bluuebunny/crossref_metadata_2025_split huggingface dataset:
THE ROLE OF THE CARBOHYDRATE MOIETY OF HUMAN FACTOR V IN COAGULATION AND TURNOVER
Authors: Bruin T , Sturk A , Ten Cate J W , Cath M 
Published: August 2018

The importance of the carbohydrate moiety of the human coagulation factor V molecule was investigated by its desialation and deglycosylation. Upon removal of 90% of the sialic acid residues a 1.5-2 fold increase in clotting activity was observed. Whereas up to 70% deglycosylation resulted in a parallel decrease in clotting activity. Thrombin induced activation of desiala-ted factor V was unchanged, whereas deglycosylated factor V activation was impaired. The importance of the carbohydrate structure was further established by lectin incubation experiments. The distribution of carbohydrate in the thrombin induced activation fragments of factor V was investigated in lectin blot experiments with sialic acid-specific LFA, galactose-specific RCA-II and mannose-specific Con A. Carbohydrate residues were demonstrated in fragments B, C1, D and F1F2 . Interestingly, sialic acid was demonstrated in C1, But galactose could not be shown. In fragment F1F2 ultimate galactose residues were found. The relevance of the carbohydrate moiety was further established by turnover experiments of native and desialated human factor V in rabbits. In contrast to the native factor V, desialated factor V was instantaneously cleared from the circulation.In summary, these findings indicate an important role for the carbohydrate moiety in human coagulation factor V.
THE COMPLETE AMINO ACID SEQUENCE OF HUMAN FACTOR V
Authors: Jenny Richard J , Pittman Debra D , Toole John J , Kriz Ronald W , Kaufman Randal J , Mann Kenneth G 
Published: August 2018

cDNA clones encoding human factor V have been isolated and sequenced. The cDNA sequence of factor V obtained from overlapping clones includes a 6672 bp coding region, a 90 bp 5'-untranslated region and a 163 bp 3’-untranslated region including a poly-A tail. The deduced amino acid sequence consists of 2224 amino acids including a 28 amino acid leader peptide. A direct comparison to human factor VIII reveals considerable homology between both proteins with respect to amino acid sequence and domain structure. A triplicated "A" domain and duplicated "C" domain show an approximate 40% identity to the corresponding domains in factor VIII. Factor V and Factor VIII both possess a heavily glycosylated B domain that separates the heavy and light chains of the activated cofactors, although no significant homology is observed in this region. The B domain of factor V contains 35 tandem and approximately 9 additional semi - conserved repeats of nine amino acids of the form (D-L-S-Q-T-T-L-S-P) and 2 additional semi-conserved repeats of 17 amino acids. Factor V contains 37 potential N-linked glycosylation sites, 25 of which are in the B domain, and a total of 19 cysteine residues. By direct comparison to amino acid sequence obtained from both human and bovine factor V, the thrombin (IIa) cleavage sites have been assigned as Arg-709/Ser-710, Arg-1018/Thr-1019, and Are-1545/Ser-1546.(Supported by NIH Grant HL-34575)
Neutrophil Elastase, Thrombin and Plasmin in Septic Shock: Influence on Prognosis
Authors: Seitz R , Wolf M , Egbring R , Havemann K 
Published: August 2018

The prognosis of septicaemia depends on the occurrence of disseminated disturbances of the microcirculation impairing organ function and haemorrhagic complications due to consumption of coagulation factors. The intravasal appearance of three potentially involved proteinases in active form can be detected by immunologic determination of their complexes with inhibitors: thrombin-antithrombin III (TAT), according to PELZER et al. (Thrombos. Haemostas. 54:24,1985); plasmin- antiplasmin (PAP), ldlE, antiserum donated by KARGES; human neutrophil elastase (HNE)- antitrypsin, ELISA, Merck, DarmstadtIn 47 patients with septic shock (19 survived, group A; 28 lethal, group B) the PAP levels were moderately elevated throughout the course without vaviations related to the outcome. TAT was initially strongly increased in both groups (18.8±6.3 ng/ml/16.5±7.2), and decreased towards the end of the course in both groups (2.7±0.5/3.7±0.8). Though the intial HNE levels were higher in group A (2458±348ng/ml) than in group B (1291±295, p=0.017), they decreased, in group A more rapidly and were at the end almost significantly lower than in group B (315±54/652&3x00B1;161, p=0,059). The decrease of TAT as well as HNE was associated with substitution of antithrombin III concentate (ATIII) and fresh frozen plasma (ffp) given with the aim of normalization of haemostasis and replacement of inhibitors. Factor XIII, a substrate of both thrombin and HNE, was initially equally low about 50% of normal in both groups, but increased only in group A (69.1±7.1/49.6±5.5, p=0.045) towards the end.Conclusions: Both TAT and HNE decreased after initial elevation under substitution of ATIII and ffp. A rapid decrease seems to be a favourable sign which is accompanied by rising levels of F XIII, while sustained elevation of HNE points to a poor prognosis
LIMITED CLEAVAGE OF HMW-KININOGEN BY PLASMIN ENHANCES RELEASE OF KININ BY PLASMA KALLIKREIN: CLEAVAGE BY LEUKOCYTE ELASTASE DOES NOT RELEASE KININ, BUT INACTIVATES ITS COAGULANT PROPERTIES
Authors: Kleniewski J , Donaldson V H 
Published: August 2018

