bluuebunny/crossref_metadata_2025 huggingface dataset:
FLUORESCENCE STUDIES OF THE BINDING OF FACTOR Va TO PHOSPHOLIPID VESICLES
Authors: Krishnaswamy S , Mann K G
Published: August 2018

The free sulphydryl groups in factor Va were analyzed under native and denaturing conditions using dithio-bis-nitrobenzoic acid (DTNB). The heavy chain (D) and the light chain (E) of factor Va were found to contain one free sulphydryl each under native conditions and following denaturation. Intact factor Va contained two free sulphydryls after denaturation while only one of these groups was accessible to DTNB under native conditions. Analysis of the rates of modification of the accessible sulphydryl in factor Va, and direct visualization of factor Va reacted with fluorescent sulphydryl modifiers and analyzed by SDS-PAGE, indicated that the accessible group was present in component D. Factor Va was labelled with the sulphydryl-directed flurophore N-pyrene-l-malemide with no loss in functional activity. Fluorescently modified Va (PYR-Va) contained between 0.83 and 0.91 moles pyrene and showed a concomittant loss in the free sulphydryls accessible to DTNB. Fluorescence polarization studies indicated that the binding of PYR-Va to phospholipid vessicles composed of phosphatidylcholine (PC) and phosphatidylserine (PS) was accompanied by a significant increase in the fluorescence polarization of the pyrene moiety. Systematic analysis of the binding of PYR-Va to PCPS (75%PC, 25%PS) indicated that the binding interaction was characterized by a Kd=2.7x10−9 M and a stoichiometry of 42 monomeric phospholipids per lipid combining site. The binding of PYR-Va to PCPS was independent of added calcium ion and could be reversed by the addition of unlabelled factor Va. Analysis of the displacement curves indicated that native factor Va and PYR-Va mutually excluded each other with identical affinities (Kd=2.5×10−9 M). Isolated component D had no effect on the interaction between PYR-Va and PCPS, while isolated component E was capable of disrupting the PYR-Va-PCPS interaction (Kd=3.1×10−9 M%) indicating that this subunit entirely mediates the interaction between Va and PCPS. No detectable binding of PYR-Va was observed when 100% PC vesicles were used and systematic variation of the PS content of the vesicles indicated that this parameter linearly influenced the stoichiometry of the PYR-Va-PCPS interaction with a minimal effect on the dissociation constant for the reaction. The data indicate that the factor Va-binding site is determined by the PS content of the lipid bilayer.

(Supported by NIH grants HL-35058 and HL-34575)
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)
IMMUNOELECTROPHORETIC CHARACTERIZATION OF SYSTEMIC FIBRINOGEN DURING THROMBOLYSIS OF PERIPHERAL ARTERIAL EMBOLI WITH tPA
Authors: Shainoff John R , Hishikawa-Itoh Youko , Lucas Fred M , Graor Robert , Healy Bernadine
Published: August 2018

Prompted by uncertainties over the possibility of ex-vivo alterations of fibrinogen in plasma samples from patients receiving thrombolytic agents, we sought to 1) use sodium dodecyl sulfate (SDS) and mild acidification to pH 5 as a general means for inhibiting post-sampling proteolysis, and 2) assess the chemical state of fibrinogen by immuno-electrophoretically profiling the molecular weight distribution of the fibrinogen-related antigens in the sample. Blood samples taken into EDTA and PPACK were immediately centrifuged, and plasma diluted 5X in 2% SDS and 0.04% monochloroacetic acid. Such samples showed no changes in molecular weight distribution of fibrinogen-related antigens over 7 days storage at room temperature when analyzed by SDS-eleetro-phoresis on 3% glyoxyl agarose foHowed by fixation with NaCNBHg3 and staining with fluorescent, affinity-purified anti-fibrinogen antibody. The method of study was applied to 16 patients with occlusive peripheral arterial emboli, all of which were successfully treated by catheter-directed administration of tissue plasminogen activator (tPA). All patients presented abnormally high levels of degraded (predominantly 170-300 kD) and dimeric forms of fibrinogen both prior and subsequent to treatment, and only 3 of the 16 patients underwent appreciable change in content or composition of fibrinogen derivatives in course of treatment. Concentrations of dimers ranged from 6 to 23 percent of the total fibrinogen. De novo elevations in degraded forms of fibrinogen observed to accompany treatment of 3 patients were reflected in prolongation of thrombin time while tests of sulfite precipitation underwent relatively minor change. The more frequent absence of change in content of derivatives, particularly the dimeric forms suggestive of a coagulopathic process, indicated that they were derived systemically rather than from the occluding thrombus that was removed by treatment. The findings raise a prospect that the clinically significant thrombosis in these patients may be symptomatic of a much more generalized vascular coagulopathic process. The absence of measurable change in molecular composition of fibrinogen-related antigens in course of successful treatment of these patients attests to the efficacy of tPA as a thrombolytic agent. Support: NIH Grants HL-19361 and HL-19767.
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, Darmstadt

