- Email: [email protected]
von Willebrand factor protein on human platelet adenylate cyclase activity
THROMBOSIS RESEARCH30; 301-308, 1983 0049-3848/83/090301-08$03.00/O Printed Copyright (c) 1983 Pergamon Press Ltd.
BRIEF
in the USA. All rights reserved.
COMMUNICATION
INHIBITORY EFFECT OF RISTOCETIN AND FACTOR VIII/VON WILLEBRAND FACTOR PROTEIN ON HUMAN PLATELET ADENYLATE CYCLASE ACTIVITY
KUO-JANG KAO, BIE-SHUNC TSAI, PATRICK A. MCKEE, ROBERT J. LEFKOWITZ and SALVATORE V. PIZZO Departments of Pathology, Biochemistry, Medicine The Howard Hughes Medical Institute Duke University Medical Center Durham, North Carolina 27710
and
(Received 21.12.1982; Accepted in revised form 15.1.1983 by Editor K.M. Brinkhous. Editorial Office 18.2.1983) Received in final form by Executive INTRODUCTION Cyclic AMP (CAMP) is a weak inhibitor of platelet aggregation (I). Inhibitors of platelet aggregation such as adenosine, PGEl, PGD2, and prostacyclin stimulate the activity of platelet adenylate cyclase or increase intraplatelet CAMP concentration (210). Intraplatelet CAMP appears to function as a second messenger for inhibitors of platelet aggregation (1 I). By contrast, platelet aggregating agents, such as ADP, epinephrine, thrombin, and collagen reduce intraplatelet CAMP concentration or inhibit basal or PGEl-stimulated adenylate cyclase activity (12-19). Since most platelet aggregating agents exert their effect through binding to specific receptors on the plasma membrane (20,211, inhibition of platelet adenylate cyclase activity is one biochemical response to receptor binding of these platelet aggregating agents. Human factor VIII/van Willebrand factor (FVIII/vWF) is a plasma glycoprotein which is essential for platelet adhesion and subsequent platelet plug formation at sites of vascular injury during primary phase hemostasis (22,23). The biological activity of FVIII/vWF can be assayed by measuring the rate of platelet aggregation at various concentrations of FVIII/vWF in the presence of a constant concentration of ristocetin-A (241, a glycopeptide antibiotic from Nocardia lurida (25). Ristocetin-A is required for the binding of human FVIII/vWF to human platelets and their subsequent aggregation (26,271. FVIII/ vWF in the presence of ristocetin-A also induces the aggregation of formaldehyde-fixed human platelets (28) suggesting a passive agglutination mechanism (28). There is, however, a significant difference in the size and physical properties of platelet clumps formed when the aggregation of fresh and fixed platelets is compared (29). This difference suggests an active platelet response after FVIII/vW binds to its platelet receptors (29). Caller has shown that agents which raise platelet CAMP inhibit platelet aggregation induced by addition of ristocetin or bovine FVIII/vWF to platelet rich plasma (30). Key Words:
Platelet
adenylate
cyclase;
Factor
301
VIII/van
Willebrand
factor;
ristocetin
302
RISTOCETINANDADENYLATE CYCLASE
Vo1.30,
No.3
Bovine FVIII/vWF is capable of binding to human platelets with a lower affinity than human FVIII/vWF but unlike the latter, it also induces aggregation. This reaction does not require the presence of ristocetin-A (28,291. It is believed that ristocetin-A functions to mask platelet negative changes which otherwise interfere with the binding of human, but not bovine FVIII/vWF (26,27,29). There is still controversy as to whether FVIII/vWF in the presence of ristocetin induces platelet aggregation by an active mechanism. We now report direct biochemical evidence demonstrating an active platelet response after binding of FVIII/vWF to human platelet. This response consists of inhibition of platelet adenylate cyclase, a result obtained with other platelet aggregating agents. These observations are consistent with the studies of Caller described above (30). MATERIALS AND M ETHODS Materials. Human FVIII/vWF was purified as previously described from intermediatepurity FVIII/vWF concentrates (American National Red Cross Laboratory, Bethesda, MD) (26). Ristocetin-A (ristocetin) was purchased from H. Lundbeck h Co. (Copenhagen, Denmark). EDTA, phosphoer#pyruvate, pyruvate kigase, PC+ myokinase, and cyclic AMP were from Sigma Co. [ a- P ]-labeled ATP and [ Hllabeled cyclic AMP were obtained from New England Nuclear Corp. Purified bovine FVIII/vWF was a gift of Dr. E.W. Davie, Department of Biochemistry, University of Washington, Seattle, WA. This preparation possessed a potency one fifth that of purified human FVIII/vWF in a platelet aggregation assay. Preparation of platelet lysates. The platelet pellet was obtained from titrated venous blood by differential centrifugation (26). The platelet pellet was washed twice for one min at room temperature by resuspending in 0.05M Tris-HCI, pH 7.4 containing 0.005M EDTA, homogenized by 20 strokes with a motor-driven, teflon-tipped pestle and centrifuged at 39,000 g for 10 min at 4’ C. The resulting pellet was washed once by resuspending in 0.03M Tris-HCI, pH 7.5. Platelet lysate prepared from 1 ml of platelet rich plasma was finally resuspended in 1OOul of 0.03M Tris-HCl, pH 7.5. Adenylate cyclase assay. Assays were performed as described by Newman et al. (17). Each incubation mixturfjfontained Tris-Hq, 30 mM, pH 7.5; MgC12, IOmM;aP, 0.1 PI-ATP, 1-2 x IO cpm; phosphoenolpyruvate, 5 mM; pyruvate