- Email: [email protected]
Arachidonic acid metabolism in the platelets of hemophiliacs with prolonged bleeding times
THROMBOSIS RESEARCH 31; 539-548, 1983 0049-3848/83 $3.00 + .OO Printed in the USA. Copyright (c) 1983 Pergamon Press Ltd. All rights reserved.
ARACHIDONIC ACID METABOLISM IN THE PLATELETS OF HEMOPHILIACS WITH PROLONGED BLEEDING TIMES
John R. Luderer, Leslie Locke, Marlene Zekoski, Laurence Demers and M. Elaine Eyster Departments of Medicine and Pathology The Milton S. Hershey Medical Center The Pennsylvania State University Hershey, Pennsylvania 17033 Received
1.11.82;
Accepted in revised Editor J.B. Smith
form 18.5.83
by
ABSTRACT The bleeding time has been said to be normal in hemophilia. However, we and others have recently reported a prolonged bleeding time unrelated to the ingestion of non-steroidal anti-inflammatory agents or to recent transfusions in 16-20% of patients with hemophilia. To determine whether this abnormality might be due to impaired biosynthesis of thromboxane (TXA2) from the cyclooxygenase pathway, or 12-hydroxy eicosotetraenoic acid (12-HETE) from the alternative lipoxygenase pathway, we studied the in vitro metabolism of arachidonic acid (AA) in platelets from four h=pmcs with repeatedly prolonged bleeding All studies were performed on an aliquot of platelet-rich times. plasma obtained from a single venepuncture, the same morning the bleeding time was measured. High pressure liquid chromtop&ams of the reaction products generated following the incubation of [ Cl-AA with washed platelets from all four subjects were identical to those obtained from two hemophiliacs with normal bleeding times and from normal controls. In addition, metabolites of the cyclooxygenase and lipoxygenase pathways were normal in these patients when assessed by prelabeling their platelets with [ I4 Cl-AA and theh stimulating maximally with thrombin. We conclude that the prolonged bleeding times observed in these hemophiliacs cannot be attributed to the presence of a cyclooxygenase inhibitor such as aspirin.
Key words:
Hemophilia, platelets, prostaglandins, arachidonic acid, bleeding time, thromboxane. 539
PLATELET FUNCTION IN HEMOPHILIA
540
Vo1.31, No.4
INTRODUCTION The bleeding time is prolonged by drugs such as aspirin which inhibit the cyclooxygenase pathway of arachidonic acid metabolism, thus blocking TXA2formation which mediates the release reaction and platelet aggregation (l-4). In addition, congenital deficiencies of cyclooxygenase and TXA2 synthetase, causing abnormal platelet function and prolonged bleeding times (5-8), and abnormal platelet responses to TXA2 presumably due to defective TXA2 receptors have been reported (9,lO). Platelets can also metabolize arachidonic acid by the alternative lipoxygenase pathway to 12-hydroxy eicosotetraenoic acid (12HETE). Although lipoxygenase deficiency has recently been reported in some patients with myeloproliferative disorders (ll), it is not yet known whether this pathway plays a physiologic role in the regulation of hemostasis. The bleeding time has been said to be normal in hemophilia (12-15). However, we and others have reported a prolonged bleeding time in 16-20% of patients with classic hemophilia (16,17). A criticism of this work has been a failure to provide documentation that these patients had not ingested aspirin or an aspirin-like product which could account for the impairment in platelet function. Documentation by means of a retrospective patient interview is probably not adequate given the ubiquitous nature of various salicylates in over-the-counter drugs and food additives. Therefore, in order to investigate the mechanism of this abnormality further, and to demonstrate conclusively that these findings are not the result of the inadvertent ingestion of a cyclooxygenase inibitor, we studied the in vitro metabolism of arachidonic acid in the platelets of four hemophiliacswimpeatedly prolonged bleeding times. METHODS AND MATERIALS Venous blood was drawn with informed consent from four previously reported males (DD, BM, MS and VG) with classic hemophilia (16). DO, BM and MS had TABLE 1 Platelet Function Studies Patient Age (yrs) D.D. 13
B.M. 8 M.S. 28 V.G. G8C. 58 M.K. 21
Bleeding Time* (2.5 - 8 min)
()18%ii,l6)
0;:;) (>17$,11) 16.0
Platelet Count (1403340 x 10 /pl)
Platelet Platelet Aggregation, % Retention change in light trans(85-98%) mission, Patient/Control Patient/Control ADP Collagen
218
92/85
80/40
60150
307
80/84
35/50
50/50
170
88185
50140
60160
272
82/68
40/65
60160
153
88184
50150
40150
180
89/85
30/50
40/60
*Previous determinations recorded in parenthesis +Receiving Indomethacin
Vo1.31, No.4
PLATELET FUNCTION IN HEMOPHILIA
541
levels of 1% or less. VG had an VIII:C level of 13% VIII coagulant (VIII:C) with a normal VIII related antigen, a normal ristocetin cofactor, a normal crossed immunoelectrophoresis, and a family history of sex-linked hemophilia. All had at least one bleeding time greater than eleven minutes on previous determinations, and all had a prolonged bleeding time with normal or nearnormal platelet aggregation studies and platelet adhesiveness at the time of the study (Table 1). Two males with severe hemophilia, (GC and MK) with previously normal bleeding times were studied for comparison. Ten normal males, ages 23-33 with normal bleeding times and normal platelet aggregations served as controls. None of the subjects had ingested aspirin or non-steroidal antiinflammatory agents for at least two weeks prior to testing. All in vitro studies of platelet function and arachidonic acid metabolism were perfGm= an aliquot of platelet-rich plasma obtained from a single venepuncture. Platelet Function Studies Blood was collected by the two-syringe technique into plastic tubes containing l/9 volume balanced citrate (2 volumes O.lM citric acid, 2 volumes 0.1 M sodium citrate). Platelet aggregation studies were performed with a ChronoLog aggregometer on platelet-rich plasma that had been centrifuged at 150 g (800 rpm) at 25' for 6 minutes, with the platelet count adjusted to approximately 200,000 cu/mn. ACP (3 PM final concentration) epinephrine 0.48 mg/ml and collagen suspension (200 pg/ml to 300 pg/ml final concentration) were used as aggregating agents. Results with epinephrine were not reported because the control failed to aggregate and insufficient material was available to repeat the test. Platelet retention on glass beads was determined by Friedberg and Zucker's modification of the method of Bowie, using 2.6 g of glass beads (18). Template bleeding times were performed using the Simplate II device (General Diagnostics, Morris Plains, N.J.) as previously described (16). Preparation of Washed Platelets Studies of arachidonic acid metabolism were