JOURNAL
OF SURGICAL
RESEARCH
51,
93-98
(19%)
Cyclosporin A Augments Human Platelet Sensitivity to Aggregating Agents by Increasing Fibrinogen Receptor Availability’s2 STEVEN
J. FISHMAN,
M.D., LAUREN J. WYLONIS, B.S., JOEL D. GLICKMAN, M.D., JACQUELYNN DAVID S. WARSAW, B.S., CAROL A. FISHER, B.A., DIANE J. JORKASKY, M.D., STEFAN NIEWIAROWSKI, M.D., PH.D., AND V. PAUL ADDONIZIO, M.D.3
Reichle
Surgical Research Laboratories, Division of Cardiac and Thoracic Surgery Thrombosis Research Center, Temple University Health Sciences Center, University of Pennsylvania School of Medicine, Philadelphia, Submitted
for publication
November
a reduction Clinical use of cyclosporin A (CsA) has been associated with platelet hypersensitivity and an increased incidence of thrombotic and vasoactive events. The purpose of this study was (1) to confirm that CsA enhances platelet sensitivity to the soluble agonists, adenosine diphosphate (ADP) and epinephrine (EPI), and (2) to determine if this enhancement is mediated by alteration in the availability of platelet surface fibrinogen receptors, a final mediator of platelet activation. Mean log dose of ADP required to achieve complete second-wave platelet aggregation in vitro decreased from 1.90 to 1.49 NM (n = 19, paired t test, P < 0.05) and 2.86 to 2.11 fl (n = 16, P < 0.05) following a 15-min and 3-hr incubation in the absence (saline) and presence of CsA (1000 rig/ml), respectively. At the threshold dose of ADP, concurrent thromboxane B, levels at 15 min were 245 f 44 rig/ml (n = 12, saline) and 265 + 54 rig/ml (n = 9, CsA; P > 0.05). At 3 hr respective levels were 333 -+ 5’7 and 442 k 81 rig/ml (P > 0.05). Similar results were obtained with EPI. The number of fibrinogen binding sites in response to 50 pM ADP was determined in washed platelets in the absence and presence of CsA by radioligand binding. In 6 of 7 volunteers, CsA increased fibrinogen receptors from 26,635 f 4841 to 35,925 -+ 7290 sites/platelet (X f SEM; P < 0.05). No change in receptor affinity was noted. In conclusion, cyclosporine does augment platelet reactivity. Furthermore, this increase in reactivity is mediated by increased exposure of surface fibrinogen receptors. Delineation of this mechanism may lead to
0 1991
Academic
the Department of Physiology, and the Department of Surgery Pennsylvania 29140
J.
COOK,
PH.D.,
and the
20, 1990
in Press,
cyclosporine-induced
complications.
Inc.
INTRODUCTION Cyclosporin A (CsA), a cyclic undecapeptide with potent immunosuppressive activity, has been used with increasing frequency since 1982. Despite its efficacy in the therapy of organ transplant recipients CsA has been associated with an increased incidence of thrombotic and adverse vasomotor phenomena [l-3]. It is well known that platelet activation is fundamental to the pathogenesis of thrombosis, and activated platelets are a source of the most potent naturally occuring vasoconstrictors, thromboxanes and cyclic endoperoxides. Consistently, glomerular capillary thrombosis and afferent arteriolar vasoconstriction may account, at least in part, for the ubiquitous nephrotoxicity seenwith cyclosporine administration [A]. The purpose of this study was twofold. To determine if CsA alters platelet reactivity, its effects on human platelet aggregation and resultant thromboxane (TX) A, release in response to the soluble agonists adenosine diphosphate (ADP) and epinephrine (EPI) were assessed.The second purpose was to determine if CsA affects the expression of platelet surface fibrinogen receptors. Regardless of agonist, fibrinogen binding is a necessary common pathway mediating platelet aggregation. MATERIALS
AND METHODS
All studies were approved by Temple University’s Institutional Review Board and the University of Pennsylvania Committee on Human Investigation. All procedures followed were in accordance with the ethical standards of the Helsinki Declaration of 1975. Verbal and written informed consent was obtained from each volun-
’ Presented at the Annual Meeting of the Association for Academic Surgery, Houston, TX, November 14-17,199O. * This work was supported in part by funds from the National Institutes of Health Grants HL-34026, HL-19055, and HL-15226. ’ Dr. Addonizio is the recipient of a National Institutes of Health Young Investigators Award HL-22346 and a Hartford Foundation Scholarship.
