A Subpopulation of Platelets Responds to Thrombin- or SFLLRN-stimulation with Binding Sites for Factor IXa

Abstract

Strong agonists cause platelets to expose a procoagulant surface supporting the assembly of two important coagulation enzyme complexes. Equilibrium binding has determined the density of high affinity saturable factor IXa binding sites to be 500–600 sites/platelet. We have now used flow cytometry to visualize the binding of factor IX and IXa to thrombin- or SFLLRN-activated platelets. Concentrations of these agonists that are half-maximal or maximal in kinetic studies resulted in only a small subpopulation (4–20%) of platelets binding factor IX or IXa with the density of binding sites for factor IX being about half of that for factor IXa, consistent with previous equilibrium binding studies. A small subpopulation (5 ± 1.5%) of platelets stimulated with either agonist also exposed annexin V binding sites, and this subpopulation of platelets also bound factor IXa. Annexin V decreased factor IXa binding in the presence or absence of factor VIIIa, and factor IXa could also decrease annexin V binding on some platelets indicating a common binding site in agreement with previous studies. All platelets binding factor IXa were positive for glycoprotein IX, at the same glycoprotein IX surface density as seen in platelets negative for factor IXa binding. These studies refine the results from equilibrium binding studies and suggest that, on average, only a small subpopulation (∼10%) of PAR 1-stimulated platelets expose ∼6000 factor IXa binding sites/platelet. Strong agonists cause platelets to expose a procoagulant surface supporting the assembly of two important coagulation enzyme complexes. Equilibrium binding has determined the density of high affinity saturable factor IXa binding sites to be 500–600 sites/platelet. We have now used flow cytometry to visualize the binding of factor IX and IXa to thrombin- or SFLLRN-activated platelets. Concentrations of these agonists that are half-maximal or maximal in kinetic studies resulted in only a small subpopulation (4–20%) of platelets binding factor IX or IXa with the density of binding sites for factor IX being about half of that for factor IXa, consistent with previous equilibrium binding studies. A small subpopulation (5 ± 1.5%) of platelets stimulated with either agonist also exposed annexin V binding sites, and this subpopulation of platelets also bound factor IXa. Annexin V decreased factor IXa binding in the presence or absence of factor VIIIa, and factor IXa could also decrease annexin V binding on some platelets indicating a common binding site in agreement with previous studies. All platelets binding factor IXa were positive for glycoprotein IX, at the same glycoprotein IX surface density as seen in platelets negative for factor IXa binding. These studies refine the results from equilibrium binding studies and suggest that, on average, only a small subpopulation (∼10%) of PAR 1-stimulated platelets expose ∼6000 factor IXa binding sites/platelet. Although normally circulating in a quiescent state, when activated with strong agonists, platelets provide an important surface for assembly of two major coagulation enzyme complexes, the intrinsic factor X-activating complex and the prothrombinase complex (1Colman R.W. Clowes A.W. George J.N. Hirsh J. Marder V.J. Colman R.W. Hirsh J. Marder V.J. Clowes A.W. George J.N. Hemostasis and Thrombosis, Basic Principles and Clinical Practice. Lippincott Williams & Wilkins, Philadelphia2001: 3-16Google Scholar). In vitro kinetic assays have demonstrated that in the presence of activated platelets (or artificial phospholipid vesicles) factor Xa generation is enhanced several million-fold, and thrombin generation is enhanced 300,000-fold over solution phase activity (2van Dieijen G. van Rijn J.L. Govers-Riemslag J.W. Hemker H.C. Rosing J. Thromb. Haemostasis. 1985; 53: 396-400Crossref PubMed Scopus (34) Google Scholar, 3Mann K.G. Prog. Hemostasis Thromb. 1984; 7: 1-23PubMed Google Scholar, 4Giles A.R. Nesheim M.E. Hoogendoorn H. Tracy P.B. Mann K.G. Br. J. Haematol. 1982; 51: 457-468Crossref PubMed Scopus (8) Google Scholar, 5Rawala-Sheikh R. Ahmad S.S. Ashby B. Walsh P.N. Biochemistry. 