THE JOURNAL OF BIOLOGICAL CHEMISTRY

Printed cn U.S.A. Vol. 257, No. 7, Issue of April 10. pp. 3963-3969, 1982

The Interaction of Human Coagulation Factor V, with Platelets* (Received for publication, October 5, 1981)

William H. Kane and Philip W. Majerus From the Division of Hematology-Oncology, Departments of Internal Medicine and Biological Chemistry, Washington University School of Medicine, St. Louis, Missouri 63110

Factor V, is necessary for prothrombin activation on the platelet surface. We have measured the binding of '251-labeled human coagulation Factor V, to unstimu- lated platelets using thrombin inhibitor 5-dimeth- ylaminonaphthalene-1-sulfonyl arginine-4-ethylpiperi- dine to prevent release of unlabeled endogenous Factor V. T w o of the three labeled peptide chains produced by thrombin activation of Factor V (Mr = 110,000 and M, = 78,000) bind to platelets and therefore comprise Fac- tor V,. Binding does not require, but is stimulated by, Factor X, with half-maximal stimulation occurring at 7 X lo-" M Factor X,. Prothrombin (1 p ~ ) also stimu- lates Factor V, binding. The binding of Factor V, in the presence of Factor X, shows a sharp dependence on the calcium ion concentration with optimal binding at 2.5 mM. '251-labeled Factor V, bound to platelets is dis- placed upon addition of unlabeled Factor V, but not when unlabeled Factor V is added, indicating that the binding sites are specific for Factor V,. We measured the Factor V, dependence of prothrombin activation in two experiments using platelets from a patient with congenital Factor V deficiency. Half-maximal pro- thrombin activation rates were achieved at 1.5 X 10"' M Factor V, and maximum rates were achieved by a concentration of 5 X 10"O M Factor V, (100 ng/ml). We also measured the binding of lZ5I-labeled Factor V, to platelets and found that binding did not saturate in the same concentration range as acceleration of prothrom- bin activation. The binding of Factor V, was identical whether or not DAPA was present. However, DAPA did inhibit the binding of Factor X, to platelets thereby precluding simultaneous measurement of the binding of Factors V, and X, to normal (Factor V-containing) platelets. The amount of Factor V, that binds to plate- lets in the range of 1000-2000 ng of added Factor V,/ml is greater (2000-3000 molecules/cell) than would be predicted from our previous studies of platelet surface prothrombin activation ( ~ 2 0 0 Factor X, binding sites/ cell). These results suggest that either platelets contain "spare" Factor V, binding sites that do not participate in prothrombin activation or alternatively that Factor V, is an oligomer composed of M,. = 188,000 subunits.

Platelet surface proteins participate with plasma proteins in several reactions which are necessary for normal hemostasis (1). Thus, platelet adherence at the site of vascular injury

* This research was supported by Grants HLBI 14147 (Specialized Center in Thrombosis) and HL 16634 from the National Institutes of Health, and in part by National Institutes of Health Research Service Award GM 07200, Medical Scientist, from the National Institute of General Medical Sciences. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisernent" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

requires the binding of the plasma protein von Willebrand factor to a membrane protein designated Glycoprotein Ib (2- 6). Subsequent aggregation of platelets to form a hemostatic plug requires the binding of fibrinogen to a platelet membrane protein designated Glycoprotein IIb-IIIa (7-12). Small amounts of thrombin formed early in hemostasis (13) serve to stimulate platelet aggregation and secretion by binding to a high affinity platelet receptor (14, 15).

Prothrombin activation also occurs on the platelet surface (16-19). Platelets bind Factor X, with high affinity (Kc! = 30- 70 PM), and bound Factor X, catalyzes the activation of prothrombin 300,000 times faster than Factor X, in solution. Factor X, binds to 200-300 sites on the platelet surface. The binding is specific for Factor X, since neither the zymogen, Factor X, nor other coagulation factors displace bound Factor X, (17). Factor X, binding requires either exogenous Factor V, or stimulation of the platelet release reaction to release platelet Factor V (which is then converted to Factor V;J. Factor X, binding is limited by some platelet component other than Factor V,. This was determined by showing that addition of exogenous Factor V, does not significantly increase the binding of Factor X, to normal thrombin-treated platelets even when Factor X, concentrations 10-fold higher than the K d were used (18).

We have isolated human plasma coagulation Factor V (21). It contains a single type of high molecular weight chain ( M , = 335,000) that is converted by thrombin to the active form Factor V,, through cleavage of three bonds within the Factor V molecule. We now report that the M, = 110,000 and the M , = 78,000 peptides bind to platelets with high affinity. The M,. = 150,000 peptide does not bind to platelets and is therefore released as an activation peptide. Maximal binding of Factor V, to platelets is seen in the presence of Factor X,, prothrom- bin, and 2.5 mM calcium ions and is not inhibited by Factor V.