93
0022-4So4/91
$1.50
Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
94
JOURNAL
OF
SURGICAL
RESEARCH:
teer prior to phlebotomy. Normal volunteers abstained from all medications, including aspirin and nonsteroidal anti-inflammatory-containing drugs, for at least 2 weeks prior to donating. Platelet Aggregation
and Thromboxane
Release
One hundred twenty milliliters of blood was drawn by venipuncture into plastic syringes containing 1 vol of 3.8% trisodium citrate for every 9 vol of blood. To one 50-ml aliquot, CsA (Sandimmune iv, Sandoz Ltd.) was added to achieve a final concentration of 1,000 rig/ml of whole blood, to another 50-ml aliquot, an equal volume of normal saline (control) was added. The dose of cyclosporine chosen represents a value equivalent to a whole blood concentration achieved within several hours after drug administration in uiuo. All samples were incubated at 37°C for 15 min and 3 hr. The remaining 20 ml of blood was divided into four equal aliquots, two containing CsA and two containing saline, in order to quantitate the potential maximum amount of releasable thromboxane. After incubation, the samples were processed for platelet aggregation studies and quantitation of resultant TxA, generation (vide infra). To perform platelet aggregation studies, platelet-rich plasma (PRP) and platelet-poor plasma (PPP) were prepared as previously described [5]. Briefly, after incubation with either CsA or saline, whole blood samples were centrifuged at 15Og for 10 min at 23°C. After gentle aspiration of the PRP (350,000 + 50,000 platelets/pi), the remaining blood was centrifuged at 12,000g for 4 min at 23°C in a microcentrifuge to obtain PPP ( 0.05) and 763 f 288 rig/ml vs 902 + 341 rig/ml (P > 0.05) in the absence and presence of CsA at 15 min and 3 hr, respectively (Fig. 6). Spontaneous thromboxane levels generated in PRP samples merely spun without the addition of agonist were 2310 +- 307 rig/ml in control platelets and 2631 +- 383 rig/ml in CsA-treated platelets following a 15-min incubation (n = 18, P > 0.05). After 3 hr respective levels were 4403 f 734 and 5693 f 2535 rig/ml (n = 18; P > 0.05) (not shown). Serum thromboxane levels generated in control and CsA-treated whole blood allowed to clot were 543 _+88 and 542 3- 81 rig/ml at 15 min and 639 f 117 and 566 f 108 rig/ml at 3 hr (not shown).
Platelet Aggregation
Fibrinogen
For control samples, mean threshold concentration of ADP decreased from 1.90 to 1.49 PM (n = 19; P < 0.05) in cyclosporine-treated platelets following a 15min incubation. After a 3-hr incubation, the threshold dose decreased from 2.86 to 2.11 PM (n = 16; P < 0.05) (Figs. IA and IB). At 15 min mean threshold dose of epinephrine decreased from 0.20 ph4 in control platelets to 0.16 p&fin CsA-incubated platelets (n = 8; P > 0.05). At 3 hr the threshold dose decreased from 1.54 to 0.79 PM (n = 8; P < 0.05) (Figs. 2A and 2B). A representative dose-response curve to ADP and EPI in the presence and absence of CsA is illustrated in Figs. 3 and 4.
Control platelets demonstrated 26,635 + 4841 (X _+ SEM) fibrinogen receptors per platelet. Cyclosporine
Thromboxane
Receptor Expression
lOOT----^-^-----
Release
Following stimulation with threshold concentrations of ADP, supernatant TxB, levels were 244 + 44 rig/ml in control platelets compared to 265 f 54 rig/ml in CsAtreated platelets (n = 12; P > 0.05) following a 15-min incubation. At 3 hr respective thromboxane generation was 333 f 57 rig/ml vs 442 + 81 rig/ml (n = 9; P > 0.05) (Fig. 5). Consistently, in EPI-stimulated platelets, TxB,
uMADP
FIG. against unteer sporine
3. Percentage aggregation varying log concentrations following a 3-hr incubation. (1000 rig/ml whole blood).
(light transmittance) is plotted of ADP from a representative vol(0) Saline (control); (0) cyclo-
96
JOURNAL
OF
SURGICAL
‘00 got
RESEARCH:
51, NO.
2, AUGUST
1991
-
1500
I
A 3 E 1200 \ F?
80i L
VOL.
701
60 -50 -40 ~30 ~20 -10 -A/-
0e 0 010
0 too
1 000
z al
900
si d Ez
600
is “I k
300
l 2 500
0
t 15 MIN
uM EPI
FIG. 4. Percentage aggregation (light transmittance) against varying log concentrations of EPI from a representative teer following a 3-hr incubation. See legend to Fig. 3.
is plotted volun-
incubation significantly increased fibrinogen receptor expression to 35,925 -t 7290 receptors per platelet (n = 7; P < 0.05). Receptor number increased in 6 of 7 volunteers. Scatchard plot analysis yielded Kd values of 3.77 + 0.74 X 10e7 and 4.57 t 0.84 X low7 in control and cyclosporine-treated platelets, respectively (P = 0.247). Thus, receptor number increased without a significant change in receptor affinity. A representative Scatchard plot, demonstrating an increase in receptor expression by cyclosporine with unchanged affinity, is depicted in Fig. 7. DISCUSSION Cyclosporin A has revolutionized the field of organ transplantation, increasing graft survival and permitting the proliferation of multiorgan transplantation. Its potent immunosuppressant properties have also been useful in the treatment of several autoimmune disorders. Unfortunately, this agent is not without side effects.
600
,
3 HR
FIG. 6. Thromboxane B, (rig/ml) release (at mean threshold dose of EPI) following a 15min (A, n = 6) and a 3-hr (B, n = 7) incubation with either saline or cyclosporine. Bars represent %+ SEM. See legend to Fig. 1.
