Exercise-induced changes of Factor VIII complex in healthy subjects and in type-I diabetics: relation between growth hormone and Von Willebrand Factor increments

This work aims to study the exercise-induced changes of Factor VIII Complex both in healthy subjects and in type I diabetics without vascular complications, and to investigate the possible relations between growth hormone and Von Willebrand's Factor response to exercise. Results show that maximal exercise performed by cycle ergometer causes a significant increment of the procoagulant subunit (VIII:C) and of Von Willebrand Factor (VIII:RiCoF) both in healthy controls and in type I diabetics, whereas a slight increment of Factor VIII-Related antigen (VIIIR:Ag) is observed only in diabetics. The shape of the mean GH response to exercise parallels the one of Von Willebrand's Factor: however, the presence of VIII:RiCoF increments also in GH non-responders supports the conclusion that growth hormone is not the only factor involved in the regulation of Von Willebrand's Factor exercise-induced increase.     

The role of Willebrand factor in platelet - blood vessel interaction, including a discussion of resistance to atherosclerosis in pigs with von Willebrand's disease

Von Willebrand pigs have all the manifestations of the severe human disease. The role of Willebrand antigen (VIII R:AG) and ristocetin cofactor (VIII: RWF) was assessed in these pigs by (1) transfusion and (2) "in vitro" bleeding time assay. The skin bleeding time became normal when the level of transfused Willebrand factor (VIII R:AG/RWF) was raised in the plasma above 30 U/dl. After single or repeated transfusions, skin capillary endothelium and platelets were still distinguished from normal by VIII R:AG deficiency. When incisions in excised porcine skin ("in vitro" bleeding time) were perfused with blood and plasma fractions, haemostasis occurred when plasmatic Willebrand factor exceeded 30 U/dl whether the skin or platelets came from normal or from von Willebrand pigs. The platelet plug occluding the skin incision contained VIII R:AG by immunofluorescence. Willebrand factor appears to coat surfaces and to serve as a platelet attachment protein. These bleeder pigs are resistant to atherosclerosis. If platelets are involved in early atherosclerotic lesions, the role of Willebrand factor in platelet - blood vessel interaction may be important.     

Multimers of von Willebrand antigen in therapeutic factor VIII concentrates

By crossed immunoelectrophoresis (XIEP), the pattern of von Willebrand factor antigen (vWF:Ag) was investigated in six commercially available factor VIII (F VIII) concentrates and in normal human plasma. At least 5 subpopulations of vWF:Ag were recognized by XIEP in the therapeutic F VIII concentrates and in normal plasma. F VIII preparations high in ristocetin cofactor activity (vWF:RICof) and low in the ratio of vWF:Ag to vWF:RiCof were found to be similar in the multimeric structure of vWF:Ag to normal plasma. However, F VIII concentrates low in activity of vWF:RiCof and high in the ratio of vWF:Ag to vWF:RiCof were found to be deficient in the slowly migrating, high molecular weight multimers of vWF:Ag present in normal plasma.     

The use of antiproteolytic mixture fails to modify the abnormal von Willebrand factor pattern in myeloproliferative disease

Abnormalities of von Willebrand factor, with reduction of higher molecular weight multimers, was described in patients with increased platelet number. We have studied twelve patients with myeloproliferative disease (9 patients with Essential Thrombocytosis and 3 patients with Polycythemia Vera). Blood samples were collected either with citrate and antiproteolytic mixture (EDTA 6 mM, Aprotinin 200 U/ml, N-ethylmaleimide 5 mM in sodium citrate 3.8%). VIIIR: RCoF was found decreased in all patients studied, using both anticoagulants, while VIIIR: Ag was within normal range. Higher molecular weight multimers were found decreased or absent both in samples collected with citrate and in those collected using the antiproteolytic mixture. These data suggested that von Willebrand factor abnormalities observed in myeloproliferative disease are not due to a proteolytic degradation that's supposed to happen in vitro during blood samples collection and manipulation.     

Factor VIII/von Willebrand Factor has potent lectin activity

Highly-purified plasma and platelet Factor VIII/von Willebrand Factor had potent lectin activity when measured in a haemagglutination assay. This lectin activity was inhibited by monoclonal and heterologous antibodies to Factor VIII/von Willebrand Factor as well as by hexosamines, mannose and net-positively charged amino acids.     

