Retroviral Infection and Selection of Culture-Derived Platelets Allows Study of the Effect of Transgenes on Platelet Physiology Ex Vivo and
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动脉硬化血栓血管生物学 2005年第8期
From the ProCorde (A.G., M.P., G.M., M.U.), Martinsried, Germany; Deutsches Herzzentrum und 1. Medizinische Klinik, Technische Universit?t München (K.L.L., S.M., I.K.), Germany; and Medizinische Klinik Innere Medizin III, Universit?t Tübingen (M.G.), Germany.
Correspondence to PD Dr M. Ungerer, Procorde GmbH, Fraunhoferstrasse 9, 82152 Martinsried, Germany. E-mail ungerer@procorde.com; or Prof Meinrad Gawaz, Medizinsche Klinik Innere Medizin III, Universit?t Tübingen, Germany. E-mail meinrad.gawaz@med.uni-tuebingen.de
Abstract
Background— We recently reported the development of culture-derived (CD) platelets with the aim to express any protein of interest in these platelets.1 We now report a specific protocol of retroviral infection into the progenitor cells and subsequent selection, which allows to generate large amounts of highly homogenous transgene-expressing CD platelets and to study transgene function rapidly and reliably at large-scale ex vivo and in vivo settings.
Methods and Results— After retroviral infection and selection, the activation-dependent expression profile of surface markers, aggregation, and granule release were investigated. The function of transgene-expressing CD platelets, the precursor cells of which had been retrovirally infected, compared well to noninfected CD platelets or freshly isolated platelets. Hence, the retroviral infection protocol did not alter platelet physiology. In contrast, adenoviral infection of precursors to CD platelets resulted in marked functional alterations that obviated their use in analytic experiments. Additionally, sufficient amounts of selected CD platelets were generated to warrant intravenous injections into living mice. This approach permitted study of their adhesive profile at endothelial lesions and their effect on thrombus formation in vivo by intravital videofluorescence microscopy.
Conclusion— The novel selection method allowed us to produce recombinant transgene-expressing platelets in sufficient amounts to study genetically modified platelets in vitro and in vivo.
A novel selection method allowed us to produce recombinant transgene-expressing culture-derived platelets in sufficient amounts to study genetically modified platelets in vitro and in vivo. These platelets maintain the physiological characteristics of freshly isolated platelets.
Key Words: platelets ? gene transfer ? pharmacology
Introduction
Considerable interest has focused on the identification and characterization of pivotal signaling proteins in platelets because they play a central role in a variety of vascular diseases. However, the study of cytosolic platelet proteins has been hampered by the fact that these proteins cannot be accessed easily. One approach consisted in the permeabilization of platelets and the subsequent administration of antibodies to inactivate various proteins, as discussed.1 Unfortunately, this permeabilization process leads to a severe disturbance of platelet physiology. Alternatively, transgenic mice were created by germline transmission, which constitutively overexpressed recombinant platelets. However, this approach is very time consuming and not feasible for the large set of platelet proteins, the overexpression or knockout of which causes lethal phenotypes.
We recently established a method to reliably generate large amounts of culture-derived (CD) platelets in vitro from megakaryocyte progenitor cells.1 However, so far, recombinant transgene-expressing platelets could not be produced homogenously in sufficient amounts for large-scale in vivo and in vivo analysis.
To further improve this system and to express any protein of interest after gene transfer into the precursors to CD platelets, we compared different gene transduction protocols and established a novel system of efficient selection of recombinant CD platelets. Previous studies investigated the possibility to transduce megakaryocytes by retroviral gene transfer.2,3 Alternatively, adenoviral gene transfer was assessed.4 However, none of these studies had established a protocol for cellular selection to purify transgene-harboring platelets to near homogeneity. At optimal conditions, the maximum percentages of transgene-expressing platelets had been reported to range between 26% and 61%,2 or rather be 19%,5 after retroviral infection and to reach 46% to 48% after adenoviral infection.4 Such inhomogeneous populations of megakaryocytes or CD platelets are inadequate for many functional in vitro and in vivo studies.
The present study investigates whether a high amount of functional recombinant CD platelets can be generated after gene transfer into the precursor cells in vitro and whether they can be selected to near homogeneity. Moreover, we evaluated various virus-based infection methods and gene transfer protocols. Subsequently, we tested the effect of overexpression of typical marker genes, such as green fluorescent protein (GFP), on various functional aspects of CD platelets.
Methods
A detailed description of all methods can be seen in the online supplement, available at http://atvb.ahajournals.org.
Human and mouse platelets were isolated as described.6,7 Also, human peripheral blood mononuclear cells and murine bone marrow cells were obtained, cultured, and differentiated to megakaryocytes as described.1
Recombinant (E1/E3-deficient) adenoviruses were generated as described.8 Retroviruses were cloned using the plasmid pLEGFP-C1 (obtained from Clontech). Transfection to obtain full retrovirus was performed by calcium phosphate coprecipitation,9 as available by Clontech with chloroquine (50 mmol/L; Sigma).10 The collected culture supernatants were concentrated by ultracentrifugation.11 These samples were used for direct infection.
Freshly prepared platelets or human or murine CD platelets were investigated by flow cytometry and aggregometry, as described.12 Serotonin release measurements were performed as described.1 ELISA assays for serotonin (DLD Diagnostics) were performed according to manufacturer instructions.
CD platelets were also investigated by intravital microscopy, as described.7 Single adhering platelets could be well distinguished from larger platelet aggregates and from thrombi by investigating their size and microscopic profile. Thrombus area at the site of vascular injury was quantified in squared micrometers, as described.7,13
Results
Adenoviral and Retroviral Infection of Megakaryocyte Precursors
Human CD34+ cells were isolated from peripheral blood and subjected to adenoviral infection. Infecting the human CD34+ cells with a recombinant adenovirus coding for GFP resulted in robust expression of the transgene, which was also maintained in the platelets shed from these megakaryocytes (see images in Figure I, available online at http://atvb.ahajournals.org). To test the infection efficacy, GFP expression in transduced CD34+ cells was determined by flow cytometry. As described previously,1 we found that transgene expression occurred 48 hours after infection (Figure IA) and persisted for >5 weeks. At a titer of 400 /cell, 40% of CD platelets were positive for GFP fluorescence and could be harvested continuously during that period. Lower titers (200 plaque-forming units [pfu]/cell) resulted in considerably reduced efficacy and could therefore not be used for further studies. In contrast, mouse precursor cells could not be infected by the available adenoviral vectors because of their human species specificity and selectivity.
