Vox Sanguinis (2015) 108, 328–339 © 2014 International Society of Blood Transfusion DOI: 10.1111/vox.12231
ORIGINAL PAPER
Riboflavin and amotosalen photochemical treatments of platelet concentrates reduce thrombus formation kinetics in vitro B. Van Aelst,1 H. B. Feys,1 R. Devloo,1 K. Vanhoorelbeke,2 P. Vandekerckhove3,4 & V. Compernolle1,3,5 1
Transfusion Research Center, Belgian Red Cross-Flanders, Ghent, Belgium Laboratory for Thrombosis Research, KU Leuven Kulak, Kortrijk, Belgium 3 Blood Service of the Belgian Red Cross-Flanders, Mechelen, Belgium 4 Department of Public Health and Primary Care, Catholic University of Leuven, Leuven, Belgium 5 Faculty of Medicine and Health Sciences, University of Ghent, Ghent, Belgium 2
Background Photochemical treatment (PCT) of platelet concentrates using photosensitizers and ultraviolet light illumination reduces the proliferation potential of pathogens by damaging biomolecules. Materials and Methods The impact of riboflavin (RF-PRT)- and amotosalen (ASPCT)-based pathogen inactivation on platelets was studied using microfluidic flow chambers on immobilized collagen using standard platelet concentrates prepared from buffy coats in additive solution. Flow cytometry, metabolic parameters and light transmission aggregometry with thrombin-related peptide, collagen and ristocetin were determined concurrently. Results Both PCTs significantly decreased the platelet surface coverage kinetics in flow chambers over the course of the 7-day study. Platelet aggregation was affected following RF-PRT in response to all agonists, while AS-PCT mainly impacted low-dose ristocetin agglutination. RF-PRT induces premature platelet activation because integrin aIIbb3 was spontaneously activated, and a-degranulation, phosphatidylserine/-ethanolamine exposure and anaerobic metabolism significantly increased following treatment, which was not the case for AS-PCT. On the other hand, AS-PCT significantly diminished thrombus growth onto von Willebrand factor under shear flow. This defect was caused by fewer integrin aIIbb3 interactions, not by defective GPIba-VWF binding as shown by adhesion experiments in the presence of tirofiban. Moreover, integrin aIIbb3 activation was also affected following the activation of platelets via GPVI-FccRIIa or PAR1. Finally, amotosalen illumination as such is sufficient to induce platelet damage, with no additional measurable effect of the chemical adsorption step. Gamma irradiation caused no significant difference compared to controls on any timepoint or for any parameter. Received: 26 June 2014, revised 12 November 2014, accepted 12 November 2014, published online 30 December 2014
Conclusion Both PCTs significantly reduce thrombus formation rate but by different biochemical mechanisms. Key words: blood components, pathogen inactivation, platelet concentrates, platelet function.