The human platelet receptor(s) for quinine/quinidine-dependent antibodies

Substantial evidence now exists to associate platelet membrane glycoprotein Ib (GP Ib) with a receptor for quinine/quinidine-dependent platelet-specific antibodies. A direct relationship between GP Ib and this receptor activity has been difficult to establish for several reasons, including: the apparent existence of additional receptor activity not directly attributable to the presence of GP Ib; the variable reactivity of different sera observed by some investigators; the instability of receptor activity in semi-purified, soluble form; and differences in methods used by various laboratories to identify and quantitate either quinine/quinidine-dependent antibodies or platelet receptor activity. Moreover, little attention has been paid to the possibility that the Bernard-Soulier syndrome may represent a more heterogeneous collection of functional and molecular platelet abnormalities than hitherto supposed. As more patients are identified and studied, this possibility can also be addressed. A role for factor VIII-related antigen (VIIIR:Ag) in platelet destruction and/or clearance by drug-antibody complexes remains controversial. The observation that VIIIR:Ag is required for platelet activation in vitro (serotonin release, aggregation and increased platelet factor 3 availability) has been made, yet recent evidence indicates that VIIIR:Ag is not required for binding of antibody to platelets in the presence of drug or for complement-mediated lysis of platelets by antibody and drug. Evidence that VIIIR:Ag participates as part of the initial immunogenic complex is intriguing, yet still unconfirmed.(ABSTRACT TRUNCATED AT 250 WORDS)     

A monoclonal antibody to factor IX that inhibits the factor VIII:Ca potentiation of factor X activation

A murine monoclonal antibody (IgG1k, Kd approximately 10(-8) M) specific for an epitope located on the heavy chain of human factor IXa was used to study structure-function relationships of factor IX. The antibody inhibited factor IX clotting activity but did not impair activation of factor IX either by factor XIa/calcium or by factor VIIa/tissue factor/calcium. The antibody also did not impair the binding of factor IXa to antithrombin III. Moreover, the antibody did not prevent calcium and phospholipid (PL) from inhibiting the binding of factor IXa to antithrombin III. The antibody also failed to impair activation of factor VII by factor IXa/calcium/PL. Furthermore, the antibody did not interfere with the very slow activation of factor X by factor IXa/calcium/PL. In contrast, the antibody did interfere with factor X activation when reaction mixtures also contained factor VIII:Ca/von Willebrand factor. The marked acceleration of factor X activation observed in control mixtures was not observed in mixtures containing the antibody. Similar results were obtained in reaction mixtures containing the Fab portion of the antibody and factor VIII:Ca free of von Willebrand factor. In additional experiments, factor VIII:Ca/von Willebrand factor was found to inhibit the binding of the antibody to 125I-factor IXa as determined using an immunosorbent assay. Moreover, the antibody displaced factor VIII:Ca from the factor X activator complex (IXa/calcium/PL/VIII:Ca) as evidenced by an altered elution pattern on gel filtration chromatography. From these observations, we conclude that the antibody impairs the clotting activity of factor IXa through interference with its binding of factor VIII:Ca. This suggests a significant role for the heavy chain (residues of 181-415) of factor IXa in binding factor VIII:Ca.     

Carbohydrate on human factor VIII/von Willebrand factor. Impairment of function by removal of specific galactose residues.

Human factor VIII/von Willebrand factor protein containing 120 +/- 12 nmol of sialic acid and 135 +/- 13 nmol of galactose/mg of protein was digested with neuraminidase. The affinity of native factor VIII/von Willebrand factor and its asialo form for the hepatic lectin that specifically binds asialoglycoproteins was assessed from in vitro binding experiments. Native factor VIII/von Willebrand factor exhibited negligible affinity while binding of the asialo derivative was comparable to that observed for asialo-alpha1-acid glycoprotein. Incubation of asialo-factor VIII/von Willebrand factor with Streptococcus pneumoniae beta-galactosidase removed only 62% of the galactose but abolished binding to the purified hepatic lectin. When the asialo derivative was incubated with purified beta-D-galactoside alpha2 leads to 6 sialyltransferase and CMP-[14C]NeuAc, only 61% of the galactose incorporated [14C]NeuAc. From the known specificites of these enzymes, it is concluded that galactose residues important in lectin binding are present in a terminal Gal/beta1 leads to 4GlcNAc sequence on asialo-factor VIII/von Willebrand factor. The relative ristocetin-induced platelet aggregating activity of native, asialo-, and agalacto-factor VIII/von Willebrand factor was 100:38:12, respectively, while procoagulant activity was 100:100:103.     

