Study of a proposed international standard for blood coagulation factor IX

An International Collaborative Study was organized to establish a standard for factor IX. Two freeze-dried concentrate preparations, C1 and C2, and one freeze-dried plasma P were compared with each other, with fresh normal plasmas and with local standards in 13 laboratories. One of the concentrate preparations (C1) contained heparin and this gave rise to non-parallel assays in laboratories testing concentrate C1 in dilutions containing more than 0.05 i.u. of heparin per ml. Assays of factor IX showed good precision for both plasma and concentrate in all laboratories; no systematic effect of method, operator or day of assay was detected. The plasma preparation P and the concentrate preparation C2 were compared with 59 individual fresh normal plasma samples, and a mean potency ratio of 0.78 (95% confidence limits 0.73-0.84) for plasma and 5.62(95% confidence limits 5.13-6.16) for the concentrate C2 obtained. Only 21 estimates of concentrate C1 in terms of fresh plasma were obtained giving a mean potency ratio of 3.85 (95% confidence limits 1.87-7.92). The estimated loss of potency for freeze-dried plasma stored at -20 degrees C is approximately 0.4% per year. The concentrate C2 is apparently more stable and only very small losses occurred even at higher storage temperatures. All participants agreed that the preparation C2 would be suitable to serve as an International Standard for factor IX; they also agreed that the figure assigned for the unitage should be based on the number of ml of 'average fresh normal plasma' estimated to contain the factor IX activity of one ampoule of the preparation. It is proposed to recommend to the World Health Organization that the preparation of factor IX concentrate C2, in ampoules coded 72/32, be considered for establishment as the International Standard for factor IX, and that the international unit for factor IX be assigned on the basis of 5.62 units per ampoule of this preparation.     

Primary structure of bovine Hageman factor (blood coagulation factor XII): comparison with human and guinea pig molecules

An understanding of the consequences of autologous vein grafting reveals both the reasons why cryopreserved allogenic veins are being used clinically and how they are most likely to be expected to fail. Autologous vein bypass grafts are characterized by a series of distinct biological properties that influences their in vivo patency. Current surgical practice ensures that the endothelium of vein grafts is preserved at the time of implantation and that there is minimal damage to the smooth muscle cells. After implantation, the endothelial cells show varying degrees of morphological changes that are maximal within the first 3 days after grafting. In autografts, extensive endothelial denudation does not appear to occur. During the initial grafting period, the smooth muscle cells change from a contractile phenotype to a synthetic phenotype, migrate from the media, proliferate in the intima, and lay down connective tissue. Thereafter, endothelial cell changes regress and the smooth muscle cells return to their contractile phenotype. Perioperative manipulation of vein grafts results in decreased endothelial cell function but preservation of smooth muscle cell responsiveness. Postoperatively endothelial cell-mediated relaxation to acetylcholine is lost and smooth muscle cell contractility is decreased. Within 7 days after implantation, smooth muscle cell contractility returns and, with time, becomes markedly greater than that of the control vein. Endothelium-mediated relaxation to acetylcholine never returns in vein grafts and this loss of endothelial cell function appears to be related to receptor-coupled G-protein defects. Smooth muscle cell contractility remains abnormal. Many of the intimal hyperplastic lesions in vein grafts progress to stenosis or become sites of accelerated atherosclerosis.(ABSTRACT TRUNCATED AT 250 WORDS)     

An activator of blood coagulation factor X from the venom of Bungarus fasciatus

A specific activator of blood coagulation factor X was purified from the venom of Bungarus fasciatus by gel filtration and by ion-exchange chromatography on a Mono-Q column (FPLC). It consisted of a single polypeptide chain, with a mol. wt of 70,000 in reducing and non-reducing conditions. The enzyme had an amidolytic activity towards the chromogenic substrates S-2266 and S-2302 but it did not hydrolyse S-2238, S2251 or S-2222, which are specific substrates for thrombin, plasmin and factor Xa, respectively. The enzyme activated factor X in vitro and the effect was Ca2+ dependent with a Hill coefficient of 7.9. As with physiological activators, the venom activator cleaves the heavy chain of factor X, producing the activated factor Xa alpha. The purified factor X activator from B. fasciatus venom did not activate prothrombin, nor did it cleave or clot purified fibrinogen. The amidolytic activity and the factor X activation activity of the factor X activator from B. fasciatus venom were readily inhibited by serine protease inhibitors such as diisopropyl fluorophosphate (DFP), phenylmethanesulfonyl fluoride (PMSF), benzamidine and by soybean trypsin inhibitor but not by EDTA. These observations suggest that the factor X activator from B. fasciatus venom is a serine protease. It therefore differs from those of activators obtained from Vipera russelli and Bothrops atrox venoms, which are metalloproteinases.     

