A transforming growth factor beta-like immunosuppressive factor in immunoglobulin G-binding factor

Immunoglobulin G-binding factors (IgG-BF), which are produced by cells of the immune system, inhibit antibody production. In this paper, we show that transforming growth factor-beta (TGF-beta) suppresses secondary in vitro anti-sheep red blood cell responses of mouse splenocytes and lipopolysaccharide- or anti-IgM-stimulated mouse B cell responses in a way similar to, and with the same kinetics as, rodent IgG-BF. Moreover, the immunosuppressive activity of IgG-BF was totally neutralized by polyclonal and monoclonal anti-TGF-beta antibodies and it eluted with TGF-beta by gel exclusion chromatography, suggesting that a TGF-beta-like immunosuppressive factor is present in IgG-BF. We also show that TGF-beta behaves as an IgG-BF since it binds to insolubilized IgG, but not to insolubilized F(ab')2 or bovine serum albumin. Altogether, the data support the concept of a biological role for TGF-beta in the IgG-mediated negative feedback of antibody responses.     

Transfer factor

The understanding of passive transfer of cell mediated-immune responses with transfer factor and other cell free materials has progressed to the point that investigators are seeking the chemical identity of the molecule(s) that are responsible for these effects and are working on their mechanisms of action. In addition, clinical trials are underway that should clarify the potential for use of transfer factor in treatment of infections, neoplastic and autoimmune diseases. This chapter will critically review the past and current data concerning the components of transfer factor and their effects on immunologic and inflammatory reactions. Some of the recently developed animal models will be described and evaluated, and the clinical studies that have provided conclusive data regarding efficacy will be reviewed.     

Impact factor

This is the first Coda I have written since Christopher Martynretired last month as editor of QJM. Christopher would no doubthave censored any compliments about himself, so this is my firstchance to praise him in these pages. He has been an extraordinarilythoughtful and literate editor, and I want to use this month'sarticle to reflect both on our relationship as columnist andeditor, and also more generally on the role of editor as muse.     

Inhibition of the activation of Hageman factor (factor XII) by platelet factor 4

Platelet factor 4 is a polypeptide constituent of platelet alpha granules that is released during platelet aggregation and inhibits heparin-mediated reactions. Hageman factor (factor XII) is a plasma proenzyme that, when activated by certain negatively charged agents, initiates clotting via the intrinsic pathway of thrombin formation. In earlier studies using crude systems, platelet factor 4 inhibited activation of Hageman factor by dextran sulfate or cerebrosides, but not activation of Hageman factor by kaolin or ellagic acid. In the present study we examined the mechanisms of inhibition by platelet factor 4, using purified reagents. Platelet factor 4 inhibited activation of Hageman factor by ellagic acid, as measured by amidolysis of a synthetic substrate of activated Hageman factor, an effect inhibited by heparin or by an anti-platelet factor 4 antiserum. Coating glass tubes with platelet factor 4 before addition of normal plasma significantly lengthened the partial thromboplastin time of normal plasma. In addition, the clot-promoting properties of kaolin were inhibited by its prior exposure to platelet factor 4. Thus, the inhibitory properties of platelet factor 4 directed against the activation of Hageman factor were confirmed in a purified system. In this purified system, in contrast to earlier studies using crude systems, platelet factor 4 inhibited activation of Hageman factor by glass, ellagic acid, or kaolin.     

Subcellular platelet factor VIII antigen and von Willebrand factor

Subcellular membrane and granule fractions derived from human platelets contain factor VIIII antigen and von Willebrand factor activity but not factor VII procoagulant activity. Circulating platelets constitute a significant reservoir of plasma factor VIII antigen, containing approximately 15% of the amount of factor VIII antigen present in platelet-poor plasma. The antibiotic ristocetin, which aggregates human platelets in the presence of von Willebrand factor, nonspecifically precipitates platelet membrane factor VIII antigen. Thus normal platelets contain surface-bound as well as internally stored von Willebrand factor, a protein synthesized by endothelial cells which is necessary for normal platelet function in vivo.     

Activation of human factor IX (Christmas factor).

Human Factor IX (Christmas factor) is a single-chain plasma glycoprotein (mol wt 57,000) that participates in the middle phase of the intrinsic pathway of blood coagulation. It is present in plasma as a zymogen and is converted to a serine protease, Factor IXabeta, by Factor XIa (activated plasma thromboplastin antecedent) in the presence of calcium ions. In the activation reaction, two internal peptide bonds are hydrolyzed in Factor IX. These cleavages occur at a specific arginyl-alanine peptide bond and a specific arginyl-valine peptide bond. This results in the release of an activation peptide (mol wt approximately equal to 11,000) from the internal region of the precursor molecule and the generation of Factor IXabeta (mol wt approximately equal to 46,000). Factor IXabeta is composed of a light chain (mol wt approximately equal to 18,000) and a heavy chain (mol wt approximately equal to 28,000), and these chains are held together by a disulfide bond(s). The light chain originates from the amino terminal portion of the precursor molecule and has an amino terminal sequence of Tyr-Asn-Ser-Gly-Lys. The heavy chain originates from the carboxyl terminal region of the precursor molecule and contains an amino terminal sequence of Val-Val-Gly-Gly-Glu. The heavy chain of Factor IXabeta also contains the active site sequence of Phe-Cys-Ala-Gly-Phe-His-Glu-Gly-Arg-Asp-Ser-Cys-Gln-Gly-Asp-SER-Gly-Gly-Pro. The active site serine residue is shown in capital letters. Factor IX is also converted to Factor IXaalpha by a protease from Russell's viper venom. This activation reaction, however, occurs in a single step and involves only the cleavage of the internal arginyl-valine peptide bond. Human Factor IXabeta was inhibited by human antithrombin III by the formation of a one-to-one complex of enzyme and inhibitor. In this reaction, the inhibitor was tightly bound to the heavy chain of the enzyme. These data indicate that the mechanism of activation of human Factor IX and its inhibition by antithrombin III is essentially identical to that previously shown for bovine Factor IX.     