Introduction Correspondence: Hendrik B. Feys, Transfusion Research Center, Ottergemsesteenweg 413, 9000 Ghent, Belgium E-mail: [email protected]
328
Transfusion-transmitted infections are a considerable threat to global blood supplies. Because the optimal conditions for safeguarding platelet integrity include storage
Photochemically treated platelets in flow chambers 329
at room temperature, platelet concentrates are in addition particularly vulnerable to bacterial contamination. Photochemical treatment [1] (PCT) efficiently tackles this problem by chemically damaging bacteria, viruses and protozoans. Three pathogen inactivation methods have been developed for platelet concentrates, one that uses exclusively ultraviolet (UV) C light [2] and two employing an exogenously added photosensitizer with ultraviolet light illumination; riboflavin (vitamin B2) pathogen reduction technology (RF-PRT, TerumoBCT, Lakewood, CO, USA) uses broad spectrum UV light [3], and amotosalen (S59) photochemical treatment (AS-PCT, Cerus Corp, Concord, CA, USA) uses UVA [4]. Damage to nucleic acids is a major driver of pathogen kill following photoexcitation of many chemical sensitizers used for blood applications. Psoralens like amotosalen intercalate in helical regions of RNA and DNA and irreversibly form adducts and cross-links with pyrimidines following photoexcitation. For RF-PRT, the pathogen reduction mechanism may be less confined to the sensitizer (dark)-binding site [5] but also includes nucleic acid damage [6]. Biostatic methodologies that specifically harm nucleic acids will evidently prevent targeted cells/ viruses from replicating, but this process by definition cannot be affected in cells without the intrinsic potential to replicate at all, like platelets. Importantly, though, this principally theoretical concept drives the notion that both PCTs are highly specific for damaging solely pathogens, which is not necessarily true. For instance, platelets do contain helical nucleic acid in the form of mitochondrial DNA and remnant RNA from their megakaryocytic precursor. Furthermore, all photosensitizers produce reactive oxygen species (ROS), and these are by no means specific in their chemistry with the surrounding milieu. Moreover, photosensitizers not only partition to nucleic acids [7], but also to other biomolecules where reaction can occur. For both PCTs, the past decade has produced extensive in vitro data on platelet storage lesion (PSL) markers such as pH and glycolytic metabolites, platelet receptor expression and/or hypotonic shock response. However, less attention has gone to functional assays like platelet aggregation [8–11] or (even less) thrombus formation in microfluidic flow chambers under controlled conditions [12]. Notwithstanding this void, the latter is important as it is the most comprehensive functional test with high sensitivity to perturbations in all steps of hemostasis [13]. A recent effort of the Biorheology subgroup of the Scientific and Standardization Committee of the International Society on Thrombosis Haemostasis has demonstrated the importance of comprehensive platelet function testing in systems with well-known hydrodynamic shear profiles supplemented with real-time analysis of platelet deposition [14]. The technique is used for screening © 2014 International Society of Blood Transfusion Vox Sanguinis (2015) 108, 328–339
patients in (pre)clinical settings [15] and has been proven valuable in drug development as well, for example to assess platelet functional inhibition [16, 17]. We have set up such an in vitro model of hemostasis with reconstituted blood perfused through microfluidic chambers combined with real-time measurement of thrombus formation on immobilized collagen and von Willebrand factor (VWF). Our data show that in vitro thrombus formation kinetics is affected immediately following both RF-PRT and AS-PCT, but that their main causative biomolecular alterations are different.