1990; 29: 2606-2611Crossref PubMed Scopus (92) Google Scholar). It is known that not all agonists are equivalent in their ability to stimulate all functional end points of platelet activation. Weak agonists (e.g. ADP and epinephrine) require feedback enhancement from secreted molecules or synthesized agonists such as thromboxane A2 to achieve stimulation of platelet aggregation and are unable to stimulate a procoagulant surface capable of supporting assembly of these enzyme complexes (6Ahmad S.S. Scandura J.M. Walsh P.N. J. Biol. Chem. 2000; 275: 13071-13081Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 7Scandura J.M. Walsh P.N. Biochemistry. 1996; 35: 8903-8913Crossref PubMed Scopus (37) Google Scholar). Strong agonists (e.g. thrombin and collagen) are capable of generating all platelet functional endpoints (8Bevers E.M. Rosing J. Zwaal R.F. Adv. Exp. Med. Biol. 1985; 192: 359-371Crossref PubMed Scopus (31) Google Scholar). Are all platelets equivalent in their response to these agonists? Visualizations of in vivo clot formation show most platelets rolling, adhering, aggregating, and serving as the foundation for fibrin clot formation (9Falati S. Gross P. Merrill-Skoloff G. Furie B.C. Furie B. Nat. Med. 2002; 8: 1175-1181Crossref PubMed Scopus (557) Google Scholar). However, circulating platelets may constitute a heterogeneous population. Their generation from megakaryocytes packages them in different sizes, with different granule loads, differing numbers of mitochondria, and other vestiges of the parent cells (10Hartwig J. Italiano Jr., J. J. Thromb. Haemostasis. 2003; 1: 1580-1586Crossref PubMed Scopus (102) Google Scholar, 11Italiano Jr., J.E. Lecine P. Shivdasani R.A. Hartwig J.H. J. Cell Biol. 1999; 147: 1299-1312Crossref PubMed Scopus (427) Google Scholar). The circulation contains platelets of different ages as well as sizes. Flow cytometry analyses detect a wide range of forward and side scatter of blood platelets, necessitating the viewing of platelets on log scales (12Shattil S.J. Cunningham M. Hoxie J.A. Blood. 1987; 70: 307-315Crossref PubMed Google Scholar). Flow cytometry provides an opportunity to view the homogeneity or heterogeneity of the incidence and density of platelet markers as well as the appearance of activation-dependent markers providing a clinical tool to probe for platelet activation in whole blood samples (13Abrams C.S. Ellison N. Budzynski A.Z. Shattil S.J. Blood. 1990; 75: 128-138Crossref PubMed Google Scholar, 14Shattil S.J. Am. J. Clin. Nutr. 1992; 56: 789S-790SCrossref PubMed Scopus (5) Google Scholar). We have formerly used both equilibrium binding studies and in vitro kinetic studies to investigate the nature of the factor IXa-factor VIIIa-catalyzed factor X-activating complex on thrombin- or SFLLRN-stimulated platelets. We determined that platelets upon activation expose 500–600 binding sites for the enzyme factor IXa (15Ahmad S.S. Rawala-Sheikh R. Walsh P.N. J. Biol. Chem. 1989; 264: 3244-3251Abstract Full Text PDF PubMed Google Scholar), 1000–1200 binding sites for the cofactor factor VIIIa (6Ahmad S.S. Scandura J.M. Walsh P.N. J. Biol. Chem. 2000; 275: 13071-13081Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar), and 1000–1200 specific binding sites for the substrate factor X (as well as 30,000 sites shared with prothrombin) (16Scandura J.M. Ahmad S.S. Walsh P.N. Biochemistry. 1996; 35: 8890-8902Crossref PubMed Scopus (64) Google Scholar). The affinity of each protein for its binding site as well as complete occupancy of sites are both optimal when all components of the enzyme complex are present. It has been assumed that the saturable binding sites determined from equilibrium binding studies are generated on all platelets as a result of agonist stimulation. We now present data from flow cytometry studies visualizing the binding of the enzyme and the zymogen that result in the surprising conclusion that although all platelets respond to the PAR 1 agonists with markers of activation, only a small subpopulation of platelets stimulated with thrombin or SFLLRN-amide responds with procoagulant surface changes allowing binding of factor IXa. Reagents—Bovine serum albumin, buffer reagents, disodium EDTA, dimethyl