After limited digestion of purified human HMW-kininogen by plasmin, the kininogen molecule consists of two disulfide-linked chains in which the bradykinin sequence resides in the "light chain" portion. Kinin was released from this molecule by plasma kallikrein at a two- to three-fold more rapid rate than from uncleaved HMW-kininogen. Similarly, when normal human plasma or prekallikrein deficient plasma was treated with sufficient streptokinase to activate plasminogen the subsequent rate of release of kinin by kallikrein was enhanced. The digestion of HMW-kininogen by plasmin as well as kinin relea^s was inhibited by epsilon aminocaproic acid at a concentration of 10−3 M, suggesting that one or more lysine residues was critical to the plasmin-HMW-kininogen interaction. Leukocyte elastase cleaved HMW-kininogen into low molecular weight fragments without releasing kinin but plasma kallikrein could then release kinin from a low molecular weight component of elastase-digested HMW-kininogen (≦50 kd mol. wt.). Elastase destroyed the coagulant properties of the kininogenWhen HMW-kininogen was converted to two-chain, disuifide-linked molecules, either by plasmin or kallikrein, the quantity of antigen detected in an ELISA with polyclonal antibody to human light chain antigens was significantly increased. Expression of antigen detectable with a monoclonal antibody to an epitope located close to the disulfide interchange in the light chain was not increased by prior limited digestion with these enzymesIt is possible that minimal activation of plasminogen in vivo may facilitate kinin release by kallikrein. In addition, in quantifying antigenic properties of HMW-kininogen in plasma, care should be taken to block in vitro activation of plasminogen or prekallikrein. Since leukocyte elastase can be released during clotting of whole blood, it might then serve as a regulator of coagulation through its inactivation of coagulant properties of HMW-kininogen
INFLUENCE OF CALCIUM FLUX ON STABILITY OF PLATELET MICROTUBULE COILS
Authors: Rao Gundu H R , White James G 
Published: August 2018

Resting human platelets have a characteristic discoid form supported by a circumferential microtubule (MT). Ultrastructural, immunocytochemical and immunofluorescence studies have shown that the circumferential MT is a stable structure which undergoes constriction following exposure of platelets to aggregating agents. However, some biochemical and morphological studies suggested that MT coils dissolved almost completely within seconds after exposure to aggregating agents, then reassembled 1-4 minutes later. One investigation suggested that disappearance of the MT coils was associated with calcium flux caused by agonist stimulation or exposure to the ionophore, A23187. The present study has examined the latter hypothesis directly. Fura 2, a calcium sensitive fluorophore, was loaded into washed platelets at a concentration of 1 μM, the cells washed once and resuspended in HEPES buffer. Fluorescence changes were monitored in a Fluorolog spectrofluorometer.Thrombin stimulation of Fura 2 loaded platelets resulted in an immediate eight fold increase in the level of cytoplasmic calcium. The calcium ionophore, ionomycin, stimulated a 15 fold rise in the cytoplasmic calcium of Fura 2 loaded cells.Samples of thrombin and ionomycin stimulated platelets were fixed in glutaraldehyde and osmic acid at the peak of calcium flux indicated by the rise in fluorescence. Examination of activated platelets in the electron microscope revealed shape change and internal transformation. MT coils were constricted, but did not disappear from platelets fixed during the maximum elevation of cytoplasmic calcium. The findings do not support the concept that the calcium rise produced in platelets following exposure to potent agonists causes disappearance of the circumferential MT.
ISOLATION AND CHARACTERIZATION OF HUMAN PLATELET MEMBRANE GLYCOPROTEINS Ia AND IIa
Authors: Bienz D , Wager T , Clemetson K J 
Published: August 2018

