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Redistribution and Hemostatic Action of Recombinant Activated Factor VII Associated with Platelets

2011; Elsevier BV; Volume: 178; Issue: 6 Linguagem: Inglês

10.1016/j.ajpath.2011.02.026

1525-2191

Irene López-Vílchez, Ulla Hedner, Carmen Altisent, Maribel Díaz‐Ricart, Ginés Escolar, Ana M. Galán,

Blood Coagulation and Thrombosis Mechanisms

Clinical evidence accumulated from hemophilic patients during prophylaxis with recombinant activated factor VII (rFVIIa) suggests that the duration of the hemostatic action of rFVIIa exceeds its predicted plasma half-life. Mechanisms involved in this outcome have not been elucidated. We have investigated in vitro the redistribution of rFVIIa in platelets from healthy donors, patients with FVII deficiency, and one patient with Bernard-Soulier syndrome. Platelet-rich plasma was exposed to rFVIIa (3 to 60 μg/mL). Flow cytometry, immunocytochemistry, and coagulation tests were applied to detect and quantify rFVIIa. The hemostatic effect of rFVIIa associated to platelets was evaluated using perfusion models. Our studies revealed a dose-dependent association of rFVIIa to the platelet cytoplasm with redistribution into the open canalicular system, and α granules. Mechanisms implicated in the internalization are multiple, involve GPIb and GPIV, and require phospholipids and cytoskeletal assembly. After platelet activation with thrombin, platelets exposed rFVIIa on their membrane. Perfusion studies revealed that the presence of 30% of platelets containing FVIIa improved platelet aggregate formation and enhanced fibrin generation (P < 0.01 versus control). Our results indicate that, at therapeutic concentrations, rFVIIa can be internalized into platelets, where it is protected from physiological clearance mechanisms and can still promote hemostatic activity. Redistribution of rFVIIa into platelets may explain the prolonged prophylactic effectiveness of rFVIIa in hemophilia. Clinical evidence accumulated from hemophilic patients during prophylaxis with recombinant activated factor VII (rFVIIa) suggests that the duration of the hemostatic action of rFVIIa exceeds its predicted plasma half-life. Mechanisms involved in this outcome have not been elucidated. We have investigated in vitro the redistribution of rFVIIa in platelets from healthy donors, patients with FVII deficiency, and one patient with Bernard-Soulier syndrome. Platelet-rich plasma was exposed to rFVIIa (3 to 60 μg/mL). Flow cytometry, immunocytochemistry, and coagulation tests were applied to detect and quantify rFVIIa. The hemostatic effect of rFVIIa associated to platelets was evaluated using perfusion models. Our studies revealed a dose-dependent association of rFVIIa to the platelet cytoplasm with redistribution into the open canalicular system, and α granules. Mechanisms implicated in the internalization are multiple, involve GPIb and GPIV, and require phospholipids and cytoskeletal assembly. After platelet activation with thrombin, platelets exposed rFVIIa on their membrane. Perfusion studies revealed that the presence of 30% of platelets containing FVIIa improved platelet aggregate formation and enhanced fibrin generation (P < 0.01 versus control). Our results indicate that, at therapeutic concentrations, rFVIIa can be internalized into platelets, where it is protected from physiological clearance mechanisms and can still promote hemostatic activity. Redistribution of rFVIIa into platelets may explain the prolonged prophylactic effectiveness of rFVIIa in hemophilia. Hemophilic patients with inhibitors to coagulation factor VIII (FVIII) or factor IX (FIX) cannot benefit from prophylaxis with these coagulation factors. Recombinant activated coagulation factor VII (rFVIIa) was developed for the treatment of bleeding episodes in these patients, facilitating their clinical management.1Hedner U. Erhardtsen E. Potential role for rFVIIa in transfusion medicine.Transfusion. 