Platelet-derived Microparticles Express High Affinity Receptors for Factor VIII*

Transcription

1 THE ~OURNAI. OF BIOLOGICAL CHEMISTRY LC 1991 by The American Society for Biochemistry and Molecular Biology, Inc. Vol. 266, No. 26, Issue of September 15, pp , 1991 Printed in U. S. A. Platelet-derived Microparticles Express High Affinity Receptors for Factor VIII* (Received for publication, April 1, 1991) Gary E. Gilbert$ Tl, Peter J. Simsll**$$, Therese Wiedmer 11, Bruce Furie$, Barbara C. FurieS, and Sanford J. ShattilGS From the $Center for Hemostasis and Thrombosis Research, Diuision of Hematology-Oncology, New England Medical Center and Departments of Medicine and Biochemistry, Tufts University School of Medicine, Boston, Massachusetts 0211 I, the Hematology-Oncology Section, Brockton- West Roxbury Veterans Administration Medical Center and Departments of Medicine, Brigham and Women s Hospital and Haruard Medical School, West Roxbury, Massachusetts 02132, the (1 Cardiouascular Biology Research Program, Oklahoma Medical Research Foundation and the **Departments of Medicine, Pathology. Microbiology and Immunology, Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, and the Hematology-Oncology Section, Departments of Medicine and Pathology and Laboratory Medicine, Uniuersity of Pennsylvania School of Medicine, Philadelphia, I ennsyluania Factor VI11 is a cofactor in the tenase enzyme com- receptors for factor VI11 are similar to those for factor plex which assembles on the membrane of activated Va. platelets. A critical step in tenase assembly is membrane binding of factor VIII. Platelet membrane factor VIII-binding sites were characterized by flow cytometry using either fluorescein maleimide-labeled recom- The enzyme reactions that lead to the formation of a fibrin binant factor VI11 or a fluorescein-labeled monoclonal clot during blood coagulation take place optimally in the antibody against factor VIII. Following activation by presence of membrane surfaces (Mann et al., 1988). Two thrombin, most platelets bound factor VI11 within 90 s. In addition, over the course of several minutes, memhomologous enzyme complexes, the tenase complex and the prothrombinase complex, assemble and function sequentially branous vesicles (microparticles) were shed from the on a suitable membrane surface. The tenase complex, factor platelet plasma membrane and each microparticle IXa and factor VIIIa, converts factor X to its active enzyme bound as much factor VI11 as a stimulated platelet. form. The product of the tenase complex, factor Xa, assembles Over 30 min, stimulated platelets (but not microparti- with factor Va to form the prothrombinase complex (Mann cles) lost the capacity to bind factor VIII. Factor VI11 et al., 1988). In the prothrombinase complex an early step in bound saturably to microparticles from platelets stimcomplex assembly is membrane binding of factor V, or factor ulated with thrombin, thrombin plus collagen, or the Va. Reasoning from homology of the enzyme complexes, an complement proteins C5b-9. The binding of factor VI11 was compared to factor V, a structurally homologous early step in the assembly of the tenase complex, involves coagulation cofactor. Analysis of microparticle binding factor VI11 or factor VIIIa binding to cell membranes. Therekinetics yielded similar on and off rates for factor VI11 fore, the nature and proximity of these receptors on cell and factor Va and KO values of 2-10 nm. In the pres- surfaces is of central importance in understanding the seence of 20 nm factor Va, the binding of factor VI11 to quential function of the two enzyme complexes. For both microparticles was increased, and there was a compa- enzymatic complexes, synthetic lipid vesicles containing phosrable increase in platelet tenase activity. At higher phatidylserine can provide a suitable surface in uitro. In vitro factor Va concentrations, factor VI11 binding and ten- and presumably in uiuo activated platelets provide a memase activity were inhibited. Conversely, factor VI11 had brane surface for both the prothrombinase and tenase reaca similar dose-dependent effect on factor Va binding tions (Rosing et al., 1985). Activated platelets bind both factor and platelet prothrombinase activity. Synthetic phos- Va (Tracy et al., 1979; Wiedmer et al., 1986b) and factor VI11 pholipid vesicles containing phosphatidylserine com- (Nesheim et al., 1988). However, properties of the membrane peted with microparticles for binding of factor VI11 receptor(s) for factor VI11 and factor V on the platelet memand factor Va. These studies indicate that activated brane are only partially defined. platelets express a transient increase in high affinity receptors for factor VIII, whereas platelet-derived microparticles express a sustained increase in receptors. The binding characteristics of platelet membrane *This work was supported by Grants HL42443, HL36946, HL40796, and HL40387 from the National Institutes of Health. 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 accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ll Recipient of Individual and Institutional National Research Serv- ice Award F32 HL07440 from the National Institutes of Health. To whom correspondence should be addressed: Dept. of Medicine, West Roxbury VA Medical Center, 1400 VFW Parkway, West Roxbury, MA $$ Established Investigator of the American Heart Association Factor VI11 and factor V are homologous in amino acid sequence (Toole et al., 1984; Gitshier et al., 1984; Church et al., 1984) and function as membrane-bound cofactors (Kane and Davie, 1988; Mann et al., 1988; Furie and Furie, 1988; Gilbert et al., 1990). The proteins share a repeating domain structure of Al-A2-B-A3-Cl-C2 in which the A domains are homologous with ceruloplasmin, the B domain is unique to each protein, and the C domains are homologous with discoidin I, a lectin. The cofactor activity of each protein is in- creased through limited proteolysis by thrombin, and this results in the removal of the B domain, with the remaining heavy and light chains interacting to form a Ca -dependent complex. Membrane binding occurs via the light chain of both factor Va and factor VIII. The binding region is reported to