Glycoproteins (GP) Ia and IIa are relatively minor components of the platelet surface with similar molecular properties. Nieuwenhuis et al. (Nature 319, 470-72, 1985) described a patient whose platelets show no response to collagen. The correlating lack of GPIa in the platelets of this patient suggests this glycoprotein being the receptor for collagen. Santoro (Cell, 46, 913-20, 1986) described a 160 kDa glycoprotein that binds to collagen in the presence of MG2 + and is possibly identical with GPIa. The role of GPIIa is still unknown but a similar molecule has also been found on endothelial cells. It has been suggested that GPIa and GPIIa are complexed with a further membrane component GPIc. The two glycoproteins show only slight difference in molecular weight, isoelectric point and in their affinity for various lectins. As a result they coisolate using most separation techniques.GPIa is usually associated with the cytoskeleton while GPIIa is mostly found in the soluble phase. GPIa is dissociated from the cytoskeleton by addition of 2% SDS (final conc.) and sonication. Performing Triton X-114 phase partition, GPIIa is found in the detergent phase. After the detergents of the GPIa and GPIIa enriched protein solutions are exchanged with the non-ionic octanoyl-N-methyl glucamide, the glycoproteins are further purified by affinity chromatography on wheat germ agglutinin-Sepharose followed by Lens culinaris lectin-Sepharose both of which bind GPIa and GPIIa. A major contaminant during the purification is GPIb. Final purification of GPIa and GPIIa was obtained by preparative SDS-PAGE using electroelution into a membrane trap. Latest results show an enrichment of GPIa and a lack of GPIb in pseudopodes, isolated by the method of Rotman et al. (Proc. Natl. Acad. Sci. USA, 4357-61, 1982).
“NEW” COAGULATION INHIBITORS LEVELS IN PNEUMONIA DISSEMINATED INTRAVASCULAR COAGULATION AND LIVER DISEASES
Authors: Andersson T R , Bell H , Sandset P M , Ødegaard O R , Aamodt L-M 
Published: August 2018

The activity levels of the “new” coagulation inhibitors, heparin cofactor II (HC II) and extrinsic pathway inhibitor (EPI),have been determined with chromogenic substrates assays, in patients with pneumonia (n=8), disseminated intravascular coagulation (DIC) (n=8) and various liver diseases (n=19). For comparison antithrombin (AT) and Protein C (PC) were also measured. In cases with DIC low values (<50%) for HC II,AT and PC were found, while EPI showed a much greater variation (60-190%). Persistent low values heralds a poor prognosis.In survivors is rapidly normalized. In pneumonia , initially low levels (except HC II),were normalized on day 7. HC II may be an acute phase reactant.Conclusion.In cirrhosis, subnormal HC II values suggests reduced synthesis.High EPI values in cirrhosis suggests extrahepatic synthesis.The mechanisms for reduced HC II in DIC,might besides consumption and reduced synthesis, be the liberation of dermatan sulfate from injured intima with increased consumption.
Changes in HC II,AT and PC are similar, whereas EPI seems to have different production and metabolism
A HIGH-YIELD FACTOR VIII CONCENTRATE SUITABLE FOR ADVANCED HEAT TREATMENT
Authors: McIntosh R V , Docherty N , Fleming D , Foster P R 
Published: August 2018