Effects of dietary fat quality and quantity on postprandial activation of blood coagulation factor VII

Acute elevation of the coagulant activity of blood coagulation factor VII (FVIIc) is observed after consumption of high-fat meals. This elevation is caused by an increase in the concentration of activated FVII (FVIIa). In a randomized crossover study, we investigated whether saturated, monounsaturated, or polyunsaturated fats differed regarding postprandial activation of FVII. Eighteen healthy young men participated in the study. On 6 separate days each participant consumed two meals (times, 0 and 1 3/4 hours) enriched with 70 g (15 and 55 g) of either rapeseed oil, olive oil, sunflower oil, palm oil, or butter (42% of energy from fat) or isoenergetic low-fat meals (6% of energy from fat). Fasting and series of nonfasting blood samples (the last at time 8 1/2 hours) were collected. Plasma triglycerides, FVIIc, FVIIa, and free fatty acids were analyzed. There were marked effects of the fat quantity on postprandial responses of plasma triglycerides, FVII, and free fatty acids. The high-fat meals caused, in contrast to the low-fat meals, considerable increases in plasma triglycerides. Plasma levels of FVIIc and FVIIa peaks were 7% and 60% higher after consumption of high-fat meals than after consumption of low-fat meals. The five different fat qualities caused similar postprandial increases in plasma triglycerides, FVIIc, and FVIIa. These findings indicate that high-fat meals may be prothrombotic, irrespective of their fatty acid composition. The postprandial FVII activation was not associated with the plasma triglyceride or free fatty acid responses.     

Differential interaction of coagulation factor VIII and factor V with protein chaperones calnexin and calreticulin

Factor VIII (FVIII) and factor V (FV) are homologous coagulation cofactors sharing a similar domain organization (A1-A2-B-A3-C1-C2) and are both extensively glycosylated within their B-domains. In mammalian cell expression systems, compared with FV, the FVIII primary translation product is inefficiently transported out of the endoplasmic reticulum. Here we show that FVIII is degraded within the cell by a lactacystin-inhibitable pathway, implicating the cytosolic 20 S proteasome machinery. Protein chaperones calnexin (CNX) and calreticulin (CRT) preferentially interact with glycoproteins containing monoglucosylated N-linked oligosaccharides and are proposed to traffic proteins through degradative and/or secretory pathways. Utilizing co-immunoprecipitation assays, intracellular FVIII was detected in association with CNX maximally within 30 min to 1 h following synthesis, whereas FV could not be detected in association with CNX. In contrast, both FVIII and FV displayed interaction with CRT during transit through the secretory pathway. B-domain deleted FVIII significantly reduced the CNX and CRT interaction, indicating the B-domain may represent a primary CNX and CRT interaction site. In the presence of inhibitors of glucose trimming, the interactions of FVIII with CNX, and of FVIII and FV with CRT, were significantly reduced whereas the secretion of FVIII, and not FV, was inhibited. In addition, transfection in a glucosidase I-deficient Chinese hamster ovary cell line (Lec23) demonstrated that both degradation and secretion of FVIII were inhibited, with little effect on the secretion of FV. These results support that CNX and CRT binding, mediated at least in part by the B-domain of FVIII, is required for efficient FVIII degradation and secretion. In contrast, FV does not require CNX interaction for efficient secretion. The results suggest a unique requirement for carbohydrate processing and molecular chaperone interactions that may limit the productive secretion of FVIII.     

Subtyping of coagulation factor XIIIA

An extended polymorphism of the coagulation factor XIIIA can routinely be detected in human plasma samples and white cell lysates by isoelectric focusing in polyacrylamide gels containing 3 M urea in the pH range 5-8. Analyses of 184 families with 513 children confirmed the formal model proposed by Suzuki et al. [Am J Hum Genet 1988;43:170-174]. Four common alleles, F XIIIA*1A, 1B, 2A, 2B, at an autosomal locus control the expression of ten phenotypes. On the basis of the population sample from southwest Germany the frequencies of the common alleles F XIIIA*1A, 1B, 2A, 2B were calculated as 0.175, 0.609, 0.011, and 0.205, respectively.     