2 17262 Factor VIII Binding to EXPERIMENTAL PROCEDURES Coagulation and Complement Proteins-Bovine factor Va, the light chain of factor Va, and prothrombin were gifts from Dr. Charles T. Esmon (Oklahoma Medical Research Foundation). The specific activity ranged from units/mg. Recombinant human factor VIII, a gift from Genetics Institute, has been extensively characterized (Kaufman et al., 1988). A stock solution of factor VI11 in 0.5 M NaC1, 5 mm CaCl,, 50 mm Tris-HC1, 0.1% Tween 80, ph 7.5, was stored in small aliquots at -80 "C and thawed only once prior to use. Specific activity of factor VI11 varied between 1400 and 5000 units of coagulant activity/mg of protein as measured using factor VIII-deficient plasma in an activated partial thromboplastin time assay (Hardisty and Macpherson, 1962). Quantitative studies were performed using factor VI11 with the highest specific activity. Human factor IX and factor XIa were provided by Dr. David Diuguid (New England Medical Center). Factor X and factor Xa were from Enzyme Research Laboratories, Inc., South Bend, IN. Human complement proteins C5b6, C7, C8, and C9 were isolated and purified as previously described (Wiedmer and Sims, 1988). Collagen from equine tendon was from Hormon-Chemie (Munich, Federal Republic of Germany), and bovine thrombin was from Organon Teknika. Protein concentrations for unlabeled proteins were estimated using the following extinction coefficients (E;;;): factor VIII, 8.8; factor IX, 13.5; murine IgG, 15; factor V, 9.6; factor Va, 15.1; factor Va light chain, 18.7; factor Xa, 12.4; factor X, 12.5; prothrombin, 15.5; C5b6, 10; C7, 9.9; C8, 15.1; and C9, 9.6. Monoclonal Antibodies-A monoclonal antibody to an epitope on the light chain of factor VI11 was obtained from Hybritech, Inc., San Diego, CA. The nominal affinity of this antibody, originally designated 1B3, is greater than 3 X 10'" l/mol (Muller et al., 1981). V237, a monoclonal antibody to an epitope on the light chain domain of factor V or factor Va, was a gift from Dr. Charles T. Esmon and has been described previously (Sims et al., 1988). Preliminary experiments established the absence of cross-reactivity of antibody V237 with factor VI11 and antibody 1B3 with factor Va. Monoclonal antibody AP1, specific for membrane glycoprotein GPIb, was a gift from Dr. Thomas J. Kunicki (Blood Center of Southeastern Wisconsin, Milwaukee, WI). Microparticles Platelet be localized to the C2 domain of factor VI11 (Arai et al., 1989; Miscellaneous Reagents-The chromogenic substrate used to meas- Foster et al., 1990). ure prothrombinase activity was Spectrozyme TH (American Diag- Efficient assembly and function of the prothrombinase and nostica, Greenwich, CT); the substrate used to measure tenase activity was S2222 (Helena Laboratories, Beaumont, TX). Bovine brain tenase complexes on platelets require cell activation. Under L-a-phosphatidylserine and egg L-a-phosphatidylcholine were purconditions in which platelets are unstirred, the most effective chased from Avanti Polar Lipids, Pelham, AL. Cholesterol and calagonists in stimulating platelet procoagulant activity are the cium ionophore A23187 were from Calbiochem, San Diego, CA. The complement proteins C5b-9, a combination of thrombin and fluorescent probes, fluorescein-5-isothiocyanate (FITC),' fluorescein collagen, and the calcium ionophore A23187 (Wiedmer and maleimide, and l,l'-dihexadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiIC16(3)), were from Molecular Probes (Eugene, Sims, 1986a; Rosing et al., 1985; Sims et al., 1989). The OR). Phycoerythrin-streptavidin conjugate was from Southern Redevelopment of platelet procoagulant activity correlates with search Associates, Birmingham, AL. the reorientation of phosphatidylserine from the inner leaflet Fluorescence Labeling of Proteins, Phospholipid Vesicles, and Plateto the outer leaflet of the plasma membrane bilayer (Bevers let Membranes-Antibodies 1B3 and V237 were labeled with FITC et al. 1983), and in the case of prothrombinase, with the (Shattil et al., 1987). Fluorescein to protein molar ratios of 3-5:l were expression of membrane-binding sites for factor Va (Tracy et achieved. Antibody AP1 was conjugated with N-hydroxysuccinimide biotin ester as described previously (Shattil et al., 1987). Factor VIII, al. 1979; Wiedmer et al., ). Furthermore, when platelets 0.2 mg in 0.5 ml of stock buffer, was labeled by the addition of solid are activated by the same agonists that stimulate procoagulant fluorescein maleimide to a final concentration of 0.5 mm. Fluorescein activity, they release small membrane vesicles derived from maleimide was dissolved by vortex mixing, and the mixture was the plasma membrane (Sims and Wiedmer, 1986; Sandberg incubated overnight at 4 "C in the dark. Free fluorescent probe was et al., 1985). These vesicles, also referred to as microparticles, removed by cation exchange chromatography on a MA7S column can be distinguished from platelets by fluorescence-gated flow (Bio-Rad). The column was pre-equilibrated in 5 mm CaC12, 0.01% Tween 80, 20 mm MES, ph 6.0. Labeled factor VI11 was diluted 10- cytometry (Sims et al., 1988; Abrams et. al., 1990). Using this fold in the column buffer and loaded onto the column. After the technique, both activated platelets and microparticles have absorbtion at 280 nm had returned to base line, indicating complete been shown to express binding sites for factor Va. The appar- removal of free fluorescein maleimide, the protein was eluted with ent density