(Submitted November 23, 1955, accepted March 20, 1956.)
Supported by grants from the National Institute of Arthritis and Metabolic Diseases, United States Public Health Service (RG A-227, CS), The Children’s Blood Foundation, Inc., and Mead Johnson & Company.
ADDRESS: (I.S.) 525 East 68th Street, New York 21, New York.
ARTICLES
347
VASCULAR
HEMOPHILIA
A
Familial
Hemorrhagic
Disease
in
Males
and
Females
Character-ized
by
Combined
Antihemophilic
Globulin
Defciency
and
Vascular
Abnormality
By Irving Schulman, M.D., Carl H. Smith, M.D., Marion Erlandson, M.D.,
Eleanor Fort, B.S., and Richard E. Lee, M.D.
De’partmenta of Pediatrics and Medicine, New York Hospital-Cornell Medical Center
I
N 1926 von Willebrand’ described afamilial hemorrhagic disease which
oc-curred in males and females and was
ap-parently transmitted as a Mendelian domi-nant. The affected patients bled excessively
and on laboratory testing exhibited mark-edly prolonged bleeding times in the
pres-ence of normal clotting times, normal
plate-let counts and normal clot retractions. The name “pseudohemophilia” was originally applied to this disorder by von Willebrand,
and was retained in his second report in
1931.2 In 1933, however, von Willebrand
and Jurgens’ adopted the name “consti-tutional thrombopathy” due to the belief that a qualitative platelet abnormality was responsible for the abnormal bleeding tend-ency. The disorder was clearly distinguish-able from the thrombasthenia of Glanz-mann4 where the bleeding time was normal while clot retraction and platelet morphol-ogy were abnormal.
Due to variations in nomenclature and in
laboratory technique it was difficult to iden-tify exactly cases of the von Willebrand syndrome which appeared in the literature
before and after the original report. In
1946, however, Estren, Medal, and Dame-shek5 collected 62 cases in the literature extending from 1900, and added 11 new cases of their own. The defined character-istics of the included cases consisted of an
hemorrhagic tendency with significantly
prolonged bleeding as the only consistent abnormality. The clotting time of whole blood was usually normal as were platelet counts, platelet morphology and clot re-traction. Estren et al. could find no evi-dence for platelet dysfunction, a conclusion also arrived at by Revol et al.6 in a review of 91 cases in 1950. Studies by Lelong and Soulier,T Cazal and Izarn8 and by Andre9 included findings of normal prothrombin consumption, constituting further evidence against platelet abnormality and disclosing no evidence of any disturbance in the coagulation mechanism. In 1954, MacFar-lane and Simpkiss’#{176} studied 11 cases in a single family and found no disturbance in coagulation even with use of prothrombin consumption and thromboplastin genera-tion tests.
A vascular basis for von Willebrand’s disease was suggested in 1941 by MacFar-lane,11 who described morphologic and
functional abnormalities of the capillaries
of the nail-beds in five cases. The capillaries
were tortuous, distorted and failed to con-strict normally on trauma. The described abnormalities were later confirmed in other reports by Levy,’2 Perkins’3 and O’Brien.’4
TABLE I
SITES OF BLEEDING AND THEIR RELATIVE SEVERITY AS DETERMINED BY NEED FOR HOSPITALIZATION
Present Series (7 Cases): (‘linical Manifestations
348
SCHULMAN VASCULAR HEMOPHILIAthe 62 cases reviewed by Estren et al. 5% demonstrated definitely prolonged clot-ting times. Of the 11 cases reported by the authors, 8 revealed clotting times which were borderline or abnormal. Jurgens and Ferli&5 found defective prothrombin
con-sumption in eight reported cases. Quick and
Hussey16 in 1953 described a girl with pro-longed bleeding time and normal clotting time who demonstrated abnormal pro-thrombin consumption and deficient
anti-hemophilic activity. Alexander and
Gold-stei&7 in 1953 reported two patients with
pseudohemophilia who demonstrated
ele-vated bleeding times, abnormal capillaries, occasionally abnormal clotting times and prothrombin consumptions, and diminished antthemophilic activities. These authors proposed a dual defect, coagulation and vascular, in pseudohemophilia. Cases ex-hibiting similar findings have also been re-ported by Bigelow,’8 Larrieu and Soulier,’9 Van Creveld et al.’#{176}and Darte.’1
The finding of definitely abnormal coagu-lation status in one of our patients prompted us to re-study our group of chil-dren classified as having pseudohemophiia.
The original diagnosis in each child had been made on the basis of a hemorrhagic diathesis existing in the presence of pro-longed bleeding time with normal clotting time.
PATIENT
MATERIAL
The patients studied consisted of seven
children, four boys and three girls, ranging
in age from 9 months to 17 years. Four chil-dren were Negro, three white. The seven chil-dren represented five families, there being two affected children in each of two families. One of the latter pairs consisted of a boy and a girl,
the other of two boys.
CLINICAL
MANIFESTATIONS
Each of the children had manifested a
definite hemorrhagic tendency since early infancy. The sites of bleeding in the seven patients are listed in Table I. The 9-month-old boy, a sibling of one of the known girls, had manifested intracranial hemorrhage at birth and had sustained a residual slight
Site of Bleeding No. of Patients
Ilospitaliza-tions (80)
Nose Skin
Gums and tongue Teeth
Joints
Muscles
CNTS
6 5 5 6
1
2
1
5
5
.5 I
1 1 1
hemiparesis. Of the remaining six children, three had initially required medical atten-tion in the first year because of excessive
bleeding from the tongue following a fall. Subsequently the most common
hemor-rhagic manifestation in all six children
was severe and spontaneous epistaxis.