The association of factor VIII with von Willebrand factor

Factor VIII (FVIII) and von Willebrand factor (vWF) are plasma glycoproteins that circulate as a tightly associated complex. Because they tend to copurify during procedures designed to isolate the biologic activities associated with them, their identity as distinct entities became unequivocally established only during the past 10 years. Improved procedures for the isolation of FVIII, the deduction of the amino acid sequences of FVIII and vWF by using molecular cloning techniques and by direct sequencing, and the use of a variety of biophysical and immunochemical techniques have enhanced the understanding of the FVIII-vWF association. Each subunit of multimeric vWF potentially can bind a single heterodimeric FVIII molecule, although in vivo most of these binding sites are empty. The binding of FVIII to vWF is primarily, if not exclusively, mediated by the light chain of FVIII to the amino-terminal region of the vWF subunit. Cleavage of a fragment from the amino-terminal region of the FVIII light chain by thrombin results in rapid dissociation of the FVIII-vWF complex, a process that apparently is necessary for development of procoagulant activity. Whether this cleavage is needed for the activation of FVIII in the absence of vWF is controversial. The extracellular association of FVIII with vWF may be necessary for efficient secretion of FVIII from its cell of origin. The thermodynamics, kinetics, and nature of the molecular contacts involved in the interaction have not been studied. The association of FVIII with vWF prolongs the lifetime of FVIII in plasma. Whether the FVIII-vWF interaction has other functional roles, such as restricting the location of procoagulant activity, remains unknown.     

Standardization of assays of factor VIII and factor IX

Summary The development of international standards over the last 15–20 years has led to improved interlaboratory agreement on assays of factor VIII and factor IX. In the most recent international collaborative study, the coefficient of variation for one-stage assays (26 laboratories) was 5.6%. However, in quality assurance surveys, carried out in the UK and USA, agreement between laboratories is much less good, with coefficients of variation ranging from 30% to over 50%. Improvements in agreement between clinical laboratories could be obtained by increasing the amount of testing on each sample, especially the number of dilutions, and reducing the number of reagent systems used. A large number of laboratories now use immunodepleted plasmas instead of congenitally deficient plasmas as substrates for one-stage assays. These plasmas may give satisfactory assays, but many of them have not been thoroughly evaluated in comparison with congenitally deficient plasma. In assessment of potency of very high purity (VHP) factor VIII concentrates, some immunodepleted plasmas were found to give lower potencies than hemophilic plasma. This is partly due to the fact that VHP concentrates contain little or no von Willebrand factor (vWF), and most immunodepleted plasmas are also deficient in vWF. In recent collaborative studies, assays of VHP factor VIII concentrate were much more variable, both within and between laboratories, than assays of intermediate purity concentrates. Standardization of these new products will require careful attention to methodological detail. Presented at the ‘2nd International Symposium on Standardization and Quality Control of Coagulation Tests: Implications for the Clinical Laboratory’, Rome, September 28–29, 1989.     

Radioimmunoassay of antihaemophilic factor (factor VIII) antigen

A simple, precise radioimmunoassay for antihaemophilic factor (AHF, factor VIII) antigen has been developed. The technique is based upon a double-antibody solid-phase (DASP) system. This assay may serve as a specific method for differentiation between Von Willebrand's disease and haemophilia A.     

Measuring tissue factor (factor III) activity in plasma

This is a method for measuring tissue factor (TF, Factor III, tissue thromboplastin) activity in plasma by using a chromogenic substrate. As pretreatment, the euglobulin fraction of plasma was prepared by removing endogenous inhibitors and heated at 60 degrees C for 3 min to remove fibrinogen. This allowed us to measure the low TF activity in plasma that could not otherwise be measured. Neither phospholipids nor coagulation factors VII, IX, X, or Xa in the samples interfere. Within-run and day-to-day reproducibility were both good. The mean value obtained by this method for normal persons was 1.02 (SD 0.91) arbitrary units/L. A markedly high plasma TF activity of 20 arb. units/L or more was observed in patients with some types of disseminated intravascular coagulation.     

Evidence that the platelet retention factor is separate from the factor VIII-related antigen and factor VIII.

Plasmas from a pregnant patient with von Willebrand's disease and from a patient with von Willebrand's disease who is taking Premarin had elevated levels of Factor VIII-related antigen and Factor VIII activity without any improvement in their platelet retention abnormality. When these were added to the blood from nonpregnant patients with von Willebrand's disease no significant improvement in platelet retention followed. This suggests that the "retention factor" is independent of the antigenic Factor VIII-like material.