of these binding sites on the microparticles is 0.75 M NaCl, 5 mm CaC % Tween 80, 20 mm MES. ph 6.0. much higher than on platelets. Similarly, the microparticle Labeling efficiency was 0.4 mol of fluorescein/mol of factor VI11 as fraction appears to contribute the bulk of the membrane judged by comparison of absorbance at 490 nm to absorbance at 280 nm (correcting for fluorescein absorbance at 280 nm). Phospholipid surface required for assembly of the functional prothrombinvesicles used in competition experiments with platelets were prepared ase complex (Sims et al., 1988, 1989; Sandberg et al., 1985). The role of microparticles in factor VI11 binding and the tenase reaction is unknown. In this study we have characterized the expression of factor VI11 receptors during platelet activation and compare the characteristics of factor VI11 binding sites to those of factor Va. by the method of Hope et al. (1985) as modified by Mayer et al. (1986) using two stacked polycarbonate membranes of 100-nm pore size (Nucleopore Corporation, Pleasanton, CA). Phospholipid concentration was determined by elemental phosphorus analysis (Chen et al., 1956). Vesicles used for direct binding experiments with factors VI11 and Va were prepared as described by Bangham (1965). Size distribution of the vesicles was limited by sedimentation at 12,000 X g for 15 min at 23 "C, resuspension by vortex mixing, and passage through a 0.6-pm pore size polycarbonate membrane. Labeling of phospholipid vesicles for fluorescence detection was achieved by addition of 0.1 mol % DiIC16(3) in ethanol to the lipid preparation prior to initial evaporation of organic solvents. Inclusion of this probe in the phospholipid membrane structure did not affect binding of factor VI11 as evaluated by resonance energy transfer (data not shown, method in Gilbert et al. 1990). All vesicle preparations were stored under a nitrogen atmosphere at 4 "C in the dark and used within 1 week of preparation. For studies of the kinetic expression of factor VIII- binding sites (see below), platelets were prelabeled by the slow dilution of 2 mm DiC16(3) in ethanol into platelet-rich plasma for a final concentration of 4 PM DiC16(3), 0.5% ethanol. Unincorporated Di- IC16(3) was removed by gel filtration. Binding of Factor VI11 and Factor Va to Platelets and Synthetic Phospholipid Vesicles-Unless stated otherwise, all procedures were performed at room temperature using plastic tubes. Venous blood from aspirin-free healthy adult volunteers was drawn into 1/7 volume of NIH formula A acid-citrate-dextrose solution, and platelet-rich plasma prepared by centrifugation for 20 min at 180 X g in the presence of apyrase (1 unit/ml) and prostaglandin El (1 pm). Platelets were then concentrated by centrifugation (15 min, 500 x g) and gelfiltered on Sepharose CL-ZB equilibrated in solution I (145 mm NaC1, 4 mm KC1, 0.5 mm MgCl,, 0.5 mm sodium phosphate, 0.1% glucose (w/v), 0.1% bovine serum albumin, 5 mm PIPES, ph 6.8). The platelets eluting in the void volume were collected, quantitated using a Coulter model ZBI particle counter, and adjusted to lo9 cells/ml. The platelets were then immediately diluted 10-fold with solution I1 (137 m, NaCl, 4 mm KC1,0.5 mm MgC12, 0.5 sodium phosphate, 0.1% glucose (w/v), 0.1% bovine serum albumin, 20 mm HEPES, ph 7.4) and incubated without stirring with various agonists for 10 min at 37 "C. When C5b-9 was the agonist, the C5b-9 complex was assembled as described previously (Sims et al., 1988). The input concentration The abbreviations used are: FITC, fluorescein isothiocyanate; DiIC16(3), l,l'-dihexadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate; MES, 4-morpholineethanesulfonic acid; PIPES, 1,4-piperazinediethanesulfonic acid; HEPES, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid.

3 of C5b-6 was 3.5 pg/lo platelets; C7, 1 pg/lox platelets; C8 and C9, each 4 pg/lox cells. For thrombin, the concentration was 0.5 unit/ml, collagen was 10 pg/ml, and A23187 was 1 pm. For equilibrium binding experiments, 2.5 X lo5 stimulated or unstimulated platelets were incubated in the dark for 10 min at 23 C in a total volume of 25 p1 in the presence of 2.5 mm CaC12, biotin AP1 (1 pg/ml), FITC-1B3 or FITC-V237 (20 pg/ml), and various concentrations of factor VI11 or factor Va. Then phycoerythrin streptavidin (2.5 pi of 0.1 mg/ml in phosphate-buffered saline) was added and the samples incubated an additional 10 min in the dark. Then, 125 pl of solution I1 with 2.5 mm CaCl, was added and the platelets analyzed by flow cytometry as described below. To examine the time course of factor VIII-binding site expression, DiC16(3)-labeled platelets (4 X 107/ml in solution 11, 1 mm CaC12) were warmed to 37 C. Fluorescein maleimide-labeled factor VI11 was added to 7 nm final concentration. Then, thrombin was added and flow cytometry evaluation was performed at various times without further dilution of the sample. To examine the time course of the loss of factor VIII-binding sites from platelet membranes, DiIC16(3)-labeled platelets were diluted to 4 X 107/ml in minimum essential medium, 0.1% bovine albumin, 0.02 M HEPES, ph 7.4, rather than solution 11. Platelets were warmed to 37 C, thrombin was added to a final concentration of 1 unit/ml, and aliquots were removed at various times. Aliquots were diluted 1:l into the same medium preparation containing 14 nm fluorescein-labeled factor VI11 and gently swirled. Flow cytometry evaluation was completed within 1 min of sample removal. To examine the time course of factor VI11 and factor Va binding, factor VI11 and FITC-1B3 or factor Va and FITC-V237 were prein- cubated for 1 h at room temperature. In separate tubes, platelets were activated with C5b-9 or thrombin plus collagen as described above. The binding reaction was started by addition of the platelets to the tubes containing factor VI11 or