MATERIALS AND METHODS

The preparation and assay of human prothrombin, thrombin, Fac- tor X,, Factor V, and thrombin-activated Factor V, have been de- scribed (17,21). Thrombin was assayed using United States standard thrombin lot J. DAPA' was synthesized from 5-dimethylaminonaph- thalene- t-sulfonyl arginine (Pierce) and 4-ethylpiperidine (gift of Mr. Edward Sowers, Reilly Tar-Chemical, Indianapolis, IN) by the method of Nesheim et al. (22). Our preparations of DAPA inhibited human thrombin with a K, of 60-120 nM as determined in an assay system containing 1 unit/ml of thrombin and 3 mg/ml of human fibrinogen. IODO-GEN (1,3,4,6-tetrachloro-3a-6a-diphenylglycouril) was obtained from Pierce. Carrier-free sodium [""Iliodide and sodium [""I]iodide were obtained from Amersham Corp. N,N'-carbonyldi- imidazole was obtained from Sigma. Apiezon oil was obtained from J. B. Biddle Co. All other reagents were purchased from Sigma, Fisher, or Mallinckrodt Chemical Works.

Sodium dodecyl sulfate-gel electrophoresis was performed (23)

I The abbreviations used are: DAPA, 5-dimethylaminonaphtha- lene-1-sulfonyl arginine-4-ethylpiperidine; SDS, sodium dodecyl sul- fate.

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3963

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3964 Binding of Human Factor V , to Platelets

using 7.5 or 10% acrylamide with a 4'7 acrylamide stacking gel. Hadioiodinated samples were detected by counting I-mm slices in a Beckman :I00 Gamma Counter or by autoradiography (24).

Factor X was iodinated using the method of Bolton and Hunter (25) or by using the IOI)O-GEN method of Fraker and Speck (26) . In the latter case 10 p g of IODO-GEN dissolved in chloroform was evaporated in the bottom of a polypropylene tube. Factor X (125 p g , 1.25 mg/ml, in 0.6 M NaCI. 10 mM benzamidine, 10 mM sodium phosphate, pH 6.0) and 1 mCi of sodium [""I]iodide or sodium ['."I] iodide were then added and the iodination was allowed to proceed for 5 min a t 4 "C. The mixture was then passed over a Sephadex G-25 column (4 X 50 mm) equilibrated with 0.15 M NaCI, 20 mM Tris, pH 7.4, to remove iodide. Protein concentration was determined by measuring absorbance at 280 nm assuming E & , = 11.6. Samples were adjusted to contain 5 mg/ml of rectystallized bovine serum albumin and stored at -70 "C. The Factor X preparations were 1ooO-3000 cpm/ng of protein. Factor X was activated using the Factor X activator from Russell's viper venom immediately before use as described previously (15). Hadioiodinated Factor X,, preparations had the same enzymatic activity as unlabeled preparations based on coagulation assay.

Human Factor V was iodinated by a modification of the method previously described (21). Human Factor V (350 p g , 1.1 mg/ml in 0.15 M NaCI, 20 mM Tris-HCI, pH 7.4, 10 mM benzamidine, 5 mM CaCI?) was added to the bottom of a polypropylene tube coated with 1 0 pg of IODO-GEN. One millicurie of sodium ["'II]iodide was added and the reaction was allowed to proceed for 5 min at 4 "C. The sample was then passed over a Sephadex G-25 column (1.5 X 3 cm) equili- brated with 0.15 M NaCI, 20 mM Tris-HCI. pH 7.4, and 5 mM CaCI?. Protein concentration was determined by absorbance at 280 nm assuming E ~ A , = 8.9 (21) . Samples were adjusted to contain 10 mM benzamidine and 5 mg/ml of recrystallized bovine serum albumin and stored at -70 "C. The Factor V preparations were 340-4000 cpm/ng of protein. '"I-labeled Factor V (200-300 pg/ml) was activated im- mediately before use by incubation for 20 min at 37 "C with 4 units/ ml of thrombin in the presence of 1 0 mM benzamidine. Hadioiodinated Factor V and Factor V,, retained the same coagulation activity as the unlabeled proteins.

Platelets were isolated as previously described (14). On two occa- sions we studied platelets from a patient (*J. l'.) who has congenital Factor V deficiency. The patient's plasma Factor V level was 1'7 of normal and his plasma Factor VI11 level was normal.

Factor V,, and Factor X,, binding to platelets was measured using methods similar to those previously described for measuring the binding of Factor X,, to platelets ( 1 7). IJnless otherwise stated, binding assay mixtures contained 10" platelets/ml, recrystallized bovine serum alhumin, 5 mg/ml. 0.15 M NaCI, 20 mM Tris, pH 7.4, 2.5 mM CaCl?, 5.5 mM glucose. and 75 p g of prothrombin/ml (where indicated). In most experiments 93 p~ DAl'A was included to prevent thrombin-induced release of endogenous platelet Factor V. In some experiments utilizing platelets from the patient with congenital Factor V deficiency, 1 unit/ ml of thrombin was included to stimulate the platelet release,reaction. Binding mixtures also contained either ""I-labeled Factor V,, and unlabeled Factor X,, or "'I-labeled Factor X,, and unlabeled Factor V,, or "..'I-labeled Factor V,, and "'I-labeled Factor X*-. Binding incu- bations were carried out for 20 min; the platelets were then sedi- mented through oil as previously described (17). Hadioactivity in platelet pellets and supernatants was detected using a Beckman 300 Gamma Counter. Nonspecific binding was determined in parallel binding mixtures where 100-fold excess of unlabeled protein was included. Nonspecific binding ranged from 1-2% of the total added radioactivity for Factor X,, binding. I'reliminary experiments indi- cated that nonspecific Factor V,, binding also increased linearly ( r = 0.998) with added ""I-labeled Factor V,, in the concentration range we examined (0-2ooO ng/ml). Factor V,, nonspecific binding was time- dependent but was not affected by added Factor X,,. In all of the experiments presented in this paper, nonspecific binding has been subtracted from the total binding to yield specific binding.

