Effect of thrombin inhibitors on thrombin-induced platelet release and aggregation

THROMBOSIS RESEARCH 49; 23-36, 1988 0049-3848188 $3.00 + .OO Printed in the USA. Copyright (c) 1988 Pergamon Journals Ltd. All rights reserved.

EFFECT OF THROMBIN INHIBITORS ON THROMBIN-INDUCED PLATELET RELEASE AND AGGREGATION

Charles L. Knupp Division of Hematology/Oncology Department of Medicine East Carolina University School of Medicine Greenville, N.C., U.S.A. (Received 3.8.1987; Accepted in revised form 14.10.1987 by Editor D.F. Masher) ABSTRACT Thrombin-induced platelet activation was interrupted with hirudin or Dansylarginine N-(3-ethyl-l-5-pentanediyl)amide (DAPA) to study the time requirement for receptor occupancy by thrombin in promoting platelet responses at low (0.25 U/ml), intermediate (0.5 U/ml) and high (1 U/ml) thrombin concentrations. Each of these thrombin inhibitors suppressed adenosine triphosphate (ATP) release and aggregation by thrombin when added either before or simultaneously with thrombin or within seconds of the initiation of these responses If the inhibitors were added later, yet before by thrombin. aggregation or release was complete, no effect was present. The period of time for which active thrombin was required in order to promote these reactions had the following characteristics: (i) it is thrombin concentration dependent for a given response; (ii) it is longer for aggregation than for ATP secretion at each thrombin concentration; (iii) it is increased in platelets modified by chymotrypsin or platelets partially inhibited by antimycin A and 2-deoxy-D-glucose, which have prolonged aggregation and ATP release In direct comparison studies, the inhibitory effects of responses. hirudin and DAPA were identical on aggregation and ATP release. Thrombin binding, under similar experimental conditions identical to those, used to measure platelet activation, was prevented by hirudin, but not by DAPA. Therefore, the effect of DAPA on thrombin must be at the proteolytic site region and not at the hirudin-inhibitable platelet binding region. It is concluded from these studies that the tight coupling requirements for thrombin to induce platelet dense granule release and aggregation are directly dependent upon both the thrombin concentration and the rate of the individual platelet responses. Catalytic site integrity is required for the duration of this period of receptor occupancy. Key Words:

thrombin, platelet aggregation, secretion 23

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INTRODUCTION Thrombin is a potent platelet agonist that promotes many platelet responses, including the release of granule constituents and cell-cell aggregation. How thrombin exerts its effect at the platelet surface is not precisely known. Aspects of the thrombin-platelet interaction suggest both a direct enzyme-substrate reaction and a ligand-receptor interaction (1). This interaction of thrombin with the platelet is believed to occur at a specific surface receptor, but the identification of the receptor is incomplete (2). thrombin receptor Continuous occupancy of the putative is required for and acid hydrolase secretion since phosphatidate synthesis, alpha granule, hirudin, the specific inhibitor of thrombin binding, can rapidly halt these Under similar experimental reactions once they have been initiated (3,4). conditions, hirudin does not limit dense granule release or aggregation after These findings have suggested that there the responses have started (3,5,6). must be either different receptors or signal generation mechanisms for these platelet responses. However, a more recent report (7) has indicated that thrombin-induced serotonin release can be immediately stopped by a large excess of hirudin. Thus, there is some disagreement about the time when thrombin must be tightly coupled to the platelet surface to promote platelet This discrepancy might be attributable to the dense granule release. different thrombin concentrations used for platelet activation or to the different methods used to assess the release reaction in those studies. This for tight coupling of thrombin to report investigates the time requirement for dense granule release and aggregation at several thrombin the . latelet concentrations and with platelets which have been modified to slow these Dynamic time-progress measurements were used under the conditions responses. maximal inhibition of thrombin by hirudin recently described as necessary fo, >lytic site and binding site domains of The roles of the active pro (7). thrombin during this receptor occup.. icy period are examined by comparing the effects of the thrombin inhibitors, hirudin and DAPA, which have different specificities for these respective functional domains. MATERIALS as sodium [ 12BI]-iodide, 17.4 Ci/mg, and Aquassure 1251 carrier free, Sodium dodecyl were purchased from New England Nuclear, Boston, Mass. 2-mercaptoethanol and glycerol were products of British Drug sulfate (SDS), from Gallard-Schleisinger, purchased Carle Place, NY. and were House bis N, N’-methylene acrylamide, Electrophoresis grade polyacrylamide, N,N,N’,N’-tetramethylene-diamine, Coomassie Brilliant ammonium persulfate, Blue R-250, high and low molecular weight SDS PAGE standards were obtained N-a-tosyl-L-lysine-chlorofrom Bio-Rad Laboratories, Rockville Centre, NY. methyl ketone hydrochloride (toslysCH2Cl) was from Research Organics, Inc., 70% clottable) and bovine albumin Cleveland, OH. Bovine fibrinogen (Pentex, Fraction V> were from Miles Laboratories, Bedford, (Pentex, fatty acid free, Silicone oils were purchased from William F. Nye, Inc., New Bedford, Mass. Iodobeads were from Pierce Chemical Co., Rockford, IL. Chrono- lume Mass. Hirudin, antireagent was obtained from (;hrono-log Corp., Havertown, PA. and Sepharose were purchased mycin A, 2-deoxy-D-glucose, ATP, chymotrypsin DAPA was synthesized by Dr. K.G. from Sigma olemical Co., St Louis, MO. and was kindly provided by Drs. F.C. Church and Mann, Rochester, Minn. (8-9) R.L. Lundblad, Chapel Hill, NC.