performed with washed human platelets prepared according to the method of Needleman (19). Fresh venous blood was collected directly into 7.5% (vol/vol) 77 mM EDTA and centrifuged at 150 x g for 10 minutes. The platelet rich plasma was then centrifuged in an EDTA-rinsed plastic tube at 1,800 g for 6 minutes and the platelet pellet resuspended in 0.15 M NaCl : 0.15 M Tris HCl (pH - 7.4) : 77 mM EDTA (90:8:2, vol/vol/vol) and subsequently recentrifuged at 600 g for 5 minutes. A calcium free Krebs-Henseleit medium was then u3ed to resuspend the washed platelet pellet in a volume adjusted to yield 10 platelets/ml. Platelet counts were done with an A0 Bright Line hemacytometer. Platelet Incubations All incubations were carried out in a Chrono-Log platelet aggrqometer at 37'C, stirred at 1,100 rpm. 0.4 ml aliquots of washed platelets (10 /ml) were added to ag regometer tubes. After a five minute pre-incubation period, 25 ~1 of sodium [94C] arachidonate (1 ug: 300,000 cpm) was added and the incubation continued for 15 minutes. Sodium arachidonate was prepared fresh each day in Tris buffer (40 pg/ml, pH = 9.0). Extraction and Chromatography The reaction mixtures were acidified to pti3.0 with 2 N formic acid and a trace amount of [3~]~~~1 ( =50,000 cpm) added for recovery analysis. The mixture was then extracted with three volumes of cold ethyl acetate. The combined organic extract was dried under a stream of nitrogen. Separation of the
542
PLATELET FUNCTION IN HEMOPHILIA
Vol.31,
No.4
reaction products was carried out using a slight modification of the high pressure liquid chromatographic method described by Whorton et al (20). HPLC was performed with a Water Associates (Milford, Mass.) chromatograph (model 6000A) on a reversed-phase P Bondapak Fatty Acid Analysis column. The packing material for the Fatty Acid Analysis column is proprietary information (Waters Associates). Samples were dissolved in 0.2 ml of the mobile phase, and 100 Pl was injected on column and initially eluted isocratically using a solvent system composed of water - acetonitrile - benzene - acetic acid (76.7:23:0.2: 0.1). After 20 minutes the percentage of acetonitrile in the mobile phase was gradually increased over a linear gradient employing a Water Associates solvent programmer (Model 660) as indicated in figure 1. The flow rate was 2 ml/ minute and one minute fractions were collected using a Gilson (B-200) automatic fraction collector. Liquid scintillation counting was performed using a Beckman (LS 7500) dual isotope counter. The identity of peaks on the chromatograms was determined by comparison with known radiolabelled standards and by mass spectroscopy as previously described (21). Platelet Phospholipid Labeling Technique The platelet phospholipid pool was labeled with [14C] arachidonic acid according to the method of Needleman (19). A platelet pellet was prepared as described above and resuspended in an albumin-phosphate buffer (33 mM, pH 6,5) containing glucose (1 mg/ml), NaCl14(6.6 mg/ml), and fatty acid poor bovine serum albumin (1 mg/ml). Sodium [ Cl arachidonate (2 x 10 cpm) was added to the platelet suspension which was incubated for 30 minutes (37°C). The labeled platelets were centrifuged (2,000 rpm, 5 minutes) and resuspended in calcium free Krebs Henseleit buffer (pH 7.4). Thin layer chromatographic analysis of the lipids extracted from these prelabeled platelets have indicated that the incorporated radioactivity is primarily in the phosphatidyl choline and phosphatidyl ethanolamine fractions, with minimal amounts of detectable free arachidonate. These prelabeled platelets were then stimulated with thrombin (\oU/ml) and after a 15 minute incubation period the samples were extracted and chromatographed as described above. This dose of thrombin had been determined in our laboratory to provide maximal stimulation of thromboxane production in normal platelets and is in agreement with that reported by others (19). C] arachidonic acid, C3HlPGEI, C3HlPGE2, C3H]PGF20, and C3H]TXB2 -14 were purchased from New England Nuclear, Boston, Mass. Unlabeled arachidonic acid, and adenosine 5'-diphosphate (sodium salts) were purchased from the Sigma Chemical Company, St. Louis, Missouri. HPLC solvents (HPLC-grade) were purchased from Fisher Scientific Company (Fair Lawn, New Jersey). Epinephrine was Purchased from Elkins-Sinn, Inc. (Cherry Hill, New Jersey). RESULTS Figure 1 shows a high pressure liquid c&omatogram of the reaction products generated following the incubation of C Cl arachidonic acid with washed platelets from one of the hemophiliacs (DD) who manifested a prolonged bleeding time. It demonstrates a normal pattern of arachidonic acid metabolism with significant synthesis of the cyclooxygenase derived products thromboxane B 2 (the inactive metabolite of TXA2) and It-hydroxyheptadecatrienoic acid (HHT), with lesser conversion of arachidonic acid to PGF2, and PGE2. This chromatogram also demonstrates that the lipoxygenase pathway of arachidonic acid metabolism is intact in this individual as evidenced by the significant
Vo1.31, No.4
PLATELET FUNCTION IN HEMOPHILIA
543
12-HETE
AA TXB2
10
20
30
40
50
FRACTIONNUMBER
FIG. 1 High pressure liquid chromatofram of the reaction products generated following the incubation of exogenous [ 4C] AA with washed platelets from a hemophiliac (DD) with a prolonged bleeding time.
production of 12-HETE. The pattern of metabolites shown in figure 1 is identical to that obtained from incubations of normal control platelets and the platelets of the other hemophiliacs (not shown) with normal and abnormal bleeding times. Figure 2 compares the pattern of arachidonic acid metabolism between the control group and the hemophiliacs with normal and prolonged bleeding times when washed platelets were incubated with exogenous arachidonic acid. The figure shows the percentage of total counts recovered as TXB 2, PGFza, PGE2, HHT (12-hydroxy heptadecatrienoic acid), and 12-HETE. The results are corrected for differences by spiking the sample with tritiated PGEl prior to extraction and chromatography. The PGEl elutes at a different point on the chromatogram (17) than the P-series pro taglandins and the tritium can be counted on a separate channel from the [ i4C] labeled products generated from arachidonic acid. Recoveries were 70 + 11% (Mean * SEM) and ranged from 62 to 84%. As shown in figure 2, there is no significant difference in platelet arachidonic acid metabolism between the groups tested when assessed by the ability of the washed platelets to metabolize exogenous arachidonate. The data for normals are presented as the mean + 2 standard deviation in order to provide an indication of the range of values obtained, as a percentage of total counts recovered.