These nosebleeds usually required nasal
packing and transfusions were frequently
necessary because of excessive blood loss.
Next in frequency was bleeding from tongue, gums, and teeth, particularly fol-lowing loss of deciduous teeth or dental extractions. In several instances bleeding had persisted for over 1 week following dental extraction despite frequent packing
with localhemostatics. Five of the children
exhibited excessive bruising on slight trauma. Bleeding into the muscles of arms and legs had occurred in two patients.
Hemarthrosis, however, occurred in only two instances. None of the patients mani-fested hemorrhage into the abdominal
vis-cera. The seven children had required a
total of 30 hospitalizations for hemorrhagic manifestations. Of these, bleeding related to loss of teeth constituted the most com-mon cause for admission to the hospital.
As was mentioned earlier, a familial in-cidence was demonstrated by the
involve-ment of two children in each of two fam-ilies. In the five family groups, however, a history of excessive bleeding in the par-ents could be elicited only in two of the fathers, one of whom was available for
ARTICLES
349
children, had suffered from severe nose-bleeds and easy bruising in childhood but had had no difficulty in adolescence or adulthood. His mother had also been con-sidered a “bleeder.” Complete studies on this parent failed to disclose any abnormal-ity. Likewise, studies on the other parents
in the five families were entirely normal.
Thus, in the group studied no positive
evi-dence of parental transmission could be
elicited.
LABORATORY
STUDIES AND
RESULTS
Investigation of
the
Coagulation StatusAlthough all of the children except the
9-month-old infant had been tested on
nu-merous occasions on the ward and in clinics, the results to be reported are limited to those performed by the authors inder
care-fully controlled laboratory conditions. In
four of the seven patients the entire series
of tests to be described below was
re-peated after an interval of not less than 1 month.
Platelet counts, platelet morphology, pro-thrombin time,22 clot retraction, and con-centrations of fibrinogen in the plasma were repeatedly and consistently normal in all seven patients. The tourniquet test wa positive in only one of the children (T.H.).
The Bleeding time (Duke) was prolonged
in all seven of the children and had, on re-peated earlier studies, constituted the most significant laboratory finding. While varia-tions in bleeding time were evident on re-peated testing of individual patients, certain limits characteristic of the particular pa-tients were also noted. Thus, in four of the patients bleeding times were almost always in excess of 15 minutes while in the other three they usually ranged from 6 to 10 minutes. No exact correlation be-tween bleeding time and clinical severity
could be detected. For example, W.D.
and B.D., with bleeding times usually less
than 10 minutes had clinically far more se-vere illnesses than S.S. whose bleeding time was usually in excess of 15 minutes.
The clotting time of whole blood was
de-termined by adding 1.0 ml of venous blood
(drawn in siiconized syringe with arquad-coated needle) to each of two dry 13 X
100
mm test tubes in a water bath at 37#{176}C.The first tube was tilted at intervals of 30 sec-onds until a firm clot was formed, at which point the second tube was tilted in similar manner. The clotting time is recorded as the time required for the second tube to clot, the upper limit of normal being 10
minutes.
The whole blood clotting time was
nor-mal in 7 of 11 individual determinations.
In the four determinations recorded as
ab-normal the values were only minimally pro-longed, the longest being 13.5 minutes. In two patients repeated determinations re-vealed normal results on one test and ab-normal results on the other. Clotting times, done by a variety of techniques, had al-most always been recorded as normal in the past records of the children.
The recalcification time was determined
by adding 0.4 ml of
.025
molar solution of calcium chloride to 0.4 ml of the patient’s plasma and determining the clotting time of the mixture at 37#{176}C.Normal values range from 90 to 180 seconds. With this test a higher incidence of abnormality was de-tected (8/11) but here too, as in the clotting time, the degree of abnormality was slight. With the exception of one value of405
sec-onds the highest value found was 255 sec-onds.The prothrombin consumption test was
performed by a modification of the method of Dreskin23 as follows: 2 ml of whole blood were allowed to clot at 37#{176}C.One hour after clotting, the blood was centrifuged and the serum reincubated at 37#{176}Cfor 10 minutes. The prothrombin time of the serum was determined by mixing 0.1 ml of serum, 0.1 ml of normal deprothrombinized plas-ma,#{176}and determining the clotting time upon addition of 0.2 ml of a calcium-thromboplastin mixture.f The normal pro-thrombin time of serum by this method is
o Plasma was deprothrombinized by adsorption with Ca3(P04)2 according to the method of Quick.”
350
in excess of 25 seconds. The prothrombin consumption of plasma, clotted by recalcifi-cation as above, was done in a similar man-ner. The normal prothrombin time of re-calcified plasma is in excess of 60 seconds.
The prothrombin consumption test on whole blood was distinctly abnormal in six of the patients, the values for serum prothrombin
time ranging from 14.5 to 23.0 seconds. The prothrombin consumption test on recalcified
plasma was likewise abnormal in the same
six patients, the values ranging from 16.0 to 51.0 seconds.
With the tests listed in Table II, definite evidence of abnormal coagulation was de-monstrated in six patients. In the seventh (R.M.) no indication of disturbed coagula-tion was evident.
Analysis of the results of the prothrombin consumption tests were of added interest. In studies of 13 cases of classical hemophilia in this laboratory it was found that the
serum prothrombin times of whole blood
ranged from 8.0 to 14.0 seconds, while that
of recalcified plasma ranged from 12.0 to 15.5 seconds. As may be seen in Table II, the six patients in the present study who demonstrated abnormal prothrombin con-sumptions had values consistently between those of classical hemophilia and the nor-mal. The seventh patient, as indicated above, had normal prothrombin
consump-tion.
