Proc. Nati. Acad. Sci. USAVol. 80, pp. 6086-6090, October 1983Medical Sciences

Inhibition of thromboxane A2 synthesis in human platelets bycoagulation factor Xa

(arachidonic acid/factor V antibody/thrombin)

A. KUMAR SINHA, A. KONETI RAO, JANET WILLIS, AND ROBERT W. COLMANThrombosis Research Center and Hematology-Oncology Section of the Department of Medicine, Temple University School of Medicine, Philadelphia, PA 19140

Communicated by Oscar D. Ratnoff, June 23, 1983

ABSTRACT Factor X. binds to platelets provided that factorVa is present on the platelet surface, an interaction that results ina striking acceleration of the conversion of prothrombin to throm-bin. Thrombin then initiates fibrin formation, induces platelet ag-gregation, and stimulates the intraplatelet synthesis of throm-boxane A2 (TXA2). Addition of thrombin (2.4-14.4 nM) to platelet-rich plasma increased the basal level of TXA2, measured asthromboxane B2, from <0.5 pmol per 108 platelets to (mean +SEM) 100 ± 22 and 250 ± 10 pmol per 108 platelets, respectively.Treatment of platelet-rich plasma with increasing concentrationsof factor X. (1-12 nM) prior to the addition of thrombin pro-gressively inhibited the production of TXA2. Thrombin (9.6 nM),which produced 93% of the maximal formation of TXA2, was in-hibited 70% by factor Xa (10 nM). To identify which of these stepsin thromboxane synthesis was inhibited by factor Xa, platelets la-beled with [14C]arachidonic acid were exposed to thrombin andproducts of prostaglandin synthesis were separated by thin-layerchromatography. In contrast to the inhibition of TXA2 synthesis,prostaglandin E2 and prostaglandin F2a synthesis were not inhib-ited suggesting that neither phospholipase(s) nor cycloxygenase wasinvolved. The inhibition of TXA2 formation by factor Xa could bereversed by increasing the molar ratio of thrombin to factor Xato 5.5. Incubation of platelets with an IgG fraction of a humanmonoclonal antifactor V antibody, previously shown to inhibit fac-tor X. binding, was found to block factor Xa inhibition of TXA2synthesis. The inhibition of TXA2 synthesis requires the presenceof the active site serine of factor Xa and is not specific for TXA2formation induced by thrombin because it is also demonstrablewhen the agonist is ADP. Further, factor Xa does not require ad-ditional plasma components for its action because its inhibitoryeffects are detected in gel-filtered platelets. The effect of factorXa was evident at physiological (1.3 mM) calcium concentrations.These results indicate that factor X. binding to platelets throughfactor Va not only stimulates thrombin formation but also has acountervailing effect by inhibiting TXA2 formation.

The interaction of blood coagulation factor Xa with the humanplatelet surface in the presence of Ca2" accelerates the gen-eration of thrombin from prothrombin at a rate 300,000 timesfaster than the rate observed with factor Xa in solution (1). Fac-tor Xa binding to platelets requires the presence of factor Va,which may be derived by release from platelets (1) from its in-tracellular site, the a granules (2), by agonists such as thrombin.Alternatively, exogenous factor Va produced by the action ofthrombin on plasma factor V (3) facilitates factor Xa binding ina coordinate manner. In addition to these two actions of throm-bin on platelets and factor V, respectively, thrombin is also apotent inducer of thromboxane A2 (TXA2) synthesis in thesecells (4).We now report that in addition to the interaction of factor Xa

with platelets that leads to the efficient formation of thrombinfrom prothrombin, a concomitant effect of this binding of factorXa is the inhibition of TXA2 synthesis in these cells. BecauseTXA2 is a platelet-aggregating agent and a potent vasoconstric-tor, the inhibition of TXA2 synthesis by factor Xa would providenegative feedback control of the synthesis of TXA2 in platelets.

