The effects of different fixatives for immunofluorescent and immunoperoxidase localization of Factor VIII-related antigen in canine carotid artery and vascular prostheses

Summary The evaluation of vascular grafts seeded with autologous endothelial cells requires a reliable method of endothelial cell identification. Previous attempts to identify positively tissue Factor VIII-related antigen, found in relatively large amounts in vascular endothelial cells, proved to be inconsistent when immunoperoxidase and immunofluorescent staining techniques were tried. Because the Factor VIII antigen is very labile, this study was performed to determine an optimal fixation technique for demonstrating this antigen in frozen sections of endothelial tissue. Unfixed, acetone-fixed, and formalin-fixed sections of canine carotid artery as well as vascular grafts fixed in 1-ethyl-3-(3-diaminopropyl)-carbodiimide were examined by an indirect immunofluorescence technique. Also, the immunoperoxidase method of Factor VIII identification was applied to unfixed, acetone-fixed, and carbodiimide-fixed endothelial cell seeded vascular grafts. Acetone was the preferred fixative, resulting in excellent antigen preservation with minimal background staining. The immunoperoxidase technique of Factor VIII-related antigen identification was found to be the method of choice because of its sensitivity.     

Physiological changes in hemostasis associated with acute exercise

Acute exercise enhances fibrinolytic (FA), factor VIII coagulant and factor VIII ristocetin cofactor activities, and increases the concentration of factor VIII-related antigen. Little is known concerning the mechanisms of these changes. To investigate possible relationships between exercise-induced changes in blood lactate, 2,3-diphosphoglycerate (DPG), and the hemostatic variables, a branching multistage treadmill protocol was used to exercise male volunteers to a maximum effort. Blood samples were drawn before, immediately post-, and 8 min postexercise. All hemostatic variables were significantly (P less than 0.05) increased postexercise. Highest values for factor VIII coagulant, factor VIII-related antigens and factor VIII ristocetin cofactor were observed at 8 min postexercise. Significant (P less than 0.001) correlations were found postexercise for lactate with factor VIII coagulant (r = 0.64), while no association between pre-, post-, or 8 min postexercise. Postexercise lactate demonstrated a significant correlation (r = +0.81), which was strengthened by including the preexercise high-density lipoprotein (HDL) concentrations (r = +0.87). Consequently, the expected postexercise FA may be calculated from the observed values for postexercise lactate and preexercise HDL. The correlations of lactate with postexercise FA and with postexercise factor VIII coagulant may reflect a common stimulus for these exercise-induced changes.     

Factor VIII and factor IX in a twin population. Evidence for a major effect of ABO locus on factor VIII level.

In order to establish the relative importance of genetic factors on the variation in plasma concentration of coagulation factors VIII and IX, these parameters were determined in 74 monozygotic and 84 like-sexed dizygotic twin pairs. The twins belonged to two age groups: 33-39 years and 57-62 years. Factor VIII was determined as factor VIII coagulant antigen (VIIICAg) and as factor VIII-related antigen (VIIIRAg). Factor IX was determined as factor IX antigen (IXAg). A higher value for each coagulation factor was found in the older-age group compared to the younger group, whereas no difference was found between the sexes. A significant correlation was found between values for VIIIRAg and VIIICAg (r = .56). For VIIICAg, it could be demonstrated that the age effect was secondary to the age effect on VIIIRAg. The concentration of VIIICAg and VIIIRAg varied among ABO blood types, being lowest in type O individuals, higher in A2 individuals, and highest in A1 and B individuals. The effect of the ABO locus on VIIICAg was secondary to an effect on VIIIRAg. Analysis of variance revealed a significant genetic influence on the variance of VIIICAg and VIIIRAg with a heritability estimate of .57 for VIIICAg and .66 for VIIIRAg. This is in agreement with a previous hypothesis of an effect of several autosomal genes on factor VIII concentration. Thirty percent of the genetic variance of VIIIRAg was due to the effect of ABO blood type. The ABO locus is therefore a major locus for the determination of factor VIII concentration. No significant genetic effect on the variation in plasma concentration of IXAg could be detected.     