Vo1.31, No.4
PLATELET FUNCTION IN HEMOPHILIA
544
5c fj NORMALS MEAN+2
S.D.
0 HEMOPHILIACS-NORMAL BLEEDING TIME
4c
??HEMOPHILIACS-PROLONGED
BLEEDING TIME
??
3a
C’ 0
m
0
20
0
m
??
10
TXB,
PGE;!
PG~Y
I
HHT
12-HETE
FIG. 2
AA metabolites generated from exogenous [ 14CJ AA utilizing washed platelets from normal male controls (B), hemophiliacs with normal bleeding times (0) and hemophiliacs with prolonged bleeding times (I). The normal controls are presented as the Mean + 2 S.D. (n = 10) and the hemophiliacs as individual data points. All results are depicted as the percentage of total counts recovered as the indicated metabolite. ??
B-NORMALS
MEAN+2 S.D.
0 HEMOPHILIACS-NORMAL BLEEDING TIME
.d II ??
??HEMOPHILIACS-PROLONGED
BLEEDING TIME
0
??
0
0
8
8
??
L
TXB;!
PGE;!
f’f3F2cx
FIG 3.
HHT
12-HETE
A44metabolites generated from thrombin stimulated platelets prelabeled with [ Cl AA. See legend for figure 2.
Vo1.31, No.4
PLATELET FUNCTION IN HEMOPHILIA
545
To determine if these patients could effectively mobilize arachidonic acid from phospholipid storage sites and subsequently metabolize it, washed platelets were prelabeled with arachidonic acid and stimulated with thrombin as described under Methods. As depicted in figure 3, there was no difference in the pattern of arachidonic acid metabolism through the cyclooxygenase pathway between hemophiliacs with prolonged bleeding times, hemophiliacs with normal bleeding times and normal controls. Twy of the four hemophiliacs with prolonged bleeding times (VG, BM) had less [ 4C] labeled 12-HETE recovered than did the normal controls, following thrombin stimulation. These were not the same patients as the bottom two in the HHT and TXB2 data. Moreover, as previously noted, the production of 12-HETE was normal in response to exogenous arachidonic acid.
mcussIor4
Arachidonic acid is released from membrane phospholipids by the enzyme phospholipase Al in response to a variety of stimuli and is metabolized in platelets by two metabolic pathways. The first pathway involves a cyclooxygenase enzyme and results in the formation of labile endoperoxide intermediates (PGG2, PGH2) which are either converted to the classical prostaglandins (PGE2, PGD2, PGF2a) and 12-L-hydroxy 5,8,10 heptadecatrienoic acid (HHT), or are transformed by thromboxane synthetase into TXA2 which is essential for normal platelet function. The second pathway involves a lipoxygenase enzyme and results primarily in the formation of 12-hydroperoxy eicosotetraenoic acid $i;; is rapidly converted to 12-hydroxy eicosotetraenoic acid (12-HETE) , Several arachidonic acid metabolites are known to affect platelet function. Of the prostaglandins synthesized by platelets through the cyclooxygenase pathway, PGE2 potentiates aggregation, whereas PGD2 inhibits it. TXA2 is a potent aggregating agent and, if prostaglandin production is blocked, aggregation in response to all aggregating agents except thrombin is markely decreased (22). The lipoxygenase produce 12-HETE is known to be chemotactic to leukocytes (23,24), but has not been shown to affect platelet reactivity. Recently Schafer has described a selective loss of platelet lipoxygenase activity in 40% of a group of patients with myeloproliferative disorders who had a tendency toward bleeding rather than thrombosis (11). It was unclear, however, whether lipoxygenase deficiency was the cause of the bleeding diathesis in these patients. We studied the metabolism of [14C1 arachidonic acid utilizing the platelets of hemophiliacs with prolonged bleeding times. The incubation conditions in these experiments allowed us to assess the maximum production of the various arachidonic acid metabolites but did not permit an assessment f initial rates of production. Using washed platelets exposed to exogenous [94 C] arachidonic acid, we found that the conversion to TXB2, PGE2, PGF2a, HHT, and 12HETE was normal in all four hemophiliacs who had prolonged bleeding times. It therefore seems unlikely that the prolonged bleeding times observed in these individuals could have resulted from the inadvertent ingestion of aspirin. This observation was not unexpected in view of the fact that in previous studies we showed that platelet aggregation with arachidonic acid using washed platelets was normal in these and two other hemophiliacs with prolonged bleeding times. However the present study extends these initial observations by quantitating the arachidonic acid meabolites and by also demonstrating that the production of the lipoxygenase product 12-HETE is normal when their platelets are exposed to exogenous arachidonic acid. This latter finding has relevance in view of the recent demonstration that some patients with myeloproliferative disorders and a bleeding tendancy manifest a platelet lipoxygenase deficiency (11). ??
