Platelet aggregation, adhesion and adenosine diphosphate release in thrombopathia (platelet factor 3 deficiency)

Platelet

Aggregation,

Diphosphate (Platelet

Adhesion

Release Factor

and Adenosine

in Thrombopathia 3 Deficiency)*

A Comparison with Glansmann’s Thrombasthenia

and

uon Willebrand’s Disease HARVEY

J. WEISS,

M.D.

New York, New York Platelet factor 3 (PF-3) release was assayed by incubating platelet-rich plasma and kaolin. Evidence is presented that the abnormal PF-3 observed in the patients studied (thrombopathia) is due to a defect in the release of platelet adenosine diphosphate (ADP). This defect may also explain the abnormal platelet adhesiveness (Salzman method) and impaired aggregation by connective tissue which was also found. In contrast to patients with Glanzmann’s thrombasthenia, the reactivity of platelets to added ADP was normal. In patients with von Willebrand’s disease, PF-3 and ADP release were normal. and Ward [6] were the first to show that when platelet-rich plasma was kept in a siliconized tube, the Stypven@ time was virtually the same as with platelet-poor plasma. When the plasma was frozen and thawed, the Stypven time became shorter, indicating that PF-3 had been released. Hardisty and Hutton [7] and Spaet and Cintron [8] recently showed that this may also be achieved by shaking the plasma with kaolin or connective tissue fragments. In previous studies Hovig [9] and Spaet and Zucker [IO] demonstrated that these substances also release adenosine diphosphate (ADP), which in turn produces platelet aggregation [77]. It has been proposed that the release of ADP is an essential step in the mechanism by which PF-3 is released (Fig. 1). In support of this hypothesis is the finding by Hardisty and Hutton [7] that adenosine and 2-chloroadenosine, which block platelet aggregation by ADP, also inhibit the release of PF-3 by kaolin. In the present study PF-3 release was measured by incubating platelet-rich plasma and kaolin. Described herein are six subjects with

participate in hemostasis by forming aggregates at the site of small vessel injury and by releasing a clotting factor, referred to as platelet factor 3 (PF-3). The term “thrombopathia” has been used to describe bleeding disorders in which the release of PF-3 is reduced. This abnormality has been detected by comparing the activity of affected and normal platelets in various in vitro clotting tests, such as prothrombin consumption [I], thromboplasactivatin generation [Z-5] and prothrombin tion [Z]. In some cases platelet morphology has been abnormal [3]. In many, but not all the patients described, the bleeding time has been prolonged, a finding which would be hard to ascribe to decreased PF-3 activity per se. Clot retraction is usually normal, thereby differentiating the disorder from Glanzmann’s thrombasthenia. Unlike most patients with von Willebrand’s disease, antihemophilic globulin (AHG, factor VIII) in patients with thrombopathia is normal. Recently, new methods for studying the release of PF-3 have been introduced. Fantl

P

LATELETS

* From the Department of Hematology, The Mount Sinai Hospital and School of Medicine, New York, New York, and The Mount Sinai Hospital Services, City Hospital Center at Elmhurst, New York, New York. This study was supported in part by U. S. Army Research and Development Contract DA-49-193-MD-2702 and the Albert A. List, Frederick Machlin and Anna Ruth Lowenberg Funds. Manuscript received October 27, 1966. 570