Identification of
the
Coagulation DefectAs a very simplified working concept the coagulation mechanism may be divided into three phases as indicated in Figure 1.
The finding of normal one-stage prothrom-bin time in all of the patients indicates normality of the prothrombin complex, i.e., normal concentrations of prothrombin, labile factor and stable factor. The finding of abnormal prothrombin consumption in
the presence of normal prothrombin com-plex indicates that the defect resides in the
TABLE II
ROUTINE COAGULATION TESTS
Patient Sex, Age
Bleeding Time (mm)
(3-5)
Clouing Time (mm) (5-10)
Serum Prolhrombin Time
Recakification TimJ(sec) (Prothrombin Consumption) (see)
(90-180) Whole Blood Recalcified Plasma
(>25) (>60)
T.H. F 13 yr
15-100 6,8 180,225 14.5, 20.5 32.5,41.5
E.J. F 12 yr
15- 80 8, 13.5 225, 255 16.0, 17.0 16.0, 22.5
D.J. M 9 mo
>15 6 240 18.0 16.0
S.S. M 6yr
12-20 9, 13 195, 200 20.0, 20.8 51.0
W.D. M 10 yr
7- 10 12.5 255 15.5 21.0
B.D. M 17 yr
6- 8 12 405 23 17
R.M. F 10 yr
FIG. 1. The normal coagulation cycle. The above concept is highly simplified but serves as a good working model in applying and interpreting the varims laboratory tests.
‘I
>-> 0
z
I-Cr, -C
a-I
COAGULATION
Ca++
Phase 1. Plasma thromboplastin precursors + pin teiets---*th roinboplastin
(AHG, PTC, PTA)
Phase 2. Thromboplastin
Prothrombin conversion factor (stable)
Prothrombin accelerator factor (labile) +prothrombin----’thrombin
Calci urn
Phase 3. Thrombin + fibrinogen---*fibrin
first phase of coagulation, namely in the
formation of thromboplastin. As will be
noted, this may result from deficiency or
abnormality of any one of four possible fac-tors: AHG, PTC,
PTA
or platelets. In order to identify the deficient factor, the thrombo-plastin generation test described by Biggsand Douglas,2’ was employed.
The thromboplastin generation test is
performed by incubating together depro-thrombinized plasma, washed platelet
sus-pension, serum and calcium. With these
reagents one has all the factors required for thromboplastin formation as demon-strated in Figure 2.
FIRST PHASE
REAGENTS FACTORS PRESENT
I. INCUBATION MIXTURE
I)DEPROTHROMBINIZED PLASMA AHG. PTA
2) WASHED PLATELET SUSPENSION PLATELET FACTORS
3) SERUM PTC, PTA
4)CALCIUM
II. SUBSTRATE
NORMAL PLATELET FREE PLASMA PROTHROMBIN
#{149}
LABLE AND STABLE FACTORS. FIBRINOGENMETHOD I ALIQUOTS OF INCUBATION MIXTURE ADDED TO ALIQUOTS OF SUBSTRATE AFTER I?,5,7,9 MINUTES OF INCUBATION AND CLOTTING
TIME DETERMINED. SPEED OF CLOTTING IS
MEASURE OF THROMBOPLASTIC ACTIVITY.
FIG. 2. The thromboplastin generation test.
Aliquots of the incubation mixture are added to aliquots of normal platelet-free
plasma after 1, 3, 5, 7 and 9 minutes
of incubation. The speed of clotting of the substrate plasma is then a measure of the thromboplastin formed in the
incuba-tion mixture. With the use of all normal
reagents a minimum clotting time of the
substrate plasma of 8 to 10 seconds is achieved after 3 to 7 minutes of incu-bation. With the use of an all normal
system, the thromboplastic activity
produc-ing a minimum clotting time of 10 seconds
was designated as 100%. Serially diluting the incubation mixture at this point and then adding further aliquots to substrate plasma allows construction of a standard
curve by means of which the clotting times of substrate plasma may be expressed in terms of per cent thromboplastin
gener-ated. In the investigation of a coagulation
NCUBATION MIXTURE: NORMAL PLASMA, PLATELETS. SERUM
INCUBATION TIME- MIN
FIG. 3. Thromboplastin generation. The solid
lines demonstrate four individual normal
PATIENTS’ PLASMA
NORMAL PLATELETS
NORMAL SERUM
I. E.J. 2. T.H. 3. W.D. 4. B.D. 5. D.J. 6. S.S. 7. R.M.
>-
I->
I-0
z
I-U) -J
a-0
0
I
I-7
6
5
2 3
4
A#{149}NORMAL PLASMA, PLATELETS. SERUM
B’ PATIENTS PLATELETS SUBSTITUTED
FIG. 5. Thromboplastin generation using plasma from each of the seven patients plus normal platelets and serum. Six of the patients are dis-tinctly abnormal. The plasma from R.M. fails to
E 10 YRS produce any abnormality in thromboplastin gen-eration.
>-
I->
I-C.)
z
I-U,
-j
a-0
0
I
I-INCUBATION TIME- MIN
352
disturbance the patient’s
deprothrombin-ized plasma, platelets, and serum are in-dividually substituted for the normal re-agents in the thromboplastin generation test. In this manner the factor responsible for abnormal thromboplastin formation may be detected. Figure 3 demonstrates four
in-dividual normal thromboplastin genera-tion tests and the range of normal in 20 children. It will be noted that normally at least 90% thromboplastic activity is reached by 7 minutes of incubation.