Effects of diet and gliclazide on the haemostatic system of non-insulin-dependent diabetics.

The effect of the sulphonylurea gliclazide on tests of haemostatic function was studied in 14 newly diagnosed non-insulin-dependent diabetics. After two months'' treatment with diet alone 11 of the 14 were given gliclazide; the three others remained on dietary treatment. Compared with pretreatment values, significant reductions in platelets retention, factor VIII-related antigen, factor VIII coagulant activity, and plasma heparin neutralising activity accompanied a fall in the plasma glucose concentration due to either diet alone or diet and gliclazide. The beneficial effects of gliclazide on platelet abnormalities seem likely to be due to its hypoglycaemic action rather than to any direct effect on haemostatic function.     

Fractionation of individual, biologically active factor VIII multimers

We have designed an electrophoretic system for the fractionation of individual, biologically active multimers of factor VIII. Human factor VIII, purified by gel filtration on Sepharose CL-2B from plasma cryoprecipitate, was submitted to electrophoresis without SDS on 2.0% polyacrylamide gels in 0.04 M Tris/0.06 M Tes buffer, pH 7.5. Staining with Coomassie blue revealed a series of protein bands. Measurement of electrophoretic mobility showed constant size intervals between adjacent bands. Electrophoresis in a second dimension, in the presence of SDS, resulted in an identical order of mobilities, suggesting that the different migration rates of factor VIII proteins in the first electrophoretic system were size- and not charge-dependent. After electrophoresis in the absence of SDS both factor VIII coagulant and ristocetin cofactor activities as well as factor VIII-related antigen were recovered by elution from gel slices. The distribution of activity peaks resembled that of Coomassie-stained factor VIII proteins found in control gels. We thus demonstrate that an electrophoretic fractionation of factor VIII multimers is possible even at neutral pH where factor VIII activities are retained.     

Problems in the detection of carriers of hemophilia A: the influence of stress and thrombin

After exposure to stress, factor VIII coagulant activity and factor VIII antigen were determined in healthy volunteers for about 3 days. Three out of four volunteers at different time showed results resembling a carrier state. In vitro incubation of plasma with thrombin in decreasing concentrations showed increased factor VIII coagulant activity, although no fibrin clot or fibrin monomers were formed. The concentration of factor VIII antigen remained constant. The action of thrombin can explain the misclassification of known carriers.     

Ultrastructural localization of factor VIII-related antigen in cultured human brain microvessel endothelial cells.

Immunogold staining of primary cultures of human brain microvessel endothelial cells demonstrated the presence of Factor VIII-related antigen within cytoplasmic vesicles in close association with the rough endoplasmic reticulum and Golgi apparatus. Immunoperoxidase staining, at the light microscopic level, revealed a similar granular, perinuclear staining. The morphology and location of these vesicular profiles indicate that they are part of the trans-Golgi region where terminal processing and short-term storage of Factor VIII-related antigen takes place. Weibel-Palade bodies, specific storage organelles for von Willebrand factor in large vessel endothelium, were not observed in cerebral microvessel endothelium. The release of Factor VIII-related antigen from the cytoplasmic vesicles was influenced by some of the factors known to stimulate or inhibit the regulated pathway of secretion from Weibel-Palade bodies. Thus, stimulation of endothelial cells with calcium ionophore A23187 resulted in almost complete loss of staining, while addition of EGTA to the culture medium led to slight increase of intracellular pools of Factor VIII-related antigen. Pre-incubation of monolayers with interferon-gamma was associated with significant increase in the number of labeled vesicles, suggesting an additional role of this cytokine in the localized immune reaction within the central nervous system.     