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I(ETHODS Thrombin Iodination and Chemical Modification. Purified human 1 a-thrombin prepared as previously described (LO) was kindly provided by Dr. R.L. Lundblad (Chapel Hill, NC). For binding studies, a-thrombin in 0.05 M sodiuu phosphate, pH. 6.8, was radio-iodinated with 1 mCi of sodium [l251]iodide using Iodobeads, as recommended by the supplier, and then dialyzed against at 0.05 M sodium phosphate, pH 7.4, at 4’C for 24 hours to remove unbound tad ioac t ive iodine . Protein determination was made by the modified using bovine serwn albumin as a standard. Lowry technique (111, Fibrinogen clotting activity was performed as previously described (LO). The radiolabeled thrombin had a specific activity of 108,000 to 144,000 cpm/unit and retained greater than 95% clotting activity radioiodination and after dialysis. Platelet Preparation. Platelets were obtained from normal healthy donors who denied recent alcohol or aspirin ingestion, as approved by the East Carolina University Policy and Review Committee on human research (12). Six parts of venous blood were collected into plastic syringes containing one Platelet rich plasma was prepared by part acid-citrate-dextrose, pH. 6.8. centrifugation of anticoagulated blood at 100 x g for 20 minutes at room Platelets were gel filtered in a plastic column containing temperature. Sepharose CL-PB equilibrated with Tyrode’s solution, pH. 7.4, containing 0.0001 M ethylenediaminetetracetic acid (EDTA) and kept at room temperature for use. Chymotrypsin Modification of Platelets. Gel filtered platelets were had chymotrypsin which been previously treated with incubated with Chymotrypsin was added to platelets at a final concentratoslysCHpCl (13). tion of 50 ug/ml for 30 minutes at room temperature. The platelets were then washed by centrifugation at 400 x g and resuspended in fresh calcium-free Tyrode’s solution prior to use. Part ial Inhibition of Platelets by Antimycin A and 2-Deoxy-D-Glucose. inhibition of platelets was achieved by adding antimycin A at a final concentration of 5 pg/ml and 2-deoxy-D-glucose at a final concentration of 6 mM as that the incubations were only for 15 described previously (141, except ATP release and aggregation studies were promptly minutes at 37°C. performed since progressive inhibition of responses continued in these inhibited platelets stored at room temperature. Measurement of ATP Secretion and Aggregation. Washed platelets, 3.0-5.0 pH 7.4, containing 0.0001 M EDTA, were stirred x lO*/mL in Tyrode’s solution, at 900 RPM in a Payton model 1015 D Lumiaggregometer at 37°C for 1 minute after the addition of 40 pl of Chronolume reagent. Chronolune was reconHuman a-thrombin was added and stituted as recommended by the manufacturer. luminescence and aggregation were simultaneously recorded. At various times, hirudin (LO antithrombin units/ml) or DAPA (2.3 pM), was added while the inhibitor concentrations These were adequate in recordings cant inued . preventing release and aggregation in preliminary studies. ATP release was quantitated using a standard ATP solution. using Iodo-[ l251]Measurement of Thrombin Binding. Thrombin binding, was measured after incubation of 0.5 ml of the stirred gel-filtered thrombin, platelets, 3.0-8.0 x lO*/ml, with labeled thrombin in the lumiaggregometer at 37°C to reproduce the conditions used to study platelet release and aggregawere removed from the aggregometer cuvettes in 0.2 ml each, Samples, tion. placed into plastic tubes containing silicone oil and immediately duplicate,, centrifuged at 7,000 x g in a Beckman Microfuge B to separate bound and free