In 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 kininogen

When 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 enzymes

It 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
IDENTIFICATION OF A NEUTROPHIL ELASTASE CLEAVAGE SITE ON THE Act -CHAIN OF PRIMATE FIBRINOGEN
Authors: Weitz J , Landman S , Birken S
Published: August 2018

Human neutrophil elastase (HNE) cleaves the Aα21-22 bond of fibrinogen thus releasing the fibrinopeptide A (FPA)-containing fragment Aαl-21. Plasma Aal-21 levels reflect in vivo HNE activity and peptide levels are increased in cigarette smokers and patients with chronic lung disease. To further explore the HNE-fibrinogen interaction, we set out to develop an animal model. The digestion of purified baboon and marmoset fibrinogen by human thrombin, HNE and extracts of baboon and marmoset neutrophils was monitored with a specific radioimmunoassay for human FPA. Thrcmbin produced quantitative release (2 mol/mol fibrinogen) of FPA. In contrast, HNE and the neutrophil extracts did not release FPA, but rather, produced quantitative release of a larger, FPA-containing fragment. Immunochemically, this fragment was clearly distinguishable from FPA in that in vitro thrombin treatment increased its immunoreactivity 1,000-fold (thrombin increasable FPA or TIFPA). TIFPA release by the neutrophil extracts was blocked by α1-proteinase inhibitor, a specific HNE inhibitor (MeO-Suc-Ala2-Pro-ValCH2Cl) and an anti-HNE IgG, indicating that elastase was the responsible proteinase and that there was homology between the human and primate enzymes. The products of HNE and neutrophil extract proteolysis of the primate fibrinogens were then separated by high performance liquid chromatography and the TIFPA-containing fractions were subjected to amino acid sequence analysis. The FPA-containing fragments each consisted of 21 amino acids, had minor substitutions when compared with human A α] -21 [Baboon: Aα(3) Ser - Thr; Marmoset Aα(l) Ala - Thr, Aα(3) Ser - Thr, Aα(ll) Glu - Ala], and exhibited complete crossreactivity with the human peptide. Using the TIFPA assay, there was good recovery of primate or human Aαl-21 added to primate blood and the mean peptide level in 8 healthy marmosets was similar to that in man (0.5 nM and 0.4 nM, respectively). In conclusion, (1) the Aα;21 -22 bond of baboon and marmoset fibrinogen is a cleavage site for human and primate elastase, (2) baboon and marmoset Aal-21 can be measured with the assay for the human peptide, and (3) the primate serves as a useful model for the study of elastase-fibrinogen interactions.
KINETICS OF THE HEPARIN-DEPENDENT INACTIVATION OF ANTITHROMBIN BY NEUTROPHIL ELASTASE
Authors: Jordan R E , Nelson R M , Kilpatrick J
Published: August 2018

The rate of inhibition of coagulation enzymes by antithrombin III is greatly increased by an interaction between the inhibitor and a limited, anticoagulantly-active subfraction of heparin molecules. We have observed that this same heparin sub-fraction also greatly stimulates the rate of inactivation of antithrombin by neutrophil elastase. Inactivation rates were increased several hundred-fold by catalytic amounts of the anticoagulantly active heparin species and were unaffected by the inactive heparin fraction or other glycosaminoglycans. Addition of catalytic amounts of elastase (1:400) to a solution of antithrombin in the presence of saturating levels of anticoagulantly-active heparin caused an approximately 30% decrease in the ultraviolet fluorescence emission of the inhibitor. The rate of fluorescence loss corresponded exactly with the loss of inhibitory activity in a parallel incubation under the same conditions. The use of the fluorescence technique for kinetic measurement of inactivation rates indicated a Km of less than 1 uM for the heparin-antithrombin complex substrate and a turnover of several hundred per minute per enzyme molecule. Although the specificity of the heparin effect appears to be at the level of its interaction with antithrombin, an elastase-heparin interaction could also be detected in kinetic analyses. Chromatographic studies employing immobilized heparin confirmed that elastase itself binds tightly to the complex carbohydrate. These results suggest a subtle mechanism, of potential relevance to thrombosis associated with inflammatory conditions, in which both heparin and elastase act catalytically to direct the inactivation of antithrombin. Since anticoagulantly-active heparin species are present on endothelial cells, the above mechanism could markedly affect the balance between procoagulant and anticoagulant processes on the usually non-thrombogenic endothelial surface.
THE IDENTIFICATION OF VIMENTIN, AN INTERMEDIATE,FILAMENT SUBUNIT PROTEIN IN HUMAN PLATELETS
Authors: Muszbek L , Adåny R , Glukhova M A , Frid M G , Kabakov A E , Kot-e-liansky V E
Published: August 2018