RESULTS

Characterization of '""I-labeled Factor V and V,-Factor V is a single chain high molecular weight procofactor as shown in Fig. 1, lane I ( M , = 335,000) that is converted by thrombin to the active form, Factor V,, through cleavage of three bonds within the Factor V molecule (21). We had previously identi- fied three activation products: two Coomassie blue-staining

components ( M , = 121,000 and a M , = 95,000-91,000 doublet); and another component ( M , = 150,000) which could only be detected using radiolabeled Factor V. Analysis of Factor V;, using 7.5%. acrylamide-SDS slab gel electrophoresis yielded lower apparent molecular weights for the major Coomassie blue-staining components (Fig. 1, lane 2 M , = 110,000 and M , = 78,000 doublet) than we previously reported (21). In addi- tion, a faintly staining M , = 30,000 activation product is also seen. This component is only detected when 10 pg or more of Factor V,, is applied to a gel. We believe that the M , = 30,000 component corresponds to the predicted fourth activation component (21), although we have not excluded the possibility that it is a degradation product of one of the other compo- nents. Human Factor V has a molecular weight of 335,000; the four activation products have an apparent molecular weight of 368,000. This difference may be due to the aberrant migra- tion of some of the components in SDS-polyacrylamide gel electrophoresis. The human Factor V activation products we have observed are quite similar to those reported by Dahlback (27) ( M , = 110,000 and M , = 72,000) and Katzmann et al. (28) (M, = 90,000, M , = 76,000, and M , = 30,000). These workers did not detect the M , = 150,000 activation product because it is not detected using Coomassie blue staining.

We iodinated human Factor V using the chloroglycouril method described previously (21). The '"'I-labeled Factor V preparations ranged from 340-4000 cpm/ng (0.025-0.3 atom of iodine/molecule of Factor V) and retained complete coag- ulation activity with activities between to 50-100 units/mg of Factor V which increased upon activation with thrombin to 1500-2100 units/mg. Analysis of '""I-labeled Factor V by SDS- polyacrylamide gel electrophoresis (Fig. 1, lane 3 ) shows a major high molecular weight component corresponding to Factor V, which comprised 60-75% of radioactivity on the gel in various experiments. The other components near the top of the gel represent partially degraded forms of Factor V. The migration of the low molecular weight components present near the tracking dye is unaffected following treatment with thrombin. These are probably radiolabeled contaminants in

1 2 3 4 5 6 7 8 9 1 0 1 1 - 200 - - 150 - 116- - - 110 - 94 - 68 - a- 78 -

43 - c_ 30 -

. . .

FIG. 1. Polyacrylamide gel electrophoresis (7.5%) with so- dium dodecyl sulfate of Factor V and thrombin-activated Fac- tor V. (23). In lanes I and 2 protein was detected by Coomassie blue staining. In lanes 3-11 protein was detected by autoradiography (24). Lane I, 10 p g of Factor V; lane 2, 10 pg of thrombin-activated Factor V.; lane 3: 170 ng of ""I-labeled Factor V. Platelets (10"/ml) were incubated in a mixture containing 0.15 M NaCI, 20 mM Tris-HCI, pH 7.4, 2.5 mM CaCI?, 5.5 mM glucose, 5 mg/ml of bovine serum albumin, 93 pM DAPA, 13 ng/ml of Factor X. and either 15, 150, or 1500 ng/ml of ""I-labeled Factor V.. After 20 min the platelets were sedimented through oil and the platelet pellet (10" platelets) and 6 pl of the platelet supernatant was dissolved in a buffer containing sodium dodecyl sulfate and 2-mercaptoethanol. Solubilized platelet pellets were electrophoresed in lanes 5-7, and platelet supernatants were electrophoresed in lanes 8-10. The migration of molecular weight standards and apparent molecular weights of Factor V,, activation components are indicated X 10. .I.

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Binding of Human Factor V, to Platelets 3965

the Factor V preparation. Following activation of Factor V with thrombin there are three radioactive products seen with apparent M , = 150,000, M, = 11O,OOO, and M , = 78,000 as shown in Fig. 1, lane 4. In this sample a residual amount of the second intermediate of thrombin activation is present (21). The M , = 30,000 component is either not labeled by the IODO-GEN method or is obscured by a radiolabeled contam- inant.

Identification of the Factor V Activation Products Corre- sponding to Factor V,-We next determined which of the three labeled components produced by thrombin activation of Factor V bind to platelets in the presence of saturating (10 X ICd) amounts of Factor X, and physiological concentrations of prothrombin. Since platelets contain a large amount of Factor V (19), it was necessary to block thrombin action to prevent release and activation of platelet Factor V, and dilution of radiolabel. We used the specific thrombin inhibitor DAPA for this purpose (22). We previously found that the concentrations of thrombin required for the release of Factor V and ["C] serotonin are similar (20). In the presence of 93 PM DAPA, at least 20,000-fold more thrombin is required to induce release of [I4C]serotonin than in its absence (data not shown). Thus, even at concentrations of thrombin as high as 100 units/ml, the maximum amount generated in our experiments, release of Factor V is less than 5% in the presence of 93 PM DAPA. We incubated platelets with 15, 150, and 1500 ng/ml of 1251 Factor V, and examined the platelet pellets and supernatants by SDS-polyacrylamide gel electrophoresis. The platelet su- pernatants (lanes 8-10) contain all three labeled components that were present in the starting material (lanes 4 and 1 1 ) . However, the platelet pellets contained only the M , = 110,000 and M, = 78,000 components (lanes 5-7). The M, = 150,000 component does not bind to the platelet surface. Since pro- thrombin activation occurs on the platelet surface the M , =