Similar to other immunosuppressive agents, CsA increases the risks of infection, lymphoproliferative disorders, and malignancy. Interestingly, CsA has also been associated with complications seemingly unrelated to its immunosuppressant actions. Although never studied in a large-scale, prospective, randomized fashion, CsA has been associated with an increased incidence of thrombotic and thromboembolic phenomena [l-3]. These phenomena include deep venous thrombosis, pulmonary embolism, renal vein thrombosis, mesenteric thrombosis, microangiopathies, and the hemolytic uremic-like syndrome. Even the most frequently troublesome toxicity of CsA, nephrotoxicity, may have origin in activation of haemostatic elements. Microangiopathy characterized by frank glomerular capillary thrombosis has been observed in some cases. In addition, decreased renal plasma flow occurs as a result of afferent arteriolar vasoconstriction [12]. It has been suggested that intrarenal
1
_1 20
M (i508) 15 MIN
FIG. 5. Thromboxane B, (rig/ml) release (at mean threshold dose of ADP) following a 15-min (A, n = 12) and 3-hr (B, n = 9) incubation with either saline or cyclosporine. Bars represent I k SEM. See legend to Fig. 1.
FIG. platelet receptor determines Control,
7. Representative Scatchard plot demonstrating increased surface fibrinogen receptor expression without alteration of affinity in the presence of cyclosporine. Abscissa-intercept receptor number, while slope determines allinity. (0) r = 0.94; (0) cyclosporin A, r = 0.99.
FISHMAN
ET
AL.:
CYCLOSPORIN
A ENHANCES
platelet activation with resulting thromboxane release may join with a functional alteration of local endothelium to mediate this vasoconstriction. Substantiating this possibility, Benigni et al. demonstrated a selective increase in urinary 2,3-dinor-TxB,, a thromboxane metabolite derived principally from formed blood elements, rather than the parent compound which, when present in urine, reflects renal thromboxane synthesis [4]. Thus, thrombosis in the broad sense and platelet activation in particular may contribute to a variety of cyclosporinemediated complications. Unfortunately, the mechanism of a CsA-related activation of formed haemostatic elements has not been completely delineated. To date Grace et al. demonstrated augmented platelet aggregation in response to soluble agonists [13]. These investigators noted that CsA enhanced in vitro platelet aggregation in response to subthreshold doses of several soluble agonists including ADP and EPI. Furthermore, a similar augmentation in responsiveness was demonstrated in platelets obtained from normal volunteers given a single oral dose of CsA. In addition, in transplant patients taking CsA, aggregation was shown to be of greater magnitude when blood was drawn at the time of peak serum levels compared to that drawn during trough levels. Finally, reduced platelet sensitivity occurred when CsA was removed from patients’ immunosuppressive regimens. Finally, Cohen et al. detected chronic platelet hyperactivity in 21 patients receiving cyclosporine [14]. Platelet aggregation responses to low doses of ADP were significantly increased up to 1 year after initiating cyclosporine therapy. Total platelet ADP and ATP content, as well as releasable quantities of these dense granule nucleotides in response to collagen were decreased, and plasma platelet factor 4 levels were increased, suggesting chronic platelet activation. Cyclosporine’s prothrombotic effects are not limited to platelets. Thomson et al. [15] and others [16, 171 found an increase in monocyte procoagulant activity. Carlsen showed enhanced cytokine-stimulated endothelial cell thromboplastin production [ 181. We and others have demonstrated alterations in endothelial arachidonate metabolism, which can disturb the delicate balance between prothrombotic thromboxane synthesis and antithrombotic prostacyclin synthesis [ 19-221. The present study unequivocally demonstrates that CsA augments platelet aggregation in uitro. Moreover, enhanced platelet responsiveness occurs to both ADP and epinephrine, common soluble agonists used to induce platelet activation. Thus, our findings corroborate and extend those of Grace et al. [13] and Cohen et al. [14]. Although it is, perhaps, predictable that ADP and epinephrine would act similarly, significant differences characterize their mechanism of action [23]. ADP but not epinephrine induces platelet shape change, and epinephrine but not ADP inhibits adenyl cyclase in broken cell preparations [24]. The two agents also differ quanti-
PLATELET
REACTIVITY
97
tatively in their ability to inhibit PGE,-stimulated formation of cyclic AMP [25]. Finally, ADP and EPI are known to initiate platelet activation by interacting with distinctly separate surface membrane receptors. Because the enhanced platelet responsiveness induced by CsA appears to be agonist independent, it would seem likely that CsA acts on a common pathway mediating platelet function. For example, platelets will not aggregate in the absence of fibrinogen binding to the membrane glycoprotein complex IIb-IIIa [26 1. Although likely continuously present, the fibrinogen receptor of this glycoprotein complex must be exposed to bind fibrinogen [27-281. This study demonstrates a cyclosporine-induced increase in platelet responsiveness to soluble agonists coincident with increased fibrinogen receptor availability, but without alteration in thromboxane production. The mechanism by which this occurs requires further investigation. However, several explanations seem plausible. First, the highly lipophilic cyclosporine molecule may insert into the platelet membrane, thus altering membrane structure and glycoprotein 1%IIIa conformation or mobility. Second, cyclosporine is known to alter intracellular calcium flux in the lymphocyte, its intended cell of action, by binding with cyclophilin. It may similarly increase cytosolic calcium concentration in platelets, which, in turn, can lead to fibrinogen receptor expression and platelet activation. Additionally, lymphocytes and platelets share several surface antigens, including CD9 and CD69, which play a role in platelet aggregation [29, 301. Indeed, CD9 has been shown to associate with the glycoprotein IIb-IIIa complex when stimulated with monoclonal antibody [29]. Cyclosporine may effect both the lymphocyte and platelet by interacting with these surface antigens. Any of these mechanisms might explain agonist-independent hypersensitivity of platelets exposed to CsA. No increase in thromboxane release was observed over and above what is normally observed at a given level of activation. Cyclosporine lowered the concentration of ADP required to completely aggregate platelets. However, once completely aggregated, platelets from both groups produced the same amount of thromboxane B,. Thus, cyclosporine-mediated increase in platelet sensitivity appeared to be independent of thromboxane synthesis. In summary, cyclosporine increases platelet sensitivity to soluble agonists and, perhaps causally, increases availability of platelet fibrinogen receptors. Whether or not this effect is specific for fibrinogen receptors or is due to a nonspecific effect on the platelet membrane remains unknown. Nevertheless, CsA-induced platelet hypersensitivity could contribute to the thrombotic and nephrotoxic effects of cyclosporine therapy. REFERENCES 1.
