An Immunoradiometric Assay for Factor III (Tissue Thromboplastin)

A solid-phase immunoradiometric assay for tissue thromboplastin (factor III) has been established based on its displacing effect on the binding of ¹²⁵I-labelled factor III-antibodies to polyvinyl tubes coated with the purified protein component of factor III (apoprotein III). By this method circulating tissue thromboplastin can be detected in experimental animals receiving infusions of crude or purified tissue thromboplastin and in patients undergoing major orthopaedic surgery.     

Anti-CD36 antibodies in thrombotic thrombocytopenic purpura

Summary. The membrane glycoprotein CD36 (GPIV, M_r 88000) is found on platelets, monocytes and endothelial cells of the microvasculature. In the present study, anti CD36 antibodies have been identified as occurring with high frequency in patients with thrombotic thrombocytopenic purpura. The presence of anti CD36 antibodies in 15 TTP plasma samples thought to contain them on the basis of an initial screening by protein blots was confirmed by re-screening against a standard of purified CD36, by immunoprecipitation from ¹²⁵I-labelled control platelets and by dot blots against purified CD36. In a further 28 random samples examined, 23/27 (85%) were CD36-positive by immunoprecipitation, 21/28 (75%) by protein blotting, and 17/28 (60%) by dot blots against purified CD36. On protein blots following SDS-PAGE, immunoprecipitates produced from normal platelets by TTP plasma gave positive reactions with the anti CD36 monoclonal antibody ¹²⁵I-Mo91. One half of the total TTP samples examined (21/42) caused 70% release in control platelets loaded with ¹⁴C-serotonin. Of samples causing release 70%, one-half (8/15) failed to cause release from Nak^a-negative platelets which constitutively lack CD36 showing that CD36 was the sole target for platelet activation in these TTP samples. These studies demonstrate that antibodies directed against CD36 occur frequently in TTP patients and could cause thrombotic complications and vascular damage by reacting with the parent antigen present in platelets and endothelial cells.     

Epitope mapping of human factor IX inhibitor antibodies

Summary. We have determined the location of epitopes on the factor IX for three haemophilia B inhibitor antibodies (HB-1, HB-3, HB-7) and a monoclonal anti-factor IX inhibitory antibody (designated 65–10). The main binding region of HB-1, HB-3 and HB-7 was ¹⁵⁵YVNSTEAETI¹⁶⁴ (residues 155–164), ¹⁶⁷NITQSTQSFN¹⁷⁶ and ¹⁵⁶VNSTEAETI¹⁶⁴, respectively. The binding region of 65–10 was ¹⁶⁸ITQSTQSFNDFTRVV¹⁸², which included the cleavage site (¹⁸⁰R-V¹⁸¹) for activation by factor XIa. By neutralization experiments using two peptides, ¹⁵⁶VNSTEAETI¹⁶⁴ and ¹⁶⁷NITQSTQSFN¹⁷⁶, the degree of neutralization of anti-factor IX IgG purified by protein A was determined. Neutralization of three antibodies, HB-1, HB-3 and HB-7, in the presence of 10m m of the peptides ¹⁵⁶VNSTEAETI¹⁶⁴ was 30.1%, 0% and 10.8%, respectively, and in the presence of 4 m m of ¹⁶⁷NITQSTQSFN¹⁷⁶ it was 0%, 13.5% and 17.3%, respectively. On the other hand, when plasmas of patients instead of purified IgG were used for neutralization, 10 m m of ¹⁵⁶VNSTEAETI¹⁶⁴ and 4 m m of ¹⁶⁷NITQSTQSFN¹⁷⁶ failed to neutralize the inhibitor in the plasmas.     

Precipitating anti-VIII:C antibodies in two patients with haemophilia A

S ummary . IgG from the plasmas of two haemophilia A patients with anti-VIII:CAg antibodies (1000 and 200 u/ml) was isolated and labelled with ¹²⁵I. The specific labelled anti-VIII:CAg IgG was further purified by binding to and elution from immobilized factor VIII/von Willebrand factor (F.VIII/vWF). When studied by immunodiffusion and autoradiography, both antibodies gave a precipitin line with normal plasma, serum, cryoprecipitate, purified F.VIII/vWF and the plasmas of two patients with haemophilia A⁺. No precipitin line was observed with the plasmas of 11 patients with haemophilia A⁻ or four patients with severe von Willebrand's disease. Levels of VIII:CAg obtained by radioelectroimmunoassay were in agreement with those obtained by immunoradiometric assay. This study demonstrates that, contrary to previous evidence, human anti-VIII:CAg antibodies are precipitating as well as neutralizing when studied by highly sensitive techniques.     