mM; ATP, 0.1 mM; [akinase, 40 &ml; myokinase, 20 &ml, and platelet lysate obtained from 0.2 ml of platelet rich plasma. The final volume of $his incubation mixture was 55 J. All incubations were carr’9 out for 10 min at 37 G and terminated by adding 1 ml of a so&ion containing C PI-CAMP (15,000 cpm), 100 pg of ATP, and 50 ug of CAMP. [ PI-CAMP was isolated using the method of Salomon et al (31). The recovery of [ 32P]-cAM P from column chromatography was monitored bying [ 3H I-CAMP. RESULTS AND DISCUSSION Ristocetin is essential for the binding of human FVIII/vWF to its receptors and the degree of binding is proportional to ristocetin concentrations between 0.2 to 1.0 mg/ml (26,27). The effect of FVIII/vWF on the activity of platelet adenylate cyclase was, Basal therefore, studied at different concentrations of ristocetin (O-O.9 mg/ml). platelet adenylate cyclase activity was significantly inhibited by increasing concenBasal activity was trations of ristocetin in the absence of FVIII/vWF (Table I). substantially inhibited by 0.9 mg/ml of ristocetin, but a further inhibition of adenylate cyclase was observed when human FVIII/vWF also was added to each incubation at a final concentration of 1.8 &ml (Table 1). Adenylate cyclase activity was not inhibited Therefore binding of FVIII/vWF to its by human FVIII/vWF in the absence of ristocetin.
Vo1.30,
No.3
303
RISTOCETIN AND ADENYLATECYCLASE
receptors is required for further inhibition of adenylate cyclase activity FVIII/vWF to its receptors requires the presence of ristocetin (26).
since
binding
of
The further of ristocetin
inhibition of adenylate cyclase activity by human FVIII/vWF in the presence was easier to detect at increasing concentrations of ristocetin from 0.36 This result is compatible with earlier observations that more mg/ml to 0.72 mg/ml. FVlII/vWF receptor binding occurs at higher concentrations of ristocetin (26,27), but the inhibition of platelet adenylate cyclase activity by ristocetin itself complicates direct However, the inhibition of platelet adenylate cyclase can interpretation of the results. be amplified after stimulation with PGE (12,14-19). The experiments, therefore, were described (12,14-19). The repeated in the presence of 10 @l PC!E as previously inhibition of platelet adenylate cyclase activity by ristocetin again was observed (Table II) and human FVIII/vWF augmented this effect. The degree of further inhibition of PCE.l-stimulated adenylate cyclase activity by human FVIII/vWF was relatively proportional to the concentration of ristocetin. These findings are consistent with earlier observations that the binding of human FVIII/vWF to its receptors is dependent on the presence of ristocetin and the degree of FVlII/vWF receptor occupancy is proportional to the ristocetin concentration. Bovine FVIII/vWF induces human platelet aggregation in the absence of ristocetin (28) by binding to the human platelet FVIII/vWF receptor (26,32). We therefore studied the effect of bovine FVIII/vWF on human platelet adenylate cyclase activity in the absence of ristocetin. Bovine FVIII/vWF inhibited basal as well as PGEl-stimulated human platelet adenylate cyclase activity (Table III). In addition, the inhibitory effect of bovine FVIII/vWF and that of ristocetin are additive. In another experiment, human FVIlI/vWF was substituted for bovine FVIII/vWF at a concentration of 20 &ml. In the absence of ristocetin, no inhibition of basal platelet adenylate cyclase was observed. These results provide additional evidence that the binding of FVIII/vWF to platelets inhibits adenylate cyclase activity. Human platelets fixed with formaldehyde retain the ability to aggregate when they are treated with FVIII/vWF in the presence of ristocetin (27). These data suggest that ristocetin-induced platelet aggregation is a process of passive agglutination. Nevertheless, a qualitative difference exists between aggregates of fresh platelets and those of fixed platelets induced by ristocetin. Formaldehyde-fixed platelets form fine granular aggregates, but large and dense platelet clumps occur when fresh platelets are exposed to ristocetin and human FVIlI/vWF. The latter form of aggregation was named “super aggregation ‘I by Kirby and Mills (29). Ultrastructural study of aggregates of fresh platelets induced by ristocetin and human FVIII/vWF demonstrated that shape change and degranulation occurred in these platelets (33). These observations suggest that interaction between human FVIlI/vWF and platelets triggers an active response. Hence, the passive agglutination of platelets induced by ristocetin and FVIII/vWF is not the only action which is responsible for ristocetin-induced platelet aggregation in platelet rich plasma or freshly washed platelets. The present study provides direct biochemical evidence, demonstrating that FVIlI/vWF binding to its receptor leads to inhibition of platelet adenylate cyclase activity, an effect which has also been observed for many other platelet aggregating agents. Moreover, ristocetin itself is an inhibitor of platelet adenylate cyclase with high efficacy. The exact mechanisms of the inhibition of platelet adenylate cyclase activity by ristocetin needs further investigation. ACKNOWLEDGEMENT This work was supported by the National 24066, HL 15615, HL 20339 and HL 16037).
