THE JOURNAL OF BI~LOGLCAL CHEMISTRY Vol. 254, No. 20. Issue of October 25, pp 10354-10361, 1979 Prmted m U.S.A

Interaction of Coagulation Factor V and Factor Va with Platelets*

(Received for publication, April 16, 1979, and in revised form, July 6, 1979)

Paula B. Tracy, James M. Peterson, Michael E. Nesheim,+ Frederic C. McDuffie, and Kenneth G. Mann8

From the Hematology and Rheumatology Research Sections, Mayo Clinic, Rochester, Minnesota 55901

Bovine platelet and plasma Factor V were compared using antisera raised to purified single chain plasma Factor V and the quantity of Factor V associated with the bovine platelet was established using a radioim- munoassay developed with the same antisera. Bovine platelet Factor V is immunochemically indistinguisha- ble from bovine plasma Factor V using both radioim- munoassay and immunodiffusion as comparative tech- niques. Radioimmunoassay quantitation of platelet Factor V indicated that 593 + 139 Factor V molecules were present/platelet. Bioassay assessment of the quantity of Factor V present in the bovine platelet gave a value similar to this (666 f 124), further suggesting that the platelet and plasma derived Factor V are iden- tical. The binding of both iz51-Factor V and -Factor Va to platelets was also measured. When incubated with washed bovine platelets, 1251-Factor Va underwent sat- urable and exchangeable binding. There are high affin- ity binding sites to which approximately 900 Factor Va molecules are bound/platelet with an apparent disso- ciation constant of 3 x lo-” M, as well as binding sites of slightly lower affinity (Z& = 3 X lo-’ M) to which as many as 3500 Factor Va molecules are bound/platelet. The binding of Factor V to platelets is also saturable and exchangeable. Approximately 800 Factor V mole- cules bind to a single class of sites with a dissociation constant of 3 x lo-’ M. Exchange studies indicated that Factor V and Factor Va both bind to the lower affinity sites; however, Factor V does not bind to the high affinity Factor Va binding sites. Thrombin-induced platelet activation was not required for, nor had any effect on, the binding of either Factor V or Factor Va. The number of Factor V molecules associated with the platelet is similar to the number of Factor V molecules bound to the low affinity platelet binding sites, and that value is also similar to the number of high affinity Factor Va binding sites.

The prothrombinase complex is composed of two proteins- the enzyme, Factor Xa, and activated Factor V (Factor Va)- as well as calcium and phospholipid. A significant feature of the prothrombinase complex is that lipid can serve to provide the surface upon which Factor Xa, and its cofactors, perform

* This research was supported by Grant HL-17430 and by the Mayo Foundation. A preliminary report of this study was presented to the VIIth International Congress on Thrombosis and Haemostasis, July 1979, London, England (37). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accord- ance with 18 USC. Section 1734 solely to indicate this fact.

$ Supported by Blood Banking and Hemostasis Training Grant HL-07069.

8 American Heart Established Investigator and to whom corre- spondence concerning this paper should be addressed.

their functions (1, 2). Since platelets have a central role in hemostasis (3-5), it has been proposed that aggregated plate- lets provide the phospholipid surface required for the activity of the prothrombinase complex (5). Subsequent to vascular injury, the adhesion, aggregation and release reactions of circulating platelets result in the formation of a platelet plug. Since the polymerization of fibrin monomers at this site leads to the formation of a clot, the final stage of the blood coagu- lation process, it appears that platelets actively participate in the reactions leading to fibrin formation and perhaps do so by providing the catalytic surface for the localized activation of the plasma clotting factors (6).

A great deal of information is now available concerning the interactions of the individual prothrombinase components with each other (1,2, 7, 8). However, until recently very little was known about the physiologically important interactions between these same components and platelets. Two groups of investigators have been unable to demonstrate the binding of prothrombin to platelets (9, lo), even though they have uti- lized techniques which ensure the rapid separation of free and platelet-bound prothrombin. Factor Xa, however, binds to a specific receptor on activated human platelets with the con- comitant generation of thrombin at the platelet surface (11, 12). This Factor Xa receptor appears to be distinct from the thrombin receptor described previously by Tollefson et al. (13). Furthermore, the Factor Xa receptor appears to be susceptible to proteolysis (11) and was absent in Factor V- deficient patients (14), suggesting that Factor V is the receptor for Factor Xa. Dahlback and Stenflo have obtained similar results in studies of bovine Factor Xa binding to bovine platelets (10).

Until recently, similar experiments with Factor V and its cofactor, Factor Va, have not been possible because purified Factor V was not available. Our laboratory has prepared homogeneous single chain bovine Factor V (15) which we have used to examine the binding of both Factor V and Factor Va to bovine platelets, as well as to develop a double-antibody radioimmunoassay to measure platelet-associated Factor V. This paper reports on the interaction of Factors V and Va with bovine platelets.

EXPERIMENTAL PROCEDURES

Materials

Tris base (Trizma), bovine serum albumin, and fat-free bovine serum albumin were obtained from Sigma. Carrier-free [‘““Iliodide was obtained from New England Nuclear. [‘%]Serotonin creatinine sulfate (55 &i/~mol) was purchased from Amersham Corp. Apiezon oil was obtained from J. B. Biddle Co., and sodium dodecyl sulfate (DodS04) was from Pierce. The thrombin inhibitor, dansylarginine N-(3-ethyl-1,5-pentanediyl)amide (DAPA) was prepared as described by Nesheim et al. (16). Prostaglandin El (PGE,) was a gift of the Upjohn Co. Bovine blood was obtained by venipuncture from the Institute Hills Farm of the Mayo Clinic.