In platelets the presence of basic subunit proteins of microtubules as well as microfilaments has been verified a long time ago and it was also shown that both of these cytoskeletal systems go through a tremendous reorganization during the activation process. Surprisingly, none of the components of intermediate filaments has so far been identified in these cells, perhaps because platelets were considered too motile to have intermediate filaments, the most static structures among the three major cytoskeletal systems. By using two different monoclonal antibodies (II C4 and II D8) here we attempted to establish if vimen-tin, an intermediate filament subunit protein present in most differentiating cells, in cells grown in tissue culture and in certain fully differentiated cells also exists in human platelets The IgM type antibodies were characterized by various immuno-morphological and immunobiochemical techniques. They labelled selectively colcemid-sensitive filamentous structures in fibroblasts, in endothelial cells and in vascular but not myometrium smooth muscle and were shown to be monospecific against epitopes on vimentin in fibroblast homogenate. When whole platelet homogenate was submitted to high resolution gradient SDS PAGE and then electroblotted to nitrocellulose sheet, both antibodies reacted with a single protein band of 55 kD that comigrated with fibroblast vimentin. By immunofluorescence microscopy an annular ring-like structure was stained for vimentin that suggests a membran skeletal role for this protein. During pseu-dopode formation a redistribution of vimentin could be observed. Parallel with the disappearance of ring-like structures vimentin appears in pseudopodia suggesting that like in the case of other filamentous systems platelet activation induces a structural reorganization of the intermediate filaments, as well.
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.
PURIFICATION AND PROPERTIES OF HUMAN PLATELET MEMBRANE GLYCOPROTEIN V (GP-V)
Authors: Zafar Rasheeda , Walz Daniel A
Published: August 2018

Platelet membrane glycoproteins function as specific ligand receptors or substrates for selected platelet agonists and antagonists. GP-V is the only such membrane glycoprotein known to be a thrombin substrate. We have purified GP-V to homogeneity in order to better characterize the nature and specificity of this thrombin proteolysis. GP-V was extracted from fresh human platelets and purified through a combination of gel filtration, hydroxylapatite, DEAE and mono S chromatographies. The resulting protein had a molecular mass of 80 kDa by both non-reduced and reduced SDS electrophoresis. AminortemtnaJ analyses of GP-V failerLto yield any detectable residues, suggesting a blocked N-terminus. Incubation of purified GP-V with 1 nM human thrombin for 20 minutes resulted in the generation of a stable (thrombin resistant) fragment of 63 kDa; smaller GP-V peptides have not been identified. The amino-terminus of the 63 kDa GP-V fragment is also blocked, indicating that thrombin proteolysis is occurring at the carboxyl-terminal region of the protein. Purified GP-V is also a substrate for human γ-thrombin, although its rate of proteolysis is much slower than that observed using γ-thrombin; the 㬱- and γ-thrombin generated GP-V fragments are electrophoretically similar. Polyclonal antibodies raised against purified GP-V react equally with the GP-V fragment. Such antibodies have been used in immunoblotting procedures to screen bovine platelets, human endothelial cells and a transformed erythroid cell line known to express other platelet proteins; all these cells failed to give a positive reaction, suggesting the absence of GP-V in these cell membranes or its presence in an amount significantly lower than that observed for human platelets.
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).
PLACENTAL ANTICOAGULANT PROTEIN
Authors: Fujikawa K , Funakoshi T , Tait J F , Heimark R L
Published: August 2018

