reduced platelet adhesiveness in patients with prosthetic ball valves: relation to adenosine...

Reduced platelet adhesiveness in patients with prosthetic ball valves: Relation to adenosine diphosphate and mechanical trauma

Jon Dale, M. D. Oslo, Norway

Patients with aortic ball valves have a disturbed platelet function, 1 and their platelet adhesiveness, as measured in native blood with Hellem's modified method, 2 is low. 1 Hellem demonstrated that platelet retention in glass bead columns was dependent upon red cells or a substance present in red cells, 3 later identified to be adenosine diphosphate (ADP)2 Recently, it has been suggested that red cells are important because they alter flow patterns within the columns, ~ and that the ADP necessary for retention is derived from blood platelets2 '7

When platelets are exposed to ADP without stirring in vitro, further addition of ADP will induce a lower degree of platelet aggregation than without preincubation, s'" the platelets have become refractory towards ADP. TM A reduced platelet adhesiveness was demonstrated in rabbits after intravenous infusion of ADP' 12 and during intravascular hemolysis initiated by water installation. '3 Disturbance of human blood in vitro or addition of ADP prior to testing reduced platelet retention in glass bead columns consider- ably2 In patients with aortic ball valve prostheses some degree of continuous intravascular hemolysis is found," which might influence platelet behavior through a similar mechanism.

This investigation was done in order to study whether the reduced adhesiveness in blood from ball-valve patients could be due to ADP liberated intravascularlY from red cells. The total content of adenine nucleotides in blood from such patients was compared with that of normals. The liberation of nucleotides during passage of blood through the glass bead columns was measured,

From the Institute for Thrombosis Research, University Hospital, Rikshospitalet, Oslo, Norway.

Received for publication June 4, 1976.

Accepted for publication July 8, 1976.

Reprint requests: Jon Dale, M.D., Institute for Thrombosis Research, University Hospital, Rikshospitalet, Oslo 1, Norway.

and a comparison made to the degree of hemolysis provoked. Further, platelet adhesiveness was determined in the same individuals and related to the ADP liberated. An a t tempt was made to detect circulating nucleotides in plasma from venous and arterial blood. Finally, the theoretical trace concentrations of ADP tha t could occur near the prosthetic valves were calculated, and the possible influence on platelet behavior was evaluated.

Materials and methods

The study was done in eleven patients with single Starr-Edwards aortic ball valves and eight healthy individuals. Blood was carefully collected in plastic tubes containing ethylene-diamine- tetra-acetate (EDTA) in order to achieve anti- coagulation and avoid degradation of adenine nucleotides. Nine parts of blood were mixed with one part of 0.077 M EDTA, and is referred to as EDTA-blood.

Whole blood contents. To determine the total blood content of hemoglobin, lactate dehydrogenase (LDH), adenosine triphosphate (ATP), and ADP, 20/~1 of EDTA-blood was diluted to two ml. in saline (pH 7.4), and frozen and thawed three times. The concentrations measured were cor- rected for dilution with saline and EDTA-solution.

Effects of passage through glass bead c o l u m n s . EDTA-blood was forced through glass bead columns at a speed of one ml. per 9.2 seconds as described by Hellem, 2 in order to study the liberation of the various substances by this slight mechanical trauma. EDTA-blood t h a t had been passed through a plastic tube at the same speed served as control. Platelet-poor plasma (PPP) was then prepared as follows: The samples of EDTA-blood were centrifuged at 1000 • g for 15 minutes, the plasma carefully pipetted off and centrifuged in new tubes at 12000 • g ~for 15

562 November, 1977, Vol, 94, No. 5, pp. 562-567

minutes, PPP for nucleotide assays was mixed with equal parts of ice-cool ethanol and frozen at -60~ until tested. 15

The concentrations of hemoglobin, LDH, and nucleotides were measured, and the differences between test and control plasmas represented the amounts derived from blood cells by passage of EDTA-blood through the columns. The proportions of the total content that was liberated was calculated for each substance. Since they were confined to the volume of plasma and EDTA- solution after the liberation and not to the volume of blood, a correction had to be made before comparison with the total blood content. This was achieved by multiplying the increase in plasma concentrations with the factor: 10/9 - Ht./100, where Ht. represents the hemato-

crit . The EDTA added to blood before passage

through the column could possible influence the degree of hemolysis produced. To study this, blood without addition of anticoagulants was forced through the column and mixed with EDTA-solution immediately afterwards. Native blood that had been passed through a tube without beads served as control. PPP was prepared and the liberation of hemoglobin was determined. A difference between the degree of hemolysis induced in native blood and in EDTA- blood would reflect the effect of EDTA on the fragility of the red cell membranes.