Vanrenterghem, V., Lerut, T., Roels, P., Gresele, P., Colucci, M., Deckmyn,
L., Gruwez, J., Michielsen, H., Amout, J., and Ver-
98
2.
-----
JUUKNAL
__.-
^-
UF
------1.-
VUL.
--^-.-I--
SUKtiIC;AL
__^_
HKQ!iAKL‘H:
mylen, J. Thromboembolic complications and haemostatic changes in cyclosporine-treated cadaveric kidney allograft recipients. Lancet 999, 1985. Innes, A., Rowe, P. A., Foster, M. C., Steiger, M. J., and Morgan, A. G. Cyclosporine toxicity and colitis. Lancet 957, 1988.
6. I.
Lewy, R. L., Smith, J. B., Silver, J., Saia, J., Walinsky, P., and Wiener, L. Detection of thromboxane B, in peripheral blood of patients with Printzmetal angina. Prostugkzndins Med. 2: 243, 1979.
a. Niewiarowski,
S., Budzynski, A. Z., Morinelli, T. A., Brudzynski, T. M., and Stewart, G. J. Exposure of fibrinogen receptor on human platelets by proteolytic enzymes. J. Biol. Chem. 266: 917, 1981.
9. Mustard,
J. F., Perry, D. W., Ardlie, N. C., and Packham, M. A. Preparation of suspensions of washed platelets from humans. Br. J. Huematol. 22: 193, 1972.
10.
11. 12.
Niewiarowski, S., Rawala, R., Lukasiewicz, H., Hershock, D., Capitanio, A. M., Kornecki, E., and Boden, G. Fibrinogen interaction with platelets of diabetic subjects. Thromb. Res. 46: 479, 1987. Scathard, G. The attraction ions. Ann. NY Acad. Sci. Remuzzi, G., and Bertani, fects of cyclosporine. Am.
of proteins
with
small
molecules
and
vascular and thrombotic Dis. 13: 261, 1989.
ef-
51: 660,1949. T. Renal J. Kidney
Grace, A. G., Barradas, M. A., Mikhailidis, D. P., Jeremy, Moorhead, J. F., Sweny, P., and Dandona, P. Cyclosporin hances platelet aggregation. Kidney Znt. 32: 889, 1987.
J. Y., A en-
14.
Cohen, H., Neild, G. H., Patel, R., Mackie, I. J., and Machin, S. J. Evidence for chronic platelet hyperaggregability and in uiuo activation in cyclosporine-treated renal allograft recipients. Thromb. Res. 49: 91, 1988.
.ll^lT”-
_^^_
lYY1
Webster, L. M., blood mononuclear Znt. Arch. Allergy
17.
Ferreira, 0. C., and Barcinski, M. A. Autologous human T-Cell-dependent monocyte procoagulant sible link between immunoregulatory phenomena ulation. Cell. Zmmunol. 101: 259, 1986.
18.
Carlsen, E., Flatmark, A., and Prydz, H. Cytokine-inducedprocoagulant activity in monocytes and endothelial cells: Further hancement by cyclosporine. Transplantation 46: 575, 1988.
1396,1985.
19.
and Thomson, A. W. Stimulation of human cell procoagulant activity by cyclosporin A. Appl. Zmmunol. 81: 371, 1986. induction of the activity: A posand blood coag-
en-
Fishman, S. J., Warsaw, D. S., Fisher, C. A., Barnathan, E. S., and Addonizio, V. P. Cyclosporin A alters endothelial arachidonate metabolism in the isolated crystalloid-perfused rabbit heart. Surg. Forum 41: 284,199O.