An anticoagulant protein was purified from the soluble fraction of human placenta by ammonium sulfate precipitation and column chromatography on DEAE-Sepharose, Sephadex G-75, and mono S (Pharmacia). Approximately 30 mg of the protein was purified from one placenta. The purified protein gave a single band on SDS polyacrylamide gel and had a molecular weight of 36,500. This protein inhibited both kaolin and thromboplastin induced clotting times of normal human plasma. It also inhibited the clotting time of platelet-rich plasma induced by factor Xa, but did not affect thrombin activity of fibrinogen-fibrin conversion. The protein neither bound factor Xa nor inhibited the amidase activity of factor Xa. This protein specifically bound to phospholipid vesicles prepared from a mixture of phosphatidylserine and phosphatidylcholine (20 to 80 weight ratio) in the presence of calcium ions. The purified protein was digested with cyanogen bromide and the resulting fragments were separated by FPLC. Partial amino acid sequences of the cyanogen bromide fragments showed that this protein was composed of at least three repeats that were homologous to the four repeats found in lipocortin I and II. Lipocortins are known to inhibit the phospholipase A2 activity, probably by binding to the phospholipid substrate. These results indicate that the placental anticoagulant protein is a member of the family of lipocortins and probably inhibits coagulation by binding to phospholipid vesicles. Supported in part by grants HL 16919 and HL 18645 from National Institute of Health.
“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.
FACTOR VIII:C PURIFIED FROM PLASMA VIA MONOCLONAL ANTIBODIES: HUMAN STUDIES
Authors: Brettler D , Forsberg A A , Levine P , Petillo J , Lamon K , Sullivan J
Published: August 2018

Factor VIII:C purified utilizing a mouse monoclonal antibody to FVIII:VWF was used exclusively for 6 months to treat hemorrhages on a demand basis in 7 patients with severe hemophilia A and no inhibitor. The mean age of the patients was 26.1 years with a range of 16-39 years. The dose administered was from 15-18 u/kg at the time of hemorrhage. The patients infused on the average of once every 5 days. One patient required 40-50 u/kg every day for 3 days secondary to an automobile accident. Laboratory assessments included: a fall-off study to determine the factor VIII half-life both when the study commenced and after 6 months on study; inhibitor levels, and ELISA assay to detect antibody to mouse protein. Additionally, immunological data including quantitative T cell subsets and skin testing on each patient was obtained on entrance to the study, at 1 month, 3 months and at conclusion in order to ascertain whether immune function in these patients would improve with the use of purer factor concentrate. The initial mean half-life of the infused concentrate was 15.4 hours ± 2.2 with a range of 12.6 to 18.4 hours. The mean half-life after the patients had been on the concentrate for 6 months was 17.5 ± 5.9 hours (range 11.0 to 29 hours). No inhibitor developed in any patient. Six of seven patients did not develop significant levels of anti-mouse IgG antibody. One patient had a rheumatoid factor which interfered with the ELISA assay for anti-mouse antibody and thus its presence could not be assessed. There were no adverse reactions to the material, and hemostatic efficacy was judged as excellent. There were no significant changes in quantitative T cell subsets. Three out of six patients lost their previous total skin test anergy and two other patients increased the number of antigens to which they reacted. This concentrate proved to be safe and efficacious, to have excellent half-life, and to be associated with apparent improvement in the immune response.
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.
MOLECULAR DEFECT IN PROTHROMBIN MADRID: SUBSTITUTION OF ARGININE 273 BY CYSTEINE PRECLUDES ACTIVATION
Authors: Rabiet M J , Furie B C , Furie B
Published: August 2018

Prothrombin Madrid, a mutant prothrombin, was detected in a patient with a excessive bleeding history. The defect was characterized by a low coagulant activity contrasting with a normal level of prothrombin antigen in plasma. Activation of the purified protein was impaired by the absence of one of the two factor Xa catalyzed cleavages, generating meizothrombin which expressed a thrombin-like activity but was inactive on fibrinogen (Guillin et al., Ann. N.Y. Acad. Sci. 370:414, 1981). Prothrombin and prothrombin Madrid were isolated directly from plasma, with high yield, by immunoaffinity chromatography using conformation specific antibodies immobilized on Sepharose. After reduction and alkylation, purified proteins were hydrolyzed by trypsin. Resulting peptides were separated by reverse phase HPLC. Comparison of the two peptide maps showed that the prothrombin Madrid digest contained an additional peptide, identified by automated Edman degradation as residues 269 to 287 in prothrombin with the substitution of cysteine for arginine at position 273. Peptide 274—287, present in the prothrombin digest, was missing in the prothrombin Madrid digest. The mutation, precluding cleavage by factor Xa and normal generation of thrombin, is identical to the one described for prothrombin Barcelona. The two patients families are not related, raising the possibility that the gene coding for the cysteine 273 mutation in prothrombin is more common than anticipated. Of the seven mutants of vitamin E-dependant blood clotting proteins structurally characterized to date, three are functionally defective due to the presence of the propeptide on the mature amino-ternfinus (factor IX Cambridge, Oxford 3 and San Dimas) and three are due to an alteration that precludes zymogen activation (faotor TX Chapel Hill, prothrombin Barcelona and Madrid). This sample remains too small to anticipate the different classes of point mutations seen in the human population but functional abnormalities of protein processing, metal and lipid binding, zymogen activation, substrate recognition and enzyme catalysis will likely be important phenotypes. However genetic defects may be limited to a discrete group of point mutations that have significant functional implication for the proteins
A SHARED EPITOPE ON HUMAN PROTEIN C, FACTOR X, FACTOR VII, AND PROTTOBIN DEFINED BY A MONOCLONAL ANTIBODY
Authors: Church W , Messier T , Howard P , Amiral J , Meyer D , Mam K
Published: August 2018