Mouse precursor cells were isolated from bone marrow and cultured under cytokine stimulation as described.1 They were then infected with recombinant retroviruses. Initial studies tested transgene expression by studying GFP expression (Figure IB). Figure IC shows that the transgene-expressing megakaryocytes shed recombinant platelets continuously. The best conditions for gene transfer were identified by testing various virus titers and infection times. Figure IIA (available online at http://atvb.ahajournals.org) shows that optimum infection time was 2 days after isolation. Therefore, the infection with purified virus and the coculture with producer cells was initiated 2 days after isolation of precursor cells in all further experiments. The maximal effect was achieved at a multiplicity of infection (MOI) of 2 to 5 cfu/cell (Figure IIB). Additionally, several chemical reagents were compared for their efficacy to enhance retroviral gene transfer. A combination of Polybrene and DEAE dextrane at a concentration of 8 μg/mL and 1 mg/mL, respectively, turned out to be the most effective adjuvant (Figure IIC). In contrast, lipofectamin (at a concentration of 10 μg/mL), fibronectin (5 μg/mL), or Polybrene or DEAE alone were clearly less effective (Figure IIC).
Retroviral infection was performed with vectors encoding the neomycin resistance gene. After testing various concentrations of geneticin (G418), a continuous supplementation of the culture medium at a concentration of 380 μg/mL turned out to be the best selection method and was used in all further experiments. Figure IID illustrates the homogenous population of recombinant platelets expressing the resistance gene (on the left), whereas the selection pressure of G418 almost eliminates noninfected cells (on the right). The counting of megakaryocytes 3 weeks after isolation clearly reconfirmed that cell number was reduced markedly in the presence of geneticin, whereas it was maintained at equal levels after retroviral infection (Figure IID, inset). Immunoblotting verified the presence of the transgene (Figure IIE). Serial assessment during in vitro culture demonstrated the increasing enrichment of transgene-expressing megakaryocytes reaching near homogeneity after 3 weeks (Figure IIF). This homogeneity was superior to 90% in all experiments.
The same homogeneities were reproduced when human isolated CD34+ cells were infected with recombinant retrovirus. Similar to mouse megakaryocytes, human transgene-expressing megakaryocytes were selected by constantly adding 380 μg/μL of G418 to the culture medium. The selection mode and time course of human megakaryocytes was very comparable to that of murine ones.
Adenoviral Infection of Precursor Cells Alters the Activation Patterns of the Derived CD Platelets
In a first set of experiments, we tested the functional effect of adenoviral infection of human CD34+ cells at a titer of 400 pfu/cell on the resulting CD platelets. Typical platelet surface markers such as CD41, CD61, PAC-1 (epitopes on GPIIb/IIIa), CD62P (P-selectin), CD63 (which indicates secretion from lysosomal vesicles), or CD-40 ligand (CD40-L) could still be detected in CD platelets, which derived from adenovirally infected precursors. Whereas CD platelets that derived from noninfected precursors showed adequate responses to several agonists with regard to fibrinogen receptor activation, externalization of activation markers and -degranulation, adenoviral infection of progenitor cells, instead, blunted these activation patterns in the derived CD platelets. As an example, Figure 1 shows the lack of activation of CD62P and PAC-1 in response to the thrombin analogue, thrombin receptor-activator peptide (TRAP), or to ADP. The increases were statistically significant for CD platelets derived from noninfected precursors versus adenovirally infected precursors (P<0.05; as determined by Mann–Whitney testing for nonparametric samples). Similar results were obtained with other release markers such as CD63 and CD40-L and after stimulation with other soluble agonists (data not shown). Lack of response did not depend on the adenoviral titers used because higher or lower titers (range 200 to 1000 pfu/cell) led to similar results.
Figure 1. Human CD platelets that derived from adenovirally infected precursors. FACS measurements of recombinant human CD platelets derived from adenovirally infected or noninfected precursors. Agonist-dependent surface expression of the fibrinogen receptor marker PAC-1 (A) and of the secretion marker P-selectin (CD62P; B). The figure shows the results from 8 independent experiments done with CD platelets from 8 independent isolations of human megakaryocytes (relative increases [%] of mean fluorescence values over basal values with SEM).
CD Platelets Derived From Retrovirus-Infected Precursors
Having found that adenovirus-mediated infection of CD34+ cells does not allow study of the effect of transgenes on the function of the derived CD platelets, we analyzed the effect of retroviral infection of the precursors on the function of CD platelets. Retroviral infection of the precursors did not alter the expression profile of fibrinogen receptor markers, nor -degranulation on the derived CD platelets compared with noninfected CD platelets or freshly isolated platelets. In contrast to CD platelets that derived from adenovirally infected cells, agonist-dependent surface recruitment of the markers CD62P and PAC-1 tended to be increased with freshly isolated as with CD platelets, the precursors of which had been retrovirally infected (Figure III, available online at http://atvb.ahajournals.org), although these trends did not reach statistical significance versus basal levels (as determined by Mann–Whitney testing). However, testing of CD platelets derived from noninfected precursors versus retrovirally infected precursors also did not result in any statistical differences. The same was observed with other markers recruited to the surface on agonist stimulation such as CD40-L and CD63 (data not shown).
Similar findings were obtained for the stimulation of murine CD platelets, the precursors of which had been infected retrovirally (Figure 2). Stimulation of these CD platelets with collagen resulted in enhanced surface expression of CD 61, CD 40-L, and CD62P in a dose-dependent manner (Figure 2). The increases were statistically significant (P<0.05) versus basal values, but CD platelets derived from noninfected precursors did not differ from those derived from retrovirally infected precursors, as determined by Mann–Whitney testing. Also, stimulation with other agonists such as ristocetin or thrombin resulted in similar activation profiles of the same surface markers (data not shown). All activation profiles also compared well to freshly isolated platelets measured under the same conditions.1 This data set indicated that mouse CD platelets, the precursors of which were retrovirally infected, also retained their full functional properties. Because of the scarcity of human CD34+ cells, a complete functional characterization had to be performed with mouse CD platelets. Therefore, we decided to focus further measurements on retrovirally infected murine CD platelets.