The relationship of biological and immunological activities of factor VIII

Factor VIII is an essential blood clotting factor which consists of two protein moieties, each with distinct biological functions and antigenic determinants. The immunological markers were originally seen as indicators of the biological activities; however this view has been increasingly challenged. We have investigated the biological and immunological properties of Factor VIII to clarify these relationships. Plasma stored at room temperature for 21 days lost biological activity, but retained immunological activity: The procoagulant activity was reduced to 35% and the ristocetin cofactor activity to 75.4% of their original levels; but the reactivities of both procoagulant antigen and Factor VIII related antigen were maintained. A dissociation of activities was also demonstrated in serum, in which the procoagulant activity was 10% and the procoagulant antigen 72% of corresponding plasma values. These results indicate that the antigenic reactivities are not appropriate markers for Factor VIII biological activity.     

Isolation, characterisation, growth and culture of endothelial cells from the rat aorta

Rat aortic endothelial cells have been isolated by the explantation technique and grown in culture. They have been identified morphologically using standard staining techniques, biochemically by identification of angiotensin convertase and have been positively stained for Factor VIII-related antigen by immunofluorescence using both anti-human and anti-rat Factor VIII antibodies. The explantation technique is a successful alternative to enzyme digestion which is not applicable to rat aortic endothelial cells because of the nature of their attachment to the subendothelial layer.     

Synthesis of factor II antigen by isolated perfused rat liver

Rat Factor II (prothrombin), isolated and purified by chromatography on Blue Dextran-agarose, was used to raise an antiserum in rabbits. On the basis of single radial immunodiffusion measurements. Factor II synthesis by isolated perfused rat liver amounted to 0.54 mg/300 cm² body surface area of the liver donor in 10 h. Corresponding measurements of Factor II coagulant activity revealed cumulative synthesis of 802 Iowa units. Coumadin added to the liver perfusate blocked production of Factor II coagulant activity, but did not change synthesis of the immunologically measured protein. In perfusions in which either heparin or citrate was used as anticoagulant, synthesis of albumin was not affected by the choice of anticoagulant but bile production and synthesis of Factor II were significantly less in citrate perfusions.     

Studies on factor VIII-related protein. I. Ultrastructural and electrophoretic heterogeneity of human factor VIII-related protein.

Human factor VIII-related protein precipitates with specific heterologous anti-bodies directed against purified factor VIII and supports ristocetin-induced aggregation of washed platelets. We purified human factor VIII from cryoprecipitate by subsequent gel filtration on crosslinked large-pore agarose. Factor VIII-related protein appeared as a large aggregate following electrophoresis on 3% polyacrylamide gels in the presence of sodium dodecyl sulfate (SDS). The same material was separated into multiple bands (molecular weight in excess of several millions) following electrophoresis on SDS-1% agarose gels. After complete disulfide reduction of factor VIII-related protein and electrophoresis on SDS-5% polyacrylamide gels a single subunit chain (Mr approximately equal to 200 000) was revealed. Analysis of this protein, in its non-reduced state, by negative contrast electron microscopy showed filaments of markedly variable size. The calculated molecular weight of such filaments ranged from about 0.6.10(6) to 20.10(6). We conclude that size heterogeneity is an essential feature of human factor VIII-related protein.     

Immunohistochemistry in the evaluation of neovascularization in tumor xenografts

Angiogenesis, or neovascularization, is known to play an important role in the neoplastic progression leading to metastasis. CD31 or Factor VIII-related antigen (F VIII RAg) immunohistochemistry is widely used in experimental studies for quantifying tumor neovascularization in immunocompromised animal models implanted with transformed human cell lines. Quantification, however, can be affected by variations in the methodology used to measure vascularization including antibody selection, antigen retrieval (AR) pretreatment, and evaluation techniques. To examine this further, we investigated the microvessel density (MVD) and the intensity of microvascular staining among five different human tumor xenografts and a mouse syngeneic tumor using anti-CD31 and F VIII RAg immunohistochemical staining. Different AR methods also were evaluated. Maximal retrieval of CD31 was achieved using 0.5 M Tris (pH 10) buffer, while maximum retrieval of F VIII RAg was achieved using 0.05% pepsin treatment of tissue sections. For each optimized retrieval condition, anti-CD31 highlighted small vessels better than F VIII RAg. Furthermore, the MVD of CD31 was significantly greater than that of F VIII RAg decorated vessels (p<0.001). The choice of antibody and AR method has a significant affect on immunohistochemical findings when studying angiogenesis. One also must use caution when comparing studies in the literature that use different techniques and reagents.     

