Factor VIII coagulant activity and factor Vlll-related antigen released from isolated perfused human spleens

Summary Eight human spleens were perfused for up to 65 h at normothermia and the coagulant Factor VIII activity measured in the perfusate. In addition, in three experiments Factor VIII-related antigen was determined in the perfusate. Although the spleens were pathologically enlarged and the normal structure involved by different diseases, all spleens released Factor VIII coagulant activity and Factor VIII-related antigen. On average the total amount of Factor VIII coagulant activity released was equivalent to that of 3.5 1 of human plasma.

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Zusammenfassung Bei der normothermen Langzeitperfusion von 8 menschlichen Milzen wurde ein Anstieg des Gerinnungsfaktors VIII im Perfusat gemessen und in 3 FÄllen die Konzentration des Faktor VIII-assoziierten Antigens bestimmt. Obwohl es sich um pathologisch verÄnderte Milzen handelte, stieg in allen FÄllen die Faktor-VIII-AktivitÄt und die Konzentration des Faktor-VIII-assoziierten Antigens an. Der Mittelwert des maximalen Gehalts an Faktor VIII entsprach der GerinnungsaktivitÄt von 3,5 1 menschlichen Plasmas.

     

In vivo dissociation of factor VII (AHF) activity and factor VII-related antigen in von Willebrand's disease.

Using monospecific rabbit antihuman factor VIII antiserum, we have examined the amounts of factor VIII-related antigen and compared these to the levels of factor VIII procoagulant activity in normal subjects and patients with von Willebrand's disease. We have observed that even without transfusion all nine probands with von Willebrand's disease and 20 of their 34 relatives possessed a significantly elevated factor VIII activity/factor VIII-related antigen ratio when compared to that of 55 normal subjects. It is suggested that an elevated factor VIII activity/factor VIII-related antigen ratio may be used for detection of the carriers of von Willebrand's disease.     

A novel congenital haemostatic defect: combined factor VII and factor XI deficiency.

Isolated deficiencies of factors VII and XI are both rare. Not surprisingly, therefore, combined factor VII and XI deficiency has not been reported previously. We report here a kindred with a combined heterozygous deficiency for both factors VII and XI. The proposita is a 28-year-old woman who had both a prolonged prothrombin time (PT) and a prolonged activated partial prothrombin time (APTT) associated with a mild bleeding tendency. Coagulation studies were performed on the six available members of this kindred. The PT and APTT were normal or mildly abnormal in five of these individuals. Factor VII coagulant activity (VII:C) varied from 0.33 to 0.77 units/ml in affected subjects. In contrast, the concentration of factor VII-related antigen for the six individuals ranged from 0.68 to 2.10 units/ml. Comparable factor VII:C levels were obtained when each subject's plasma was tested with either a rabbit or a human thromboplastin reagent. Factor XI coagulant activity was less than 0.5 units/ml in three of the six subjects and normal (approximately 1.0 units/ml) in the other three. The concentrations of thrombin-antithrombin-III and prothrombin fragment 1.2 were within normal limits for all individuals. In addition to being associated with heterozygous factor XI deficiency, the abnormal factor VII molecule in the plasma of affected individuals in this kindred appears to represent a newly described mutation. This is suggested by the pattern of reactivity with thromboplastin from different species, the normal tissue factor binding and the bleeding tendency in heterozygous individuals in this kindred.     

Von Willebrand disease associated with familial thrombocytopenia and increased ristocetin-induced platelet aggregation

Two cases of von Willebrand disease (vWD) associated with familial thrombocytopenia were reported. The proband (daughter) and her father showed thrombocytopenia with large platelets and decreased von Willebrand factor activity (VIIIR:WF). Factor VIII procoagulant activity (VIII:C) and factor VIII-related antigen (VIIIR:AG) were normal, but both patients revealed an increased ristocetin-induced platelet aggregation and a qualitative abnormality of the factor VIII protein, which was characterized by fast electrophoretic mobility of VIIIR:AG and an abnormal elution of factor VIII-related activities on Sepharose 2B. DDAVP was hemostatically effective even in this thrombocytopenic patient undergoing a dental extraction.     