MATERIALS AND METHODSChemicals and Reagents. [1-`4C]Arachidonic acid (55.8 mCi/

mmol; 1 Ci = 3.7 x 1010 Bq) and [5,6,8,9,11,12,14,15-3H(N)]thromboxane B2 (TXB2) (100 Ci/mmol) were purchased fromNew England Nuclear. [5,6,8,11,12,14,15-3H(N)]Prostaglan-,din E2 (PGE2) (160 Ci/mmol) was bought from Amersham. Di-isopropyl fluorophosphate (iPr2P-F) was bought from Sigma.Antisera to PGE2 was obtained from Miles. All other chemicalsused were of analytical grades.

Coagulation Factors. Bovine factor X was prepared by themethod of Bajaj and Mann (5). The purified factor X was ac-tivated by using the Russell's viper venom and the resultingfactor Xa purified according to Jesty and Esnouf (6). Polyacryl-amide gel (8.6%) electrophoresis of factor Xa in the presenceof dithiothreitol showed the presence of three bands corre-sponding to heavy chain of aXa (Mr = 34,500), heavy chain of(3Xa (Mr = 29,500), and light chain of I3Xa (Mr = 16,000) (7),the major component accounting for 95%. The prepared factorXa had a specific activity of 5,500 units per mg of protein de-termined according to Cole et al. (8). The molarity of factor Xawas obtained by adding the molecular weights of heavy chainof PXa and the light chain (Mr = 45,000).

Both bovine factors X and Xa were also obtained from Sigma.Factor X was found to be a single component (Mr = 54,000) on8.6% polyacrylamide gel electrophoresis in NaDodSO4 (0.1%)but in the absence of reducing agent. The specific activity offactor X was 100-150 units/mg of protein. Factor Xa used hada specific activity of 4,000-5,000 units/mg of protein. Poly-acrylamide gel electrophoresis of factor Xa in the presence ofNaDodSO4 and dithiothreitol showed the presence of three bandscorresponding to the heavy chain of aXa (Mr = 34,500, 10%),heavy chain of /3Xa (Mr = 29,500, 90%), and light chain (Mr =16,000). Except that the commercial factor Xa was somewhatless active than the proteinase prepared as described above, theenzyme was essentially identical in both preparations.Human thrombin was purified by the method of Fenton et

al. (9) by using commercial thrombin as the starting materialfrom Sigma. The purified material was a homogenous proteinin gel electrophoresis and had a specific activity of 2,480 units/mg. The molarity of the thrombin preparation was calculatedfrom the molecular weight of the enzyme, Mr 33,500.

Abbreviations: TXA2, thromboxane A2; TXB2, thromboxane B2; PGF2a,prostaglandin F2a; PGE2, prostaglandin E2; iPr2P-F, diisopropyl fluo-rophosphate; RIA, radioimmunoassay.

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iPr2P-F-inactivated factor Xa was prepared by incubating 1.0mg of factor Xa, prepared as above, in 25 mM Tris'HCl buffer(pH 8.0) with 15 mM iPr2P-F in a total volume of 0.5 ml for 2.5hr at 370C as described by Leveson and Esnouf (10). Treatmentof factor Xa by iPr2P-F does not result in total loss of activity,but under these conditions we found that factor Xa lost >85%of its original activity. The reaction mixture was then dialyzedagainst 0.15 M NaCl for 16 hr at 40C.Human factor X was prepared according to DiScipio et al.

(11), was activated and purified as described above (6), and hada specific activity of 4,000 units/mg of protein.

Platelet Preparation. All blood donors had not taken anymedication at least 2 wk before blood donation. Venous bloodwas collected through siliconized needles into plastic syringes;coagulation was prevented by mixing 9 vol of blood with 1 volof trisodium citrate (final concentration, 13 mM). Platelet-richplasma was obtained by centrifugation of samples for 10 min at170 x g. The supernatant platelet-rich plasma (2-3 X 10' cellsper ml) was collected. Platelet counts were performed in aCoulter Counter model ZB equipped with a 50-Mum aperturetube. Gel-filtered platelets were prepared as described (12).The labeling of gel-filtered platelets with [I4C]serotonin and itsrelease were studied according to Walsh et al. (13).

Platelet Aggregation Studies. Platelet-rich plasma (0.5 ml)was placed in a cylindrical cuvette 8 mm in diameter containinga silicone-coated stirring bar. Aggregation was studied by add-ing thrombin or ADP and measuring the increase in light trans-mittance as platelet aggregates formed in an aggregometer(Chronolog, Broomall, PA) with a stirring rate of 1,200 rpm at37°C. The apparatus was calibrated so that the difference inlight transmittance between platelet-rich and platelet-poor plasmawas defined as 100% (14).