Figure 2. Murine CD platelets that derived from retrovirally infected precursors. FACS measurements of recombinant, murine CD platelets derived from retrovirally infected or noninfected precursors. Collagen-dependent surface expression of the fibrinogen receptor markers CD40-L (A) and CD61 (B), and of the secretion marker P-selectin (CD62P; C). The figure shows the results from 15 independent experiments done with CD platelets from 5 independent isolations of mouse megakaryocytes (relative increases [%] of mean fluorescence values over basal values with SEM).
Next, we investigated whether the aggregation profile of CD platelets, the precursors of which had been infected retrovirally, was comparable to that of noninfected platelets. In response to thrombin (1.5 U/mL) and ADP (5 μmol/L), a typical time course of aggregation was observed with GFP-expressing CD platelets derived from retrovirally infected precursors (Figure 3A). This signal corresponded well to the transmission signal obtained with noninfected CD platelets (Figure 3A) or freshly isolated washed platelets.1 Similar results were obtained with ristocetin (1.6 mg/mL). Figure 3B shows the mean maximal aggregation of CD platelets, the precursors of which had been infected retrovirally, in response to thrombin. The results compared well to those obtained with noninfected CD platelets or freshly isolated platelets.1
Figure 3. Aggregation of murine CD platelets which derived from retrovirally infected precursors. A, Original recordings of representative aggregometric experiments of murine CD platelets derived from retrovirally infected precursors (on the left) in response to agonists ADP (5 μmol/L) and thrombin (1.5 U/mL). For comparison, the corresponding curves of noninfected CD platelets are shown on the right. B, Mean maximum aggregation of murine CD platelets derived from retrovirally infected or noninfected precursors in response to 2 different concentrations of thrombin (results from 5 independent experiments with SEM). The indicated % values refer to the initial calibration of the aggregometric device with platelet suspension or buffer. C, Agonist-dependent release of serotonin from murine CD platelets derived from retrovirally infected or noninfected precursors, as determined by ELISA (results from 3 experiments with SDs). In these experiments, each well contained 107 cells.
Also, secretion of vasoactive agents from CD platelets, the precursors of which had been retrovirally infected, compared well to that from noninfected CD platelets or freshly isolated platelets. Figure 3C shows that stimulation with thrombin clearly induced the release of serotonin from CD platelets, the precursors of which had been retrovirally infected, well comparable to that from noninfected platelets.
Adhesion and Thrombus Formation In Vivo
The adhesion of recombinant platelets to endothelial lesions was investigated in mice in vivo by intravital videofluorescence microscopy. CD platelets, of which the precursors had been infected retrovirally, were injected into either healthy mice or animals that had been subjected to a carotid injury by external ligation of the carotid artery and assessed for adhesive properties and thrombus formation at arterial endothelial lesions in vivo.
Figure 4 shows that firm adhesion (A) or thrombus formation (B) was virtually absent in noninjured carotid arteries, both when freshly isolated platelets or transgene-expressing CD platelets derived from retrovirally infected precursors were analyzed. In contrast, adhesion of CD platelets, the precursors of which precursors had been retrovirally infected and their incorporation into the newly formed thrombus, occurred at injury sites of the carotid artery. The top panels of Figure 4C show single adhering platelets. The bottom right panel of Figure 4C shows the image of a typical thrombus in the left carotid artery of a mouse in vivo composed of transgene-expressing CD platelets.
Figure 4. Intravital microscopy detects platelet adhesion and thrombus formation at endothelial lesions in vivo. After injection of freshly isolated platelets ("freshly isolated")13 or GFP-expressing CD platelets derived from retrovirally infected precursors ("CD platelets"), average platelet adhesion at the vessel wall (A) and thrombus formation (B) was determined in the carotid arteries of mice that had been subjected to an arterial lesion and compared with the results in uninjured mice ("no lesion"). Results of 7 (A) and 4 (B) independent animals with SEMs (C). Top panels, Single adhering platelets in the carotid artery of mice in vivo (freshly isolated platelets on the left; CD platelets derived from retrovirally infected precursors on the right). Bottom panels, Image of a typical thrombus in the carotid artery of a mouse in vivo composed of transgene-expressing CD platelets in response to local endothelial injury (right), whereas no thrombus formation is observed in uninjured arteries (left panel). The borders of the blood vessels have been delineated with dashed lines in all panels of C. Arrows indicate the direction of flow.
Discussion
In the present study, we show that a highly homogenous pool of recombinant functional platelets can be generated in vitro by infecting CD34+ progenitor cells at specific conditions (concerning retroviral titers, infection time, and others) and by applying a continuous selection pressure based on a coinfected neomycin resistance gene. These resulting recombinant CD platelets are characterized by similar functional features as CD platelets derived from noninfected precursors or freshly isolated platelets. Retroviral gene transfer into the CD34+ progenitor cells caused robust expression of green fluorescent protein (GFP) in both mouse and human CD platelets. The expression of GFP as a marker protein by retroviral gene transfer did not have an effect on the physiological platelet functions.
After having established a novel protocol for the preparation of CD platelets,1 we sought to identify the best viral transduction system for the creation of recombinant, transgene-expressing CD platelets, which offered the highest efficacy and least side effects. Adenoviral4,14 and retroviral2,3,5,15 transduction of megakaryocytes had been described previously, and positive results on the feasibility of both approaches had been published. However, the functional consequences of using either viral system and the best transduction protocols had not been investigated in detail previously. No direct comparison of the different approaches had been performed previously.
Studies on Adenoviral Infection
Efficiency of adenoviral gene transfer had been estimated previously at up to 45% at an MOI of 200 pfu/cell,4 which is largely corroborated by our present data. In the same study,4 a single functional investigation of recombinant CD platelets had consisted in the microscopic analysis of the activation marker PAC-1, which was based on an in situ immunostaining for PAC-1 and subsequent X-gal staining for Lac-Z expression (transgene detection). This experiment resulted in the identification of single isolated recombinant PAC-1–binding megakaryocytes after previous incubation with the thrombin receptor agonist TRAP.4 When studying PAC-1 activation in a large pool of GFP-expressing CD platelets that had been adenovirally infected at the same titer, we did not detect any difference of GFP-expressing platelets (gated by fluorescence-activated cell sorter [FACS]) in terms of their PAC-1 signal in presence or absence of TRAP (Figure 1). Therefore, in our eyes, it seems unlikely that a physiological PAC-1 activation of adenovirally infected CD platelets occurs. The relative discrepancy of findings might be caused by functional differences between megakaryocytes and platelets or by an overestimation of effects when investigating single cells in the absence of an equally formed control group. Accordingly, we did not detect any other signs of agonist-dependent activability in a broad data set on adenovirally infected CD platelets, using several agonists and activation markers.