110,000 and M, = 78,000 components comprise Factor V, by definition and the M , = 150,000 component is an activation peptide. The M , = 30,000 component is not labeled by the IODO-GEN or Bolton-Hunter labeling techniques; thus, we do not know whether it binds to the platelet surface and constitutes part of Factor V..

In order to calculate the amount of Factor V, binding to platelets it was necessary to estimate the molecular weight and specific radioactivity of the labeled Factor V, preparation. We assumed the molecular weight of Factor V, to be equal to the sum of the apparent molecular weights of the two radio- labeled components that bind to platelets, 188,000. The spe- cific radioactivity of Factor V, was determined by multiplying the specific activity of the Iz5I-labeled Factor V preparation by the fraction of the radioactivity in the M, = 110,000 and M, = 78,000 components as determined by SDS-polyacryl- amide gel electrophoresis and then by multiplying by the ratio of the molecular weight of Factor V/Factor V, (335,000/ 188,000). Since we do not know whether the M, = 30,000 component binds to the platelet surface and there is some uncertainty in the molecular weights and therefore in the specific activities of various components, our estimates are probably accurate within 20%.

Binding of Factor V, to Platelets in the Presence of DAPA-Fig. 2 shows a time course of the binding of Factor V, to platelets utilizing 150 ng/ml of Iz5I-labeled Factor V, in the presence of saturating amounts of Factor X, and 1 PM prothrombin. Thrombin-induced release of endogenous Fac- tor V was prevented by the inclusion of 93 PM DAPA in the binding mixtures. Binding approached a steady state within 20 min. When 25 p / d of unlabeled Factor V, was added, there was rapid displacement of labeled Factor V,, indicating that the binding is reversible.

The binding of Factor V, to platelets in the presence of Factor X, (13 ng/ml) shows a sharp dependence on the calcium ion concentration (Fig. 3). Optimal binding is seen at 2.5 mM calcium which is the concentration for that ion in plasma. Inhibition of Factor V, binding occurs at lower or higher calcium concentrations. Similar results were obtained when prothrombin plus Factor X, was added except that total Factor V, binding increased as shown below. In contrast, binding of Factor V, to platelets in the absence of Factor X,

r

I I L 1

IO 20 30 40 TIME (Mlnutes)

FIG. 2. Time course of '"'I-labeled Factor V, binding to plate- lets. Platelets (lOR/ml) were incubated with a mixture containing 0.15 M NaCl, 20 mM Tris-HCI, pH 7.4, 2.5 mM CaC12, 5.5 mM glucose, 5 mg/ml of bovine serum albumin, 93 ELM DAPA, 13 ng/ml of Factor X,, 1 p~ prothrombin, and 150 ng/ml of '2sI-labeled Factor V. (400 cpm/ng). The experiment was started with the addition of platelets. At the indicated times samples were taken and specific Factor V. binding was determined. Nonspecific binding determined in a parallel experiment was time-dependent ranging from 0.9 to 2.5% of the added radioactivity. After 25 min of incubation 25 pg/ml of unlabeled Factor V, was added to a parallel reaction mixture and specific Factor V, binding was determined at the indicated times.

1 I 1 I 5 10 15 20

CaClz (mM)

FIG. 3. Effect of Cas+ concentration on Factor V, binding to platelets. Reaction mixtures contained lo8 platelets/ml, 0.15 M NaCl, 20 mM Tris-HCI, pH 7.4, 5.5 mM glucose, 5 m g / d of bovine serum albumin, 93 p~ DAPA, 150 (0) or 15 n g / d (0) of '251-labeled Factor V,; and varying concentrations of CaCL. Nonspecific binding was 0.7% of the added radioactivity.

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3966 Binding of H ~ m a n

and prothrombin was maximal at the lowest concentration used (0.13 mM) and decreased to nearly zero at 20 mM calcium (data not shown). All subsequent experiments were performed using 2.5 mM calcium ions. '"SI-labeled Factor V, binds to platelets in the absence of other coagulation factors as shown in Fig. 4. When prothrombin is included, Factor V, binding increased approximately 2-fold. Maximal Factor V, binding to platelets was observed when both prothrombin and Factor X, were used. Factor X, enhances Factor V, binding to platelets when prothrombin is omitted from the binding assay as shown in both Figs. 5 and 6. Factor V (5 pg/ml) has essentially no effect on the binding of Factor V, to platelets either in the absence or in the presence of added Factor X, as shown in Fig. 5. This indicates that the binding sites are specific for Factor V,.