Exposure of platelet binding sites in von Willebrand factor by adsorption onto polystyrene latex particles

Von Willebrand factor molecules are flexible linear polymers composed of repeating protomeric polypeptide subunits. In the process of primary hemostasis, von Willebrand factor promotes platelet adhesion and platelet plug formation at the site of vascular injury. This biologic activity is apparently related to the multimeric size of von Willebrand factor. We simulated von Willebrand factor binding to the subendothelial surface by adsorbing purified human von Willebrand factor onto polystyrene latex particles of two different diameters, i.e., 0.312 μm and 2.02 μm. The rate and extent of ¹²⁵I-labeled von Willebrand factor binding to polystyrene was similar with both size classes of latex particles. The von Willebrand factor-coated latex beads of 2.02 μm diameter, in contrast to the smaller size, induced rapid agglutination of formalin-fixed human platelets in the absence of any other aggregating agent. Von Willebrand factor was also adsorbed from human plasma onto latex particles coated with anti-von Willebrand factor antibodies. Again, only the large beads, carrying the von Willebrand factor-antibody complex, induced agglutination of fixed platelets. Shear stress promoted the rate of von Willebrand factor adsorption to latex particles. Our results suggest that adsorption to surface exposes binding sites in human von Willebrand factor for platelets.     

Thrombin-activated and factor Xa-activated human factor VIII: differences in cofactor activity and decay rate.

The decay of human coagulation factor VIIIa has been studied by kinetic methods that ensure no interference through proteolytic feedback. The rate of decay of factor VIIIa activity was found to vary with the activator used to activate factor VIII. Thrombin-activated factor VIII-von Willebrand factor complex (fVIII-vWf) decayed at a rate of 0.31 min-1, whereas factor Xa-activated fVIII-vWf decayed at 0.11 min-1 under the same conditions. Factor VIII free of von Willebrand factor (factor VIII: C), although decaying at a generally slower rate after activation, still showed a dependence of decay rate on activator: thrombin-activated factor VIII:C decaying at a rate of 0.06 min-1, and factor Xa-activated factor VIII: C at 0.01 min-1. Readdition of von Willebrand factor (18 micrograms/ml) to factor VIII:C did not alter the observed activity or decay rate. The decay of the two species of factor VIIIa was studied, using the fVIIIa-vWf complex, in the presence of varying levels of factor IXa. Plots of reciprocal decay rates vs factor IXa concentration were linear, and nearly parallel for the two factor VIIIa species, with a mean slope of 0.56 min.nM-1. In addition to these studies, we have confirmed previous studies showing that the two forms of factor VIIIa differ in cofactor activity, but they do so in the same ratio as in their decay rates. We suggest that this difference and that observed in decay rate have a common cause, and incorporate this into a potential kinetic model of factor VIII activation and decay.     

von Willebrand factor is a cofactor for thrombin-catalyzed cleavage of the factor VIII light chain

The proteolytic activation of highly purified, heterodimeric porcine factor VIII and factor VIII-von Willebrand factor complex by thrombin was compared at I 0.17, pH 7.0, 22 degrees C. During the activation of factor VIII, heavy-chain cleavage is necessary to activate the procoagulant function, whereas light-chain cleavage is required to dissociate factor VIII from von Willebrand factor. The kinetics of activation of free factor VIII and factor VIII-von Willebrand factor complex were identical. The steady-state kinetics of thrombin-catalyzed heavy-chain cleavages and light-chain cleavage of factor VIII either free or in complex with von Willebrand factor were studied using sodium dodecyl sulfate-polyacrylamide gel radioelectrophoresis and scanning densitometry of fragments derived from 125I-labeled factor VIII. Association of factor VIII with von Willebrand factor resulted in an 8-fold increase in the catalytic efficiency (kcat/Km) of light-chain cleavage (from 7 x 10(6) to 54 x 10(6) M-1 s-1). The catalytic efficiencies of heavy-chain cleavage at position 372 (approximately 6 x 10(6) M-1 s-1) and position 740 (approximately 100 x 10(6) M-1 s-1) were not affected by von Willebrand factor. We conclude that von Willebrand factor promotes cleavage of the factor VIII light chain by thrombin which is followed by rapid dissociation of the complex, so that the rate-limiting step becomes heavy-chain cleavage at position 372. This accounts for the observation that von Willebrand factor has no effect on the kinetics of activation of factor VIII by thrombin.     