AMERICAN

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Thrombopathia

(Platelet

bleeding disorders in whom PF-3 release was abnormal. The studies suggest that this is due to defective release of ADP. Platelet adhesiveness to glass beads, as measured by the direct method of Salzman, and aggregation by conADP nective tissue, both of which require release, were also abnormal. By contrast, aggregation and adhesion by ADP itself was normal. The results obtained in these patients are compared with those in patients with Glanzmann’s thrombasthenia and von Willebrand’s disease. CASE MATERIAL Thrombopathia. There were six patients whose PF-3 release was abnormal and who had bleeding of variable severity. CASE 1. This patient (G.P.), a forty-one year old woman, was described previously (Case VI, [5]). She bled excessively after tooth extractions at age thirty-two, after excision of a mammary cyst at age thirty-six, and during a hysterectomy at age thirtynine, although she had previously undergone tonsillectomy and appendectomy at age twenty-two without difficulty. In addition to her bleeding symptoms she has had multiple allergies to foods and drugs since age thirty, and intermittent swelling of knees and ankles, without residual deformity, since age thirtyone. Results of lupus erythematosus tests were negative. CASE 2. This patient, (B.P., a forty year old woman) was also described previously (Case VII, [5]). She bled excessively after tooth extractions at age twenty-four and thirty-one, after tonsillectomy at age twenty-three and after rhinoplasty at twenty-four. In addition, she has had asthma and hay fever since childhood and, more recently, allergic rhinitis. Skin testing revealed allergies to many pollens. year old woman (F.A.) CASE 3. This fifty-three had frequent epistaxis as a child. She bled excessively after a tonsillectomy at age five and, more recently, after three tooth extractions. She bruises easily and states that bleeding after minor cuts is often difficult to stop. In addition to her bleeding symptoms she has had recurrent bouts of muscle and joint pains for twenty years. Studies for collagen disease have been negative. A brother is said to have “almost died” of hemorrhage after tonsillectomy and a sister bled excessively after a hysterectomy.

CASE 4.

This sixty-five year old woman (M.Y.) was told she had pseudohemophilia at age thirty-five. At age twenty she bled excessively immediately after a tonsillectomy and had renewed bleeding ten days later. Postpartum hemorrhage occurred after the birth of her three children. VOL.

43,

OCTOBER

1967

Factor

3 Deficiency)-Weiss

571

? ,J I

i

AGGREGATION

PF- 3 t SEROTONIN,

ETC.

FIG. 1. Platelet factor 3 (PF-3) release. Incubation of platelet-rich plasma (PRP) with kaolin or connective tissue (C.T.) releases ADP and PF-3. Although ADP is required, it is not established that it accounts for all the PF-3 released. CASE 5. This twenty-nine year old woman (M.G.) bled excessively, requiring transfusion, at age seven, but not after a rhinoplasty at age eighteen. She received 10 transfusions because of postpartum hemorrhage at age twenty-six and has always bruised easily. She has had ankylosing spondylitis for many years. This twenty year old woman (S.N.) has CASE 6. always bruised easily. She bled excessively after a tooth extraction at age eight and after a tonsillectomy at age twenty. M.G. (Case 5) was taking indomethacin and M.Y. (Case 4) was receiving digoxin therapy when studied. None of the other patients was receiving medication of any kind. van Willebrand’s Disease (Table I). Ten patients with the diagnosis of von Willebrand’s disease had a prolonged bleeding time and decreased AHG [72], with the following exceptions: In B.S., the father of H.S., AHG was 70 per cent despite a bleeding time which exceeded twenty minutes. Since the AHG value in his son and mother was 30 and 45 per cent, it was assumed that the family had von Willebrand’s disease, with incomplete expression of the AHG defect in B.S. Glanzmann’s Thrombasthenia. Three patients with classic Glanzmann’s thrombasthenia were studied. All had a markedly prolonged bleeding time, absent clot retraction, isolated platelets on peripheral smear and no aggregation when ADP was added to platelet-rich plasma in a final concentration of 10 y per ml. They are described in greater detail elsewhere [ 731. METHODS Venous blood, collected in plastic syringes, was mixed with 3.2 per cent sodium citrate (9: 1) and centrifuged for three minutes at 2,500 r.p.m. and 20°c. in an International PR-2 centrifuge to obtain platelet-rich plasma, which was kept at room temperature. When platelet-poor plasma was required, the remaining blood was centrifuged for sixty minutes at

572

Thrombopathia

(Platelet

Factor TABLE

PATIENTS

Case No.

Patient, Age (yr.) and Sex

Severity of Bleeding (0 to 4f)

3 Deficiency)-

I

STUDIED

Ivy Bleeding Time (min.)