Macromolecular factor VIII complex: functional and structural heterogeneity observed in von Willebrand swine with transfusion.

The physiologic activities concerned with hemostasis and associated with the Factor VIII macromolecular complex were investigated in swine with von Willebrand's disease after infusion of cryoprecipitate, a lyophilized Factor VIII concentrate, or porcine serum. Immediately after each infusion the various activities antihemophilic factor, von Willebrand platelet aggregating factor, and Factor VIII-related antigen, were elevated in approximate proportion to dose and the bleeding time was shortened.There was a late secondary rise in antihemophilic factor. During the period after infusion, there was a differential fall-off of the various activities, with the bleeding time effect lost first, followed by the von Willebrand platelet aggregating factor and then by the Factor VIII-related antigen. The plasma from swine with von Willebrand's disease late after infusion contained high levels of antihemophilic factor without other detectable activities of the complex. Antihemophilic factor, free of the other components, obtained from plasma from swine with von Willebrand's disease either before or late after infusion eluted from agarose gel columns both as high and lower molecular weight material, unlike normal antihemophilic factor, which had a high molecular weight. In contrast, on ultracentrifugation the antihemophilic factor in these plasma sedimented slowly, even though chromatographically the plasmas contained both high and low molecular weight factor. All of the Factor VIII complex activities in normal porcine plasma sedimented rapidly. These studies demonstrate the heterogeneity of the Factor VIII complex and the apparent dependence of its chromatographic and sedimentation behavior on the functional activities associated with the complex.     

Combined factor VIII and factor XI congenital deficiency: a case report

Combined congenital defect involving both Factor VIII and XI is a very rare disorder. We describe a case of combined Factor VIII and XI deficiency in which the propositus has a mild hemophilia A and inherited a Factor XI deficiency from the father. Moreover, we report on the benefit of DDAVP administration to the patient during a bleeding episode.     

Actions of ristocetin on platelets.

The absence of ristocetin-induced platelet aggregation appears to correlate with the platelet defect in von Willebrand's disease, suggesting that this reaction mimics a physiological process. The effect of ristocetin on plasma and on the residual levels of the von Willebrand factor (vWF), Factor VIII procoagulant activity, and Factor VIII-related protein in plasma after aggregation of platelet rich plasma by this agent has been studied in order to further elucidate the mechanism and requirements of this reaction. Ristocetin-induced platelet aggregation causes a consumption of vWF, Factor VIII procoagulant activity, and Factor VIII antigen from the supernatant plasma which is proportional to the number of platelets aggregated. Such a consumption of these factors does not appear to occur after aggregation by other agents. Factor VIII procoagulant activity does not appear necessary for ristocetin-induced platelet aggregation, yet is utilized in this process. These findings support the hypothesis that the molecule associated with Factor VIII procoagulant activity is carried by the molecule necessary for ristocetin-induced platelet aggregation.     

Antihaemophilic factor A activity, F VIII-related antigen and von Wilebrand factor in hepatic cirrhosis.

F VIII activity, F VIII-related antigen and von Willebrand factor were measured in 46 patients with hepatic cirrhosis and in 30 normal individuals. These parameters were significantly higher in hepatic cirrhosis than in the controls. Linear relationships between F VIII activity and F VIII-related antigen and between F VIII-related antigen and von Willebrand factor were found in patients with hepatic cirrhosis as well as in normal individuals. However, in both groups no relationship between F VIII activity and von Willebrand factor was present. The existence of a low-grade intravascular coagulation in hepatic cirrhosis may be postulated but more information about the metabolism of F VIII protein is needed before such a statement can be proven.     

In vivo and in vitro effects of thrombin and plasmin on human factor VIII (AHF).