Materials and methods Study design The two studies for RF-PRT and AS-PCT have been conducted separately, but with comparable design. Within each study, the samples were paired, but not between studies. For both RF-PRT and AS-PCT, leucocyte-depleted platelet concentrates were prepared by pooling of five and six buffy coats, respectively. In all cases, the buffy coats were derived from voluntary non-remunerated whole-blood donations after giving written informed consent. For RF-PRT, three platelet concentrates in additive solution (SSP+, Macopharma, Tourcoing, FR) were pooled, gently mixed and then split into three equivalent products (n = 6). One product remained untreated (control), another was RF-PRT treated following a 1-h resting period, and the last one was treated with 25–50 Gray of gamma irradiation. In this particular study, gamma-treated concentrates were included alongside untreated controls to include products that follow the current routine production. For RF-PRT, 35 ml of riboflavin (RF) (500 lM in saline) was added followed by expelling air bubbles and illumination with a dose of ultraviolet light (265– 370 nm) optimized by the provider for treating products in additive solution [8]. The RF-PRT concentrates were protected from ambient light, and all concentrates were kept in the same type of storage container (TerumoBCT). For AS-PCT, two platelet concentrates in additive solution (SSP+) were pooled, gently mixed and split into two equivalent products (n = 6). One product remained untreated (control), and the other was treated with AS-PCT according to the guidelines of the manufacturer. In brief, the product was transferred to an illumination bag while 175 ml amotosalen (final ~150 lM) was added, followed by expelling excess air bubbles and UVA illumination (320–400 nm). Following overnight incubation (
ORIGINAL PAPER
Riboflavin and amotosalen photochemical treatments of platelet concentrates reduce thrombus formation kinetics in vitro B. Van Aelst,1 H. B. Feys,1 R. Devloo,1 K. Vanhoorelbeke,2 P. Vandekerckhove3,4 & V. Compernolle1,3,5 1
Transfusion Research Center, Belgian Red Cross-Flanders, Ghent, Belgium Laboratory for Thrombosis Research, KU Leuven Kulak, Kortrijk, Belgium 3 Blood Service of the Belgian Red Cross-Flanders, Mechelen, Belgium 4 Department of Public Health and Primary Care, Catholic University of Leuven, Leuven, Belgium 5 Faculty of Medicine and Health Sciences, University of Ghent, Ghent, Belgium 2
Background Photochemical treatment (PCT) of platelet concentrates using photosensitizers and ultraviolet light illumination reduces the proliferation potential of pathogens by damaging biomolecules. Materials and Methods The impact of riboflavin (RF-PRT)- and amotosalen (ASPCT)-based pathogen inactivation on platelets was studied using microfluidic flow chambers on immobilized collagen using standard platelet concentrates prepared from buffy coats in additive solution. Flow cytometry, metabolic parameters and light transmission aggregometry with thrombin-related peptide, collagen and ristocetin were determined concurrently. Results Both PCTs significantly decreased the platelet surface coverage kinetics in flow chambers over the course of the 7-day study. Platelet aggregation was affected following RF-PRT in response to all agonists, while AS-PCT mainly impacted low-dose ristocetin agglutination. RF-PRT induces premature platelet activation because integrin aIIbb3 was spontaneously activated, and a-degranulation, phosphatidylserine/-ethanolamine exposure and anaerobic metabolism significantly increased following treatment, which was not the case for AS-PCT. On the other hand, AS-PCT significantly diminished thrombus growth onto von Willebrand factor under shear flow. This defect was caused by fewer integrin aIIbb3 interactions, not by defective GPIba-VWF binding as shown by adhesion experiments in the presence of tirofiban. Moreover, integrin aIIbb3 activation was also affected following the activation of platelets via GPVI-FccRIIa or PAR1. Finally, amotosalen illumination as such is sufficient to induce platelet damage, with no additional measurable effect of the chemical adsorption step. Gamma irradiation caused no significant difference compared to controls on any timepoint or for any parameter. Received: 26 June 2014, revised 12 November 2014, accepted 12 November 2014, published online 30 December 2014
Conclusion Both PCTs significantly reduce thrombus formation rate but by different biochemical mechanisms. Key words: blood components, pathogen inactivation, platelet concentrates, platelet function.