Serum Prothrombin Time (sec. )

<6*

>25*

AHG

50-200’

1 2 3 4 5 6

G.P.,41,F B.P.,40,F F.A.,53,F M.Y.,65,F M.G.,29,F S.N.,21,F

2f 2+ 2+ 1+ 2f 2+

7 8 9 10 11 12 13 14 15 16

D.R.,20,F J.H.,46,F A.S.,34,F J.L.,$41,F E.L.,13,F F.L.,15,M D.L.,7,M B.S.,$35,M H.S.,14,M R.G.,Zl,F

4+ 3+ 2f f 2f 2+ f 1+ 2+ 1+

>30, >30, >30, >30, >20, >20, >15 7 10,>15 9 >30 >30, >30, 10

If 3+ 3+

Patients with Thrombasthenia >30, >30, >30 29 >30, >30, >30 18 >ll 26

Patients with uon W&-brand’s

17 18 19

L.W.,24,M M.C.,28,M K.K.,l,F

>30

Miscellaneous

(%)

Patients with Thrombopathia 9, 3, 14t 40 3, 5 28 18, 15, 8 21, 53 13, 6 17 16 27 >30, >30, >30 30 >30 >30 >20

Weiss

100 90 120 100 100 75

Allergies Allergies Arthralgias, myositis 4 PTA

. . ; ankylosing spondylitis

Disease

16 16 28 22 19 22 18

5 12 30 8 7 8 9 70 35 33

::: .

100 85 180

. . . . No clot retraction ...

.

* Normal values. t Bleeding time values were obtained on different days. $ Mother of E.L. (Case ll), F.L. (Case 12) and D.L. (Case 13). $ Father of H.S. (Case 15). 3,000 r.p.m. and 4’~. All test tubes and transfer pipettes were siliconized.* Platelet factor 3 (PF-3) was determined by a modi-

fication of the methods of Hardisty and Hutton [7] and Spaet and Cintron [B]. In principle, platelet-rich plasma is incubated with kaolin (tube l), thereby releasing PF-3 and activating the plasma contact factors. At various intervals an aliquot is removed and added to a second tube containing kaolin-activated normal platelet-poor plasma and a recalcification time immediately determined. Since the plasma factors are maximally activated in the second tube, a progressive shortening of the recalcification times reflects the amount of PF-3 released in the first tube. In practice, 1.0 ml. of platelet-rich plasma, adjusted to a platelet count of 300,000 per cu. mm. with the subject’s platelet-poor plasma, is incubated at 37’~. with 0.5 ml. of 8 per cent (w/v) kaolin (Fischer) in 0.05M imidazole buffered normal saline solution, pH 7.3 (tube 1). At intervals, 0.05 ml. of the incubation mixture is added, with 0.4 ml. of * GE SC-87 DRI-FILM,

15 Per cent in toluene.

0.025M calcium chloride, to a second tube containing activated normal platelet-poor plasma and the clotting time determined. The platelet-poor plasma is kept in an ice bath until needed and activated prior to testing by incubating 0.3 ml. with 0.15 ml. of 2 per cent kaolin at 37’~. for four minutes (the kaolin is not removed). The progressive shortening of the recalcification time in three normal subjects is shown in Figure 2. Studies were performed within four hours of venipuncture. It was found during the study that the most striking differences between normal subjects and patients with thrombopathia were obtained by comparing the clotting times after six minutes of incubation. Platelet aggregation in platelet-rich plasma was determined by the photometric method of Born [74] as modified by Mustard and co-workers [75]. Plateletrich plasma is stirred by magnet in the cuvette of an EEL titrator* and the transmittance, relative to a platelet-poor plasma blank, of light from a source in the titrator

is recorded

automatically

on a Bausch and

* Sales Associates, Box 361, Langehorne, AMERICAN

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Pennsylvania. MEDICINE

Thrombopathia

(Platelet

Factor

3 Deficiency)-Weiss

573

Lomb VOM-6 recorder. When ADP or connective tissue is added, the formation of increasingly large platelet aggregates is accompanied by an increase in the transmittance (Fig. 6). The initial (I) and maximal (M) transmittance are converted to optical density

(O.D.)

