Factor VIIc Research Articles

Technical aspects of different donor plasmapheresis systems and biological results obtained in collected plasma.

Four plasmapheresis systems were comparatively studied: a centrifugation system (Haemonetics Model 50) and three filtration ones (Dideco BT 810, Organon Teknika PLASMAPUR and HemaScience Autopheresis Plasmacell). For each separator we studied the technical conditions for plasma separation, the biological characteristics of the collected plasma and the in vitro recovery of factor VIIc after preparation of cryoprecipitates. Concerning the plasma extraction rate, the HemaScience apparatus was the most efficient but values were quite similar to those obtained with the Organon Teknika machine. Contaminating cells were only found in plasma separated with the Haemonetics. Total protein and immunoglobulin levels were higher in plasma collected with the Haemonetics and HaemaScience systems. Factor VIIIc activity was comparable in plasma separated by filtration or by centrifugation while fibrinopeptide A levels were higher in plasma collected by Haemonetics. Whatever the machine, no statistical difference was observed when in vitro recovery of factor VIII was studied.

Technical Aspects of Different Donor Plasmapheresis Systems and Biological Results Obtained in Collected Plasma

Four plasmapheresis systems were comparatively studied: a centrifugation system (Haemonetics Model 50) and three filtration ones (Dideco BT 810, Organon Teknika PLASMAPUR and HemaScience Autopheresis Plasmaceli). For each separator we studied the technical conditions for plasma separation, the biological characteristics of the collected plasma and the in vitro recovery of factor VIIIc after preparation of cryoprecipitates. Concerning the plasma extraction rate, the HemaScience apparatus was the most efficient but values were quite similar to those obtained with the Organon Teknika machine. Contaminating cells were only found in plasma separated with the Haemonetics. Total protein and immunoglobulin levels were higher in plasma collected with the Haemonetics and HaemaScience systems. Factor VIIIc activity was comparable in plasma separated by filtration or by centrifugation while fibrinopeptide A levels were higher in plasma collected by Haemonetics. Whatever the machine, no statistical difference was observed when in vitro recovery of factor VIII was studied.

Chirurgie et inhibiteur acquis du facteur VIII coagulant

A case is reported of an 80 year old woman with old sarcoidosis and acquired factor VIIIc inhibitor undergoing orthopaedic surgery. This was successfully carried out after she had been given a total of 48,420 units of factor VIII concentrate; there were no haemorrhagic complications. The main pathological states in which acquired factor VIIIc inhibitor may be found are related; principal treatments available nowadays are discussed : immuno-suppressors, human factor VIII, animal factor VIII, prothrombin complex concentrate, plasma exchange.

Physical mapping of the factor VIII gene proximal to two polymorphic DNA probes in human chromosome band Xq28: implications for factor VIII gene segregation analysis.

Genomic DNA segments for the coagulation factor VIIIc gene (F8C), which exhibits only limited restriction length polymorphism, map to the proximal region of band Xq28 by somatic cell hybridization analysis and in situ hybridization. Using somatic cell hybrids, we have obtained data which place probes DX13 (used to detect locus DXS15) and St14 (used to detect DXS52) distal to F8C, within band Xq28. Previous studies have mapped the factor IX gene (F9) and probe 52A (used to detect DXS51) proximal to F8C, in Xq26----q27 and Xq27, respectively (Camerino et al., 1984; Drayna et al., 1984; Mattei et al., 1985). Thus, the relative order of genetic marker loci in the Xq27----qter region is most likely cen-F9-DXS51-F8C-(DXS15, DXS52)-Xqter. The collection of these molecular probes is thus potentially useful in three-factor crosses of factor VIII gene segregation.

Effect of propranolol, barbiturate anesthesia and splenectomy on factor VIIIC release induced by DDAVP. An experimental study in the dog

Effect of propranolol, barbiturate anesthesia and splenectomy on factor VIIIC release induced by DDAVP. An experimental study in the dog

Artificially depleted plasma for Factor VIIIC assays

Artificially depleted plasma for Factor VIIIC assays

Human von Willebrand factor (vWF): isolation of complementary DNA (cDNA) clones and chromosomal localization.