We also examined the dependence of Factor V, binding on varying Factor X, concentration as shown in Fig. 6. As the Factor X, concentration is increased, both the amount of'

t & H 1 I I L 1 I I 1 J

100

FACTOR V, FREE (ng/ml) 200 300 A00

FIG. 4. Effect of prothro~bin and Factor X, on Factor V, binding to platelets. All reaction mixtures contained lo8 platelets/ ml, 0.15 M NaC1, 20 mM Tris-HC1, pH 7.4, 2.5 mM CaCL, 5.5 mM glucose, 5 mg/ml of bovine serum albumin, 93 PM DAPA, and increas- ing concentrations of lz5I-1abeled Factor V, (2246 cpm/ng). 0, no additional proteins included; A, added prothrombin (75 pg/ml); 0, added prothrombin (75 pg/ml) and Factor X, (17 ng/ml). Nonspecific binding corrections were: 0,0.65% of the total radioactivity, (1.9 ng of Factor V,/la" platelets at 300 ng of Factor VJml added); A and 0, 0.8% of the total radioactivity (2.4 ng of Factor V,/lO') platelets a t 300 ng of Factor V,/ml).

FACTOR-V, FREE (ngrml]

FIG. 5. Effect of Factor V on Factor V, binding to platelets. Reaction mixtures contained IO8 platelets/ml, 0.15 M NaC1, 20 mM Tris-HC1, pH 7.4, 2.5 mM CaC13, 5.5 mM glucose, 5 mg/ml of bovine serum albumin, 93 p~ DAPA, and varying concentrations of "'1- labeled Factor V, (1050 cpm/mg). D, no additional proteins; 0, plus unlabeled Factor V (5 pg/ml); 0, plus 13 ng of Factor X,/@ 0, plus 5 pg of unlabeled Factor V/ml and 13 ng of Factor X,/ml. Nonspecific binding was 0.5% of the added radioactivity corresponding to 1.5 ng of Factor V,/108 platelets at 300 ng of Factor V,/ml.

i Factor V, to Platelets

Factor X, and of Factor V, bound to platelets increases. In this experiment a maximum of 81 Factor X, molecules and 223 Factor V, molecules were bound/platelet. The amount of Factor X, binding is less than maximal both because the concentration of Factor V, used in this experiment, 150 ng/ ml, is less than an optimal amount and because DAPA inter- feres with Factor X, binding (see below). However, the con- centration of Factor X, required for a half-maximal increase in Factor V, binding (7 X lo-" M) was similar to the concen- tration of Factor X, required for half-maximal Factor X, binding to normal platelets (17). We found the same Factor X, concentration dependence of Factor V, binding at Factor V, concen~ations ranging from 100-300 ng of Factor V,/ml. The experiment shown in Fig. 6 was performed without pro- thrombin, indicating that the increase in Factor V, binding is due directly to Factor X, and is not a consequence of pro- thrombin activation. Similar results were obtained when pro- thrombin (75 pg/ml) was included in the binding assay except that Factor V, binding increased to a m a ~ m u m of 600 mole- cules/platelet with a half-maximal increase in binding occur- ring at 7.5 X lo-" M Factor X,.

We repeated the experiment shown in Fig. 6 using platelets that were preincubated at a concentration of 10B/ml with 20 ng/ml of 13'I-labeled Factor X,, 93 p~ DAPA, 2.5 mM CaC12, 0.15 M NaC1, 20 mM Tris, pH 7.4, and 5 mg/ml of bovine serum albumin for 20 min and then centrifuged and washed to remove Factor X,. No Factor X, binds in the absence of Factor V, (20) and the Factor X, dependence of Factor V, binding was the same in these platelets as in those not previously exposed to Factor X,. Thus, the enhancement of Factor V, binding by Factor X, requires the continued pres- ence of Factor X, and is not due to a change in the platelets induced by Factor X.

Although the binding of Factor V, to platelets is stimulated by Factor X, platelets can bind Factor V, in the absence of Factor X, (Figs. 5 and 6). In the converse experiment where Factor X, is added to platelets in the absence of Factor V,, no Factor X, binding is observed (18, 20).

~nte~ac t ion of Factor V, with Platelets from a Patient with Congenital Factor V Deficiency-It is apparent that there are more binding sites for Factor V, on platelets than for

FACTOR X, FREE ( n g l m l ) FIG. 6. Effect of increasing Factor X, on Factor V, and Fac-

tor X, binding to platelets. Mixtures contained platelets (108/ml), 0.15 M NaC1, 20 mM Tris-HC1, pH 7.4, 2.5 mM CaCl,, 5 mg/ml of bovine serum albumin, 93 p~ DAPA, and either 150 ng/ml of 1251- labeled Factor V, (570 cpm/ng) and increasing concentrations of unlabeled factor X, (0), or 150 ng/ml of unlabeled Factor V, and increasing amounts of '2sI-labeled Factor X. (5100 cpm/mg) (0). Nonspecific binding was 0.76 and 0.9% of the radioactivity added for Factor V, and Factor X, binding, respectively. Factor V, nonspecific binding was 1.1 ng/108 platelets.

