HEMOPHILIA A AND VON WILLEBRAND’S DISEASE: THE RELATIONSHIP EXAMINED

Bruce Bennett

Department of Medicine Aberdeen University A berdeen, Scotland

Von Willebrand’s disease as it was first recognized about 40 years ago shared with classic hemophilia only the symptoms of bleeding and the fact that the disorder was heritable. Even in these two areas differences were apparent between the disorders. Troublesome bleeding in von Willebrand’s disease tended to be from mucus surfaces, from the gut, or from the uterus, whereas spontaneous bleeding in classic hemophilia was predominantly in muscles and joints. The pattern of inheritance in hemophilia was that of an X-chromosome- linked recessive disorder, whereas in von Willebrand’s disease it was that of an autosomal dominant trait. In addition to these features, the disorders differed in that bleeding times were normal in hemophilic patients and abnormally long in those with von Willebrand’s disease.

In the 1950s, observations seeming to link the two disorders more closely were made. Nilsson and her colleagues 1 observed that plasma of patients with von Willebrand’s disease appeared to lack the same clotting factor as did plasma of patients with classic hemophilia. This was widely confirmed, but very shortly thereafter even in this respect a further difference between the two disorders became apparent. Whereas, in the laboratory, both disorders were charac- terized by low plasma levels of AHF, the change in AHF levels after trans- fusion was radically different. It was observed that hemophilic patients re- sponded to transfusion with a rapid and fairly predictable rise in levels of circulating AHF and that levels then declined rapidly, with an apparent half-life of about 12 hours. In contrast, von Willebrand’s disease patients responded with a rise in AHF followed by a further rise not predictable from the amount infused and a much slower rate of decline of levels of AHF?. (FIGURE 1) . This delayed rise in AHF levels occured even if inactive hemophilic plasma was infused into von Willebrand’s disease subjects. Thus, von Willebrand’s disease patients could utilize hemophilic plasma in some manner; but hemo- philiacs could not respond in a similar way to infusions of von Willebrand’s disease plasma. So the apparent link between the two disorders did not remain a simple one but became complex almost at once. A further complication was added by the observation that the bleeding time was not corrected after trans- fusion of von Willebrand’s disease patients for a period comparable with correc- tion of AHF levels but for a much shorter period, suggesting that an agent correcting the bleeding time and discrete from AHF was present in normal and hemophilic plasmas.

At this stage, therefore, one might reasonably have regarded hemophilia as the simple disorder with deficiency of a single agent, a normal bleeding time, and a predictable response to transfusion, and von Willebrand’s disease as the complex one with a long bleeding time due to one plasma deficiency, a clotting disorder due to another, and a complex response to transfusion correcting the

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P

FIGURE 1. Changes in blood levels of AHF clot-promoting activity after transfu- sion of patients with classic hemophilia and von Willebrand’s disease.

two abnormalities for differing lengths of time and apparently allowing new production or release of AHF. Several theories to explain the complex features of von Willebrand’s disease arose : A “von Willebrand’s disease factor,” differ- ent from AHF, was proposed to account for the bleeding-time abnormality, and a second one was talked about that promoted the new production of AHF after transfusion. Further, a number of proposals were made as to how normal AHF might be synthesized under control of autosomal and X chromosomal genes and how the findings in the two diseases might be accounted for by abnormalities of these mechanisms. All these theories depended upon agents not then isolated from the blood. We still do not know precisely which of the many proposals is correct, but recent developments have presented us with new data on which hypotheses may be built. Zimmerman and Ratnoff prepared a precipitating antiserum in rabbits against purified normal AHF and observed precipitin lines between antiserum and hemophilic plasma (FIGURE 2), a not wholly expected finding. In contrast, von Willebrand’s disease plasma did not appear to contain an antigen similar to AHF detectable by this antiserum. Here, then, was the first demonstration of a specific easily demonstrable deficiency in von Willebrand’s disease plasma setting it apart from both normal and hemo- philic plasmas, and similar results have now been reported by many workers. How has this fundamental observation led to further knowledge about the two

Bennett: Hemophilia A and Von Willebrand’s Disease 111

disorders and their possible relationship? For the purposes of discussion I should like to consider two general areas, the first concerning the bleeding time and the second the mode of production of AHF.

