Proc. Nat! Acad. Sci. USA Vol. 80, pp. 2380-2384, April 1983 Medical Sciences

Prothrombinase complex assembly on the surface is mediated through the 74,000-dalton component of factor Va (factor Xa/ activation/prothrombin activation) PAULA B. TRACY AND KENNETH G. MANN* Hematology Research Section, Mayo Clinic/Foundation, Rochester, Minnesota 55905 Communicated by Oscar D. Ratnoff, January 24, 1983 ABSTRACT The blood protein factor Va forms the for factor Va activity (14, 15, 18). Ca2" is required to maintain receptor for the serine protease factor Xa on the platelet surface. the subunit interaction and resultant factor Va activity (7, 15). This membrane-bound complex of factor Va and . plus Ca2+ Autoradiography of "2I-labeled factor Va bound to comprises the prothrombinase complex, the enzyme that catalyzes indicates that components D and E are the factor Va peptides the proteolytic conversion of prothrombin to the clotting enzyme associated with the platelet membrane (6, 19). With time, how- . Factor Va is a two-subunit protein composed of com- ever, another peptide appears which we have labeled D' be- ponent D (Mr = 94,000) and component E (Mr = 74,000); subunit cause it arises as a result of a platelet-associated protease cleav- interaction is Ca2+ dependent. Factor Va bound to platelets con- age ofcomponent D (19). The protease appears sists of three peptides: component D, component E, and com- platelet-associated ponent D' (Mr = 90,000) which appears as the result of a platelet- to be similar to the Ca2+-dependent sulfhydryl platelet pro- associated protease cleavage of component D. The present studies tease (20-22) because its activity is inhibited by protease in- were undertaken to determine which peptide(s) mediates the hibitors such as leupeptin and pepstatin A and by inhibitors of binding of factor Va to the platelet membrane surface and which platelet activation such as prostaglandin E1 (19). Kane et al. (23) peptide(s) serves as the binding site for factor Xa. These inter- recently reported that disrupted platelets release another pro- actions were assessed by direct measurements of radiolabeled fac- tease which cleaves the procofactor factor V into peptides dif- tor V. and factor Xa binding to platelets as well as autoradio- ferent than those derived from thrombin activation. The pro- graphic visualization of the factor Va peptides associated with the teolytic activity they observed was not associated with intact platelet. Experiments were performed to determine the inter- platelets and was not inhibited by EDTA. The platelet-asso- action of components D and E with platelets under reaction con- ciated protease reported by our group (19) cleaves membrane- ditions in which components D and E were present as either the bound factor Va on intact platelets and is inhibited by EDTA, intact, functional two-subunit protein or as nonfunctional discrete which suggests that these two platelet-associated or derived peptides dissociated by the addition of Na2EDTA. The results sug- proteases are likely to be different. gest that component E mediates the binding of factor Va to the Factor Va bound to platelets consist of three peptides-com- platelet and also serves as the binding site for the interaction of ponents D, D', and E. However, whether any single peptide factor Xa with platelet-bound factor Va. or combination of peptides mediates factor Va-platelet inter- action and subsequent factor Xa binding has not yet been re- The blood protein coagulation factor Va is an essential, nonen- ported. zymatic component of the prothrombinase complex (1, 2). This Experiments in our laboratory suggest that component E plays complex-composed of the enzyme factor Xa, factor Va, plate- a significant role in the binding of factor Xa (24, 25). The bind- lets (or an equivalent phospholipid surface), and Ca2+-effec- ing offactor Xa to platelet-bound factor Va results in a factor Xa- tively catalyzes the hydrolysis of the zymogen