and

the value

0.D.y m

x 100

may

be taken as an estimate of the platele: aggregation. ADP* was frozen in aliquots containing 100 pg. per ml. and thawed immediately prior to use. Connective tissue suspensions were prepared by the method of Zucker and Borelli [ 761 from human subcutaneous tissue obtained from radical mastectomy specimens. Aliquots were frozen. Release of ADP, estimated as platelet aggregating activity, was determined on platelet-rich plasma, 100 to 120 minutes after venipuncture, as follows: a mixture of 1 ml. of platelet-rich plasma and 0.5 ml. of 8 per cent kaolin was shaken every two minutes for ten minutes at 37’~. The mixture was centrifuged rapidly for five minutes and the amount of aggregating activity released was determined by adding 0.5 ml. of the supernatant to 2.5 ml. of fresh normal platelet-rich plasma (substrate) and determining the per cent aggregation, as previously defined. The concentration of ADP which produces an equivalent amount of aggregation in the same substrate platelet-rich plasma was determined from a standard curve and the results expressed as mpM of ADP released per 109 platelets in the platelet-rich plasma. The standard curve was prepared by adding varying concentrations of ADP to the substrate platelet-rich plasma and plotting the per cent maximal aggregation versus the ADP concentration on log-log paper. The minimum concentration of ADP which could be determined for the substrate platelet-rich plasma used was determined from the curve by extrapolation. When no aggregation was obtained with a test sample, ADP was recorded as “less than” (<) the minimal concentration. Platelet adhesiveness, using platelet-rich plasma, was determined by the method of Hellem and coworkers [/7]. ADP was kept frozen, thawed immediately prior to use, and added to the test plasma in a final concentration of 0.05 pg. per ml. Using an infusion-withdrawal pump,t the mixture was then filtered for thirty seconds, at the rate of 1.4 ml. per minute, through 5 gm. of glass beads.1 Three aliquots of the platelet-rich plasma were tested in this manner, using three separate bead filters, and the platelet adhesiveness values were averaged. In twenty-four normal subjects the values ranged from 21 to 63 per cent. Platelet adhesiveness was also determined by filtering venous blood directly through glass beads. * Cal-Biochem, Berkeley, California. t Harvard Apparatus, Dover Massachusetts (Model 1100,8 RPM). $ Superbrite, Minnesota Mining and Manufacturing, Type 070-5005. VOL.

43,

OCTOBER

1967

I 0

j

I

2

3

4

I

I

5

6

INCUBATION

TIME

.I

,

7

8

3

13

II

I2

(MINUTES!

FIG. 2. Platelet factor 3 (PF-3) assay. Platelet-rich plasma is incubated with kaolin. Aliquots are removed at intervals and added, with calcium chloride, to a substrate of kaolin activated platelet-poor normal plasma. As PF-3 is released, clotting (recalcification) time becomes shorter. In Salzman’s original method [ 181the effluent from a filter containing 1.0 gm. of glass beads is collected into a Vacutainer tube. Using this method we obtained zero values in many apparently normal subjects. More satisfactory results were obtained by drawing the effluent into a 30 ml. syringe, affixed to the infusion-withdrawal pump, at the rate of 3.0 ml. per minute for thirty seconds. Using a scalp vein infusion set,* venous blood issuing from the adapter end was first sampled directly (first “no-glass” specimen), then passed successively through three separate filters containing 0.7 gm. of beads (“glass specimens”) and finally sampled directly a second time. The platelet counts in the two “no glass” and three “glass” specimens were averaged and the platelet adhesiveness was calculated. The range of values obtained on fifty-two normal subjects was 25 to 82 per cent. Other Studies. Bleeding time was performed by the standard Ivy method in a stab puncture 3 to 4 mm. deep produced with a No. 11 Bard-Parker blade. The sphygmomanometer cuff was maintained at 40 mm. Hg. Platelets were counted by phase microscopy. AHG (factor VIII), PTC (factor IX) and PTA (factor XI) were assayed by a one stage method, as described elsewhere [19], using plasma from patients congenitally deficient in these factors. RESULTS

Preliminary Studies (Table I). The Ivy bleeding time in S.N. (Case 6) was greater than thirty minutes on all occasions and in F.A. (Case 3) it was consistently prolonged. In three other thrombopathic subjects the bleeding time was variably prolonged ; in B.P. (Case 2) it was normal. AHG, clot retraction and platelet morphology were normal in all patients. One * Abbott, 19-G Thinwall Needle (18-G Bore).