2002; 42: 114-124Crossref PubMed Scopus (205) Google Scholar The rFVIIa, which has the same structure and activity as the human coagulation factor, restores hemostasis by favoring thrombin generation.2Monroe D.M. Further understanding of recombinant activated factor VII mode of action.Semin Hematol. 2008; 45: S7-S11Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar Notably, rFVIIa has proven useful to control active bleeding episodes not only in hemophilia, but also in other hemostatic deficiencies, including platelet and coagulation disorders.1Hedner U. Erhardtsen E. Potential role for rFVIIa in transfusion medicine.Transfusion. 2002; 42: 114-124Crossref PubMed Scopus (205) Google Scholar, 3Hedner U. Ezban M. Tissue factor and factor VIIa as therapeutic targets in disorders of hemostasis.Annu Rev Med. 2008; 59: 29-41Crossref PubMed Scopus (46) Google Scholar, 4Hers I. Mumford A. Understanding the therapeutic action of recombinant factor VIIa in platelet disorders.Platelets. 2008; 19: 571-581Crossref PubMed Scopus (15) Google Scholar The main mechanism by which rFVIIa exerts its hemostatic action in the control of active bleeding in congenital and acquired disorders of hemostasis could be explained by an enhanced thrombin generation at damaged vessels.5Hoffman M. Monroe 3rd, D.M. The action of high-dose factor VIIa (FVIIa) in a cell-based model of hemostasis.Semin Hematol. 2001; 38: 6-9Abstract Full Text PDF PubMed Scopus (85) Google Scholar, 6Roberts H.R. Hoffman M. Monroe D.M. A cell-based model of thrombin generation.Semin Thromb Hemost. 2006; 32: 32-38Crossref PubMed Google Scholar Tissue factor (TF) exposed at sites of vascular damage would help to localize the hemostatic response, favoring fibrin generation and platelet recruitment in more stable thrombi.7Galan A.M. Tonda R. Altisent C. Maragall S. Ordinas A. Escolar G. Recombinant factor VIIa (NovoSeven®) restores deficient coagulation: experience from an ex vivo model.Semin Hematol. 2001; 38: 10-14Abstract Full Text PDF PubMed Scopus (16) Google Scholar, 8Lisman T. De Groot P.G. Mechanism of action of recombinant factor VIIa.J Thromb Haemost. 2003; 1: 1138-1139Crossref PubMed Scopus (93) Google Scholar, 9Galán A.M. Tonda R. Pino M. Reverter J.C. Ordinas A. Escolar G. Increased local procoagulant action: a mechanism contributing to the favorable hemostatic effect of recombinant FVIIa in PLT disorders.Transfusion. 2003; 43: 885-892Crossref PubMed Scopus (56) Google Scholar Pharmacokinetic studies performed on rFVIIa by different groups have established a half-life of 2.7 hours in adults and 1.3 hours in children.10Lindley C.M. Sawyer W.T. Macik B.G. Lusher J. Harrison J.F. Baird-Cox K. Birch K. Glazer S. Roberts H.R. Pharmacokinetics and pharmacodynamics of recombinant factor VIIa.Clin Pharmacol Ther. 1994; 55: 638-648Crossref PubMed Scopus (258) Google Scholar, 11Girard P. Nony P. Erhardtsen E. Delair S. Ffrench P. Dechavanne M. Boissel J.P. Population pharmacokinetics of recombinant factor VIIa in volunteers anticoagulated with acenocoumarol.Thromb Haemost. 1998; 80: 109-113PubMed Google Scholar, 12Erhardtsen E. Pharmacokinetics of recombinant activated factor VII (rFVIIa).Semin Thromb Hemost. 2000; 26: 385-391Crossref PubMed Google Scholar Clinical experience from exploratory phase II trials, however, suggests that the hemostatic action of rFVIIa exceeds its predicted plasma half-life in patients subjected to prophylaxis.13Ludlam C.A. The evidence behind inhibitor treatment with recombinant factor VIIa.Pathophysiol Haemost Thromb. 