prothrombin to induced cleavage of component E (74,000-dalton peptide) to the blood clotting enzyme thrombin (3, 4). The platelet mem- yield peptides of approximately 48,000 and 30,000 daltons (19). brane (or a phospholipid surface) is required for proper assem- Furthermore, an inhibitory murine monoclonal anti-bovine fac- bly of the protein components (1, 2, 4-6), and Ca2+ stabilizes tor Va antibody specific for component E blocks the binding of several of the binding interactions (7-10). Through direct bind- factor Xa to lipid-bound factor Va (24). However, these exper- ing measurements, as well as inferences drawn from the ki- iments do not eliminate the possibility that component D plays netics of prothrombin activation, factor Va has been shown to a significant role in the interaction of factor Xa with factor Va. fulfill its cofactor role by forming the receptor for factor Xa on In this report we describe experiments that indicate that the the surface of either platelets (4-6) or well-defined phospho- interaction offactor Va with the platelet surface is mediated pri- lipid vesicles (11-13). A high-affinity interaction between factor marily through component E and, furthermore, that this same Xa and platelet-bound or lipid-bound factor Va results in a stoi- peptide (in the absence of component D) provides the binding chiometric (1:1), Ca2+-dependent, enzymatic complex with a site for the interaction of factor Xa with platelet-bound factor dissociation constant of approximately 4 X 10-10 M (4, 12). Va. Factor Va is obtained by thrombin-catalyzed hydrolysis of the procofactor, factor V (14, 15), a single-chain glycoprotein of ap- proximately 330,000 daltons (16, 17). Thrombin activation of EXPERIMENTAL PROCEDURES factor V ultimately results in a two-subunit protein consisting Materials. Tagit reagent [N-succinimidyl 3-(4-hydroxyphe- ofcomponents D and E (Mr = 94,000 and 74,000, respectively) nyl)propionate] and fat-free bovine serum albumin were ob- as well as activation peptides whose association is not required tained from Sigma. Carrier-free Na'"I was obtained from

The publication costs ofthis article were defrayed in part by page charge Abbreviation: ACD, acid/citrate/dextrose solution (0.15 M sodium ci- payment. This article must therefore be hereby marked "advertisement" trate/citric acid/2% glucose, pH 4.5). in accordance with 18 U. S. C. §1734 solely to indicate this fact. * To whom reprint requests should be addressed. 2380 Downloaded by guest on September 29, 2021 Medical Sciences: Tracy and Mann Proc. Natl. Acad. Sci. USA 80 (1983) 2381 Amersham. Apiezon oil was obtained from J. B. Biddle Co. (Blue platelet binding reaction mixtures as detailed above were re- Bell, PA), and NaDodSO4 was from Pierce. Dansylarginine N- moved at various times and centrifuged through oil on a Brink- (3-ethyl-1,5-pentanediyl)amide, a potent inhibitor of thrombin, mann Microfuge for 1 min at 12,000 X g. The supernatant and was prepared as described by Nesheim et al. (26). Na2EDTA oil layer were rapidly removed and the platelet pellet was sol- was from Baker. Bovine blood was obtained by venipuncture ubilized in 10% acetic acid, frozen, and lyophilized. Lyophi- from healthy dairy animals. All other reagents and chemicals lized samples were solubilized in 2% NaDodSO4, subsequently were of analytical grade. reduced with 2% 2-mercaptoethanol, and subjected to electro- Bovine factor V was isolated and assayed for procoagulant phoresis in polyacrylamide gradient slab gels (5-15%) according activity as described by Nesheim et al. (16, 18). Factor Va was to Neville (35). The dried gels were subjected to autoradiog- prepared by incubation of factor V with thrombin as described raphy at -700C on Kodak XR-1 film. (5). Bovine prothrombin and bovine factor X were prepared as described by Bajaj and Mann (27). Factor Xa was prepared by a modification of the method of Downing et al. (28) with factor RESULTS X-activator from Russell's viper