Adeno-associated viral vector-mediated gene transfer of human blood coagulation factor IX into mouse liver

Recombinant adeno-associated virus vectors (AAV) were prepared in high titer (10(12) to 10(13) particles/mL) for the expression of human factor IX after in vivo transduction of murine hepatocytes. Injection of AAV-CMV-F.IX (expression from the human cytomegalovirus IE enhancer/promoter) into the portal vein of adult mice resulted in no detectable human factor IX in plasma, but in mice injected intravenously as newborns with the same vector, expression was initially 55 to 110 ng/mL. The expression in the liver was mostly transient, and plasma levels decreased to undetectable levels within 5 weeks. However, long-term expression of human F.IX was detected by immunofluorescence staining in 0.25% of hepatocytes 8 to 10 months postinjection. The loss of expression was likely caused by suppression of the CMV promoter, because polymerase chain reaction data showed no substantial loss of vector DNA in mouse liver. A second vector in which F.IX expression was controlled by the human EF1alpha promoter was constructed and injected into the portal vein of adult C57BL/6 mice at a dose of 6.3 x 10(10) particles. This resulted in therapeutic plasma levels (200 to 320 ng/mL) for a period of at least 6 months, whereas no human F.IX was detected in plasma of mice injected with AAV-CMV-F.IX. Doses of AAV-EF1alpha-F. IX of 2.7 x 10(11) particles resulted in plasma levels of 700 to 3, 200 ng/mL. Liver-derived expression of human F.IX from the AAV-EF1alpha-F.IX vector was confirmed by immunofluorescence staining. We conclude that recombinant AAV can efficiently transduce hepatocytes and direct stable expression of an F.IX transgene in mouse liver, but sustained expression is critically dependent on the choice of promoter.     

Dietary fatty acids and blood coagulation

Factor VII coagulant activity (VIIc) is a risk factor for coronary heart disease (CHD). Plasma VIIc is positively associated with dietary fat intake, suggesting that fat-rich diets are accompanied by a hypercoagulable state. Reduction in total fat consumption is followed by a decrease in VIIc within 24 h. In adults taking diets rich in long-chain saturated fatty acids, a postprandial increase in VIIc occurs after a fatty meal irrespective of its fat composition. This effect has dose-response characteristics, persists for several hours, and is due to activation of factor VII. There is no acute effect of dietary fat on factor VII antigen (VIIag) concentration, but VIIag is positively related to dietary fat intake. More studies are needed on the effects of dietary fat composition on fasting and postprandial factor VII. Dietary fat appears to influence both the atherosclerotic and thrombogenic components of CHD.     

Structural organization and chromosomal localization of the human hepatocyte growth factor activator gene--phylogenetic and functional relationship with blood coagulation factor XII, urokinase, and tissue-type plasminogen activator

BACKGROUND: This study determined the dose-response relation of intrathecal fentanyl for labor analgesia and described the onset, duration, and quality of analgesia when used as the sole analgesic. METHODS: Eighty-four parturients in active labor who requested analgesia were randomized to one of seven treatment groups. They received 5-45 microg intrathecal fentanyl as part of a combined spinal-epidural technique. Visual analog pain scores were recorded before and at intervals after injection patients requested additional analgesia. The occurrence and severity of pruritus, nausea, and vomiting were also recorded. Maternal blood pressure was recorded before injection and at intervals after injection. Fetal heart rate was recorded before and 30 min after injection. RESULTS: By 5 min after injection, pain scores were significantly different among groups (P < 0.001). Mean duration of analgesia increased to 89 min as the dose increased to 25 microg. Maternal diastolic blood pressure was significantly lower 10 and 30 min after injection. There was no difference among groups in the incidence of pruritus; nausea and vomiting were uncommon. Fetal heart rates did not change after injection. A dose-response curve indicates that the median effective dose of intrathecal fentanyl for labor analgesia is 14 microg (95% confidence interval, 13-15 microg). CONCLUSIONS: Intrathecal fentanyl produces rapid, profound labor analgesia with minimal side effects. These data indicate that there is little benefit to increasing the dose beyond 25 microg when it is used as the sole agent for intrathecal labor analgesia.     

Activation of blood coagulation in patients with mitral stenosis and sinus rhythm

The aim of this study was to assess whether there is blood coagulation activation in patients with mitral stenosis (MS) and sinus rhythm (SR) and to investigate the value of left atrial spontaneous echo contrast (LASEC) as a predictive sign of increased coagulation activity. Using thrombin-antithrombin III complexes and prothrombin fragment 1+2 as in vivo hemostatic markers, we concluded that there is a hypercoagulable state in patients with MS and SR when LASEC is present.     