Figure 5 demonstrates the results of the thromboplastin generation test in one of the
patients (E.J.) who had exhibited impaired prothrombin consumption. It will be noted that substitution of patient’s platelets and patient’s serum resulted in normal thrombo-plastin generation, thus indicating
normal-ity of platelet function and PTC and PTA
CD
#{149}. SERUMD#{149} “ PLASMA
ED HEMOPHILIC PLASMA
Fic. 4. Thromboplastin generation test in pa-tient E.J. Patient’s platelets and serum reveal no defect. Patient’s plasma however, produces markedly abnormal thromboplastin generation similar to that obtained with plasma from a pa-tient with classical hemophilia.
INCUBATION TIME
-
MINactivity. When the patient’s plasma is sub-stituted, however, thromboplastin genera-tion became markedly abnormal. In the
latter situation deficiency of antihemophilic
globulin is strongly suggested since the
nor-ma! serum and platelets supply all of the
other factors needed for normal thrombo-plastin formation. This is shown in Figure
4 whi,ch includes the thromboplastin genera-tion curve when the plasma of a patient
with classical hemophilia is incubated with normal platelets and normal serum.
Complete thromboplastin generation tests were performed on all of the patients in the group. In all seven, platelets and serum ac-tivities were entirely normal. In six of the
patients, substitution of patient’s plasma
into the incubation mixture resulted in dis-tinctly abnormal thromboplastin generation
II
INCUBATION TIME
-
MINFIG. 6. Thromboplastin generation tests dem-onstrating failure of mutual correction between patient’s plasma and hemophilic plasma.
I. E.J.
2. T.H.
3. W.D.
4, B.D. 5. D.J. 6. S.S. 7. R.M.
7
>-
I->
I-0
.
z
I-U)
-J
a-0
0
I
I-INCUBATION TIME -MIN
Fic. 7. Thromboplastin generation tests dem-onstrating failure of mutual correction between six of the patients’ plasmas and hemophilic plasma. Plasma of the seventh patient (R.M.) completely corrects the defect in hemophilia.
ARTICLES 353
A’ NORMAL PLASMA.PLATELETS. SERUM
B’ 50% HEMOPHILIC PLASMA SUBSTITUTED
Qo 50% PATIENTS
50% HEMOPHILIC
D. 50% PATIENT’S
>.
I->
I-C-)
z I-U) -C a-0
0 I
I-normal (R.M.) was the same girl who had
been found to have no abnormality in pro-thrombin consumption.
Demonstration of Deficiency of
Antihemophilic Globulin
That the deficient coagulation factor was indeed antihemophilic globulin was shown in two ways: by demonstrating failure of pa-tient’s plasma to correct the coagulation ab-normalities in classical hemophilia, and by
direct assay of antihemophilic activity. This effect of the patient’s plasma on the coagu-lation defects of classical hemophilia was studied with the use of the thromboplastin generation and the prothrombin consump-tion tests.
Figure 6 illustrates the use of the throm-boplastin generation test in determining the antihemophilic activity of the patient’s plasma. Incubation of hemophilic plasma
with normal platelets and normal serum results in markedly abnormal thrombo-plastin generation (Fig. 5). A mixture of
50% hemophilic plasma and 50% normal plasma leads to normal thromboplastin gen-eration due to the known corrective effect of
normal plasma (Fig. 7, curve B). Similarly
a mixture of 50% patient’s plasma and 50%
normal plasma results in normal thrombo-plastin generation, thus demonstrating the corrective effects of normal plasma on the
coagulation defect in the patient under
study (curve C). However when equal parts
EJ9IOYRs
.
of the patients plasma and hemophihc plasma are mixed, no evidence of mutual correction is evident (curve D).
Figure 7 demonstrates the thromboplastin
generation curves obtained when the
plas-ma of each of the seven patients was mixed with equal parts of hemophilic plasma. It may be seen that plasma of six of the
pa-50% PATIENTS’ PLASMA
50% HEMOPHILIC PLASMA
NORMAL PLATELETS
TABLE III
THE EFFEC’r OF PATIENT’S PLASMA AND OF NORMAL PLASMA ON THE RECALCIFICATION TIME AND
PROTHROMBIN C0N5UMP’rIoN IN CLASSICAL HEMOPHILIA
Hemophilic plasma, ml .4 .3 .0 .0 .3 .1
Patient plasma, ml 0 0 .4 .3 .1 .3
Normal plasma, ml 0 .1 .0 .1 0 0
CaCI, .025M, ml .4 .4 .4 .4 .4 .4
Recalcification time (see) 435 135 180 135 215 180
Prothrombin consumption time (see) 15.0 >120 32.5 100 23.5 27.0
C.)
LU
U)
LU
I-z
0 I I.-0
a.
LU U)
U)
a-0 LU
‘I-C.) -J C.) LU
AHG ACTIVITY
S OF NORMAL
354 SCHULMAN
tients fail to correct the defect in
hemo-philia, whereas the plasma of R.M., as noted before, corrects the hemophilic abnormality completely.
Similar results were obtained with a
somewhat different technique, i.e., by
deter-mining the corrective effect of patient’s plasma on the abnormal recalcification time and prothrombin consumption of a patient
with classical hemophilia. An example of
this technique is given in Table III. It may
be seen that normal plasma corrects to nor-mal the recalcification time and prothrom-bin consumption of both the hemophiliac and the patient (T.H.). However, with mix-tures of patient’s plasma and hemophilic plasma the values for recalcification time and prothrombin consumption remain dis-tinctly abnormal. Similar studies were car-ried out in all seven patients and confirmed the results observed above with the use of the thromboplastin generation test.