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Binding of Human Factor V, to Platelets 3967

Factor X,. In previous experiments we showed that Factor X, binds to a single class of sites on platelets that can bind 200- 300 molecules/platelet (16-20). Furthermore, the rate of pro- thrombin activation correlates with the binding of Factor X, (17). We were unable to measure the rate of prothrombin activation as a function of added Factor V, using normal platelets because of the fact that the thrombin formed during the assay causes the release and activation of large amounts of endogenous platelet Factor V. In order to estimate the participation of Factor V, in prothrombin activation we used platelets from a patient with congenital Factor V deficiency. Factor V, accelerated prothrombin activation in a hyperbolic fashion as shown in Fig. 7 with half-maximal rates at 30 ng of Factor V,/ml (1.5 X 10"" M). We repeated this experiment on another occasion and again found that Factor V, stimulated prothrombin activation in a saturable manner with a half- maximal prothrombin activation rate occurring at 1.9 x 10"" M Factor V, (Fig. 8). In this experiment we measured Factor V, binding simultaneously and found that it did not strictly parallel the rate of prothrombin activation. In the experiments where Factor V,-dependent prothrombin activation was meas- ured, rates of 80-100% of maximum were achieved by 100 ng of Factor VJml (Figs. 7 and 8). When Factor V, binding was measured directly there was a 2-fold increase in the amount of Factor V, bound as the concentration of added Factor V, was increased from 100 to 300-400 ng/ml. Therefore, the binding of Factor V, to platelets, although required for accel- erated prothrombin activation, does not correlate precisely with the rates of thrombin formation. This is also apparent from the fact that Factor V, binds to platelets even in the absence of Factor X,. It appears that platelet activation has no effect on Factor V, binding, since, as shown in Fig. 8, the binding of Factor V, to platelets from the Factor V-deficient subject was the same whether or not DAPA was present. However, when we simultaneously measured the binding of

I-Factor X, under the same conditions, we found that 131

I 0

l/FACTOR V, ADDED (ng/ml)-'

I I I I I J 0 100 200 300 400 500

FACTOR V, ADDED (ng/mI) FIG. 7. Dependence of the rate of prothrombin activation on

added Factor V. using platelets from a patient with congenital Factor V deficiency. Reaction mixtures contained platelets (lon/ ml), 0.15 M NaCI, 20 mM Tris-HCI, pH 7.4, 2.5 mM CaC12, 5 mg/ml of bovine serum albumin, 5.5 mM glucose, 20 ng of Factor X,/ml, 75 pg of prothrombin/ml, 1 unit of thrombin/ml, and varying concentra- tions of Factor V,. The Lineweaver-Burk plot obtained from the data is shown as an inset. The calculated K d and VmaX values were 1.4 X 10"" M Factor V, and 12 units of thrombin/ml/min, respectively.

I 1' I

1 I I I 100 200 300

FACTOR V, ADDED FIG. 8. Factor V. binding to platelets from a patient with

congenital Factor V deficiency. Reaction mixtures contained platelets (loR/ml), 0.15 M NaC1, 20 mM Tris-HCI, pH 7.4, 2.5 mM CaC12, 5 mg/ml of bovine serum albumin, 5.5 mM glucose, and 20 ng of '"I-labeled Factor X./ml (2750 cpm/ng), 75 pg of prothrombin/ml, and varying concentrations of '2sII-labeled Factor V, (240 cpm/ng). 0, contained 93 p~ DAPA to prevent thrombin stimulation of the platelet release reaction; 0, contained thrombin (1 unit/ml) to stim- ulate the platelet release reaction. After a 20-min incubation platelet Factor V, binding and Factor X, binding (not shown) were deter- mined. Factor V, nonspecific binding was 0.6 and 0.9% of the total I2'I radioactivity added for unstimulated platelets (0) and stimulated (O), respectively. At a Factor V, concentration of 300 ng/ml, 1.8 ng of Factor V,/lOR platelets were bound nonspecifically to unstimulated platelets while 2.7 ng of Factor V,/lOH platelets were bound nonspe- cifically to stimulated platelets. The rate of prothrombin activation measured in parallel assay mixtures carried out in the absence of DAPA is also shown (M).

DAPA interfered with Factor X, binding in a manner that suggests inhibition of the Factor X,-V, interaction. Thus, the binding of '"I-labeled Factor X, to platelets in the experiment of Fig. 8 a t 50, 10G, and 200 ng of Factor V,/ml was 0.77, 1.25, and 2.0 ng of Factor X,/ml, respectively, when binding was measured without DAPA. In the presence of DAPA the corresponding values were 0.34, 0.64, and 1.25 ng of Factor X,/ml. In this experiment the concentration of added Factor X, was 20 ng/ml which would be sufficient to give maximum Factor X, binding to platelets from normal subjects. The degree of inhibition of Factor X, binding diminished from 56% at 50 ng of Factor V,/ml to 37% at 200 ng of Factor V,/ml. In the converse experiment where Factor X, binding was meas- ured as a function of Factor X, concentration at a single Factor V, concentration (160 ng/ml), DAPA again inhibited binding of Factor X, by approximately 35% at concentrations ranging from 1-50 ng of Factor X,/ml. Since DAPA interferes with Factor X, binding to platelets, we are unable to measure the binding of both Factors X, and V, in the same experiment to normal (Factor V-containing) platelets.