2002; 32: 13-18Crossref PubMed Scopus (19) Google Scholar, 14Morfini M. Auerswald G. Kobelt R.A. Rivolta G.F. Rodriguez-Martorell J. Scaraggi F.A. Altisent C. Blatny J. Borel-Derlon A. Rossi V. Prophylactic treatment of haemophilia patients with inhibitors: clinical experience with recombinant factor VIIa in European Haemophilia Centres.Haemophilia. 2007; 13: 502-507Crossref PubMed Scopus (77) Google Scholar, 15Konkle B.A. Ebbesen L.S. Erhardtsen E. Bianco R.P. Lissitchkov T. Rusen L. Serban M.A. Randomized, prospective clinical trial of recombinant factor VIIa for secondary prophylaxis in hemophilia patients with inhibitors.J Thromb Haemost. 2007; 5: 1904-1913Crossref PubMed Scopus (281) Google Scholar Recent publications have highlighted the potential role of rFVIIa in prophylaxis of hemophilic patients with inhibitors.14Morfini M. Auerswald G. Kobelt R.A. Rivolta G.F. Rodriguez-Martorell J. Scaraggi F.A. Altisent C. Blatny J. Borel-Derlon A. Rossi V. Prophylactic treatment of haemophilia patients with inhibitors: clinical experience with recombinant factor VIIa in European Haemophilia Centres.Haemophilia. 2007; 13: 502-507Crossref PubMed Scopus (77) Google Scholar, 16Yilmaz A.A. Yalcin S. Serdaroglu H. Sonmezer M. Uysalel A. Prophylaxis with recombinant-activated factor VII (rFVIIa) for minimally invasive surgery in a patient with congenital factor VII deficiency: a case report with a single-low dose of rFVIIa.Blood Coagul Fibrinolysis. 2008; 19: 693-695Crossref PubMed Scopus (2) Google Scholar, 17Jiménez-Yuste V. Alvarez M.T. Martín-Salces M. Quintana M. Rodriguez-Merchan C. Lopez-Cabarcos C. Velasco F. Hernández-Navarro F. Prophylaxis in 10 patients with severe haemophilia A and inhibitor: different approaches for different clinical situations.Haemophilia. 2009; 15: 203-209Crossref PubMed Scopus (49) Google Scholar Although the mechanisms of action of rFVIIa in the correction of active bleeding have been widely studied, those involved in the apparent long-lasting effects of rFVIIa for prophylactic treatment remain to be clarified. It has been speculated that a portion of the rFVIIa infused into patients could diffuse to the extravascular space and, once there, become available at the site of injury.18Hedner U. Potential role of recombinant factor FVIIa in prophylaxis in severe hemophilia patients with inhibitors.J Thromb Haemost. 2006; 4: 2498-2500Crossref PubMed Scopus (28) Google Scholar Several research groups have already suggested the presence of TF in platelets.19Müller I. Klocke A. Alex M. Kotzsch M. Luther T. Morgenstern E. Zieseniss S. Zahler S. Preissner K. Engelmann B. Intravascular tissue factor initiates coagulation via circulating microvesicles and platelets.FASEB J. 2003; 17: 476-478PubMed Google Scholar, 20Mackman N. Tilley R.E. Key N.S. Role of the extrinsic pathway of blood coagulation in hemostasis and thrombosis.Arterioscler Thromb Vasc Biol. 2007; 27: 1687-1693Crossref PubMed Scopus (461) Google Scholar, 21Schwertz H. Tolley N.D. Foulks J.M. Denis M.M. Risenmay B.W. Buerke M. Tilley R.E. Rondina M.T. Harris E.M. Kraiss L.W. Mackman N. Zimmerman G.A. Weyrich A.S. Signal-dependent splicing of tissue factor pre-mRNA modulates the thrombogenicity of human platelets.J Exp Med. 2006; 203: 2433-2440Crossref PubMed Scopus (290) Google Scholar Indeed, recent investigations from our own group have demonstrated that platelets possess mechanisms to internalize TF-rich microvesicles.22Lopez-Vilchez I. Escolar G. Diaz-Ricart M. Fuste B. Galan A.M. White J.G. Tissue factor-enriched vesicles are taken up by platelets and induce platelet aggregation in the presence of factor VIIa.Thromb Haemost. 