Thrombopathia

574

(Platelet

Factor

3 Deficiency)-

Iv&s

v, s9

55.

2 2

50.

.4

45

-

F k u’

. 15 .I9

.......

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40

35 -

FIG. 3. Platelet factor 3 (PF-3) in patients with Glanzmann’s thrombasthenia (TS), thrombopathia (TP) and van Willebrand’s disease (VW). PF-3 was assayed as described under Methods and shown in Figure 2. Clotting (recalcification) times obtained after six minutes of incubation are shown, Case number accompanies each symbol.

.T

j.

c P

.**

1% . .910

***

30.

, NORMAL

TS

TP

80

Effect of adding ADP on platelet factor 3 (PF-3) release in three subjects with thrombopathia and three with thrombasthenia. Platelet-rich plasma is incubated with kaolin and either buffer or ADP. Clotting (recalcification) time, reflecting PF-3 release, is determined as described under Methods and shown in Figure 2. FIG. 4.

.:. ... .,.... ._..

I

40

-NO ---

..._.... ... .

....

ADP ADP. Zr/ml.

40

..

ADF 2g/ml.

-NO ---ADP,

t :f

THA;BOPAT;A

0

2

stage prothrombin time, platelet count, fibrinogen, partial thromboplastin time and specific assays for PTC and PTA were within normal limits in all subjects, with the exception of M.G. (Case 5). In this patient PTA* was 30 per cent and the serum was abnormal in the thromboplastin generation test. Platelet

Studies.

Platelet

factor

3 release

(Fig.

Values obtained in normal subjects on all days throughout this study demonstrate a wide range, at least in part due to variation in the substrate plasma from day to day. In von Willebrand’s disease PF-3 was normal, similar to previous findings obtained with the thromboplastin generation test [20,27]. In three * Kindly assayed by Dr. J. Niemetz. 3 and 4):

,

4

, NORMAL

6 INCUBATION

TIME

,

30

0 i MINUTES)

THROMBASTHENIA

2

4

6

with Glanzmann’s thrombasthenia patients the release of PF-3 was abnormal (Fig. 3), in accordance with the findings of Hardisty and co-workers [22], and addition of ADP to the incubation mixture had no effect (Fig. 4). In all but one patient with thrombopathia (Case 4) the release of PF-3 was similarly abnormal. By contrast, when ADP in a final concentration of 27 per ml. was added at the beginning of the incubation, there was a marked increase in PF-3 release (Fig. 4). The addition of normal platelet-poor plasma had no effect. ADP release: These results suggested that the basic defect in thrombopathia is a decreased ability to release ADP, whereas in thrombasthenia the defect is, at least in part, an inAMERICAN

JOURNAL

OF

MEDICINE

(Platelet

Thrombopathia

Factor

3 Deficiency)-

575

FVeiss AOP

coyyTcTvE

6OF 55-

.

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. 40 35 I 30 I i

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VW

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Amount of ADP released when platelet-rich plasma is incubated with kaolin. Results were obtained in patients with thrombasthenia (TS), thrombopathia (TP) and van Ft’illebrand’s disease (VW). < . 17 means that the amount of .4DP released in Case 17 was less than 5 rn~M per 109 platelets.

THRdMBO

I

x/m,.

