IX RECEPTORS ON HUMAN PLATELETS

Thrombosis Research, Vol. 85, No. 1, pp. 23-31, 1997 Copyright @ 1996 Elsevier Science Ltd Printed in tk USA. All rights reserved OC49-3848/97 $17.(XI + .00

Pergamon

PII SO049-3848(%)fMZ18-6

INFLUENCE OF IONIZED CALCIUM ON THROMBIN-lNDUCED DOWN REGULATION OF GPIb/IX RECEPTORS ON HUMAN PLATELETS

Gundu H. R. Rae, Janet D. Peller, James G. White Departments of Laboratory Medicine and Pathology, Pediatrics, University of Minnesota Medical School, Minneapolis,Minnesota55455 USA

(Received24 July 1996 by Editor D. Triplett; revised/accepted21 October 7996)

Abstract The influenceof ionized calciumon the down-regulationof GPIb/IX receptors on human plateletswas evaluatedby flow cytometryusing monoclinal antibodies. Additionof EDTA alone to a washed platelet suspensiondid not cause decreased monoclinal antibodybindingto the cells. However, introductionof thrombin to the washed platelets containingEDTA resulted in a marked decrease in bindingof monoclinalantibodiesto the GPIb/IXreceptors. If calciumat 1-3mmol/Lwas added to bufferedplateletsinsteadof EDTA beforethrombin,down-regulationwas prevented or significantlyreduced. Restoringcalciumto EDTA plateletsafter the thrombinstimulateddown-regulationhad been in progress for 1-3 minutescaused reversal of decreased antibodybindingby GPIb/IX to near resting levels. Results demonstrate that extracellularcalcium is a major factor regulatingthrombin-induceddownregulation. Copyright @ 1997 Elsevier Science Ltd Grant et al (1) appear to have been the first workers to describe the phenomenon of GPIb/IX down-regulationfollowing exposure of platelets to activating agents in suspension. Their studyshowedthatprior exposureto ADP inhibitedvon Willebrandfactor (vWF)- mediated platelet agglutination. However, the down-regulationobserved by Grant et al could only be demonstrated in the presence of EDTA or EGTA. Hagen and her colleagues (2) made a virtually identicalobservationin their early study. Human platelets separated from blood Key Words: Platelets, GlycoproteinIb/IX, Calcium, Down-regulation,Clearance

Corresponding Author: Gundu H. R. Rae, Ph.D., Department of Laboratory Medicine and Pathology, University of MinnesotaMedical School, UMHC Box 609, 420 Delaware Street, S.E., Minneapolis,Minnesota55455 USA, Fax: 612-626-2883

23

24

REGULATION OF GPlb/lX RECEPTORS

v

85, N

1

collected in EDTA anticoagulantand activatedby thrombin, agglutinatedpoorly when stirred subsequentlywith bovine vWF. Mills et al (3), in a more recent publication, confirmed and extendedthe findingsreportedby the two earlier groups. The abilityof ADP and other agonists to inhibit agglutinationby bovine vWF appeared to be related to membrane changes that accompany loss of discoid shape, but not to shape change per se. It is of interest that their platelet agglutinationstudieswere also carried out in the presence of EDTA. George et al (4) and George and Torres (5), despitethe earlier observationscited above, are generally credited with the finding that thrombin-activatedplatelets bind significantlyless vWF and monoclinal antibodiesto GPIb/IX than resting platelets. However, they found that the binding of AP1, a monoclinal antibodyto GPIb, was affected by the presence of calcium ions in the buffer (5). As a result, in most experimentsAP1 binding was measured in the presence of EDTA. The sensitivityof down-regulationto calciumion concentrationindicatedin these studies has stimulatedus to re-examinethe markeddecreasein monoclinalantibodybindingto GPIb/IX observed when thrombin-activatedplatelets are examinedby flow cytometry. Results of this investigation indicate that calcium ion concentration is critical to decreased binding of monoclinal antibodiesto GPIb/IX on thrombin-activatedplatelets. MATERIALS AND METHODS 1. Reagents Bovine thrombin was obtainedfrom Parke-DavisCompany, Detroit, MI. The GPIbspecific monoclinal antibody, 6D1, was a gift from Dr. Barry Coller, Mount Sinai Medical School, New York, NY. FITC coupledantimouseIgG was from Organon Teknika Company, Durham, NC. Unless otherwise indicated, all other chemicals were purchased from Sigma ChemicalCompany, St. Louis, MO. 2. Platelet metxiration Blood for these studieswas obtainedfrom adult volunteersafter informed consent who had not taken any medicationfor two weeks. Fresh blood was mixed immediatelywith citratecitric acid-dextrose(CCD; citrate 0.1 Mel/L; citric acid 7.0 mMol/L; dextrose0.14 Mel/L, pH 6.5) anticoagulantin a ratio of 9 parts blood to 1 part CCD (6). Platelet-richplasma (PRP) was obtainedby centrifugingwholeblood at room temperaturefor 20 minutesat 100x g. To obtain washed platelets, PRP was dilutedwith 1:9 CCD. Plateletswere sedimentedby centrifugation at 800 g for 10 minutesat room temperature(7). The supernatantwas removedand the platelet pellet suspendedin 7 parts of modifiedTyrode’sbuffer (137 mMol/L NaCl, 2.8 mMol/L KCL, 1 mMol/L MgClz, 12 mMol/L NaHCO~,0.4 mMol/LNazHPOi, 0.35% bovine serumalbumin, 10 mMol/L HEPES, 5.5 mMol/L glucose, pH 7.4.) (8, 9). All experimentswere repeated“at least three to sixteentimes with plateletsobtainedfrom differentdonors. Tracings presented as figures represent typical observations. 3.

