aggregation and reach a maximum value after 5 min. The increase in HETE is much slower with a maximum after 20 min.These data indicate that the AA, used in the formation of TxB2 and HHT is probably derived from PI after degradation by a phospholipase C-like enzyme to diglycerides.On the contrary, the time course suggests that the AA transformed via the lipoxygenase pathway to HETE is originally present in PC and released by the action of phospholipase A2. This would explain the differences in the formation.
Experience Of Control Of The Initiation Of Coumarol Therapy With An Amidolytic Assay Of Factor X
Authors: Egberg N , Johnsson H 
Published: July 2018

Most authors agree that probably any of the vitamin K dependant factors can be used for control of coumarol therapy at steady state of treatment. However, during initiation of coumarol therapy poor correlations have been found between one stage prothrombin assays and specific factor analyses (II and X) and thus the usefulness of the specific assays in the initial phase of treatment has been questioned.Purpose of study To investigate if a specific factor X analyses could be used for control of the initiation of coumarol (warfarin) therapy.Methods Thrombotest (Nyegaard AS, Oslo). Amidolytic factor X assay according to Bergström and Egberg.Patients admitted to the hospital because of venous thrombosis and/or pulmonary embolism. At present 13 patients (9 men, 4 women), age 36-81 years.Design of study Heparin therapy was started as soon as the diagnosis was assigned. Warfarin treatment was commenced on the following day. Thrombotest and factor X was determined before and daily for 6 days after coumarol therapy was started. Only the results from the factor X analyses were given to the doctor in charge of the patients for monitoring of the coumarol therapy.Results Thrombotest and factor X values generally fell in parallell except when high concentrations of heparin in the blood samples inhibited the Thrombotest method. The correlation between Thrombotest and factor X in 10 patients was good (r 0.81-0.99, mean 0.94).(Three patients were excluded for different reasons). The doctor in the ward found it as easy to monitor coumarol treatment with the factor X assay as with the Thrombotest. No bleeding or thromboembolic complications occurred.Conclusion The amidolytic factor X assay could be used for monitoring coumarol treatment in the initial phase of treatment. The absence of sensitivity to heparin was regarded an advantage to the Thrombotest method.
Diagnostic value Of Ethanol Gelation Test (EGT) And Fibrin- (OGEN) Degradation Products (FDP) In Suspected Deep Vein Thrombosis (DVT), Correlated To The 125-1-Fibrinogen Uptake Test (FUT)
Authors: Hamborg T , Skjennald A , Godal H C 
Published: July 2018

The study was performed in order to investigate the correlation between EGT, respectively FDP, and a positive FUT (FUT+), confirmed by venography. Control groups: patients 1) with a negative FUT (FOT−) in spite of DVT, 2) without DVT, because a negative venography also implies FUT−. Accordingly, FUT was not accomplished in this group.The material consisted of 41 patients with clinically suspected DVT; 20 with and 21 without DVT. Of the patients with DVT, 9 were definitely FUT+ and 11 definitely FUT−.EGT was applied for detection of soluble fibrin, Thrombo- Wellcotest for FDP and the Chi-squared test for statistical analysis. Blood specimens were analysed 0-1 days before isotope injection in patients with DVT, or 0-2 days before a negative venography.Conclusions: In this material, 1) Concurrently abnormal EGT and FDP was specific for the FUT+ patients. 2) On the other hand, a positive FUT could be excluded when both parameters were normal. 3) In patients with DVT, a positive EGT was found only in the FUT+ patients. (P values in the table refers to comparison with the FUT+ group).
Automated Coagulation Factor Assays Using A Thrombin Specific Chromogenic Substrate
Authors: Duncan A , Bowie E J W , Owen C A 
Published: July 2018