Although the ability of Factor V, to accelerate prothrombin activation is saturable with respect to Factor V,, its binding to the platelet does not reach saturation at similar concentra- tions as shown in Figs. 4 and 8. We also studied the binding of Factor V, at even higher concentrations that do not affect rates of prothrombin activation as shown in Fig. 9. It is apparent that Factor V, binding approaches saturation only at concentrations of 2000 ng of Factor V,/ml. This is most clearly apparent when the data are plotted as suggested by

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3968 Binding of Human Factor V, to Platelets

100 r

9 - * e e

.E

-...L-d

0 1.000 2,000

FIG. 9. Factor V. binding to platelets. Reaction mixtures con- tained I d p~atele~/mi, 0.15 M NaCI, 20 m~ Tris-HCI, pH 7.4, 2.5 mM CaC12, 5.5 mM glucose, 5 m g / d of bovine serum albumin, 75 pg of prothrombin/ml, 25 ng of Factor X,/ml, 93 PM DAPA, and increasing concentrations of '251-labeled Factor V, (1400 cpm/ng). Nonspecific binding corrections were 0.88 of the total radioactivity (4.8 ng of Factor V,/lO* platelets at 600 ng of Factor V./ml). Znset, data plotted as Factor V, bound uersus log Factor V. concentration according to the method of Klotz and Hunston (29).

Klotz and Hunston where saturation is indicated by a plateau in the binding isotherm (29). Our inability to demonstrate clear saturation of Factor V, binding precludes further anal- ysis of the binding data.

FACTOR V, FREE (ng/ml)

DISCUSSION

Our previous studies have implicated Factor V, 8s part of the Factor X, binding site on human platelets (17-20). In this paper we demonstrate directly the binding of Factor V, to human platelets. Factor V consists of a high molecular weight polypeptide chain when analyzed by S~S-polyacrylamide gel electrophoresis and cleavage of Factor V by thrombin gener- ates Factor V. producing four noncovalently linked poly- peptide components (21). Dahlback (27) and Katzmann et al. (28) have also isolated human Factor V. The properties of their preparations including activation by thrombin are essen- tially identical to our Factor V preparation although they did not show the M, = 150,000 component since they used only Coomassie blue staining to detect activation products. Our results indicate that the M, = 110,000 and Mr = 78,000 peptides bind to platelets. Thus, we conclude that these two compo- nents comprise Factor V, and that the M, = 150,000 compo- nent is released as an activation peptide. Since the M, = 3 0 , ~ activation product is not labeled by our method, we do not know if it binds to platelets or whether it is released as an activation peptide as is the M, = 150,000 peptide. A two-chain structure of Factor V, would be consistent with the studies of Esmon (30), who isolated Mr = 110,000 and M, = 73,000 components from bovine Factor V, and showed that while neither peptide had Factor V, activity, the two peptides mixed together in the presence of calcium ions regenerated complete Factor V, activity as measured in a coagulation assay. Further, Esmon's work suggests that the two chains are present in a I/ 1 proportion since maximum activity was achieved with an equimolar mixture of peptides.

In order to determine the number of Factor V, molecules bound to platelets, the molecular weight, of Factor V, must be estimated. The molecular weight of human Factor V, has not yet been rigorously determined by sedimentation equilibrium or sedimentation velocity studies. The results of Dahlback indicate that bovine and human Factors V and V, are similar in structure as deduced by SDS-polyacrylamide gel electro- phoresis. Mann et al. (31) have estimated the molecular weight of bovine Factor V, to be between 151,000 and 185,000. Their analysis was complicated by the fact that there was evidence for association equilibria in the sedimentation equi-

librium studies. Several laboratories have found that the molecular weights of bovine and human Factor V, determined by gel fdtration are in the range of 300,000 to 500,000, based on globular protein standards (30, 32, 33). These higher esti- mates could be due to the asymmetry of the Factor V, molecule (31) or alternatively to self-association of Factor V, under the experimental conditions employed. For our studies we have assumed that the molecular weight of Factor V, is 188,000 based on the molecular weights of the two peptides which bind to platelets,

Whether measuring the binding of a ligand to a cell surface is of physiological significance is often difficult to establish. When binding is found to correlate with some functional response, relevance is inferred. In our previous work we showed that platelets have 200-300 Factor X, binding sites/ cell and that Factor X, binding was hyperbolic, required Factor V,, and correlated precisely with rates of Factor X,- dependent prothrombin activation. Analysis of Factor V, binding to platelets is more complex in that: fa) Factor V, binds to platelets without Factor X,, (b) the binding is not simply hyperbolic and saturation is not reached even at con- centrations above those that accelerate prothrombin activa- tion, (c) platelets contain large amounts of endogenous Factor V (approximately 300 ng/108 cells),2 which is released and activated during prothrombin activation thereby requiring addition of DAPA to prevent release of platelet Factor V when studies are performed using platelets from normal sub- jects. Stimulation of the platelet release reaction does not affect the binding of Factor V, to platelets, and the binding is specific in that Factor V does not inhibit binding. Maximal binding of Factor V, to platelets requires prothrombin, Factor X,, and 2.5 mM calcium ions. Although we were unable to determine the number of Factor V, binding sites on platelets, it is apparent that they exceed the number of Factor X, binding sites. We previously found that the amount of Factor V contained in normal platelets ( ~ 3 0 0 ng/108 platelets) was sufficient to support maximum rates of prothrombin activa- tion; additional Factor V, did not further accelerate thrombin formation (19, 20). Thus, under conditions of maximal accel- eration of prothrombin activation and maximal Factor X, binding (17),3 we find 25-35 ng of Factor V, bound/lO' plate- lets (Figs. 4,8, and 9). Assuming a molecular weight of 188,000 this corresponds to approximately 700-1000 Factor V, mole- cules bound whereas the number of Factor X, binding sites is only 2 ~ 3 ~ . We have considered three possible explanations for these "extra" Factor V, binding sites. 1) Our Factor V, preparation contains two-thirds inactive Factor V, that can bind to some nonfunctional site unrelated to prothrombin activation thereby obscuring the physi~lo~cally significant binding. This explanation seems unlikely since two other laboratories have isolated human Factor V by methods differ- ent from ours and they obtain the same specific activity by coagulation assay as we do (27, 28). Further, the experiment in Fig. 1 indicates that even at 1500 ng of Factor VJml only the 2 peptides of Factor V, bind to the platelet surface. 2) Factor V, is an oligomer of M, = 188,000. If, for example, Factor V, polymerizes on the platelet surface forming com- plexes of 2-4 units, this could explain both the complex binding pattern as well as the apparent increase in binding