Assay of antthemophilic activity was per-formed in the following manner: Plasma from a patient with severe classical hemo-philia was taken and designated as having zero antthemophilic activity. Plasma from
one of the authors was designated the standard normal plasma. Several dilutions
of the normal plasma were made and 0.1
ml of each dilution added to 0.3 ml of the
hemophilic plasma. Each mixture was
re-calcified with 0.4 ml of .025 molar solution
of calcium chloride and the prothrombin
consumption time determined. The undi-luted normal plasma was designated as
containing 100% antihemophilic activity. (It
should be noted that this actually corre-sponds to 25% AHG in the mixture which
contains three parts of hemophilic plasma
and one part normal plasma.) A standard curve was then constructed relating the
percentage of normal plasma added to the resulting prothrombin consumption time of
FIG. 8. Standard curve for antihemophilic globu-lin assay; 0.1 ml amounts of serial dilutions of normal plasma were added to 0.3 ml amounts of
known hemophilic plasma and the serum pro-thrombin times determined 1 hour after
recalci-fication of the mixture. The hemophilic plasma was considered to have zero AHG activity and the normal plasma 100% AHG activity. The serum prothrombin times were plotted against the per-centage of normal plasma added to the 0.3 ml of hemophilic plasma. For example 0.1 ml undi-luted normal plasma was designated as 100%; 0.1 ml of a 1:10 dilution of normal plasma was desig-nated as 10% when added to 0.3 ml of hemophilic
ST MIS ) 15 6 ) IS
CT MIS 10 5 9!4
‘SEC 15 57 42 ATIN (IFMFRATI(Th&
IOC
TH9 12YRS _______________________
TH _______________________
PATIENT PLASMA. NORMAL PLATELETS AND SERUM
‘I
> I-0
4
z
I-U) 4 -C
a.
0
0 I
I-INCUBATION TIME- MIN
the mixture (Fig. 8). It is of interest that as
little as .48% normal plasma was capable of inducing normal prothrombin consumption.
Assays of antihemophilic activity were carried out on 10 boys with classical hemo-philia and on the seven patients in the
pres-ent series. The results are shown in Figure 9. It will be seen that patients with classical hemophilia demonstrate AHG activity from 0 to .06% of normal. In the seven patients under study six disclosed AHG activity ranging from .12 to .26% of normal. The seventh patient (R.M.) again demonstrated normal AHG activity.
PLASMA
AHG ACTIVITY
IN S OF NORMAL CLASSICAL HEMOPHILIA
10 CASES
0.0 - 0.06 5
PRESENT CASES I.EJ 2.BD 3.DJ 4.WD 5.TH 6. SS 7. RM
0.12% 0.12% 0.16% 0.18% 0.26 S 0.26 S 100.0 5
FIG. 9. AHG assay of patients’ plasma and of plasmas from patients with classical hemophilia. Assays were performed by adding 0.1 ml of test
plasma to 0.3 ml of a standard hemophilic plasma. The mixture was recalcified and the serum pro-thrombin time determined 1 hour after clotting. The AHG activity as a percentage of normal was read from the standard curve shown in Figure 8.
The Effect of Transfusion of Plasma.
Be-cause in-vitro testing had demonstrated the corrective effect of normal plasma, the effect of transfusion of plasma was investigated in one of the patients (T.H.) who exhibited clearly defined coagulation abnormalities. The effects of plasma transfusion on bleed-ing time, clotting time, prothrombin con-sumption was determined. Two hundred twenty-five milliliters of “fresh frozen”
nor-mal plasma were administered
intravenous-ly in about 60 minutes. Studies were per-formed immediately before and 1 hour and 22 hours after completion of the infusion of plasma. The results are shown in Figure 10.
It will be seen that prior to transfusion there
was marked prolongation of bleeding time, impaired prothrombin consumption and de-fective thromboplastin generation. One hour after administration of plasma all abnormalities were corrected to normal. At 22 hours after plasma infusion the coagula-tion tests were still normal but the bleeding time had returned to the abnormal pre-transfusion value.
TEST BEFOREPLASMA AFTERI HRI22NRSPLASMA
ROvBOPL.... .. .
Fic. 10. The effect of transfusion of fresh-frozen normal plasma (225 ml) on bleeding time, clotting time, prothrombin consumption, and thromboplas-tin generation of patient T.H.
Demonstration of the Vascular
Abnormality
As was noted initially the predominant laboratory finding in the von Willebrand
syndrome is a prolonged bleeding time, a situation which does not exist in classical hemophilia where the bleeding time is
nor-mal. Since the AHG deficiency in our cases
was shown to be less severe than in classical
hemophilia it appeared unlikely that the
hemorrhagic diathesis could be explained
solely on the basis of the demonstrated AHG deficiency. Therefore, attention was
next directed to the vascular system. To
investigate this aspect capillary microscopy
lI(. I 1 11/)J)r 1(ft). III( flOIlIhtl cOI1
tiiictit 21()). \. trtril, (;
t1II( (1p1IIar1(. 1T V(1u11I(. \()t( tlu
((k ((1 \((S(l (litO1t1OI( (11(1 t()It(I((lt\.
I right). Bl!1l)al (flh(1I11((tiV(
Pitt 1.11. ‘,t)tr jiitikd oiliiig (11(1
t()ItlIO’itV f (aI)lll(1i(S (11(1 \(11IIl(S.
1I(. 1: (TOtt(’I 1(ft). 111ll)L1 cijiiiitivt
I l)U\ vitIi (l(’’i(.dl l((Ii(OI)lIllli(. ( 1I)hhIu1
)( (11 ‘(1((( ti (11(1 1(_( liii!.