sulfoxide (Me2SO), calcium chloride, and Sepharose 2B-CL, were obtained from Sigma. Electrophoresis reagents were from Bio-Rad Laboratories, Inc. Chromogenic substrates N-α-benzyloxycarbonyl-d-arginyl-l-glycyl-l-arginine-p-nitroanaline-dihydrochloride (S-2765) and H-d-phenylalanyl-l-pipecolyl-l-arginine-p-nitroanaline dihydrochloride (S-2238) were purchased from DiaPharma (West Chester, OH). The thrombin receptor agonist peptide, SFLLRN-amide, was synthesized using Fmoc (9-fluorenylmethyloxycarbonyl) chemistry on an Applied Biosystems 430A synthesizer and reverse-phase high pressure liquid chromatography purified to greater than 99% homogeneity. A fluorescein isothiocyanate (FITC) 1The abbreviations used are: FITC, fluorescein isothiocyanate; GPIX, glycoprotein IX; GPIbα, glycoprotein Ibα; PE, phycoerythrin; FL, fluorescence channel. labeling kit was purchased from Molecular Probes, Inc. (Eugene, OR), and the biotinylation kit was from Pierce. FITC-labeled rabbit anti-human fibrinogen was from ICN Biochemicals, Inc. (Costa Mesa, CA). FITC-labeled CD42a against glycoprotein IX (GPIX), FITC-labeled CD42b against glycoprotein Ibα (GPIbα), FITC-labeled CD62P against P-selectin and streptavidin-phycoerythrin (PE)-cy5 (Quantum Red) were purchased from Pharmingen. Unlabeled CD42b was purchased from DakoCytomation (Carpinteria, CA). Proteins—Factors IX, X, and XIa were obtained from Enzyme Research Laboratories, Inc. (South Bend, IN) or Hematologic Technologies, Inc. (Burlington, VT). Coagulation proteins were obtained in or dissolved in 20 mm Tris-hydroxymethane, 150 mm NaCl and dialyzed against the same buffer to remove inhibitors. Concentrations were determined by bicinchoninic acid assay (Pierce), and purity was assessed by SDS-PAGE visualized with Coomassie Brilliant Blue staining or by Western blot using specific antibodies. Baxter Healthcare Corporation (Duarte, CA) generously supplied recombinant factor VIII. Human α-thrombin was purchased from Sigma and was active-site titrated against human antithrombin III, the residual activity was assayed chromogenically with S-2238. The monoclonal antibody C10D raised against the catalytic domain of factor IX (17Frazier D. Smith K.J. Cheung W.F. Ware J. Lin S.W. Thompson A.R. Reisner H. Bajaj S.P. Stafford D.W. Blood. 1989; 74: 971-977Crossref PubMed Google Scholar) was fluoresceinated using manufacturer's instructions (Molecular Probes) or was biotinylated using manufacturer's instructions (Pierce). Protein modifications did not interfere with the ability of the antibody to inhibit the clotting of normal human plasma. Platelet Preparation—Washed, aspirin-treated, gel-filtered platelets were prepared from human whole blood as described previously (18London F. Walsh P.N. Biochemistry. 1996; 35: 12146-12154Crossref PubMed Scopus (20) Google Scholar) with the following modifications. Platelet-rich plasma was incubated with 1 mm acetylsalicylic acid for 30 min to inhibit feedback activation through prostaglandin synthesis, with prostaglandin E1 (2 μm) added 5 min before layering over the bovine serum albumin gradient for the wash step. Factor Xa Generation—Factor Xa was generated as described previously (18London F. Walsh P.N. Biochemistry. 1996; 35: 12146-12154Crossref PubMed Scopus (20) Google Scholar) with the following modifications. Washed, aspirin-treated, gel-filtered platelets (108/ml) were added to wells of a microtiter plate. Factor IXa and varying concentrations of agonists (thrombin or SFLLRN-amide) were added, and activation proceeded for 10 min before the addition of the cofactor and substrate. Factor VIII (200 units/ml) was incubated 1 min at 37 °C with thrombin (0.1 units/ml) and an additional minute with hirudin (5 units/ml) immediately before addition to reaction mixtures. Reaction mixtures were stopped after 3 min and were for factor Xa generated by substrate as described F. Ahmad S.S. Walsh P.N. Biochemistry. 