Human factor VIII--von Willebrand factor (vWF) is a large, multimeric glycoprotein that plays a central role in the blood coagulation system, serving both as a carrier for factor VIIIC (antihemophilic factor) and as a major mediator of platelet-vessel wall interaction. Diminished or abnormal vWF activity results in von Willebrand's disease (vWD), a common and complex hereditary bleeding disorder. Overlapping vWF cDNA clones that span 8.2 kilobases of the vWF messenger RNA have been obtained. vWF accounts for approximately 0.3 percent of endothelial cell messenger RNA and was undetectable in several other tissues examined. A large single copy gene for vWF is located on the short arm of chromosome 12 (12p12----12pter). No gross gene rearrangement or deletion was detected in the DNA of two patients with severe vWD.

Oral administration of factor VIII concentrate preparation in von Willebrand's disease

We investigated on how to effectively administer orally factor VIII preparation to two patients with von Willebrand's disease as volunteers, and the following results were obtained: (1) Bleeding time and aPTT were shortened, but no change of PT was observed. (2) Factor VIII:C increased slightly, but no significant increase was observed in factor VIIIC:Ag. The recovery of factor VIII:C was 19–40 %. (3) Factor VIIIR:Ag increased slightly, but no changes of factor VIIIR:RCo were observed. (4) In the Ristocetin induced platelet aggregation ( 1.5 mg/ml of ristocetin), the collected aggregabilities were not clear. The factor VIII concentrate preparations, entrapped with liposome, were absorbed from the intestines.

Danazol and Hemophilia

Danazol is a 2,3-isoxazol derivative of 17 α-ethinyl testosterone; it is known to be an anabolic steroid with mild androgenic activity. The administration of the drug is associated with raised plasma levels of several proteins including prealbumin, haptoglobulin, transferrin, antithrombin III, prothrombin, plasminogen, α₁-antitrypsin, and Cl esterase inhibitor.^1,2Last year Gralnick and Rick¹reported evidence that danazol was associated with increased levels of factor VIIIc and factor IX and decreased transfusion requirements in patients with hemophilia. Subsequent studies by Gralnick and associates⁴as well as other studies by Garewal et al⁵are reported in this issue ofThe Journaland form the basis for this commentary. The readers of the aforementioned articles are faced with a dilemma in judging the usefulness of danazol in management of hemophilia patients. Gralnick et al report on three of their initial patients plus eight French patients with hemophilia A.

Epidemiological Study on Factor VII, Factor VIII and Fibrinogen in an Industrial Population - II. Baseline Data on the Relation to Blood Pressure, Blood Glucose, Uric Acid, and Lipid Fractions

In a preceding paper the baseline data in the Münster Arteriosclerosis Study (PROCAM study) of the levels of fibrinogen, factor VIIc and factor VIIIc were described, and their correlation of age, body weight, smoking, alcohol, pill-using and menopause discussed. In this part the relationship of these coagulation factors to blood pressure, blood glucose, uric acid and lipid parameters, which were examined in 4186 apparently healthy individuals, is presented. The correlations are described using two different statistical models, first the Pearson correlation coefficients after allowing each clotting factor for the effects of age, cigarette and alcohol consumption, body weight, menopausal state, pill using and the other clotting factors, and secondly by a multiple regression analysis. The data suggest that there are multiple interrelationships between hemostatic factors and the variables under consideration. The most striking positive correlations were found for factor VIIc to blood glucose and triglycerides in men and women and to HDL-cholesterol in women.