Assays. Hemoglobin concentrations were determined by the method of Crosby and Furth. TM

This method was fairly accurate for determination in plasma, the coefficient of variation between duplicate samples being 5.0 The plasma heine levels were expressed in terms of oxyhemo- globin standards, determined with the cyanmet- hemoglobin method." LDH measurements were done according to Wroblewski and LaDue at 25~ TM using commercial reagents.*

Microdetermination of ATP and ADP was done by the firefly luciferase method, TM using a DuPont 760 Luminescence Biometer. To make the method sufficiently sensitive, 100 ttl of ethanol-EDTA extracts of PRP and standards were used instead of 20 /~l. With this modification a linear relationship between light emission and nucleotide concentrations in the actual range was obtained, and it allowed determination of ADP levels lower than 0.05 ttM. The ADP was

*Kabi AB, Stockholm, Sweden.

Reduced platelet adhesion with prosthetic ball valves

measured after conversion to ATP by a pyruvate kinase system, 15 and the combined amounts of ATP and ADP were therefore determined in one plasma sample. In another, the conversion was not induced, and ATP alone was measured. ADP was then calculated as the difference between the two values. Standard curves were made from ADP.*

Platelet adhesiveness. Native blood from the same subjects was collected in 5 ml. plastic syringes and immediately used for platelet adhesiveness measurements by Hellem's modified method. 2 Thus, native blood was forced through the glass bead columns at the same speed that had been applied to the EDTA-blood, Platelets were counted in a hemocytometer according to Nygaard's method. 3

ADP in arterial plasma: measurements and calculations. To study whether trace amounts of ADP could be detected in arterial plasma, blood was collected from the ascending aorta during catheterization in one and from puncture of the radial artery in three valve patients. Arterial blood from four individuals who were catheter- ized because of coronary heart disease was used as control. The blood was collected in EDTA- solution, PPP prepared and analyzed for hemoglobin, LDH, and adenine nucleotides.

In order to evaluate the possible influence on platelet retention of ADP liberated intravascularly, an estimate of the maximal concentrations of ADP that could theoretically be expected to occur as a hemolysis-related phenomenon near the valve was made from the following data: From the mean plasma-LDH values and the blood volume the abnormal red cell breakdown could be calculated, s~ Assuming tha t the cells were all destroyed by the prosthetic valve, the intravascular breakdown per heart beat could be estimated. Knowing the total ADP content, and assuming further that the ADP was immediately diluted by the volume of a stroke output, the maximal theoretical ADP levels near the valve could be calculated. Since the elimination of ADP is very rapid, 21 accumulation would not occur.

Results

Whole blood contents. The total contents o f hemoglobin, LDH, ATP and ADP were quite similar in blood from patients with prosthetic aortic valves and healthy subjects (Table I), none

*Sigma Chemical Company, St. Louis, Mo.

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Dale

Table I. The whole blood content of hemoglobin (Hb), lactate dehydrogenase (LDH), adenosine triphosphate (ATP), and adenosine diphosphate (ADP). Mean values and standard error of the mean in 8 healthy subjects and 11 patients with aortic ball valve prostheses

Healthy individuals

Total [ content of Mean S.E.M.

Hb. (G./100 ml.) 12.64 0.51 LDH (U./L.) 13,851 991 ATP + ADP (/LM) 605.1 27.1 ATP (p.M) 453.1 30.2 ADP (/~M) 15210 10.4

Ball valve patients

Mean S.E.M.

12.74 0.42 14,694 798 627.8 17.6 474.6 15.7 153.2 14.0

of the differences were statistically significant. The mean ATP:ADP ratio was 3.6 and 3.2 in the two groups.

Effects of passage through glass bead columns. A slight increase in the plasma concentrations of hemoglobin, LDH, and adenine nucleotides was found after passage of EDTA- blood through the columns (Table II), and it was only possible to determine the ADP levels by increasing the sensitivity of the method as described. The effect of the t rauma was almost identical on blood from the two groups of subjects. ATP and ADP appeared in proportions that did not differ significantly from those of whole blood.