1996; 35: PubMed Scopus Google Scholar). from enzyme assays were to factor Xa by to a of factor Xa of and using to kinetic from were and for and Flow aspirin-treated, gel-filtered platelets were incubated with FITC-labeled CD42b against platelet to the platelet population. were incubated with or annexin V at concentrations or with concentrations of factor IXa or IX, and half of each was activated with thrombin or units/ml) or with SFLLRN-amide μm) for 10 samples factor VIIIa at 10 factor IX or IXa were incubated with either FITC-labeled C10D or biotinylated C10D for and the was with samples were to detect V binding and factor IXa binding samples were to detect platelet and factor IXa binding. All samples were and on a flow with a were using either or for forward scatter and a of fluorescence was to were to fluorescence 1 fluorescence or 3 and and or platelets were by their forward side scatter as well as by positive staining for CD42a for 10 mm for or and with (or the of biotinylated C10D and in the absence of factor platelets were for fluorescein as as or as and on log scales as density or or markers were with negative samples to the of positive and negative were that density from could be as platelet and and for forward scatter with buffer were from with platelet from different the same were and activated with of thrombin or PAR intrinsic factor Xa generation R. Ahmad S.S. Ashby B. Walsh P.N. Biochemistry. 1990; 29: 2606-2611Crossref PubMed Scopus (92) Google Scholar) and expose high affinity saturable binding sites for factor X-activating complex components (6Ahmad S.S. Scandura J.M. Walsh P.N. J. Biol. Chem. 2000; 275: 13071-13081Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 7Scandura J.M. Walsh P.N. Biochemistry. 1996; 35: 8903-8913Crossref PubMed Scopus (37) Google Scholar, S.S. Rawala-Sheikh R. Walsh P.N. J. Biol. Chem. 1989; 264: 3244-3251Abstract Full Text PDF PubMed Google Scholar, S.S. Rawala-Sheikh R. Ashby B. Walsh P.N. J. Clin. 1989; PubMed Scopus Google Scholar). We have now used flow cytometry studies to visualize the of PAR 1 stimulation on both factor IXa binding and on of procoagulant and this with the ability of PAR 1 agonists to stimulate platelet for factor Xa stimulated with of thrombin or SFLLRN-amide for 10 min at 37 °C factor Xa generation at ± in response to thrombin at 1 and at ± in response to 150 PAR 1 agonist The for thrombin was ± and for SFLLRN-amide was ± incubated with an FITC-labeled antibody to P-selectin platelets stimulated with SFLLRN-amide a fluorescence of the of platelets indicating that all platelets to the agonist with from and of P-selectin the plasma The same on P-selectin was in response to thrombin at and these platelets fibrinogen when stimulated with as as 10 or thrombin at not indicating that all platelets are capable of aggregation as well as in response to PAR 1 stimulation. The assembly of the factor X-activating complex on platelets was by flow cytometry for factor IXa binding. The monoclonal antibody C10D to the of factor IX was either fluoresceinated with for in the or was biotinylated and with to a at and by from to in the Factor IXa binding results were equivalent using either binding results determined in the presence and absence of the cofactor factor VIIIa units/ml) when platelets were stimulated with half-maximal concentrations of SFLLRN-amide factor IXa resulted in an of platelets binding factor in both the presence and absence of the The presence of the cofactor did not the of platelets for a factor IXa did the density of binding sites at than concentrations of 10 factor IXa, the of binding sites in the presence and absence of the cofactor were The of platelets binding Factor IXa in the presence of 10 factor IXa in the absence of the cofactor was ± with a fluorescein of ± platelets incubated with biotinylated C10D and in the absence of factor IXa positive population. than of platelets density binding in the presence of 10 factor IXa. the binding of the enzyme with that of the zymogen that of the platelets were positive for factor IXa, and were positive for factor IX, with the density of binding sites for factor IXa to than for factor IX is in agreement with the equilibrium binding studies (15Ahmad S.S. Rawala-Sheikh R. Walsh P.N. J. Biol. Chem. 1989; 264: 3244-3251Abstract Full Text PDF PubMed Google Scholar) that half the of sites for factor IX as were for factor of factor IXa binding to activated platelets prepared as described were incubated with factor IXa or factor IX at the concentrations and activated