2007; 97: 202-211PubMed Google Scholar Of note, one of the TF preparations used in these studies was known to contain traces of FVII.23Orvim U. Roald H.E. Stephens R.W. Roos N. Sakariassen K.S. Tissue factor-induced coagulation triggers platelet thrombus formation as efficiently as fibrillar collagen at arterial blood flow conditions.Arterioscler Thromb. 1994; 14: 1976-1983Crossref PubMed Google Scholar It was therefore hypothesized that platelets may be able to incorporate FVIIa or even TF-FVIIa complexes. Redistribution of rFVIIa into platelets could protect this factor from physiological clearance mechanisms and thus explain the prolonged hemostatic action of rFVIIa under some clinical conditions. In the present study, we investigated the possible redistribution of rFVIIa into intravascular compartments, with specific focus on platelets. To detect the possible traffic of rFVIIa into platelets, and to evaluate its potential implications on its hemostatic capacity, we applied a combination of flow cytometry, electron microscopy, coagulometry, and perfusion techniques. This study was approved by the Ethics Committee of the Hospital Clinic in Barcelona. (2008/4624). Whole blood was anticoagulated with citrate/phosphate/dextrose buffer (CPD) to a final concentration of citrate of 19 mmol/L, or with low molecular weight heparin (Fragmin, Pharmacia, Madrid, Spain) at a final concentration of 20 U/mL. rFVIIa was supplied as NovoSeven by Novo Nordisk (Bagsvaerd, Denmark). PBS was from Gibco BRL Life Technologies (Paisley, UK). Antibody against CD41a was from BD Biosciences (San Jose, CA). Antibody to CD62-P (clone CLBThromb/6) was from Immunotech (Marseille, France). The Alexa Fluor 488 microscale protein labeling kit, was from Invitrogen Molecular Probes (Eugene, OR). IntraPrep permeabilization kit was from Beckman Coulter (Fullerton, CA). For detection of unlabeled human rFVIIa, a polyclonal rabbit anti-human antibody was used (Agrisera, Vännäs, Sweden). The secondary antibody was a goat anti-rabbit IgG fluorescein isothiocyanate-conjugated (BD Biosciences San Jose, CA). For immunocytochemical techniques, the same antibody against FVIIa previously described was used (polyclonal rabbit anti-human antibody; Agrisera, Vännäs, Sweden), which in turn was detected with protein A coupled to colloidal gold particles 10 nm in diameter (from the Cell Microscopy Center, Department of Cell Biology, University Medical Center Utrecht, The Netherlands). Bovine serum albumin, Cohn V fraction, was from Sigma-Aldrich (Steinheim, Germany). Thrombin from human plasma used for platelet activation was from Sigma-Aldrich (Buchs SG, Switzerland). The Staclot VIIa-rTF kit was from Diagnostica Stago (Asnieres, France). The monoclonal mouse anti-human CD36 antibody (clone 185-1G2) was from LifeSpan BioSciences (Seattle, WA). The monoclonal mouse anti-human CD42b antibody (clone SZ2) was from Immunotech. For the inhibitory strategies, we used Fab fragments of the chimeric anti-GIIbIIIa MoAb (clone 7E3, ReoPro, Abciximab) from Lilly (Madrid, Spain). The recombinant human activated protein C (rhAPC, activated Drotrecogin α) was from Lilly Pharma (Giessen, Germany). Annexin V was prepared as described previously.24Vanheerde W.L. Sakariassen K.S. Hemker H.C. Sixma J.J. Reutelingsperger C.P. de Groot P.G. Annexin v inhibits the procoagulant activity of matrices of TNF- stimulated endothelium under blood flow conditions.Arterioscler Thromb. 