venom immobilized on CNBr- Factor Va bound to platelets consists of components D, D', and activated agarose. The factor X-activator was generously sup- E (94,000, 90,000, and 74,000 daltons, respectively) (19). Com- plied by Walter Kisiel (University of Washington, Seattle). Bo- ponent D' appears to arise as a result of proteolysis of com- vine thrombin was prepared as described by Lundblad et al. ponent D mediated by the Ca2'-dependent sulfhydryl platelet (29). protease described in both the bovine and human systems (20- mlI-Labeled factor V ("2I-factor V), '25I-labeled factor Va (125I- 22). Because the activity ofthis protease is dependent also upon factor Va), "2I-labeled factor X ('25I-factor X), and I'I-labeled millimolar concentrations of Ca2+, experiments were per- factor Xa ("WI-factor Xa) were prepared by using Bolton-Hunter formed initially to determine if platelets isolated in the pres- reagent as detailed (5, 18). The 125I-factor V obtained had a spe- ence of Na2EDTA would no longer exhibit proteolytic activity cific radioactivity of 550 cpm/ng (0.04 mol of 125I per mol of toward factor Va. Platelets were isolated from blood in which factor V) and retained 85-100% of its coagulant activity. The the anticoagulant was 5 mM Na2EDTA ("EDTA platelets"). At "2I-factor X had a specific radioactivity of 1,200 cpm/ng (0.03 various times, aliquots were removed and processed to deter- mol of 125I per mol of factor X) and retained 90-100% of its co- mine the amount offactor Va bound to the platelets and the fac- agulant activity. 1"I-factor Va (61 nM) was functionally inacti- tor Va peptides associated with the platelets as visualized by vated and subunit dissociation was accomplished by addition of NaDodSO4/polyacrylamide gel electrophoresis and autoradi- 5 mM Na2EDTA (final concentration) and incubation at am- ography. In addition, EDTA platelets that had been recalcified bient temperature (approximately 10 min) until no factor Va ac- with 2.5 mM CaC12 ("recalcified EDTA platelets") for 10 min tivity remained as determined by bioassay (16). The molecular prior to the addition offactor Va were studied. Platelets isolated weights and extinction coefficients (el`cm, 28 nm) of the respec- from blood in which ACD was the anticoagulant ("ACD plate- tive bovine proteins were taken as follows: factor V (16), 330,000, lets") were used as the control because these platelets had pre- 9.6; prothrombin (30, 31), 72,000, 14.4; factorXa (32, 33), 55,000, viously been found to possess proteolytic activity toward factor 12.4; and thrombin (29), 37,400, 19.5. Va (19). Bovine blood (13 vol) was drawn into 2 vol ofacid-citrate dex- The time course of factor Va binding to these three different trose (ACD) solution (0.15 M sodium citrate/citric acid/2% platelet preparations is shown in Fig. 1 Left; an autoradiograph glucose, pH 4.5). Platelets were isolated and washed to remove depicting the factor Va peptides associated with these same plasma by the method of Orloff and Michaeli (34) as described platelets is shown in Fig. 1 Right. Of the three platelet prep- (5). Washed platelets were stored at ambient temperature in a arations studied, the ACD platelets bound the greatest amount medium at pH 6.6 and containing 0.127 M sodium chloride, 4 of factor Va (24.6% bound at equilibrium) and exhibited pro- mM potassium chloride, 0.03 M glucose, 0.01 M imidazole, teolytic activity toward bound factor Va as shown by the ap- 0.35% bovine serum albumin, and they were used within 5 hr pearance of component D' with the concomitant loss of com- of preparation. Alternatively, blood was drawn into 5 mM ponent D. When no additional Ca2+ was added to the reaction Na2EDTA (final concentration) and platelets were isolated and mixture, the amount of factor Va bound or the appearance of washed as indicated above except that 5 mM Na2EDTA was component D' was not affected. With EDTA platelets (no Ca2+ included in the first two washes. The final wash and