by SFLLRN-amide μm) for 10 min at 37 °C before the of factor VIII with a data were and as described are as the of two or as the of the of in the factor factor in a factor IXa was incubated with platelets in the presence of half-maximal or of thrombin and units/ml) or SFLLRN-amide and 150 was on fluorescence ± ± in both The of platelets from ± to ± with thrombin and from ± to ± with for of annexin V binding sites after stimulation with thrombin or at either their or at only a small subpopulation of platelets was positive with a high density of annexin V binding Annexin V binding in platelets and as platelets were activated with half-maximal to maximal of both PAR 1 agonists, thrombin and SFLLRN-amide In with V and with V with the concentrations of the agonists, the of platelets positive with thrombin stimulation of annexin V binding was ± and with SFLLRN-amide stimulation was ± maximal of SFLLRN-amide μm) resulted in ± activation with PAR 1 agonists was by a in the positive for annexin V binding with a density of binding studies were to investigate the annexin V binding and factor IXa binding to SFLLRN-activated platelets. were stimulated with SFLLRN-amide μm) and exposed to 10 factor IXa factor units/ml) in the presence of varying concentrations of annexin annexin V concentrations from 1 to resulted in density of bound annexin not that the density of bound factor IXa decreased with annexin V in the absence or presence of factor The presence of both on the of platelets positive for either annexin V or for factor IXa It was that of all a decrease in factor IXa binding in the presence of annexin only in annexin V binding in the presence of 10 factor IXa. the data for samples incubated with annexin V in the absence or presence of The on the all data for annexin V binding. in annexin V bound in the presence of that annexin V binding could be decreased by the presence of These results were using V with of factor IXa and using V with of factor IXa. Factor IXa in the absence of factor VIIIa the same The ability of factor IXa to binding sites for annexin V was with annexin V concentrations at or the previously determined kinetic F. Ahmad S.S. Walsh P.N. Biochemistry. 1996; 35: PubMed Scopus Google Scholar). In of this for was that the platelet to PAR 1 stimulation with binding of either annexin V or factor IXa were that all positive in the in the that was to platelets. in binding was consistent with previously kinetic studies F. Ahmad S.S. Walsh P.N. Biochemistry. 1996; 35: PubMed Scopus Google Scholar) of annexin V of factor factor X activation, an in platelet binding sites for both platelets to thrombin with the appearance of that annexin V are the same platelets capable of binding the enzyme factor IXa. of platelets to R.F. P. E.M. 1992; PubMed Scopus Google Scholar), in procoagulant exposed that coagulation activation of platelets with the of agonists to platelet procoagulant data from and platelet samples from factor V were for positive and of the platelet population. It was that of all positive for factor IXa of all positive for annexin V and of all positive for both were the platelet population. data R.F. P. E.M. 1992; PubMed Scopus Google Scholar) formation in platelets stimulated with thrombin agonist that factor IXa binding stimulated by PAR 1 activation on the activated platelet that all positive for factor IXa binding were platelets, staining was with IXa and FITC-labeled A is in The were to the negative of platelets incubated with both in the presence of and with biotinylated C10D and in the absence of factor IXa of activated platelets were positive for factor IXa all platelets were positive for that all platelets positive for factor IXa the same density of as the activated platelet that negative for factor IXa binding In these that platelet activation with of PAR 1 agonists to at platelet procoagulant activity resulted in only a subpopulation of platelets binding sites for factor IXa and for annexin the same surface density of complex as seen in the of activated platelets negative for factor IXa binding. for specific binding on over the of cells these was determined that factor IXa to thrombin- or platelets with 500–600 and that the zymogen to that of sites (15Ahmad S.S. Rawala-Sheikh R. Walsh P.N. J. Biol. Chem. 1989; 264: 3244-3251Abstract Full Text PDF PubMed Google Scholar). Flow cytometry provides the opportunity to the as and all cells provide binding sites or activation are specific to of the population. It also are the binding Although has been demonstrated that a strong agonist for platelet activation is a agonist for activation-dependent of procoagulant E.M. P. Zwaal R.F. PubMed Scopus Google Scholar), have previously used thrombin or SFLLRN-amide to stimulate platelet of factor Xa generation at to the concentrations that ability to stimulate of procoagulant Walsh P.N. Blood. 