ARTICLES
the fingers and the bulbar conjunctivae. Examination of nail-bed capillaries was per-formed according to the method of Leader.25
Good visualization of the capillaries was achieved with a standard binocular
micro-scope under 100X magnification. Nail-bed
capillaries were examined in four of the chil-dren who had demonstrated coagulation abnormalities and in the one patient (R.M.) who was consistenfly normal on coagulation testing. Of great significance was the fact that marked morphologic abnormalities were noted in all of the patients examined, i.e., in the four children with coagulation defect and in the one who had only pro-longed bleeding time. Whereas the nail-bed capillaries in normal children form very smooth, regular, parallel “hair-pin” loops, the nail-bed capillaries of the children in the
present series revealed marked distortion of
arrangements with striking coiling, tortuos-ity and irregularity of individual loops. Many capillaries were noted which ap-peared quite dilated and which followed a serpentine course toward the base of the nail. The changes seen were identical with
those demonstrated and illustrated by Mac-Farlane.11 No abnormalities were found in the nail-bed capillaries of the parents of the aforementioned children. The nail-bed
capillaries were examined in three boys with classical hemophilia and were found to be entirely normal, thus confirming the findings of MacFarlane.11
The vasculature of the bulbar conjunc-tivae was examined by the method of Lee
and Holzel6 using a slit-lamp microscope
with a magnification of 60 X. Studies were carried out in three patients in the present group, two (E.J., T.H.) having demonstrable coagulation defects, the third (R.M.) being the patient with normal coagulation status. In addition to examination of entirely nor-mal individuals, additional controls in-cluded three boys with classical hemophilia.
Subjects were referred to the examiner
(R.L.) at random with no information as to
diagnosis. Immediately after examination a verbal report on the findings was
re-corded. In addition, photomicrographs were
taken in each case for further examination. Figure 11 shows the normal bulbar con-junctiva, and demonstrates the capillaries forming smooth, regular ioops among the
larger vessels. Figure 12 reveals the typical features noted in all three of the patients in the present series who were examined. It may be seen that there is marked coiling of the venules and striking tortuosity of almost all visible capillaries with practically no regular capillary loops present. Under di-rect observation a marked three-dimensional coiling was also noted and, finally, a signif-icant slowing of blood flow throughout the conjunctival vessels. The changes seen were very similar to those described in patients with hypertensive vascular disease.27 (All patients in the present series were normo-tensive.) The bulbar conjunctivae in the
pa-tients with classical hemophilia, on the other hand, were entirely normal.
DISCUSSION
There appears to be little doubt, from the
present studies, that deficiency of anti-hemophiic globulin may occur in a manner distinct from the sex-linked recessive in-heritance of classical hemophilia. The oc-currence of true hemophilia in the female may be anticipated as a result of union
between a hemophilic male and hemophilia
carrier female, and has been reported in several instances.283#{176} In the present series, however, there was no suggestion of true
hemophilia in the family background. In
addition, the reported cases of hemophilia
in the female were characterized by normal
bleeding times and
prolonged
clotting times, in direct opposition to the findingsin the present cases. It has also become
and was transmitted as a sex-linked reces-sive characteristic. In addition, the bleeding
times were normal.
While the present cases demonstrate the
AHG deficiency to be familial, they offer no positive evidence concerning genetic transmission. In only two of the five families
studied were positive family histories ob-tamed. Only one parent who gave a
posi-tive history was available for study and was
found to be entirely normal. Similar
diffi-culty is found in the other reported cases of
the von Willebrand syndrome in which
AHG deficiency was found. The patients reported by Quick and Hussey16 and Lar-rieu and Soulier19 had negative family his-tories and their parents were found to be
normal. In the case report of Van Creveld
et al.2#{176}the patient’s mother was stated to
have had hypermenorrhea but was not
available for study. In the case of Alexander
and Goldstein,’7 the patient’s mother was a non-bleeder but did reveal abnormal capil-laries. The only study presenting definite evidence of genetic transmission of the
AHG deficiency is the recent report of Darte” where a mother and daughter were found to be affected. In this study, how-ever, capillary morphology was not deter-mined.
That the von Willebrand syndrome may result even when antihemophilic activity is
entirely normal was clearly demonstrated
by the recent investigations by MacFarlane
and Simpkiss.’#{176} These authors investigated
a large family with 64 members in five
generations. Twenty-one members of the
family had hemorrhagic diatheses, with 10
females and 11 males in the group.
Com-plete coagulation studies, including
pro-thrombin consumption and thromboplastin generation tests, were performed on 11
pa-tients and were found to be entirely
nor-mal. The disease in this family, it appears,
corresponds to that of the patient, R.M.,
who demonstrated prolonged bleeding time
and abnormal capillaries but completely normal coagulation status. While all re-ported cases of the von Willebrand
syn-drome, taken as a single group, reveal
posi-tive family histories in more than 75% of
the cases, and suggest strongly the inherit-ance as a simple dominant, sufficient
evi-dence is not yet at hand to define clearly
the mode of transmission in those cases demonstrating AHG deficiency.
The nature of the vascular abnormality in the von Willebrand syndrome is of dis-tinct interest. The presence of a prolonged bleeding time indicates at least functional
abnormality in all cases, while the results of the present study demonstrate morpho-logic abnormalities of the capillaries both in the cases with AHG deficiency and those
with perfectly normal AHG activity. It is generally believed that the capillaries are congenitally abnormal and that the func-tional disturbance results from their failure to constrict normally on trauma. Yet, simi-lar morphologic changes have been noted in a variety of acquired conditions such as
vas-cular hypertension,27 idiopathic thrombo-cytopenic purpura,1’ and PTA deficiency.34 Because both a prolonged bleeding time
and morphologic abnormalities of the
capil-laries occur in thrombopenia, the possibility
of some platelet abnormality is again
sug-gested. At present the platelets are
con-sidered to regulate at least three distinct
phases in the hemostatic mechanism: a
vaso-constriction, due to release of serotonin (5-hydroxytryptomine); clot retraction, due to
release of a theoretical substance
“retrac-tozyme”; and coagulation, due to release of
plasma thromboplastic factors.6 In the
pa-tients in the present series the role of the
platelets in the coagulation mechanism was
conclusively demonstrated to be normal by
the thromboplastin generation test.