Factor IX Amagasaki: a new mutation in the catalytic domain resulting in the loss of both coagulant and esterase activities

Factor IX Amagasaki (AMG) is a naturally occurring mutant of factor IX having essentially no coagulant activity, even though normal levels of antigen are detected in plasma. Factor IX AMG was purified from the patient's plasma by immunoaffinity chromatography with an anti-factor IX monoclonal antibody column. Factor IX AMG was cleaved normally by factor VIIa-tissue factor complex, yielding a two-chain factor IXa. Amino acid composition and sequence analysis of one of the tryptic peptides isolated from factor IX AMG revealed that Gly-311 had been replaced by Glu. We identified a one-base substitution of guanine to adenine in exon VIII by amplifying exon VIII using the polymerase chain reaction method and sequencing the product. This base mutation also supported the replacement of Gly-311 by Glu. In the purified system, factor IXa AMG did not activate factor X in the presence of factor VIII, phospholipids, and Ca2+, and no esterase activity toward Z-Arg-p-nitrobenzyl ester was observed. The model building of the serine protease domain of factor IXa suggests that the Gly-311----Glu exchange would disrupt the specific conformational state in the active site environment, resulting in the substrate binding site not forming properly. This is the first report to show the experimental evidence for importance of a highly conserved Gly-142 (chymotrypsinogen numbering) located in the catalytic site of mammalian serine proteases so far known.     

The binding of factor IXa to cultured bovine aortic endothelial cells. Induction of a specific site in the presence of factors VIII and X

Previous studies have demonstrated a Factor IX and IXa binding site on the endothelial cell surface for which both the zymogen and enzyme compete with equal affinity. In this report, we demonstrate that the affinity of Factor IXa, but not Factor IX, for the cell surface is increased in the presence of both Factors VIII and X. When Factor Xa formation was studied in the presence of saturating concentrations of Factors VIII and X, the half-maximal rate was observed at a Factor IXa concentration of 151 +/- 12 pM. Active site-blocked Factor IXa, 5-dimethylaminonaphthalene-1-sulfonyl-Glu-Gly-Arg-Factor IXa, was a more effective inhibitor of Factor X activation (Ki = 124 pM) than was Factor IX (Ki = 3.0 nM). Radioligand binding studies carried out in the presence of Factors VIII and X confirmed the presence of a selective endothelial cell Factor IXa binding site with Kd = 127 +/- 27 pM. In contrast, when Factor IXa binding was studied in the absence of other coagulation factors, or in the presence of Factor VIII (thrombin-activated or unactivated) alone, this new high affinity site was not observed. Competitive binding studies indicated that Factor IXa was 12 times more effective as an inhibitor of Factor IX-endothelial cell binding in the presence of Factors VIII and X. Consistent with the increased affinity of Factor IXa binding in the presence of factors VIII and X, cell-associated Factor IXa coagulant activity decayed 7 times more slowly in the presence of these coagulation factors. These results demonstrate selective Factor IXa-endothelial cell binding in the presence of Factors VIII and X, suggesting this interaction could be a physiologic occurrence.     