Studies on Retroviral Infection
Wilcox et al had described previously the efficiency of retroviral infection of human CD34+ cells and megakaryocytes as being 19% when the transgene was driven by a cytomegalovirus (CMV) promotor5 (ie, a maximum of 19% of transduced stem cells expressed the transgene) and 34% after retroviral infection with a human -IIb promotor3 (however, incurring a low expression level).
Similarly, Castellino et al reported an average 34% transduction efficacy.15 Using a coculture approach for retroviral infection, Burstein et al described a range of 41% to 82% transgene-expressing megakaryocytes and 26% to 59% transgene-expressing CD platelets.2 A similar range was reported in a recent technical overview article.16 With coculture and infection of purified virus, the maximum infectivity was 30% in our hands. In contrast to these data and to the above-reported efficiencies on retroviral infection, we were able to markedly enhance efficiency and reproducibility by combining retroviral gene transfer with a selection pressure and reliably reached efficacies of >90% (Figure IIF).
Functional measurements of retrovirally infected megakaryocytes had studied the surface recruitment of CD62P by phorbol 12-myostate 13-acetate (PMA) on the derived transgene-expressing CD platelets,2 as determined by FACS, and had also found signs of activability. We now reconfirm this finding on a larger database. In the literature, retroviral infection of precursor cells had been described either by coculture of the retrovirus-producing cells with isolated megakaryocytes2,16,17 or after previous virus isolation, purification, and subsequent infection of the isolated cells at defined titers.18 We were able to optimize both approaches by standardizing the conditions. For example, early initiation of a continuous coculture (starting 1 to 2 days after isolation) turned out to be superior to an initiation after 5 days and a coculture for the 3 subsequent days, as described.2 Although the coculture approach generally allows for the easier generation of large amounts of CD platelets, it incurs the disadvantage that the CD platelets are harder to purify. Therefore, infection of purified virus and coculture are feasible and should be used according to the specific requirements of each experiment.
Therefore, the direct comparison of CD platelets derived from retrovirally infected precursors and adenovirally infected precursors showed several advantages of the retroviral selection approach:
Adenoviral infection led to a fairly profound alteration of the surface recruitment of typical markers in response to classical agonists. This finding occurred across a large range of infectious titers.
In contrast, retroviral infection and selection of the precursors did not result in changes of the agonist-dependent expression of surface markers on the derived transgene-expressing CD platelets compared with noninfected CD platelets or freshly isolated platelets.
Additionally, we found acceptable aggregation characteristics of recombinant CD platelets derived from retrovirally infected precursors, as determined by light-transmission aggregometry. Submaximal and maximal concentrations of the typical agonists ADP and thrombin resulted in aggregation levels that were very comparable to those of noninfected CD platelets or freshly isolated platelets.
Also, the agonist-dependent release of vasoactive agents (such as serotonin) from the dense granules of transgene-expressing CD platelets derived from retrovirally infected precursors corresponded well to that of CD platelets derived from noninfected precursors or freshly isolated platelets. These findings corroborate the typical agonist-dependent externalization of markers that indicate granule secretion from -granules, such as CD 62P (Figure III; Figure 2). These measurements show that functional storage granules exist in CD platelets and that they are physiologically regulated.
Additionally, the maximum infection efficacy to be obtained after adenoviral infection was 40%, whereas it approached 100% after retroviral infection and selection.
To define the role of specific platelet target proteins for platelet adhesion to the vascular wall under physiological and pathophysiological conditions in vivo, recombinant platelets were analyzed in a model of vascular injury of the mouse common carotid artery. This approach allowed to study their effects in cerebral vessels, which play a pivotal role in the pathophysiology of stroke. It was clearly seen that transgene-expressing recombinant platelets that derived from retrovirally infected precursors showed a similar in vivo adhesion profile to endothelial lesions as freshly isolated platelets. Also, the thrombus formation of CD platelets at the local site of injury was very comparable to that obtained with freshly isolated platelets (Figure 4).
Another possible improvement of the system would be to use megakaryocyte-specific viral promotors instead of the currently used CMV promotor. However, this approach did not seem urgently necessary because almost all isolated stem cells differentiate to CD41-expressing megakaryocytes during progressive culture by virtue of the described cytokine cocktail. Additionally, CD platelets can be size-gated for all FACS experiments and sufficiently washed for all other investigations.
In summary, we established a novel technology to study the functional relevance of any protein in CD platelets. We were able to produce recombinant transgene-expressing platelets in sufficient amounts to study the most relevant parameters that determine platelet function in cardiovascular physiology and disease.
Acknowledgments
This work was supported by a grant from the Bavarian Research Foundation (Bayr. Forschungsstiftung), No. 446/01.
References
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Burstein SA, Dubart A, Norol F, Debili N, Friese P, Downs T, Yu X, Kincade PW, Villeval JL, Vainchenker W. Expression of a foreign protein in human megakaryocytes and platelets by retrovirally mediated gene transfer. Exp Hematol. 1999; 27: 110–116.
Wilcox DA, Olsen JC, Ishizawa L, Bray PF, French DL, Steeber DA, Bell WR, Griffith M, White GC III. Megakaryocyte-targeted synthesis of the integrin ?3-subunit results in the phenotypic correction of Glanzmann thrombasthenia. Blood. 2000; 95: 3645–3652.
Faraday N, Rade JJ, Johns DC, Khetawat G, Noga SJ, DiPersio JF, Jin Y, Nichol JL, Haug JS, Bray PF. Ex vivo cultured megakaryocytes express functional glycoprotein IIb-IIIa receptors and are capable of adenovirus-mediated transgene pression. Blood. 1999; 94: 4084–4092.
Wilcox DA, Olsen JC, Ishizawa L, Griffith M, White GC II. Integrin alpha IIb promotor-targeted expression of gene products in megakaryocytes derived from retrovirus-transduced human hematopoietic cells. Proc Natl Acad Sci U S A. 1999; 96: 9654–9659.
Gawaz M, Neumann FJ, Dickfeld T, Reininger A, Adelsberger H, Gebhardt A, Sch?mig A. Vitronectin receptor (alpha(v)beta3) mediates platelet adhesion to the luminal aspect of endothelial cells: implications for reperfusion in acute myocardial infarction. Circulation. 1997; 96: 1809–1818.