Homogeneous single chain bovine Factor V was purified as de-

10354

by guest on Novem

ber 16, 2020http://w

ww

.jbc.org/D

ownloaded from

Platelet Interaction with Factor V and Va 10355

scribed previously by Nesheim et al. (15). Factor Va was prepared by thrombin activation of Factor V as described previously (17). Briefly, Factor V (20 to 200 pg/ml) was incubated with 2 to 3 units of thrombin (200 NIH units/ml) for a minimum of 2 min. The mixture then was assayed to determine if complete activation had occurred or if addi- tional incubation time was necessary. Factor Va solutions, stored on ice, were used for binding measurements within 3 h of preparation. Factor V (Va) protein concentrations were determined based on a molecular weight of 330,000 and an extinction coefficient uEln, 280 nm ) of 9.6. Bovine thrombin was prepared as described by Lundblad et al. (18). The protein preparations used were at least 95% homogeneous as determined by DodSOd’ gel electrophoresis (19) as modified by Mann (20).

‘*“I-Factor V was prepared using Bolton-Hunter reagent (21) with a ratio of 2 mCi of ‘“?/mg of protein. The ““I-Factor V was separated from the other labeled products of the conjugation reaction by chro- matography on Cibacron blue Sepharose.’ The specific radioactivity of ‘*?-Factor V was from 250 to 1000 cpm/ng. The characterization of labeled Factor V is described under “Results.”

Platelets were isolated from bovine blood and washed to remove plasma according to the method of Orloff and Michaeli (22). The washed platelets were suspended in cold wash medium (0.127 M NaCl, 0.004 M KCl, 0.03 M glucose, 0.01 M imidazole, 0.35% bovine serum albumin, pH 6.6) at a concentration of 2 to 5 X IO9 platelets/ml and stored on ice. All platelet counts were done in a Coulter Counter. When platelets were isolated in the presence of 5 pM PGEI, to prevent any activation whatsoever, appropriate aliquots of a concentrated PGE, solution (1 mg/ml in absolute ethanol) were added to both the anticoagulant and the wash medium.

Washed platelets (1 -X 10’ platelets/ml) were incubated with 5 pM

[‘Y!]serotonin at 37°C for 15 min, washed twice by centrifugation at 1200 X g for 15 min, and resuspended in a buffer containing 20 mM

Tris-HCl, 0.14 M NaCl, 2.5 mM CaCl2, 0.1% glucose, 0.5% fat-free albumin, pH 7.4, to a concentration of 1 x 10s platelets/ml. This same buffer was used for the protein binding and aggregometer studies described below.

Specific antisera to bovine Factor V and to burro IgG were pro- duced by immunization of a burro and a goat, respectively, with the antigens in complete Freund’s adjuvant. The animals received weekly subcutaneous injections of 0.2 ml of a given antigen preparation containing either 0.05 mg of Factor V or 0.2 mg of burro IgG weekly, for 8 weeks. The specificity and purity of the antisera were evaluated by double immunodiffusion in agar according to the method of Au- ernheimer and Atchley (23) which is capable of detecting as little as 4 ag of antibody/ml (24).

Methods

Bioassay of Factor V-Factor V was converted to Factor Va by catalytic amounts of thrombin and then assayed for activity using Factor V-deficient plasma as described in detail previously (15). In this assay, fully activated Factor V (Factor Va) has a specific activity of 1250 units/mg of protein based on a bovine plasma standard which, by definition, contains 1 unit of Factor Va activity/ml. Therefore, the activity, determined by assay after thrombin treatment, was a meas- ure of total procofactor content, or Factor V. Platelets (2 to 5 x lo”/ ml) were assayed for Factor V activity following thorough washing as described above and solubilization in 0.2% Triton X-100. This concen- tration of Triton X-100 had no effect on the Factor V bioassay.

Factor V Radioimmunoassay-The amount of platelet-associated Factor V was also determined by radioimmunoassay. The platelet suspensions to be tested were diluted with platelet wash medium to yield approximately 1.0 to 2.5 x lo8 platelets/ml. The platelets were lysed by the addition of 10% Triton X-100 (final concentration, 0.2%). To plastic tubes (1.0 x 6.0 cm) were added 0.4 ml of the lysed platelet suspensions, 0.2 ml of “‘I-Factor V (4 to 25 ng) and 0.2 ml of a 1:8000 dilution of burro anti-bovine Factor V which had been diluted in normal burro serum 1:40. (All reagents were diluted in assay buffer which was 0.075 M Tris, 0.075 M NaCl, pH 7.0, containing 1% bovine serum albumin and 0.2% Triton X-100.) The 1:40 normal burro serum was added to provide sufficient burro IgG to form a manageable precipitate after the addition of goat anti-burro IgG in the following step. Following a l-h incubation at 37’C, 0.2 ml of goat anti-burro

’ The abbreviations used are: DodSOd, sodium dodecyl sulfate; DAPA, dansylarginine N-(3-ethyl-1,5-pentanediyl)amide; PGEI, pros- taglandin El; IgG, immunoglobulin G.

’ L. Hibbard and K. G. Mann, manuscript in preparation.

IgG, of sufficient titer to precipitate all the burro IgG present, was added. The tubes then were incubated for an additional 16 h at 4°C after which time they were centrifuged. The precipitate was washed twice with assay buffer and then counted in a Packard 5110 gamma counter. A standard curve was prepared with known amounts of purified Factor V.

Protein Binding Measurements-The binding of Factor V and Factor Va to platelets was measured by centrifugation through oil as described by Miletich et al. (11) for Factor Xa binding. Binding experiments were conducted at ambient temperature, 22-24°C. A 0.5- ml aliquot of the reaction mixture (1 x 10” platelets/ml plus added Factor V or Va) was carefully layered onto 0.5 ml of an oil mixture (1 part Apiezon, 9 parts 1-butyl phthalate) in 1.5~ml Eppendorf conical centrifuge tubes. After centrifugation at 12,000 x g for 2 min in a Brinkmann Microfuge, the entire supernatant was removed for deter- mination of unbound “‘1 activity. The remaining oil layer was re- moved and the bottom of the tube, containing the platelet pellet, was cut off and the radioactivity measured in a Beckman gamma counter.

