Leon W. Hoyer

Von Willebrand factor: dissociation from antihemophilic factor procoagulant activity.

Factor VIII corrects both the clotting defect in hemophilia A and an abnormality of platelet aggregation in von Willebrands disease. These two activities of factor VIII (antihemophilic factor and von Willebrand factor) are both detected in the void volume when human plasma or cryoprecipitate is chromatographed on Bio-Gel 5M under conditions of isotonic salt concentration. In contrast, antihemophilic factor procoagulant activity is detected with proteins of lower molecular weight when the chromatography is performed with a buffer containing 0.8M NaCl. In this way, the two activities of factor VIII can be dissociated. It remains to be determined whether these components are separate molecules associated as a complex of high molecular weight in plasma or whether they are subunits of a complex macromolecule.

Synthesis and Release of Factor VIII by Cultured Human Endothelial Cells

Summary. Endothelial cells (ECs) derived from human umbilical veins were cultured in order to study the physiological control of factor VIII synthesis and release. The culture media were studied from multiple replicate cultures at confluence. Factor VIII related antigen (VIIIR:Ag) and factor VIII coagulant antigen (VIII:CAg) were measured by sensitive immunoradiometric assays. De novo synthesis of factor VIII related protein (VIII:R) was quantitated by incorporation of labelled amino acids into specific protein subunits. The following agents were added to the culture medium in a range of concentrations from physiological to pharmacological: adrenaline, 5 hydroxytryptamine, 2,3‐DPG, cyclic AMP, thyroxine, hydrocortisone, and human growth hormone. None of them had any effect at any concentration on the rate of accumulation of VIIIR:Ag in the culture medium. Addition of exogenous factor VIII had no effect on do novo synthesis of VIII:R. VIII:CAg was found to be stable under the conditions of culture but none was released from the ECs. Long‐term monocyte cultures also failed to release VIII:CAg. It appears that VIII:R is a constitutive gene product of umbilical vein endothelial cells and that VIII:CAg is not made by these cells.

Molecular weight of human factor VIII procoagulant activity

Abstract Low molecular weight factor VIII procoagulant activity has been prepared by agarose gel chromatography of highly purified human factor VIII using 0.24 M CaCl 2 buffer. The sedimentation properties of this procoagulant activity in sucrose density gradient centrifugation studies carried out at physiologic ionic strength were consistent with a 6.7S protein. These experiments demonstrate that the elution pattern of factor VIII activity in agarose gel chromatography with high ionic strength buffers is due to separation of low molecular weight material — rather than artifactual retention of large molecules within the gel bed — and that the low molecular weight portion of the large factor VIII complex is sufficient for procoagulant function.

Coagulation activities in perfused organs: regulation by addition of animal plasmas.

Summary. Production of factor‐VIII and ‐IX activities was studied in isolated rabbit livers and spleens perfused for I hr to release stored coagulation activity and then for another 4 hr. At 30 min of the second perfusion period, 5 ml citrated dog or rabbit plasma from normal animals or from those with congenital or artificially induced coagulation defects was added to the perfusates. Subsequent production of factor‐VIII and ‐IX activities was monitored at intervals for 3.5 hr. Results indicated that following the addition of animal plasmas to perfused organs, production of coagulation activity was inversely proportional to the level of these coagulation factors in the initial perfusate. In addition, development of clotting factor activities in the liver and spleen perfusates was inhibited by puromycin, cycloheximide, and actinomycin D. Several interpretations of the data have been considered, and the results are compatible with regulation by a negative feedback effect on organ protein synthesis and/or release of previously formed molecules.

Hemophilia A in a female: Use of factor VIII antigen levels as a diagnostic aid

A genotypic female with severe hemophilia A and a negative family history for bleeding dyscrasias is described. Diagnosis was made by determining levels of antihemophilic factor (AHF) procoagulant activity and AHF antigen; by this method her father was shown to be normal and her mother a carrier of hemophilia A. Possible mechanisms which might have resulted in severe AHF deficiency in the patient are discussed.

IMPLICATIONS OF IMMUNOLOGIC METHODS FOR MEASURING ANTIHEMOPHILIC FACTOR (FACTOR VIII)

Recent immunologic studies have provided important new information about the nature of AHF deficiency diseases and the structure and synthesis of this protein. They provide, moreover, a considerably improved ability to identify the carrier state in hemophilia A. Additional progress may be possible in the identification of carriers and of hemophilic patients at risk of antibody formation, but it will require a different approach to these problems. While preliminary goals have been obtained by using antibodies that identify antigenic determinants shared by normal and nonfunctional AHF, further progress will require the capacity to identify differences between the normal protein and the nonfunctional AHF-like molecules in hemophilic plasma.

RESPONSE OF PROTEIN C AND PROTEIN C INHIBITOR TO WARFARIN THERAPY IN PATIENT WITH COMBINED DEFICIENCY OF FACTORS V AND VIII

The role of Protein C in combined factor V/VIII deficiency was examined by reducing the Protein C concentration using warfarin therapy in a patient with the combined deficiency. The factor VIII deficiency was like Hemophilia-A, with deficiency of VIII:C and VIII:C(Ag), but normal VIIIR:Ag and VIIIR:cof. The factor V deficiency was due to loss of the V antigen. During warfarin therapy the Protein C level was reduced, but concentrations of factors V and VIII did not change. Protein C Inhibitor was normal throughout. Thus combined factor V/VIII deficiency is not related to Protein C levels.

DISCUSSION PAPER: FACTOR VIII SUBUNITS

The discussion of factor VIII subunits and the controversy about factor VIII dissociation in the salt buffers is a very important matter. Rick and I have performed approximately 60 dissociation experiments using 0.24 M calcum chloride and 1 M sodium chloride and we have regularly detected the dissociation. The top panel of FIGURE 1 shows the gel chromatography pattern of purified AHF on Sepharose 6B using a normal ionic strength buffer. The bulk of the protein is eluted at the void volume, as is AHF procoagulant activity (open circles) and AHF antigen (closed circles). This study was done with material prepared by gel chromatography of a commercial concentrate using Sepharose 6B. That material was also separated using high salt conditions, as shown in the lower panel. In 0.24 M CaCI, there is a slightly lower protein peak; the AHF antigen still appears at the void volume, although some trailing is seen; and the AHF procoagulant activity appears in later fractions. I think the differences between these experiments and those presented by McKee reflect differences in amount of protein added to the columns and differences in the separation conditions. In order to be certain that the factor VIII procoagulant activity measured after gel filtration in 0.25 M CaC12 is related to factor VIII activity present in plasma, it .was necessary to have some sort of independent verification apart from the clotting time assays. The best evidence that the two procoagulant activities are similar is the immunological demonstration that both can be inactivated by antibodies to factor VIII. FIGURE 2 shows the inactivation of the low-molecular-weight subunit compared to inactivation of plasma factor VIII. Inactivation is observed with a human antibody to factor VIII obtained from a patient with a spontaneous anticoagulant and with a rabbit antibody to “whole” factor VIII. This observation strongly suggests that the low-molecular-weight procoagulant activity is not an artifact. We have also separated the subunits by sucrose density centrifugation and have obtained the same results; this argues against the possibility that the results of filtration reflect protein interactions with the agarose. It should be noted that the different results which have been reported for dissociation studies involved highly purified factor VIII as the starting material. It is possible that nondissociable factor VIII is selected by some of the purification steps.