Assay of Anti-D Activity by Two Radioimmunoassay Techniques Using Purified 125I-Labelled Anti-D

Abstract. Two methods are described for the estimation of anti-D activity in immunoglobulin preparations which are based on the radioimmunoassay inhibition techniques. In one of the methods, estimates were made under equilibrium conditions in a solution of normal ionic strengh. In this method, the assay is affected both by the concentration of the anti-D and the value of its equilibrium constant. This method did not correlate well with the standard assay method using ¹²⁵I-labelled anti-IgG. In the second method, conditions were modified so that measurements were made at non-equilibrium conditions and in a solution of low ionic strength. Under these conditions, the assay is mainly dependent on concentration of anti-D, although it is influenced by the rate of association of anti-D with red cells. The second method correlated well in 24 out of 27 instances with the concentration of anti-D measured by the method using ¹²⁵I-labelled anti-IgG.     

A monoclonal antibody binding to human medulloblastoma cells and to the platelet glycoprotein IIb-IIIa complex

S ummary. A monoclonal antibody, designated M148, produced by the hybridoma technique from spleen cells of mice immunized with human medulloblastoma, was found by indirect immunofluorescence to bind to normal human platelets (both Pl^Al positive and Pl^Al negative) and megakaryocytes, as well as to some medulloblastoma and neuroblastoma cells and cell lines and certain other solid tumours. No binding was observed to other marrow constituents, nor to any other normal tissue examined. The antibody bound to platelets from a patient with the Bernard-Soulier syndrome but not to thrombasthenic platelets. It immunoprecipitated glycoproteins IIb and IIIa from ¹²⁵I-labelled normal platelet membranes, and completely inhibited ADP-induced fibrinogen binding and aggregation of platelets. Aggregation was also inhibited in response to adrenaline, collagen, thrombin, sodium arachidonate and the ionophore A23187; clot retraction was partially inhibited. The antibody was without effect on thromboxane formation or 5-hydroxytryptamine (5HT) secretion in response to thrombin, but inhibited 5HT secretion in response to arachidonate. It did not inhibit factor VIII binding or agglutination in response to ristocetin, but completely inhibited factor VIII binding in response to thrombin. These findings suggest that the epitopes are close to the fibrinogen and factor VIII binding sites on glycoproteins IIb/IIIa, and that the lack of these glycoproteins is sufficient explanation for the pattern of dysfunction observed in thrombasthenic platelets, without invoking any other membrane abnormality.     

125I-Transferrin Turnover in Rabbits with Haemolytic Anaemia

The metabolism of purified ¹²⁵I-labelled homologous transferrin in rabbits with haemolytic anaemia was compared to that in control rabbits and no significant differences were found. During 3 weeks of haemolysis plasma transferrin concentrations did not change appreciably. The fractional turnover rate was 43% of the intravascular pool per day. The intravascular pool was 45% of the total exchangeable transferrin.     

Transferrin Receptors in Developing Murine Erythroid Cells

S ummary . Techniques of cell separation were used to isolate murine erythroid cells at different stages of maturation. The number of transferrin receptors in these cell populations was assayed by measuring binding of ¹²⁵I-labelled transferrin. Nearly 23 times as many receptors were found in the least mature cells, chiefly pronormoblasts, as in reticulocytes. Iron transport, determined by measurement of the rate of ⁵⁹Fe uptake from ⁵⁹Fe-labelled transferrin, was proportional to the number of receptors at all stages of differentiation. Electron microscope radioautographic studies of the interaction of ¹²⁵I-labelled transferrin with erythroid precursor cells demonstrated that 15–35% of cell associated transferrin was intracellular in erythroid precursors.     

Low molecular weight dermatan sulphate (Desmin 370) does not cross the ovine placenta