Flow cytometricanalvsisof the platelet surface exmession of GPIb/IX complex Washed plateletswere suspendedin modifiedTyrode’s buffer and aliquoted (15 uI) for

vol. 85, No. 1

REGULATION OF GPlb/lX RECEPTORS

25

various experimentaltreatments. Sampleswere prepared with 1 mM EDTA, Quin 2/BAPTAfree acids or Ca2+. To follow the influence of different concentrations(O.1 -1.0 U/ml) of thrombin, samples were incubated with the agonist at 37° C for different time periods. Thrombin action was stoppedby the additionof 15 U1of 2% paraformaldehydeand incubated for 30 minutes at room temperature. Samples were washed and resuspended in modified Tyrode’s buffer (60 u]) and incubated with a saturating concentration of a GPIb-specific monoclinal antibody, 6D1, for 60 minutes. After incubation, unreacted antibodies were removed by washingand the plateletsexposedto FITC-conjugatedIgG for 60 minutes. All of the reaction mixtureswere washedagain, resuspendedin modifiedTyrode’sbuffer and analyzed for fluorescencein a BecktonDickinsonFACScan flow cytometer (8, 9). The instrumentused a 15W argon laser for the power source. FITC fluorescencewas monitoredusinga 530 nm band pass filter. Samplescontainingfluorescentlabeledligandswere passed through tbe detector at a flow rate of 10,000 cells per second, Light scatter and fluorescencedata were collectedand analyzedusing a Hewlett Packard computer (30 H-P 217) (PaloAlto, CA). Fluorescencevaluesobtainedfor resting-EDTAplateletswere used to compute relative agonist-mediateddown-regulationof GPIb/IX receptors (10-13). The percent loss of fluorescence caused by platelet activation was calculated by using contour plots of the fluorescencedata. RESULTS

Exposure of washed platelets suspendedin modifiedTyrode’s buffer containing 1 mM EDTA to thrombinat a concentrationof 0.1 -0.2 U/ml revealedtime-dependentdown-regulation of GPIb/IXreceptorsas measuredby flowcytometry(Fig. 1). The thrombin-mediateddecrease

Influence of Calcium Chelation on Thrombin-Mediated Down Regulation of GPlb/lX on Platelets ,? 300

—

1

R ......... E

; ; : ;

-5

Cells +T

..

..

o 100

10’

10*

103

Log Flourescence Fig. 1 Platelets in modified Tyrode’s buffer containingEDTA revealed significantdownregulationof GPIb/IX followingexposureto thrombin (O.1 U/ml) in suspension.

26

REGULATION OF GPlb/lX RECEPTORS

v

85, N

1

Table 1. INFLUENCE OF CALCIUM AND CALCIUM CHELATION (EDTA, 1 mM) ON THROMBIN-MEDIATEDDOWN-REGULATIONOF GPIb/IX

Treatment P1 + P1 + P1 + P1 + P1 +

HBSS EDTA EDTA + Thrombin (0.1 U/ml) Ca2 (1 mM) + Thrombin (0.1 U/ml) EDTA + Thrombin (0.1 U/ml) + Ca2+(1 mM)

o 0 77.6 + 3* 11.4 * 3.1** 19.4 * 4.3**

* Mean and standard deviation(n=49; P > 0.0001) ** Man and s~ndard deviation(n= 11; P . 0.0001)