Epidemiological Study on Factor VII, Factor VIII and Fibrinogen in an Industrial Population: I. Baseline Data on the Relation to Age, Gender, Body-Weight, Smoking, Alcohol, Pill-Using, and Menopause

The Münster Arteriosclerosis Study (MAS) is a prospective, longitudinal epidemiological study on an industrial population in Westfalia aimed to establishing clinical and laboratory data with possible relationship to cardiovascular events. The data presented here describe the baseline measurements of fibrinogen, factor VIIc and factor VIIIc from the recruitment of 2880 male and 1306 female persons and their relationship to age, gender, bodyweight, smoking, alcohol, pill-using and menopause. The correlations were made by means of a multiple regression analysis. We found an increase of those coagulation factors with age, a correlation of F VII and fibrinogen with body-weight index and of fibrinogen with cigarette smoking. No correlation was found for alcohol consumption. F VIII and F VII were significantly higher after onset of menopause and F VII and fibrinogen in women using the pill.

Effect of Stanozolol on Factors VIII and IX and Serum Aminotransferases in Haemophilia

The treatment of haemophilia has been dramatically improved since the introduction of factor VIII and IX concentrates, however these concentrates have brought new problems such as hepatitis and A.I.D.S. An oral agent which could raise endogenous levels of factor VIII and IX would be of great benefit. Danazol, an anabolic steroid, has recently been shown to increase levels of factors VIII and IX in haemophilia. We therefore studied the effect of stanozolol, a closely related anabolic steroid, in 15 patients with haemophilia A or Christmas disease over a 2-4 week period. There was no consistent change in factor VIIIc or factor IX, and fibrinolysis was significantly enhanced. No effect was apparent on the incidence of spontaneous bleeds. However serum aminotransferases which were abnormal in 11 of the 15 patients at the start of the study fell significantly with stanozolol therapy. This raises the interesting possibility that anabolic steroids may be beneficial in patients with chronic liver diseases.

Hypotensive and hemostatic properties of rattlesnake (Crotalus viridis helleri) venom and venom fractions in dogs: Richard C. Schaeffer Jr, Carolyn Briston, Shawn-Marie Chilton and Richard W. Carlson (Department of Medicine, Wayne State University, Detroit, MI, U.S.A.)

Hypotensive and hemostatic properties of Southern Pacific rattlesnake (Crotalus viridis helleri) venom (100 micrograms/kg; i.v. bolus) or venom fractions (40 micrograms/kg) were studied in mongrel dogs (n = 27, 15-27 kg). Venom was separated by gel filtration (Sephadex G-100) into three lethal fractions (FR I, II and III). Nonreduced crude venom contained 11 main protein bands on sodium dodecyl sulfate-10% polyacrylamide gel electrophoresis with a molecular weight range of 104 to 13 kD. FR I contained nine of these prominent bands (104-27 kD) and FR II contained five bands (34-13 kD). A 19-13 kD band comprised 92% of FR III. Crude venom rapidly produced hypotension, thrombocytopenia, hemolysis, the generation of fibrin monomers and a decrease in Factor VIIIC activity. Fibrin/fibrinogen degradation products appeared at +15 min and fibrinogen concentration was significantly depressed at +120 min. There was no significant change in prothrombin time or concentration of Factors VII, X and XII activity. However, the partial thromboplastin time was increased (P less than .05) by +30 min. Our data show that a high molecular weight thrombin-like venom component in FR I (greater than 27 kD) directly digests fibrinogen without activation of extrinsic and intrinsic coagulation factors. In addition, this fraction leads to the activation of the fibrinolytic system. It appears to be likely that the thrombocytopenia is primarily the result of ca. 27 kD platelet aggregating protein found in similar amounts in FR I and II. Venom components in FR II (ca. 34 and 29 kD,) were associated with hypotension and hemolysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of three abnormal factor IX variants (Bm Lake Elsinore, Long Beach, and Los Angeles) of hemophilia-B. Evidence for defects affecting the latent catalytic site.