Assay of TXA2. TXA2 is a very unstable compound and israpidly converted into more stable derivative TXB2 (4). In thepresent study TXA2 was determined by TLC or radioimmu-noassay (RIA) in its TXB2 form.TLC of TXB2. Platelet-rich plasma was incubated with

[I4C]arachidonic acid (5 ,Ci/1O ml of platelet-rich plasma) for30 min at 37C. Ten percent of the [14C]arachidonic acid wasincorporated under these conditions. The labeled platelet-richplasma was then treated with thrombin in the presence or ab-sence of factor Xa. After aggregation, the plasma was acidifiedwith cold (0°C) formic acid (final concentration, 1%) and thelipids were extracted twice with 3 vol of cold (0°C) ethyl ace-tate. The extract was concentrated and analyzed by TLC on asilica gel G plate by using CHCl3/CH3OH/CH3COOH/H20,90:8:1:0.8 (vol/vol) (15). The plates were subsequently scannedin a Vanguard Scanner for the radioactivity. The arachidonicacid metabolites representing 19% of the incorporated fatty acidwere identified by using authentic samples of prostaglandin F2,(PGF2), TXB2, PGE2, and PGA2 in the silica gel platelets. Thesecompounds were visualized in the chromatographic plate bystaining with iodine vapor.RIA of TXB2 and PGE2. RIA of TXB2 was performed by us-

ing the antiserum against TXB2 as described by Lewy et al. (16)with antiserum provided by J. Bryan Smith (Thrombosis Re-search Center). Each sample is assayed in triplicate and the meanvalue reported. The RIA of PGE2 was carried out by using com-mercial antibody following the instruction of the manufacturer.

Assay of Coagulation Factors. Factor Xa was assayed ac-cording to Denson (17) by using congenitally factor X-deficientplasma, except that the activation step with Russell's viper venomwas omitted. Factor V was assayed by the one-stage method ofLewis and Ware (18). One unit of factor V is defined as theamount in 1 ml of normal human plasma.

Antifactor V Antibody. Antifactor V antibody used in thesestudies was a IgG fraction of a human monoclonal IgG4 A an-tifactor V antibody (19) kindly supplied by Helen Glueck (Uni-versity of Cincinnati). An IgG fraction prepared by (NH4)2SO4precipitation and DEAE-Sephadex chromatography of the an-tibody was prepared and furnished by H. C. Chiu (ThrombosisResearch Center). Approximately 40 Mg of the IgG fraction perml neutralized >99% of factorV activity in 1 ml of normal plasma.

RESULTSStimulation of TXA2 Formation by Thrombin. Addition of

increasing concentrations of thrombin to platelet-rich plasmainduced the synthesis of higher levels of platelet TXA2, as de-termined by RIA of TXB2 (Fig. 1). Although as little as 0.2 unitof thrombin per ml (2.4 nM) would aggregate 80% of the plate-lets (not shown), the formation of TXB2 continued to increase,up to a thrombin concentration of 14.4 nM. The basal level ofTXB2 (<0.5 pmol per 108 platelets) increased -300-fold (100± 22 pmol per 108 cells; mean ± SEM) and 800-fold (250 ± 10pmol per 108 cells) in the presence of 2.4 nM and 14.4 nMthrombin, respectively.

Inhibition of TXB2 Production by Factor Xa. When platelet-rich plasma was treated with 9.6 nM thrombin, the TXA2 levelincreased from the basal level of <0.5 pmol per 108 plateletsto (mean ± SEM) 224 ± 15.6 pmol per 108 platelets. Treatmentof platelet-rich plasma with increasing concentrations of factorXa (1-12 nM) prior to the addition of thrombin progressivelyinhibited the production of TXA2 in these cells, to a value 20%of the level with thrombin alone (Fig. 2).