Influence of EDTA and calcium on the down-regulationof platelet GPIb/IX after exposureto thrombin. There was no differencein the bindingof monoclontiantibodies(MoAb) to GPIb/IX on resting plateletsin the presence of EDTA or calciumions. Thrombin caused a marked decrease in bindingof MoAb in the presenceof EDTA, but much less down-regulation in the presence of calcium. Addition of calcium after thrombin-induceddown-regulationof GPIb on EDTA plateletswas in progress caused almost completereversal. in monoclinal antibody binding to GPIb/IX could be observed as early as 30 seconds after additionof the agonist. Maximumloss of FITC fluorescencewas detectedafter exposureof the cells to the agonist for 3 minutes and remained stable for at least 5 minutes. No effort was made to examine later intervalsto see if the process underwentspontaneousreversal (14). The degree of down-regulationwas estimatedby comparing the difference between the fluorescenceof restingplateletsand thrombin-activatedcells. Maximumdown-regulationcaused by 0.1 -0.2 U/ml of thrombinin the presenceof 1 mM EDTA varied from 60-80% (Table 1).

Exposure of platelets to 0.1 U/ml thrombin in low calcium buffers caused significant down-regulationof GPIb receptors (data not shown). Calcium completing agents Quin 2 and BAPTA-free acids also facilitated down-regulationof GPIb receptors following addition of thrombin (Fig. 2).

vol. 85, No. 1

REGULATION OF GPlb/lX RECEPTORS

27

Influenceof BAPTAand Quin-2 Free Acidson ThrombinMediatedDownRegulationof GPlb/lX Receptors 2

2 , ,. :.

BAPTA

Q

— w

-

Resting Cells Thrombin

m . G z

;

&

...

e 2

...’ ,.”” ... ... 0

100

1

101

0

102

103

, ,,,,,, , 100

L

10’

.....

... ... ... ...

...

,,1,,, *

102

103

F

l

Fig. 2 Additionof thrombin (0.1 u/ml) to platelets suspendedin modifiedTyrode’s buffer contianing free acids of BAPTA or Quin 2 caused significantdown-regulationof GPIb/IX receptors. 2.

Influenceof addedextracellularcalciumon thrombin-induceddown-remdationof GPIb/IX receDtors

Platelets suspendedin modifiedTyrode’s buffer containing 1-5 mM CaClz, rather thim EDTA, responded in a different manner when thrombin was added. Instead of the 60-80% decrease in bindingof the monoclinal antibodyto GPIb/IX seen in the presence of EDTA the thrombin-induceddown-regulationwas significantlyless in the presence of calcium ions (Fig. 3, Table 1).

Thrombin Mediated Down Regulation of GPlb/lX on Platelets in the Presence of Calcium 300

1

— Resting Cells ......... &2+ + Thr~mbin

Log Flourescence

Fig. 3 Additionof calcium (l-3 mM) to plateletssuspendedin modifiedTyrode’s buffer before additionof thrombin(0,1 -0.2 U/ml) almost completelyblocked the down-regulationof GPIb/IX. The decrease measuredby FACS was usually 12% or less (Table I).

28

3.

REGULATION OF GPlb/lX RECEPTORS

Vol.

85,

No.

1

Influenceof decalcificationon down-regulationof GPIb/IX

The absenceof thrombin-induceddown-regulationof GPIb/IXin the presenceof calcium ions comparedto the markeddecreaseobservedwhenEDTA was addedsuggestedthe possibility that the phenomenon might be reversible. Addition of calcium (l-3 mM) at 2-5 minutes following stimulationof EDTA plateletsby thrombin restored 6D1 binding to a level 75% to 80% of that observed with unstimulatedplatelets(Fig. 3, Table 1). Additionof 1 mM calcium 10 minutesafter thrombinto EDTA plateletsdid not cause reversal of down-regulation. DISCUSSION

The present study has explored the importance of extracellular calcium ions on the thrombin-induceddown-regulationof GPIb/IX, the receptor for vWF on humanplatelets(1, 15, 16). Resultsof the investigationindicatethat the concentrationof calciumoutsideof thrombinactivated platelets can have a marked influence on the decreased binding of monoclinal antibodies to the cells as measured by flow cytometry (8, 9, 11, 13). Addition of EDTA to chelate calcium ions in the buffer used to suspend platelets greatly facilitated reduction in binding of the monoclinal antibody, 6D1, to the receptor, GPIb/IX. Similar, though not as marked, lowering of antibody binding could be demonstratedusing calcium-specificprobes, including Quin 2 or BAPTA-free acids, or multiple washings of the platelet suspension in calcium-free buffers (data not shown). EDTA was the most efficient facilitator of downregulation. Addition of thrombin to EDTA platelets resulted in a 60-80% decrease ‘in monoclinal antibodybinding.