Abnormal factor IX variant proteins were isolated from the plasmas of three unrelated severe hemophilia-B families that had been previously shown to contain functionally impaired molecules immunologically similar to normal factor IX. The families studied were: (1) a patient with markedly prolonged ox brain prothrombin time, designated factor IX Bm Lake Elsinore (IXBmLE); (b) three patients (brothers) with moderately prolonged ox brain prothrombin time, designated factor IX Long Beach (IXLB); and (c) a patient with normal ox brain prothrombin time designated factor IX Los Angeles (IXLA). Each variant molecule comigrates with normal factor IX (IXN) both in the sodium dodecyl sulfate and in the nondenaturing alkaline gel electrophoresis. All three variant proteins are indistinguishable from IXN in their amino acid compositions, isoelectric points, carbohydrate distributions and number of gamma-carboxyglutamic acid residues. Each variant protein undergoes a similar pattern of cleavage by factor XIa/Ca2+ and by factor VIIa/Ca2+/tissue factor, and is activated at a rate similar to that observed for IXN. All of the three variant proteins also react with an anti-IXN monoclonal antibody that interferes with the binding of activated IXN(IXaN) to thrombin-treated factor VIIIC. However, in contrast to IXaN, the cleaved IXBmLE has negligible activity (approximately 0.2%), and cleaved forms of IXLA and IXLB have significantly reduced activity (approximately 5-6%) in binding to antithrombin-III/heparin, and in activating factor VII (plus Ca2+ and phospholipid) or factor X (plus Ca2+ and phospholipid) +/- factor VIII. These data, taken together, strongly indicate that the defect in these three variant proteins resides near or within the latent catalytic site. This results in virtually a complete loss of catalytic activity of the cleaved IXBmLE molecule and approximately 95% loss of catalytic activity of the cleaved IXLA and IXLB molecules.

In vivo effectiveness of a high-yield factor VIII concentrate prepared in a blood bank.

1YOPHILIZED factor VIII concentrates have beJ come the treatment of choice for factor VIII-deficient patients with hemophilia. The cost of such treatment is high, primarily because under normal conditions of collection and preparation, factor VIII procoagulant activity (VIIIC, sec Table 1 for terms and definitions1) is unstable and as much as 80 per cent of the activity is lost during the purification procedure. Recently, we have reported that factor VIIIC activity is stabilized when blood is collected into heparin rather than citrate anticoagulant, and we have shown that the cryoprecipitates obtained from heparinized blood contain 80 per cent of the VIIIC . . .

Localization of factor VIIIC: antigen in guinea‐pig tissues and isolated liver cell fractions

Factor VIIIC:antigen (VIII:CAg) was estimated in guinea-pig tissues by an immunoradiometric assay using a human inhibitor antibody. In homogenized guinea-pig tissues, VIII:CAg was shown to be stable and to be predominantly located in the liver (9 +/- 1.2 units; mean +/- SEM, n = 8). Lesser amounts were detected in spleen (1.3 +/- 0.02 units), lung (0.6 +/- 0.07) and kidney (0.4 +/- 0.06). In isolated liver cell fractions separated by centrifugal elutriation VIII:CAg was mainly detected in the hepatocyte fraction (0.3 +/- 0.07 units/10(8) cells;mean +/- SEM, n = 5) and in lesser amounts in the endothelial (0.02 +/- 0.01 units/10(8) cells) and the Kupffer cell fractions (0.05 +/- 0.02 units/10(8) cells). The liver concentration of VIII:CAg was (0.17 +/- 0.02 units/g) which was 20% of the plasma concentration (0.96 +/- 0.01 units/ml, n = 8) suggesting that VIII:CAg may not be stored in the liver but is rapidly exported following synthesis.

Sedimentation analysis of von Willebrand and factor VIIIC protein using partition cells in the analytical ultracentrifuge

Sedimentation analysis of factor VIII complex was performed in the analytical ultracentrifuge using partition cells. This method allowed for the calculation of three different sedimentation coefficients from each run: one based on ristocetin agglutination activity for von Willebrand protein, SWF; one based on coagulant activity for factor VIIIC, SVIIIC; and one based on the schlieren or adsorption data for protein concentration, Sconc. In most cases, there was no agreement between the three values calculated from the same run, indicating a heterogeneous system. The calculated functional sedimentation coefficients give values that require the molecules to be highly asymmetric to be consistent with a glycoprotein of high molecular weight, which is in agreement with results observed in electron microscope studies. The dissociation of VIIIC into a smaller form can be demonstrated by this method. Determination of the three sedimentation coefficients in a series of fractions from gel filtration indicates a uniform size for the VIIIC activity but not for the WF activity. These observations are in agreement with the concept of a copolymer between WF and VIIIC and also with the concept of separate polymers for the two activities.