!Gi--

r------i

FIG. 5.

to react to it. To test this hypothesis the amount of ADP released during the incubation of platelet-rich plasma with kaolin was determined. The results are shown in Figure 5. Less ADP was released in the thrombopathic subjects (including Case 4) than in sixty of sixtytwo normal subjects tested. Low values were also obtained in three patients with Glanzmann’s thrombasthenia and in two apparently normal subjects. The results were normal in all patients with von Willebrand’s disease. Aggregation oj platelets by connective tissue and ADP: When normal platelet-rich plasma is stirred in the presence of either ADP [l I] or such as connective tissue substances, [ 761, which release ADP, platelet aggregation occurs. In thrombopathic subjects, ADP in all concentrations produced the same amount of aggregation as in normal subjects. With a standard concentration of connective tissue, however, aggregation was consistently decreased in patients S.N. B.P. and G.P. (Cases 6, 2 and 1, respectively) and intermittently decreased in the F.A., M.Y. and M.G. (Cases 3, 4 and 5). The impaired aggregation by connective ability

VOL.

43,

OCTOBER

1967

-Le.---

THROMBASTHENIA

T’ME ( !iiGE) FIG. 6. Aggregation produced in platelet-rich plasma (PRP) by connective tissue and by ADP in a normal subject, patient S.N. (Case 6) with thrombopathia and patient M.C. (Case 18) with thrombasthenia. 0.05 ml. of connective tissue suspension or 0.13 ml. of ADP solution was added ( t ) to 2.5 ml. of platelet-rich plasma. The platelet-rich plasma was continuously stirred in the cuvette of a spectrophotometer and the formation of platelet aggregates was accompanied by an increase in light transmission. ADP concentration shown is the final concentration in the platelet-rich plasma.

tissue, similar to the impaired PF-3 release, was due to defective release of ADP. The most marked and persistent abnormality was observed in patient S.N. (Case 6) (Fig. 6), whose bleeding time was also the most prolonged. The defect was not absolute. When ten times the standard concentration of connective tissue was added, significant aggregation was observed. In von Willebrand’s disease, aggregation by both connective tissue and ADP was normal; in thrombasthenia (Fig. 6), no aggregation occurred with either substance. (1) Modified Adhesion of platelets to glass beads: Salzman technic (direct method, Fig. 7). The results in six of eight patients with von Willebrand’s disease were abnormal, as they were in

Thrombopathia

576

(Platelet

Factor

go-

1:: *. ?

. 13

- “i’ ...

z w 50-

‘i

6 5 40w 1 z30-

‘:’

a2

I2

.

IO

5 ::

:a

*n

‘“: NORMAL

17

:: .4

Al5 .I4 .7

TP

VW

A8

A9

Fro. 7. Platelet adhesiveness to glass beads in patients with thrombasthenia (TS), thrombopathia (TP) and von Willebrand’s disease (VW). In the Salzman method, venous blood is filtered immediately through the beads; in the Hellem method, ADP is added to platelet-rich plasma (PRP) and the mixture is then filtered. Case number accompanies each symbol.

A9

,

TS

2

A?

A8

96

IO-

0'

.

.

+ w d 20t d

(HELLEM)

90

: ..:..

% g 70a & 60

IV&

PRP

BLOOD (SALZMANI

90-

3 Deficiency)-

” NORMAL

both of the patients with Glanzmann’s thrombasthenia who were tested. The results are in accord with the findings of Salzman [78]. Platelet adhesiveness was decreased in four of five patients with thrombopathia. (2) Hellem method (Fig. 1). By this method, in which ADP is added to platelet-rich plasma prior to testing adhesiveness, the results in patients with Glanzmann’s thrombasthenia were abnormal. In von Willebrand’s disease, however, the results were normal, similar to the recent findings of Cronberg and co-workers [23]. RECALClFlCATlON TiME OFCONTACTEOPPP r

STYPVEN

TIME

r

TS

TP

VW

In all patients with thrombopathia, platelet adhesiveness was normal by this method.