Heart
Lung and Blood
Institute
(HL
c)
b)
a)
+
+
+
+
+
+
Addeda
FVIII/vWF
2.0
3.5 1.8
5.7 3.6
7.8 6.8
18.0 16.7
27.9 27.8
29.2’ 30.8
pmole/lO min %
6.8
12.0 6.2
19.5 12.3
26.7 23.3
61.6 57.2
95.5 95.2
100 105
CAMP Synthesized
Cyclase Activity
and Human FVIII/vWF
48.9
36.9
12.7
7.1
0.3
0
Each value is the mean of duplicated incubations. The variation The same results were obtained in three separate experiments.
between duplicates
was less than 7%.
of 1.8 &ml.
FVIII/vWF (%Ib
Inhibition of Adenylate Cyclase Activity by
(-1: no FVIII/vWF was added; (+): 0.1 M of FVIII/vWF was added in each incubation to a final concentration CAMP synthesized in the CAMP synthesize in the absence of FVIII/vWF presence of FVIII/v WF Inhibition of adenylate cyclase = CAMP synthesized in the absence of FVIII/vWF
0.9
0.72
0.54
0.36
0.18
0.09
0
(mg/ml)
Ristocetin Concentration
on Basal Adenylate
Effect of Ristocetin
TABLE 1
P
incubations. were obtained
Each value is the mean of duplicated was less than 5%. The same results
d)
activity
was calculated
according
to’the
of Adenylate Activity by
b
35.3
30.4
41.6
24.3
5.5
1.5
of 1.8 ug,/ml.
FVIII/vW F (%I=
Inhibition Cyclase
to a fin& concentration
of 10 ~&l PCE1.
22.4 16.7
39.4 22.6
48.0 34.0
53.8 40.7
83.5 78.9
100 98.5
%
was added in each incubation
in the presence
127.8 95.5
185.6 129.3
274.0 160.1
307.1 232.6
476.9 450.5
571.1d 562.5
min
The variation between duplicates in two separate experiments.
(+): 0.1 pg of FVIII/vWF cyclase
was added;
The percent inhibition of adenylate equation shown in Table IX.
was studied
c)
cyclase
(-1: no FVIII/vWF
adenylate
b)
of platelet
+
+
+
+
+
+
pmole/lO
CAMP Synthesized
Activitya
FVIII/vWF
Cyclase
and Human
2
Adenylate
The activity
0.90
0.72
0.54
0.36
0.18
0
Addedb
FVIII/vWF
Platelet
of Ristocetin
a)
(mghl)
Ristocetin Concentration
on PGEl-Stimulated
Effect
TABLE
306
vo1.30, No.3
RISTOCETINAND ADENYLATE CYCLASE
TABLE 3 Effect of Bovine FVIII/vWF on the Activity of Platelet
Adenylate
\Cd
Cyclase
CAMP Synthesized Basal
PGEl (1qM) stimulated %
pmole/lOmin
pmole/lOmin 571.1
100
Control
29.2a
Bovine FVIII/vWF (18 &ml)
20.1
68.8
440.7
77.2
Ristocetin (0.18 mg/ml)
18.0
61.6
477.0
83.5
Bovine FVIII/vWF + Ristocetin
12.0
41.1
___
___
a)
100
%
Each value is the mean of duplicate incubations. The variation between duplicates was less than 3%.
- 600
- 400
0
0.25 0.5 0.75 Ristocetin (mg/ml)
FIG. 1
I.0
vol.
30,
RISTOCETIN
No.3
AND ADENYLATE
CYCLASE
307
REFERENCES 1.
MARCUS, A.J. and ZUCKER, M.B. In “The Physiology of Blood Platelets: Recent Biochemical, Morphological and Clinical Research.*’ New York: Grune & Stratton, 1965, p. 53.
2.
ABDULLA, Y.H. B-Adrenergic 9 171-177, 1969. -Res. -9
3.
CORMAN adenylate
4.
CORMAN, R.R., FITZPATRICK, F.A. and MILLER, O.V. Reciprocal regulation of human platelet cyclic AMP levels by thromboxane A2 and prostacyclin. & Cyclic Nucleotide Res. 2, 597-609, 1978.
5.
HASLAM, adenylate 1972.
6.
MARQUIS, N.R., VIGDAHL, R.L. and TAVORMINA, P.A. regulation by cyclic AMP and prostaglandin El. Biochem. )6, 965-972, 1969.
7.
MILLS, D.C.B. Zucker, teds.). 504-512.
8.
MILLS, D.C.B. and MACFARLANE, D.E. Stimulation of human platelet cyclase by prostaglandin D2. Thromb. Res. 2, 401-412, 1974.
9.
TATESON, J.E., MONCADA, 5. and VANE, J.R. Effects of prostacyclin or cyclic AMP concentrations in human platelets. Prostaglandins 13, 389-397, 1977.
10.
WOLFE, S.M. and SHULMAN, N.R. Adenyl cyclase Biochem. Biophys. Res. Comm. 5, 265-272, 1969.
Il.
HASLAU, R.J., platelet function 1978.
12.
MARQUIS, N.R., BECKER, J.A. and VUGDAHL, R.L. Platelet aggregation III. An epinephrine induced decrease in cyclic A%IP synthesis. Biochem. Biophys. Res. Comm. ,9, 783-789, 1970.
13.
SALZMAN, 363.
14.
HASLAM, S/v!. tion Symposium,
15.