The relationship between factor VIII (AHF) procoagulant activity and factor VIII-related antigen were examined in patients with disseminated intravascular coagulation (DIC), pulmonary embolism (PE), and coronary artery disease with or without myocardial infarction (MI). It was found that 13 of 13 patients with DIC, 17 of 17 patients with PE, and 10 of 12 patients with MI possessed a significantly elevated factor VIII-related antigen to factor VIII activity ratio (VIII-ratio). The VIII-ratio returned to normal in each of 2 patients with DIC and 1 paitent with PE after treatment with heparin, heparin and alpha-amino-caproic acid, and heparin and coumadin respectively. In contrast, the VIII-ratio was slightly elevated only in 1 of 15 patients with coronary artery insufficiency without MI. In in vitro studies, after treatment of plasma with thrombin or plasmin, factor VIII activity was lost, whereas the amount of factor VIII-related antigen remained the same or was even increased when measured by agarose quantitative immunoelectrophoresis. These observations have led us to conclude that an elevated VIII-ratio is a very sensitive indicator of intravascular coagulation.     

Selective absence of large forms of factor VIII/von Willebrand factor in acquired von Willebrand's syndrome. Response to transfusion.

A previously healthy elderly man with mucocutaneous bleeding was found to have a benign monoclonal IgG gammapathy associated with criteria for severe von Willebrand disease (Factor VIII procoagulant activity, Factor-VIII-related antigen, and ristocetin cofactor activity, less than 10% of normal). Associated qualitative abnormalities of factor VIII/von Willebrand factor were demonstrated by radiocrossed immunoelectrophoresis and immunoradiometric assay. The late clinical onset and negative family history are in favor of an acquired form of vWD. The monoclonal gammapathy and abnormalities of factor VIII/von Willebrand factor have been stable over a 10-yr period. No inhibitor to Factor VIII procoagulant activity, ristocetin cofactor activity, or Factor-VIII-related antigen could be demonstrated. Following transfusion of cryoprecipitate (with a normal cross immunoelectrophoretic pattern), there was a rapid removal of the large forms of Factor.-VIII-related antigen, paralleled by a decay of ristocetin cofactor activity. The transfusion study of this patient with acquired von Willebrand disease type II (variant of von Willebrand disease) serves to emphasize the relationship between polydispersity of Factor VIII/von Willebrand Factor and functional heterogeneity.     

A new variant of dominant type II von Willebrand's disease with aberrant multimeric pattern of factor VIII-related antigen (type IID)

A new type II variant form of von Willebrand's disease has been recognized in a mother and daughter who have bleeding manifestations typical of von Willebrand's disease. Laboratory findings include consistently prolonged bleeding times, with normal levels of factor VIII procoagulant and antigen, but decreased ristocetin cofactor activity. Electrophoresis in SDS 1.5% agarose gel and reaction with 125I-labeled anti-factor VIII-related antigen rabbit IgG, followed by autoradiography, revealed that both plasma and platelets lack the large multimers of factor VIII-related antigen. In 2.5% gel, the propositus plasma lacked the normal "triplet" pattern. In 3.0% gel, a 5-band pattern was observed in normal, type IIA, and type IIB plasma, whereas type IIC plasma revealed a 2-band pattern. The patient's plasma revealed a 4-band pattern distinctly different from normal or other type II variants. We suggest that this new variant be labeled type IID, until a more appropriate nomenclature is developed.     

Management of cesarean section under replacement therapy with factor VIII concentrates in a pregnant case with congenital combined deficiency of factor V and factor VIII

The congenital combined deficiency of Factor V and Factor VIII, a rare bleeding disorder, was identified in a 25-year-old woman. She was admitted to our hospital with a complaint of genital bleeding. Her prothrombin time and activated partial thromboplastin time were prolonged. She had low levels of Factor V coagulant activity (F. V:C) 14%, and Factor VIII coagulant activity (F. VIII:C), 12%, and normal levels of von Willebrand factor antigen (vWF:Ag), ristocetin cofactor (Rcof) and Protein C antigen. Her Protein C inhibitor level was slightly low. Her Rcof, vWF:Ag and F. VIII:C were elevated following administration of 1-deamino-8-D-arginine-vasopressin (DDAVP), but her F. V:C remained unchanged. Four years later, her F. VIII:C rose to 70% during the course of her pregnancy, but her F. V:C value remained low. It was expected that the vaginal delivery would be possible at the termination of pregnancy. Premature rupture of the membranes and an anomaly of rotation appeared in the course of delivery, however, and cesarean section was accomplished without excess bleeding under replacement therapy with Factor VIII concentrates. These findings suggested that DDAVP and Factor VIII concentrates were useful for management of her delivery. However the mechanisms of the rise of plasma F. VIII:C during pregnancy in a case with congenital combined deficiency of Factor V and Factor VIII are unclear.     

