Biomaterials, Artificial Cells and Immobilization Biotechnology
ISSN: 1055-7172 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/ianb18
Effects of X-Linked Hemoglobin oph in-Vitro Platelet Function Robert F. Reiss, Ruben Caballero & John Hess To cite this article: Robert F. Reiss, Ruben Caballero & John Hess (1992) Effects of X-Linked Hemoglobin oph in-Vitro Platelet Function, Biomaterials, Artificial Cells and Immobilization Biotechnology, 20:2-4, 651-655, DOI: 10.3109/10731199209119696 To link to this article: http://dx.doi.org/10.3109/10731199209119696
Published online: 11 Jul 2009.
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Article views: 1
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Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=ianb18 Download by: [New York University]
Date: 17 March 2016, At: 19:32
BIWP., ART. CELLS h ImOB. BIOTECH.,
20(2-4),
651-655 (1992)
EFFECTS OF X-LINKED HEMOOLOBIN ON IN-VITRO PLATELET FUNCTION
Downloaded by [New York University] at 19:32 17 March 2016
Robert F. Reiss, Ruben Caballero, and John Hess The Blood Research Division, Lctterman Army Institute of Research, Presidio of San Francisco, CA, USA and The Transfusion Service, Columbia-Presbyterian Medical Center, New York, NY, USA
ABBLRIICT: The in-vitro functim of platelets in 1:l mixtures of fresh whole blood and 10% DBSLF a-a cross-linked hamglobin in Ringer's acetate buffer was assessed by quantitative aggregatim after challage with c ~ m agonists m and carpard to the f m d i o n of platelets in similar dilutions of whole blood in saline. Whole blood aggregamtry was p e r f o d after addition of ADP, collagen, and ristocctin. Aggregation was quantified by mrasuring the change in inpsdance as drawn m the chart recording. No differcnce in man inpedance change was noted betweem the groups of blood simples which were incubated and tested in the hamglobin solution and those incubated and tested in saline. In addition, incubation and stirring of the above mixtures over a period of six minutes without the addition of agfmists did not result in spontaneous platelet aggregation. We cmclude that O-Q cross-linked -lobin, in the concentratian studid, does not affect in-vitro platelet fmction, as measured by whole blood amregamtry.
IITRODUCTION: Infusion of crude hemolysates into experimental animals is associated with disturbances in normal hemostasis, most commonly disseminated intravascular coagulation,(DIC), and is generally believed to be due to contaminating stroma and stromal phospholipids,(l,2,3,4). More recently, endotoxin and large hemoglobin polymers were also shown to cause similar hemostatic abnormalities,(4,5). Although DIC was noted in dogs infused with purified stroma free preparations,(6), studies in other species utilizing crystallized, stroma free or pyridoxylated polymerized hemoglobins show that extensive transfusion is not associated with changes in the platelet count or coagulation screening tests, other than simple hemodilution,(6,7,8,9). Only one study has examined the effect of these materials on platelet function. It showed that exposure of rat platelets, in platelet rich plasma, to an elevated concentration of stroma free or pyridoxylated polymerized hemoglobin did not alter their aggregation response to increasing quantities of ADP, compared to the same platelet rich plasma mixed with autologous platelet poor plasma,(lO). Although the infusion of relatively small amounts of partially purified, stroma free, and modified hemoglobin solutions in humans has not been associated with bleeding or thrombosis, only limited information is available about their effects on hemostatic mechanisms. In particular, very little has been reported about their effects on platelet function in humans. This study examines the effect of a 10 gm./dl. solution of DBBF (Bis [ 3 , 5 dibromosalicyl] fumarate) Q - a cross-linked hemoglobin solution in Ringer's acetate buffer on invitro human platelet function, in a mixture with whole blood similar to that seen in massive transfusion situations.
651
Copydght 0 1992 by Mucel D e e r , lac.
REISS, CABALLERO, AND HESS
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652
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EFFECTS OF X-LINKED HEMOGLOBIN ON PLATELET FUNCTION
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m: Compodte a m g a l o n patems obwned after &ding nDtocwtin (maconcentratlon of 1.25 mg/mL). to 1:1 mlaunr of lmsh mtated whob blood with both 10% a - a X-linked M O X I ~ Saggmgsbon ~ a~80 seconds is ~ndicatrd Hb. (-), ud n o n d UYM.+) for each mixhln of blood with X-Hb ( 0 ) and sallne (*). Mean aggmgaUon lor each gmuo IS 18ohms. (n-3ineachgmup).