546
PLATELET FUNCTION IN HEMOPHILIA
Vo1.31, No.4
Neither the demonstration that platelets from these hemophiliacs metabolize exogenous arachidonic acid normally nor the finding of normal platelet aggregation in response to arachidonic acid, rules out the possibility of a defect in the mobilization of arachidonic acid from phospholipid storage sites.14 The present study addressed this question by prelabeling platelets with [ C] arachidonic acid and then stimulating the platelets with thrombin. When this was done, the production of TXB2, PGE2, PGF2o, and HHT was normal in all of the hemophiliacs with prolonged bleeding times. The production of 12-HETE seemed to be somewhat depressed in two of the patients (V.G. and B.M.) and the significance of this finding is not clear. Both of these individuals generated normal amounts of 12-HETE when their platelets were exposed to exogenous arachidonic acid so that a relative lipoxygenase deficiency seems unlikely. In conclusion, these findings indicate that the biosynthesis of TXA2 is normal in these patients, and further supports the concept that the abnormal bleeding times observed in some hemophiliacs maybe due to a plasma rather than a platelet defect. ACKNOWLEDGEMENTS We wish to thank Bonnie Kern, Virginia McGarvey and Debra Riley for their assistance in the study of these families. This work was supported by a Faculty Department Award in Clinical Pharmacology (Dr. Luderer) from the Pharmaceutical Manufacturers Association Foundation, the Delaware Valley Chapter of the National Hemophilia Foundation, Contract No. 632741 from the Pennsylvania Department of Health, and Grant No. MCB-420001-05-0 from the Department of Health and Human Services. REFERENCES 1.
MONCADA, S. and VANE, J.R. Arachidonic acid metabolites and the interactions between platelets and blood-vessel walls. E., Eng. J. Med., 300, 1142-1147, 1979.
2.
MUSTARD, J.F. and KINLOUGH-RATHBONE, R.L. Ann. Rev. Med 31 89-96, 1980. _-_----*~ -9
3.
MIELKE, C.H., KANESHIRO, M.M., HAHER I.A., WEINER, J .M., and RAPAPORT, S.I. The standardized normal Ivy bleeding time and its prolongation by 1969. aspirin. sltz.od, 34, 024-215,
4.
SUTOR, A.H., BOWIE, E.J.W., AND OWEN, Jr., C.A. Effect of aspirin, sodium salicylate and acetaminophen on bleeding. Mayo Clinic Proc.,.s, 178-181, 1971.
5.
NYMAN, D., ERIKSSON, A.W., LEHMANN, W., and BLOMBACK, M. Inherited defective platelet aggregation with arachidonate as the main expression of a defective metabolism of arachidonic acid. Thrombosis Research, 14, - . 739-746, 1979.
6.
PARETI, F.I., MANNUCCI, P.M., D'ANGELO, A., SMITH, J.B., SAUTEBIN, L., and GALLI, G. Congenital deficiency of thromboxane and prostacyclin. Lancet, L, 898-900, 1980.
Prostaglandins and platelets.
Vo1.31, No.4
547
PLATELET FUNCTION IN HEMOPHILIA
7.
WU, K.K., MINKOFF, I.M., ROSSI, E.C., and CHEN, Y.C. Hereditary bleeding disorder due to a primary defect in platelet release reaction. Br. J. Haematol., 47. 241-249, 1981.
8.
DEFRY, N.G., MACHIN, S.J., CARRERAS, L.O., DAUDEN, M.B., CHAMONE, D.A.F., Familial bleeding tendency with partial platelet and VERMYLEN, J. thromboxane synthetase deficiency: reorientation of cyclic endoperoxide metabolism. Br. J. Haematol., 3, 29-41, 1981.
9.
Abnormal platelet WIJ, K.K., LEBRETON G.C., TAI, H.H., CHEN, Y.C. response to thromboxane Ap. J. Clin. Invest., 67, 1801-1804, 1981.
10.
BALLARD, J.O., AL-MONDHIRY, H.A., LUDERER, J .R., LOCKE, L., GORDON, R.A., and EYSTER, M.E. Bleeding disorder associated with thrombocytopathy due to defective response of platelets to thromboxane A2. Blood (Suppl l), 58, 187a, 1981.
11.
SCHAFER, A.I. Deficiency of platelet lipoxygenase activity myeloproliferative disorders. N. Engl. J. Med., 386, 381-386, 1982.
12.
DUKE, W.W. The relation of blood platelets to hemorrhagic disease. J*, 55, 1185-1192, 1910.
13.
BORCHGREVINK, C.F. Platelet adhesion -in vivo in patients with bleeding disorders. Acta. Med. Stand., I7D, 231-243, 1961.
14.
KANESHIRO, M.M., MIELKE, C.H., KASPER, C.K., and RAPAPORT, S.I. Bleeding time after aspirin in disorders of intrinsic clotting. N. Engl. J. Med., m, 1039-1042, 1969.
15.
BOWIE, E.J., and OWEN, C.A. Tha., _2, 249-271, 1974.
16.
EYSTER,. M.E., GORDON, R.A., and BALLARD, J.O. The bleeding time longer than normal in hemophilia. -Blood, 58(4), 719-723, 1982.
17.
BUCHANAN, G.R., and HOLTKAMP, C.A. Prolonged bleeding time in children and young adults with hemophilia. Pediatrics, 66, 951-955, 1980.
18.
FRIEDBERG, N.M., and ZUCKER, M.B. ADP as the cause of reversible inhibition of platelet retention in glass-bead columns. J. Lab. Clin. &I, 603-612, 1972. @.,
19.
NEEDLEMAN, P., WYCHE, A., and RAZ, A. Platelet and arachidonate metabolism and interactions. J. Clin. Invest., 1979.
20.
WHORTON, A.R., CARR, K., SMIGEL, M., WALKER, L., ELLIS, K., and OATES, J.A. Reversed phase high-performance liquid chromatography of J. Chromatogr., 163, 64-71, prostaglandins biological applications. 1979.
21.
LUDERER, J.R., DEMERS, L.M., JANSON, R.W., NOMIDES, C.T., and HAYES, Jr., A.H. The effect of hydralazine on arachidonic acid metabolism in isolated washed human platelets. Res. Cotnn.Chem. Path. and Pharm., 28, 43-52, 1980.
The
bleeding time.
in
Prog. Haemostas. is
blood vessel 63, 345-349,
548
PLATELET FUNCTION IN HEMOPHILIA
Vo1.31, No.4
Interactions with and hemostasis
22.
Prostaglandins: WEKSLER, B.B., and GOLDSTEIN, I.M. polymorphonuclear leukocytes in and platelets inflammation. Am. J. Med., 68, 419-428, 1980.
23.
TURNER, S.R., TAINER, J.A. and LYNN, W.S. Biosynthesis of chemotactic molecules by the arachidonate lipoxygenase system of platelets. Nature, m, 680-681, 1975.
24.