Massberg S, Brand K, Grüner S, Page S, Muller E, Müller I, Bergmeier W, Richter T, Lorenz M, Konrad I, Nieswandt B, Gawaz M. A critical role of platelet adhesion in the initiation of atherosclerotic lesion formation. J Exp Med. 2002; 196: 887–896.
He TC, Zhou S, da Costa LT, Yu J, Kinzler KW, Vogelstein B. A simplified system for generating recombinant adenoviruses. Proc Natl Acad Sci U S A. 1998; 95: 2509–2514.
Graham FL, and van der Eb AJ. Transformation of rat cells by DNA of human adenovirus 5. Virology. 1973; 54: 536–539.
Warren S, Nolan GP, Scott ML, Baltimore D. Production of high-titer helper-free retroviruses by transient transfection. Proc Natl Acad Sci U S A. 1993; 90: 8392–8396.
Burns JC. Vesicular stomatitis virus G glycoprotein pseudotyped retroviral vectors: concentration to very high titer and efficient gene transfer into mammalian and nonmammalian cells. Proc Natl Acad Sci U S A. 1993; 90: 8033–8037.
Gawaz M, Neumann FJ, Sch?mig A. Evaluation of platelet membrane glycoproteins in coronary artery disease:consequences for diagnosis and therapy. Circulation. 1999; 99: E1–E11.
Massberg S, Konrad I, Bültmann A, Schulz C, Münch G, Peluso M, Lorenz M, Schneider S, Besta F, Müller I, Hu B, Langer H, Kremmer E, Rudelius M, Heinzmann U, Ungerer M, Gawaz M. Soluble glycoprotein VI dimer inhibits platelet adhesion and aggregation to the injured vessel wall in vivo. FASEB J. 2004; 18: 397–399.
Shayakhmetov DM, Papayannopoulou T, Stamatoyannopoulos G, Lieber A. Efficient gene transfer to human CD34+ cells by a retargeted adenovirus vector. J Virol. 2000; 2567–2583.
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Correspondence to PD Dr M. Ungerer, Procorde GmbH, Fraunhoferstrasse 9, 82152 Martinsried, Germany. E-mail ungerer@procorde.com; or Prof Meinrad Gawaz, Medizinsche Klinik Innere Medizin III, Universit?t Tübingen, Germany. E-mail meinrad.gawaz@med.uni-tuebingen.de
Abstract
Background— We recently reported the development of culture-derived (CD) platelets with the aim to express any protein of interest in these platelets.1 We now report a specific protocol of retroviral infection into the progenitor cells and subsequent selection, which allows to generate large amounts of highly homogenous transgene-expressing CD platelets and to study transgene function rapidly and reliably at large-scale ex vivo and in vivo settings.
Methods and Results— After retroviral infection and selection, the activation-dependent expression profile of surface markers, aggregation, and granule release were investigated. The function of transgene-expressing CD platelets, the precursor cells of which had been retrovirally infected, compared well to noninfected CD platelets or freshly isolated platelets. Hence, the retroviral infection protocol did not alter platelet physiology. In contrast, adenoviral infection of precursors to CD platelets resulted in marked functional alterations that obviated their use in analytic experiments. Additionally, sufficient amounts of selected CD platelets were generated to warrant intravenous injections into living mice. This approach permitted study of their adhesive profile at endothelial lesions and their effect on thrombus formation in vivo by intravital videofluorescence microscopy.
Conclusion— The novel selection method allowed us to produce recombinant transgene-expressing platelets in sufficient amounts to study genetically modified platelets in vitro and in vivo.
A novel selection method allowed us to produce recombinant transgene-expressing culture-derived platelets in sufficient amounts to study genetically modified platelets in vitro and in vivo. These platelets maintain the physiological characteristics of freshly isolated platelets.
Key Words: platelets ? gene transfer ? pharmacology
Introduction
Considerable interest has focused on the identification and characterization of pivotal signaling proteins in platelets because they play a central role in a variety of vascular diseases. However, the study of cytosolic platelet proteins has been hampered by the fact that these proteins cannot be accessed easily. One approach consisted in the permeabilization of platelets and the subsequent administration of antibodies to inactivate various proteins, as discussed.1 Unfortunately, this permeabilization process leads to a severe disturbance of platelet physiology. Alternatively, transgenic mice were created by germline transmission, which constitutively overexpressed recombinant platelets. However, this approach is very time consuming and not feasible for the large set of platelet proteins, the overexpression or knockout of which causes lethal phenotypes.
We recently established a method to reliably generate large amounts of culture-derived (CD) platelets in vitro from megakaryocyte progenitor cells.1 However, so far, recombinant transgene-expressing platelets could not be produced homogenously in sufficient amounts for large-scale in vivo and in vivo analysis.
To further improve this system and to express any protein of interest after gene transfer into the precursors to CD platelets, we compared different gene transduction protocols and established a novel system of efficient selection of recombinant CD platelets. Previous studies investigated the possibility to transduce megakaryocytes by retroviral gene transfer.2,3 Alternatively, adenoviral gene transfer was assessed.4 However, none of these studies had established a protocol for cellular selection to purify transgene-harboring platelets to near homogeneity. At optimal conditions, the maximum percentages of transgene-expressing platelets had been reported to range between 26% and 61%,2 or rather be 19%,5 after retroviral infection and to reach 46% to 48% after adenoviral infection.4 Such inhomogeneous populations of megakaryocytes or CD platelets are inadequate for many functional in vitro and in vivo studies.
The present study investigates whether a high amount of functional recombinant CD platelets can be generated after gene transfer into the precursor cells in vitro and whether they can be selected to near homogeneity. Moreover, we evaluated various virus-based infection methods and gene transfer protocols. Subsequently, we tested the effect of overexpression of typical marker genes, such as green fluorescent protein (GFP), on various functional aspects of CD platelets.
Methods
A detailed description of all methods can be seen in the online supplement, available at http://atvb.ahajournals.org.
Human and mouse platelets were isolated as described.6,7 Also, human peripheral blood mononuclear cells and murine bone marrow cells were obtained, cultured, and differentiated to megakaryocytes as described.1
Recombinant (E1/E3-deficient) adenoviruses were generated as described.8 Retroviruses were cloned using the plasmid pLEGFP-C1 (obtained from Clontech). Transfection to obtain full retrovirus was performed by calcium phosphate coprecipitation,9 as available by Clontech with chloroquine (50 mmol/L; Sigma).10 The collected culture supernatants were concentrated by ultracentrifugation.11 These samples were used for direct infection.