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

Summary 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 or 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 the 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.This study was supported in part by a grant from the C.N.R. (grant CT. 74.00189.04).     

Structural analysis of factor VIII antigen in von Willebrand disease.

The Factor VIII antigen molecules in the plasma of patients with classical type 1 and variant type 2A von Willebrand disease were compared to the Factor VIII antigen molecules in normal plasma. Factor VIII antigen was isolated from plasma by solid-phase immunoprecipitation and analyzed by NaDodSO4/polyacrylamide gel electrophoresis; the stained Factor VIII antigen bands were removed, radioiodinated, and subjected to tryptic digestion. Computerized analysis of autoradiographs revealed that the two-dimensional peptide maps of the different Factor VIII antigens were remarkably similar. The results suggest that the Factor VIII antigen molecules in these two forms of von Willebrand disease are probably identical to the Factor VIII antigen molecules present in normal plasma. It is thus likely that the differences observed in plasma Factor VIII antigen in classical and variant von Willebrand disease are not due to qualitatively abnormal molecules but rather represent quantitative shifts in the metabolism of normal Factor VIII antigen molecules.     

Immunological Studies in Combined Factor V and Factor VIII Deficiency

Plasma samples from patients with inherited combined factor V and factor VIII deficiency were examined by immunological methods for the presence of factor V and Factor VIII-related antigens. A factor V-related antigen was consistently demonstrated in all plasma samples by inhibitor neutralization assay using a non-precipitating rabbit antibody. Factor VIII-related antigens were detected by inhibitor neutralization using human antibody and by electroimmunoassay using a precipitating rabbit antibody. The amounts of factor V and factor VIII-related antigens present in the patient's samples were similar to those found in normal human plasma. The findings confirm the presence of normal levels of factor VIII-related antigen in the plasma of these patients and suggest that inactive antigenic determinants of procoagulant factor V and procoagulant factor VIII are also present. The results are consistent with the possibility that a common precursor of porcoagulant factor V and factor VIII is defective in these patients.     

In vitro survival studies of a factor VIII concentrate (Kryobulin)

Summary Factor VIII: C activity decay was studied in lyophilized concentrates (Kryobulin, Immuno) stored at room temperature and in reconstituted preparations frozen and stored at –20°C. A slow, pratically identical, decay of Factor VIII: C was found in lyophilized concentrates kept at room temperature, in reconstituted lyophilized concentrates frozen and stored at –20°C and in lyophilized concentrate kept at +4°C.At the end of the experiment, which was several months after expiration date of the product, the concentrates maintained Factor VIII: C levels of approximately 45% with respect to a pool of normal human plasmas.This study was supported by grants from the M.P.I., Rome (Grant 1592, 1979) and from the Veneto Region, Venice     

Circulating anticoagulant in a family with prolonged bleeding time and factor VIII deficiency

A circulating anticoagulant against factor VIII activity was demonstrated in the plasma of a boy from a family with both factor VIII deficiency and prolonged bleeding time. However, the factor VIII- related antigen, ristocetin-induced platelet aggregation activity, platelet retention in glass bead columns, platelet aggregation with adenosine 5'-diphosphate, collagen and epinephrine, and clot retraction among affected members were normal. The electrophoretic mobility of factor VIII-related antigen on crossed immunoelectrophoresis was normal. The inactivation of factor VIII activity by the inhibitor was time dependent and was nonlinear as the concentration of the inhibitor was increased. Immunotyping showed that the inhibitor was IgG with k light chains.     