resuspen- added to the reaction mixture), approximately 6.8% of the added sion contained no Na2EDTA. factor Va was bound after 2 min; then there was a gradual loss in factor Va bound, resulting in 5.5% bound at equilibrium. The METHODS autoradiograph depicting the factor Va peptides associated with Factor Va Binding Measurements. The binding of 12'I-factor the EDTA platelets indicates that at the time of maximal factor Va to platelets was measured by centrifugation through oil as Va binding (2 min), components D and E were associated with described (5). The pH 7.4 reaction mixtures contained 0.02 M the EDTA platelets; however, with passage oftime, component Tris HCI, 0.14 M NaCl, 0.1% glucose, 0.5% fat-free albumin, D dissociated from the platelet surface and only component E 2.5 mM Ca2+, 30 AM dansylarginine N-(3-ethyl-1,5-pentane- remained bound. Even though component D can be observed diyl)amide, 2.0 x 108 platelets per ml, and, 0.40 nM 125I-factor to be associated with the platelet membrane over a 5-min pe- Va. When Na2EDTA-inactivated factor Va was used, additional riod, there is no evidence ofplatelet proteolytic activity toward Ca2+ was not included in the reaction mixtures. (Our buffers bound factor Va. Component D' is not seen on the autoradio- routinely contain approximately 0.1 ,uM Ca2' from our water graph. source.) Aliquots were removed from the reaction mixture at These results are interpreted as indicating that residual EDTA various times and centrifuged through oil on a Brinkmann Mi- within the platelet preparation was inhibiting the activity of the crofuge for 1 min at 12,000 x g and assayed for radioactivity or Ca2+-dependent platelet protease. In addition, it appears that processed for autoradiography as described below. local concentrations of EDTA were chelating the Ca2+ required Electrophoresis and Autoradiography of '2SI-Factor Va Bound to maintain factor Va integrity, thus resulting in the loss of com- to Platelets. The 125I-factor Va peptides associated with platelets ponent D from the, platelet' surface. From this observation, it were visualized by autoradiography. Aliquots of "2I-factor Va may be postulated that the binding of factor Va to the platelet Downloaded by guest on September 29, 2021 2382 Medical Sciences: Tracy and Mann Proc. Natl. Acad. Sci. USA 80 (1983) Mr X 10-3 D ____94 -l~ - ~,, -l O_ zri- r_ _t -----W ...... 90 Mu E - -~.-- - -74 16- 12 a0 w. Ie XCDg0 8 cJ l usA 4I min 2 5 10 30 60 2 5 10 30 2 5 10 30 60 0 10 20 30 40 50 60 Time, min ACD 4 Va EDTA + Ca2 + Va EDTA + Va FIG. 1. Interaction of factor Va with platelets determined by direct binding measurements (Left) and electrophoresis and autoradiography of factor Va bound to platelets (Right). 125I-Factor Va (0.4 nM) was incubated with washed platelets (2 x 108/ml) isolated from blood collected in either ACD or 5 mM Na2EDTA and washed. At the time points indicated, aliquots were removed from the reaction mixture, centrifuged through oil, and processed for either amount of isotope bound (Left) or electrophoresis (an equal amount of isotope, 2,500 cpm, was applied to each lane) and au- toradiography (Right). (Left) Factor Va binding to platelets isolated from blood in which the anticoagulant was: o, ACD; *, 5 mM Na2EDTA;e, 5 mM Na2EDTA but the platelets were recalcified with CaC12 (2.5 mM) for 10 min prior to the addition of '251-factor V8. membrane is mediated primarily through component E and that activated factor V. [factor V8,(EDTA)] or active factor V., at 0.4 component D interacts with the platelet through its association nM was incubated with ACD platelets (2 X 108 platelets per with component E. ml). At various times, aliquots were removed and processed for Recalcification of EDTA platelets for 10 min prior to the ad- determining both the amount of factor V8 bound (Fig. 2 Left) dition of factor Va partially restored the ability of the EDTA and the factor V. peptides associated as visualized by autora- platelets to bind