An automated method for specific factor assays has been developed using a novel thrombin-specific chromogenic substrate (developed by A. Aiyappa, Ph. D., Abbott Labs). For factors VIII, IX, XI and XII, the test system includes an ellagic acid activator and brain phospholipid in a calcium containing tris buffer pH 7.4. These are mixed with a plasma sample consisting of a mixture of specific factor deficient substrate and test plasma for 5 minutes and then the chromogenic substrate is added. After another 5 minutes the increase in optical density ΔOD is recorded. Normal plasma serially diluted in specific factor deficient substrate is used to construct a standard graph and test plasma results are interpolated from this.Fifty-four comparative assays have been done for factors VIII and IX and correlated with the results obtained from conventional clotting assays. For factor VIII the correlation coefficient (r)=0.935 (N=35) and for factor IX r=0.941 (N=19). While insufficient data exists to date for factors XI and XII, these assays appear to be comparable to the VIII and IX results.The assays for factors V, VII and X use a simplified system in that the chromogenic substrate and phospholipid are added together. The ΔOD is again recorded after 5 minutes. Test plasma results are interpolated from a standard graph constructed using serially diluted normal plasma as before. To date only the factor V assay has enough data to do a correlation with the clotting assays and r=0.953 (N=13) which is similar to the r value for the VIII and IX assays.Because of the widespread availability of the automated analyzer used in the assay and the standardization of end point determination it provides, specific factor assays might be made more widely available outside specialized coagulation centers.
Measurement Of Platelet Factor 3 Availability By Using Chromogenic Substrate
Authors: Hársfalvi J , Chmielewska J , Latallo Z S , Muszbek L 
Published: July 2018

The measurement of platelet factor 3 (Pf 3) availability (the prothrombin converting procoagulant activity of platelet membran phospholipids) induced by collagen, ristocetin, arachidonic acid or other activating agents is a potentially valuable test in the diagnosis of platelet disorders. Its diagnostic potential, however is greatly impaired by the fact that RW clotting time method usually used for Pf 3 determination is influenced by many factors, its reproducibility is rather poor, and results are difficult to express in quantitative well defined biochemical terms.In the present paper a new method measuring Pf 3 activity by using a chromogenic substrate was developed. Platelet rich plasma(PRP) was incubated by various activating agents, then prothrombin thrombin conversion was started by the addition of purified factor Xa. After a certain interval the reaction was stopped by soybean trypsin inhibitor and thrombin formed during the reaction was quantified by the chromogenic substrate S-2238 (Kabi). By varying incubation time, prothrombin, factor V concentrations, the optimal conditions, where platelet phospholipid was the rate limiting factor, were determined. In these conditions Pf 3 activity was a linear function of the number of activated platelets. The results are expressed as the ratio of thrombin generated in activated and unactivated PRP. The method is a rather simple quantitative photometric technique, has an improved reproducibility, and results showing the increase in thrombin generation have well defined biochemical meaning.
Interaction Of Plasma Proteins With Artificial Surfaces With Reference To Platelet Adhesion
Authors: Malhotra O P , Helmus M N , Gibbons D F 
Published: July 2018

On occasion, fewer platelets from platelet rich plasma (PRP), adhered to hydrophilic (glass) surfaces exposed to platelet poor plasma (PPP) for 3 min than areas exposed for 3 s. The decrease was dramatic and consistent when platelet suspension (gel-PLS, obtained from PRP by 2B Sepharose gel filtration) was used instead of PRP. To further explore the factors which influence platelet adhesion, we used the following: for surfaces, a) sparkleen-cleaned glass (hydrophilic), b) acid-washed (somewhat hydrophobic), and c) siliconized (hydrophobic); for proteins, a) PPP, b) fibrinogen (96% clottable), c) defi-brinogenated (defib.) plasma, and d) defib. plasma plus fibrinogen; for platelet suspension, a) PRP, b) gel-PLS, and c) platelets in defib. plasma (defib. PLS).From gel-PLS, non-siliconized surfaces exposed to fibrinogen for 3 s attached more platelets (F<0.05) than those exposed to PPP or defib. plasma plus fibrinogen. The latter two attracted more platelets (P<0.01) than defib. plasma. Hydrophilic sparkleen-cleaned glass previously exposed to PPP (or defib. plasma plus fibrinogen) attached a minimum of 10-fold as many platelets from gel-PLS than from PRP. Under similar conditions acid-cleaned surfaces attached 2-fold from gel-PLS, while hydrophobic glass did not show any change. By exposing the surfaces to PPP followed by gel-PLS, the sparkleen-cleaned glass showed the greatest decrease (P<0.001) in the number of platelets attached to areas exposed to PPP for 3 min as compared to 3 s, while siliconized showed no such decrease. If, however, the surfaces were exposed to defib. plasma, they all showed decreases in platelet attraction at 3 min.