1994; 14: 824-830Crossref PubMed Scopus (41) Google Scholar Cytochalasin B, tyrphostin-47, and wortmannin were from Sigma-Aldrich (Steinheim, Germany). Fibrillar collagen type I was from Chrono-Log (Havertown, PA). The embedding kit JB-4 was from Polyscience (Warrington, PA). The present study was designed to evaluate the possible redistribution of rFVIIa into platelets. Platelet-rich plasma samples from normal donors or from four patients diagnosed with FVII deficiency (one with mild deficiency, 57% FVII, and three with severe deficiency, <2% FVII) and one patient with Bernard-Soulier syndrome were exposed for 2 hours to rFVIIa (0, 3, 6, 12, 30, and 60 μg/mL, final concentration). Presence of intraplatelet rFVIIa and membrane rFVIIa was evaluated using flow cytometry and immunolabeling techniques. The amount of FVIIa in platelets was determined with a coagulometry assay specifically designed to evaluate only the activated form of FVII (FVIIa). The ability of platelets to expose previously internalized rFVIIa was measured using flow cytometry after platelet activation with thrombin. Inhibitory strategies were introduced in several experiments, to determine the possible mechanisms through which FVIIa could be internalized into platelets. Some of these focused on the involvement of major glycoproteins, such as GPIIbIIIa and GPIb (the latter has been suggested as a possible receptor for FVIIa25Weeterings C. de Groot P.G. Adelmeijer J. Lisman T. The glycoprotein Ib-IX-V complex contributes to tissue factor-independent thrombin generation by recombinant factor VIIa on the activated platelet surface.Blood. 2008; 112: 3227-3233Crossref PubMed Scopus (63) Google Scholar). Moreover, the role of the scavenger receptor GPIV (CD36) was evaluated. Other strategies were aimed at investigating the binding of FVIIa to anionic phospholipids or the involvement of the cytoskeleton arrangement. The involvement of a protein C receptor was also assessed, because it was suggested in endothelial cells.26Ghosh S. Pendurthi U.R. Steinoe A. Esmon C.T. Rao L.V. Endothelial cell protein C receptor acts as a cellular receptor for factor VIIa on endothelium.J Biol Chem. 2007; 282: 11849-11857Crossref PubMed Scopus (126) Google Scholar, 27López-Sagaseta J. Montes R. Puy C. Diez N. Fukudome K. Hermida J. Binding of factor VIIa to the endothelial cell protein C receptor reduces its coagulant activity.J Thromb Haemost. 2007; 5: 1817-1824Crossref PubMed Scopus (78) Google Scholar, 28Gopalakrishnan R. Hedner U. Ghosh S. Nayak R.C. Allen T.C. Pendurthi U.R. Rao L.V. Bio-distribution of pharmacologically administered recombinant factor VIIa (rFVIIa).J Thromb Haemost. 2010; 8: 301-310Crossref PubMed Scopus (58) Google Scholar Finally, a combination of wortmannin and tyrphostin-47,29Diaz-Ricart M. Brunso L. Pino M. Navalon F. Jou J.M. Heras M. White J.G. Escolar G. Preanalytical treatment of EDTA-anticoagulated blood to ensure stabilization of the mean platelet volume and component measured with the ADVIA counters.Thromb Res. 2010; 126: e30-e35Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar was used to page the main intracellular signaling pathways involved in the internalization and traffic of particles independently of the mechanism of binding to the membrane. The preservation of the hemostatic activity of intraplatelet FVIIa was tested using flow devices. The effects on thrombus formation and fibrin generation were assessed. Moreover, the effect of intraplatelet rFVIIa on viscoelastometric properties of the forming clot was evaluated using the ROTEM thromboelastometry analyzer (Pentapharm, Munich, Germany). In some experimental procedures the use of a fluorescent-labeled rFVIIa was required. Labeling was performed with the Alexa Fluor 488 (AF488) microscale protein labeling kit according to the manufacturer's instructions. The final concentration was determined with a NanoDrop ND-1000 spectrophotometer (Isogen, IJsselstein, The Netherlands). Blood samples were collected from healthy donors (n = 10) who had not taken any drug known to affect platelet function in the previous 10 days. Blood samples were anticoagulated with citrate/phosphate/dextrose (CPD) at a final citrate concentration of 19 mmol/L. Platelet-rich plasma was