GOETZL, E.J., and GORMAN, R.R. Chemotactic and chemokinetic stimulation of human eosinophil and neutrophil polymorphonuclear leukocytes by HHT. J. Immunol_.,120, 526-531, 1978.
ARACHIDONIC ACID METABOLISM IN THE PLATELETS OF HEMOPHILIACS WITH PROLONGED BLEEDING TIMES
John R. Luderer, Leslie Locke, Marlene Zekoski, Laurence Demers and M. Elaine Eyster Departments of Medicine and Pathology The Milton S. Hershey Medical Center The Pennsylvania State University Hershey, Pennsylvania 17033 Received
1.11.82;
Accepted in revised Editor J.B. Smith
form 18.5.83
by
ABSTRACT The bleeding time has been said to be normal in hemophilia. However, we and others have recently reported a prolonged bleeding time unrelated to the ingestion of non-steroidal anti-inflammatory agents or to recent transfusions in 16-20% of patients with hemophilia. To determine whether this abnormality might be due to impaired biosynthesis of thromboxane (TXA2) from the cyclooxygenase pathway, or 12-hydroxy eicosotetraenoic acid (12-HETE) from the alternative lipoxygenase pathway, we studied the in vitro metabolism of arachidonic acid (AA) in platelets from four h=pmcs with repeatedly prolonged bleeding All studies were performed on an aliquot of platelet-rich times. plasma obtained from a single venepuncture, the same morning the bleeding time was measured. High pressure liquid chromtop&ams of the reaction products generated following the incubation of [ Cl-AA with washed platelets from all four subjects were identical to those obtained from two hemophiliacs with normal bleeding times and from normal controls. In addition, metabolites of the cyclooxygenase and lipoxygenase pathways were normal in these patients when assessed by prelabeling their platelets with [ I4 Cl-AA and theh stimulating maximally with thrombin. We conclude that the prolonged bleeding times observed in these hemophiliacs cannot be attributed to the presence of a cyclooxygenase inhibitor such as aspirin.
Key words:
Hemophilia, platelets, prostaglandins, arachidonic acid, bleeding time, thromboxane. 539
PLATELET FUNCTION IN HEMOPHILIA
540
Vo1.31, No.4
INTRODUCTION The bleeding time is prolonged by drugs such as aspirin which inhibit the cyclooxygenase pathway of arachidonic acid metabolism, thus blocking TXA2formation which mediates the release reaction and platelet aggregation (l-4). In addition, congenital deficiencies of cyclooxygenase and TXA2 synthetase, causing abnormal platelet function and prolonged bleeding times (5-8), and abnormal platelet responses to TXA2 presumably due to defective TXA2 receptors have been reported (9,lO). Platelets can also metabolize arachidonic acid by the alternative lipoxygenase pathway to 12-hydroxy eicosotetraenoic acid (12HETE). Although lipoxygenase deficiency has recently been reported in some patients with myeloproliferative disorders (ll), it is not yet known whether this pathway plays a physiologic role in the regulation of hemostasis. The bleeding time has been said to be normal in hemophilia (12-15). However, we and others have reported a prolonged bleeding time in 16-20% of patients with classic hemophilia (16,17). A criticism of this work has been a failure to provide documentation that these patients had not ingested aspirin or an aspirin-like product which could account for the impairment in platelet function. Documentation by means of a retrospective patient interview is probably not adequate given the ubiquitous nature of various salicylates in over-the-counter drugs and food additives. Therefore, in order to investigate the mechanism of this abnormality further, and to demonstrate conclusively that these findings are not the result of the inadvertent ingestion of a cyclooxygenase inibitor, we studied the in vitro metabolism of arachidonic acid in the platelets of four hemophiliacswimpeatedly prolonged bleeding times. METHODS AND MATERIALS Venous blood was drawn with informed consent from four previously reported males (DD, BM, MS and VG) with classic hemophilia (16). DO, BM and MS had TABLE 1 Platelet Function Studies Patient Age (yrs) D.D. 13
B.M. 8 M.S. 28 V.G. G8C. 58 M.K. 21
Bleeding Time* (2.5 - 8 min)
()18%ii,l6)
0;:;) (>17$,11) 16.0
Platelet Count (1403340 x 10 /pl)
Platelet Platelet Aggregation, % Retention change in light trans(85-98%) mission, Patient/Control Patient/Control ADP Collagen
218
92/85
80/40
60150
307
80/84
35/50
50/50
170
88185
50140
60160
272
82/68
40/65
60160
153
88184
50150
40150
180
89/85
30/50
40/60
*Previous determinations recorded in parenthesis +Receiving Indomethacin
Vo1.31, No.4
PLATELET FUNCTION IN HEMOPHILIA
541
levels of 1% or less. VG had an VIII:C level of 13% VIII coagulant (VIII:C) with a normal VIII related antigen, a normal ristocetin cofactor, a normal crossed immunoelectrophoresis, and a family history of sex-linked hemophilia. All had at least one bleeding time greater than eleven minutes on previous determinations, and all had a prolonged bleeding time with normal or nearnormal platelet aggregation studies and platelet adhesiveness at the time of the study (Table 1). Two males with severe hemophilia, (GC and MK) with previously normal bleeding times were studied for comparison. Ten normal males, ages 23-33 with normal bleeding times and normal platelet aggregations served as controls. None of the subjects had ingested aspirin or non-steroidal antiinflammatory agents for at least two weeks prior to testing. All in vitro studies of platelet function and arachidonic acid metabolism were perfGm= an aliquot of platelet-rich plasma obtained from a single venepuncture. Platelet Function Studies Blood was collected by the two-syringe technique into plastic tubes containing l/9 volume balanced citrate (2 volumes O.lM citric acid, 2 volumes 0.1 M sodium citrate). Platelet aggregation studies were performed with a ChronoLog aggregometer on platelet-rich plasma that had been centrifuged at 150 g (800 rpm) at 25' for 6 minutes, with the platelet count adjusted to approximately 200,000 cu/mn. ACP (3 PM final concentration) epinephrine 0.48 mg/ml and collagen suspension (200 pg/ml to 300 pg/ml final concentration) were used as aggregating agents. Results with epinephrine were not reported because the control failed to aggregate and insufficient material was available to repeat the test. Platelet retention on glass beads was determined by Friedberg and Zucker's modification of the method of Bowie, using 2.6 g of glass beads (18). Template bleeding times were performed using the Simplate II device (General Diagnostics, Morris Plains, N.J.) as previously described (16). Preparation of Washed Platelets Studies of arachidonic acid metabolism were performed with washed human platelets prepared