factor Va, and the samples were incubated at room temperature and analyzed at various times by flow cytomtery. To examine the rates of dissociation of factor VI11 and factor Va from platelets, the final incubation mixtures including activated platelets were equilibrated for 80 min at room temperature. Samples were then diluted 100-fold in solution I1 containing 2.5 mm CaCly and analyzed at various times by flow cytometry. For binding experiments with synthetic phospholipid vesicles, DiC16(3)-labeled vesicles (0.2-2 mm phospholipid) and the indicated concentrations of factor VI11 or factor Va were incubated with FITC-1B3 or FITC- V237 for 20 min in the dark. Dilution and analysis by flow cytometry were as for platelets. Flow Cytometry-Platelet samples were analyzed in a Becton- Dickinson FACStar or FACScan flow cytometer formatted for twocolor analysis as described previously (Shattil et al., 1987). The light scatter and fluorescence channels were set at logarithmic gain. Platelets and platelet-derived microparticles were distinguished from background light scatter by gating acquisition to include only those particles staining positive either for DiC16(3) or GPIb (defined as positive for biotin-ap1 and phycoerythrin streptavidin) within the FL2 fluorescence gate (585 f 42 nm). FITC fluorescence was determined in the FL1 fluorescence gate (530 f 30 nm). Any contribution of FITC fluorescence in the FL2 gate of or phycoerythrin fluorescence in the FL1 gate was eliminated by electronic compensation of the instrument. Ten-thousand phycoerythrin-positive particles from each sample were analyzed for forward and right angle light scatter and for FITC and phycoerythrin fluorescence intensities. Microparticles and platelets were identified and independently analyzed on the basis of their characteristic light scatter profiles on particle dot plots of forward light scatter versus right angle light scatter (Sims et al., 1988). To analyze the binding of factors VI11 and Va to synthetic phospholipid vesicles labeled with DiIC16(3), acquisition was gated to include only those particles positive for DiIC16(3) fluorescence in the FL2 gate. Factor Platelet VIII to Binding Microparticles into solution I1 containing 5 mm EDTA at 0 C and sampled for factor Xa activity. Amidolytic activity was determined in microtiter enzyme-linked immunosorbent assay plates using a 1:l mixture of diluted reaction mixture and 1.5 mm S The amidolytic activity of the test samples was quantitated by comparing the rate of increase in absorbance at 405 nm to that of serial dilutions of a standard factor Xa preparation. RESULTS Microparticle Formation and Factor VZIZ Binding-Following stimulation, platelets release plasma membrane vesicles that are responsible for a large proportion of platelet procoagulant activity. These vesicles, called microparticles, have a high density of factor Va-binding sites (Sims et al., 1988, 1989). In order to determine whether factor VIII-binding sites are also concentrated on microparticles, the binding of factor VI11 to platelets and to microparticles was measured by flow cytometry using a noninhibitory, fluorescein-labeled, antibody to factor VIII, FITC-1B3. Unstimulated platelets, detected by an antibody to GPIb, appeared a single as population by light-scattering criteria (Fig. 1A). Following stimulation by thrombin, two particle populations expressing GPIb were detected (Fig. 1B). The smaller particles, with lower lightscattering values, were the newly formed microparticles. Platelets and microparticles were distinguished based upon,;.;i Forward Scatter (arbitrary Units) - 0 $120,;,- Mp,-*,,.-. t,. 60.-,<., I.,..... L,. ;..,.., 3,j u i FITC anti-factor vlll Fluorescence (arbitrary units) FIG. 1. Platelet activation results in the formation of membrane vesicles (microparticles) which contain binding sites for factor VIII. The upper panels (A and B) are contour plots of 10,000 GPIb-containing particles that demonstrate the relative number of platelets and platelet membrane-derived particles (microparticles) observed before (A) and after (B) a 30-min exposure of platelets to thrombin, 1 unit/ml. The factor VI11 concentration was 6 nm. Polygons in the upper right panel indicate the forward and side light scatter values chosen to distinguish microparticles (MP) and Prothrombinase and Tenase Assays-Platelet prothrombinase ac- platelets (Plt). Lines on contourplots indicate 15% linear increments tivity was measured as described (Sims et al., 1988) with the modifi- of maximum event density with lines drawn at the middle of each cation that varying amounts of factor VI11 were added to the incu- 15% increment i.e. 7.5%, 22.5%, etc. The lowerpanels are histograms bation mixture prior to initiation of the reaction. Platelet tenase of the factor-vi11 dependent FITC fluorescence of platelets and activity was measured with a two-step amidolytic substrate assay. microparticles monitored with FITC-lB3 as described under Exper- Factor IXa was prepared by incubation of factor IX with factor XIa. imental Procedures. In panel C, factor VI11 binding to platelets Resting or activated platelets were diluted to 1-2 x 107/ml in solution incubated with buffer (solid line) is compared to platelets exposed to 11 containing 2.5 mm Ca. Factor X was added to a final concentration thrombin (dotted line). On the right, factor VI11 binding to microparof 0.5 pm and factor Va was added as indicated. The mixture was ticles resulting from thrombin exposure is displayed. Bars indicate then incubated at 37 C for 2 min and the reaction started by rapid the range of values averaged to obtain the numeric binding data addition of factors VI11 and IXa to final concentrations of 12 nm and displayed in Fig. 2. Note that FITC fluorescence is displayed on a log 24 nm, respectively. At 1-min intervals, aliquots were diluted 1:lO scale.