Freshly prepared platelets or human or murine CD platelets were investigated by flow cytometry and aggregometry, as described.12 Serotonin release measurements were performed as described.1 ELISA assays for serotonin (DLD Diagnostics) were performed according to manufacturer instructions.
CD platelets were also investigated by intravital microscopy, as described.7 Single adhering platelets could be well distinguished from larger platelet aggregates and from thrombi by investigating their size and microscopic profile. Thrombus area at the site of vascular injury was quantified in squared micrometers, as described.7,13
Results
Adenoviral and Retroviral Infection of Megakaryocyte Precursors
Human CD34+ cells were isolated from peripheral blood and subjected to adenoviral infection. Infecting the human CD34+ cells with a recombinant adenovirus coding for GFP resulted in robust expression of the transgene, which was also maintained in the platelets shed from these megakaryocytes (see images in Figure I, available online at http://atvb.ahajournals.org). To test the infection efficacy, GFP expression in transduced CD34+ cells was determined by flow cytometry. As described previously,1 we found that transgene expression occurred 48 hours after infection (Figure IA) and persisted for >5 weeks. At a titer of 400 /cell, 40% of CD platelets were positive for GFP fluorescence and could be harvested continuously during that period. Lower titers (200 plaque-forming units [pfu]/cell) resulted in considerably reduced efficacy and could therefore not be used for further studies. In contrast, mouse precursor cells could not be infected by the available adenoviral vectors because of their human species specificity and selectivity.
Mouse precursor cells were isolated from bone marrow and cultured under cytokine stimulation as described.1 They were then infected with recombinant retroviruses. Initial studies tested transgene expression by studying GFP expression (Figure IB). Figure IC shows that the transgene-expressing megakaryocytes shed recombinant platelets continuously. The best conditions for gene transfer were identified by testing various virus titers and infection times. Figure IIA (available online at http://atvb.ahajournals.org) shows that optimum infection time was 2 days after isolation. Therefore, the infection with purified virus and the coculture with producer cells was initiated 2 days after isolation of precursor cells in all further experiments. The maximal effect was achieved at a multiplicity of infection (MOI) of 2 to 5 cfu/cell (Figure IIB). Additionally, several chemical reagents were compared for their efficacy to enhance retroviral gene transfer. A combination of Polybrene and DEAE dextrane at a concentration of 8 μg/mL and 1 mg/mL, respectively, turned out to be the most effective adjuvant (Figure IIC). In contrast, lipofectamin (at a concentration of 10 μg/mL), fibronectin (5 μg/mL), or Polybrene or DEAE alone were clearly less effective (Figure IIC).
Retroviral infection was performed with vectors encoding the neomycin resistance gene. After testing various concentrations of geneticin (G418), a continuous supplementation of the culture medium at a concentration of 380 μg/mL turned out to be the best selection method and was used in all further experiments. Figure IID illustrates the homogenous population of recombinant platelets expressing the resistance gene (on the left), whereas the selection pressure of G418 almost eliminates noninfected cells (on the right). The counting of megakaryocytes 3 weeks after isolation clearly reconfirmed that cell number was reduced markedly in the presence of geneticin, whereas it was maintained at equal levels after retroviral infection (Figure IID, inset). Immunoblotting verified the presence of the transgene (Figure IIE). Serial assessment during in vitro culture demonstrated the increasing enrichment of transgene-expressing megakaryocytes reaching near homogeneity after 3 weeks (Figure IIF). This homogeneity was superior to 90% in all experiments.
The same homogeneities were reproduced when human isolated CD34+ cells were infected with recombinant retrovirus. Similar to mouse megakaryocytes, human transgene-expressing megakaryocytes were selected by constantly adding 380 μg/μL of G418 to the culture medium. The selection mode and time course of human megakaryocytes was very comparable to that of murine ones.
Adenoviral Infection of Precursor Cells Alters the Activation Patterns of the Derived CD Platelets
In a first set of experiments, we tested the functional effect of adenoviral infection of human CD34+ cells at a titer of 400 pfu/cell on the resulting CD platelets. Typical platelet surface markers such as CD41, CD61, PAC-1 (epitopes on GPIIb/IIIa), CD62P (P-selectin), CD63 (which indicates secretion from lysosomal vesicles), or CD-40 ligand (CD40-L) could still be detected in CD platelets, which derived from adenovirally infected precursors. Whereas CD platelets that derived from noninfected precursors showed adequate responses to several agonists with regard to fibrinogen receptor activation, externalization of activation markers and -degranulation, adenoviral infection of progenitor cells, instead, blunted these activation patterns in the derived CD platelets. As an example, Figure 1 shows the lack of activation of CD62P and PAC-1 in response to the thrombin analogue, thrombin receptor-activator peptide (TRAP), or to ADP. The increases were statistically significant for CD platelets derived from noninfected precursors versus adenovirally infected precursors (P<0.05; as determined by Mann–Whitney testing for nonparametric samples). Similar results were obtained with other release markers such as CD63 and CD40-L and after stimulation with other soluble agonists (data not shown). Lack of response did not depend on the adenoviral titers used because higher or lower titers (range 200 to 1000 pfu/cell) led to similar results.
Figure 1. Human CD platelets that derived from adenovirally infected precursors. FACS measurements of recombinant human CD platelets derived from adenovirally infected or noninfected precursors. Agonist-dependent surface expression of the fibrinogen receptor marker PAC-1 (A) and of the secretion marker P-selectin (CD62P; B). The figure shows the results from 8 independent experiments done with CD platelets from 8 independent isolations of human megakaryocytes (relative increases [%] of mean fluorescence values over basal values with SEM).
CD Platelets Derived From Retrovirus-Infected Precursors
Having found that adenovirus-mediated infection of CD34+ cells does not allow study of the effect of transgenes on the function of the derived CD platelets, we analyzed the effect of retroviral infection of the precursors on the function of CD platelets. Retroviral infection of the precursors did not alter the expression profile of fibrinogen receptor markers, nor -degranulation on the derived CD platelets compared with noninfected CD platelets or freshly isolated platelets. In contrast to CD platelets that derived from adenovirally infected cells, agonist-dependent surface recruitment of the markers CD62P and PAC-1 tended to be increased with freshly isolated as with CD platelets, the precursors of which had been retrovirally infected (Figure III, available online at http://atvb.ahajournals.org), although these trends did not reach statistical significance versus basal levels (as determined by Mann–Whitney testing). However, testing of CD platelets derived from noninfected precursors versus retrovirally infected precursors also did not result in any statistical differences. The same was observed with other markers recruited to the surface on agonist stimulation such as CD40-L and CD63 (data not shown).