We find that a mixture of 1 part normal to 9 parts Factor V- deficient. platelets at IO* platele~/ml binds one-half the amount of Factor X, that binds to lo8 normal platelets in the presence of 10 ng of Factor X,/ml(19). Since 30 ng/ml of Factor V, gives half-maxima1 rates of prothrombin activation as shown in Figs. 7 and 8, then lo7 normal platelets must contain approximately 30 ng of Factor V.

1 p~ prot~ombin (17), 20 ng of Factor XJml 1 ~ 1 0 X K d , and a300 ng of Factor V,/rnl.

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Binding of Human Factor V, to Platelets 3969

sites over that observed for Factor X,. It is conceivable that p o l ~ e ~ a t i o n is required for biological activity. 3) There are “spare” receptors for Factor V, that are not required for prothrombin activation. These might serve to prevent escape of excess Factor V, from a hemostatic plug thereby localizing prothrombin activation. There is no evidence to favor either of these latter hypotheses. Factors X, and V, appear to interact with each other in binding to platelets since each stimulates the binding of the other. Since Factor V, can bind without Factor X, but not the converse, it appears that Factor V,, must be bound to the platelet before Factor X, can bind.

Factor X, interacts not only with Factor V, but also with a separate site on platelets? We have prepared a derivative of bovine Factor X,, des (1-44) Factor X,, that lacks the NH2- terminal portion (44 amino acid residues) of the molecule which contains the y carboxyglu~myl residues. Although des (1-44) Factor X, is equivalent to Factor X, in its ability to interact with Factor V, in solution and catalyze prothrombin activation, its ability to bind to the human platelet Factor X, receptor is less than 1% of that of human or bovine Factor X,. Thus, binding of Factor X, to the Factor X,, receptor on human platelets requires both the interaction between plate- let-bound Factor V, and an interaction between a Factor X, domain at its NH2 terminus and some platelet component. The nature of the components on the platelet membrane to which Factor V, and X, bind remains obscure. Many workers assume that these components represent negatively charged phospholipids which are analogous to the phospholipid used in in uitro coagulation assays. An alternative possibility is that Factors V, and X, bind to specific protein receptors in a manner similar to fibrinogen and von Willebrand factor. Sup- porting this latter possibility are the studies of Thiagarajan et al. (34), who demonstrated that a monoclonal antibody reac- tive towards negatively charged phospholipids inhibited pro- thrombin activation in the presence of phospholipids, but not when platelets were substituted for phospholipid. They found that the antibody did not block Factor X, binding to human platelets. The patient who developed this in uitro coagulation inhibitor had no bleeding diathesis. In contrast, we have described a patient with a moderately severe bleeding disorder who had normal plasma coagulation factors and platelet Fac- tor V, but abnormal platelet Factor X. binding and Factor X,- catalyzed prothrombin activation (19). This patient’s platelets are deficient in some platelet component required for the Factor X, receptor. This deficiency suggests that a protein component is required.

Tracy et al. (35, 36) have recently presented studies on the interaction between bovine Factors V, and X, and platelets. These authors also found that the rate of prothrombin acti- vation saturates at lower bovine Factor V, concentrations than does Factor V, binding, although there are many differ- ences between their results and our results in the human system. The major differences include: I) The affinity of Factor X, for bovine platelets is 10- to 20-fold less than in the human system. 2) Not all of the Factor X, bound to bovine platelets participates in platelet surface prothrombin activa- tion. 3 ) Significant amounts of Factor X, bind to unstimulated bovine platelets (200-400 molecules/ceU in the absence of exogenous Factor V,; in contrast, there is no Factor X, binding to human platelets in the absence of Factor V,. 4) Bovine platelets have the same number of Factor X, binding sites as Factor V, binding sites, while human platelets have more Factor V, binding sites than Factor X, binding sites. 5 ) Bovine platelets contain limiting amounts cf Factor V while human platelets have an excess of Factor V compared to the amount ‘ T. Morita, W. H. Kane, P. W. Majerus, and C. M. Jackson,

manuscript in preparation.

required to saturate prothrombin activation (19). 6) The bind- ing of Factor V, to bovine platelets is unaffected by prothrom- bin and Factor X, while these proteins stimulate Factor V, binding to human platelets. Thus, the bovine system may not be a good model to study platelet-dependent prothrombin activation as it might relate to human disorders.

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W H Kane and P W MajerusThe interaction of human coagulation factor Va with platelets.

1982, 257:3963-3969.J. Biol. Chem. 

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