4 17264 Factor VIIZ Binding to Platelet Microparticles their differing light-scattering characteristics for further analysis. Thirty min after agonist exposure, thrombin-stimulated platelets exhibited 50% more green fluorescence than the background fluorescence of unstimulated platelets, indicating a small amount of binding of factor VI11 (Fig. 1C). By comparison, microparticles demonstrated 50 times as much green fluorescence/particle as unstimulated platelets (Fig. ld), indicating 30-fold more factor VI11 binding/microparticle than per stimulated platelet. Background fluorescence of cence above background. However, within 60 s after exposure microparticles in the presence of FITC-1B3 but without added to thrombin, a large fraction of platelets exhibited green factor VI11 was less than that of platelets (data not shown). fluorescence, signifying binding of factor VIII-fluorescein The same pattern of preferential factor VI11 binding to mi- (Fig. 3). Emergence of microparticles expressing factor VIIIcroparticles over stimulated platelets was seen when platelets binding sites was also evident by 60 s. The number of small were stimulated with thrombin plus collagen, the complement proteins C5b-9, and the calcium ionophore A23187 (data not shown). As the concentration of factor VI11 was increased up to 230 nm, the quantity of factor VI11 bound to microparticles increased but appeared to approach saturation at 100 nm (Fig. 2). In preliminary experiments performed at nm factor VIII, increasing the concentration of the FITC-1B3 did not lead to an increase in platelet or microparticle fluorescence, indicating that the amount of anti-factor VI11 antibody used throughouthese experiments to detect membranebound factor VI11 was not limiting. Factor VI11 binding to stimulated platelets also increased with increasing factor VI11 concentration. However, the quantity of FITC fluorescence remained low, within 3-fold the value for unstimulated platelets. Furthermore, the fluorescence of unstimulated platelets did not increase with factor VIII, confirming the observation of Nesheim et al. (1989) that unstimulated platelets do not bind factor VIII. In those experiments in which thrombin was the platelet agonist, factor VI11 was converted to the more active form of factor VIII, factor VIIIa, since thrombin rapidly activates factor VI11 at the concentrations employed (Lollar et al., 1984; Neuenschwander and Jesty, 1988). The results of the functional platelet tenase assays described below are consistent with this assumption. Binding of bovine factor Va was compared directly to binding of factor VI11 using FITC- V237, an antibody specific for the light chain domain of factor V (Faioni et al., 1988; Sims et al., 1988). As reported previously, under similar conditions, where factor Va binding was observed 30 min after platelet stimulation (Sims et al., 1988, 1989), the majority of factor Va-binding sites were located on microparticles in the same pattern observed with factor VI11 binding. Factor Va binding to microparticles was saturated at 125 nm factor Va (data not shown). Because microparticles originate from the plasma mem- brane of platelets, we postulated that soon after stimulation platelets might bind factor VI11 but that the binding capacity would be lost during or after microparticle formation. In order to follow the time course of the expression of factor VIIIbinding sites, factor VI11 was directly labeled with fluorescein maleimide. No loss of procoagulant activity resulted from protein modification. Unstimulated platelets, incubated with 7 nm factor VIII-fluorescein, did not exhibit green fluores- particles that bound factor VI11 continued to increase over the next 30 s so that by 90 s the number of microparticles detected approximated the number of platelets. The amount of factor VI11 fluorescence/microparticle was comparable to 1047 L 2 10 O Id.- c, lo4 $ Y ril 104 U E: 3 2 lo s I I o [lactor Vlll] nm FIG. 2. Factor VI11 binds preferentially to microparticles. Platelets were incubated for 30 min with increasing concentrations of factor VI11 and FITC-labeled monoclonal antibody in the presence of buffer (A) thrombin plus collagen (0), or the complement proteins C5b-9 (0). Platelets and microparticles were distinguished for analysis on the basis of forward and side light scatter as indicated in Fig. 1 and average fluorescence/platelet or microparticle was calculated. Solid symbols indicate platelet-derived microparticles; open symbols indicate platelets O Side Scatter (arbitrary units) FIG. 3. Kinetic expression of factor VIII-binding sites on platelets and microparticles. Platelets were prelabeled with Di- IC16(3) and gel filtered to remove excess dye. Fluorescein-labeled factor VI11 (7 nm) was added to platelets at 37 "C and measurements of particle fluorescence were obtained before and at various times after the addition of thrombin (1 unit/ml). Prior to thrombin addition, background green fluorescence was measured (upper panel). Sixty s after thrombin addition many platelets exhibited increased green fluorescence signifying factor VI11 binding (center panel). Emergence of a new particle population with less side scatter than platelets but comparable factor VI11 binding was evident. Ninety s after thrombin addition most platelets bound factor VI11 and the size of the micro- particle population had increased (lower panel). Lines on contourplots indicate 20% linear increments of maximum event density.