JAKOBS, K.H., SAUR, W. and SCHULTZ, G. Reduction of adenylate activity lysates of human platelets by the alpha-adrenergic component of epinephrine. Cyclic Nucleotide Res. 2, 381-392, 1976.
16.
hlILLER, O.V., JOHNSON, R.A. and CORMAN, R.R. stimulated CAMP accumulation in human platelets by Prostaglandins g, 599-609, 1977.
receptors
in human platelets.
R.R., BUNTING, S. and MILLER, O.V. Modulation cyclase by prostacyclin. Prostaglandins 11, 377-388,
J.
Atheroscler.
of human 1977.
platelet
R.J. and LYNHAM, J.A. Activation and inhibition of blood platelet cyclase by adenosine or by 2-chloroadenosine. ~Life Sci. -11 1143-1154,
E.W.
In “Platelet Washington,
Function Testing. H.J. Day, l-i. Holmsen D.C.: U.S. DHEW Publication No. 78-1087,
DAVIDSON, M.M.L., by cyclic nucleotides.
Cyclic
Platelet aggregation I. Biophys. Res. Comm.
activity
and M.B. 1976, pp.
adenylate
in human platelets.
Regulation of blood DAVIES, T. et al Adv. Cyclic Nucleotide Res. 2, 533-552,
AMP and platelet
function.
New Eng. J. Med.
In “Biochemistry and Pharmacology of Platelets”, Ciba vol. 35. Amsterdam: Elsevier Co., 1975, pp. 121-151.
286, 358-
Founda-
in &
Inhibition of PCElthrornboxane A2. -
RISTOCETIN
308
AND ADENYLATE
CYCLASE
Vo1.30,
17.
NEWMAN, K.D., WILLIAM, L.T., BISHOPRIF, N.H. et al. Identification of aadrenergic receptors in human platelets by [ H] dihydroergocryptine binding. L Clin. Invest. 6i, 395-402, 1978.
18.
GORMAN, R.R. thromboxane AZ.
Modulation of human platelet Fed. Proc. 38, 83-88, 1979.
19.
COOPER, D.M.F. plasma membrane
and RODBELL, M. adenylate cyclase.
20.
MILLS, D.C.B. and MACFARLANE, D.E. In “Platelets in Biology and Pathology.” 1.1. Gordon, ted.). Amsterdam: North-Holland Co., 1976, pp. 159-202.
21.
NICHOLS, W.L., GERRARD, J.M. and DIDISHEIM, P. In “Recent L. Poller ted.). Edinburgh: Churchill-Livingstone Blood CoaguIation”. pp. l-39.
22.
HOVIG, T. and STORMORKEN, H. Ultrastructure studies on the platelet plug formation in bleeding time wounds from normal individuals and patients with von Willebrands disease. Acta Path. IMicrobiol. Stand. 248, 105-122, 1974.
23.
SAKARIASSEN, J.J., BOLHUIS, P.A. and SIXMA, J.J. Human platelet adhesion to artery subendothelium is mediated by factor VIII von Willebrand factor bound to the subendothelium. Nature 279, 636-638, 1979.
24.
WEISS, H.J., HOYER, L.W., RICKLES, F.R. et al. Quantitative assay of a plasma factor deficient in von Willebrand’s disease that is necessary for platelet aggregation. J. Clin. Invest. 52, 2708-2716, 1973.
25.
The chemical basis for the action PERKINS, H.R. and NIETO, ?.!. Ann. N.Y. Acad. Sci. 235, 348-363, 1974. vancomycin group of antibiotics.
26.
KAO, K.J., PIZZO, S.V. and MCKEE, P.A. Demonstration specific binding sites for factor VIIl/von Willebrand factor Clin. Invest. 63, 656-664, 1979.
27.
KAO, K.J., PIZZO, S.V. and MCKEE, P.A. Platelet functional correlation VIIl/von Willebrand factor: Proc. Natl. ristocetin-induced platelet aggregation. 5320, 1979.
28.
ALLAIN, J.P., COOPER, H.A., WAGNER, R.H. and BRINKHOUS, K.M. Platelets a new reagent for assay of von Willebrand factor fixed with paraformaldehyde: J. Lab. Clin. hAed. g, 318-328, 1975. and platelet aggregating factor.
29.
KIRBY, E.P. and MILLS, D.C.B. The interaction platelets. J. Clin. Invest. 2, 491-502, 1975.
30.
COLLER, B.S. Inhibition of von Willebrand factor-dependent platelet function increased platelet cyclic AMP and its prevention by cytoskeleto*disrupting agents. --Blood 57, 846-855, 1981.
31.
SALOMON, Y., LONDOS, C. and RODBELL, u. cyclase assay. Anal. Biochem. 8, 541-548, 1974.
32.
SUZUKI, platelets Thromb.
33.
by prostacyclin
and
ADP is a potent inhibitor of human platelet Nature 282, 517-518, 1979.
K., NISHIOKA, J. and HASHIMOTO, for von Willebrands factor and Res. 11, 215-223, 1980.
TS’AO, C.-H., GREEN, gated human platelets !iaematol. 11, 287-297,
function
D. and ROSSI, E.C. tortuous boundaries 1973.
of
the
and characterization on human platelets.
of &
receptors for human factor of receptor occupancy and Acad. Sci. U.S.A. 76, 5317-
of bovine factor
S.
bovine
Advances in Co., 1981,
A highly
Identical
platelet
Ultrastructure and swollen
VIII with human
sensitive
by
adenylate
binding site on human aggregating factor.
of ristocetin aggreStand. J. granules.