Nonspecific binding due to isotope entrapment, etc., was estimated in parallel platelet ““I-Factor V or -Va reaction mixtures in the presence of a lOO-fold excess of unlabeled protein. Specific Factor V and Factor Va platelet binding data were subjected to Scatchard analysis (25) using the following equation:

b N.P, 1

f K,, -TTdb’vo

in which b and f are fractions of Factor V bound and free, respectively, N is the number of Factor V binding sites/platelet, PO is the molar concentration of platelets, Kd is the apparent dissociation constant for the Factor V binding site interaction, and [VJJ is the nominal concentration of Factor V. The apparent dissociation constant and the number of binding sites were calculated from the slope and vertical intercept, respectively.

Z’hrombin-induced Platelet Actiuation-Platelet activation was induced by 1 unit of thrombin/ml of platelets and studied by both aggregation and [?Z]serotonin release. Platelet aggregation was stud- ied with a turbidimetric device (Payton Aggregometer). A 0.5-ml aliquot of platelets (2.5 x 10” platelets/ml) was treated with 0.5 unit of thrombin and the reaction mixture was stirred at 1000 rpm at ambient temperature for approximately 15 min. The increase in light transmittance was recorded.

Platelet serotonin release was measured by determining the [‘?]serotonin in aliquots of supernatants from parallel incubations in the protein binding measurements from which iodinated proteins were omitted. Radioactivity was measured in the scintillation fluid described in Packard Instrument Bulletin 405E.” The vials containing 15 ml of fluid and 206~1 aliquots of the sample were counted in a Beckman scintillation counter (LSC-1OOC) in the 14C channel. One hundred per cent release was estimated by determining the radioac- tivity in an aliquot of the platelet suspension prior to centrifugation.

RESULTS

Characterization of Burro Anti-bovine Factor V Anti- body-The purity and specificity of the burro anti-bovine Factor V antibody determined by double diffusion in agar is shown in Fig. 1. A single precipitin band was formed against both purified bovine plasma Factor V (Wells 1 to 3), as well as bovine plasma (Wells 4 to 6). Immunoelectrophoresis of the antibody against whole bovine plasma also produced a single precipitin arc, another indication that the antibody was specific for bovine plasma Factor V. This antibody, in con- junction with goat anti-burro IgG, was used to develop a double antibody radioimmunoassay for plasma- and platelet- derived Factor V.

The competitive inhibition curve obtained using purified plasma Factor V in the radioimmunoassay is shown in Fig. 2. When used in the radioimmunoassay, dilutions of platelet lysates produced an inhibition curve identical in slope to the curve obtained with plasma Factor V, indicating that plasma and platelet Factor V are immunochemically the same. In addition, the antibody to plasma Factor V formed a line of

J Packard Instrument Bulletin 405E (1979) Packard Instrument Co., Downers Grove, Ill.

by guest on Novem

ber 16, 2020http://w

ww

.jbc.org/D

ownloaded from

10356 Platelet Interaction with Factor V and Vu

FIG. 1. Double immunodiffusion in agar analysis of burro anti-bovine Factor V antibody. The center well contained the antiserum raised against bovine Factor V. Wells 2 to 3 contained purified bovine Factor V and Wells 4 to 6 contained whole bovine plasma.

0.001 0.004 0.016 0.064 0.256 1.024

Factor V added Ipg per assay)

FIG. 2. Standard competitive inhibition curve obtained with Factor V radioimmunoassay and used to quantitate platelet derived Factor V. The ordinate represents “‘I-Factor V precipi- tated by the antibody. In the absence of unlabeled Factor V, the system contained fIOO0 cpm of “?-Factor V. Therefore, approximately 30% of the radioactivity added was bound and precipitated by the antibody.

immunochemical identity with platelet-derived and plasma Factor V in double-diffusion in agar analysis.4 Both results indicate that plasma and platelet-derived Factor V are im- munochemically the same and that the radioimmunoassay based on plasma Factor V could be used to measure platelet- derived Factor V as well.

Measurement of Platelet-associated Factor V-The amount of Factor V associated with washed bovine platelets was determined by both radioimmunoassay and bioassay. Quantification of platelet Factor V using the radioimmunoas-

’ S. Eid, M. E. Nesheim and K. G. Mann, manuscript in preparation.

say employed the standard curve shown in Fig. 2. Bioassay data were quantitated based on the activity of purified plasma Factor V, i.e. 1.25 units of Factor V was equivalent to each microgram of protein. The radioimmunoassay data for eight different preparations of Triton X-100 lysed, washed platelets revealed that 593 f 139 Factor V molecules were present/ platelet. Factor V clotting assays of these same Triton extracts were consistent with the radioimmunoassay data for any given platelet preparation with statistical analysis of the data indi- cating the presence of 666 f 124 Factor V molecules/platelet. The average of all the data indicated that approximately 630 + 134 Factor V molecules were present/platelet.

Characterization of Zodinated Factor V-Factor V (0.5 mg) was iodinated using 1 mCi of Bolton-Hunter reagent (21), resulting in the incorporation of 10 to 50% of the radioactivity into the protein. Following chromatography on Cibacron blue- Sepharose the Factor V preparations had specific radioactiv- ities of 250 to 1000 cpm/ng (0.03 to 0.12 mol of ?/mol of Factor V).