MATERIALS AND METHODS: Whole blood was collected by venapuncture from one of the investigators, (RFR), into 3.8% sodium citrate in a ratio of 9:l on three successive days. This individual’s platelet count was 238,OOO/mm’ and 228.0001 mm3 on days one and two respectively. DBBF a - a cross-linked hemoglobin, (X-Hb), was produced in the Letterman Army Institute of Research pilot plant. The lot utilized in these experiments had a final concentration of 10 gm./dl. in Ringer‘s acetate buffer. Whole blood aggregometry was performed within three hours of preparation of a 1:l mixture of the freshly collected whole blood, (WB), and either X-Hb or normal saline, utilizing a Whole Blood Aggregometer (Model 540, Chronolog Corp., Haverstown, PA). The strip recorder was calibrated so that 60 horizontal lines corresponded to 20 ohms of impedance. Aggregation patterns were recorded following the addition of ADP (Sigma Chemical Co., St. Louis, MO), to a final Concentration of 4uM, ristocetin (Chronolog Corp.), to a final concentration of 1.25 mg/ml, and collagen (Chronolog Corp.) to a final concentration of 5ug/ml. Maximal aggregation was calculated in ohms as the observed difference in impedance at three minutes, 90 seconds, and 15 seconds following the addition of each agonist respectively. Composite aggregation patterns were drawn to be representative of the individual tracings obtained with multiple aliquots of the WB-XHb and WB-saline mixtures following addition of the agonists. The means of the maximal aggregation impedance difference for each group of mixtures, following the reaction times indicated above, were also calculated.
6 54
REISS, CABALLERO, AND HESS
Downloaded by [New York University] at 19:32 17 March 2016
In addition, both the WB-XHb and WB-saline mixtures were incubated and stirred in aggregometer cuvettes for six minutes, without the addition of agonists, to detect spontaneous aggregation. RESULTS: The composite aggregation patterns obtained with multiple aliquots of both WB-XHb and WB-saline were similar after addition of each agonist utilized, (Figures 1, 2, 3). Furthermore, the mean values of maximal aggregation showed no difference between the groups of WB-XHb and WB-saline mixtures following addition of each agonist. The mean differences in impedance observed were as follows: ADP - 17 and 19 ohms at three minutes with the WB-XHb and WB-saline mixtures respectively; collagen - 23 ohms at 75 seconds with both mixtures: and ristocetin -18 ohms at 90 seconds with both mixtures. In addition, no spontaneous aggregation occurred during incubation and stirring of both mixtures for six minutes in aggregometer cuvettes
.
DISCUSSION: Although experimental data suggest that the infusion of stroma free and modified hemoglogin solutions in rats and some other animals is without adverse effects on hemostasis, these conclusions may not be applicable to all species. Infusion of stroma free hemoglobin in dogs was associated with DIC,(6). Furthermore, little experience has been reported with other larger mammals and laboratory primates. In-vivo experience in humans is limited to early safety studies in which relatively small amounts of hemoglobin solutions were administered. In one study, the infusion of an only partially purified hemoglobin solution was not accompanied by clinical or laboratory evidence of DIC,(11). On the other hand, the infusion of a stroma free preparation has been associated with a disproportionate prolongation of the partial thromboplastin time,(l2). In the recent clinical trials of pyridoxylated polymerized hemoglobin, none of the subjects infused with this material demonstrated changes in coagulation screening tests, platelet count, or bleeding time,(l3). Given the incompleteness of our current knowledge, it remains possible that the infusion of larger quantities of these solutions to humans during resuscitation might be associated with significant hemostatic abnormalities other than hemodilution. For this reason, i t is important that their effects on hemostatic mechanisms be more completely understood. In this study, we have demonstrated that human platelets maintain normal whole blood aggregation responses to ADP, collagen, and ristocetin following exposure to concentrations of an a - Q cross-linked hemoglobin solution which would be reached during massive transfusion.