The heat treatment of Factor VIII concentrates in the freeze-dried state has been widely adopted to inactivate Human Immunodeficiency Virus (HIV) which may be present. However, the degree of heating that can be applied has been limited by adverse effects on product yield and quality (eg.solubility).The established SNBTS product (1) can be heated for up to 2 hours at 68°C in the absence of stabiliser and for up to 24 hours at 68°C in the presence of 2% sucrose.These conditions are believed to inactivate HIV but are not considered sufficient to destroy the hepatitis viruses.Therefore, a new product has been developed which can be heated more severely (eg.80°C for 72 hours) with the objective of destroying all potential viral contaminants while retaining good product solubility and a yield consistent with the maintenance of national self-sufficiency.The process combines Zn++ precipitation of cryoprecipitate (2) and Ca++ stabilisation of VIII:C (2,3) with developments in extraction, adsorption, formulation, freezing and freeze drying.Cryoprecipitate is extracted in an equal volume of tris buffer; further purification is achieved by precipitation with cold zinc acetate and adsorption with Al(OH)3.The supernatant solution is stabilised by adding Na3 citrate, CaC12, NaCl and sucrose; then concentrated and diafiltered to a final formulation of 20mM tris, 130mM NaCl, 30mM Na3 citrate, 4mM CaC12 and 3% sucrose.The product is dispensed at 15ml (VIII:C 20iu/ml) for reconstitution in 20ml.A critical part of the process is a special 2-stage freezing procedure where the product is supercooled to -5°C before freezing to -50°C. Sublimation is completed with the product temperature held below -30°C at a pressure of 0.08 - O.10mbar.This process has been carried out at full-scale (750 litres plasma) giving a readily soluble, improved, intermediate -purity product with an VIII:C yield of 300iu/litre plasma after heat treatment.
RELATIVE THERAPEUTIC EFFICACY AND MOLECULAR WEIGHT DISTRIBUTION OF DRY-HEATED AND n-HEPTANE-HEATED PREPARATIONS OF FACTOR VIII
Authors: Abramson S B , Yang J , Gomperts E D , Kasper C K , Fedor E J 
Published: August 2018

Recent reports by Kemoff et al. (1985) and Gomperts et al. (1987; a multicentered multinational trial) showed that Factor VIII (Antihemophilic Factor, or AHF) “wet” heat-treated in n-hep-tane (Profilate Heat-TreatedR) presented a lower risk of transmitting non-A, non-B hepatitis and HIV than AHF products heated as lyophilized powders. No direct comparison has been reported, however, of the therapeutic efficacy of these products. We compared recovery and half-life in vivo for Profilate Heat-Treated11 with those of the dry-heated products HT ProfilateR and Koate HTR. Two sets of six subjects with severe hemophilia A were infused with either Profilate Heat-TreatedR or a dry-heated AHF in a crossover trial, and blood samples were drawn at times from 10 min to 24 hr. Half-life was determined from a linear regression plot of log (plasma AHF) vs. time from 1 hr to 24 hr. The table gives the mean ± one standard deviation of initial recovery and half-life for each product comparison. Unpaired t-tests showed no significant differences between products. Spearman’s rank analysis showed a high degree of correlation for both the initial recovery and half-life of each product pair.Analysis of molecular weight (MW) distributions of Factor VIII:C polypeptides in several commercial products using the method of Weinstein showed the majority of AHF in all products tested to have MW = 100,000 - 110,000. H.T. FactorateR, which exhibited a substantial amount of the AHF polypeptide whose MW approximates 210,000, is reported by the manufacturer to have a half-life = 11 ± 3.9 hr. We thus conclude that the 210,000 MW form of AHF is
not required for therapeutic efficacy.
EVIDENCE FOR SELF-ASSOCIATION OF PROTHROMBIN FRAGMENT 1 IN THE ABSENCE OF CALCIUM IONS: IMPLICATIONS FOR THE INTERPRETATION OF COOPERATIVITY OF CALCIUM BINDING
Authors: Jackson Craig M , Brenckle George M , Hogg Philip J , Winzor Donald J 
Published: August 2018

The necessity to consider the binding of metal ion by prothrombin and its fragment 1 in terms of two entirely different mechanistic models has been removed. Cooperativity of Ca+T binding to prothrombin and prothrombin fragment 1 may reflect isomerization and/or self association of the protein. Sedimentation equilibrium studies have demonstrated that both prothrombin and its fragment 1 reversibly dimerize in the absence of Ca++.Based on this pre-existing equilibrium, a model for preferential binding of Ca++ to the dimer has been found capable of accounting quantitatively for the interaction of Ca2+ with fragment 1. This phenomenon is described by the relationship
r ={pkA[A][S] (1 + kA[S])p-1 + qkc X [A]2[S](1 + kc [S])q-1}/[A]
in which X (1,000 M-1) denotes the association constant for the pre-existing monomer-dimer equilibrium, and p, kA (10, 100 M-1) and q, kc (20, 2,000 M-1) are the respective stoichiometries and intrinsic binding constants for the interactions of Ca++ with monomeric and dimeric fragment 1, A. There is also evidence from exclusion chromatography and sedimentation velocity experiments that different isomeric states of prothrombin and its fragment 1 exist. It is therefore proposed that a model based on co-existence of isomeric and dimeric protein states will enable quantitative differences in the Ca++-mediated responses of fragment 1 and prothrombin to be rationalized solely in terms of differences in the relative magnitudes of equilibrium constants for the same interactions in the two systems.
A FUSION PROTEIN OF THE A-CHAIN OF t-PA WITH LOW Mr scu-PA COMBINES THE FIBRIN-SPECIFICITY OF BOTH MOLECULES
Authors: Lijnen H R , Nelles L , Lemmens G , Collen D , Holmes W E 
Published: August 2018