Factor V had the same electrophoretic mobility on DodS04- gel electrophoresis prior to and after labeling. Fig. 3 shows a radioelectropherogram of ‘251-Factor V after electrophoresis on a 5% polyacrylamide gel in the presence of DodSOa. The major peak of radioactivity corresponded to the protein band of homogeneous unlabeled Factor V and usually contained at least 85% of the total isotope applied to the gel. Even though the Bolton-Hunter method is the mildest protein iodination procedure available, some activated Factor V (Factor Va) was always produced during the process of labeling. This Factor Va contamination in any labeled Factor V preparation varied from 5 to 10% of the total isotope as determined by polyacryl- amide gel electrophoresis. However, ?-Factor V retained 80 to 100% of its cofactor activity and gave products indistin- guishable from unlabeled Factor V following thrombin acti- vation, indicating that labeled Factor V was still a substrate for thrombin. DodSO1-gel electrophoresis of ‘?-Factor Va also indicated that 85% or more of the isotope applied to the gel was recovered in the peaks corresponding to Factor Va.

Characterization and Inhibition of Thrombin-induced

I I

0 10 20 30

Gel slice

FIG. 3. Sodium dodecyl sulfate/polyacrylamide gel radioe- lectropherogram of ‘*‘I-Factor V. The gel (5% acrylamide, 2.5% cross-link) was run in 0.1 M sodium phosphate buffer (pH 7.1). An aliquot corresponding to 1.5 c(g of ‘251-Factor V was electrophoresed. The ‘UI-Factor V had a specific activity of 5.6 x ld cpm/pg.

by guest on Novem

ber 16, 2020http://w

ww

.jbc.org/D

ownloaded from

Platelet Interaction with Factor V and Va

Platelet Actiuation-The release reaction (monitored by [‘*C]serotonin release), followed by platelet aggregation, was induced by the addition of 1 unit of thrombin/ml of platelets. Incubation of prelabeled washed bovine platelets with throm- bin for 10 min resulted in almost complete serotonin release (85 to 95%). Furthermore, the aggregometer tracing shown in Fig. 4A indicates that aggregation of the stirred platelets began within 2 to 3 min following thrombin addition. DAPA, a competitive inhibitor of thrombin (& = 5 X lo-’ M) (16), completely blocked serotonin release and aggregation (Fig. 4B) when included in the reaction mixture at a concentration of 30 PM. Consequently, to study Factor V binding to throm- bin-activated platelets, washed platelets were incubated with thrombin for 10 min followed by the addition of 30 pM DAPA in order to inhibit thrombin activation of added Factor V to Factor Va. Platelets treated in this manner released [‘“Cl- serotonin; however, the platelet suspensions showed no signs of aggregation during the first 40 to 50 min because they were not stirred. DAPA was also included in studies of Factor V and Factor Va binding to nonactivated platelets.

Factor Va Binding to Platelets-The binding of Factor Va to nonactivated platelets was measured as a function of Factor Va concentration. The amount of Factor Va added to the reaction mixture ranged from 20 ng to 2 pg/lO* platelets. Binding measurements were made following a 30-min incu- bation at ambient temperature since measurements of the kinetics of Factor Va binding indicated that, even though initial binding was very rapid, steady state binding was not reached until 30 min had elapsed. In addition, a 30-min incubation did not appear to be detrimental to the platelets since they could be fully activated by thrombin treatment following the incubation. The concentration dependence of steady state Factor Va binding to platelets is shown in Fig. 5, in which Factor Va bound (closed circles) is plotted versus Factor Va added. Nonspecific binding due to isotope entrap- ment, etc. (open circles), was estimated in parallel platelet ‘251-Factor Va reaction mixtures in the presence of a loo-fold excess of unlabeled Factor Va. Specific binding of Factor Va (dashed line) was obtained by subtraction of nonspecifically bound ?-Factor Va from the total ‘251-Factor Va bound. The percentage of added Factor Va bound ranged from 30% at the lowest Factor Va concentration to 5% at the highest Factor Va concentration. At the highest Factor Va concentration added, 93 ng of Factor Va was bound/lo’ platelets, indicating that at least 1700 Factor Va molecules were bound/platelet.

100, I I

0 0 1 2 3 4 5 6 7

Length of incubation fmin)

FIG. 4. Tracing of the relative change in light transmittance as a function of time of platelet suspensions incubated differ- ently. A, incubated with thrombin (1 unit/ml); B, incubated with thrombin (1 unit/ml) and 30 ,uM DAPA.

Factor Va added Ipg /lo* platelets1

FIG. 5. Binding of ““I-Factor Va to platelets as a function of Factor Va concentration after a 30-min incubation. The “‘I- Factor Va had a specific activity of 526 cpm/ng. Nonspecific binding was determined in parallel reaction mixtures containing IOO-fold excess of unlabeled Factor Va. The ““I-Factor Va specific binding was determined by subtraction of nonspecific binding from total binding. 0, binding of Factor Va; 0, nonspecific binding; - - -, specific Factor Va binding.

1 I I I I

u, o- 0 10 20 30 40 50 60

Time (minutes)

FIG. 6. Displacement of bound “‘I-Factor Va by unlabeled Factor Va. o---<), binding of ““I-Factor Va (250 ng added/lo’ platelets); M, displacement of labeled Factor Va by a IOO-fold excess of unlabeled Factor Va (2.5 pg/ml) added at 30 min to a portion of the reaction mixture. The data were corrected for nonspecific binding as described in Fig. 5.

Repetitive experiments using Factor Va preparations of differing specific radioactivities yielded nearly identical re- sults, indicating that Factor Va binding was not affected by the amount of iodine incorporated in the Factor V prepara- tions and that labeled Factor Va bound the same as unlabeled Factor Va.

Factor Va binding was completely reversible (Fig. 6). The open circles show the time course of ‘251-Factor Va binding. If, at 30 min, a loo-fold excess of unlabeled Factor Va was added, the labeled Factor Va was rapidly and completely displaced (closed circles).