REFERENCES:
1. Rabiner SF, Friedman LH: The role of intravascular hemolysis and the reticuloendothelial system in the production of a hypercoagulable state. Brit J Haematol 14: 105, 1968. 2. Spector J I , Lang E, Crosby WH: Coagulation changes in baboons during acute experimental hemoglobinemia and dextran infusion. Am J Pathol 78: 469, 1975. 3 . Rabiner SF, Helpert JR, Lopas HL, Friedman LH: Evaluation of stroma free hemoglobin as a plasma expander. J Exp Med 126: 1127, 1967. 4 . Feola M, Simoni J , Tran R, Canizaro PC: Mechanism of toxicity of hemoglobin solutions. Biomat Art Cells Art Org 16: 217, 1988. 5 . White CT, Murray AJ, Smith DJ, Qreene JR, Bolin RB: Synergistic toxicity of endotoxin and hemoglobin. J Lab Clin Med 108: 132, 1986. 6. Bolin R, Smith D, Moore G, Boewell a, DeVenuto F: Hematologic effects of hemoglobin solutions in animals, in Bolin RB, Geyer RP,
Downloaded by [New York University] at 19:32 17 March 2016
EFFECTS OF X-LINKED HEMOGLOBIN ON PLATELET FUNCTION
655
Nemo 0 , (ads.): Advances in Blood Substitute Research. New York, Liss, 1983, pg. 117. 7. DeVenuto F, Moores WY, Zegna AI, Zuck TF: Total and partial blood exchange in the rat with hemoglobin prepared by crystallization. Transfusion 17: 555, 1971. 8. Ning J, Chang TMS : Effects of stroma-free hemoglobin and polyhemoglobin on complement activation, blood cells and coagulation factors in rats. Biomat Art Cells Art Org 16: 651, 1988. 9. Ning J, Chang TMS: In vivo effects of stroma-free hemoglobin and polyhemoglobin on coagulation factors in rats. Int J Art Organs 13: 509, 1990. 10.Lalla FR, Ning J, Chang TMS: Effects of pyridoxylated polyhemoglobin and stroma free hemoglobin on ADP induced platelet aggregation. Biomat Art Cells Art Org 17: 363, 1989. 11.Spector J I , Crosby WM: Coagulation studies during experimental hemoglobinemia in humans. J Appl Physiol 38: 195, 1975. 12.Savitsky JP, Doczi J, Black J, Arnold JD: A clinical safety trial of stroma free hemoglobin. Clin Pharmacol Ther 23: 73, 1978. 13.Moss GS, Gould SA, Rosen AL, et al.: Results of the first clinical trial with a polymerized hemoglobin solution (abs). Biomat Art Cells Art Org 17: 633, 1989.
ISSN: 1055-7172 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/ianb18
Effects of X-Linked Hemoglobin oph in-Vitro Platelet Function Robert F. Reiss, Ruben Caballero & John Hess To cite this article: Robert F. Reiss, Ruben Caballero & John Hess (1992) Effects of X-Linked Hemoglobin oph in-Vitro Platelet Function, Biomaterials, Artificial Cells and Immobilization Biotechnology, 20:2-4, 651-655, DOI: 10.3109/10731199209119696 To link to this article: http://dx.doi.org/10.3109/10731199209119696
Published online: 11 Jul 2009.
Submit your article to this journal
Article views: 1
View related articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=ianb18 Download by: [New York University]
Date: 17 March 2016, At: 19:32
BIWP., ART. CELLS h ImOB. BIOTECH.,
20(2-4),
651-655 (1992)
EFFECTS OF X-LINKED HEMOOLOBIN ON IN-VITRO PLATELET FUNCTION
Downloaded by [New York University] at 19:32 17 March 2016
Robert F. Reiss, Ruben Caballero, and John Hess The Blood Research Division, Lctterman Army Institute of Research, Presidio of San Francisco, CA, USA and The Transfusion Service, Columbia-Presbyterian Medical Center, New York, NY, USA
ABBLRIICT: The in-vitro functim of platelets in 1:l mixtures of fresh whole blood and 10% DBSLF a-a cross-linked hamglobin in Ringer's acetate buffer was assessed by quantitative aggregatim after challage with c ~ m agonists m and carpard to the f m d i o n of platelets in similar dilutions of whole blood in saline. Whole blood aggregamtry was p e r f o d after addition of ADP, collagen, and ristocctin. Aggregation was quantified by mrasuring the change in inpsdance as drawn m the chart recording. No differcnce in man inpedance change was noted betweem the groups of blood simples which were incubated and tested in the hamglobin solution and those incubated and tested in saline. In addition, incubation and stirring of the above mixtures over a period of six minutes without the addition of agfmists did not result in spontaneous platelet aggregation. We cmclude that O-Q cross-linked -lobin, in the concentratian studid, does not affect in-vitro platelet fmction, as measured by whole blood amregamtry.