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ligand as previously described (12). This procedure was repeated three to and the results averaged to minimize five times sampling error for each The effect of DAPA or hirudin on thrombin binding was assessed measurement. by the addition of these inhibitors to the stirred platelets prior to the addition of Iodo-[1251]-thrombin. Non-specific binding was determined as that amount bound in the presence of hirudin (15-16). RRSULTS Platelet Release. Thrombin-induced ATP release from gel-filtered platelets, using low (0.25 U/ml), intermediate (0.5 U/ml) and high (1 U/ml) thrombin concentrations to induce platelet responses, could be inhibited by at least a ten-fold excess of hirudin or DAPA. Release was prevented when with the addition of thrombin (Fig. hirudin or DAPA was added simultaneously When either inhibitor was added after thrombin, release was suppressed 1). The effect on release was specific for thrombin but not prevented (Fig. 1). as there was no inhibition of release by these inhibitors with platelets stimulated by A23187 or arachidonate.

C

%&

k

Luminescence recordin s demonstrating the effects of hindi% and tbrorbirinduced ATP &WC. Gel-filtered plapt,l..tsL3.3.,; :zlLmn;,

were exposed to thrombin 1 unit/ml for 90 seconds. Panel B shows hirudin 10 antithrombin unitslml with no inhibitors added. (upper firre) and DAPA 2.3 uM {lower flfure) added 5 seconds after tbrombln. figure) added squltanPanel C s ows hrrudrn (up er figure) an DAPA (lower deflec.tions or interruptions In the 1R e negative eously with thrombin. tracings indicate when thrombin or inhibitor IS added.

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‘The inhibition of release varied according to thrombin concentration and time of exposure of platelets to thrombin before the addition of hirudin This time-dependent inhibitory effect of hirudin was most evident (Fig. 2). at the lowest thrombin concentrations when the rate of ATP release was slowest (Fig. 3).

1

u/ml

3 0.5

u/ml

0.25

u/ml

1

10

20

30

40

50

set

The tim-dependeat effect of hiredin OIL the extent of throebis Gel-filtered latelets, in ucccdATP releew. 7.6 x 108/ml, were exposed to

iv *-

Pi

thrombin at 0.25, 0.5 or 1 .O units Pml for 1 minute. At various times after the addition of thrombin, hirudin, 10 antithrombin units/ml final concentrawas added to interrupt ATP release measured by lumiaggregometry. The t ion, extent of ATP release at the end of the 1 minute incubation was then plotted as a function of the time from thrombin to hirudin addition for each sample.

c\ ATP lrg/ml

J

P

?i

IT 1 min

30 set

15 sac

10 set

5rec

2.5 8ec

Control

Lemiaeecence recordings demoaetretilrgthe inhibition of thrombiatr.ac.ings of ATP. release by 0.25 ArP releeee by hirudin. Luminescence