Reversal of Thrombin-inducedDown-Regulation of GPlb/lX on EDTA Platelets by Calcium

—

Resting Cells

. . . — -

EDTA + Thrombin EDTA + Thrombin + Calcium

10°

1

1 L

1

F

Fig. 4 Additionof 1-5 mM calcium to the platelet suspensionin EDTA 1-3 minutesafter addition of thrombin (O.1 - 0.2 U/ml) almost completely reversed the down-regulationin progress. Instead of the 60-80% decrease in bindingof monoclinal antibodieswhen thrombin was added in the presenceof EDTA, additionof calciumrestored the level of decreasedbinding of monoclinal antibodiesto levels near those observed when calcium was present, instead of EDTA.

l

Voi.85, No. 1

REGULATION OF GPlb/lX RECEPTORS

29

On the other hand, additionof 1-5 mM calcium chloride to the buffer before thrombin almostcompletelypreventeddecreasesin the bindingof API or 6D1. Also, additionof 1-3mM calcium within 2-5 minutes after thrombin-induceddown-regulationof EDTA platelets was already underway resulted in significantreversal of GPIb/IX down-regulation. The influenceof calciumions on the phenomenonof agonist-induceddown-regulationof GPIb/IX has not been exploredin detailpreviously. Grant et al (l), Hagen and colleagues(2), and Mills and associates (3) all noted that prior exposure to aggregating agents inhibited the ability of plateletsto agglutinateon subsequentstirring with vWF. However, EDTA or EGTA was required in the several studies, even thoughthe importanceof chelationwas not discussed. Subsequentinvestigationsemployingflow cytometryhave used EDTA in their platelet buffers or prepared plateletsby washingrepeatedlyin calcium-freesalt solutions(9, 12, 13, 17-20). Recently, Nurden et al (13) have shown that thrombin-induceddown-regulation of GPIb/IX could take place in the presenceof normallevels of calcium. While that has not been our experienceas shown in the present study, it is noteworthythat down-regulationinducedby thrombin in the presence of EDTA was significantlygreater than occurred when calcium ions were present. The authors had no explanationfor the difference. It seems unusual that a phenomenon considered to have physiologicalimportance in vitro and in vivo can only be demonstratedon single platelets in suspensionand is significantlyenhanced by the absence, rather than the presence of calcium. A conclusionsimilar to ours was reached in the study by Fujimoto et al (15). They demonstratedthat thrombin activation caused increased binding of vWF to human platelets. Excess fibrinogenin the medium had no effect on the result, whereas stripping off GPIb by exposure of the platelets to chymotrypsinbefore exposureto thrombin completelyblocked upregulation. EDTA and EGTA blockedthe thrombin-inducedincreasein vWF binding. Though presentedin reverse, these findingsare very similarto ours. Normal levelsof calciumfostered up-regulationor increased bindingof vWF, while EDTA or EGTA blocked it. In our studies normal levels of calcium prevented or minimized down-regulation, while EDTA markedly enhancedthe phenomenon. In conclusion, our studiesdemonstratethat the thrombin-mediateddown-regulationof platelet GPIb receptors is modulatedby the concentrationand availabilityof ionized calcium. Stimulationof plateletsby thrombinin the presence of potent chelatingagents such as EDTA, resulted in significant down-regulation. However, similar studies done in the presence of calcium concentrationsof 1 mM or more failed to show marked down-regulationof these receptors. Furthermore, providing calcium to plateletsalready stimulatedby thrombin in the presence of EDTA, significantlyreversed the agonist-induceddown-regulation. Resultsof our study suggestthat, under physiologiczdconditionsagonist-mediatedstimulationof plateletsdoes not result in marked down-regulationor clearanceof the GPIb receptorsthat play a critical role in cell surface interactions. Acknowledgements This work was supportedby NIH grants HL11880 and HL49556 and a grant from the Center for Interracial Engineeringat the Universityof Minnesota.

30

REGULATION OF GPlb/lX RECEPTORS

vol. 85,

No.