Summary. Transplacental passage of the low molecular weight dermatan sulphate Desmin 370 was investigated in pregnant sheep, using ¹²⁵I-labelled Desmin 370 to optimize the sensitivity of the study. Chronically catheterized crossbred pregnant ewes at approximately 120 d gestation received 3700 kBq ¹²⁵I-labelled Desmin 370 with 1 mg/kg carrier unlabelled Desmin 370 intravenously. Early clearance from the maternal circulation was biexponential, and the volume of distribution corresponded closely with the theoretical value for distribution in total body water. Soon after injection low levels of radioactivity were detected in the fetal circulation and accumulated over the next 2 h, so that as the concentration of ¹²⁵I-Desmin 370 in the maternal circulation declined with time the fetal level of radiolabel rose to represent a significant concentration in relation to that in the mother. Radioactivity was also excreted into the fetal urine. However, while 50% of the radiolabeled material present in maternal plasma 150 min post-injection was intact Desmin 370 and the remaining 50% represented degradation products, fetal urine contained only these fragments. By contrast, intact Desmin 370 was readily excreted into fetal urine after direct introduction to the fetal circulation. Thus molecules of intact Desmin 370 with anticoagulant activity cannot cross the ovine placenta, and low molecular weight dermatan sulphates may be valuable for prophylaxis and treatment of thrombotic disease during pregnancy.     

The Anti-D Content of IgG Preparations for Use in the Prevention of Rh Haemolytic Disease

Summary. Anti-D concentrations in IgG preparations for use in the prevention of haemolytic disease of the newborn have been estimated by direct labelling of the IgG with ¹²⁵I and by an indirect method using ¹²⁵I-labelled antiglobulin.
The concentrations ranged from 50–2000 μg anti-D/ml for total protein concentrations of 10–16 g/100 ml in the IgG preparations.     

Relationship of Factor VIII-like Antigen (VIII AGN) and Clot Promoting Activity (VIII AHF) as Measured by One-and Two-Stage Assays in Patients with Liver Disease

S ummary . We recently observed an increase in factor-VIII clot promoting activity as measured by a one-stage assay (VIII AHF₁) in a haemophiliac with hepatitis. However, VIII AHF as measured by a two-stage assay (VIII AHF₂) was 0.013 u/ml at a time when VIII AHF₁ measured 0.38 u/ml.
We then studied seven non-haemophiliacs with liver disease, and attempted to correlate the levels of VIII AHF₁ and VIII AHF₂ with factor VIII-like antigen (VIII AGN) as measured by quantitative immunoelectrophoresis. In four of the seven patients, disproportionate elevations of VIII AHF₂ compared to VIII AHF₁ were found. Furthermore, VIII AHF₂ values correlated well with VIII AGN values. No such discrepancy was apparent in four normal control subjects.
These findings emphasize the necessity for performing two-stage assays in haemophiliacs as well as non-haemophiliacs with liver disease to assess factor-VIII levels. In addition, they suggest that confirmation of the diagnosis of haemophilia may not be possible in the haemophiliac with hepatitis unless VIII AHF₂ determinations are performed. The reason for the disparity between VIII AHF₁ and VIII AHF₂ levels is not apparent. However, the correlation of VIII AGN and VIII AHF₂ levels in the non-haemophiliacs with liver disease provides further support for the concept that VIII AGN and VIII AHF are closely related or identical molecular entities.     

Development of an Immunoassay for the Detection of Minute Amounts of IgG-Coated Erythrocytes in Whole Blood and Its Application for the Assessment of Fc-Mediated Clearance of Anti-D-Coated Erythrocytes in vivo

Abstract. A rapid and sensitive immunoassay for the detection of minute quantities of IgG-coated erythrocytes in whole blood was developed. Washed red blood cells were incubated in two steps with anti-human IgG antiserum followed by ¹²⁵I-labelled protein A. The assay was able to detect amounts of sensitized erythrocytes as small as 0.5 ml of packed erythrocytes in a total blood volume of 5 liters and hematocrit 40%. A linear relation between increasing amounts of IgG-coated red cells in whole blood and the binding of ¹²⁵I-labelled protein A was obtained. We applied the technique on the assessment of the removal of IgG anti-D-coated erythrocytes from the circulation of test individuals. T_1/2 for the elimination of approximately 4 ml packed red cells sensitized with 62 μg of anti-D in 14 normal subjects was 20±5 min (mean±SEM). A splenectomized person did not clear the injected cells from the circulation during the test period of 70 min. If a standard curve was constructed the total blood volume in the test subjects could be calculated. This value correlated well (r = 0.99) with the blood volume calculated from the height and weight of the test individuals.     

Molecular ferrokinetics in the rabbit

S ummary . Using urea-polyacrylamide gel electrophoresis it has been possible to distinguish the molecular forms of transferrin in rabbit serum. When ⁵⁹Fe-labelled diferric transferrin is injected into normal, anaemic or hypertransfused, polycythaemic rabbits, iron is removed from diferric transferrin in essentially pairwise fashion. Exchange of iron between transferrin and tissues was also studied using predominantly monoferric transferrin labelled with ⁵⁹Fe or ¹²⁵I, and with ¹²⁵I-labelled apotransferrin. The return of iron from tissue stores to circulating transferrin occurs one atom at a time to either site of the protein and, possibly, in pairwise fashion as well. The rate of clearance of iron from diferric transferrin differs from that of monoferric transferrins, and the rates at which iron is returned to empty sites of transferrin also differ, so that serum iron is not a kinetically homogeneous pool in the rabbit.     