5 the amount of factor VI11 fluorescence/platelet, implying that a microparticle contained as many factor VIII-binding sites as a platelet at the time of maximum binding site exposure. The increase in platelet fluorescence, observed between 60 and 90 s in Fig. 3, suggests that exposure of factor VIIIbinding sites may be relatively slow, with a half-time of s. However, experiments in which the rate of factor VI11 binding was augmented by swirling the sample gently between measurements demonstrated that within 30 s of agonist exposure a few platelets exhibited maximum factor VI11 binding and that within the next 45 s most of the platelets exhibited maximal factor VI11 binding. Very few platelets exhibited intermediate fluorescence at any time point (data not shown). This suggests that expression of the binding sites for an individual platelet is rapid and that under unstirred conditions binding of factor VI11 is rate-limiting. Exposure to a lower thrombin concentration, 0.1 unit/ml, led to the same temporal pattern of factor VIII-binding site exposure, but fewer platelets exhibited enhanced factor VI11 binding and fewer microparticles were released (data not shown). Loss of Platelet Factor VIII-binding Sites-The above experiments showed that platelet membrane-binding sites for factor VI11 were expressed within 2 min of thrombin stimulation but had largely disappeared by 30 min. To characterize this process further, platelets were incubated with thrombin at 37 "C, and their capacity to bind factor VI11 was evaluated at various times (Fig. 4). At each designated time point, an aliquot of platelets was removed from the incubation mixture, mixed with an equal volume of medium containing fluorescein-labeled factor VIII, and evaluated by flow cytometry within 1 min of mixing. This experimental design minimized the possibility that proteolytic modification of factor VI11 on the platelet membrane caused the loss of binding over time. Factor VIII-binding site expression was maximal by 2 min but then decreased over the next 25 min to within 1.5 times background fluorescence. Since the expression of binding sites for factor VI11 on platelet-derived microparticles was substantial and not lost over 30 min, additional experiments were performed to characterize these binding sites. Kinetics of Factor VIII Binding to Microparticles-The rate of association of factor VI11 with platelet-derived microparticles was estimated by measuring the time-dependent increase in microparticle fluorescence after the addition of factor VI11 (Fig. 5A). For these experiments, activated plate- 56 Factor Microparticles Platelet VIII to Binding m YInutes FIG. 5. Factor VI11 binds rapidly to microparticles and dissociates more slowly. Activated platelets and corresponding microparticles resulting from stimulation with thrombin plus collagen were added to preformed factor VI11 (15 ~ M)-FITC-~B~ antibody complex, and serial fluorescence measurements were obtained (upper panel). Data were fitted to a pseudo-first order model (inset) for estimated association constant. For dissociation experiments, 80 min of incubation of activated platelets and microparticles with factor VI11 (15 nm)-fitc 1B3 was followed by a rapid 100-fold dilution and serial fluorescence measurements were obtained (lower panel). Data were fitted to a first order dissociation model (inset) to obtain dissociation rates (Table I). TABLE I Rate and binding constants of factor VIII and factor Va binding to microparticles agonist Platelet k2 " kl k j min" M" min" (k,/k,) Factor VI11 C5b x lo6 5 x Thrombin plus Collagen X lo6 1 X lo-" Factor Va C5b X lo7 2 X lo-' Thrombin plus Collagen X lo6 3 X lo-' a Analysis was performed assuming that platelet- and microparticle-binding sites were at a much lower concentration than factor VI11 or factor Va, that fluorescence/particle, F1, was proportional to bound protein/particle, and that association reached equilibrium by 80 min Aln(Fl/Fleq) Fl,, = Fl,, min. The off constant, k, =, and the observed At Minutes FIG. 4. Platelet factor VI11 binding capacity is lost over 30 min. Platelets were prepared in minimum essential media and warmed to 37 "C. Following addition of thrombin, 1 unit/ml, aliquots were removed at various times and diluted 1:l into media containing 4 nm fluorescein-labeled factor VIII. Samples were evaluated by flow cytometry, and the platelets were distinguished for analysis using scatter criteria as depicted in Fig. 1. Mean log fluorescence was calculated and converted to a linear scale for display. lets and microparticles were incubated with preformed antibody-factor VI11 complexes, as described under "Experimental Procedures." The data were fitted to a pseudo-first order binding model (Fig. 5A, inset) and yielded an association rate constant, kl, of2-4 x lo6 M" min" (Table I). This rate constant was similar to that obtained when the binding of factor Va was measured using FITC-labeled V237 fluorescence (Table I). After ligand binding had reached equilibrium, the rates of dissociation of factor VI11 and factor Va from microparticles were estimated by measuring the decrease in particle fluorescence following a 100-fold dilution of the sam- ples (Fig. 5B). For factor VIII, the first order dissociation rate constant, kz, was 2 x lo-' min". Similar results were obtained for factor Va (Table I). The calculated equilibrium constants

6 17266 Factor VIII Binding to K,,, ( k2/kl), for factor VI11 and factor Va were comparable. Effect of Phospholipid Vesicles on the Binding of Factor VIII and Factor Vu to Microparticles-Since phosphatidylserinecontaining lipid vesicles bind factor VI11 and factor Va (Gilbert et al., 1990; Krishnaswamy and Mann, 1988), we examined the ability of these vesicles to compete with the membranes of microparticles for these proteins. Large unilamellar phospholipid vesicles composed of phosphatidylserine, phosphatidylcholine, and cholesterol were prepared. To detect these vesicles by flow cytometry, DiIC16(3), a fluorescent membrane probe, was incorporated during vesicle preparation. These vesicles bound factor VI11 and factor Va, and binding of each was dependent on the content of phosphatidylserine over the range 0-25 mol %. To examine whether these phospholipid vesicles could compete with microparticles for factor VI11 or factor Va, platelets were activated and then Microparticles Platelet binding of factor VI11 to microparticles. However, higher concentrations of factor Va caused a progressive decrease in factor VI11 binding such that, at 1 PM factor Va, factor VI11 binding was inhibited by approximately 75%. The effect of factor Va on factor VI11 binding to the surface of phospholipid vesicles containing 15 mol % phosphatidylserine was also examined (Fig. 6B). As with microparticle membranes, the binding of factor VI11 to lipid vesicles was increased at low concentrations of factor Va and inhibited at higher concen- trations. If factor Va affects the binding of factor VI11 to membrane surfaces, then