DDAVP administration in a case of congenital combined factor V and factor VIII deficiency

Combined deficiency of factor V and factor VIII, a rare bleeding disorder, was found in a 43 year-old male. He had often presented manifestations of easy bruising since childhood, but none of his family had shown evidence of a bleeding tendency. We examined him and his family as far as we could and his abnormality of blood coagulation was apparent, but the members of his family were normal. The prothrombin time and activated partial thromboplastin time of this patient were prolonged, but his thrombin time was normal. Factor V and factor VIII coagulant activity were low, but von Willebrand factor antigen and activity (ristocetin cofactor activity) levels were normal. Protein C and Protein C inhibitor antigen and activity levels were also found to be normal. Following 1-deamino-8-D-arginine vasopressin (DDAVP) injection, he had immediate increases in factor VIII coagulant activity, but both von Willebrand factor antigen, activity levels and factor V coagulant activity remained low. Moreover, there was no rapid decline in factor VIII complex activity. These findings suggest that the endogenous factor VIII in this patient is metabolized normally and that at least the deficiency of factor VIII does not result from accelerated degradation in plasma.     

Pseudo-factor-XI deficiency: effect of an inhibitor of factor XI adsorption to surface

Recently we have described a normal plasma activity that modulates contact activation by inhibiting adsorption of factor XI to activating surfaces. Here we report the first identified case in which a patient has abnormal clotting tests due to an excess of a similar activity. The patient's plasma had a prolonged partial thromboplastin time and low apparent factor XI assay. His plasma prolonged the partial thromboplastin time of normal plasma and partially neutralized normal factor XI activity in vivo and in vitro. Analysis in dilute plasma revealed normal amounts of factor XI activity and antigen. Factor XI adsorption from plasma to activating surfaces was tested by adding a small amount of 125I-labeled purified factor XI to plasma, exposing the mixture to a glass tube or kaolin, and determining the amount of factor XI adsorbed to the surface. Whereas normal plasma and plasmas deficient in factor XII, factor XI, or Fletcher factor yielded about 4% adsorption to glass, factor XI adsorption from patient's plasma was less than 1%, indicating the presence of an adsorption inhibitor. This inhibitor did not affect factor XI activation or the activity of preformed factor XIa. It was not adsorbed by AI(OH)3 and was present in serum and the macroglobulin peak on gel filtration of the plasma through Sephadex G-200. The patient's history does not allow a definitive conclusion as to whether this inhibitor was associated with abnormal bleeding.     

Changes in the factor VIII complex in diabetic ketoacidosis: evidence of endothelial cell damage?

Summary Factor VIII-related antigen and von Willebrand factor are synthesised by and released from vascular endothelium. Acute increases in the plasma concentration of these proteins may reflect endothelial cell damage. We have thus measured the plasma concentration of factor VIII-related antigen and von Willebrand factor, together with procoagulant factor VIII, during the course of acute diabetic ketoacidosis in seven patients. In addition, evidence for qualitative changes in the factor VIII complex was sought. Plasma factor VIII-related antigen and von Willebrand factor were markedly increased (plasma factor VIII-related antigen at presentation, median 2.75 U/ml; von Willebrand factor 2.95 U/ml) and returned toward normal with clinical and biochemical resolution (plasma factor VIII-related antigen at clinical recovery, median 1.80 U/ml; von Willebrand factor 2.05 U/ml). Plasma procoagulant factor VIII followed a similar pattern, but levels were less elevated (plasma procoagulant factor VIII, at presentation, median 1.6U/ml; at clinical recovery, 1.2U/ml). Crossed immunoelectrophoresis and sodium dodecyl sulphate-acrylamide electrophoresis with autoradiographic identification of multimeric structure revealed no evidence of structurally abnormal factor VIII-related antigen in diabetic ketoacidosis. However, an extra peak on crossed immunoelectrophoresis (pre-peak) was a feature in the acute phase ketoacidotic plasma in six subjects, and may represent aggregated factor VIII. Changes in plasma factor VIII are a feature of diabetic ketoacidosis and, whilst not specific to this condition, may be the result of endothelial cell damage.