Scatchard analyses of the individual binding experiments could be interpreted to indicate the existence of two classes of binding sites. The Scatchard plot of the specific Factor Va binding data depicted in Fig. 5 is shown in Fig. 7. There were high affinity binding sites to which 900 Factor Va molecules were bound/platelet, with an apparent dissociation constant of 3 X 10-l’ M, as well as binding sites of slightly lower affinity, Kd = 4 X 1O-9 M to which as many as 3400 Factor Va molecules were bound.

The binding of Factor Va to thrombin-activated platelets

by guest on Novem

ber 16, 2020http://w

ww

.jbc.org/D

ownloaded from

10358 Platelet Interaction with Factor V and Va

0’ \, I 0 0.5 1.0 1.5 2.0 2.5 3.0

[Factor Va bound] IM x lo'*1

FIG. 7. Scatchard plot of specific Factor Va binding data shown in Fig. 5. Two classes of binding sites were evident. The dissociation constants (Kd) and number of binding sites (n) calculated are: ---, Kd = 3.2 x lo-‘” M, n = 896; p, K,, = 4.2 x lo-’ M, n = 3382.

was also studied. Platelets were treated with thrombin (1 unit/lo’ platelets) for 10 minutes. DAPA (30 PM) was then added to inhibit any further thrombin-induced effects. Plate- lets treated in this way showed no signs of aggregation for the first 40 to 50 min; therefore, valid binding measurements such as those described above could be made. The binding curves and Scatchard plots of Factor Va binding to thrombin-acti- vated platelets were completely independent of thrombin- induced activation. Table I lists the dissociation constants and number of Factor Va binding sites obtained with nonactivated and thrombin-activated platelets.

The binding data were also analyzed and fitted for two classes of binding sites as described by Klotz and Hunston (26). This analysis did not appreciably alter the dissociation constants or the number of high affinity sites since the num- bers obtained were consistent within experimental error (& high affinity sites = 2.9 to 3.4 X 10-l’ M, n = 600 to 730; Kd low affinity sites = 5.0 to 6.6 x lo-’ M, n = 2400 to 2700). The total number of sites obtained, however, was lower than that determined from Scatchard analysis. In any event, the number of low affinity sites is subject to some error in view of the extrapolation required.

Factor Va binding studies were also carried out with plate- lets collected in the presence of PGEl (5 PM). Bovine blood was drawn into anticoagulant containing PGEl and all subse- quent platelet washings and incubations contained PGEl in order to prevent any platelet activation. The binding data obtained with Factor Va and platelets collected in the pres- ence of PGE, were identical to the data obtained with non- activated and thrombin-activated platelets.

In all these analyses the amount of Factor Va bound was calculated using a molecular weight of 330,000 for Factor Va, even though activation of Factor V by thrombin results in the formation of a variety of activation components which can be resolved by DodS04 electrophoretic analysis (17). When sol- ubilized platelet pellets previously incubated with ‘251-Factor Va were analyzed by polyacrylamide-DodS04 gel electropho- resis, all of the activation components could be identified. This result is not surprising since electrophoretic analysis of Factor Va on gels lacking detergent results in a single band, suggesting that the thrombin-induced products noncovalently associate in dilute aqueous buffer (17).

Factor V Binding to Platelets-Similar binding studies were conducted with Factor V. The binding of Factor V to nonactivated and thrombin-activated platelets and platelets collected in the presence of PGEl was studied as a function

of Factor V concentration over the range from 20 ng to 2 pg of Factor V/lo8 platelets. Factor V binding to these three types of platelets was similar, suggesting that thrombin-induced activation was not a prerequisite for Factor V binding. The fraction of Factor V bound ranged from 14% at the lowest concentration of Factor V added to 2% at the highest Factor V concentration. At saturation, 30 ng of Factor V was bound/ 10R activated or nonactivated platelets. This value is equiva- lent to 550 Factor V molecules bound/platelet.

Initial experiments suggested that Factor V binding was not completely reversible. Fig. 8 shows the time course of lz51- Factor V to platelets (open circles). I f a loo-fold excess of unlabeled Factor V was added at 30 min, 70% of the bound Factor V was rapidly displaced (closed circles). The remaining label which corresponded to 5.8 ng of Factor V bound, or 100 Factor V molecules, was not due to isotope entrapment since the data have been corrected for nonspecific binding. How- ever, addition of a IOO-fold excess of unlabeled Factor Va to an aliquot of these same platelets resulted in the complete and rapid displacement of the bound label (dotted line), suggesting that the label bound represented Factor Va. Since each iodinated Factor V preparation contained a small amount of contaminating Factor Va it was possible that the platelet was selecting out the Factor Va present, in addition to binding Factor V. Since Scatchard analyses of several sets of Factor V binding data showed the existence of two classes of binding sites similar to those described for Factor Va, it was postulated that Factor V was binding to only one class of sites, the lower affinity sites, and that the apparent high affinity sites were due to Factor Va binding. Table II lists the dissociation

TABLE I

Properties of Factor Vu binding to nonactivated and thrombin activated platelets

The average of four separate Scatchard analyses of binding studies done on different davs is given + standard deviation.

High affXty sites

K, (XIOLO n” M)

Low affinity sites

Kd (X109 M) nCL

Nonactivated 4.0 + 2.1 837 + 48 3.6 k 0.8 3403 k 30 platelets

Thrombin-acti- 3.4 f 1.7 827 +- 230 2.9 + 1.3 3496 + 104 vated nlatelets

’ Number of factor Va sites/platelet.

Time lminutesl

FIG. 8. Displacement of bound ‘%Factor V by unlabeled Factor V and Factor Va. W, binding of ‘?-Factor V (400 ng/ 10” platelets). After 30 min of incubation a lO@fold excess (4 pg/ml) of unlabeled Factor V (M) or unlabeled Factor Va (X---X) was added to portions of the reaction mixture and the displacement of the labeled Factor V measured. The data were corrected for nonspecific binding as described in Fig. 5.

by guest on Novem

ber 16, 2020http://w

ww

.jbc.org/D

ownloaded from

Platelet Interaction with Factor V and Vu 10359

TABLE II

Properties of Factor V binding to nonactivated and thrombin- activatedplatelets

The average of five separate Scatchard analyses of binding studies done on different days is given + standard deviation.