No.3
BRIEF
in the USA. All rights reserved.
COMMUNICATION
INHIBITORY EFFECT OF RISTOCETIN AND FACTOR VIII/VON WILLEBRAND FACTOR PROTEIN ON HUMAN PLATELET ADENYLATE CYCLASE ACTIVITY
KUO-JANG KAO, BIE-SHUNC TSAI, PATRICK A. MCKEE, ROBERT J. LEFKOWITZ and SALVATORE V. PIZZO Departments of Pathology, Biochemistry, Medicine The Howard Hughes Medical Institute Duke University Medical Center Durham, North Carolina 27710
and
(Received 21.12.1982; Accepted in revised form 15.1.1983 by Editor K.M. Brinkhous. Editorial Office 18.2.1983) Received in final form by Executive INTRODUCTION Cyclic AMP (CAMP) is a weak inhibitor of platelet aggregation (I). Inhibitors of platelet aggregation such as adenosine, PGEl, PGD2, and prostacyclin stimulate the activity of platelet adenylate cyclase or increase intraplatelet CAMP concentration (210). Intraplatelet CAMP appears to function as a second messenger for inhibitors of platelet aggregation (1 I). By contrast, platelet aggregating agents, such as ADP, epinephrine, thrombin, and collagen reduce intraplatelet CAMP concentration or inhibit basal or PGEl-stimulated adenylate cyclase activity (12-19). Since most platelet aggregating agents exert their effect through binding to specific receptors on the plasma membrane (20,211, inhibition of platelet adenylate cyclase activity is one biochemical response to receptor binding of these platelet aggregating agents. Human factor VIII/van Willebrand factor (FVIII/vWF) is a plasma glycoprotein which is essential for platelet adhesion and subsequent platelet plug formation at sites of vascular injury during primary phase hemostasis (22,23). The biological activity of FVIII/vWF can be assayed by measuring the rate of platelet aggregation at various concentrations of FVIII/vWF in the presence of a constant concentration of ristocetin-A (241, a glycopeptide antibiotic from Nocardia lurida (25). Ristocetin-A is required for the binding of human FVIII/vWF to human platelets and their subsequent aggregation (26,271. FVIII/ vWF in the presence of ristocetin-A also induces the aggregation of formaldehyde-fixed human platelets (28) suggesting a passive agglutination mechanism (28). There is, however, a significant difference in the size and physical properties of platelet clumps formed when the aggregation of fresh and fixed platelets is compared (29). This difference suggests an active platelet response after FVIII/vW binds to its platelet receptors (29). Caller has shown that agents which raise platelet CAMP inhibit platelet aggregation induced by addition of ristocetin or bovine FVIII/vWF to platelet rich plasma (30). Key Words:
Platelet
adenylate
cyclase;
Factor
301
VIII/van
Willebrand
factor;
ristocetin
302
RISTOCETINANDADENYLATE CYCLASE
Vo1.30,
No.3
Bovine FVIII/vWF is capable of binding to human platelets with a lower affinity than human FVIII/vWF but unlike the latter, it also induces aggregation. This reaction does not require the presence of ristocetin-A (28,291. It is believed that ristocetin-A functions to mask platelet negative changes which otherwise interfere with the binding of human, but not bovine FVIII/vWF (26,27,29). There is still controversy as to whether FVIII/vWF in the presence of ristocetin induces platelet aggregation by an active mechanism. We now report direct biochemical evidence demonstrating an active platelet response after binding of FVIII/vWF to human platelet. This response consists of inhibition of platelet adenylate cyclase, a result obtained with other platelet aggregating agents. These observations are consistent with the studies of Caller described above (30). MATERIALS AND M ETHODS Materials. Human FVIII/vWF was purified as previously described from intermediatepurity FVIII/vWF concentrates (American National Red Cross Laboratory, Bethesda, MD) (26). Ristocetin-A (ristocetin) was purchased from H. Lundbeck h Co. (Copenhagen, Denmark). EDTA, phosphoer#pyruvate, pyruvate kigase, PC+ myokinase, and cyclic AMP were from Sigma Co. [ a- P ]-labeled ATP and [ Hllabeled cyclic AMP were obtained from New England Nuclear Corp. Purified bovine FVIII/vWF was a gift of Dr. E.W. Davie, Department of Biochemistry, University of Washington, Seattle, WA. This preparation possessed a potency one fifth that of purified human FVIII/vWF in a platelet aggregation assay. Preparation of platelet lysates. The platelet pellet was obtained from titrated venous blood by differential centrifugation (26). The platelet pellet was washed twice for one min at room temperature by resuspending in 0.05M Tris-HCI, pH 7.4 containing 0.005M EDTA, homogenized by 20 strokes with a motor-driven, teflon-tipped pestle and centrifuged at 39,000 g for 10 min at 4’ C. The resulting pellet was washed once by resuspending in 0.03M Tris-HCI, pH 7.5. Platelet lysate prepared from 1 ml of platelet rich plasma was finally resuspended in 1OOul of 0.03M Tris-HCl, pH 7.5. Adenylate cyclase assay. Assays were performed as described by Newman et al. (17). Each incubation mixturfjfontained Tris-Hq, 30 mM, pH 7.5; MgC12, IOmM;aP, 0.1 PI-ATP, 1-2 x IO cpm; phosphoenolpyruvate, 5 mM; pyruvate mM; ATP, 0.1 mM; [akinase, 40 &ml; myokinase, 20 &ml, and platelet lysate obtained from 0.2 ml of platelet rich plasma. The final volume of $his incubation mixture was 55 J. All incubations were carr’9 out for 10 min at 37 G and terminated by adding 1 ml of a so&ion containing C PI-CAMP (15,000 cpm), 100 pg of ATP, and 50 ug of CAMP. [ PI-CAMP was isolated using the method of Salomon et al (31). The recovery of [ 32P]-cAM P from column chromatography was monitored bying [ 3H I-CAMP. RESULTS AND DISCUSSION Ristocetin is essential for the binding of human FVIII/vWF to its receptors and the degree of binding is proportional to ristocetin concentrations between 0.2 to 1.0 mg/ml (26,27). The effect of FVIII/vWF on the activity of platelet adenylate cyclase was, Basal therefore, studied at different concentrations of ristocetin (O-O.9 mg/ml). platelet adenylate cyclase activity was significantly inhibited by increasing concenBasal activity was trations of ristocetin in the absence of FVIII/vWF (Table I). substantially inhibited by 0.9 mg/ml of ristocetin, but a further inhibition of adenylate cyclase was observed when human FVIII/vWF also was added to each incubation at a final concentration of 1.8 &ml (Table 1). Adenylate cyclase activity was not inhibited Therefore binding of FVIII/vWF to its by human FVIII/vWF in the absence of ristocetin.