2002; PubMed Scopus Google Scholar). for the stimulation of factor Xa generation with the of factor IXa binding sites and of procoagulant by flow We have that although thrombin or all platelets to and to expose P-selectin through of only of the platelets respond to thrombin or with of factor IXa binding These platelets with factor IXa binding sites with the with annexin V binding. The subpopulation in platelets, the for are and Tracy P. Tracy P. Blood. 1 Scholar) that factor Xa binding was to a subpopulation of stimulated platelets, be to is a a and the of platelets to thrombin stimulation with of factor IXa or Xa binding flow of factor IXa binding to platelets data supporting the of two sites, a high affinity site with was and a affinity site with factor IXa concentrations M. Thromb. Haemostasis. 1992; PubMed Scopus (92) Google Scholar). Factor IXa binding results were for two different fluorescein of a monoclonal antibody to the factor IX catalytic domain and biotinylation of that antibody with of factor IXa in the presence of the cofactor and a of that in the absence of the from equilibrium binding 10 factor IXa a density was not by the presence of the The was not in these flow cytometry was that although the presence of both cofactor and substrate in equilibrium binding studies the from to S.S. Rawala-Sheikh R. Ashby B. Walsh P.N. J. Clin. 1989; PubMed Scopus Google Scholar), an of the cofactor in the absence of substrate did not result in half-maximal binding density at factor IXa. may either a of at density or the for the substrate to the However, the presence of the cofactor the density of binding most at a the equilibrium from the of agonists in kinetic maximal concentrations of both SFLLRN-amide and thrombin the factor IXa binding as seen with half-maximal The additional binding resulted from of the of platelets to factor IXa than from in the of factor IXa binding sites/platelet. In these factor IX bound to the same of platelets with to of the density of binding sites/platelet. is in agreement with equilibrium binding studies. in agreement with previous studies F. Ahmad S.S. Walsh P.N. Biochemistry. 1996; 35: PubMed Scopus Google Scholar) is the in binding for annexin V and factor IXa. In kinetic annexin V was to inhibit factor Xa generation with a of In these binding although the density of factor IXa was by the presence of annexin with annexin V present at concentrations its for kinetic annexin V density could also be by the presence of factor IXa. These results suggest that is for binding with factor IXa as a for annexin V binding sites than annexin V is for factor IXa binding sites for factor IXa and for annexin V were the platelet is in agreement with the previous that of factor IXa binding was to platelet M. Thromb. Haemostasis. 1992; PubMed Scopus (92) Google Scholar). was from the ability of thrombin to from activated platelets R.F. P. E.M. 1992; PubMed Scopus Google Scholar). The studies both and factor IXa binding that all platelets binding factor IXa an equivalent surface density of complexes as the of the platelet that negative for factor IXa binding. These flow cytometry studies visualizing factor IXa binding to thrombin- or SFLLRN-stimulated platelets suggest a of the previous about intrinsic factor X-activating complex binding It from the present results that of all platelets factor IXa binding an of of activated platelets an ∼6000 factor IXa binding sites/platelet. Flow cytometry of platelet binding of other factor Xa complex components data to and refine obtained from equilibrium binding studies. We for and and for with flow