The. lnltral of thrombin noted in Fi.gure 2. each tracing indicates the additron of thrombrn and tracing indicates when hlrudln was added.

negative defection the rnterruptlon of

on the

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At higher thrombin concentrations, the release reaction was quite stable and nearly complete within seconds of the addition of thrombin and to have any effect, hirudin had to be added almost immediately after thrombin. Extent of release was inhibited 50 percent when hirudin was added 2 seconds after 1 unit/ml of thrombin, 6 seconds after 0.5 units/ml of thrombin, and 9.5 seconds after 0.25 units/ml of thrombin. This inhibition of release by hirudin was not instantaneous, as the release reaction continued for a brief time after the addition of hirudin at all thrombin concentrations (see Fig. 3, top portion of each tracing beyond the interruption of each tracing). The subsequent rate of ATP release was not changed after hirudin had been added in any of these studies as evidenced by the lack of change in the slope of the tracings following hirudin addition. The time which was required to turn to remain constant at off the response after hirudin was added appeared approximately 12 to 15 seconds, unless hirudin was added as the release response neared completion (Fig 3, 30 second tracing). The time required to terminate the response after hirudin addition appeared to remain constant at thrombin concentrations. However, the different the rapid responses at higher thrombin doses and the discontinuation of the tracings caused by the addition of hirudin made a more precise analysis impossible. DAPA had a similar inhibitory effect on thrombin-induced ATP release (Fig. 4).

1

0

10

20

30 set

40

u/ml

so

FL-filtered on thrombin~induced 4TP release. Pig. 4. Effect 0fB MPA platelets,D2;i x 10 /ml, *were treated wl;th thrombln at 0.5 or 1 unit/ml for 1 was then added at various times 2.3 UM final concentration, minute . Extent of ATP release was plotted after thrombin) to interrupt ATP release. as a function of the time from thrombin to DAPA addition.

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Suppression of release by MPA was also dependent on thrombin concentration (Pig. 4). Half maximal inhibition of ATP release occurred when DAPA was after 0.5 unit/ml added 3 seconds after 1 unit/ml of thtombin and 7 seconds of thrombin . As with hirudin, release continued for a brief time after the addition of DAPA at all thrombin concentrations. In view of the rapid platelet responses at all thrombin concentrations, various methods were used to slow the platelet responses in order to amplify the effect of DAPA and hirudin and to emphasize any differences between them. Treatment of platelets with chymotrypsin prior to the addition of thrombin markedly altered the time-progress curves for thrombin-induced ATP release When the platelets were activated with 1 unit/ml of thrombin, the (Pig. 5). lag phase before the onset of ATP release was lengthened from less than 2 seconds to 20 seconds, the time required for the completion of release was increased from 30 seconds to 90 seconds, and the extent of release was decreased 45 percent (Fig. 58, SC, top tracings) as compared to the unmodified platelets (Fig. 5A, top tracing). Addition of hirudin or DAPA at various times after thrombin resulted in suppression of both the rate and extent of ATP release in these modified platelets (Fig. 5B,C). The effects Half maximal ATP release and the half of DAPA or hirudin appeared equal. maximal rate of ATP release occurred when either of the thrombin inhibitors was added 30 seconds after the stimulation of platelets by 1 unit/ml of thrombin.

Corn arisoa Pig. 5. of the inhi%*Itory effect of hirudin DAPA and thrambia-induced &i

and ATP release was for 2 recorded minutes. A, upr,e; shows panel, res onse of unmodiB fie B platelets. and C, upper ;“,o,v the .ef. Pe?te’zk addlt ion of 10 antihirudin thrombin unit s/ml B or DAPA 2.3 PM I!C at the specified times after thrombin had been added. The bottom tracing in each’ panel (control) demonstrates the to buffer response instead of thrombin.