Nonactivated platelets

Apparent high affinity sites Low affinity sites

Kd (x10'" Kg (~10~ M) no M)

na

3.2 f 1.2 256 -c 140 2.8 f 1.5 816 -c 92

Thrombin-acti- 3.3 + 1.7 239 + 102 3.6 + 2.4 919 + 283 vated platelets

a Number of factor V sites/platelet

constants and number of binding sites for the two classes of sites obtained from Factor V binding data. A comparison between the data shown in both Table I and Table II reveals that thrombin-activated and nonactivated platelets possess Factor V and Factor Va binding sites with an apparent dis- sociation constant of 4 X lo-’ M. However, the number of available binding sites varies significantly between Factor V and Factor Va. In marked contrast to Factor Va, only 900 Factor V molecules occupy these lower affinity sites.

Selective Binding of Factor Vu by Platelets-The exchange experiment depicted in Fig. 8 suggested the occurrence of either preferential binding of the small amount of Factor Va present in the iodinated Factor V preparation, or activation of Factor V to Factor Va by the platelets. When platelet pellets obtained from binding experiments conducted with Factor V were subjected to electrophoretic analysis on 2% agarose gels in the presence of DodSOa (27) (Fig. 9, Panel A), the ratio of Factor Va bound to the platelets was increased relative to the amount of Factor Va originally present in the Factor V prep- aration (Panel C, solid line). The first peak represents Factor V, the second peak Factor Va. In order to determine whether activation or selective binding was occurring adsorption ex- periments were conducted. Adsorption of a Factor V prepa- ration with two aliquots of washed platelets and subsequent electrophoretic analyses of both platelet pellets (Panels A and B, respectively) show clearly that the ratio of bound Factor Va/bound V is significantly lower in the second platelet pellet (A, 1.42:1; B, O.&l). The composite electrophoretic data show that there is selective binding of the Factor Va present in the Factor V preparation and that the platelets are not activating the Factor V to Factor Va since, if this event were occurring, the distribution of Factor V and Factor Va would presumably have remained constant.

Characterization of Factor V and Factor Vu Binding Sites-The exchange experiment depicted in Fig. 8 indicates that Factor V would not displace Factor Va from its high affinity sites, suggesting that Factor V does not bind to the high affinity Factor Va binding sites. The similarity of the apparent dissociation constants for Factor V and Factor Va bound to platelets, however, suggests that these proteins are binding to the same lower affinity sites. Therefore, additional exchange experiments were performed to examine Factor V displacement of bound Factor Va (Table III). Factor Va (250 or 600 ng of Factor Va/lO* platelets) was incubated with platelets so that most of the Factor Va would be bound to the high affinity sites. The addition of a loo-fold excess of unla- beled Factor V to aliquots of these platelets would not displace the bound Factor Va from these sites. When sufficient Factor Va was added to partially fill the lower affinity sites the addition of unlabeled Factor V caused the displacement of 350 of the 1400 bound Factor Va molecules. These results indicate that Factor V can displace Factor Va from at least some of

the lower affinity binding sites, suggesting that those sites may accommodate both Factors V and Va. The high affinity binding sites are specific for Factor Va since Factor V is not able to displace Factor Va from those sites.

5 A

I 4-

3- i

2-

l- \

o-- J '

Gel slice

FIG. 9. Sodium dodecyl sulfate/agarose gel radioelectro- pherogram of solubilized platelet pellets after incubation with ‘251-Factor V. A, following a 30-min incubation with ‘?-Factor V (1 pg/lO’ platelets), 5 x lo8 platelets were pelleted, washed three times to remove occluded isotope, and solubilized with 10% DodS04. The equivalent of 1 x 10’ platelets were electrophoresed on 2% agarose- DodS04 gels as described by Fass et al. (27), then sliced, and counted for radioactivity. B, the supernatant from the reaction mixture de- scribed above was used in another reaction mixture with 5 x 10” platelets and incubated for 30 min. The platelet pellet was treated as described above. C, a composite of “‘I-Factor V (U) and 12’1- Factor Va (X . . . .x) radioelectropherograms showing relative mobil- ities in this gel system. Both proteins were electrophoresed in the presence of 1 x 10” untreated solubilized platelets.

TABLE III

Displacement of platelet- bound Factor Vu by excess Factor V ‘251-Factor Va was incubated with platelets for 30 min. A lOO-fold

molar excess of Factor V then was added to a portion of the reaction mixtures and the displacement of Factor Va was measured.

Factor Va added Factor Va bound Factor Va displaced

by a 100 molar Excel of Factor V

ng/lff platelets 250

600 IO90

molecules/platelet molecules/platelet

442 42 870 a2

1390 348

by guest on Novem

ber 16, 2020http://w

ww

.jbc.org/D

ownloaded from

10360 Platelet Interaction with Factor V and Va

DISCUSSION

The preparation of a specific antibody directed against plasma Factor V has enabled our laboratory to determine the relationship between plasma Factor V and platelet-derived Factor V. Platelet Factor V activity was fist described by Ware in 1948 (28). It was postulated that platelet Factor V activity represented plasma Factor V bound to the platelet surface (29). More recently, however, other investigators have indicated that platelet-derived Factor V may be distinct from plasma Factor V (30, 31). Our results indicate that plasma Factor V and platelet-derived Factor V are identical in their immunochemical properties and procoagulant activities/unit mass, suggesting that the Factor V activity associated with platelets may be derived from circulating plasma Factor V. Through the development and use of a radioimmunoassay for Factor V, we have been able to quantitate the amount of Factor V activity associated with platelets. The radioimmu- noassay data indicated that 593 f 139 Factor V molecules were present/platelet. Clotting assay data were consistent with the radioimmunoassay data for any given platelet prep- aration and indicated that 666 f 124 Factor V molecules were present/platelet. Therefore, studies were conducted to exam- ine the specific binding of plasma Factor V and Factor Va to platelets.