20. Zoja, C., Furci, 21.
L., andchilardi, F. Cyclosporine inducedendothelial cell injury. Lab. Znuest. 55: 455, 1986. Voss, B. L., Hamilton, K. K., and Samara, E. N. S. Cyclosporine suppression of endothelial prostacyclin generation. Transplantation 45: 793, 1988.
22. Lau, D. C. W., Wong, ity on cultured
K-L., and Hwang, W. S. Cyclosporine toxicrat microvascular endothelial cells. Kidney Znt.
35:6041,1989. 23. Charo, I. F., Feinman, 24. 25.
R. D., andDetwiler, T. C. Interrelations of platelet aggregation and secretion. J. Clin. Znuest. 60: 866,1977. Zieve, P. D., and Greenough, W. B., III. Adenyl cyclase in human platelets: Activity and responsiveness. Biochem. Biophys. Res. Commun. 35: 462,1969. Haslam, R. J., and Taylor, A. Role of cyclic 3’:5’-adenosine monophosphate in platelet aggregation. In J. P. Caen, (Ed.), Platelet Aggregation, Paris, Masson et Cie, 1971. Pp 85-93.
26. Nurden,
A. T., and Caen, J. P. The different glycoprotein abnormalities in thrombosthenic and Bernard-Soulier platelets. Semin. Hematol. 16: 234, 1979.
27. Bennett, 28. 29.
13.
^
2, AUtilJS’l’
16.
5. Addonizio,
V. P., Macarak, E. J., Niewiarowski, S., Colman, R. W., and Edmunds, L. H. Preservation of human platelets with prostaglandin E, during in uitro simulation of cardiopulmonary bypass. Circ. Rex 44: 350, 1979. Carvalho, A. C. A., Colman, R. W., and Lees, R. J. Clofibrate reversal of platelet hypersensitivity in hyperbetalipbproteinemia. Circulation 50: 570, 1974.
.-,.
NU.
Thomson, A. W., Webster, L. M., Aldridge, R. D., and Morrice, L. M. Cyclosporine and leukocyte procoagulant activity. Lancet
4. Benigni,
A., Chiabrando, C., Piccinelli, A., Perico, N., Gavinelli, M., Furci, L., Patino, O., Abbate, M., Bertani, T., and Renuzzi, G. Increased urinary excretion of thromboxane B, and 2,3-dinorTxB, in cyclosporin A nephrotoxicity. Kidney Int. 34: 1264, 1988.
-_
15.
3. Holler,
E., Kolb, H. J., Hiller, E., Mraz, W., Lehmacher, W., Gleixner, B., Seeber, C. H., Jehn, U., Gerhartz, H. H., Brehm, G., and Wilmanns, W. Microangiopathy in patients on cyclosporine prophylaxis who developed acute graft-versus-host disease after HLA-identical bone marrow transplantation. Blood 73: 2018, 1989.
51,
J. S., and Vilaire, G. Exposure of platelet fibrinogen receptors by ADP and epinephrine. J. Clin. Znuest. 64: 1393, 1979. Peerschke, E. I. B. The platelet fibrinogen receptor. Semin. Hematol. 22: 241, 1985. Slupsky, J. R., Seehafer, J. G., Tang, S-C., Masellis-Smith, A., and Shaw, A. R. E. Evidence that monoclonal antibodies against CD9 antigen induce specific association between CD9 and the platelet glycoprotein IIb-IIIa complex. J. Biol. Chem. 2 1: 12289, 1989.
30. Testi,
R., Pulcinelli, F., Frati, L., Gazzaniga, P. P., and Santoni, A. CD69 is expressed on platelets and mediates platelet activation and aggregation. J. Exp. Med. 172: 701, 1990.
OF SURGICAL
RESEARCH
51,
93-98
(19%)
Cyclosporin A Augments Human Platelet Sensitivity to Aggregating Agents by Increasing Fibrinogen Receptor Availability’s2 STEVEN
J. FISHMAN,
M.D., LAUREN J. WYLONIS, B.S., JOEL D. GLICKMAN, M.D., JACQUELYNN DAVID S. WARSAW, B.S., CAROL A. FISHER, B.A., DIANE J. JORKASKY, M.D., STEFAN NIEWIAROWSKI, M.D., PH.D., AND V. PAUL ADDONIZIO, M.D.3
Reichle
Surgical Research Laboratories, Division of Cardiac and Thoracic Surgery Thrombosis Research Center, Temple University Health Sciences Center, University of Pennsylvania School of Medicine, Philadelphia, Submitted
for publication
November
a reduction Clinical use of cyclosporin A (CsA) has been associated with platelet hypersensitivity and an increased incidence of thrombotic and vasoactive events. The purpose of this study was (1) to confirm that CsA enhances platelet sensitivity to the soluble agonists, adenosine diphosphate (ADP) and epinephrine (EPI), and (2) to determine if this enhancement is mediated by alteration in the availability of platelet surface fibrinogen receptors, a final mediator of platelet activation. Mean log dose of ADP required to achieve complete second-wave platelet aggregation in vitro decreased from 1.90 to 1.49 NM (n = 19, paired t test, P < 0.05) and 2.86 to 2.11 fl (n = 16, P < 0.05) following a 15-min and 3-hr incubation in the absence (saline) and presence of CsA (1000 rig/ml), respectively. At the threshold dose of ADP, concurrent thromboxane B, levels at 15 min were 245 f 44 rig/ml (n = 12, saline) and 265 + 54 rig/ml (n = 9, CsA; P > 0.05). At 3 hr respective levels were 333 -+ 5’7 and 442 k 81 rig/ml (P > 0.05). Similar results were obtained with EPI. The number of fibrinogen binding sites in response to 50 pM ADP was determined in washed platelets in the absence and presence of CsA by radioligand binding. In 6 of 7 volunteers, CsA increased fibrinogen receptors from 26,635 f 4841 to 35,925 -+ 7290 sites/platelet (X f SEM; P < 0.05). No change in receptor affinity was noted. In conclusion, cyclosporine does augment platelet reactivity. Furthermore, this increase in reactivity is mediated by increased exposure of surface fibrinogen receptors. Delineation of this mechanism may lead to
0 1991
Academic
the Department of Physiology, and the Department of Surgery Pennsylvania 29140
J.