25 s.c

A

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Since chymotrypsin might proteolyze important signal generating surface proteins, platelets were also inhibited metabolically by treatment with antimycin A and 2-deoxy-D-glucose to decrease the rate of ATP release in response to thrombin. Incubation of gel filtered platelets with antimycin A and 2-deoxy-D-glucose for 15 minutes prior to the addition of thrombin resulted in a 34% reduction of the initial rate and 29% reduction of the extent of ATP release induced by thrombin (Fig. 6B, far right tracing) as compared to the control platelets (Fig. 6A, far right tracing). Hirudin or DAPA equally suppressed the extent, but not the subsequent rate of release in these metabolically inhibited platelets (Fig. 6B, left and center tracings), and the buffer treated controls (Fig. 6A, left and center tracings), when these thrombin inhibitors were added shortly after the induction of release. Although the duration of exposure to thrombin before the addition of inhibitors was equal, the metabolically compromised platelets exhibited only 52% of the total amount of ATP released by the control platelets.

I

A

Al-P l)rg/mi

d -

B f

r--

d

d-----

Effect of tbrqbin inhibitors on ATP rcl~re by metabolically Fig. 6. platelets 3.2 x 10 /ml, were exposed to im aired platelets. Gel-filtered buffer(A) or antimycin A 5 ng/ml and 2-dioxg-D-glucose 6 mM for (B) 15

minutes tracings tracings hirudin

at 37°C and then treated with throm in 1 unit/ml The control without DAPA or hirud’in added. The show the 3 minute responses to the left and center show the responses when DAPA 2.3 nM or 10 antithrombin units/ml was added 10 seconds after thrombin.

Platelet Aggregation. DAPA and hirudin also similarly The rate and the extent induced platelet aggregation.

of

Limited thrombinaggregation was

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suppressed

THROMBIN-INDUCED PLATELET ACTIVATION

by the

addition

of

either

thrombin

inhibitor

(Pig.

31

7).

30 set

Pi Inhibitory efP' tct '* of birudia on thrombin-induced Latclct CoY$EX P.xoa. aggregation tracings for the ATP release studies shown in Pi ure 2. % eflecThe initial tion in each tracing indicates the addition of thrombin and the second indicates deflecttYe” addition hirudin. A: : tracings were for 1 minute. The control sampi: dheat buffer of instead thrombin.

15 set

10 set

5sec

2.5 set Control

This response was also not immediately halted by the inhibitors if they were This inhibition was dependent on the thrombin concenadded after thrombin. tration used to initiate platelet aggregation. Half maximal aggregation resulted when hirudin was added 2.5 seconds after 1 unit/ml of thrombin, 9 seconds after 0.5 units/ml of thrombin and 13 seconds after 0.25 units/ml of Hirudin or DAPA had a greater effect on aggregation than on ATP thrombin. Similar release when directly compared (Table 1) in a given experiment. inhibitory effects were also seen in the chymotrypsin modified platelets and the metabolically inhibited platelets (results not shown). TMLg

1. % Maximal Aggregation

Thrombin Binding. A non-equilibrium technique was used for these the early critical binding before “receptor studies to attempt to measure processing” (15) and the release of alpha granule thrombin binding proteins In preliminary studies, hirudin suppressible binding, or the had occurred. specific binding, was comparable to that which was inhibited by a 50 to 100

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fold excess of native unlabeled thrombin, under these experimental condit ions. The measured specific thrombin binding was quantitatively similar to that assessed previously under equilibrium conditions (12). DAPA pretreatment of gel-filtered platelets had no apparent effect on the specific, Iodo-[ 1251]-thrombin binding (Table 21, at thrombin concentrations up to 1 unit/ml, the concentrations used for the release and aggregation studies. The specific binding was also unaltered by pretreatment with DAPA in similar studies with chymot rypsin modified platelets and platelets inhibited by ant imycin A and 2-deoxy-D-glucose. TABLE 2. Thrombin

SPBCIPIC TSRONBIN BINDING TO GEL-FILTERED PLATELETS IN THE PBBSENCB OR ABSENCE OF TBROktBIN INBIBITOBS.