The binding of Factor Va to platelets is saturable, exchange- able, and of high affinity. There are two classes of binding sites. The highest affinity sites (& = 3 x lO-‘O M) accommo- date approximately 900 Factor Va molecules; however, as many as 3500 Factor Va molecules are bound to the slightly lower affinity sites (& = 3 X lo-” M). Thrombin pretreatment of the platelets is not required for, nor had any effect on, Factor Va binding since thrombin-activated platelets, as well as platelets collected in the presence of PGE1, showed Factor Va binding characteristics identical to those of nonactivated platelets.

The binding of Factor V to nonactivated and thrombin- activated platelets, as well as PGEl-treated platelets, indicates that platelets most likely possess a single class of Factor V binding sites which will accommodate 800 to 900 Factor V molecules/platelet (Kd = 3 x lo-’ M). Exchange studies indi- cate that both Factor V and Factor Va bind to these same lower affinity sites. Factor V does not bind to the high affinity Factor Va binding sites, suggesting that Factor Va interacts with platelets significantly better than the procofactor, Factor V. However, since in the bovine system Factor V circulates at a concentration of approximately 1.5 X 10m7 M, it is likely that all of the exchangeable Factor V sites are filled because the dissociation constant for the platelet-Factor V interaction examined in this study is 3 x lOmy M. Furthermore, the number of exchangeable Factor V binding sites is similar to both the number of Factor V molecules associated with washed plate- lets and the number of high affinity Factor Va binding sites. These observations suggest that Factor V is always associated with the platelet, but activation of Factor V to Factor Va results in its binding more tightly to the platelet membrane.

Factor V(Va), in addition to calcium, phospholipid, and the enzyme, Factor Xa, is involved in the activation of prothrom- bin to thrombin, which most likely occurs on the platelet surface in order to promote fibrin formation at a site of vascular injury. Miletich et al. (11, 12) have reported that thrombin-activated human platelets possess approximately 200 to 300 high affinity Factor Xa binding sites, as well as a larger, undetermined number of lower affinity, nonspecific Factor Xa binding sites. The data of Dahlback and Stenflo (10) can also be interpreted to indicate that thrombin-acti- vated bovine platelets possess two classes of Factor Xa binding

sites, even though these investigators have only reported a single class of high affinity Factor Xa binding sites.

Factor V(Va) has been postulated to be the Factor Xa receptor (11, 12, 14). Miletich et al. found that patients with a congenital Factor V deficiency have decreased Factor Xa binding sites on their platelets (14). Thrombin-treated control platelet supernatants, possessing Factor V activity, corrected the decreased Factor Xa binding, but did not increase binding of Factor Xa to normal platelets. In addition, they were able to inhibit both Factor Xa binding and thrombin generation by incubating thrombin-treated platelets with a spontane- ously occurring human antibody to Factor V before adding Factor Xa. Osterud et al. (31) have demonstrated that plate- lets appear to release an activated Factor V after exposure to collagen and have hypothesized that this may be a physiolog- ically significant early step in hemostasis.

The results obtained in this study indicate that platelets possess sites specific for Factor Va. Thrombin-induced platelet activation does not appear to be required for the expression of these sites. Other investigators have reported that Factor V does not appear to bind prothrombin (2) or Factor Xa (32) until it has been activated by thrombin to Factor Va. In addition, the difference in magnitude between Factor V and Factor Va procoagulant activity in promoting prothrombin activation has only recently been appreciated. Earlier reports of experiments utilizing clotting assays suggested that Factor Va possessed from 2 (33) to 8 (34) and as high as 30 (35) to 80 (17) times the activity of Factor V. However, more recent results using a well defined assay system of purified compo- nents, indicated that Factor Va may possess 400 times the procoagulant activity of Factor V (36). Therefore, the marked contrast observed between Factor V and Factor Va platelet binding characteristics is not surprising when one considers that Factor Va (and not Factor V) is the more physiologically significant cofactor in the prothrombinase complex.

That Factor V and Factor Va both bind to the lower affinity sites may, in fact, reflect a lipid-protein interaction since both Factor V and Factor Va bind fairly tightly to negatively charged phospholipid vesicles (Kd = 5 x lo-* M) (8). In both the platelet binding studies described here, as well as the lipid binding studies, the number of Factor Va molecules bound exceeds the number of Factor V molecules bound, even though the dissociation constants for these interactions are identical. Factor V binding may be significant since Factor V is most likely always associated with the platelet, but activation of Factor V to Factor Va results in its binding more tightly and, hence, its ability to bind Factor Xa and to function in the prothrombinase complex.

Our studies indicate that Factor V is associated with plate- lets and that Factor Va binds tightly to platelets. Whether or not Factor V(Va) is the receptor for Factor Xa needs to be established. Even though platelets appear to possess two classes of Factor Va and Factor Xa binding sites, the number of high affinity Factor Va binding sites indicated by our studies exceeds the number of high affinity Factor Xa binding sites determined by other investigators. Furthermore, the appearance of Factor Xa binding sites is somehow dependent upon thrombin-induced platelet release, whereas Factor Va binding is not dependent on, nor affected by, thrombin-in- duced platelet activation. Future studies examining the inter- action of Factor Xa with Factors V and Va with platelets will clarify these points and perhaps elucidate the synergistic participation of these components at the platelet surface in order to reconstruct the workings of the prothrombinase com- ponents in normal hemostasis.