Increased factor VIII/vWf levels in patients with reduced platelet number

Factor VIII/von Willebrand factor (VIII/vWf) related properties were studied in twenty six patients with thrombocytopenia. Fifteen patients were affected by idiopathic thrombocytopenic purpura (ITP) and 11 patients by thrombocytopenia of a different nature or non-ITP (n-ITP). All patients showed an enhancement of platelet associated IgG (PAIgG). A significant increase of factor VIII ristocetin cofactor (VIII R: RCoF) and factor VIII related antigen (VIII R:Ag) was found in ITP patients while normal values were observed for factor VIII coagulant (VIII:C). All factor VIII/vWf components, on the contrary, were increased in n-ITP group with a prevalence of VIII R:RCoF as observed in ITP group even though with lower mean values. Multimeric analysis of VIII/vWf demonstrated a higher concentration of all multimeric components, with major representation of higher molecular weight multimers (HMWM) in patients of both groups. Two patients were studied before and after improvement in platelet count. A decrease of vWf related properties (VIII R:RCoF and VIII R:Ag) concomitant with the increase in platelet count was found. In n-ITP patients a statistical correlation between VIII R:RCoF and PAIgG was also observed while no correlation was found between other factor VIII/vWf components and PAIgG both in ITP and n-ITP patients.     

Identification of a factor IX/IXa binding protein on the endothelial cell surface

Endothelium provides a specific binding site for Factor IX/IXa which can propagate activation of coagulation by promoting Factor IXa-VIII-mediated activation of Factor X. In this report the endothelial cell Factor IX/IXa binding site has been identified and the coagulant function of the receptor blocked. Studies using [3H]Factor IX derivatized with the photoaffinity labeling agent N-succinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate (SANPAH) and cultured bovine endothelial cells demonstrated cross-linking to a trypsin-sensitive cell surface protein of Mr approximately equal to 140,000. Immunoprecipitation of metabolically labeled endothelium with Factor IX derivatized with the cleavable cross-linking agent N-succinimidyl(4-azidophenyl)-1,3'-dithiopropionate and antibody to Factor IX demonstrated the endothelial cell origin of the Mr 140,000 cell surface protein. Blockade of the Factor IX/IXa binding protein by covalently linking SANPAH-5-dimethylaminonaphthalene-1-sulfonyl-Glu-Gly-Arg-Factor IXa or SANPAH-Factor IX prevented both specific Factor IXa binding and effective Factor IXa-VIII-mediated activation of Factor X on endothelium. Following extraction of endothelium with detergents, Factor IX/IXa binding activity was solubilized and could be assayed using a polyvinyl chloride plate binding assay. Western blots of cell extracts demonstrated binding of 125I-Factor IX at Mr approximately equal to 140,000 which was blocked by excess Factor IX, but not antisera to Factor VIII, von Willebrand factor, alpha 2-macroglobulin, or epidermal growth factor receptor. These data indicate that endothelium provides a distinct binding site for Factor IX/IXa consisting, at least in part, of a membrane protein which can modulate the coagulant activity of Factor IXa on the cell surface.     

Influence of fixation and decalcification on the immunohistochemical staining of cell-specific markers in paraffin-embedded human bone biopsies

A number of fixation and decalcification procedures were evaluated to determine their suitability for immunohistochemistry on trephine samples of bone marrow after paraffin embedding. In particular, the immunoreactivity of antigens characteristic for various hematopoietic cell lines (immunoglobulin heavy and light chains for plasmacytoid cells; elastase for neutrophil myeloid cells; lysozyme, alpha-1-antitrypsin and alpha-1-antichymotrypsin for hystiocytic cells; leukocyte common antigen for lymphocytes; hemoglobin and glycophorin A for erythroid cells; Factor VIII-related antigen for thrombocytoid cells) as well as some antigens specific for epithelial tumors (CEA, 115D8, and keratin) were investigated. Fixation in a mercuric chloride-formaldehyde mixture followed by decalcification in acetic acid-formaldehyde-saline proved to be the best procedure for antigen preservation and retention of morphologic detail. Moreover, there is no need of trypsinization when using this procedure. The only exception was Factor VIII-related antigen in megakaryocytes, which was best demonstrated in trypsin-digested sections of formalin-fixed and acetic acid-decalcified biopsies.     

Combined deficiency of factor V and factor VIII. A report of another case.