COOK,
PH.D.,
and the
20, 1990
in Press,
cyclosporine-induced
complications.
Inc.
INTRODUCTION Cyclosporin A (CsA), a cyclic undecapeptide with potent immunosuppressive activity, has been used with increasing frequency since 1982. Despite its efficacy in the therapy of organ transplant recipients CsA has been associated with an increased incidence of thrombotic and adverse vasomotor phenomena [l-3]. It is well known that platelet activation is fundamental to the pathogenesis of thrombosis, and activated platelets are a source of the most potent naturally occuring vasoconstrictors, thromboxanes and cyclic endoperoxides. Consistently, glomerular capillary thrombosis and afferent arteriolar vasoconstriction may account, at least in part, for the ubiquitous nephrotoxicity seenwith cyclosporine administration [A]. The purpose of this study was twofold. To determine if CsA alters platelet reactivity, its effects on human platelet aggregation and resultant thromboxane (TX) A, release in response to the soluble agonists adenosine diphosphate (ADP) and epinephrine (EPI) were assessed.The second purpose was to determine if CsA affects the expression of platelet surface fibrinogen receptors. Regardless of agonist, fibrinogen binding is a necessary common pathway mediating platelet aggregation. MATERIALS
AND METHODS
All studies were approved by Temple University’s Institutional Review Board and the University of Pennsylvania Committee on Human Investigation. All procedures followed were in accordance with the ethical standards of the Helsinki Declaration of 1975. Verbal and written informed consent was obtained from each volun-
’ Presented at the Annual Meeting of the Association for Academic Surgery, Houston, TX, November 14-17,199O. * This work was supported in part by funds from the National Institutes of Health Grants HL-34026, HL-19055, and HL-15226. ’ Dr. Addonizio is the recipient of a National Institutes of Health Young Investigators Award HL-22346 and a Hartford Foundation Scholarship.
93
0022-4So4/91
$1.50
Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
94
JOURNAL
OF
SURGICAL
RESEARCH:
teer prior to phlebotomy. Normal volunteers abstained from all medications, including aspirin and nonsteroidal anti-inflammatory-containing drugs, for at least 2 weeks prior to donating. Platelet Aggregation
and Thromboxane
Release
One hundred twenty milliliters of blood was drawn by venipuncture into plastic syringes containing 1 vol of 3.8% trisodium citrate for every 9 vol of blood. To one 50-ml aliquot, CsA (Sandimmune iv, Sandoz Ltd.) was added to achieve a final concentration of 1,000 rig/ml of whole blood, to another 50-ml aliquot, an equal volume of normal saline (control) was added. The dose of cyclosporine chosen represents a value equivalent to a whole blood concentration achieved within several hours after drug administration in uiuo. All samples were incubated at 37°C for 15 min and 3 hr. The remaining 20 ml of blood was divided into four equal aliquots, two containing CsA and two containing saline, in order to quantitate the potential maximum amount of releasable thromboxane. After incubation, the samples were processed for platelet aggregation studies and quantitation of resultant TxA, generation (vide infra). To perform platelet aggregation studies, platelet-rich plasma (PRP) and platelet-poor plasma (PPP) were prepared as previously described [5]. Briefly, after incubation with either CsA or saline, whole blood samples were centrifuged at 15Og for 10 min at 23°C. After gentle aspiration of the PRP (350,000 + 50,000 platelets/pi), the remaining blood was centrifuged at 12,000g for 4 min at 23°C in a microcentrifuge to obtain PPP ( 0.05) and 763 f 288 rig/ml vs 902 + 341 rig/ml (P > 0.05) in the absence and presence of CsA at 15 min and 3 hr, respectively (Fig. 6). Spontaneous thromboxane levels generated in PRP samples merely spun without the addition of agonist were 2310 +- 307 rig/ml in control platelets and 2631 +- 383 rig/ml in CsA-treated platelets following a 15-min incubation (n = 18, P > 0.05). After 3 hr respective levels were 4403 f 734 and 5693 f 2535 rig/ml (n = 18; P > 0.05) (not shown). Serum thromboxane levels generated in control and CsA-treated whole blood allowed to clot were 543 _+88 and 542 3- 81 rig/ml at 15 min and 639 f 117 and 566 f 108 rig/ml at 3 hr (not shown).