obtained by centrifugation at 120 × g for 15 minutes. Platelet-rich plasma aliquots were incubated with PBS, unlabeled rFVIIa (3, 6, 12, and 60 μg/mL, final concentration), or with the same concentrations of AF488-rFVIIa, for 2 hours at 37°C, in agreement with the calculated half-life of this drug. Afterward, platelet aliquots were fixed (0.3% paraformaldehyde) and washed three times with PBS and processed for flow cytometry. Fixed platelets were incubated with a CD41a-PerCP antibody to detect the platelet population through the glycoprotein GPIIbIIIa. Interaction of AF488-rFVIIa with platelets was evaluated directly by flow cytometry. To assess whether unlabeled rFVIIa interacting with platelets was located at the platelet membrane or inside platelets, we applied a permeabilization procedure to arrive at the intraplatelet antigen. The method of permeabilization used has proved to be feasible and is widely used for this purpose.30Sedlmayr P. Grosshaupt B. Muntean W. Flow cytometric detection of intracellular platelet antigens.Cytometry. 1996; 23: 284-289Crossref PubMed Scopus (20) Google Scholar, 31Tenedini E. Fagioli M.E. Vianelli N. Tazzari P.L. Ricci F. Tagliafico E. Ricci P. Gugliotta L. Martinelli G. Tura S. Baccarani M. Ferrari S. Catani L. Gene expression profiling of normal and malignant CD34-derived megakaryocytic cells.Blood. 2004; 104: 3126-3135Crossref PubMed Scopus (67) Google Scholar Labeling of FVIIa was performed in parallel for surface and intraplatelet rFVIIa, according to the instructions provided in the IntraPrep kit. To determine membrane rFVIIa, solution 2 (saponin) from the kit was replaced by PBS. Samples were incubated with 14 μg/mL of primary rabbit anti-human FVII/VIIa antibody for 30 minutes at room temperature. Because the anti-FVII/VIIa primary antibody was not fluorescent, a second step of incubation was performed with a secondary goat anti-rabbit IgG FITC-conjugated, in dark conditions (30 minutes at room temperature). This second incubation was performed with PBS for the membrane antigen or solution 2 (saponin), because saponin induces a reversible permeabilization, to reach the intracellular antigen. The final pellet was resuspended in paraformaldehyde (1%) and was kept at room temperature in darkness until analysis by flow cytometry. Platelets were analyzed by flow cytometry using a FACScan flow cytometer (BD Biosciences, Mountain View, CA) at an excitation wavelength of 488 nm. Labeled platelets were analyzed using dual-color labeling as described previously.32Lozano M. Estebanell E. Cid J. Diaz-Ricart M. Mazzara R. Ordinas A. Escolar G. Platelet concentrates prepared and stored under currently optimal conditions: minor impact on platelet adhesive and cohesive functions after storage.Transfusion. 1999; 39: 951-959Crossref PubMed Scopus (34) Google Scholar Platelets were differentiated by their characteristic forward versus side scatter. Histograms were generated with fluorescence data obtained in the logarithmic mode from 10,000 events analyzed in each sample. Data were expressed as the mean of fluorescence measured per platelet or as percentage of positive platelets. For the latter, an analytical marker was set in the corresponding fluorescence channel to define 2% of the resting platelet population with the highest membrane fluorescence at baseline level. This marker was used as a threshold to determine the proportion of platelets exhibiting immunofluorescence above this level in all subsequent samples. For other experimental techniques, such as immunolocalization, quantification and perfusion studies, after the incubation with PBS or rFVIIa for 2 hours at 37°C, platelet-rich plasma was washed three times with equal volumes of citrate-citric acid-dextrose (93 mmol/L sodium citrate, 7 