Studies on Platelet Glycoproteins in Glanzmann's Thrombasthenia using 125I-labelled Lectins

S ummary . Glycoproteins ((2) of Glanzmann's thrombasthenia (GT) and normal platelets were separated on sodium dodecyl sulphate (SDS) polyacrylamide slab gels and identified by their binding of ¹²⁵I-labelled lectins having different sugar specificities. This highly sensitive techniquc showed that in thrombasthenic platelets, glycoproteins in the positions of IIb and III (IIIa) were greatly reduced when compared to normal platelets. After incubation with neuraminidase, IIb became undectable. GT platelet glycoproteins I, IV (IIIb), and high molecular weight GPs bound ¹²⁵I-lectins more strongly than those in normal platelets, showing that there are drastic changes in the carbohydrate moieties of thcse major GPs.     

Structure-Function of the Factor VIII Complex Studied with an Immobilized Heteroantisera to VIII:C

An antibody was raised in rabbits to the small active fragment of human factor VIII, obtained by Ca²⁺ dissociation of a human factor VIII preparation made from a multidonor plasma pool. After absorption, the antibody neutralized the factor VIII coagulant activity of normal human plasma, but did not precipitate with any plasma or plasma fractions or neutralize von Willebrand factor (vWF) activity as measured by ristocetin aggregation of fixed washed platelets. Immune beads were prepared by CNBr binding of the partially purified rabbit antibody to 1% agarose beads. Non-immune beads were prepared with IgG fractions obtained from the rabbits before immunization and used throughout as a control. The amount of factor VIII coagulant activity (VIII:C) removed from plasma by immune beads was time-dependent and proportional to the amount of beads used, but all of the VIII:C could not be readily removed. Removal of VIII:C by immune beads parallelled removal of factor VIII:antigen, but less vWF activity was removed. Immune beads could be blocked or saturated by treatment with large amounts of normal plasma, but not by von Willebrand disease plasma and only by some haemophilic plasmas.     

Plasma Clearance of 125I-labelled Haemopexin in Normal and Haem-loaded Rabbits

S ummary . Plasma clearance of ¹²⁵I-labelled rabbit haemopexin has been studied in six normal control rabbits and in a test group of rabbits haem-loaded with 25 mg of haem.
Following intravenous injection, equilibration of ¹²⁵I-haemopexin between intravascular and extravascular compartments appeared to be complete within 24 hr and the decline in plasma activity then became constant in rate. The plasma activity curve was resolved into two exponential functions of time and from the data, fractional catabolic rate, intravascular:extravascular distribution of haemopexin and total haem binding capacity were calculated.
Control group. Mean plasma half-clearance time of haemopexin was 20.3 hr; fractional catabolic rate was 1.8 (SD 0.19) times the intravascular haemopexin pool per day. Extravascular haemopexin was 0.6 (SD 0.11) times the mass of haemopexin in the intravascular pool.
Test group. 90–95% of injected ¹²⁵I-activity was eliminated from the plasma with a mean half-clearance time of 1.57 hr and was associated with a significant urinary excretion of labelled iodine. Fractional catabolic rate was 5.84 (SD 0.3) times the intravascular haemopexin pool per day and extravascular haemopexin was 3.38 (SD 1.03) times the mass of haemopexin in the intravascular pool.
Haemopexin concentration in normal rabbit plasma was 31–52 mg/100 ml; intravascular plus extravascular haemopexin was 54.5 mg (mean value) and total haem binding capacity of haemopexin was 0.53 mg (mean value).
Intravascular haem rapidly complexed with haemopexin and the data suggested that this complex was promptly eliminated from the plasma and the haemopexin catabolized.     

Reactivity to Factor-VIII Concentrates of Lymphocytes from Patients with Haemophilia and von Willebrand''s Disease

S ummary . T o tcst the antigenicity of two materials used in the management of factor-VIII deficiency, lymphocytes from normal volunteers and from patients with haemophilia and with von Willebrand's disease (vWd) were exposed in vitro to cryoprecipitate and to glycine-precipitated factor VIII (Hemofil). As measured by incorporation of ¹⁴C-thymidine, the factor-VIII concentrates stimulated lymphocytes from some haemophiliacs and nontransfused normal donors, whereas lymphocytes from patients with vWd were unreactive. The data provide further immunological support for the concept that haemophilia and vWd are two separate and distinct disorders.     