Converting blood coagulation factor IXa into factor Xa: dramatic increase in amidolytic activity identifies important active site determinants

The coagulation factors IXa (fIXa) and Xa (fXa) share extensive structural and functional homology; both cleave natural substrates effectively only with a cofactor at a phospholipid surface. However, the amidolytic activity of fIXa is 10(4)-fold lower than that of fXa. To identify determinants of this poor reactivity, we expressed variants of truncated fIXa (rf9a) and fXa (rf10a) in Escherichia coli. The crystal structures of fIXa and fXa revealed four characteristic active site components which were subsequently exchanged between rf9a and rf10a. Exchanging Glu219 by Gly or exchanging the 148 loop did not increase activity of rf9a, whereas corresponding mutations abolished reactivity of rf10a. Exchanging Ile213 by Val only moderately increased reactivity of rf9a. Exchanging the 99 loop, however, dramatically increased reactivity. Furthermore, combining all four mutations essentially introduced fXa properties into rf9a: the amidolytic activity was increased 130-fold with fXa substrate selectivity. The results suggest a 2-fold origin of fIXa's poor reactivity. A narrowed S3/S4 subsite disfavours interaction with substrate P3/P4 residues, while a distorted S1 subsite disfavours effective cleavage of the scissile bond. Both defects could be repaired by introducing fXa residues. Such engineered coagulation enzymes will be useful in diagnostics and in the development of therapeutics.     

State of blood coagulation and capillary permeability in patients with lung cancer

Cinematography was used to biomechanically compare individually selected overground jogging and running velocities with equated treadmill jogging and running by adult males and females. All subjects were regular joggers but not competitive track or cross-country runners. No significant differences were recorded in stride length, stride rate, support time or non-support time for males or females when jogging at velocities of between 3.33 and 4.78 m/s (x = 3.70) or 3.45 and 4.80 m/s (x=3.97) respectively. However, it was demonstrated that at velocities of 4.82 - 6.2 m/s for males (x - 5.41) and 4.85 - 5.76 m/s for females (x = 5.29) significant differences did occur between overground and treadmill running. For both males and females stride length decreased, stride rate increased, and the period of non-support was also significantly less when running on a treadmill as compared to running overground.     

Purification and characterization of human coagulation factor V

We have purified human coagulation Factor V 6,000-fold to homogeneity from citrated plasma using polyethylene glycol 6000 precipitation, adsorption of Factor V to barium citrate, DEAE-Sepharose chromatography, and gel filtration on Ultrogel AcA 34 (yield 21%). Human Factor V is a single polypeptide chain before and after disulfide bond reduction with an apparent Mr = 335,000 as determined by electrophoresis on 5% acrylamide sodium dodecyl sulfate gels. Human Factor V is a glycoprotein containing 13% of weight carbohydrate and there is a high content of sialic acid (86 residues/mol) compared to the other sugars. When human Factor V is treated with thrombin, coagulation activity increases 25- to 30-fold to a specific activity of 1.7 to 2.0 units/microgram. Thrombin activation is accompanied by the cleavage of three bonds in the Factor V molecule. We have detected activation intermediates with apparent Mr = 295,000 and 248,000 and final products with apparent Mr = 150,000, 121,000, and a doublet at 95,000-91,000 as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The final products of thrombin activation of human Factor V and bovine Factor V are similar, yet the intermediates observed are different. This suggests that cleavages are made at similar locations in bovine and human Factor V, but that they occur in a different sequence. When human Factor V is treated with the Factor V activator from Russell's viper venom, it is split into two components with apparent Mr = 303,000 and 95,000-91,000 and is fully activated. The increase in coagulation activity observed upon treatment of human Factor V with thrombin or the Factor V activator from Russell's viper venom seems to correlate with the generation of the doublet Mr = 95,0090-91,000 component.     

Labeling of a major fibroblast surface protein (fibronectin) catalyzed by blood coagulation factor XIIa

Incubation of cultured human fibroblasts with blood coagulation factor XIIIa (plasma transglutaminase, fibrinoligase) and the fluorescent primary amine, N-(5-aminopentyl)-5-dimethylaminonaphthalene-1-sulfonamide, resulted in fluorescent labeling of three cellular polypeptides. The molecular weights of the labeled polypeptides, estimated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate after reduction, were: greater than 1.2-10(6), 2.2-10(5), and 1.3-10(5). The labeled 2.2-10(5) dalton polypeptide was susceptible to mild trypsinization and not present in cultures of SV-40 transformed fibroblasts, indicating that it is the subunit of cell-surface fibronectin and identical with the external transformation-sensitive polypeptide of similar molecular weight described by others. Upon coelectrophoresis, the labeled 2.2-10(5) dalton polypeptide migrated slightly behind the subunit of plasma fibronectin (cold-insoluble globulin), indicating that the immunologically cross-reactive forms of fibronectin in human plasma and cultured human fibroblasts differ slightly in molecular weight. The identities of the labeled greater than 1.2-10(6) and 1.3-10(5) dalton polypeptides are not known. The XIIa-reactive glutamine residues of fibroblast cell-surface proteins are potential sites for intermolecular cross-linking (by xi-(gamma-glutamyl)lysyl linkages) to other proteins of connective tissue.     