The amounts of hemoglobin, LDH, and nucleotides liberated were calculated in per cent of the

t o t a l content (Table III). None of the differences between the two groups were statistically significant. The liberation of hemoglobin illustrated that passage through the columns represented only a minimal trauma to the blood cells, since it corresponded to rupture of only one out of 2,000 erythrocytes. Further, a rather similar degree of hemolysis was produced in EDTA-blood from the different individuals of both groups, demonstrat- ing that the effect of the t rauma was quite constant. Slightly more LDH than hemoglobin was liberated in the healthy individuals, p < 0.05.

The ATP and ADP appeared in proportions that did not differ from those of hemoglobin within each of the groups.

The addition of EDTA to blood before expsoure to the glass beads did not affect the fragility of the red cell membranes, since the

Table If. Increase in plasma concentrations of hemoglobin, lactate dehydrogenase, adenosine triphosphate, and adenosine diphosphate by passage of EDTA-blood through glass bead columns. A comparison between 8 healthy subjects and 11 patients with aortic ball valves

Healthy individuals

Liberation I Mean ] S.E.M. of

i

Hb. (mg./100 ml.) 10.1 0.7 LDH (U./L.) 18.0 2.5 ATP + ADP, (/LM) 0.54 0.08 ATP (/zM) 0.44 0.09 ADP (#M) 0.10 0.02

Ball valve patients

Mean I S.E.M.

9.4 0.6 14.3 2.9 0.55 0.05 0.45 0.05 0.10 0.04

liberation of hemoglobin from red cells of native blood by passage through the columns was similar to that of EDTA-blood {Table IV}, and the inter-individual variation was equally small.

Platelet adhesiveness. The retention of platelets when native blood was forced through the columns was markedly lower than normal in the ball valve patients {Table IV), the reduction being highly significant (p < 0.001).

Intravascular hemolysis. The elevation of the Plasma-LDH levels in the patients reflected a considerable degree of hemolysis.

Adenine nucleotides in arterial plasma. Low concentrations of hemoglobin and adenine nucleotides were measured in PPP from arterial and venous blood. Thus, the mean hemoglobin concentrations in arterial plasma from normals and valve patients were 5.2 and 6.8 mg./100 ml., while the corresponding adenine nucleotide levels were 0.26 and 0.29 ~M, the mean ATP:ADP ratio being 1.3 in each group. In venous plasma, similar levels were found. It is, however, unlikely that free nucleotides should be circulating in plasma, as discussed below.

The results of the calculations on hemolysis- related liberation of ADP were: A mean plasma LDH level of 381 U./L. corresponds to a red cell half-life of 20 days, as compared to 27.5 days in normals. ~~ Considering the blood volume to be 5.3 LY, the daily red cell breakdown would be equiv- alent to 96 ml. of blood in the healthy subjects, and to 132 ml. in the ball valve patients. Assuming that the cells of the extra 36 ml. were destroyed by the prosthetic valve, and that the heart rate was 70, cells of 0.36 #1 of blood were ruptured by each heart stroke. Since the whole

5 6 4 November, 1977, Vol. 94, No. 5

blood contained on average 138 ~M of ADP, and since the ADP liberated was supposed to be diluted by the 40 ml. o~ plasma of a stroke output, ~- the maximal ADP concentrations derived from red cells would be in the range of 0.0012 #M. Accumulation would not occur, because ADP would rapidly be diluted and eliminated? -I These calculations do not include ADP that might possibly appear from blood platelets by the release reaction.

Discussion

The whole blood content of adenine nucleotides, hemoglobin, and LDH was not lower in patients with prosthetic ball valves than in healthy individuals, in spite of increased red cell turnover, as reflected by the elevated plasma LDH levels. 2~ The total nucleotide content compares well with the results from other studies, ~-~" ~ while a higher ATP:ADP ratio has been reported..~3.2~ The bulk of adenine nucleotides in whole blood is located in the red cells, and the platelets contribute only approximately 12 to 20 #M of ATP and 6 to 10 ~M of ADP. ~

The total concentrations of LDH in blood from ball valve patients compared well with the results from a previous study, whereas somewhat higher normal levels then were found? 5 The determination of the LDH content was, however, more accurate in the present investigation, since the measurements were done in less diluted blood.