“Re-tractozyme” activity was also apparently normal for clot retraction was consistently normal in all of the patients. Concentration of serotonin in the serum was analyzed in two of the patients with the most severe hemorrhagic diatheses (E.J., T.H.) and was
found to be 0.25 and 0.18 g/ml, values at
the lower limits of normal.6 Bigelow,’8
how-* We are greatly indebted to Dr. Mario
ARTICLES 359
ever, reported normal concentrations of serotonin in two girls similar to those de-scribed in the present series. Thus none of the known platelet factors could be demon-strated to be significantly deficient.
That the AHG deficiency itself was not responsible for the vascular abnormality is evident by the fact that the bleeding time is normal in classical hemophilia where the deficiency of AHG is much greater than in our patients. The fact that transfusion of normal plasma produced correction of bleeding time as well as of coagulation ab-normalities suggests the possibility that the vascular dysfunction may result from de-ficiency of still another factor required for normal vasoconstriction. Whether adminis-tration of normal plasma will correct the bleeding time in the patients showing only the vascular abnormality awaits further study.
From a therapeutic point of view the use of fresh plasma, at least in those patients demonstrating AHG deficiency, promises to be of great value. In one of our patients the administration of plasma before and after dental extraction resulted in an almost bloodless procedure whereas previously tooth extractions had always been followed by very severe hemorrhage. Likewise, in a second instance, the administration of fresh plasma resulted in cessation of bleeding fol-lowing tooth extraction after repeated trans-fusions of stored blood had failed during a
period of 2 weeks. Cortisone has, in our hands, not resulted in cessation of hemor-rhage or in shortening of bleeding time. Vitamins P and C, rutin, adrenochrome and adrenocorticotropin have been reported to be ineffective by Biggs and MacFarlane.’5 Splenectomy is definitely contraindicated.
As a matter of classification, it would appear that the von Willebrand syndrome must now be divided into at least two dis-tinct groups. Both groups demonstrate functional and morphologic capillary ab-normalities; one of the groups, in addition, demonstrates marked deficiency in anti-hemophilic globulin. For the group
mani-festing the combined vascular defect and
AHG deficiency we suggest the name “vas-cular hemophilia.” For the group manifest-ing only the vascular abnormality, we recommend retention of the name “pseudo-hemophilia.”
SUMMARY
The coagulation and vascular aspects of the von Willebrand syndrome have been studied in seven children.
Six of the children demonstrated a severe defect in the coagulation mechanism which was found to result from a marked de-ficiency in antthemophiic globulin. The latter deficiency was found in boys and girls, was familial, and apparently occuned in a manner distinct from the sex-linked re-cessive inheritance of classical hemophilia.
Morphologic capillary abnormalities were noted in the nail-beds of the fingers and in the bulbar conjunctivae of children with and without associated deficiency of anti-hemophilic globulin.
Administration of fresh plasma resulted in correction of both bleeding time and coagulation defects in a patient with the combined vascular-coagulation abnormality.
The vascular abnormality was found to be independent of deficiency of antihemo-philic globulin or any of the known platelet factors.
Classification of the von Willebrand syndrome into at least two groups was re-commended: patients with vascular defect plus AHG deficiency (vascular hemophilia), and patients with vascular defect and
nor-mal coagulation status (pseudohemophilia).
REFERENCES
1. von Willebrand, E. A.: Heredit#{228}re Pseu-dohemofili. Finska l#{228}k.-s#{228}llsk.handl., 68:87, 1826.
2. Ibid.: Uber heredit#{228}re Pseudohamophilie.
Acta med. scandinav., 76:521, 1931.
3. von Willebrand, E. A., and Jurgens, R.:
Uber eine neue Bluterkrankheit, die konstitutionelle thrombopathie. Klin. Wchnschr., 12:414, 1933.
4. Glanzmann, E.: Heredit#{228}re hamorrha-gisehe thrombasthenie. Jahrb. Kinderh., 88:113, 1918.
VASCULAR HEMOPHILIA
W. : Pseudohemophilia. Blood, 1:504,
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6. Revol, L., Favre-Gilly,
J.,
and Ollagnier,C.: La maladie de Willebrand (throm-bopathie constitutionelle ou pseudo-hemophilie). Rev. hemat., 5:24, 1950. 7. Lelong, M., and Soulier,
J.
P. : Sur unemaladie h#{233}morragique constitutionelle caracterisde par l’allongement isole du temps de saignement (maladie de Wile-brand. Etude de neuf cas). Rev. h#{233}mat., 5:13, 1950.
8. Cazal, P., and Izarn, P. : Consideration sur la pseudo-h#{233}mophilie de Willebrand, a propos de deux noxeaux cas. Acta haemat., 4:357, 1950.
9. Andre, A.: Contribution
a
l’#{233}tudede la thrombasth#{233}nie. Sang, 23:54, 1952. 10. MacFarlane,J.
C. W., and Simpkiss, M.J.:
The investigation of a large family affected with von Willebrand’s Disease. Arch. Dis. Childhood, 29:483, 1954.
11. MacFarlane, R. G.: Critical review: Mech-anism of haemostasis. Quart.
J.
Med., 10:1, 1941.12. Levy, L.: Non-hemophilic hereditary
hemorrhagic diathesis; report of a family of bleeders. Ann.
mt.
Med., 27:96, 1947.13. Perkins, W.: Pseudohemophilia; case study. Blood, 1:497, 1946.