Vo1.30,
No.3
303
RISTOCETIN AND ADENYLATECYCLASE
receptors is required for further inhibition of adenylate cyclase activity FVIII/vWF to its receptors requires the presence of ristocetin (26).
since
binding
of
The further of ristocetin
inhibition of adenylate cyclase activity by human FVIII/vWF in the presence was easier to detect at increasing concentrations of ristocetin from 0.36 This result is compatible with earlier observations that more mg/ml to 0.72 mg/ml. FVlII/vWF receptor binding occurs at higher concentrations of ristocetin (26,27), but the inhibition of platelet adenylate cyclase activity by ristocetin itself complicates direct However, the inhibition of platelet adenylate cyclase can interpretation of the results. be amplified after stimulation with PGE (12,14-19). The experiments, therefore, were described (12,14-19). The repeated in the presence of 10 @l PC!E as previously inhibition of platelet adenylate cyclase activity by ristocetin again was observed (Table II) and human FVIII/vWF augmented this effect. The degree of further inhibition of PCE.l-stimulated adenylate cyclase activity by human FVIII/vWF was relatively proportional to the concentration of ristocetin. These findings are consistent with earlier observations that the binding of human FVIII/vWF to its receptors is dependent on the presence of ristocetin and the degree of FVlII/vWF receptor occupancy is proportional to the ristocetin concentration. Bovine FVIII/vWF induces human platelet aggregation in the absence of ristocetin (28) by binding to the human platelet FVIII/vWF receptor (26,32). We therefore studied the effect of bovine FVIII/vWF on human platelet adenylate cyclase activity in the absence of ristocetin. Bovine FVIII/vWF inhibited basal as well as PGEl-stimulated human platelet adenylate cyclase activity (Table III). In addition, the inhibitory effect of bovine FVIII/vWF and that of ristocetin are additive. In another experiment, human FVIlI/vWF was substituted for bovine FVIII/vWF at a concentration of 20 &ml. In the absence of ristocetin, no inhibition of basal platelet adenylate cyclase was observed. These results provide additional evidence that the binding of FVIII/vWF to platelets inhibits adenylate cyclase activity. Human platelets fixed with formaldehyde retain the ability to aggregate when they are treated with FVIII/vWF in the presence of ristocetin (27). These data suggest that ristocetin-induced platelet aggregation is a process of passive agglutination. Nevertheless, a qualitative difference exists between aggregates of fresh platelets and those of fixed platelets induced by ristocetin. Formaldehyde-fixed platelets form fine granular aggregates, but large and dense platelet clumps occur when fresh platelets are exposed to ristocetin and human FVIlI/vWF. The latter form of aggregation was named “super aggregation ‘I by Kirby and Mills (29). Ultrastructural study of aggregates of fresh platelets induced by ristocetin and human FVIII/vWF demonstrated that shape change and degranulation occurred in these platelets (33). These observations suggest that interaction between human FVIlI/vWF and platelets triggers an active response. Hence, the passive agglutination of platelets induced by ristocetin and FVIII/vWF is not the only action which is responsible for ristocetin-induced platelet aggregation in platelet rich plasma or freshly washed platelets. The present study provides direct biochemical evidence, demonstrating that FVIlI/vWF binding to its receptor leads to inhibition of platelet adenylate cyclase activity, an effect which has also been observed for many other platelet aggregating agents. Moreover, ristocetin itself is an inhibitor of platelet adenylate cyclase with high efficacy. The exact mechanisms of the inhibition of platelet adenylate cyclase activity by ristocetin needs further investigation. ACKNOWLEDGEMENT This work was supported by the National 24066, HL 15615, HL 20339 and HL 16037).