this effect should be observable in a functional assay involving the tenase complex (membrane-bound factor IXa and factor VIIIa) of activated platelets and microparticles. Factor VI11 and factor IXa were added to a solution containing platelets preactivated by thrombin plus collagen, mixed with varying concentrations of non-fluorescent vesicles factor X, and varying concentrations of factor Va. Consistent containing 15 mol % phosphatidylserine. The lipid vesicles with the factor VIII-binding data, platelet tenase activity was effectively competed with activated platelets and platelet- increased in the presence of 1-40 nm factor Va and was derived microparticles for either factor VI11 or factor Va. At inhibited at higher concentrations of factor Va (Fig. 6C). a vesicle concentration of 2.5 PM phospholipid, factor VI11 Effect of Factor VIII on the Binding of Factor Vu to Microand factor Va binding to microparticles was reduced by greater particles-if factor Va influences the binding of factor VI11 than 75%. Binding was inhibited completely at a vesicle to membrane surfaces, would factor VI11 reciprocally influconcentration of 10 PM phospholipid. ence the binding of factor Va to membrane surfaces? To Effect of Factor Vu on the Binding of Factor VIII to Micro- address this question, the interaction of factor Va with microparticles-the structural homology between factors VIIIa and particles (Fig. 7A) and phospholipid vesicles (Fig. 7B) was factor Va and their ability to bind to acidic phospholipid evaluated in the presence of factor VIII. Factor VI11 concensuggested that these cofactors might bind to the same class trations in the range of 1 to 30 nm increased the binding of of receptors on platelet microparticles. Therefore, the influ- factor Va to microparticles and phospholipid vesicles. At ence of each cofactor on the binding of the other cofactor was higher factor VI11 concentrations, factor Va binding was studied. The addition of factor Va had a complex effect on inhibited. To study the effects on the prothrombinase reacthe binding of factor VI11 (Fig. 6A). In the presence of 12 nm tion, factor Va and factor Xa were added to incubation mixfactor VIII, the addition of factor Va at concentrations betures containing prothrombin, increasing concentrations of tween l and 35 nm caused an increase of10-50% in the [Factor Val nm [Factor VIII] nm C [Factor Val (nu) FIG. 6. Factor VI11 binding is enhanced, then inhibited, by increasing concentrations of factor Va. Factor Va was added to an incubation mixture of factor VI11 (12 nm for platelet experiments, 15 nm for phospholipid vesicle) and FITC-1B3 prior to the addition of platelets (A) or vesicles (B). Phospholipid vesicle composition was phosphatidylserine/phosphatidylcholine/cholesterol/diicl6(3), 15:75:10:0.1. Platelet agonists were complement proteins C5b-9 (D), thrombin plus collagen (O), and thrombin (A). In A, factor VI11 binding to microparticles is depicted. The effect of factor Va on factor VIII-dependent tenase activity was studied in a platelet-based assay, C. Factor VI11 and factor IXa were added to activated platelets (1 X 107/ml) and factor X which had been preincubated with factor Va at the indicated concentration. The rate of factor Xa formation was measured with an amidolytic substrate assay [Faelor Vlll] (nm) FIG. 7. Factor Va binding is enhanced, then inhibited, by increasing concentrations of factor VIII. Factor VI11 was added to an incubation mixture of factor Va (2.5 nm for platelet experiments, 10 nm for phospholipid vesicle experiment) and FITC-V237. The effect upon factor Va binding to microparticles (A) or to phospholipid vesicles (B) were observed. Phospholipid vesicle composition and platelet agonists were as described for Fig. 6. The effect of factor VI11 on factor Va-dependent prothrombinase activity was studied in a platelet-based assay, C. Factor Va (2 nm)was added to activated platelets which had been preincubated with factor VI11 at the indicated concentration. Factor Xa (2 nm) was added to start the reaction, and the formation of thrombin was measured by an amidolytic substrate assay. The platelet concentration was 5 X 106/ml, and the platelets had been preactivated with the complement proteins C5b-9 (X) or thrombin plus collagen (W).

7 lets but not microparticles have been shown to lose the capacity to support prothrombinase activity over min (Bevers et al., 1989; Comfurius et al., 1990). Thisuggests that the factor VI11 and factor V receptors are expressed via a common pathway. The physiologic importance of the transiently expressed platelet membrane-binding site versus the more stable microparticle-binding sites remains to be determined. However, we took advantage of the relative stability of microparticle-binding sites to characterize them more throroughly in vitro. Kinetic binding studies indicated that the apparent KD for factor VI11 or factor Va binding to microparticles was approximately 5 nm. This value for factor VI11 is close to the Kn obtained in equilibrium binding studies using intrinsically radiolabeled factor VI11 and thrombin-activated platelets (Nesheim et al., 1988). Although the surface area of a microparticle is substantially less than that of a platelet (Sims et al., 1988), the amount of factor VI11 bound/microparticle was comparable to the amount of factor VI11 bound/activated platelet. This suggests an efficient transfer of binding sites from platelet to microparticle during the vesiculation process with a resulting high density of microparticle-binding sites. Factor VIII, rather than factor VIIIa, was used in these experiments. Factor VI11 circulates in plasma complexed to von Willebrand factor, and this complexed form of factor VI11 is not capable of interacting with binding sites on activated platelets (Nesheim et al., 1989).' Upon activation by thrombin, however, factor VI11 is converted to factor VIIIa, which dissociates from von Willebrand factor (Hill-Eubanks et al., 1989) and is presumably available for binding to activated platelets. The instability of fluid-phase factor VIIIa influenced our decision to avoid its use as a starting ligand in these studies. Our approach to this problem was to perform each type of platelet experiment in the presence of enough thrombin to activate factor VI11 as well as the platelets. Of note, no difference in results were observed when platelets were activated with the complement proteins, C5b-9, or the calcium ionophore A Although these agonists do not directly activate factor VIII, we cannot exclude the possibility that proteases from activated platelets may be able to convert factor VI11 to an active form (Nesheim, 1988). In this context, proteolytic conversion of platelet-derived factor V has been demonstrated upon platelet activation by C5b-9 (Faioni et al., 1988). Furthermore, in the present study, no lag phase in the generation of factor Xa was observed in the platelet tenase assay, suggesting prior conversion of factor VI11 to its active ' G. E. Gilbert, P. J. Sims, T. Wiedmer, B. C. Furie, B. Furie, and S. J. Shattil, unpublished observations. Factor VIII Binding to Microparticles Platelet factor VIII, and platelets preactivated with thrombin plus form during the course of platelet activation. collagen. Consistent with the factor Va binding data in Fig. The binding of factor VI11 and factor Va to activated 7, prothrombinase activity was increased in the presence of up to 20 nm factor VI11 and inhibited at higher concentrations platelets and microparticles shared several features in common with the binding of these cofactors to synthetic phos- (Fig. 7C). Similar results were obtained when platelets acti- pholipid vesicles. The calculated dissociation constant for vated with C5b-9 were employed in the prothrombinase assay. factor VI11 binding to microparticles (approximately 5 nm) is similar to the value obtained by equilibrium binding of factor DISCUSSION VI11 to phosphatidylserine-containing phospholipid vesicles This study demonstrates that within 1 min after platelet (Gilbert et al., 1990). Further, the calculated dissociation stimulation by thrombin, factor VIII-binding sites are ex- constant for factor Va binding to microparticles is similar to pressed on the platelet plasma membrane and that within 90 the equilibrium Kr, for factor Va binding to phospholipid s plasma membrane vesicles (microparticles) are shed which vesicles (Krishnaswamy and Mann, 1988). These similarities have a high density of these sites. Platelet binding capacity suggest that platelet membrane phospholipid could be the for factor VI11 is subsequently diminished within 30 min of source of binding sites for both factors VI11 and Va. This is agonist exposure while microparticles continue to bind factor consistent with the view put forward by Bevers and CO- VIII, whether these microparticles are generated by thrombin, workers (1983) who showed that phosphatidylserine is transthrombin plus collagen, or the complement proteins, C5b-9. located from the inner leaflet to the outer leaflet of the platelet In this context it is interesting to note that stimulated plate- plasma membrane upon cell activation. Moreover, in the present study we demonstrated reciprocal competition for binding between factors VI11 and Va on platelets, microparticles, and phospholipid vesicles. We also found that low concentrations of synthetic, phosphatidylserine-containing lipid vesicles competed effectively with both platelets and microparticles for binding of factors VI11 and Va. In contrast to the similarities noted above, the rates of association and dissociation that we observed between factor VI11 or factor Va and microparticles were considerably slower than those reported for factor Va and phospholipid vesicles (Abbott and Nelsestuen, 1987). Interpretation of this difference is difficult because prior kinetic studies examined membrane association over a short period of time and do not exclude a slower second component to phospholipid membrane binding. Thus, further study remains to identify the molecular determinants of the factor VI11 and factor Va receptors. We observed that concentrations of factor Va between 1 and 30 nm caused an increase in the binding of 12 nm factor VI11 to microparticles. This same effect was observed when synthetic phospholipid vesicles were used instead of platelets. Conversely, factor VI11 at 1-30 nm caused an increase in the binding of factor Va. Furthermore, this reciprocal increase in cofactor binding was associated with a parallel increase in cofactor function. The presence of factor Va increased factor VIIIa-dependent platelet tenase activity, and factor VI11 increased factor Va-dependent prothrombinase activity. At this time explanations of the phenomenon remain speculative. It is possible that factor Va and factor VI11 compete for limited phosphatidylserine-containing binding sites and also bind to one another. It is also possible that the enhanced binding is a less specific effect of one membrane-binding protein on another. Evidence against this possibility includes the absence of cooperativity in the binding of factor VI11 alone (Fig. 2), the absence of binding enhancement in a membrane-binding experiment including human placental anticoagulant protein- I and prothrombin or factor Xa (Tait et al., 1989), and the absence of binding enhancement in a membrane-binding experiment including factor Va and a peptide derived from the putative membrane-binding domain (Krishnaswamy and Mann, 1988). Neither enhancement of factor VI11 binding to platelet membranes by factor V nor competition for factor VI11 binding to platelet membranes by factor V was observed by Nesheim, et al. (1988). However, there is no apparent discrepancy between their results and this work. In that study, competition for factor VI11 binding to platelets was studied in the presence of 170 nm factor V. Our results indicate that factor VI11 binding is enhanced at factor Va concentrations substantially below 170 nm, and that factor VI11 binding is

8 17268 Factor VIII Binding to inhibited at factor Va concentrations above170 nm. Both studies agree that at factor V or factor Va concentrations in the range of 170 nm, the net effect on factor VI11 binding to platelets is small. The normal concentration of factor VI11 in plasma is about 0.3 nm and that of factor V is 30 nm. While the plasma concentration of factor VI11 is at least 10-fold lower than the concentration of factor VI11 that maximally enhanced factor Va binding in these studies, the plasma concentration of factor V is in the concentration range where factor Va maximally enhanced factor VI11 binding. It is possible that the local concentration of factor VI11 at the membrane surface may be higher than the fluid-phase concentration (Mann et al., 1988). Thus, it is conceivable that interactions between factor VI11 and factor Va influence the net procoagulant activity of platelet membranes under physiological circumstances. For example, immediate proximity of factor VI11 and factor Va could represent an efficient mechanism for the translocation of newly activated factor Xa from the tenase complex to the nascent prothrombinase complex. In addition, the reciprocal enhancement of binding could lead to more efficient partitioning of each of the cofactors from the fluid-phase to the platelet membrane. In conclusion, this work indicates that factor VIII-binding sites are expressed within 60 s after platelet stimulation both upon the platelet membrane and upon microparticles shed from the platelet membrane. Binding sites are concentrated upon the microparticle so that a microparticle, whose mem- brane surface area represents only a small fraction of the surface area of a platelet, binds a quantity of factor VI11 equivalent to that of a stimulated platelet. While the binding sites on the platelet membrane are depleted within 30 min, those on the microparticle are expressed for a longer time. 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