Considering the bleeding time, it is often assumed that its prolongation in von Willebrand‘s disease reflects the inability of platelets of these patients to adhere normally to foreign surfaces, an abnormality not present in normal or hemophilic individuals’ blood. It has been shown 6* that von Willebrand’s disease platelets adhere poorly to glass beads and that this abnormality tends to correlate with prolongation of the bleeding time. Could this abnormality be due to the protein deficiency demonstrated by the rabbit antiserum that similarly separates von Willebrand’s disease patients from both normal and hemophilic individuals? Workers in the Netherlands first explored this possibility.’ They purified normal AHF on Sepharose columns, took the void-volume fraction containing AHF clot-promoting activity and antigen, and showed that it re- stored normal adhesiveness to von Willebrand’s disease platelets. They then fractionated hemophilic plasma in a similar way and again took the void- volume fraction. This contained the antigenic agent detected by the antisera but, of course, no AHF clot-promoting activity. This fraction, too, corrected the adhesiveness of von Willebrand’s disease platelets. Finally, they showed that antisera to AHF itself could destroy adhesiveness of normal platelets. More recently, using the fact that von Willebrand’s disease platelets do not aggregate normally on exposure to ristocetin, comparable correction of this phenomenon by similar fractions of normal and hemophilic plasmas has been observed.*# I) All these findings suggested that the AHF-like antigen, whether associated with clot-promoting activity or not, might be the platelet adhesiveness factor. If these observations on the in vitro behavior of von Willebrand’s disease platelets can be transposed to the bleeding time in patients, a consider- able advance in understanding of the disorder has been made. A further obser- vation is relevant here. I have mentioned that after transfusion of von Wille- brand’s disease patients, correction of bleeding time and platelet adhesiveness tended to be very transient and not concurrent with the rise in AHF clot- promoting activity in the plasma. Study of the von Willebrand’s disease response to transfusion in Ratnoffs laboratory showed that the level of the antigenically detected AHF did not follow that of clot-promoting activity.’” There was a rapid rise and fall of antigenically detectable material after trans-

FIGURE 2. Precipitin lines between rab- bit antiserum to purified normal, hemophi- NORM lic. and von Willebrand’s disease plasmas.

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112 Annals New York Academy of Sciences

fusion and clot-promoting activity showed a slower rise and fall (FIGURE 3). This dissociation of levels of AHF clot-promoting activity from those of antigen has now been observed by other workers.ll9 Now, in normal subjects we have not noted such dissociation of antigenic from clot-promoting activity after various stresses.13 Somehow the von Willebrand's disease patient can separate these entities. If the antigenic protein is the von Willebrand's disease factor or is associated with it, this observation would indicate why correction of the bleeding time is relatively short-lived. The situation in vivo may not always be as simple as this explanation suggests, however, since in some patients with von Willebrand's disease whose plasma levels of the antigenic protein have risen to normal as a result of pregnancy or other factors, the bleeding time has remained pr~longed. '~ In addition, some workers have reported that a

2.0

a - + d 1.5

LL 5 a5

r yoprecipitate 50ml fresh Mood

0 I 2 3 4 5 6 7 8 TIME (DAYS)

FIGURE 3. Changes in levels of AHF clot-promoting activity and AHF-like antigen after transfusion of a patient with von Willebrand's disease.

small proportion of patients, regarded as having von Willebrand's disease, have normal amounts of the AHF-like antigen.". 15, 18 Obviously the proposal I have outlined is not applicable to all patients who would formerly have been regarded as having von Willebrand's disease. One must presume that those patients with antigen produce a protein deficient in both clot-promoting and adhesiveness-promoting properties. One further question that might be con- sidered in relation to the bleeding time is that of distribution of the antigenic protein in the body. Immunofluorescent studies have identified the AHF-like antigen on the endothelium of blood vessel^.^^*'^ Perhaps in this situation it can influence the bleeding time in a manner not measured by tests of platelet function; as yet we have no information on whether any AHF-like antigen produced by or transfused into patients with von Willebrand's disease is dis- tributed on their vascular endothelium.

Bennett: Hemophilia A and Von Willebrand's Disease 113

FIGURE 4. Representation of possible structure of normal AHF complex; antigenic (larger) and clot-promoting (smaller)

components.