Heart
Lung and Blood
Institute
(HL
c)
b)
a)
+
+
+
+
+
+
Addeda
FVIII/vWF
2.0
3.5 1.8
5.7 3.6
7.8 6.8
18.0 16.7
27.9 27.8
29.2’ 30.8
pmole/lO min %
6.8
12.0 6.2
19.5 12.3
26.7 23.3
61.6 57.2
95.5 95.2
100 105
CAMP Synthesized
Cyclase Activity
and Human FVIII/vWF
48.9
36.9
12.7
7.1
0.3
0
Each value is the mean of duplicated incubations. The variation The same results were obtained in three separate experiments.
between duplicates
was less than 7%.
of 1.8 &ml.
FVIII/vWF (%Ib
Inhibition of Adenylate Cyclase Activity by
(-1: no FVIII/vWF was added; (+): 0.1 M of FVIII/vWF was added in each incubation to a final concentration CAMP synthesized in the CAMP synthesize in the absence of FVIII/vWF presence of FVIII/v WF Inhibition of adenylate cyclase = CAMP synthesized in the absence of FVIII/vWF
0.9
0.72
0.54
0.36
0.18
0.09
0
(mg/ml)
Ristocetin Concentration
on Basal Adenylate
Effect of Ristocetin
TABLE 1
P
incubations. were obtained
Each value is the mean of duplicated was less than 5%. The same results
d)
activity
was calculated
according
to’the
of Adenylate Activity by
b
35.3
30.4
41.6
24.3
5.5
1.5
of 1.8 ug,/ml.
FVIII/vW F (%I=
Inhibition Cyclase
to a fin& concentration
of 10 ~&l PCE1.
22.4 16.7
39.4 22.6
48.0 34.0
53.8 40.7
83.5 78.9
100 98.5
%
was added in each incubation
in the presence
127.8 95.5
185.6 129.3
274.0 160.1
307.1 232.6
476.9 450.5
571.1d 562.5
min
The variation between duplicates in two separate experiments.
(+): 0.1 pg of FVIII/vWF cyclase
was added;
The percent inhibition of adenylate equation shown in Table IX.
was studied
c)
cyclase
(-1: no FVIII/vWF
adenylate
b)
of platelet
+
+
+
+
+
+
pmole/lO
CAMP Synthesized
Activitya
FVIII/vWF
Cyclase
and Human
2
Adenylate
The activity
0.90
0.72
0.54
0.36
0.18
0
Addedb
FVIII/vWF
Platelet
of Ristocetin
a)
(mghl)
Ristocetin Concentration
on PGEl-Stimulated
Effect
TABLE
306
vo1.30, No.3
RISTOCETINAND ADENYLATE CYCLASE
TABLE 3 Effect of Bovine FVIII/vWF on the Activity of Platelet
Adenylate
\Cd
Cyclase
CAMP Synthesized Basal
PGEl (1qM) stimulated %
pmole/lOmin
pmole/lOmin 571.1
100
Control
29.2a
Bovine FVIII/vWF (18 &ml)
20.1
68.8
440.7
77.2
Ristocetin (0.18 mg/ml)
18.0
61.6
477.0
83.5
Bovine FVIII/vWF + Ristocetin
12.0
41.1
___
___
a)
100
%
Each value is the mean of duplicate incubations. The variation between duplicates was less than 3%.
- 600
- 400
0
0.25 0.5 0.75 Ristocetin (mg/ml)
FIG. 1
I.0
vol.
30,
RISTOCETIN
No.3
AND ADENYLATE
CYCLASE
307
REFERENCES 1.
MARCUS, A.J. and ZUCKER, M.B. In “The Physiology of Blood Platelets: Recent Biochemical, Morphological and Clinical Research.*’ New York: Grune & Stratton, 1965, p. 53.
2.
ABDULLA, Y.H. B-Adrenergic 9 171-177, 1969. -Res. -9
3.
CORMAN adenylate
4.
CORMAN, R.R., FITZPATRICK, F.A. and MILLER, O.V. Reciprocal regulation of human platelet cyclic AMP levels by thromboxane A2 and prostacyclin. & Cyclic Nucleotide Res. 2, 597-609, 1978.
5.
HASLAM, adenylate 1972.
6.
MARQUIS, N.R., VIGDAHL, R.L. and TAVORMINA, P.A. regulation by cyclic AMP and prostaglandin El. Biochem. )6, 965-972, 1969.
7.
MILLS, D.C.B. Zucker, teds.). 504-512.
8.
MILLS, D.C.B. and MACFARLANE, D.E. Stimulation of human platelet cyclase by prostaglandin D2. Thromb. Res. 2, 401-412, 1974.
9.
TATESON, J.E., MONCADA, 5. and VANE, J.R. Effects of prostacyclin or cyclic AMP concentrations in human platelets. Prostaglandins 13, 389-397, 1977.
10.
WOLFE, S.M. and SHULMAN, N.R. Adenyl cyclase Biochem. Biophys. Res. Comm. 5, 265-272, 1969.
Il.
HASLAU, R.J., platelet function 1978.
12.
MARQUIS, N.R., BECKER, J.A. and VUGDAHL, R.L. Platelet aggregation III. An epinephrine induced decrease in cyclic A%IP synthesis. Biochem. Biophys. Res. Comm. ,9, 783-789, 1970.
13.
SALZMAN, 363.
14.
HASLAM, S/v!. tion Symposium,
15.
JAKOBS, K.H., SAUR, W. and SCHULTZ, G. Reduction of adenylate activity lysates of human platelets by the alpha-adrenergic component of epinephrine. Cyclic Nucleotide Res. 2, 381-392, 1976.