Cryoprecipitate of intermediate purity produced in a closed thaw-siphon system from DDAVP stimulated blood donor plasma.

The effects of intravenous administration of DDAVP to blood donors and the use of DDAVP plasma for the production of cryoprecipitate in the closed thaw-siphon system were evaluated. DDAVP treatment produced on the average a 3.2-fold rise in plasma levels of factor VIII. Von Willebrand factor antigen increased to a lesser extent. Cryoprecipitate prepared from 220-280 ml aliquots of DDAVP stimulated donor plasma contained 472 +/- 210 units of factor VIII and 276 +/- 130 units of von Willebrand factor antigen. The average yield of factor VIII was 57% of that in the prefrozen plasma. The specific activity of factor VIII in cryoprecipitate was 0.77 +/- 0.44 U/mg protein, comparable to that for intermediate purity concentrates. Thus, by the use of DDAVP and the thaw-siphon technique it is possible to produce cryoprecipitate 4-7 times as potent as conventionally manufactured preparations.     

The domain of platelet glycoprotein I as recognized by various antibodies, both monoclonal and polyspecific

Platelet membrane glycoprotein Ib plays a major role in the binding of factor VIII/von Willebrand factor to allow platelet adhesion to subendothelium. We have used polyspecific and monoclonal antibodies to glycoprotein Ib and have demonstrated that both antibodies were directed to glycoprotein Is, a soluble fragment of glycoprotein Ib. By showing an inhibition of the binding of factor VIII/von Willebrand factor to control platelets in presence of the antibodies, it can be concluded that glycoprotein Is is involved in these binding sites.     

Intranasal desmopressin (DDAVP) by spray in mild hemophilia A and von Willebrand's disease type I

Desmopressin acetate (1-desamino-8-D-arginine vasopressin, DDAVP) has mostly been given by the parenteral route for the treatment of mild hemophilia A and von Willebrand's disease type I. In the present study the hemostatic effects of desmopressin acetate administered intranasally by spray in a dose of 300 micrograms and intravenously 0.3-0.4 micrograms/kg were assessed and compared in 8 patients with hemophilia A and 22 patients with von Willebrand's disease type I. A bioequivalent response to intravenous and intranasal desmopressin acetate was found in Factor VIII coagulant activity (VIII:C) in the hemophilia patients. In the von Willebrand patients, an equivalent shortening of the bleeding time was seen after the two modes of administration, even though intravenous injection gave a higher increase in plasma levels of VIII:C and vWF:Ag. In five patients with von Willebrand's disease the duration of the spray effect on VIII:C and vWF:Ag was followed for 24 h. After 12 h the mean level of VIII:C was 1.4, and of vWF:Ag 1.5, times the basal level. The findings suggest that the spray can be recommended for home or prophylactic treatment of patients with mild hemophilia A and von Willebrand's disease.     

The interaction of the factor VIII/von Willebrand factor complex (VIII/vWf), with guanidinium-derivatized matrices.

Five different guanidinium (Gu)-derivatized agarose matrices were investigated for their potential in chromatographically resolving the Factor VIII/von Willebrand complex, VIII/vWf, fibrinogen, Fg, and fibronectin, Fn, from cryoprecipitate. Using conventional NaCl gradient methodology it was found that the order of elution of specific plasma proteins, and the yield of VIII/vWf, varied with the methods used to derivatize the agarose beads. Good yields of VIII:C (generally 30-45%) were obtained with Gu-matrices prepared by bis-oxirane coupling procedures. Cryoprecipitate binding studies showed that the capacity of Gu-Sepharose 4B, prepared by isourea modification of amino-Sepharose 4B, was 36 units VIII/vWf per ml matrix. The product, depleted of both Fg and Fn, had a specific activity of 2 units VIII:C per mg total protein, (yield 100% vWf:Ag and 47% VIII:C).     

Reconstitution of human factor VIII from isolated subunits

Human factor VII heterodimers were fractionated into component heavy and light chains using an anti-light chain specific monoclonal antibody immunosorbant. Neither the light chain nor the heavy chain alone possessed activity. Factor VII activity was reconstituted by recombining the subunits in the presence of Mn2+ or Ca2+. Reconstitution of activity also showed ionic strength dependence suggesting the importance of hydrophobic and electrostatic interactions. All factor VIII heavy chains (93 to 210 kDa) recombined with the 83 kDa light chain as judged by retention of all reconstituted heterodimeric forms by the monoclonal immunosorbant. Maximum specific activity (3 units/micrograms) was obtained at a 1:1 molar ratio of light chain:heavy chain. The presence of von Willebrand factor enhanced the rate of factor VIII reconstitution as much as 5-fold. This effect was both ionic strength-dependent and dose-dependent up to a 25-fold weight excess of von Willebrand factor over factor VIII.     