according to the method of Needleman (19). Fresh venous blood was collected directly into 7.5% (vol/vol) 77 mM EDTA and centrifuged at 150 x g for 10 minutes. The platelet rich plasma was then centrifuged in an EDTA-rinsed plastic tube at 1,800 g for 6 minutes and the platelet pellet resuspended in 0.15 M NaCl : 0.15 M Tris HCl (pH - 7.4) : 77 mM EDTA (90:8:2, vol/vol/vol) and subsequently recentrifuged at 600 g for 5 minutes. A calcium free Krebs-Henseleit medium was then u3ed to resuspend the washed platelet pellet in a volume adjusted to yield 10 platelets/ml. Platelet counts were done with an A0 Bright Line hemacytometer. Platelet Incubations All incubations were carried out in a Chrono-Log platelet aggrqometer at 37'C, stirred at 1,100 rpm. 0.4 ml aliquots of washed platelets (10 /ml) were added to ag regometer tubes. After a five minute pre-incubation period, 25 ~1 of sodium [94C] arachidonate (1 ug: 300,000 cpm) was added and the incubation continued for 15 minutes. Sodium arachidonate was prepared fresh each day in Tris buffer (40 pg/ml, pH = 9.0). Extraction and Chromatography The reaction mixtures were acidified to pti3.0 with 2 N formic acid and a trace amount of [3~]~~~1 ( =50,000 cpm) added for recovery analysis. The mixture was then extracted with three volumes of cold ethyl acetate. The combined organic extract was dried under a stream of nitrogen. Separation of the
542
PLATELET FUNCTION IN HEMOPHILIA
Vol.31,
No.4
reaction products was carried out using a slight modification of the high pressure liquid chromatographic method described by Whorton et al (20). HPLC was performed with a Water Associates (Milford, Mass.) chromatograph (model 6000A) on a reversed-phase P Bondapak Fatty Acid Analysis column. The packing material for the Fatty Acid Analysis column is proprietary information (Waters Associates). Samples were dissolved in 0.2 ml of the mobile phase, and 100 Pl was injected on column and initially eluted isocratically using a solvent system composed of water - acetonitrile - benzene - acetic acid (76.7:23:0.2: 0.1). After 20 minutes the percentage of acetonitrile in the mobile phase was gradually increased over a linear gradient employing a Water Associates solvent programmer (Model 660) as indicated in figure 1. The flow rate was 2 ml/ minute and one minute fractions were collected using a Gilson (B-200) automatic fraction collector. Liquid scintillation counting was performed using a Beckman (LS 7500) dual isotope counter. The identity of peaks on the chromatograms was determined by comparison with known radiolabelled standards and by mass spectroscopy as previously described (21). Platelet Phospholipid Labeling Technique The platelet phospholipid pool was labeled with [14C] arachidonic acid according to the method of Needleman (19). A platelet pellet was prepared as described above and resuspended in an albumin-phosphate buffer (33 mM, pH 6,5) containing glucose (1 mg/ml), NaCl14(6.6 mg/ml), and fatty acid poor bovine serum albumin (1 mg/ml). Sodium [ Cl arachidonate (2 x 10 cpm) was added to the platelet suspension which was incubated for 30 minutes (37°C). The labeled platelets were centrifuged (2,000 rpm, 5 minutes) and resuspended in calcium free Krebs Henseleit buffer (pH 7.4). Thin layer chromatographic analysis of the lipids extracted from these prelabeled platelets have indicated that the incorporated radioactivity is primarily in the phosphatidyl choline and phosphatidyl ethanolamine fractions, with minimal amounts of detectable free arachidonate. These prelabeled platelets were then stimulated with thrombin (\oU/ml) and after a 15 minute incubation period the samples were extracted and chromatographed as described above. This dose of thrombin had been determined in our laboratory to provide maximal stimulation of thromboxane production in normal platelets and is in agreement with that reported by others (19). C] arachidonic acid, C3HlPGEI, C3HlPGE2, C3H]PGF20, and C3H]TXB2 -14 were purchased from New England Nuclear, Boston, Mass. Unlabeled arachidonic acid, and adenosine 5'-diphosphate (sodium salts) were purchased from the Sigma Chemical Company, St. Louis, Missouri. HPLC solvents (HPLC-grade) were purchased from Fisher Scientific Company (Fair Lawn, New Jersey). Epinephrine was Purchased from Elkins-Sinn, Inc. (Cherry Hill, New Jersey). RESULTS Figure 1 shows a high pressure liquid c&omatogram of the reaction products generated following the incubation of C Cl arachidonic acid with washed platelets from one of the hemophiliacs (DD) who manifested a prolonged bleeding time. It demonstrates a normal pattern of arachidonic acid metabolism with significant synthesis of the cyclooxygenase derived products thromboxane B 2 (the inactive metabolite of TXA2) and It-hydroxyheptadecatrienoic acid (HHT), with lesser conversion of arachidonic acid to PGF2, and PGE2. This chromatogram also demonstrates that the lipoxygenase pathway of arachidonic acid metabolism is intact in this individual as evidenced by the significant
Vo1.31, No.4
PLATELET FUNCTION IN HEMOPHILIA
543
12-HETE
AA TXB2
10
20
30
40
50
FRACTIONNUMBER
FIG. 1 High pressure liquid chromatofram of the reaction products generated following the incubation of exogenous [ 4C] AA with washed platelets from a hemophiliac (DD) with a prolonged bleeding time.
production of 12-HETE. The pattern of metabolites shown in figure 1 is identical to that obtained from incubations of normal control platelets and the platelets of the other hemophiliacs (not shown) with normal and abnormal bleeding times. Figure 2 compares the pattern of arachidonic acid metabolism between the control group and the hemophiliacs with normal and prolonged bleeding times when washed platelets were incubated with exogenous arachidonic acid. The figure shows the percentage of total counts recovered as TXB 2, PGFza, PGE2, HHT (12-hydroxy heptadecatrienoic acid), and 12-HETE. The results are corrected for differences by spiking the sample with tritiated PGEl prior to extraction and chromatography. The PGEl elutes at a different point on the chromatogram (17) than the P-series pro taglandins and the tritium can be counted on a separate channel from the [ i4C] labeled products generated from arachidonic acid. Recoveries were 70 + 11% (Mean * SEM) and ranged from 62 to 84%. As shown in figure 2, there is no significant difference in platelet arachidonic acid metabolism between the groups tested when assessed by the ability of the washed platelets to metabolize exogenous arachidonate. The data for normals are presented as the mean + 2 standard deviation in order to provide an indication of the range of values obtained, as a percentage of total counts recovered.