1

REFERENCES 1. GRANT, R.A., ZUCKER, M.B., MC PHERSON, J. ADP-induced inhibition of von Willebrand factor-mediatedplatelet agglutination. Am J Physiol230, 1406-1410, 1976. 2. HAGEN, I., SOLUM, N.O., OLSEN, T. Membrane alterations in connection with the releasereaction in humanplateletsas studiedby the lactoperoxidase-iodinationtechniqueand by agglutinationwith bovine factor VIII-relatedprotein. BiochimBiophysActa 468, 1-10, 1977. 3. MILLS, D.C.B., HUNCHAK, K., KARL, D.W., KIRBY, E.P. Effect of plateletactivation on the agglutinationof plateletsby von Willebrandfactor. Molec Pharm 37, 271-277, 1990. 4. GEORGE, J.N., PICKETT, E.B., SAUCERMAN, S., MC EVER, R.P., KUNICKI, T.J., KIEFFER, N., NEWMAN, P.J. Plateletsurfaceglycoproteins. Studieson restingand activated plateletsand platelet membranemicroparticlesin normal subjectsand observationsin patients during adult respiratory distress syndromeand cardiac surgery. J Clin Invest 78L, 340-348, 1986. 5. GEORGE, J.N., TORRES, M.M. Thrombin decreases von Willebrand factor binding to glycoproteinIb. Blood 71, 1253-125M9, 1988. 6. RAO, G.H.R., ESCOLAR, G., WHITE, J.G. Influenceof heat on platelet biochemistry physiologyand function. J Lab Clin Med 122, 455-465, 1993. 7. RAO, G.H.R., FIELDS, C.M., WHITE, J.G. and FIELDS, G. Promotionof humanplatelet adhesionand aggregationby a synthetic,triple-helical“minicollagen. ” J BiolChem 269, 1389913903, 1994. 8. MICHELSON, A.D., BENOIT, S.E., KROLL, M.H., LI, J., ROHRER, M.J., KESTIN, A.S., BARNARD, M.R. The activation-induceddecrease in the platelet surface expressionof the glycoproteinIb-IX complex is reversible. Blood 83, 2562-3573, 1994. 9. MICHELSON, A.D. Plateletactivationby thrombincan be directlymeasuredin wholeblood through the use of the peptide GPRP and flow cytometry: methods and clinical applications. Blood Coag Fibrinol 5, 121-131, 1994. 10. MICHELSON, A.D., BARNARD, M.R. Plasmin-induced redistribution of paltelet glycoproteinIb. Blood 76, 2005-2010, 1990. 11. MICHELSON., A.D. F1OWcytometric analysis of platelet surface glycoproteins: phenotypicallydistinct subpopulationsof platelets in children with chronic myeloid leukemia. J Lab Clin Med 110, 346-354, 1987. 12. MICHELSON, A.D. Thrombin-induced down-regulation of the platelet membrane glycoproteinIb-IX complex. Sem Thromb Hemost 18, 18-27, 1992. 13. NURDEN, A., CAZER, E., BIHOUR, C., HUMBERT, M., COMBRIE, R., PAPONNEAU, A., WINCKLER,J., NURDEN, P. Confirmationthat GPIb/IXcomplexeshave a reduced surface distributionon plateletsactivatedby thrombinand TRAP-14-merpeptide. Br J Haematol 90, 645-654, 1995. 14. LU, H., MENASHI, S., GARCIA, L, CRAMER, E.M., LI, H., TENZA, D., DE ROMEUF, C., SORIA, J. and SORIA, C. Reversibilityof thrombin-induceddecrease “in platelet glycoproteinIb function. Br J Haematol 85, 11-6123, 1993. 15. FUJIMOTO, T., OHARA, S., HAWIGER,J. Thrombin-inducedexposureand prostacyclin inhibitionof the receptor for factor VIII/von Willebrand factor on human platelets. J. Clin. Invest. 69, 1212-1222,1982. 16. CLEMETSONK.J., CLEMETSON,J.M. PlateletGPIb-V-IXcomplexstructure, function, physiologyand pathology. Sem Thromb Hemost 21, 130-136, 1995. 17. ADELMAN, B., MICHELSON, A.D., HANDIN, R.I. and AULT, K.A. Evaluation of platelet glycoproteinIb by fluorescenceflow cytometry. Blood 66, 423-427, 1985.

Vol.85, No. 1

REGULATION OF GPlb/lX RECEPTORS

31

18, MICHELSON, A.D. and BARNARD, M.R. Thrombin-induced changes in platelet membraneglycoproteinsIb, IX and Hb-IIIacomplex. Blood 70, 1673-1678,1987. 19.HOURDILLE, P, , HEILMANN, E., COMBRIE,R. , WINCKLER,J, , CLEMETSON, K.J. and NURDEN, A.T. Thrombininducesa rapid redistributionof glycoproteinIb-IX complexes within the membrane systemsof activatedhuman platelets. Blood 76, 1503-1513,1990, 20. HOURDILLE, P,, BIHOUR, C., COMBRIE, R., GRALNICK, H. and NURDEN, A.T. Glycoprotein Ib-IIa (VLA-2) and glycoproteinIb-IX complexes are processed independently during thrombin-inducedplatelet activation. J. Lab. Clin. Meal, 124, 579-588, 1994.