Concentration

Ruf fer

DAPA

Hirudin

fj 5 z//m”: 0.25 U/ml Gel-filtered platelets, 5.6 x 108/ml, were ex osed to iodo-[1251]-thrombin for 30 seconds in the presence of buffer, DAPI 2.3 nM or hirudin 10 antithrombin unit s/ml. Samples were removed, centrifuged through silicone oil, and the radioactivity of the platelet . [ellet was measured. Binding in the nonspeci.ic binding and calculates to zero at presence of hirudin represents each thrombin concentration. The numerical values shown represent CPM bound to the pellet and are the average of four separate measurements. DISCUSSION These studies were performed to determine the period of receptor occupancy or tight coupling of thrombin to the platelet surface necessary for platelet dense granule release and aggregation responses. The wider range of thrombin concentrations and the methodology used allowed greater quantification of these reactions than prior studies. My results confirm earlier studies which demonstrated that only a very brief exposure to thrombin is necessary for dense granule release and aggregation to occur (3,5). Either hirudin OK DAPA is effective in preventing or limiting the platelet responses to thrombin. However, with each inhibitor, the effect is only instantaneous when the inhibitors are added before or simultaneously with, but not after, thrombin. This indicates that there is a period of time required to “turn off” the responses. Thus, what has been called the receptor occupancy time is only an early portion of the total time the responses . At higher thrombin concentrations, required to complete thrombin exerts its effect more quickly. As the thrombin concentration is increased, the requisite thrombin exposure time is shortened in parallel with the time required to reach the maximal extent of the response. Since the appears to remain constant at different thrombin concentime to “turn off” this would suggest that the receptor occupancy time is shortened at trations, this requisite period of thrombin higher thrombin concentrations. Therefore, exposure appears to vary with thrombin concentrations. Chymotrypsin-treated platelets exhibit a decrease in the rate of aggregation and dense granule release following a long lag period in response to This treatment does not alter the specific thrombin thrombin (6,12,17). binding (6). Therefore, chymotrypsin treatment has been proposed to uncouple the stimulus-response mechanism during thrombin-induced platelet activation when hirudin or DAPA is added during the lag period or In my studies, (5). soon after the initiation of release or aggregation by thrombin, the rate and the extent of these responses are both suppressed. Ant imycin A and 2-deoxy-

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D-glucose disturb oxidative phosphorylation and anaerobic glycolysis, and induce partial depletion of intracellular ATP in washed platelets (18). Incubation of platelets with these metabolic inhibitors results in a progressive decrease in the aggregation and release responses induced by thrombin and prolongs the time required to complete the responses (14). Hirudin or DAPA limit the responses to thrombin to a greater extent in the energy depleted platelets than in the control platelets in my studies.. Howappears to remain unchanged. the time to “turn off” Therefore, the ever, time of thrombin exposure required to induce maximal responses in the metamust be lengthened under these experimental bolically inhibited platelets These findings with the chymotrypsin-treated and the metaboliconditions. indicate that post-receptor events are also inhibited platelets cally critical in determining the “receptor occupancy time” for a given response. The effect of hirudin or DAPA was greater on thrombin-induced aggregation than on ATP release at a given thrombin concentration (Table 1). Thus, time must be longer for aggregathe tight coupling period and the “turn-off” in order to promote the full response. tion than for dense granule release aggregation proceeds more slowly This is not surprising since than dense granule release (19). The interpretation of the effects of these inhibitors on ATP release and aggregation responses are complex as thrombin is only required for a portion Previous studies have shown that some of the duration of the responses. unresponsive at maximal concentrations of individual platelets are even Other recent data have suggested that there is heterogeneity thrombin (20). to thrombin when individual platelet of platelet secret ion in response responses are assessed using flow cytometry (21). Therefore, these aggregate the variability in individual responses reflect the platelet may not reactions. The other aim of these studies was to probe the thrombin structure necessary to promote these platelet responses during the period of receptor by directly comparing the effect of hirudin to that of DAPA. occupancy thrombin at a platelet binding region, Because of its ability to inhibit hirudin has been used to measure specific binding of thrombin to the putative Hirudin also inhibits thrombin-induced proteolysis (22). receptor (15-16). It is generally assumed that the effect of hirudin is to disturb thrombin However, hirud in al so binding in order to prevent its enzymatic activity. to react with the proteolytic region of the thrombin molecule (221, appears which is distinct from the platelet binding region (23). Thus, it is unclear whether the effect of hirudin on thrombin-induced platelet activation is regions of the interference with the binding or the proteolytic through DAPA is a recently described thrombin inhibitor with high thrombin molecule. This inhibitor has been affinity for the proteolytic site of thrombin (8). My studies shown to prevent thrombin-induced platelet aggregation (9). demonstrate that DAPA is as effective as hirudin in preventing or halting ATP In contrast release or aggregation under the experimental conditions used. DAPA does not prevent the specific thrombin binding measured by to hirudin, under the same non-equilibrium conditions conventional binding techniques Thus, DAPA appears to inhibit thrombin used to assess platelet activation. at its proteolytic site and not at its measurable platelet binding site. The studies with DAPA indicate that catalytic site integrity is required not only for the initiation of these responses but is necessary for the duration of These that time when active thrombin must be present on the platelet. that the crucial interaction of thrombin with platelets findings suggest during this receptor occupancy period may be mediated through the proteolytic