Human Factor IX in Animals: Kinetics from Isolated, Radiolabelled Protein and Platelet Destruction following Crude Concentrate Infusions

S ummary Crude, concentrated and isolated, labelled human factor IX was prepared and infused into animals. Initially, two dogs with severe haemophilia B received both preparations and factor IX clotting activity and label survived well. Kinetic parameters fit a two-compartment open model.
For 12 infusions of concentrates into normal baboons, clotting and antigen data again fit the model. Similar kinetics, with less variability, were found for nine infusions of [¹²⁵I]factor IX. Radioactivity of the first post-infusion samples was >90% precipitated in the double antibody system with an average of 84% of counts adsorbable to barium citrate. Compared to plasma, barium adsorbable counts gave a shorter t _1/2β-elimination phase suggesting a small recirculating ¹²⁵iodide pool. When [¹²⁵I]factor IX was rechromatographed over heparin-agarose, variable amounts of altered, labelled protein were present; this species was more rapidly cleared from plasma post-infusion.
Twelve infusions of crude concentrates into baboons were given to animals whose platelets had been labelled with ⁵¹Cr. Platelet counts as well as survival were then followed. For seven of these animals, mean counts decreased and the labelled platelets were destroyed, to an average of 21%, during the first 4 h post-infusion. Subsequent platelet survival was normal. Three infusions of gel-filtered, crude concentrates and two infusions of 'activated'commercial concentrates did not significantly alter platelet survival.     

Binding and Release of Factor VIII/von Willebrand''s Factor by Human Endothelial Cells

The uptake and release of factor VIII/von Willebrand's protein by cultured human umbilical vein endothelial cells have been examined using highly purified 125I-factor VIII possessing von Willebrand's factor activity. 125I-factor VIII/vWF was taken up by the cells, reaching maximum binding within 4 h with a t1-2 of binding of 15 min. Endothelial cell binding of 125I-factor VIII/vWF reached saturation at a concentration of 1.5 mg/l. Binding was inhibited by coincubation of excess unlabelled factor VIII/vWF. Most of the cell-associated radioactivity was released by treatment of the cells with trypsin. Internalization of bound protein was evidenced by the incorporation into the cells of radioactivity which could not be released by trypsin. Human vascular smooth muscle cells did not bind 125I-factor VIII/vWF. Addition of 0.1 microM epinephrine to the 125I-factor VIII/vWF labelled endothelial cultures induced the release of cell bound, protein-associated radioactivity into the medium. Propranolol inhibited completely epinephrine-induced release, whereas phenylephrine had no effect. Endothelial cells maintained in medium partially depleted of factor VIII/vWF by tricalcium citrate cellulose treatment of plasma did not release factor VIII antigen into the culture medium during subsequent incubation. Although [3H]proline was incorporated into proteins released by endothelial cells under these experimental conditions, specific incorporation of label into factor VIII/vWF antigen was not detectable by a sensitive solid-phase immunoradiometric assay. We conclude that factor VIII/vWF binds to endothelial cells and that this cell-bound protein is mobilized by epinephrine through beta-adrenergic stimulation.     

Purification and characterization of human platelet von Willebrand factor

Summary. Platelet von Willebrand factor (vWf) was purified from human platelet concentrates. The multimeric structure of the purified platelet vWf was similar to that observed in the initial platelet lysate, and, like the platelet lysate, the purified platelet vWf contained higher molecular weight multimers than plasma vWf. The apparent molecular weight of the reduced platelet vWf subunit was similar to the plasma vWf subunit. The N-terminal amino acid of the purified platelet and plasma vWf was blocked. In concentration dependent binding to botrocetin- or ristocetin-stimulated platelets, ¹²⁵I-plasma vWf bound with a higher affinity than platelet. The ristocetin cofactor activity per mg of purified plasma vWf was 5-fold greater than the platelet vWf activity. Platelet and plasma vWf bound to collagen with similar affinities; however, platelet vWf bound to thrombin-stimulated platelets and to heparin with a higher affinity than plasma vWf. The differences in the binding affinity(s) of plasma and platelet vWf to platelet GPIb and GPIIb/IIIa and extracellular matrix proteins may reflect different roles for plasma and platelet vWf in the initial stages of haemostasis and thrombosis.