16.
hlILLER, O.V., JOHNSON, R.A. and CORMAN, R.R. stimulated CAMP accumulation in human platelets by Prostaglandins g, 599-609, 1977.
receptors
in human platelets.
R.R., BUNTING, S. and MILLER, O.V. Modulation cyclase by prostacyclin. Prostaglandins 11, 377-388,
J.
Atheroscler.
of human 1977.
platelet
R.J. and LYNHAM, J.A. Activation and inhibition of blood platelet cyclase by adenosine or by 2-chloroadenosine. ~Life Sci. -11 1143-1154,
E.W.
In “Platelet Washington,
Function Testing. H.J. Day, l-i. Holmsen D.C.: U.S. DHEW Publication No. 78-1087,
DAVIDSON, M.M.L., by cyclic nucleotides.
Cyclic
Platelet aggregation I. Biophys. Res. Comm.
activity
and M.B. 1976, pp.
adenylate
in human platelets.
Regulation of blood DAVIES, T. et al Adv. Cyclic Nucleotide Res. 2, 533-552,
AMP and platelet
function.
New Eng. J. Med.
In “Biochemistry and Pharmacology of Platelets”, Ciba vol. 35. Amsterdam: Elsevier Co., 1975, pp. 121-151.
286, 358-
Founda-
in &
Inhibition of PCElthrornboxane A2. -
RISTOCETIN
308
AND ADENYLATE
CYCLASE
Vo1.30,
17.
NEWMAN, K.D., WILLIAM, L.T., BISHOPRIF, N.H. et al. Identification of aadrenergic receptors in human platelets by [ H] dihydroergocryptine binding. L Clin. Invest. 6i, 395-402, 1978.
18.
GORMAN, R.R. thromboxane AZ.
Modulation of human platelet Fed. Proc. 38, 83-88, 1979.
19.
COOPER, D.M.F. plasma membrane
and RODBELL, M. adenylate cyclase.
20.
MILLS, D.C.B. and MACFARLANE, D.E. In “Platelets in Biology and Pathology.” 1.1. Gordon, ted.). Amsterdam: North-Holland Co., 1976, pp. 159-202.
21.
NICHOLS, W.L., GERRARD, J.M. and DIDISHEIM, P. In “Recent L. Poller ted.). Edinburgh: Churchill-Livingstone Blood CoaguIation”. pp. l-39.
22.
HOVIG, T. and STORMORKEN, H. Ultrastructure studies on the platelet plug formation in bleeding time wounds from normal individuals and patients with von Willebrands disease. Acta Path. IMicrobiol. Stand. 248, 105-122, 1974.
23.
SAKARIASSEN, J.J., BOLHUIS, P.A. and SIXMA, J.J. Human platelet adhesion to artery subendothelium is mediated by factor VIII von Willebrand factor bound to the subendothelium. Nature 279, 636-638, 1979.
24.
WEISS, H.J., HOYER, L.W., RICKLES, F.R. et al. Quantitative assay of a plasma factor deficient in von Willebrand’s disease that is necessary for platelet aggregation. J. Clin. Invest. 52, 2708-2716, 1973.
25.
The chemical basis for the action PERKINS, H.R. and NIETO, ?.!. Ann. N.Y. Acad. Sci. 235, 348-363, 1974. vancomycin group of antibiotics.
26.
KAO, K.J., PIZZO, S.V. and MCKEE, P.A. Demonstration specific binding sites for factor VIIl/von Willebrand factor Clin. Invest. 63, 656-664, 1979.
27.
KAO, K.J., PIZZO, S.V. and MCKEE, P.A. Platelet functional correlation VIIl/von Willebrand factor: Proc. Natl. ristocetin-induced platelet aggregation. 5320, 1979.
28.
ALLAIN, J.P., COOPER, H.A., WAGNER, R.H. and BRINKHOUS, K.M. Platelets a new reagent for assay of von Willebrand factor fixed with paraformaldehyde: J. Lab. Clin. hAed. g, 318-328, 1975. and platelet aggregating factor.
29.
KIRBY, E.P. and MILLS, D.C.B. The interaction platelets. J. Clin. Invest. 2, 491-502, 1975.
30.
COLLER, B.S. Inhibition of von Willebrand factor-dependent platelet function increased platelet cyclic AMP and its prevention by cytoskeleto*disrupting agents. --Blood 57, 846-855, 1981.
31.
SALOMON, Y., LONDOS, C. and RODBELL, u. cyclase assay. Anal. Biochem. 8, 541-548, 1974.
32.
SUZUKI, platelets Thromb.
33.
by prostacyclin
and
ADP is a potent inhibitor of human platelet Nature 282, 517-518, 1979.
K., NISHIOKA, J. and HASHIMOTO, for von Willebrands factor and Res. 11, 215-223, 1980.
TS’AO, C.-H., GREEN, gated human platelets !iaematol. 11, 287-297,
function
D. and ROSSI, E.C. tortuous boundaries 1973.
of
the
and characterization on human platelets.
of &
receptors for human factor of receptor occupancy and Acad. Sci. U.S.A. 76, 5317-
of bovine factor
S.
bovine
Advances in Co., 1981,
A highly
Identical
platelet
Ultrastructure and swollen
VIII with human
sensitive
by
adenylate
binding site on human aggregating factor.
of ristocetin aggreStand. J. granules.
No.3