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region. What role the hirudin sensitive binding domain has in the overall platelet-thrombin interaction is uncertain from these studies. The manner in which the proteolytic region of thrombin interacts with the platelet surface to promote these platelet responses is not known. It is generally assumed that proteolysis of a specific surface receptor or substrate (24) is required for platelet activation. This conclusion is supported by the finding that thrombin modified at the proteolytic site does not promote platelet activation (251, but still binds to platelets (26-27). Thrombin does hydrolyze a surface associated platelet glycoprotein, termed Glycoprotein V, during platelet activation (28-29). recent studies However, have cast doubt about the role of Glycoprotein V hydrolysis in modulating platelet An alternative mechanism for the action of responses (12,301. thrombin is that thrombin might not directly proteolyze a surface substrate but might bind to the platelet through this active site region and induce another enzyme, surface-associated or intracellular possibly the calcium activated protease (31), or another protease, phosphatase, or phospholipasc This alternative mechanism would be reactions. (32) to promote these consistent the dose-response kinetics of thrombin-induced platelet with which both a receptor-agonist and enzyme-mediated activation suggest The present data does not allow a determination about which reaction. hypothesis most accurately describes the effect of thrombin at the platelet surface. ACKNOWLEDGENENTS

I thank Dianna Dean for her expert technical assistance, Mrs. Diane Paramore and Mrs. Sue Chapman of the Word Processing Center for their skillful preparation of this manuscript, and Drs. Gilbert C. White, II, and D. Kirk Ways for their helpful suggestions regarding this manuscript. These studies were supported in part by the North Carolina United Way, REFERENCES

MARTIN, B.M., FEINMAN, R. D., DETWILER, T. C. 1. thrombin and other proteases. Biochemistry, 14,

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The interaction of KNUPP, C.L. and LUNDBLAD, R.L. WHITE, II, G.C., 2. In The Thrombin, ed. Machovich, R.L., CRC Press, thrombin with platelets. New York, pp. 25-41, 1985. 3. HOLMSEN, H., DANGELMAIER, C.A. and HOLMSEN, H.-J. platelet responses differ in requirement for receptor for tight coupling of occupancy and compartmentalized format ion. J. Biol. Chem., 256, 9393-9396, 1981.

Thrombin-induced occupancy. Evidence phosphatidic acid

Tight HOLMSEN, H. , DANGELMAIER, C.A. and RONGVED, S. 4. thrombin-induced acid hydrolase secretion and phosphatidate Biochem. J., 222, receptor occupancy in human platelets. DETWILER, T.C. 5. release of calcium

and (II)

FEINMAN, R. D. by platelets.

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the thrombin-induced 12, 282-289, 1973.

Platelet-thrombin TAM, S.W., FENTON, II, J.W. and DETWILER, T.C. 6. Binding of a-thrombin is coupled to signal generation by a receptor 8. chymotrypsin-sensitive mechanism. J. Biol. Chem., 255, 6626-6632, 1980.

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