In a separate experiment ['4C]arachidonic acid-labeledplatelets were treated with thrombin in the presence and ab-sence of factor Xa. The platelet lipids were extracted and thepresence of ['4C]TXB2 in the extract was determined by TLC.As in the case of the RIA, the conversion of ['4C]arachidonicto radiolabeled TXA2 by thrombin was inhibited by factor Xabecause the peak of radioactivity corresponding to TXB2 is al-most completely ablated (Fig. 3). Comparison of the TLC ofextracts after thrombin treatment alone and in the presence offactor Xa indicated that the formation of radiolabeled TXA2 wasspecifically decreased in the presence of factor Xa, whereas theconversion to PGE2 or PGF2, was unaffected. The latter ob-servation was quantified by using a RIA for PGE2. In three ex-periments, platelets in plasma contained (mean ± SEM) 14.6± 4.5 pg per 109 cells. In the presence of thrombin (2.4 nM)the PGE2 concentration rose to 55.6 ± 10.6 pg per 109 plate-

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FIG. 1. Thrombin stimulation of thromboxane formation by plate-lets. Platelets were aggregated by adding different concentration ofthrombin to platelet-rich plasma (0.5 ml, 2.5 x 108 cells per ml). Afteraggregation, the platelet preparations were immediately centrifugedand the plasma was collected. Concentration ofTXB2 in the plasma wasdetermined by RIA. Values are mean ± SEM for 5-10 different donors.

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FIG. 2. Inhibition ofthrombin-inducedthromboxane productionbyfactor Xa. Platelet-rich plasma (0.5 ml, 2.5 x 108 cells per ml) was in-cubated with increasing concentrations of factor Xa as indicated for 30sec at 37°C before addition of 9.6 nM thrombin (0.8 unit/ml). After ag-gregation TXB2 concentration was determined by RIA. Values are mean± SEM of experiments on three different donors.

lets. However, factor Xa (4.0 nM) failed to inhibit the thrombin-stimulated formation of PGE2 because the concentration was58.2 ± 12.8 pg per 109 platelets.

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Reversal of Factor Xa Inhibition of TXB2 Formation byThrombin. The inhibition of TXA2 formation by factor Xa wasfound to be reversible. Thrombin at 9.6 nM, a concentrationthat produced 93% of a maximal response, was inhibited 70%by factor Xa (10 nM) (Fig. 4). As the concentration of thrombinwas increased in the presence of a constant amount of factor Xa,the inhibitory effect of the protease was overcome. At a con-centration of 52.8 nM thrombin, the concentration of TXA2 wasnot different from that produced by 9.6 nM thrombin in theabsence of factor Xa.

Although the addition of factor Xa to platelet-rich plasma in-hibited thrombin-induced TXA2 synthesis in platelets, the fac-tor Xa at the above concentration has no effect on the aggre-gation of these cells induced by thrombin. However, byincreasing the factor Xa concentration by 10-fold (24 nM) overthrombin (2.4 nM), inhibition of platelet aggregation could beshown in platelet-rich plasma (not shown).

Effect of Antifactor V Antibody on the Inhibition of TXB2Formation by Factor Xa. Factor Xa binding to the platelet sur-face requires the presence of factor Va molecules, which arethought to behave as receptors for such interactions (1). If theeffects of factor Xa on the inhibition of TXA2 were mediatedthrough the interaction of the coagulant enzyme with factor Va,it might be expected that the addition of antibody against factorV to the platelet suspension would be able to counteract theinhibitory effect of factor Xa. As shown in Fig. 5, incubation ofplasma with increasing concentrations of the IgG fraction of ahuman monoclonal antibody against factor V caused progressiveinhibition of factor V coagulant activity until, at 40 ,g/ml, <10%of the original activity was present. The antibody had no effecton the formation of TXA2 in platelet by thrombin. However,the antibody was able to reverse the inhibition of thrombin-stimulated TXA2 formation by factor Xa. The neutralization offactor V coagulant activity by the antibody closely parallels theability of the antibody to reverse the inhibitory effects of factorXa on thrombin-stimulated TXB2 formation.