factor Va. At equilibrium the recalcified EDTA diography (Fig. 2 Right). When active factor Va was incubated platelets had bound 21% of the added factor Va compared to with platelets, approximately 22.7% of the isotope was bound 24.6% bound by the ACD platelets. In addition, both com- to the platelets. In contrast, 15.3% was bound when factor ponents D and E were bound to the recalcified EDTA plate- Vai(EDTA) was added to the platelets. Autoradiographs of the lets. The appearance of component D' was not apparent until factor V. peptides associated with these platelets indicated that 10 min after the addition of factor Va to these platelets, indi- only component E bound to the platelet surface even though cating that recalcification did not completely restore the plate- a mixture of both components D and E was incubated with let Ca2+-dependent proteolytic activity. platelets. There was no indication ofcomponent D being bound These experiments suggested that the binding of factor Va to even after a 30-min incubation. the platelet surface is mediated primarily through the 74,000- Incubation of factor V8J(EDTA) with ACD platelets resulted dalton peptide, component E. Additional experiments were in the binding of more isotope than we would have anticipated performed to test this hypothesis. We compared the interaction on the basis of the results shown in Fig. 1 Left when only com- offactor Va with platelets when factor Va was present as the two- ponent E remained bound to EDTA platelets. Comparison of subunit functional molecule or as an inactive mixture of both the amount of isotope associated with EDTA platelets (when components D and E whose subunit interaction had been de- only component E was bound) to the amount with recalcified stroyed by preincubation with Na2EDTA. Either EDTA-in- EDTA platelets (when both components D and E were bound)

D I_- -O E

I6a

o -oxx

t 0 co x min 2 5 10 30 0.5 1 2 5 10 30

2 4 6 8 10 30 Time, min ACD + Va ACD + Vai(EDTA) FIG. 2. Comparison of the interaction of factor V8 and EDTA-inactivated factor V. [factor V8 (EDTA)I with platelets as determined bydirect binding measurements (Left) and electrophoresis [an equal amount (2,000 cpm) of isotope was applied to each lane] and autoradiography of factor V. bound to platelets (Right). Reaction conditions and methods were as in Fig. 1 except that Ca2+ was not added when lMI-factor Va. (EDTA) was used in the reaction mixture. (Left) e, Factor Va binding to ACD platelets; A, factor Vji(EDTA) binding to ACD platelets. (Right) Factor V. peptides associated with platelets described in Left visualized by autoradiography. Downloaded by guest on September 29, 2021 Medical Sciences: Tracy and Mann Proc. Nati. Acad. Sci. USA 80 (1983) 2383 suggests that 33% of the isotope is associated with component periment, unlabeled factor Xa (1.5 nM) was added to the ra- E for this particular iodinated factor V preparation. Therefore, diolabeled component E/platelet reaction mixture and aliquots we might anticipate that the binding of only component E to were removed at various times for autoradiographic visualiza- ACD platelets would be approximately 33% of the isotope bound tion of the factor Xa-induced cleavage products of component when both components D and E were bound. However, ap- E (Fig. 3 Left). For the second experiment, '25I-factor Xa (1.5 proximately 63% of the isotope was platelet-associated when nM) was added to the unlabeled component E/platelet reaction only component E was bound to ACD platelets. mixture and the time-dependent binding of radiolabeled factor All of the isotope was displaced by a 100-fold molar excess Xa to the platelet-component E complex was determined (Fig. of unlabeled factor Va; thus, component E binds to the factor 3 Right). These data were corrected for nonspecific binding of Va binding site. The increased binding ofcomponent E alone- factor Xa. The autoradiograph indicates that addition of factor in the absence of complex