Introduction Correspondence: Hendrik B. Feys, Transfusion Research Center, Ottergemsesteenweg 413, 9000 Ghent, Belgium E-mail: [email protected]
328
Transfusion-transmitted infections are a considerable threat to global blood supplies. Because the optimal conditions for safeguarding platelet integrity include storage
Photochemically treated platelets in flow chambers 329
at room temperature, platelet concentrates are in addition particularly vulnerable to bacterial contamination. Photochemical treatment [1] (PCT) efficiently tackles this problem by chemically damaging bacteria, viruses and protozoans. Three pathogen inactivation methods have been developed for platelet concentrates, one that uses exclusively ultraviolet (UV) C light [2] and two employing an exogenously added photosensitizer with ultraviolet light illumination; riboflavin (vitamin B2) pathogen reduction technology (RF-PRT, TerumoBCT, Lakewood, CO, USA) uses broad spectrum UV light [3], and amotosalen (S59) photochemical treatment (AS-PCT, Cerus Corp, Concord, CA, USA) uses UVA [4]. Damage to nucleic acids is a major driver of pathogen kill following photoexcitation of many chemical sensitizers used for blood applications. Psoralens like amotosalen intercalate in helical regions of RNA and DNA and irreversibly form adducts and cross-links with pyrimidines following photoexcitation. For RF-PRT, the pathogen reduction mechanism may be less confined to the sensitizer (dark)-binding site [5] but also includes nucleic acid damage [6]. Biostatic methodologies that specifically harm nucleic acids will evidently prevent targeted cells/ viruses from replicating, but this process by definition cannot be affected in cells without the intrinsic potential to replicate at all, like platelets. Importantly, though, this principally theoretical concept drives the notion that both PCTs are highly specific for damaging solely pathogens, which is not necessarily true. For instance, platelets do contain helical nucleic acid in the form of mitochondrial DNA and remnant RNA from their megakaryocytic precursor. Furthermore, all photosensitizers produce reactive oxygen species (ROS), and these are by no means specific in their chemistry with the surrounding milieu. Moreover, photosensitizers not only partition to nucleic acids [7], but also to other biomolecules where reaction can occur. For both PCTs, the past decade has produced extensive in vitro data on platelet storage lesion (PSL) markers such as pH and glycolytic metabolites, platelet receptor expression and/or hypotonic shock response. However, less attention has gone to functional assays like platelet aggregation [8–11] or (even less) thrombus formation in microfluidic flow chambers under controlled conditions [12]. Notwithstanding this void, the latter is important as it is the most comprehensive functional test with high sensitivity to perturbations in all steps of hemostasis [13]. A recent effort of the Biorheology subgroup of the Scientific and Standardization Committee of the International Society on Thrombosis Haemostasis has demonstrated the importance of comprehensive platelet function testing in systems with well-known hydrodynamic shear profiles supplemented with real-time analysis of platelet deposition [14]. The technique is used for screening © 2014 International Society of Blood Transfusion Vox Sanguinis (2015) 108, 328–339
patients in (pre)clinical settings [15] and has been proven valuable in drug development as well, for example to assess platelet functional inhibition [16, 17]. We have set up such an in vitro model of hemostasis with reconstituted blood perfused through microfluidic chambers combined with real-time measurement of thrombus formation on immobilized collagen and von Willebrand factor (VWF). Our data show that in vitro thrombus formation kinetics is affected immediately following both RF-PRT and AS-PCT, but that their main causative biomolecular alterations are different.
Materials and methods Study design The two studies for RF-PRT and AS-PCT have been conducted separately, but with comparable design. Within each study, the samples were paired, but not between studies. For both RF-PRT and AS-PCT, leucocyte-depleted platelet concentrates were prepared by pooling of five and six buffy coats, respectively. In all cases, the buffy coats were derived from voluntary non-remunerated whole-blood donations after giving written informed consent. For RF-PRT, three platelet concentrates in additive solution (SSP+, Macopharma, Tourcoing, FR) were pooled, gently mixed and then split into three equivalent products (n = 6). One product remained untreated (control), another was RF-PRT treated following a 1-h resting period, and the last one was treated with 25–50 Gray of gamma irradiation. In this particular study, gamma-treated concentrates were included alongside untreated controls to include products that follow the current routine production. For RF-PRT, 35 ml of riboflavin (RF) (500 lM in saline) was added followed by expelling air bubbles and illumination with a dose of ultraviolet light (265– 370 nm) optimized by the provider for treating products in additive solution [8]. The RF-PRT concentrates were protected from ambient light, and all concentrates were kept in the same type of storage container (TerumoBCT). For AS-PCT, two platelet concentrates in additive solution (SSP+) were pooled, gently mixed and split into two equivalent products (n = 6). One product remained untreated (control), and the other was treated with AS-PCT according to the guidelines of the manufacturer. In brief, the product was transferred to an illumination bag while 175 ml amotosalen (final ~150 lM) was added, followed by expelling excess air bubbles and UVA illumination (320–400 nm). Following overnight incubation (