Specificity of Factor Xa on the Inhibition of TXA2 For-mation. The inhibition of thrombin-stimulated TXA2 formationby factor Xa (80%) was not demonstrable after factor Xa was in-activated by iPr2P-F (9%) (Table 1). Factor X in high concen-tration showed slight inhibition of TXA2 synthesis (18%). How-ever, the minimal inhibition was probably due to the presenceof a small amount of factor Xa in the factor X preparation. When

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FIG. 3. Thin-layer radiochromatogram scan of ["4C]arachidonic acid-labeled platelet lipid extract in the presence of thrombin with or with-out factor Xa. Platelet-rich plasma was incubated with ['4C]arachi-donic acid (0.5 ,Ci/ml) for 60 min at 37°C. The labeled platelets werethen treated with either 9.6 nM thrombin or 10 nM factor Xa followedby 9.6 nM thrombin. After the aggregation of the platelets, lipids wereextracted and analyzed by TLC by using a CHC13/CH3OH/CH3COOH/H20, 90:8:1:0.8 (vol/vol), solvent system. (A) Platelet-rich plasma andthrombin. (B) Platelet-rich plasma and factor Xa with thrombin. Thearrows indicate the origin.

0 Ll-0 9.6 19.2 28.8 38.4 48.0 57.6

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FIG. 4. Reversal of inhibitory effect of factor Xa by increasing con-centration ofthrombin. Platelet-rich plasma (0.5 ml, 2.5 x 10i cells perml) was treated with a constant quantity of factor Xa (10 nM) and in-creasing amounts of thrombin (o). The value was compared with thefunction of TXA2 by thrombin (9.6 nM) alone (o). After platelet ag-gregation was completed, TXB2 in the supernatant fraction was mea-sured by RIA. Values are mean ± SEM for three different experiments.

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FIG. 5. Effect of antifactor V antibody on the inhibition of throm-bin-induced thromboxane formation by factor X.. Platelet-rich plasma(0.5 ml, 2.5 x 10' cells per ml) was incubated at various concentrationswith IgG fractions of monoclonal antifactor V antibody for 15 min. Afterincubation platelets were treated with factor Xa (10 nM) followed bythrombin (9.6 nM) (A). In the control experiment (A) addition of factorXa to platelet-rich plasma was omitted. After platelet aggregation theplasma supernatant was analyzed for the determination ofTXB2 by RIA.Parallel experiments were run to determine the neutralization offactorV by the antibody (o). Values are mean ± SEM for three different ex-periments.

ADP is added at low concentrations (4 ,uM), little TXA2 is formed(TXB2 = 8.0 pmol/ml, where 1 ml contains 2.5 X 108 plate-lets). However, at high concentrations of ADP (0.5 mM), moreextensive TXA2 formation is observed (Table 1). Substantial in-hibition of the thromboxane synthesis is observed when factorXa is added and this increases at higher concentrations of factorXa.

Effect of Factor Xa on Thrombin- and ADP-Induced Ag-gregation in Gel-Filtered Platelets and Serotonin Release. Fac-tor Xa inhibited thrombin-induced aggregation and ['4C]sero-tonin release in gel-filtered platelets (Table 2). Concentrationsof both thrombin and factor Xa needed to induce and inhibitplatelet aggregation are much lower than that in the case ofplatelet-rich plasma. This discrepancy is probably due to thepresence of antithrombin in the plasma, which inactivates boththrombin and factor Xa (20). Factor Xa also inhibited ADP-in-duced aggregation and release, although the effect was muchmore marked on serotonin release.

Table 1. Specificity of factor Xa-induced inhibition of TXA2formation in platelets

TXB2,Addition to platelet-rich plasma pmol/mlNone <0.5Bovine X., 10 nM <0.5Thrombin, 9.6 nM 590 ± 70+ Bovine X., 10 nM 120 ± 10+ Bovine X, 100 nM 485 ± 25+ iPr2P-F bovine Xa, 100 nM 538 ± 30+ Human Xa, 10 nM 230 ± 25

ADP, 0.5 mM 89.0 ± 10.2+ Bovine Xa, 4 nM 33.6 ± 5.9+ Bovine Xa, 10 nM 18.2 ± 6.5

Platelet-rich plasma (0.5 ml; 2.5 x 108 cells per ml) was treated withfactor X, with factor Xa, or with iPr2P-F factor Xa and the platelet ag-gregation was initiated with thrombin or ADP as indicated. Factor Xand factor Xawere prepared as described in the text. After aggregation,TXB2 concentration in the plasma supernatant was determined by RIA.Values are mean ± SEM for three different experiments in differentdonors.