formation with component D-may Xa to a platelet-component E complex resulted in the factor Xa- be the result of various factors. The manipulation of the two induced cleavage ofcomponent E to yield the factor Xa-derived platelet preparations was not identical in that they were pre- cleavage products at 48,000 and 30,000 daltons. Furthermore, pared from blood anticoagulated with either ACD or EDTA. the platelet-component E complex provided a binding site for Steric hindrance may be involved, facilitating more binding of the interaction offactor Xa with the platelet surface (Fig. 3 Right). component E. In addition, the affinity constant for component Radiolabeled factor Xa became associated with the platelet sur- E binding may be different from that for component D and E face in a time-dependent manner and was displaced by unla- (factor Va) binding. beled factorXa. Approximately 7.5% ofthe added factorXa bound A previous report from our laboratory indicated that factor specifically to the platelet-component E complex. These stud- Va forms a 1:1 complex with factor Xa at the platelet surface (4). ies do not allow calculation of the specific radioactivity of com- Upon binding to platelet-bound factorVa, factor Xa rapidly cleaves ponent E on a molar basis; therefore, the present level of ex- component E into two peptides of approximately 48,000 and perimental development does not allow calculation ofthe binding 30,000 daltons. (A secondary time-dependent cleavage occurs parameters governing the factor Xa-component E-platelet in- in components D and D'.) This observation was used to design teraction. experiments to determine if factor Xa would interact with com- ponent E bound to platelets in the absence of component D. DISCUSSION This potential interaction was studied in two ways: (a) by de- The blood coagulation cofactor factor Va is a two-subunit protein termination of whether factor Xa would cleave platelet-bound consisting of components D and E (94,000 and 74,000 daltons, component E and (b) by direct measurements of binding of ra- respectively) (14, 15, 18) which are associated by tightly bound diolabeled factor Xa to the platelet-component E complex. calcium (7). Factor Va appears to fulfill its cofactor role in the EDTA platelets were prepared and incubated (2 X 108 plate- factor Xa-catalyzed conversion of prothrombin to thrombin in lets per ml) with either unlabeled factor Va or 125I-factor Va (0.4 at least two ways. Factor Va concentrates the enzyme and the nM) for 30 min. Under these conditions, only component E was substrate into a smaller catalytic volume by forming the recep- bound to the platelet membrane (Fig, 3 Left). The platelets were tor for factor Xa on the surface of either platelets (4-6) or phos- then washed to remove any unbound components D and E and pholipic vesicles (11-13). Apart from its concentration effect, resuspended in a buffer containing 2.5 mM Ca2' (because Ca2+ factor Va appears to increase the catalytic efficiency of the en- is required for factor Xa binding to platelets). For the first ex- zyme (13, 36).

X Mr_iO-3 94 D __ -'90 DE E - MWM_ Nm -- 74 ,,2 8 -B 7 -,_ -V - - _ 48 . o 5 x

30 1> 3 0 min 2 5 10 30 60 0.5 1 2 5 10 5 % 6 2 x 1 EDTA + Va EDTA + V, (30 min) Va 0. Q 5 10 15 20 25 30 +Ca'- - Xa Xa Time, min FiG. 3. Interaction of factor X. with component E bound to platelets. (Left) Electrophoresis and autoradiography of the factor X.