mmol/L citric acid, and 140 mmol/L dextrose, pH 6.5, containing 5 mmol/L adenosine and 3 mmol/L theophylline) to eliminate the rFVIIa present in the solution and to obtain a platelet suspension containing only the rFVIIa internalized by platelets. The final platelet pellet was resuspended in Hanks balanced salt solution supplemented with dextrose and NaHCO3 (pH 7.2), and rested for 45 minutes at 37°C before use. Platelet suspensions were chemically fixed at 4°C with a mixture of 2% paraformaldehyde and 0.1% glutaraldehyde in PBS buffer. After a washing with PBS containing 50 mmol/L glycine, cells were embedded in 12% gelatin and were infused in 2.3 mol/L sucrose. Mounted gelatin pages were frozen in liquid nitrogen. Thin sections were prepared in an ultracryomicrotome (Leica EM Ultracut UC6/FC6; Leica, Vienna, Austria). Ultrathin cryosections were collected with 2% methylcellulose in 2.3 mol/L sucrose. Cryosections were incubated at room temperature on drops of 2% gelatin in PBS for 20 minutes at 37°C, followed by 20 mmol/L glycine in PBS during 15 minutes, and 1% BSA in PBS during 15 minutes. Subsequently, the sections were incubated with anti-FVIIa (20 μg/mL) in 1% BSA in PBS for 60 minutes. After three washes with drops of 0.1% BSA in PBS for 10 minutes, sections were incubated for 20 minutes using protein A coupled to colloidal gold (10 nm in diameter), using a 1:40 dilution with 1% BSA. This was followed by three washes with drops of PBS for 10 minutes and two washes with distilled water. As a control for nonspecific binding of the colloidal gold-conjugated antibody, the primary polyclonal antibody was omitted. Observations were performed using a transmission electron microscope (Tecnai Spirit; FEI, Eindhoven, The Netherlands) with a charge-coupled device camera SIS Megaview III. Quantification of FVIIa was performed according to the instructions provided in the Staclot VIIa-rTF kit (Diagnostica Stago, Asnieres, France). The Staclot is a clotting assay designed to quantify FVIIa. The test is based on a recombinant soluble tissue factor (rsTF) that possesses a cofactor function specific for FVIIa. The rsTF binds only to FVIIa, and does not activate FVII into FVIIa. We applied this assay using as sample not plasma aliquots but platelet lysates obtained through three rapid cycles of freeze/thaw of washed platelet suspensions (1.2 × 106 platelets/μL) containing or not containing rFVIIa. Washed platelet suspensions were obtained from control platelet-rich plasma aliquots incubated for 2 hours with different concentrations of rFVIIa (0, 3, 6, 12, 30, and 60 μg/mL). To evaluate the ability of platelets previously incubated with rFVIIa to expose FVIIa on their membrane after activation with a physiological agonist: 225 μL of washed platelets previously incubated with rFVIIa were activated with 0.1 U/mL final concentration of thrombin at 37°C for 0, 1, 5, 10, and 15 minutes. Presence of FVIIa on activated platelets was evaluated using flow cytometry techniques, as described above. Concomitant exposure of CD62-P (P-Selectin), an activation-dependent marker of platelets, was also evaluated at the same time intervals. To determine the potential mechanisms through which rFVIIa is internalized, some inhibitory strategies were performed in the flow cytometry models. Some of them focused on the involvement of major glycoproteins, such as GPIb and GPIIbIIIa. For this purpose, washed platelets, at 0.3 × 106 platelets/μL, were incubated with antibodies against the glycoproteins [ie, the clone SZ2 (20 μg/mL), which binds to GPIb, or abciximab (20 μg/mL), which binds to GPIIbIIIa]. The involvement of the scavenger receptor GPIV (also known as CD36) was also evaluated, using an antibody (20 μg/mL) against this glycoprotein. To investigate