Secondly, have the observations led to better understanding of the produc- tion of AHF? This is an area in which Graham, in the early 1960s, proposed a number of mechanisms by which the presence of abnormal genes on X or autosomal chromosomes might possibly produce hemophilia or von Wille- brand's di~ease. '~ Knowledge of the presence of an antigen related to AHF in hemophilic plasma has recently provided an opportunity for new specula- tions; most have been variations on the earlier themes of Graham and appeared in the correspondence columns of the Lancet over a period of some months in 1973. Most speculations assume that the antigenic agent is not identical with the AHF clot-promoting agent. This is clearly reasonable, as the AHF-like antigen in hemophilic plasma or normal serum has no clot-promoting activity. However, in fresh whole plasma, clot-promoting activity and antigen appear closely associated; they remain together throughout purification procedures, they have similar physical characteristics, and antisera raised against purified normal AHF or the inactive hemophilic protein both destroy AHF clot- promoting activity.'O? Recently separation of the antigen from clot-promoting activity in the laboratory has been reported,22 but this does not imply that they are not closely associated in plasma. Recent suggestions as to how AHF is synthesized have therefore been variations on the following themes: Normal AHF is seen as a molecule comprising two parts; that represented in FIGURE 4 as being the larger is regarded as possessing the antigenic sites detected by the heterologous antisera, while that represented as the smaller possesses AHF clot- promoting activity. This allows at least two possible conceptions of the abnor- mality in hemophilic plasmas. These are illustrated in FIGURE 5. The antigenic part of the complex is present in both, but the X chromosomal gene abnormality may result either in a failure to add the clot-promoting agent to it or in the addition of an abnormal form as yet undefined. Both forms probably occur and represent, in fact, the CRM- and CRM+ types of hemophilia identified some years ago by means of human antibodies to AHF presumably directed specifically at the clot-promoting part of the complex.23 The new component in this concept is the presence of the agent detected by the heterologous antisera, and the suggestion is that it is a precursor molecule to or carrier molecule for normal AHF that may possess the ability to influence platelet behavior and the

FIGURE 5. Representation of possi- sible structure of agents produced by hemophilic patients: Antigenic com- ponent with either no clot-promoting agent or a functionally defective one.

114 Annals New York Academy of Sciences

bleeding time. Can these ideas be extended to explain the findings in von Willebrand‘s disease? Well, as yet, they can explain some, but not all of them. It is suggested that the abnormal autosomal gene expresses itself by reduced production of the antigenic agent, precursor molecule, carrier molecule, call it what you will, and that this results in reduced delivery of clot-promoting activity to the plasma. If the antigen is the agent influencing platelet function and the bleeding time, this is a satisfactory hypothesis by which to explain the findings in the majority of patients with von Willebrand’s disease, namely those with low levels of antigen and clot-promoting activity (modification of this idea is necessary to explain the disorder in those few patients who appear to have von Willebrand’s disease clinically but have normal amounts of antigen; see FIGURE 6). Applying this concept to the situation observed after transfusion of von Willebrand’s patients is difficult, however. One might expect that the infusion of the antigen, if it is a carrier protein, would allow the patient to add the clot-promoting fragment to it and that, during the posttransfusion period, both clot-promoting activity and antigen levels would be raised in the blood. As I have indicated, this has not been observed, and three laboratories have

FIGURE 6. Representation of possible structures produced by patients with von Willebrand’s disease. Reduced amounts of antigenic component associated in a nor- mal way with clot-promoting agent. A small minority of patients may produce normal amounts of antigenic material that associates poorly with the clot-promoting agent and does not promote platelet adhesion. After transfusion, the clot-promoting fragment may enter the blood without the antigenic agent.

noted that changes in levels of antigen and clot-promoting activity are not con- current.’&’? An adequate explanation of the changes occurring after transfu- sion is crucial to any theory attempting to explain the molecular disorder i n von Willebrand’s disease, and the suggestion that the antigenic agent is a carrier protein does not easily do this. One must regard this concept as remaining on probation at present, therefore. Explaining the findings after transfusion may well be difficult, but until it is achieved we shall be unable to relate convincingly the molecular disorders in hemophilia and von Willebrand’s disease.

REFERENCES

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2. NILSON, I. M., M. BLOMBACK & B. BLOMBACK. 1959. Von Willebrand’s dis- ease in Sweden: its pathogenesis and treatment. Acta. Med. Scand. 164: 263- 278.

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3. CORNU, P., M. J. LARRIEU, J. CAEN & J. BERNARD. 1963. Transfusion studies in von Willebrand’s disease; effect on bleeding time and factor VIII. Brit. J. Haemat. 9: 189-202.

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11 . HOLMBERG, L. & I. M. NILSSON. 1973. Two genetic variants of von Wille- brand’s disease. New Eng. J. Med. 288: 595-598.

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