A monoclonal antibody prepared against hunan protein C (HPC) was found to react with several other vitamin K-dependent blood proteins. Using a competitive inhibition solid-phase radioinminoassay with HPC, binding of 125I-HPC to the antibody was inhibited by purified prothrombin, Factor X, and Factor VII in addition to protein C. Other vitamin K-dependent proteins including Factor IX, protein S, and bone-GLA protein did not compete for binding of 125I-HPC to the antibody. The effect of calciun ion on the binding of antibody to 125I-HPC was examined in a solid-phase imnunoassay system with the antibody bound to rabbit anti-mouse inminoglobulin adsorbed to microtiter plates. In the presence of 5 mM calciun ion, radiolabeled protein C did not bind to the antibody; radiolabeled protein C did bind, however, in the presence of 5 nM EDTA suggesting that the epitope is expressed only after removal of calciun ion. The antibody bound to prothrombin and to decarboxylated prothrombin after adsorption of the antigens onto nitrocellulose indicating that the presence of GLA was not required for antibody binding. Iimunoblotting of proteins which were reduced, the peptides separated by SDS-PAGE, and transferred to nitrocellulose showed that the antibody reacts with a determinant found on the light chains of protein C and Factor X and with prothrombin Fragment 1. Comparison of the protein sequences of protein C light chain, Factor X light chain, Factor VII, and prothrombin Fragment 1 identified a segment of amino acid sequence that is highly conserved in all four proteins and might contain the antigenic site. The monoclonal antibody thus defines an antigenic determinant which is masked by calcium ion and is found on the surface of several related, yet different coagulation proteins. This antibody should prove useful in understanding the evolutionary relationships amongst the vitamin K-dependent proteins and also in understanding the effect of calcium ion on the structure of protein C, Factor X, prothrombin, Factor VII and possibly other related proteins. (Supported by NIH grant MHLBI HL35058)
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.
IMAGING OF THROMBI USING ANTI-PADGEM ANTIBODIES SPECIFIC FOR ACTIVATED PLATELETS
Authors: Palabrica Theresa M , Furie Barbara C , Konatam Marvin A , Brockway Barbara , Aronovitz Mart , Furie Bruce
Published: August 2018

PADGEM protein is a platelet-specific alpha grannie membrane protein that is translocated to the plasma membrane during platelet activation and secretion. Monoclonal EC4 and polyclonal anti-PADGEM antibodies are specific for PADGEM on activated platelets. Because PADGEM is internal in unstimulated platelets* these antibodies do not bind to resting platelets. Since activated platelets are concentrated in thrombi, we used radiolabeled anti-PADGEM antibodies to image thrombi in baboons.

In vitro: Dacron graft material incubated with thrombin-activated platelet-rich plasma and 131I-KC4, 131I-anti-PADGEM, 131I-nonimmune IgG or 131-BSA bound 95%, 70%, 9% and 1% of the radiolabel, respectively, after exhaustive washing.

In vivo: Imaging experiments were carried out in baboons with an external Dacron shunt between the femoral artery and vein. Gamma camera images following 123I-anti-PADGEM infusion (1.0 mCi; 300 μg) demonstrated intense uptake in the thrombus induced by the Dacron vascular graft within 10 minutes. The graft:blood pool activity ratio was 33:1. Control experiments with 123I-nonimmune IgG showed minimal uptake.

123I-anti-PADGEM cleared the circulating blood pool with an initial half-disappearance time of 2-6 minutes. The primary routes of metabolism were hepatic uptake, dehalogenation and urinary excretion of free iodine. There was no evidence that the anti-PADGEM antibodies had any adverse effects on the normal processes of hemostasis in any of these experiments.

Anti-PADGEM antibodies, both monoclonal and polyclonal, are directed against activated platelets in thrombi or in areas of tissue injury; they do not bind to resting platelets in the circulation. Radioimmunoscintigraphy with these agents provides a novel approach to the noninvasive detection and localization of thrombi in vivo, with potential application to human disease. Furthermore, linkage of these antibodies to fibrinolytic enzymes offers a strategy for the specific lysis of thrombi in vivo.