Similar findings were obtained for the stimulation of murine CD platelets, the precursors of which had been infected retrovirally (Figure 2). Stimulation of these CD platelets with collagen resulted in enhanced surface expression of CD 61, CD 40-L, and CD62P in a dose-dependent manner (Figure 2). The increases were statistically significant (P<0.05) versus basal values, but CD platelets derived from noninfected precursors did not differ from those derived from retrovirally infected precursors, as determined by Mann–Whitney testing. Also, stimulation with other agonists such as ristocetin or thrombin resulted in similar activation profiles of the same surface markers (data not shown). All activation profiles also compared well to freshly isolated platelets measured under the same conditions.1 This data set indicated that mouse CD platelets, the precursors of which were retrovirally infected, also retained their full functional properties. Because of the scarcity of human CD34+ cells, a complete functional characterization had to be performed with mouse CD platelets. Therefore, we decided to focus further measurements on retrovirally infected murine CD platelets.
Figure 2. Murine CD platelets that derived from retrovirally infected precursors. FACS measurements of recombinant, murine CD platelets derived from retrovirally infected or noninfected precursors. Collagen-dependent surface expression of the fibrinogen receptor markers CD40-L (A) and CD61 (B), and of the secretion marker P-selectin (CD62P; C). The figure shows the results from 15 independent experiments done with CD platelets from 5 independent isolations of mouse megakaryocytes (relative increases [%] of mean fluorescence values over basal values with SEM).
Next, we investigated whether the aggregation profile of CD platelets, the precursors of which had been infected retrovirally, was comparable to that of noninfected platelets. In response to thrombin (1.5 U/mL) and ADP (5 μmol/L), a typical time course of aggregation was observed with GFP-expressing CD platelets derived from retrovirally infected precursors (Figure 3A). This signal corresponded well to the transmission signal obtained with noninfected CD platelets (Figure 3A) or freshly isolated washed platelets.1 Similar results were obtained with ristocetin (1.6 mg/mL). Figure 3B shows the mean maximal aggregation of CD platelets, the precursors of which had been infected retrovirally, in response to thrombin. The results compared well to those obtained with noninfected CD platelets or freshly isolated platelets.1
Figure 3. Aggregation of murine CD platelets which derived from retrovirally infected precursors. A, Original recordings of representative aggregometric experiments of murine CD platelets derived from retrovirally infected precursors (on the left) in response to agonists ADP (5 μmol/L) and thrombin (1.5 U/mL). For comparison, the corresponding curves of noninfected CD platelets are shown on the right. B, Mean maximum aggregation of murine CD platelets derived from retrovirally infected or noninfected precursors in response to 2 different concentrations of thrombin (results from 5 independent experiments with SEM). The indicated % values refer to the initial calibration of the aggregometric device with platelet suspension or buffer. C, Agonist-dependent release of serotonin from murine CD platelets derived from retrovirally infected or noninfected precursors, as determined by ELISA (results from 3 experiments with SDs). In these experiments, each well contained 107 cells.
Also, secretion of vasoactive agents from CD platelets, the precursors of which had been retrovirally infected, compared well to that from noninfected CD platelets or freshly isolated platelets. Figure 3C shows that stimulation with thrombin clearly induced the release of serotonin from CD platelets, the precursors of which had been retrovirally infected, well comparable to that from noninfected platelets.
Adhesion and Thrombus Formation In Vivo
The adhesion of recombinant platelets to endothelial lesions was investigated in mice in vivo by intravital videofluorescence microscopy. CD platelets, of which the precursors had been infected retrovirally, were injected into either healthy mice or animals that had been subjected to a carotid injury by external ligation of the carotid artery and assessed for adhesive properties and thrombus formation at arterial endothelial lesions in vivo.
Figure 4 shows that firm adhesion (A) or thrombus formation (B) was virtually absent in noninjured carotid arteries, both when freshly isolated platelets or transgene-expressing CD platelets derived from retrovirally infected precursors were analyzed. In contrast, adhesion of CD platelets, the precursors of which precursors had been retrovirally infected and their incorporation into the newly formed thrombus, occurred at injury sites of the carotid artery. The top panels of Figure 4C show single adhering platelets. The bottom right panel of Figure 4C shows the image of a typical thrombus in the left carotid artery of a mouse in vivo composed of transgene-expressing CD platelets.
Figure 4. Intravital microscopy detects platelet adhesion and thrombus formation at endothelial lesions in vivo. After injection of freshly isolated platelets ("freshly isolated")13 or GFP-expressing CD platelets derived from retrovirally infected precursors ("CD platelets"), average platelet adhesion at the vessel wall (A) and thrombus formation (B) was determined in the carotid arteries of mice that had been subjected to an arterial lesion and compared with the results in uninjured mice ("no lesion"). Results of 7 (A) and 4 (B) independent animals with SEMs (C). Top panels, Single adhering platelets in the carotid artery of mice in vivo (freshly isolated platelets on the left; CD platelets derived from retrovirally infected precursors on the right). Bottom panels, Image of a typical thrombus in the carotid artery of a mouse in vivo composed of transgene-expressing CD platelets in response to local endothelial injury (right), whereas no thrombus formation is observed in uninjured arteries (left panel). The borders of the blood vessels have been delineated with dashed lines in all panels of C. Arrows indicate the direction of flow.
Discussion
In the present study, we show that a highly homogenous pool of recombinant functional platelets can be generated in vitro by infecting CD34+ progenitor cells at specific conditions (concerning retroviral titers, infection time, and others) and by applying a continuous selection pressure based on a coinfected neomycin resistance gene. These resulting recombinant CD platelets are characterized by similar functional features as CD platelets derived from noninfected precursors or freshly isolated platelets. Retroviral gene transfer into the CD34+ progenitor cells caused robust expression of green fluorescent protein (GFP) in both mouse and human CD platelets. The expression of GFP as a marker protein by retroviral gene transfer did not have an effect on the physiological platelet functions.