Sedimentation Analysis of von Willebrand and Factor VIIIC Protein Using Partition Cells in the Analytical Ultracentrifuge

AbstractSedimentation analysis of factor VIII complex was performed in the analytical ultracentrifuge using partition cells. This method allowed for the calculation of three different sedimentation coefficients from each run: one based on ristocetin agglutination activity for von Willebrand protein, SWF; one based on coagulant activity for factor VIIIC, SVIIIC; and one based on the schlieren or adsorption data for protein concentration, Sconc. In most cases, there was no agreement between the three values calculated from the same run, indicating a heterogeneous system. The calculated functional sedimentation coefficients give values that require the molecules to be highly asymmetric to be consistent with a glycoprotein of high molecular weight, which is in agreement with results observed in electron microscope studies. The dissociation of VIIIC into a smaller form can be demonstrated by this method. Determination of the three sedimentation coefficients in a series of fractions from gel filtration indicates a uniform size for the VIIIC activity but not for the WF activity. These observations are in agreement with the concept of a copolymer between WF and VIIIC and also with the concept of separate polymers for the two activities.

Abnormal Haemostasis and Blood Viscosity in Malignant Hypertension

We have previously shown abnormalities of haemostasis suggestive of intravascular coagulation in patients with malignant hypertension, a condition associated with retinopathy and renal fibrin deposition. To determine whether such abnormalities are specific to malignant hypertension, we have measured several haemostatic and haemorheological variables in 18 patients with malignant hypertension (Group 1), 18 matched healthy controls (Group 2), and 18 patients with non-malignant hypertension (Group 3) matched for renal pathology, blood pressure and serum creatinine with Group 1. Both Groups 1 and 3 had increased mean levels of fibrinogen, factor VIIIc, beta-thromboglobulin, plasma viscosity and blood viscosity (corrected for haematocrit); and decreased mean levels of haematocrit, antithrombin III and platelet count. Mean levels of fast antiplasmin and alpha2-macroglobulin were elevated in Group 1 but not in Group 3. We conclude that most blood abnormalities are not specific to malignant hypertension; are also present in patients with non-malignant hypertension who have similar levels of blood pressure and renal damage; and might result from renal damage as well as promoting further renal damage by enhancing fibrin deposition. However increased levels of fibrinolytic inhibitors in malignant hypertension merit further investigation in relation to removal of renal fibrin.

How to measure factor VII and factor VII activation.

This communication describes the different techniques that can be used to evaluate the activity state of factor VII in plasma samples. At the present time direct methods for quantitation of activated factor VII (factor alpha-VIIa) are not available. Combined methods are therefore used to measure the degree of factor VII activation. These methods can be summarized in the following way: a regular factor VII clotting assay measuring factor VII coagulant activity (factor VIIc) is carried out simultaneously with: (1) a factor VII antigen assay (factor VIIag): (2) a coupled amidolytic factor VII assay (factor VIIam), or (3) a factor VII clotting assay utilizing bovine tissue thromboplastin (VIIbt). The activity state of factor VII can then be calculated from either of the following ratios: f.VIIc/f.VIIag; f.VIIc/f.VIIam, or f.VIIbt/f.VIIc. A study of the potency of a 30-fold activated factor VII to activate factor X in the presence of phospholipids is also included. This experiment demonstrates that even a low factor VII concentration (0.01 U/ml in the coupled amidolytic assay and 0.30 U/ml in the factor VII clotting assay) causes a significant activation of factor X when incubated together in the presence of lipids and calcium ions. Activated factor VII may therefore possess potential thrombotic properties even in the absence of exposed tissue thromboplastin in the blood circulation.