the possible binding of FVIIa to anionic phospholipids or the involvement of the cytoskeleton arrangement, platelets were incubated, respectively, with annexin V at 250 nmol/L in the presence of 2.5 mmol/L ionic calcium, or with cytochalasin B (25 μmol/L). Moreover, because it has been described26Ghosh S. Pendurthi U.R. Steinoe A. Esmon C.T. Rao L.V. Endothelial cell protein C receptor acts as a cellular receptor for factor VIIa on endothelium.J Biol Chem. 2007; 282: 11849-11857Crossref PubMed Scopus (126) Google Scholar, 27López-Sagaseta J. Montes R. Puy C. Diez N. Fukudome K. Hermida J. Binding of factor VIIa to the endothelial cell protein C receptor reduces its coagulant activity.J Thromb Haemost. 2007; 5: 1817-1824Crossref PubMed Scopus (78) Google Scholar, 28Gopalakrishnan R. Hedner U. Ghosh S. Nayak R.C. Allen T.C. Pendurthi U.R. Rao L.V. Bio-distribution of pharmacologically administered recombinant factor VIIa (rFVIIa).J Thromb Haemost. 2010; 8: 301-310Crossref PubMed Scopus (58) Google Scholar that FVIIa interacts with the endothelial cells through the endothelial protein C receptor (EPRC) and that platelets could interact with surfaces exposing activated protein C (APC), despite the lack of evidence of a receptor for APC in platelets, we performed a previous incubation using recombinant human APC (rhAPC) at 50 nmol/L to page this possible receptor before exposure to rFVIIa. Finally, a combination of wortmannin and tyrphostin-4729Diaz-Ricart M. Brunso L. Pino M. Navalon F. Jou J.M. Heras M. White J.G. Escolar G. Preanalytical treatment of EDTA-anticoagulated blood to ensure stabilization of the mean platelet volume and component measured with the ADVIA counters.Thromb Res. 2010; 126: e30-e35Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar was used to page the main intracellular signaling pathways involved in the internalization and traffic of particles, independently of the mechanism of binding to the membrane. Tyrosine phosphorylation was prevented with tyrphostin-47 (100 μmol/L). Endo- and exocytosis processes were pageed using wortmannin (11.6 μmol/L). The combination of both inhibitors is known to preserve platelet stability during blood manipulation.29Diaz-Ricart M. Brunso L. Pino M. Navalon F. Jou J.M. Heras M. White J.G. Escolar G. Preanalytical treatment of EDTA-anticoagulated blood to ensure stabilization of the mean platelet volume and component measured with the ADVIA counters.Thromb Res. 2010; 126: e30-e35Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar In all these inhibitory strategies, platelets were incubated with the inhibitors for 30 minutes at 37°C. After incubation with cytochalasin B, platelets were washed. To determine the mechanisms involved in FVIIa internalization, inhibitory strategies were tested using two different approaches: in the presence of a supratherapeutic rFVIIa dose (30 μg/mL) and in the presence of a therapeutic dose equivalent to 6 μg/mL. The aim of this strategy was to evaluate the effect of different concentrations on the process of internalization. In the experiments with unlabeled FVIIa, platelet samples were processed after incubation for flow cytometry analysis as described above, including permeabilization to measure the intraplatelet antigen, whereas in the experiments with AF488-rFVIIa the samples were fixed in 0.3% paraformaldehyde, labeled with CD41a-PerCP, and directly analyzed. Results were expressed as mean fluorescence ± SEM (n = 3) or as percentage of inhibition. Adhesive and cohesive properties of platelets containing rFVIIa were assessed in perfusion models. Aliquots of blood samples anticoagulated with citrate (19 mmol/L) and with low molecular weight heparin (20 U/mL) were obtained from the same donor. Platelet-rich plasma from citrated bloo