Acknowledgments-We wish to thank David and Daniel Tweite

by guest on Novem

ber 16, 2020http://w

ww

.jbc.org/D

ownloaded from

Platelet Interaction with Factor V and Va 10361

for the use of their animals and for their assistance in procuring the 18. Lundblad, R. L., Uhteg, R. C., Vogel, C. N., Kingdon, H. S., and blood samples used in this study. We also wish to thank Ms. Cecilia Mann, K. G. (1975) Biochem. Biophys. Res. Commurz. 66,482- M. Droege for her excellent assistance in the binding studies. Finally, 489 we thank Ruth Kendall for her patience while repeatedly typing the 19. Weber, K., and Osborn, M. (1969) J. Viol. Chem. 244,4406-4412 manuscript. 20. Mann, K. G., Heldebrant, C. M., and Fass, D. N. (1971) J. Biol.

1. 2. 3. 4.

5.

6.

7.

8. 9.

10. 11.

12.

13.

14.

15.

16.

17.

REFERENCES

Chem. 246,5994-6001 21. Bolton, A. E., and Hunter, W. M. (1973) Biochem. J. 133, 529-

539 Mann, K. G. (1976) Methods Enzymol. 45, 123-156 Suttie. J. W.. and Jackson. C. M. (1977) Phvsiol. Rev. 57. l-70 Weiss; H. J. (1975) N. En&. J. Med. 293, 531-541 Gordon, J. L., and Mimer, A. J. (1976) in Platelets in Biology

and Pathology (Gordon, J. L., ed) pp. 3-22, North-Holland Publishing Co., Amsterdam

22. Orloff, K. G., and Michaeli, D. (1976) Am. J. Physiol. 231, 344- 350

23.

24.

25. 26.

Auernheimer, A. B., and Atchley, F. 0. (1962) Am. J. Clin. Pathol. 38, 548-553

Macy, N. E., O’Sullivan, M. B., and Gleich, G. J. (1968) Proc. Sot. Exp. Biol. Med. 128, 1098-1102

Scatchard, G. (1949) Ann. N. Y. Acad. Sci. 51, 660-672 Klotz, I. M., and Hunston, D. L. (1971) Biochemistry 10, 3065-

3069

Biggs, R. (1976) in Human Blood Coagulation, Haemostasis and Thrombosis (Biggs, R., ed) pp. 66-80, Blackwell Press, Oxford

Walsh, P. N. (1977) in Platelets and Thrombosis (Mills, D. C. B., and Pareti, F. I., eds) pp. 125-136, Academic Press, New York

Nelsestuen. G. L.. and Broderius. M. (1977) Biochemistry 16, 4172-4177 28.

Bloom, J. W., and Mann, K. G. (1979) Biochemistry, in press Tollefsen, D. M., Jackson, C. M., and Majerus, P. W. (1975) J.

Clin. Znuest. 56,241-245 29.

DahIback, B., and Stenflo, J. (1978) Biochemistry 17,4938-4945 Miletich, J. P., Jackson, C. M., and Majerus, P. W. (1977) Proc.

Natl. Acad. Sci. U. S. A. 74,4033-4036 Miletich, J. P., Jackson, C. M., and Majerus, P. W. (1978) J. Biol.

Chem. 253,6908-6916

30.

31.

32. Tollefsen, D. M., Feagler, J. R., and Majerus, P. W. (1974) J. Biol.

Chem. 249,2646-2651 Miletich, J. P., Majerus, D. W., and Majerus, P. W. (1978) J. Clin.

Invest. 62,824-831

33. Colman, R. W. (1969) Biochemistry 8, 1438-1445 34. Saraswathi. S.. Rawala. R.. and Colman. R. W. (1978) J. Biol.

Nesheim, M. E., Myrmel, K. H., Hibbard, L., and Mann, K. G. (1979) J. Biol. Chem. 254,508-517

Nesheim, M. E., Prendergast, F. G., and Mann, K. G. (1979) Biochemistry 18, 996-1003

Nesheim, M. E., and Mann, K. G. (1979) J. Biol. Chem. 254, 1326-1334

35.

36.

37.

27. Fass, D. N., Knutson, G. J., and Bowie, E. J. W. (1978) J. Lab. Clin. Med. 91, 307-320

Ware, A. G., Fahey, J. L., and Seegers, W. H. (1948) Am. J. Physiol. 154, 140-147

Hjort, P., Rapaport, S. I., and Owren, P. A. (1955) Blood 10, 1139-1150

Breederveld, K., Giddings, J. C., TenCate, J. W., and Blood, A. L. (1975) Br. J. Hematol. 29,405-412

Osterud, B., Rapaport, S. I., and Lavine, K. K. (1977) Blood 49, 819-834

Freeman, J. P., Guillan, M. C., Begeand, A., and Jackson, C. M. (1977) Fed. Proc. 36,675

Chem. 253, 1024-1029 Bajaj, S. P., Butkowski, R. J., and Mann, K. G. (1975) J. Biol.

Chem. 250,2150-2156 Nesheim, M. E., Taswell, J. B., and Mann, K. G. (1979) J. Biol.

Chem., in press Tracy, P. B., Peterson, J. M., Nesheim, M. E., McDuffie, F. C.,

and Mann, K. G. (1979) Thromb. Haemostas. 42(l), 325

by guest on Novem

ber 16, 2020http://w

ww

.jbc.org/D

ownloaded from

P B Tracy, J M Peterson, M E Nesheim, F C McDuffie and K G MannInteraction of coagulation factor V and factor Va with platelets.

1979, 254:10354-10361.J. Biol. Chem. 

  http://www.jbc.org/content/254/20/10354.citation

Access the most updated version of this article at

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

  http://www.jbc.org/content/254/20/10354.citation.full.html#ref-list-1

This article cites 0 references, 0 of which can be accessed free at

by guest on Novem

ber 16, 2020http://w

ww

.jbc.org/D

ownloaded from