Table 2. Effects of factor Xa on ADP- and thrombin-induced gel-filtered platelet aggregation and release of ["4C]serotonin

Thrombin, ADP, Factor Xa, Aggregation, [14ClSerotoninnM AM nM % release, %0.5 - 80 39.00.5 4 4 0.0

5 - 80 27.45 4 72 6.85 16 10 1.1

10 - 80 27.410 4 76 9.2

Gel-filtered platelets suspended in Tyrode's buffer without added Ca2+(1.5 x 10" cells per ml) were labeled with [14C]serotonin. Platelet ag-gregation was initiated with thrombin orADP as indicated. The resultsare representative of three experiments with three different donors.

Effect of Calcium on the Inhibition of Thrombin-InducedTXA2 Formation by Factor Xa. The previously described ex-periments were performed in either citrated platelet-rich plasma(estimated Ca2+ = 30 AM) or Tyrode's buffer with no addedcalcium, in which Ca2+ measured by atomic absorption spec-troscopy is 40 AtM (21). Therefore, it is possible that at the cal-cium concentration in blood, 1.3 mM (22), the inhibition ob-served at depressed ionic calcium concentration might not obtain.Therefore, the effect of added calcium on the formation of TXA2stimulated by thrombin (2.4 nM) in the presence and absenceof factor Xa (4 nM) was studied (Fig. 6). No effect on thrombinstimulation of TXA2 formation was noted when calcium (1 mM)was added and even at 2 mM added calcium only 11% decreasewas noted. Similarly, the inhibitory effect of factor Xa was un-changed at 1 mM added calcium and only decreased 7% at 2mM. Thus, at 1.3 mM calcium added (Fig. 6, arrows) the in-hibitory effect of factor Xa on thrombin-stimulated TXA2 wasessentially unchanged. Only at the unphysiologic concentrationof 3 mM added calcium was there a significant alteration of theinhibitory effect of factor Xa. Addition of EDTA (1.0 mM) eitherto platelet-rich plasma or to gel-filtered platelets in Tyrode'sbuffer completely inhibited the formation of TXA2 (<0.5 pmolper 10' cells) in the presence of 2.4 nM thrombin, indicatingthe necessity of divalent cations in the synthesis of TXA2.

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FIG. 6. Effect of addition of CaCl2 on the inhibition of TXA2 for-mation in gel-filtered platelets. Varying amounts of CaC12 were addedto the gel-filtered platelets before the addition of factor Xa (4 nM) fol-lowed by the addition of thrombin (2.4 nM). After aggregation of theplatelets (3.0 min), at 1,200 rpm at 37°C, TXA2 was measured in thesupernatant. Values are mean ± SEM for three different experiments.*, Thrombin; A, thrombin and factor Xa. The arrows indicate the for-mation of TXA2 at plasma Ca2+ concentration (1.33 mM).

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DISCUSSIONThis study demonstrates that thrombin-induced formation ofTXA2 in human platelets (Fig. 1) is inhibited by factor Xa. Thisconclusion is supported by further experiments using both RIA(Fig. 2) and measurements of arachidonic acid incorporation(Fig. 3) into TXB2 by TLC. The inhibition by factor Xa of TXA2synthesis is reversed by high concentrations of thrombin (Fig.4) or by inhibiting factor Va, the putative receptor of factor Xaon platelets, with a human monoclonal antibody (Fig. 5). Theinhibition of TXA2 synthesis requires the presence of the activesite serine of factor Xa and is not specific for thrombin-inducedTXA2 formation (Table 1) because TXA2 synthesis stimulatedby high concentrations of ADP is inhibited by factor Xa. Theinhibition by factor Xa is not mediated by other components ofplasma because it is also demonstrated in gel-filtered platelets(Table 2). Although TXA2 formation can be stimulated by closeplatelet contact, as in centrifugation at low ionic calcium levels,the inhibition by factor Xa appears to be similar at physiologiccalcium concentrations as at the ionic calcium concentration ex-isting in citrated plasma (Fig. 6).TXA2 that is synthesized from arachidonic acid during plate-