The passage of EDTA-blood through the glass bead columns exposed the red cells to a quite constant trauma, since the variation in the degree of hemolysis induced was small. Further, the same degree of hemolysis was produced in both groups of subjects, indicating that the stress inflicted by the ball valves had not weakened the red cell membranes. Normal fragility of erythrocytes from ball valve patients has previously been revealed, ~6 as has a slightly higher liberation of LDH than of hemoglobin by mechanical trauma. 2~

The increase in plasma concentrations of ATP and ADP after passage of EDTA-blood through the column was most probably related to the hemolysis produced, since the nucleotides and hemoglobin appeared in comparable proportions. This is in accord with the results from a previous study from this institute, in which a different method for hemoglobin determination was used. 23 Zucker's group 6 found a similar liberation of ADP from heparinized blood, but failed to reveal a


Table III. Liberation of hemoglobin, lactate dehydrogenase, adenosine triphosphate, and adenosine diphosphate by passage of EDTA-blood through glass bead columns in per cent of the whole blood content

Percent liberation of

Hb. LDH ATP + ADP ATP ADP

Healthy individuals

Mean [ S.E.M.

0.052 0.004 0.085 0.011 0.058 0.010 0.064 0.011 0.042 0.011

Ball valve patients

Mean S.E.M.

0.051 0.005 0.067 0.018 0.061 0.0O5 0.066 0.007 0.045 0.018

Table iV. Retention of platelets by passage of native blood through glass bead columns, total platelet counts, hematocri t values, and plasma LDH levels, with level of significance on the differences between the values in 8 heal thy subjects and 11 ball-valve patients (N.S. -- not significant). Liberation of hemoglobin in columns from native blood of 8 heal thy subjects

Healthy Ball valve individuals patients

Mean l S.E.M. Mean l S.E.M.

Level of signi/~- vance

Platelet retention 76.1 2.3 34.2 5.0 p < 0.001 (per cent)

Platelets per ~l 255.300 9.000 247.500 12.500 N.S. Hematocrit 45.6 1.5 42.6 1.2 N.S.

(per cent) Plasma LDH 104.0 10.6 381.4 91.4 p < 0.01

(U./L.) Liberation of Hb. 9.3 1.0

(mg./100 ml.)

corresponding increase in hemoglobin concentrations. Less ADP was found to appear after passage of blood from subjects with low platelet retention, 7 and it was suggested tha t the ADP was derived by release from platelets during the passage through the column. 6' 7 They stated, however, tha t the classic release reaction may not have been responsible, since the liberation occurred at room temperature and was not prevented by acetylsalicylic acid or accompanied by measurable release of serotonin. 7 Furthermore, no indication of release has been revealed by electronmicroscopic studies of retained platelets. '-'T If release had occurred to any extent, much higher ADP concentrations would appear, as

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Dale

found after addition of thrombin to platelet-rich plasma. ~3

In the present study, the added EDTA would markedly inhibit platelet retention and release of ADP from platelets. Moreover, the ATP:ADP ratio was as high as in whole blood, which confirms tha t the nucleotides were derived mainly from red cells. The hemolysis induced cannot be attributed to the addition of EDTA, since plasma hemoglobin increased to the same extent when native blood was passed through the columns.

Zucker's group ~8 have demonstrated that the first step in platelet retention is adhesion of platelets to the glass beads, and that this reaction is independent of ADP. Subsequent retention is caused by platelet-platelet interactions requiring ADP, supposed to be derived from the reacting platelets. 28 This study demonstrates, however, that comparable amounts of ADP are liberated from red cells. Lower ADP levels are able to induce platelet retention in platelet-rich plasma, 2' and although red cells facilitate retention by enhancing physical interactions, ~ the require- ment for ADP has been well documented2" 7 The results therefore support the hypothesis that ADP necessary for normal platelet retention to occur in glass bead columns is derived from red cells.