Platelet Aggregation
Fibrinogen
For control samples, mean threshold concentration of ADP decreased from 1.90 to 1.49 PM (n = 19; P < 0.05) in cyclosporine-treated platelets following a 15min incubation. After a 3-hr incubation, the threshold dose decreased from 2.86 to 2.11 PM (n = 16; P < 0.05) (Figs. IA and IB). At 15 min mean threshold dose of epinephrine decreased from 0.20 ph4 in control platelets to 0.16 p&fin CsA-incubated platelets (n = 8; P > 0.05). At 3 hr the threshold dose decreased from 1.54 to 0.79 PM (n = 8; P < 0.05) (Figs. 2A and 2B). A representative dose-response curve to ADP and EPI in the presence and absence of CsA is illustrated in Figs. 3 and 4.
Control platelets demonstrated 26,635 + 4841 (X _+ SEM) fibrinogen receptors per platelet. Cyclosporine
Thromboxane
Receptor Expression
lOOT----^-^-----
Release
Following stimulation with threshold concentrations of ADP, supernatant TxB, levels were 244 + 44 rig/ml in control platelets compared to 265 f 54 rig/ml in CsAtreated platelets (n = 12; P > 0.05) following a 15-min incubation. At 3 hr respective thromboxane generation was 333 f 57 rig/ml vs 442 + 81 rig/ml (n = 9; P > 0.05) (Fig. 5). Consistently, in EPI-stimulated platelets, TxB,
uMADP
FIG. against unteer sporine
3. Percentage aggregation varying log concentrations following a 3-hr incubation. (1000 rig/ml whole blood).
(light transmittance) is plotted of ADP from a representative vol(0) Saline (control); (0) cyclo-
96
JOURNAL
OF
SURGICAL
‘00 got
RESEARCH:
51, NO.
2, AUGUST
1991
-
1500
I
A 3 E 1200 \ F?
80i L
VOL.
701
60 -50 -40 ~30 ~20 -10 -A/-
0e 0 010
0 too
1 000
z al
900
si d Ez
600
is “I k
300
l 2 500
0
t 15 MIN
uM EPI
FIG. 4. Percentage aggregation (light transmittance) against varying log concentrations of EPI from a representative teer following a 3-hr incubation. See legend to Fig. 3.
is plotted volun-
incubation significantly increased fibrinogen receptor expression to 35,925 -t 7290 receptors per platelet (n = 7; P < 0.05). Receptor number increased in 6 of 7 volunteers. Scatchard plot analysis yielded Kd values of 3.77 + 0.74 X 10e7 and 4.57 t 0.84 X low7 in control and cyclosporine-treated platelets, respectively (P = 0.247). Thus, receptor number increased without a significant change in receptor affinity. A representative Scatchard plot, demonstrating an increase in receptor expression by cyclosporine with unchanged affinity, is depicted in Fig. 7. DISCUSSION Cyclosporin A has revolutionized the field of organ transplantation, increasing graft survival and permitting the proliferation of multiorgan transplantation. Its potent immunosuppressant properties have also been useful in the treatment of several autoimmune disorders. Unfortunately, this agent is not without side effects.
600
,
3 HR
FIG. 6. Thromboxane B, (rig/ml) release (at mean threshold dose of EPI) following a 15min (A, n = 6) and a 3-hr (B, n = 7) incubation with either saline or cyclosporine. Bars represent %+ SEM. See legend to Fig. 1.
Similar to other immunosuppressive agents, CsA increases the risks of infection, lymphoproliferative disorders, and malignancy. Interestingly, CsA has also been associated with complications seemingly unrelated to its immunosuppressant actions. Although never studied in a large-scale, prospective, randomized fashion, CsA has been associated with an increased incidence of thrombotic and thromboembolic phenomena [l-3]. These phenomena include deep venous thrombosis, pulmonary embolism, renal vein thrombosis, mesenteric thrombosis, microangiopathies, and the hemolytic uremic-like syndrome. Even the most frequently troublesome toxicity of CsA, nephrotoxicity, may have origin in activation of haemostatic elements. Microangiopathy characterized by frank glomerular capillary thrombosis has been observed in some cases. In addition, decreased renal plasma flow occurs as a result of afferent arteriolar vasoconstriction [12]. It has been suggested that intrarenal
1
_1 20
M (i508) 15 MIN
FIG. 5. Thromboxane B, (rig/ml) release (at mean threshold dose of ADP) following a 15-min (A, n = 12) and 3-hr (B, n = 9) incubation with either saline or cyclosporine. Bars represent I k SEM. See legend to Fig. 1.
FIG. platelet receptor determines Control,
7. Representative Scatchard plot demonstrating increased surface fibrinogen receptor expression without alteration of affinity in the presence of cyclosporine. Abscissa-intercept receptor number, while slope determines allinity. (0) r = 0.94; (0) cyclosporin A, r = 0.99.