let aggregation is believed to be an important mediator of sec-ondary aggregation and the release reaction (4). This arachi-donic acid metabolite, in addition to being an inducer of plateletaggregation, is a potent vasoactive agent, which causes con-traction of a wide variety of vascular and smooth muscles (23).The factors regulating synthesis of TXA2 are incompletelyunderstood. The present study indicates that the synthesis ofTXA2 stimulated by thrombin is inhibited by a component (fac-tor Xa) of the enzyme complex that catalyzes the generation ofthe agonist itself. Thus, this inhibitory effect of factor Xa couldcontrol the synthesis of the vasoactive compound TXA2 withoutaffecting the generation of thrombin or the aggregation ofplatelets induced by high concentrations of that enzyme.The conversion of ['4C]arachidonic acid to various prosta-

glandin metabolites in the labeled platelets induced by throm-bin was inhibited by factor Xa specifically at the step of TXA2formation without affecting the production of either PGF2. orPGE2. Because these stable prostaglandins require both phos-pholipase(s) and cyclooxygenase for their syntheses (24) it ap-pears that neither of these enzymes is inhibited by factor Xa.However, the inhibitory effect of factor Xa is limited to the in-tact platelet. The coagulant enzyme has no effect on the for-mation of TXA2 from arachidonic acid by a platelet homogenatein the presence or absence of added factor Va (unpublished data).

Previous investigators have suggested that the factor Xabinding sites in platelets are distinct from the thrombin bindingsites, because no competition was observed for binding toplatelets (25). The results of our investigation indicate that theinhibitory effect of factor Xa could be overcome by increasingthe concentration of the agonist. Further evidence that this ap-parent competition between thrombin and factor Xa is not atthe receptor level is provided by the finding that factor Xa caninhibit the stimulation of TXA2 production by high concentra-tions of ADP (Table 1). It should be noted that at physiologicalcalcium concentrations in the presence ofADP even close platelet

contact does not induce TXA2 formation. The lack of effect offactor Xa on the inhibition of TXA2 synthesis in platelet ho-mogenate indicates the participation of a common mediator forthe stimulation of thrombin and ADP of TXA2 formation andinhibition of that process by factor Xa. However, the nature ofthe mediator is currently unknown.

We thank Ms. Cheryl Beckett for her assistance, J. B. Smith for sup-plying us with the TXB2 antibody, H. Glueck for the human monoclonalantibody to factor V, and R. Rawala for supplying us with purified bo-vine factor Xa. Human factor Xa was kindly donated by the late J. Pi-perno. This work was supported by National Institutes of Health GrantsHL24674 and HL27189 and by a grant for a Specialized Center of Re-search in Thrombosis (HL14217).

1. Miletich, J. P., Jackson, C. M. & Majerus, P. W. (1978)J. Biol.Chem. 253, 6908-6916.

2. Chesney, C. M., Pifer, D. & Colman, R. W. (1981) Proc. Natl. Acad.Sci. USA 78, 5180-5184.

3. Tracy, P. B., Peterson, J. M., Hosherm, M. E., McDuffie, F. C.& Mann, K. G. (1979)J. Biol. Chem. 254, 10354-10361.

4. Hamberg, M., Svensson, J. & Samuelsson, B. (1975) Proc. Natl.Acad. Sci. USA 72, 2994-2998.

5. Bajaj, S. P. & Mann, K. G. (1973)J. Biol. Chem. 248, 7729-7741.6. Jesty, J. & Esnouf, M. P. (1973) Biochem. J. 131, 791-799.7. Jesty, J., Spencer, A. K. & Nemerson, Y. (1974)J. Biol. Chem. 249,

5614-5622.8. Cole, E. R., Marciniak, E. & Seegers, W. H. (1962) Thromb. Diath.

Haemorrh. 8, 434-441.9. Fenton, J. W., II, Fasco, M. J., Stackrow, A. B., Aaronson, D.

L., Young, A. M. & Finlayson, J. S. (1977)J. Biol. Chem. 252, 3587-3598.

10. Leveson, J. E. & Esnouf, M. P. (1969) Br. J. Haematol. 17, 173-178.

11. DiScipio, R. G., Hermodson, M. A., Yates, S. G. & Davie, E. W.(1977) Biochemistry 16, 698-706.

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