A patient with combined factor V and factor VIII deficiency is presented. The bleeding manifestations were: easy bruising, post-traumatic bleeding, bleeding after tooth extractions. The main laboratory feature was a prolonged partial thromboplastin time which was corrected by the addition of adsorbed normal plasma but not by the addition of normal serum, hemophilia A plasma of another patient with combined factor V and factor VIII deficiency. The thromboplastin generation test was clearly abnormal and was corrected by the addition of adsorbed normal plasma but not by addition of normal serum. Prothrombin consumption was also defective. Prothrombin time was slightly prolonged too, Thrombin time, platelet and vascular tests were within normal limits and there was no hyperfibrinolysis. Factor VIII was 8% of normal, whereas factor V was 14% of normal. Factor VIII associated antigen was normal. All other clotting factors were within normal limits. The parents of the propositus were consanguineous (first cousins) but had normal factor V and factor VIII activity and normal factor VIII antigen. The same was true for other family members. The hereditary transmission of the condition appears autosomal recessive.     

The binding of 35S-labeled recombinant factor VIII to activated and unactivated human platelets

Recombinant-derived human Factor VIII was labeled intrinsically with [35S]methionine, and its binding to washed human platelets was studied. Binding measurements were performed by incubating Factor VIII and platelets for 15 min at room temperature in Tyrode's solution supplemented with Ca2+ (5.0 mM), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (5.0 mM), 0.50% bovine serum albumin, and the Factor Xa and thrombin inhibitors 5-dimethylaminonaphthalene-1-sulfonylglutamylglycinylarginyl chloromethyl ketone and 5-dimethylaminonaphthalene-1-sulfonyl-arginine-N-(3-ethyl-1, 5-pentanediyl)amide. Separation of free from bound Factor VIII was accomplished by centrifugation through oil, and nonspecific binding was determined with excess unlabeled Factor VIII. Binding was saturable, reversible, and stimulated 20-fold after platelet activation with thrombin. Furthermore, binding was specific in that bound labeled Factor VIII could be displaced by excess unlabeled Factor VIII, but not by Factor V. Scatchard analysis indicated a single class of binding sites with Kd = 2.9 nM and 450 sites/activated platelet. The time course of displacement indicated a t1/2 of bound Factor VIII of approximately 5 min. When platelets were incubated in Ca2+, both the heavy and light chains of Factor VIII were bound, whereas exposure to EDTA resulted in the binding of the light chain only. These results demonstrate the specific reversible binding of Factor VIII to human platelets, likely mediated through the light chain.     

Proteolytic processing of human factor VIII. Correlation of specific cleavages by thrombin, factor Xa, and activated protein C with activation and inactivation of factor VIII coagulant activity

Human factor VIII was isolated from commercial factor VIII concentrates and found to consist of multiple polypeptides with molecular weights ranging from 80 000 to 210 000. Immunological and amino acid sequence data identified these polypeptides as subunits of factor VIII. N-Terminal amino acid sequence analysis determined that the Mr 210 000 and 80 000 proteins are derived from the N- and C-terminal portions of factor VIII, respectively; Mr 90 000-180 000 polypeptides are derived from the Mr 210 000 polypeptide by C-terminal cleavages. Treatment of purified factor VIII with thrombin resulted in proteolysis of Mr 80 000-210 000 proteins and the generation of polypeptides of Mr 73 000, 50 000, and 43 000. Maximum coagulant activity of thrombin-activated factor VIII was correlated with the generation of these polypeptides. The proteolysis as well as activation of factor VIII by thrombin was found to be markedly dependent on CaCl2 concentration. Proteolysis of factor VIII with activated protein C (APC) resulted in degradation of the Mr 90 000-210 000 proteins with the generation of an Mr 45 000 fragment. This cleavage correlated with inactivation of factor VIII by APC. The Mr 80 000 protein was not degraded by APC. Factor Xa cleaved the Mr 80 000-210 000 factor VIII proteins, resulting in the generation of fragments of Mr 73 000, 67 000, 50 000, 45 000, and 43 000. Factor Xa was found to initially activate and subsequently inactivate factor VIII.(ABSTRACT TRUNCATED AT 250 WORDS)