Vo1.31, No.4
PLATELET FUNCTION IN HEMOPHILIA
544
5c fj NORMALS MEAN+2
S.D.
0 HEMOPHILIACS-NORMAL BLEEDING TIME
4c
??HEMOPHILIACS-PROLONGED
BLEEDING TIME
??
3a
C’ 0
m
0
20
0
m
??
10
TXB,
PGE;!
PG~Y
I
HHT
12-HETE
FIG. 2
AA metabolites generated from exogenous [ 14CJ AA utilizing washed platelets from normal male controls (B), hemophiliacs with normal bleeding times (0) and hemophiliacs with prolonged bleeding times (I). The normal controls are presented as the Mean + 2 S.D. (n = 10) and the hemophiliacs as individual data points. All results are depicted as the percentage of total counts recovered as the indicated metabolite. ??
B-NORMALS
MEAN+2 S.D.
0 HEMOPHILIACS-NORMAL BLEEDING TIME
.d II ??
??HEMOPHILIACS-PROLONGED
BLEEDING TIME
0
??
0
0
8
8
??
L
TXB;!
PGE;!
f’f3F2cx
FIG 3.
HHT
12-HETE
A44metabolites generated from thrombin stimulated platelets prelabeled with [ Cl AA. See legend for figure 2.
Vo1.31, No.4
PLATELET FUNCTION IN HEMOPHILIA
545
To determine if these patients could effectively mobilize arachidonic acid from phospholipid storage sites and subsequently metabolize it, washed platelets were prelabeled with arachidonic acid and stimulated with thrombin as described under Methods. As depicted in figure 3, there was no difference in the pattern of arachidonic acid metabolism through the cyclooxygenase pathway between hemophiliacs with prolonged bleeding times, hemophiliacs with normal bleeding times and normal controls. Twy of the four hemophiliacs with prolonged bleeding times (VG, BM) had less [ 4C] labeled 12-HETE recovered than did the normal controls, following thrombin stimulation. These were not the same patients as the bottom two in the HHT and TXB2 data. Moreover, as previously noted, the production of 12-HETE was normal in response to exogenous arachidonic acid.
mcussIor4
Arachidonic acid is released from membrane phospholipids by the enzyme phospholipase Al in response to a variety of stimuli and is metabolized in platelets by two metabolic pathways. The first pathway involves a cyclooxygenase enzyme and results in the formation of labile endoperoxide intermediates (PGG2, PGH2) which are either converted to the classical prostaglandins (PGE2, PGD2, PGF2a) and 12-L-hydroxy 5,8,10 heptadecatrienoic acid (HHT), or are transformed by thromboxane synthetase into TXA2 which is essential for normal platelet function. The second pathway involves a lipoxygenase enzyme and results primarily in the formation of 12-hydroperoxy eicosotetraenoic acid $i;; is rapidly converted to 12-hydroxy eicosotetraenoic acid (12-HETE) , Several arachidonic acid metabolites are known to affect platelet function. Of the prostaglandins synthesized by platelets through the cyclooxygenase pathway, PGE2 potentiates aggregation, whereas PGD2 inhibits it. TXA2 is a potent aggregating agent and, if prostaglandin production is blocked, aggregation in response to all aggregating agents except thrombin is markely decreased (22). The lipoxygenase produce 12-HETE is known to be chemotactic to leukocytes (23,24), but has not been shown to affect platelet reactivity. Recently Schafer has described a selective loss of platelet lipoxygenase activity in 40% of a group of patients with myeloproliferative disorders who had a tendency toward bleeding rather than thrombosis (11). It was unclear, however, whether lipoxygenase deficiency was the cause of the bleeding diathesis in these patients. We studied the metabolism of [14C1 arachidonic acid utilizing the platelets of hemophiliacs with prolonged bleeding times. The incubation conditions in these experiments allowed us to assess the maximum production of the various arachidonic acid metabolites but did not permit an assessment f initial rates of production. Using washed platelets exposed to exogenous [94 C] arachidonic acid, we found that the conversion to TXB2, PGE2, PGF2a, HHT, and 12HETE was normal in all four hemophiliacs who had prolonged bleeding times. It therefore seems unlikely that the prolonged bleeding times observed in these individuals could have resulted from the inadvertent ingestion of aspirin. This observation was not unexpected in view of the fact that in previous studies we showed that platelet aggregation with arachidonic acid using washed platelets was normal in these and two other hemophiliacs with prolonged bleeding times. However the present study extends these initial observations by quantitating the arachidonic acid meabolites and by also demonstrating that the production of the lipoxygenase product 12-HETE is normal when their platelets are exposed to exogenous arachidonic acid. This latter finding has relevance in view of the recent demonstration that some patients with myeloproliferative disorders and a bleeding tendancy manifest a platelet lipoxygenase deficiency (11). ??
546
PLATELET FUNCTION IN HEMOPHILIA
Vo1.31, No.4
Neither the demonstration that platelets from these hemophiliacs metabolize exogenous arachidonic acid normally nor the finding of normal platelet aggregation in response to arachidonic acid, rules out the possibility of a defect in the mobilization of arachidonic acid from phospholipid storage sites.14 The present study addressed this question by prelabeling platelets with [ C] arachidonic acid and then stimulating the platelets with thrombin. When this was done, the production of TXB2, PGE2, PGF2o, and HHT was normal in all of the hemophiliacs with prolonged bleeding times. The production of 12-HETE seemed to be somewhat depressed in two of the patients (V.G. and B.M.) and the significance of this finding is not clear. Both of these individuals generated normal amounts of 12-HETE when their platelets were exposed to exogenous arachidonic acid so that a relative lipoxygenase deficiency seems unlikely. In conclusion, these findings indicate that the biosynthesis of TXA2 is normal in these patients, and further supports the concept that the abnormal bleeding times observed in some hemophiliacs maybe due to a plasma rather than a platelet defect. ACKNOWLEDGEMENTS We wish to thank Bonnie Kern, Virginia McGarvey and Debra Riley for their assistance in the study of these families. This work was supported by a Faculty Department Award in Clinical Pharmacology (Dr. Luderer) from the Pharmaceutical Manufacturers Association Foundation, the Delaware Valley Chapter of the National Hemophilia Foundation, Contract No. 632741 from the Pennsylvania Department of Health, and Grant No. MCB-420001-05-0 from the Department of Health and Human Services. REFERENCES 1.