EJect of ADP alone on PF-3 release in normal PRP: PRP was incubated with ADP, kaolin or buffer and a Stypven time (which is PF-3 dependent) determined on an aliquot (Fig. 8). ADP alone released PF-3, but was less effective than kaolin. In another experiment, an aliquot of the incubation mixture was added to a second tube containing activated platelet-poor plasma (kaolin removed) and the recalcification time was determined. Since the plasma factors were activated separately (tube 2), a shortening of the recalcification time reflects a release of PF-3 in the incubation tube 1. Again, ADP alone released PF-3, but even in a final concentration of 30-y per ml. was less effective than kaolin. The amount of intrinsic ADP released by the kaolin was only 5 to 10 gamma per ml. COMMENTS

,L 0

20 40 ,NCUBATlON

,L 0

TIME

IO

20

t MINUTES)

FIG. 8. Platelet factor 3 (PF-3) release by kaolin and by ADP. Platelet-rich plasma was incubated with either kaolin, ADP or buffer (IBS). At intervals, PF-3 release was estimated by (a) transferring 0.1 ml. aliquot to a tube platelet-poor containing 0.2 ml. of kaolin-activated plasma (kaolin removed) and determining the recalcification time (b) removing 0.1 ml. and determining the Stypven time (Russel Viper venom, 1:50,000).

In the patients with thrombopathia (PF-3 deficiency), platelet adhesiveness by the direct method (Salzman) and aggregation by connective tissue were also abnormal. The studies suggest that all the abnormalities may be attributed to a defect in the release of platelet ADP. In the presence of added ADP, platelet adhesiveness, aggregation and factor 3 release were normal, in contrast to the marked abnormality observed in patients with Glanzmann’s thrombasthenia. Assuming that ADP is required to form a platelet hemostatic plug in uz’uo, a defect in its release would provide a more reasonable explanation for the prolonged bleeding time AMERICAN

JoURN.&,.

OF

MEDICINE

Thrombopathia

(Platelet

observed in some of these patients than would the defect in platelet clotting activity (PF-3). In patients with von Willebrand’s disease, the release of platelet ADP and PF-3 was normal. The abnormal platelet adhesiveness obtained by the Salzman method in this disease remains unexplained, but may be related to a deficiency of the plasma “antibleeding” factor [20,24]. The findings in the patients with thrombopathia lend further support to the theory that activation of PF-3 requires the prior release of platelet ADP. Whether or not ADP is the direct cause of PF-3 release is not clear. Affirmative evidence has been presented by several investigators [ 75,25,26]. Hardisty and Hutton recently confirmed these observations, using a Stypven time method [27], although they had previously been unable to do so using the recalcification time [7]. In the present study release of PF-3 by ADP alone was demonstrated by both methods. However, since ADP, even in high concentration, is less effective than kaolin, it seems unlikely that the kaolin effect is entirely due to the ADP which is released. Whatever the mechanism in vitro, the physiologic role of ADP in PF-3 release is even less clear. Whether the predominant site of PF-3 activity resides in the membrane [28], granules [29] or a nonsedimentable fraction [30] is also the subject of some dispute. The thrombopathic patients described herein appear to comprise a heterogeneous group. The patients with multiple allergies are clinically similar to those reported by Kupfer and Clark 1371. Their patients, chiefly women, had a prolonged bleeding time, an allergic diathesis and hypogammaglobulinemia. The patients described herein differ somewhat in that their serum gamma globulin concentration was normal. The combination of a prolonged bleeding time and PTA deficiency, as found in M.G. (Case 5), has been described by others [32,33]. In the patient described by White and coworkers [33] the bleeding time was shortened by platelet transfusions, even though platelet function tests appeared to be normal. To our knowledge this is the first report in which platelet abnormalities have been demonstrated in vitro. The cases reported herein may also be similar to those with “athrombia” described by Inceman and co-workers [34,35]. In their patients, clot retraction was also normal but the platelets did not spread or agglomerate normally on a glass slide, possibly analogous to VOL.

43,

OCTOBER

1967

Factor

3 Deficiency)-l&‘&s

577

the decreased glass adhesiveness obtained by the Salzman method in the present study. Finally, patients who have an abnormal Salzman test without other evidence of von Willebrand’s disease, as described by Strauss and Bloom [36], may be similar to those described herein. I wish to thank Mr. John Acknowledgment: Rogers for his technical assistance, Doctors Peter Vogel, Mary C. Tyson, Julian Niemetz, Nathaniel Wisch, Ralph Nachman and Janet Cuttner for referral of patients, and the Walter Reed Army Medical Center for followup studies on Cases 1 and 2. REFERENCES

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