Proteolytic activity of alpha 2-macroglobulin-enzyme complexes toward human factor VIII/von Willebrand factor

The low level of enzymatic activity of certain alpha 2-macroglobulin-proteinase complexes could be important to the function of factor VIII/von Willebrand glycoprotein since it is especially sensitive to proteolytic cleavage. To test this possibility, complexes of alpha 2-macroglobulin with plasmin, trypsin, and thrombin were formed in at least a 2:1 molar ratio of alpha 2-macroglobulin:proteinase and tested for effects on the factor VIII procoagulant activity of the factor VIII/von Willebrand glycoprotein. Neither the alpha 2-macroglobulin-trypsin complex nor the alpha 2-macroglobulin-plasmin complex affected factor VIII procoagulant activity. The behavior of the alpha 2-macroglobulin-thrombin complex was different. When alpha 2-macroglobulin and thrombin were incubated in a mole ratio of 3:1 or less, factor VIII procoagulant activity was enhanced to about the same extent as with free thrombin. Even at a 24:1 mole ratio, the mixture could produce 45% of the increase in factor VIII activity obtained with free thrombin. The isolated alpha 2-macroglobulin-thrombin complex could also activate the factor VIII procoagulant function to about 45% of the level obtained with an identical amount of uncomplexed thrombin. Analysis of the alpha 2-macroglobulin-125I-labeled thrombin complexes by rechromatography or by polyacrylamide gel electrophoresis in sodium dodecyl sulfate indicated that this activation was not due to free thrombin. We conclude that the alpha 2-macroglobulin-thrombin complex retains sufficient proteolytic activity to activate the procoagulant function of factor VIII/von Willebrand glycoprotein despite the latter being a very large substrate, having an estimated molecular weight of 1-20 million.     

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.     

Definition of the affinity of binding between human von Willebrand factor and coagulation factor VIII

Factor VIII and von Willebrand factor are two plasma proteins essential for effective hemostasis. In vivo, they form a non-covalent complex whose association appears to be metal ion dependent. However, a precise definition of the nature of the molecular forces governing their association remains to be defined, as does their binding affinity. In this paper we have determined the dissociation constant and stoichiometry for Factor VIII binding to immobilized von Willebrand factor. The data demonstrate that these proteins interact saturably and with relatively high affinity. Computer assisted analyses of the Scatchard data favour a two site binding model. The higher affinity site was found to have a Kd of 62 (+/- 13) x 10(-12) M while that of the lower affinity site was 380 (+/- 92) x 10(-12) M. The density of Factor VIII binding sites (Bmax) present on von Willebrand factor was 31 (+/- 3) pM for the high affinity binding site and 46 (+/- 6) pM for the lower site, corresponding to a calculated Factor VIII: von Willebrand factor binding ratio of 1:33 and 1:23, respectively.     

Ontogeny of reactivity to endothelial cell markers during development of the embryonic and fetal rat lung.

The reactivity of endothelial cells to putative endothelial cell-specific markers varies with species, with vessel size and with the organ studied. To determine their value in studies of fetal rat lung, and whether organ immaturity would also influence reactivity, we studied endothelial cell immunoreactivity to antibodies against Factor VIII/von Willebrand factor (VIII/vWF), and binding reactivity to Bandeiraea (Griffonia) simplicifolia 1 lectin (BSL 1) during rat fetal lung development. Using an indirect immunofluorescent technique to detect Factor VIII/von Willebrand factor (VIII/vWF), endothelial cells lining the aortic arches were identified as early as day 11 of gestation (term = 22 days), prior to lung development. Immunoreactivity to VIII/vWF was subsequently localized to intrapulmonary endothelial cells and was not dependent on vessel size. In contrast, binding reactivity of FITC-conjugated BSL 1 was observed to both endothelial cells and to the basement membrane of developing airways, thus limiting its value as endothelial cell marker. During very early lung development solitary angioblasts could not be identified by reactivity to either VIII/vWF antibodies or to BSL 1, and neither marker appears to be of value for studies of early angiogenic events.