MONCADA, S. and VANE, J.R. Arachidonic acid metabolites and the interactions between platelets and blood-vessel walls. E., Eng. J. Med., 300, 1142-1147, 1979.
2.
MUSTARD, J.F. and KINLOUGH-RATHBONE, R.L. Ann. Rev. Med 31 89-96, 1980. _-_----*~ -9
3.
MIELKE, C.H., KANESHIRO, M.M., HAHER I.A., WEINER, J .M., and RAPAPORT, S.I. The standardized normal Ivy bleeding time and its prolongation by 1969. aspirin. sltz.od, 34, 024-215,
4.
SUTOR, A.H., BOWIE, E.J.W., AND OWEN, Jr., C.A. Effect of aspirin, sodium salicylate and acetaminophen on bleeding. Mayo Clinic Proc.,.s, 178-181, 1971.
5.
NYMAN, D., ERIKSSON, A.W., LEHMANN, W., and BLOMBACK, M. Inherited defective platelet aggregation with arachidonate as the main expression of a defective metabolism of arachidonic acid. Thrombosis Research, 14, - . 739-746, 1979.
6.
PARETI, F.I., MANNUCCI, P.M., D'ANGELO, A., SMITH, J.B., SAUTEBIN, L., and GALLI, G. Congenital deficiency of thromboxane and prostacyclin. Lancet, L, 898-900, 1980.
Prostaglandins and platelets.
Vo1.31, No.4
547
PLATELET FUNCTION IN HEMOPHILIA
7.
WU, K.K., MINKOFF, I.M., ROSSI, E.C., and CHEN, Y.C. Hereditary bleeding disorder due to a primary defect in platelet release reaction. Br. J. Haematol., 47. 241-249, 1981.
8.
DEFRY, N.G., MACHIN, S.J., CARRERAS, L.O., DAUDEN, M.B., CHAMONE, D.A.F., Familial bleeding tendency with partial platelet and VERMYLEN, J. thromboxane synthetase deficiency: reorientation of cyclic endoperoxide metabolism. Br. J. Haematol., 3, 29-41, 1981.
9.
Abnormal platelet WIJ, K.K., LEBRETON G.C., TAI, H.H., CHEN, Y.C. response to thromboxane Ap. J. Clin. Invest., 67, 1801-1804, 1981.
10.
BALLARD, J.O., AL-MONDHIRY, H.A., LUDERER, J .R., LOCKE, L., GORDON, R.A., and EYSTER, M.E. Bleeding disorder associated with thrombocytopathy due to defective response of platelets to thromboxane A2. Blood (Suppl l), 58, 187a, 1981.
11.
SCHAFER, A.I. Deficiency of platelet lipoxygenase activity myeloproliferative disorders. N. Engl. J. Med., 386, 381-386, 1982.
12.
DUKE, W.W. The relation of blood platelets to hemorrhagic disease. J*, 55, 1185-1192, 1910.
13.
BORCHGREVINK, C.F. Platelet adhesion -in vivo in patients with bleeding disorders. Acta. Med. Stand., I7D, 231-243, 1961.
14.
KANESHIRO, M.M., MIELKE, C.H., KASPER, C.K., and RAPAPORT, S.I. Bleeding time after aspirin in disorders of intrinsic clotting. N. Engl. J. Med., m, 1039-1042, 1969.
15.
BOWIE, E.J., and OWEN, C.A. Tha., _2, 249-271, 1974.
16.
EYSTER,. M.E., GORDON, R.A., and BALLARD, J.O. The bleeding time longer than normal in hemophilia. -Blood, 58(4), 719-723, 1982.
17.
BUCHANAN, G.R., and HOLTKAMP, C.A. Prolonged bleeding time in children and young adults with hemophilia. Pediatrics, 66, 951-955, 1980.
18.
FRIEDBERG, N.M., and ZUCKER, M.B. ADP as the cause of reversible inhibition of platelet retention in glass-bead columns. J. Lab. Clin. &I, 603-612, 1972. @.,
19.
NEEDLEMAN, P., WYCHE, A., and RAZ, A. Platelet and arachidonate metabolism and interactions. J. Clin. Invest., 1979.
20.
WHORTON, A.R., CARR, K., SMIGEL, M., WALKER, L., ELLIS, K., and OATES, J.A. Reversed phase high-performance liquid chromatography of J. Chromatogr., 163, 64-71, prostaglandins biological applications. 1979.
21.
LUDERER, J.R., DEMERS, L.M., JANSON, R.W., NOMIDES, C.T., and HAYES, Jr., A.H. The effect of hydralazine on arachidonic acid metabolism in isolated washed human platelets. Res. Cotnn.Chem. Path. and Pharm., 28, 43-52, 1980.
The
bleeding time.
in
Prog. Haemostas. is
blood vessel 63, 345-349,
548
PLATELET FUNCTION IN HEMOPHILIA
Vo1.31, No.4
Interactions with and hemostasis
22.
Prostaglandins: WEKSLER, B.B., and GOLDSTEIN, I.M. polymorphonuclear leukocytes in and platelets inflammation. Am. J. Med., 68, 419-428, 1980.
23.
TURNER, S.R., TAINER, J.A. and LYNN, W.S. Biosynthesis of chemotactic molecules by the arachidonate lipoxygenase system of platelets. Nature, m, 680-681, 1975.
24.
GOETZL, E.J., and GORMAN, R.R. Chemotactic and chemokinetic stimulation of human eosinophil and neutrophil polymorphonuclear leukocytes by HHT. J. Immunol_.,120, 526-531, 1978.