-induced cleav- age of the factor V. light chain (component E) bound to platelets. '25I-Factor Va (0.4 nM) was incubated for 30 min with platelets (2.0 x 108/ml) collected from blood anticoagulated with 5 mM Na2EDTA and then washed. Autoradiography ofthe 125I-factor Va peptides associated with platelets indicates that only the 74,000-dalton component E is bound to platelets after a 30-min incubation. After the 30-min incubation, the platelets were washed twice and resuspended in a buffer containing 2.5 mM Ca2+. Factor X. (1.5 nM) was then added, and aliquots were removed at the time points indicated and processed for electrophoresis [an equal amount (2,500 cpm) of isotope was applied to each lane] and autoradiography. A standard showing the factor Xa-induced cleavages of platelet-bound factor V. (components D, D', and E) in which component E is rapidly cleaved to peptides of approximately 48,000 and 30,000 daltons with secondary cleavages occurring in components D and D' is shown at the extreme right. (Right) Time course of binding of factor Xa to the 74,000-dalton peptide (component E) of factor Va bound to platelets. Reaction conditions were as in Left except that 125I-factor Xa (1.5 nM) was added to a reaction mixture containing unlabeled factor V.. Aliquots of the reaction mixture were removed and assayed for the amount of isotope associated with the platelets. These data were corrected for nonspecific factor X. binding. Downloaded by guest on September 29, 2021 2384 Medical Sciences: Tracy and Mann Proc. Natl. Acad. Sci. USA 80 (1983) The data presented in this report indicate that: (i) the binding 4. Tracy, P. B., Nesheim, M. E. & Mann, K. G. (1981)J. Biol. Chem. offactor Va to the platelet membrane is mediated through com- 256, 743-751. ponent E, and (ii) component E, in the absence of component 5. Tracy, P. B., Peterson, J. M., Nesheim, M. E., McDuffie, F. C. D, williform the receptor for factor Xa at the platelet surface. & Mann, K. G. (1979) J. Biol. Chem. 254, 10354-10361. 6. Kane, W. H. & Majerus, P. W. (1982)J. Biol. Chem. 257, 3963- Component-E -will bind to the platelet surface independently 3969. of component D; however, component D is bound to platelets 7. Hibbard, L. S. & Mann, K. G. (1980)J. Biol. Chem. 255, 638-645. only through its Ca2"-mediated association with component E. 8. Stenflo, J. & Suttie, J. W. (1977) Annu. Rev. Biochem. 46, 157- Because component E alone will bind factor Xa, we have iden- 172. tified component E as the factor Va peptide that facilitates the 9. Howard, J. B. & Nelsestuen, G. L. (1975) Proc. Natl. Acad. Sci. assembly and.localization of the prothrombinase USA 72, 1281-1285. complex on 10. Nelsestuen, G. L. & Lim, T. K. (1977) Biochemistry 16, 4164- the platelet surface. Quantitative studies of the binding inter- 4171. actions of component E with platelets, in addition to the factor 11. Bloom, J. W., Nesheim, M. E. & Mann, K. G. (1979) Biochem- Xa interactions with platelet-component E complexes, will yield istry 18, 4419-4425. the binding parameters governing these. interactions. 12. Nesheim, M. E., Taswell, J. B. & Mann, K. G. (1979)J. Biol. Chem. These observations are consistent with results ofother stud- 254, 10952-10962. 13. Nesheim, M.. E., Eid, S. & Mann, K. G. (1981)J. Biol. Chem. 256, ies in our laboratory using defined phospholipid vesicles. Hig- 9874-9882. gins and Mann (25),have shown that component E alone is re- 14. Nesheim, M. E. & Mann, K. G. (1979)1. Biol. Chem. 254, 1326- sponsible for the binding of factor Va to phospholipid vesicles. 1334. Tucker et al. (24) observed that a murine antibovine factor V 15. Esmon, C. T. (1979) J. Biol. Chem. 254, 964-973. monoclonal antibody directed against an epitope on component 16. Nesheim, M. E., Myrmel, K. H., Hibbard, L. & Mann, K. G. E completely blocked the binding offactor X.a to factor Va bound (1979) J. Biol. Chem. 254, 508-517. to vesicles. Although this 17. Mann, K. G., Nesheim, M. E. & Tracy, P. B. (1981) Biochem- monoclonal antibody had no effect on istry 20, 28-33. factor Va binding to phospholipid vesicles, it prevented the 18. Nesheim, M. E., Katzmann, J. A., Tracy, P. B. & Mann, K. G. binding of factor Va to platelets (unpublished data). Thus, even (1981) Methods Enzymol. 80, 245-274. though component E mediates the binding of factor Va to syn- 19. Tracy, P. B., Nesheim, M. E. & Mann, K. G. (1983)J. Biol. Chem. thetic vesicles and platelets, factor Va binding in these two sys- 258, 662-669. tems is qualitatively different and lends additional support to 20. Truglia, J. A. & Stracher, A. (1981) Biochem. Biophys. Res. Com- the idea that the interaction of factor Va with mun. 100, 814-822. platelets is spe- 21. Phillips, D. R. & Jakabova, M. (1977) J. Biol. Chem. 252, 5602- cifically receptor mediated (4-6, 37). 