FISHMAN
ET
AL.:
CYCLOSPORIN
A ENHANCES
platelet activation with resulting thromboxane release may join with a functional alteration of local endothelium to mediate this vasoconstriction. Substantiating this possibility, Benigni et al. demonstrated a selective increase in urinary 2,3-dinor-TxB,, a thromboxane metabolite derived principally from formed blood elements, rather than the parent compound which, when present in urine, reflects renal thromboxane synthesis [4]. Thus, thrombosis in the broad sense and platelet activation in particular may contribute to a variety of cyclosporinemediated complications. Unfortunately, the mechanism of a CsA-related activation of formed haemostatic elements has not been completely delineated. To date Grace et al. demonstrated augmented platelet aggregation in response to soluble agonists [13]. These investigators noted that CsA enhanced in vitro platelet aggregation in response to subthreshold doses of several soluble agonists including ADP and EPI. Furthermore, a similar augmentation in responsiveness was demonstrated in platelets obtained from normal volunteers given a single oral dose of CsA. In addition, in transplant patients taking CsA, aggregation was shown to be of greater magnitude when blood was drawn at the time of peak serum levels compared to that drawn during trough levels. Finally, reduced platelet sensitivity occurred when CsA was removed from patients’ immunosuppressive regimens. Finally, Cohen et al. detected chronic platelet hyperactivity in 21 patients receiving cyclosporine [14]. Platelet aggregation responses to low doses of ADP were significantly increased up to 1 year after initiating cyclosporine therapy. Total platelet ADP and ATP content, as well as releasable quantities of these dense granule nucleotides in response to collagen were decreased, and plasma platelet factor 4 levels were increased, suggesting chronic platelet activation. Cyclosporine’s prothrombotic effects are not limited to platelets. Thomson et al. [15] and others [16, 171 found an increase in monocyte procoagulant activity. Carlsen showed enhanced cytokine-stimulated endothelial cell thromboplastin production [ 181. We and others have demonstrated alterations in endothelial arachidonate metabolism, which can disturb the delicate balance between prothrombotic thromboxane synthesis and antithrombotic prostacyclin synthesis [ 19-221. The present study unequivocally demonstrates that CsA augments platelet aggregation in uitro. Moreover, enhanced platelet responsiveness occurs to both ADP and epinephrine, common soluble agonists used to induce platelet activation. Thus, our findings corroborate and extend those of Grace et al. [13] and Cohen et al. [14]. Although it is, perhaps, predictable that ADP and epinephrine would act similarly, significant differences characterize their mechanism of action [23]. ADP but not epinephrine induces platelet shape change, and epinephrine but not ADP inhibits adenyl cyclase in broken cell preparations [24]. The two agents also differ quanti-
PLATELET
REACTIVITY
97
tatively in their ability to inhibit PGE,-stimulated formation of cyclic AMP [25]. Finally, ADP and EPI are known to initiate platelet activation by interacting with distinctly separate surface membrane receptors. Because the enhanced platelet responsiveness induced by CsA appears to be agonist independent, it would seem likely that CsA acts on a common pathway mediating platelet function. For example, platelets will not aggregate in the absence of fibrinogen binding to the membrane glycoprotein complex IIb-IIIa [26 1. Although likely continuously present, the fibrinogen receptor of this glycoprotein complex must be exposed to bind fibrinogen [27-281. This study demonstrates a cyclosporine-induced increase in platelet responsiveness to soluble agonists coincident with increased fibrinogen receptor availability, but without alteration in thromboxane production. The mechanism by which this occurs requires further investigation. However, several explanations seem plausible. First, the highly lipophilic cyclosporine molecule may insert into the platelet membrane, thus altering membrane structure and glycoprotein 1%IIIa conformation or mobility. Second, cyclosporine is known to alter intracellular calcium flux in the lymphocyte, its intended cell of action, by binding with cyclophilin. It may similarly increase cytosolic calcium concentration in platelets, which, in turn, can lead to fibrinogen receptor expression and platelet activation. Additionally, lymphocytes and platelets share several surface antigens, including CD9 and CD69, which play a role in platelet aggregation [29, 301. Indeed, CD9 has been shown to associate with the glycoprotein IIb-IIIa complex when stimulated with monoclonal antibody [29]. Cyclosporine may effect both the lymphocyte and platelet by interacting with these surface antigens. Any of these mechanisms might explain agonist-independent hypersensitivity of platelets exposed to CsA. No increase in thromboxane release was observed over and above what is normally observed at a given level of activation. Cyclosporine lowered the concentration of ADP required to completely aggregate platelets. However, once completely aggregated, platelets from both groups produced the same amount of thromboxane B,. Thus, cyclosporine-mediated increase in platelet sensitivity appeared to be independent of thromboxane synthesis. In summary, cyclosporine increases platelet sensitivity to soluble agonists and, perhaps causally, increases availability of platelet fibrinogen receptors. Whether or not this effect is specific for fibrinogen receptors or is due to a nonspecific effect on the platelet membrane remains unknown. Nevertheless, CsA-induced platelet hypersensitivity could contribute to the thrombotic and nephrotoxic effects of cyclosporine therapy. REFERENCES 1.
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HKQ!iAKL‘H:
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