Whole blood platelet aggregation and coagulation factors in patients with systemic sclerosis

It is unclear whether the changes in platelet function which are observed in systemic sclerosis are a primary characteristic of this disease or whether they occur secondary to vascular changes. Whole blood platelet aggregation was studied in 26 patients with systemic sclerosis, normal subjects matched for age, sex and secondary characteristics, 19 patients with Raynaud's disease and 19 patients with systemic lupus erythematosus. Plasma levels of fibrinogen, von Willebrand factor antigen and factor VIII:C were also measured. Systemic sclerosis was associated with a significant (P > 0.001) enhancement of the sensitivity of platelets to collagen. In contrast, significant enhancement of the response to either ADP or adrenaline was not observed. Enhanced sensitivity to collagen was not associated with the presence of either Raynaud's disease or systemic lupus erythematosus. Systemic sclerosis was associated with significantly raised levels of von Willebrand factor antigen and fibrinogen. On an individual patient basis, von Willebrand factor antigen was related to the severity of the disease whereas platelet sensitivity to collagen was not. In conclusion, this study suggests that the enhanced sensitivity to collagen which occurs in systemic sclerosis is due to a primary change in the platelet and that this change can combine with elevated levels of adhesive proteins.     

Effects of tannins from Geum japonicum on the catalytic activity of thrombin and factor Xa of blood coagulation cascade

Bioassay-guided fractionation of the MeOH extract of the whole plant of Geum japonicum led to the isolation of seven known tannins. They were identified by spectroscopic methods as penta-O-galloyl-beta-glucoside (1), pedunculagin (2), 2, 3-(S)-hexahydroxydiphenoyl-D-glucose (3), tellimagrandin II (4), 2, 6-di-O-galloyl-D-glucose (5), casuariin (6), and 5-desgalloylstachyurin (7). Compounds 1, 2, 4, 6, and 7 showed potent anticoagulant activity by significantly prolonging the clotting of rabbit plasma. The inhibitory effect of 2 was competitively directed against thrombin. Its IC50 values for inhibition of the enzymatic activity of thrombin on synthetic substrate and fibrinogen were 0.18 and 0.15 microM, respectively. On the other hand, compounds 1, 4, 6, and 7 are mixed noncompetitive inhibitors of thrombin. Their IC50 values for inhibition of fibrinogen hydrolysis were twofold to sevenfold lower than those for the inhibition of synthetic substrate hydrolysis. Factor Xa was competitively inhibited by compounds 1, 2, 4, 6, and 7. The phenolic hydroxyl groups of the active tannins appear to play an important role in their inhibitory effect on the enzymes.     

Characterization of recombinant human coagulation factor XFriuli

Naturally occurring plasma factor XFriuli (pFXFr) is marginally activated by both the extrinsic and intrinsic coagulation pathways and has impaired catalytic potential. These studies were initiated to obtain confirmation that this molecule is multi-functionally defective due to the substitution of Ser for Pro at position 343 in the catalytic domain. By the Nelson-Long site-directed mutagenesis procedure a construct of cDNA in pRc/CMV was derived for recombinant factor XFriuli (rFXFr) produced in human embryonic (293) kidney cells. The rFXFr was purified and shown to have a molecular size identical to that of normal plasma factor X (pFX) by gel electrophoretic, and amino-terminal sequencing revealed normal processing cleavages. Using recombinant normal plasma factor X (rFXN) as a reference, the post-translational gamma-carboxy-glutamic acid (Gla) and beta-hydroxy aspartic acid (beta-OH-Asp) content of rFXFr was over 85% and close to 100%, respectively, of expected levels. The specific activities of rFXFr in activation and catalytic assays were the same as those of pFXFr. Molecular modeling suggested the involvement of a new H-bond between the side-chains of Ser-343 and Thr-318 as they occur in anti-parallel beta-pleated sheets near the substrate-binding pocket of pFXFr. These results support the conclusion that the observed mutation in pFXFr is responsible for its dysfunctional activation and catalytic potentials, and that it accounts for the moderate bleeding tendency in the homozygous individuals who possess this variant procoagulant.