After having established a novel protocol for the preparation of CD platelets,1 we sought to identify the best viral transduction system for the creation of recombinant, transgene-expressing CD platelets, which offered the highest efficacy and least side effects. Adenoviral4,14 and retroviral2,3,5,15 transduction of megakaryocytes had been described previously, and positive results on the feasibility of both approaches had been published. However, the functional consequences of using either viral system and the best transduction protocols had not been investigated in detail previously. No direct comparison of the different approaches had been performed previously.
Studies on Adenoviral Infection
Efficiency of adenoviral gene transfer had been estimated previously at up to 45% at an MOI of 200 pfu/cell,4 which is largely corroborated by our present data. In the same study,4 a single functional investigation of recombinant CD platelets had consisted in the microscopic analysis of the activation marker PAC-1, which was based on an in situ immunostaining for PAC-1 and subsequent X-gal staining for Lac-Z expression (transgene detection). This experiment resulted in the identification of single isolated recombinant PAC-1–binding megakaryocytes after previous incubation with the thrombin receptor agonist TRAP.4 When studying PAC-1 activation in a large pool of GFP-expressing CD platelets that had been adenovirally infected at the same titer, we did not detect any difference of GFP-expressing platelets (gated by fluorescence-activated cell sorter [FACS]) in terms of their PAC-1 signal in presence or absence of TRAP (Figure 1). Therefore, in our eyes, it seems unlikely that a physiological PAC-1 activation of adenovirally infected CD platelets occurs. The relative discrepancy of findings might be caused by functional differences between megakaryocytes and platelets or by an overestimation of effects when investigating single cells in the absence of an equally formed control group. Accordingly, we did not detect any other signs of agonist-dependent activability in a broad data set on adenovirally infected CD platelets, using several agonists and activation markers.
Studies on Retroviral Infection
Wilcox et al had described previously the efficiency of retroviral infection of human CD34+ cells and megakaryocytes as being 19% when the transgene was driven by a cytomegalovirus (CMV) promotor5 (ie, a maximum of 19% of transduced stem cells expressed the transgene) and 34% after retroviral infection with a human -IIb promotor3 (however, incurring a low expression level).
Similarly, Castellino et al reported an average 34% transduction efficacy.15 Using a coculture approach for retroviral infection, Burstein et al described a range of 41% to 82% transgene-expressing megakaryocytes and 26% to 59% transgene-expressing CD platelets.2 A similar range was reported in a recent technical overview article.16 With coculture and infection of purified virus, the maximum infectivity was 30% in our hands. In contrast to these data and to the above-reported efficiencies on retroviral infection, we were able to markedly enhance efficiency and reproducibility by combining retroviral gene transfer with a selection pressure and reliably reached efficacies of >90% (Figure IIF).
Functional measurements of retrovirally infected megakaryocytes had studied the surface recruitment of CD62P by phorbol 12-myostate 13-acetate (PMA) on the derived transgene-expressing CD platelets,2 as determined by FACS, and had also found signs of activability. We now reconfirm this finding on a larger database. In the literature, retroviral infection of precursor cells had been described either by coculture of the retrovirus-producing cells with isolated megakaryocytes2,16,17 or after previous virus isolation, purification, and subsequent infection of the isolated cells at defined titers.18 We were able to optimize both approaches by standardizing the conditions. For example, early initiation of a continuous coculture (starting 1 to 2 days after isolation) turned out to be superior to an initiation after 5 days and a coculture for the 3 subsequent days, as described.2 Although the coculture approach generally allows for the easier generation of large amounts of CD platelets, it incurs the disadvantage that the CD platelets are harder to purify. Therefore, infection of purified virus and coculture are feasible and should be used according to the specific requirements of each experiment.
Therefore, the direct comparison of CD platelets derived from retrovirally infected precursors and adenovirally infected precursors showed several advantages of the retroviral selection approach:
Adenoviral infection led to a fairly profound alteration of the surface recruitment of typical markers in response to classical agonists. This finding occurred across a large range of infectious titers.
In contrast, retroviral infection and selection of the precursors did not result in changes of the agonist-dependent expression of surface markers on the derived transgene-expressing CD platelets compared with noninfected CD platelets or freshly isolated platelets.
Additionally, we found acceptable aggregation characteristics of recombinant CD platelets derived from retrovirally infected precursors, as determined by light-transmission aggregometry. Submaximal and maximal concentrations of the typical agonists ADP and thrombin resulted in aggregation levels that were very comparable to those of noninfected CD platelets or freshly isolated platelets.
Also, the agonist-dependent release of vasoactive agents (such as serotonin) from the dense granules of transgene-expressing CD platelets derived from retrovirally infected precursors corresponded well to that of CD platelets derived from noninfected precursors or freshly isolated platelets. These findings corroborate the typical agonist-dependent externalization of markers that indicate granule secretion from -granules, such as CD 62P (Figure III; Figure 2). These measurements show that functional storage granules exist in CD platelets and that they are physiologically regulated.
Additionally, the maximum infection efficacy to be obtained after adenoviral infection was 40%, whereas it approached 100% after retroviral infection and selection.
To define the role of specific platelet target proteins for platelet adhesion to the vascular wall under physiological and pathophysiological conditions in vivo, recombinant platelets were analyzed in a model of vascular injury of the mouse common carotid artery. This approach allowed to study their effects in cerebral vessels, which play a pivotal role in the pathophysiology of stroke. It was clearly seen that transgene-expressing recombinant platelets that derived from retrovirally infected precursors showed a similar in vivo adhesion profile to endothelial lesions as freshly isolated platelets. Also, the thrombus formation of CD platelets at the local site of injury was very comparable to that obtained with freshly isolated platelets (Figure 4).
Another possible improvement of the system would be to use megakaryocyte-specific viral promotors instead of the currently used CMV promotor. However, this approach did not seem urgently necessary because almost all isolated stem cells differentiate to CD41-expressing megakaryocytes during progressive culture by virtue of the described cytokine cocktail. Additionally, CD platelets can be size-gated for all FACS experiments and sufficiently washed for all other investigations.
In summary, we established a novel technology to study the functional relevance of any protein in CD platelets. We were able to produce recombinant transgene-expressing platelets in sufficient amounts to study the most relevant parameters that determine platelet function in cardiovascular physiology and disease.
Acknowledgments
This work was supported by a grant from the Bavarian Research Foundation (Bayr. Forschungsstiftung), No. 446/01.
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