A hybrid human cDNA was constructed by ligation of a cDNA fragment of tissue-type plasminogen activator (t-PA), encoding 5∲-untranslated, the pre-pro region and amino acids Ser 1 through Thr 263, with a cDNA fragment of urokinase-type plasminogen activator (u-PA), encoding amino acids Leu 144 through Leu 411. The hybrid cDNA was expressed in Chinese Hamster Ovary Cells and the translation product purified from the conditioned cell culture media in the presence of aprotinin. On SDS-gel electrophoresis under reducing conditions, the protein migrated as a single band with approximate Mr 70,000 and on immunoblot-ting, it reacted with rabbit antisera raised against human t-PA and against human u-PA. The urokinase-like amidolytic activity (S-2444) of the protein’ was 320 IU/mg but increased to 43,000 IU/mg after treatment with plasmin, which resulted in conversion of the single chain molecule (t-PA/scu-PA) to a two-chain molecule (t-PA/tcu-PA).Both proteins activated plasminogen directly with Michaelis constant (Km) 1.5 μM and catalytic rate constant (km2) 0.0058 s-1 for t-PA/scu-PA and with K = 80 μM and = 5.6 s-1 for t-PA/tcu-PA. CBNr-digested fibrinogen stimulated the activation rate of plasminogen with t-PA/tcu-PA (increase of k2/Km of 88-fold).Both t-PA/scu-PA and t-PA/tcu-PA bound specifically to fibrin albeit ^np$re weakly than t-PA. In an in vitro system composed of a human I-fibrin labeled plasma clot immersed in human plasma, the t-PA/tcu-PA hybrid has a higher fibrin-selectivity of clot lysis than tcu-PA, but this difference was not evident between t-PA/scu-PA and scu-PA. The stability of the t-PA/scu-PA hybrid in plasma was much higher than that of the t-PA/tcu-PA hybrid, a difference comparable to that between scu-PA and tcu-PA.It is concluded that these t-PA/u-PA hybrid proteins combine fibrin-affinity of t-PA with the enzymatic properties of u-PA (either scu-PA or tcu-PA), resulting in improved fibrin-mediated plasminogen activation.
PROCOAGULANT ACTIVITY OF PLATELET LIPCKYGENASE PRODUCTS
Authors: Gray E , Kerry P J , Edwards S J , Barrowcliffe T W 
Published: August 2018

Arachidomc acid is metabolised by cyclo-oxygenase and lipoxygenase enzymes in platelets. Cyclo-oxygenase produces the highly pro-aggregatory thromboxane A2 but the physiological significance of the lipoxygenase pathway in platelets remains uncertain. Arachidonic acid can also be converted to biologically active peroxides by free radical induced autoxidation, and previous studies have shown that such products generate large amounts of thrombin in platelet-free plasma, via their interaction with plasma lipoproteins.In the present study, we have investigated the procoagulant activities of platelet lipoxygenase products and compared them with autoxidised arachidonic acid. Platelet concentrates were incubated with arachidonic acid and indomethacin for 30 minutes and the products extracted with ethyl acetate. After drying down, reconstitution in ethanol and partitioning with petrolehm ether to remove unchanged acid, contaminating platelet phospholipid was removed by TLC. By inclusion of 14C-arachidonic acid, average conversion was estimated as 17% in six experiments.The products promoted the generation of large amounts of thrombin in platelet-free plasma; the peak thrombin averaged 23.1 iu/ml with three different batches. The activity was similar to that of a procoagulant phospholipid (PL) but, unlike PL, the lipoxygenase products did not shorten the kaolin recalcification time, confirming the absence of platelet PL contamination, and were virtually inactive in lipoprotein-free plasma. The activity of the lipoxygenase products was therefore similar to that of autoxidised arachidonic acid in its requirement for plasma lipoproteins. Further TLC separation of the products showed that the activity was associated with a minor component of the mixture which was active at plasma concentrations below 10 μg/ml.These results suggest a possible role for platelet lipoxygenase products in the coagulation system and provide a novel link between platelets, lipoproteins and coagulation which could be inportant in the pathogenesis of atherosclerosis.