The platelet retention was considerably reduced in the valve patients in spite of normal hemolysis-related ADP liberation. Why did not the ADP made available cause normal platelet retention? A tempting explanation is tha t the ptatelets were refractory because they were influenced by ADP appearing during continuous intravascular hemolysis, as demonstrated in rabbits by water infusions. 1~ The minimum concentrations of ADP that are required to induce refractoriness have not been established, but the water infusions resulted in reduced retention only when a considerable acute hemolysis had developed. 13 To induce refractoriness in vitro, ADP concentrations of 0.1 ~M have been used. ~

The ADP measured in plasma from venous and arterial blood was most probably derived from blood cells during the sampling and the prepara- tion of plasma, and also from the few platelets that remained in spite of the two-step centrifuga- tion. Thus, proportionally more ADP than hemoglobin was found, although ADP is rapidly eliminated from the circulation, 21 and hemoglobin more slowly. 3~

The calculations on the intravascular liberation of ADP from red cells have established tha t only very low concentrations could theoretically occur near the prosthetic valves. Moreover, the rapid breakdown of ADP would prevent accumulation and make the time of contact with a limited number of platelets short: I t is therefore quite inconceivable tha t the ADP liberated by intravascular hemolysis could influence platelet behavior.

Two other possible mechanisms for the disturbed platelet function in patients with prosthetic valves have also been discussed previously. 1 Consumption of adhesive platelets by thrombus formation h a s been rejected, while damage inflicted on the platelets by the prosthetic valve could explain all the phenomena observed. The valve could reduce platelet adhesiveness by af- fecting their membranes, either by the impact of the ball or by the turbulence induced in the blood, or it might disturb platelet function by other mechanisms. Thus, it has recently been demonstrated that platelets tha t have undergone release and aggregated after exposure to thrombin may disaggregate and survive in the circulation, and even have some hemostatic effect21 It is therefore possible that the t rauma might induce release to some extent, and leave platelets 'with a reduced functional ability in the blood. Release could explain the raised plasma levels of platelet factor 4 found in some patients with prosthetic valves22 Some ADP would then also be released, but in far too small amounts to induce refractoriness in other platelets.

In conclusion, the reduced platelet adhesiveness in ball-valve patients is most probably due to the effect of the prosthesis on the platelets them- selves, and is unlikely to be secondary to intravascular hemolysis.

Summary The whole blood content of ADP and ATP was

normal i n p a t i e n t s with prosthetic aortic ball valves, in spite of increased red cell breakdown. During passage of EDTA-anticoagulated blood through glass bead columns a slight, but quite constant degree of hemolysis was produced, similar in. blood from healthy individuals and ball-valve patients. The adenine nucleotides were liberated largely in the same proportions as hemoglobin, and ADP appeared in mean concentrations of 0.10 ~M in each group of subjects. The results indicate that the ADP necessary for

566 November, 1977, Vol. 94, No. 5


n o r m a l p l a t e l e t r e t e n t i o n is m a i n l y d e r i v e d f r o m red cel ls a n d n o t f rom p l a t e l e t s .

T h e r e t e n t i o n o f p l a t e l e t s b y p a s s a g e o f n a t i v e b l o o d t h r o u g h t h e c o l u m n s w a s s i g n i f i c a n t l y r e d u c e d in t h e p a t i e n t s in sp i t e o f t h e n o r m a l l i b e r a t i o n of A D P . T h i s low p l a t e l e t a d h e s i v e n e s s cou ld p o s s i b l y be d u e to r e f r a c t o r i n e s s t o w a r d s A D P l i b e r a t e d f r o m red cel ls d u r i n g i n t r a v a s c u l a r hemo lys i s . T h e A D P c o n c e n t r a t i o n s t h a t c o u l d be de r i ved f r o m r e d cel ls were , h o w e v e r , c a l c u - l a t e d to be fa r l o w e r t h a n t h e l eve l s k n o w n t o a f fec t p l a t e l e t b e h a v i o r , a n d A D P is r a p i d l y e l i m i n a t e d f r o m p l a s m a . I t is c o n c l u d e d t h a t t h e r e d u c e d p l a t e l e t r e t e n t i o n in b l o o d f r o m b a l l - va lve p a t i e n t s was m o s t p r o b a b l y a r e s u l t o f t r a u m a to t h e p l a t e l e t s i n f l i c t ed b y t h e va lve , a n d was n o t s e c o n d a r y to i n t r a v a s c u l a r h e m o l y s i s .

The skilled technical assistance of Mrs. A.-L. Almaas is greatly appreciated.

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reduced platelet adhesiveness in patients with prosthetic ball valves: relation to adenosine...

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