14. O’Brien,
J.
R.: Familial capillary fragility (diffuse capillary telangiectasia). Proc. Third Intemat. Congress of Hemat. New York, Grune & Stratton, 1950. 15. Jurgens, R., and Ferlin, A.: Uber densog. Prothrombin-konsumptionstest bei Hamophilie (Hamophilie, Kondukatarin-nen) und bei Konstitutioneller throm-bopathie (v. Willebrand-Jurgens).
Schweiz. med. Wchnschr., 80:1098, 1950.
16. Quick, A.
J.,
and Hussey, C. V.: Hemo-philic condition in the female (abstract).J.
Lab. & Clin. Med., 42:928, 1953. 17. Alexander, B., and Goldstein, R.: Dualhemostatic defect in pseudohemophilia (abstract).
J.
Clin. Investigation, 32:551, 1953.18. Bigelow, F. S.: Serotonin activity in blood.
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Lab. & Clin. Med., 43:759, 1954. 19. Larrieu, M.J.
and Soulier,J.
P.: Deficiten facteur antih#{233}mophilique A chez une file, associ#{233}
a
un trouble du saigne-ment. Rev. h#{233}mat.,8:361, 1953.20. van Creveld, S., Jordan, F. L.
J.,
Punt, K.,and Veder, H. A. : Deficiency of anti-hemophilic factor in a woman, corn-bined with a disturbance in vascular function. Acta med. scandinav., 151: 381, 1955.
21. Darte,
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M. M. : Defect of antihernophilic globulin in von Willebrand’s disease (abstract). Am.J.
Dis. Child., 90:561, 1955.22. Quick, A.
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: The Physiology and Path-ology of Hemostasis. Philadelphia, Lea, 1951.23. Dreskin, 0. H. : The prothrornbin con-sumption test: a simplified technic. Am.
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Clin. Path., 22:140, 1952.24. Biggs, R., and Douglas, A. S. : The throm-boplastin generation test.
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Clin. Investigation, 29:146, 1950.27. Ibid.: Peripheral vascular hemodynamics
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35. Biggs, R., and MacFarlane, R. G.: Human
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Oxford, Blackwell, 1953.
ARTICLES
SUMMARIO
IN INTERLINGUA
Hemophilia Vascular: Un Morbo
He-morrhagic Familial in Maseulos e
Femi-ninas, Characterisate per le
Combina-tion de Carentia de Globulina
Anti-hemophilic con Anormalitate Vascular
Pseudohemophilia (syndrome de von Wille-brand) es definite como un familial morbo hemorrhagic que es characterisate per
pro-longate tempores de sanguination in le pre-sentia de normal tempores de coagulation, normal contos de plachettas, e normal
tern-pores de retraction del coagulo. Le functional e morphologic anormalitates capillari,
cons-tatate in iste morbo per MacFarlane in 1941, generava le opinion currente que
pseudohemo-philia resulta ab un anormalitate vascular
hereditari plus tosto que ab anormalitates del
coagulation sanguinee. Nonobstante, in tern-pores plus recente plure reportos ha indicate que disturbationes coagulatori pote occurrer in
casos del syndrome de von Wilebrand.
Le presente studio esseva concernite con 7 juveniles, 4 mascule e S feminin, 4 negre e S blanc. Duo fratres esseva afficite in un familia, un fratre e un soror in un altere. Omne
le patientes habeva manifestate distincte ten-dentias hemorrhagic ab le tempore de br prime infantia, con sanguination ab naso, lingua, gingivas e dentes, e pelle como
symp-tomas be plus comrnun. Sanguination a in musculos, articulationes, e le systema nervose central occurreva rarmente. Omne be patientes habeva probongate tempores de sanguination, durante que be tempores de coagulation esseva normal o rninimabrnente probongate. Le contos
del plachettas, be retraction de coagulo, le tempore prothrombinic, e be fibrinogeno del plasma esseva normal in omne casos. Le
tern-pore de coagulation de plasma recalcificate e
be test del consumption de prothrombina revelava distinc,te anormalitates in 6 del 7
casos. Le test del generation de thrombo-plastina confirrnava be presentia de un
anor-malitate del coagulation in 6 del 7 patientes e supportava be conclusion que il se tractava del effecto de un carentia de gbobulina antihemo-philic. Le confirmation del carentia de gbobu-lina anti-hemophilic esseva effectuate per ex-perimentos de correction in plasma cognoscite-mente hemophilic e per essayos directe del hemogbobina antihemophilic in be patientes. Un del 7 patientes, ben que su caso esseva
cinicamente identic al altere 6, revelava un normal stato de coagulation e un normal con-centration de gbobubina anti-hemophilic.
Le examine del capillares de lecto ungular e conjunctiva bulbar revelava marcate
anor-malitates in be palientes con carentia de gbobu-lina anti-hernophilic sed etiam in be patiente unic in qui be stato de coagulation esseva normal.
Le capillares esseva rolate, tortuose, e irregular. Nubbe tab abterationes esseva trovate in ver hemophilia. Le administration de plasma re-frigerate fresc a un del patientes resubtava in
retornos al norma in omne tests de coagulation e etiam del tempore de sanguination.
Le studios demonstrava que sever carentias de gbobulina antihemophilic pote occurrer in
mascubos e femininas e que be hereditation es distincte ab be hereditation recessive a associ-ation sexual que occurre in ver hemophilia. II etiam deveniva evidente que pseudohemophilia (syndrome de von Willebrand) debe esser sub-dividite in al minus 2 forrnas. Ambes exhibi functional e morphologic anorrnalitates capib-ban, sed be un exhibi in plus marcate carentia