5605. None of our results precludes the possibility that component 22. Sakon, M., Kambayashi, J., Ohno, H. & Kosaki, G. (1981) Thromb. D contributes to the binding offactor Xa and contributes to the Res. 24, 207-214. function of the prothrombinase complex. The importance of 23. Kane, W. H., Mruk, J. S. & Majerus, P. W. (1982)J. Clin. Invest. component D is apparent for several reasons. (i) The inacti- 70, 1092-1100. vation of 24. Tucker, M. M., Foster, W. B., Katzmann, J. A. & Mann, K. G. phospholipid or platelet-bound factor Va by activated (1983) J. Biol. Chem. 258, 1210-1214. is accomplished by cleavage ofcomponents D (19, 38, 25. Higgins, D. L. & Mann, K. C. (1983)J. Biol. Chem., in press. 39) and D' (19). (ii) An interaction between factor Xa and com- 26. Nesheim, M. E., Prendergast, F. G. & Mann, K. G. (1979) Bio- ponent D is implied from the observation that factor Xa protects chemistry 18, 996-1003. factor Va from activated protein C inactivation (19, 39-41). (iii) 27. Bajaj, S. P. & Mann, K. G. (1973)J. Biol. Chem. 248, 7729-7741. The binding of factor Xa to platelet-bound factor Va results in 28. Downing, M. R., Butkowski, R. J., Clark, M. M. & Mann, K. G. a time-dependent cleavage (1975)J. Bil. Chem.. 250, 8897-8906. of both components D and D' (19). 29. Lundblad, R. L., Uhteg, L. C., Vogel, C. N., Kingdon, H. S. & (iv) The platelet membrane-associated protease cleaves bound Mann, K. G. (1975) Biochem. Biophys. Res. Commun. 66, 482-489. component D to component D' which remains associated with 30. Heldebrant, C. M., Butkowski, R. J., Bajaj, S. P. & Mann, K. G. the platelet (19). However, the present report clearly indicates (1973)J. Biol. Chem. 248, 7149-7163. that these component D interactions are not essential for the 31. Owen, W. G., Esmon, C. T. & Jackson, C. M. (1974)J. Biol. Chem. component E-platelet receptor interaction or factor Xa binding. 249, 594-605. 32. Fujikawa, K. & Davie, E. W. (1976) Methods Enzymol. 45, 89- Because component D is not required for the assembly of the 95. prothrombinase complex, it is reasonable to speculate that the 33. Jackson, C. M., Johnson, T. F. & Hanahan, D. J. (1968) Bio- function of component D is to increase the catalytic efficiency chemistry 7, 4492-4497. of the enzymatic complex. 34. Orloff, K. G. & Michaeli, D. (1976) Am.J. Physiol. 231, 344-350. 35. Neville, D. M., Jr. (1971)J. Biol. Chem. 246, 6328-6334. We thank Lisa L. Eide for her excellent technical assistance. We also 36. Rosing, J., Tans, G., Govers-Riemslag, J. W. P., Zwaal, R. F. A. thank Mary Lou Stewart and Gregory Heiler for their assistance in pro- & Hemker, H. C. (1980)J. Biol. Chem. 255, 274-283. curing the blood samples used in this study and William Rabehl for the 37. Miletich, J. P., Kane, W. H., Hofmann, S. C., Stanford, N. & use ofhis animals. Finally, we thank Jeanne Nemitz for her patience and Majerus, P. W. (1979) Blood 54, 1015-1022. assistance in the preparation ofthis manuscript. The work was supported 38. Canfield, W., Nesheim, M., Kisiel, W. & Mann, K. G. (1978) by Grants HL-17430D and HL-07069 from the National Heart, Lung, Circulation 58, II-210 (abstr.). and Blood Institute and by the Mayo Foundation. 39. Walker, F. J., Sexton, P. W. & Esmon, C. T. (1979) Biochim. Biophys. Actd 571, 333-342. 1. Suttie, J. W. & Jackson, C. M. (1977) Physiol. Rev. 57, 1-70. 40. Nesheim, M. E., Canfield, W., Kisiel, W. & Mann, K. G. (1979) 2. Mann, K. G. (1976) Methods Enzymol. 45, 123-156. Blood 54, Suppl. 1, 293 (abstr.). 3. Davie, E. W. & Fujikawa, K. (1975) Annu. Rev. Biochem. 44, 799- 41. Nesheim, M. E., Canfield; W., Kisiel, W. & Mann, K. G. (1982) 829. J. Biol. Chem. 257, 1443-1447. Downloaded by guest on September 29, 2021