IITRODUCTION: Infusion of crude hemolysates into experimental animals is associated with disturbances in normal hemostasis, most commonly disseminated intravascular coagulation,(DIC), and is generally believed to be due to contaminating stroma and stromal phospholipids,(l,2,3,4). More recently, endotoxin and large hemoglobin polymers were also shown to cause similar hemostatic abnormalities,(4,5). Although DIC was noted in dogs infused with purified stroma free preparations,(6), studies in other species utilizing crystallized, stroma free or pyridoxylated polymerized hemoglobins show that extensive transfusion is not associated with changes in the platelet count or coagulation screening tests, other than simple hemodilution,(6,7,8,9). Only one study has examined the effect of these materials on platelet function. It showed that exposure of rat platelets, in platelet rich plasma, to an elevated concentration of stroma free or pyridoxylated polymerized hemoglobin did not alter their aggregation response to increasing quantities of ADP, compared to the same platelet rich plasma mixed with autologous platelet poor plasma,(lO). Although the infusion of relatively small amounts of partially purified, stroma free, and modified hemoglobin solutions in humans has not been associated with bleeding or thrombosis, only limited information is available about their effects on hemostatic mechanisms. In particular, very little has been reported about their effects on platelet function in humans. This study examines the effect of a 10 gm./dl. solution of DBBF (Bis [ 3 , 5 dibromosalicyl] fumarate) Q - a cross-linked hemoglobin solution in Ringer's acetate buffer on invitro human platelet function, in a mixture with whole blood similar to that seen in massive transfusion situations.
651
Copydght 0 1992 by Mucel D e e r , lac.
REISS, CABALLERO, AND HESS
Downloaded by [New York University] at 19:32 17 March 2016
652
O !
0.0
I
0.5
1.0
2.0
1.5
25
3.0
Minutes
0.0
03
1A
Minutes
1.5
20
653
Downloaded by [New York University] at 19:32 17 March 2016
EFFECTS OF X-LINKED HEMOGLOBIN ON PLATELET FUNCTION
0.0
0.5
1.o
1.5
20
Minutes
m: Compodte a m g a l o n patems obwned after &ding nDtocwtin (maconcentratlon of 1.25 mg/mL). to 1:1 mlaunr of lmsh mtated whob blood with both 10% a - a X-linked M O X I ~ Saggmgsbon ~ a~80 seconds is ~ndicatrd Hb. (-), ud n o n d UYM.+) for each mixhln of blood with X-Hb ( 0 ) and sallne (*). Mean aggmgaUon lor each gmuo IS 18ohms. (n-3ineachgmup).
MATERIALS AND METHODS: Whole blood was collected by venapuncture from one of the investigators, (RFR), into 3.8% sodium citrate in a ratio of 9:l on three successive days. This individual’s platelet count was 238,OOO/mm’ and 228.0001 mm3 on days one and two respectively. DBBF a - a cross-linked hemoglobin, (X-Hb), was produced in the Letterman Army Institute of Research pilot plant. The lot utilized in these experiments had a final concentration of 10 gm./dl. in Ringer‘s acetate buffer. Whole blood aggregometry was performed within three hours of preparation of a 1:l mixture of the freshly collected whole blood, (WB), and either X-Hb or normal saline, utilizing a Whole Blood Aggregometer (Model 540, Chronolog Corp., Haverstown, PA). The strip recorder was calibrated so that 60 horizontal lines corresponded to 20 ohms of impedance. Aggregation patterns were recorded following the addition of ADP (Sigma Chemical Co., St. Louis, MO), to a final Concentration of 4uM, ristocetin (Chronolog Corp.), to a final concentration of 1.25 mg/ml, and collagen (Chronolog Corp.) to a final concentration of 5ug/ml. Maximal aggregation was calculated in ohms as the observed difference in impedance at three minutes, 90 seconds, and 15 seconds following the addition of each agonist respectively. Composite aggregation patterns were drawn to be representative of the individual tracings obtained with multiple aliquots of the WB-XHb and WB-saline mixtures following addition of the agonists. The means of the maximal aggregation impedance difference for each group of mixtures, following the reaction times indicated above, were also calculated.
6 54
REISS, CABALLERO, AND HESS
Downloaded by [New York University] at 19:32 17 March 2016
In addition, both the WB-XHb and WB-saline mixtures were incubated and stirred in aggregometer cuvettes for six minutes, without the addition of agonists, to detect spontaneous aggregation. RESULTS: The composite aggregation patterns obtained with multiple aliquots of both WB-XHb and WB-saline were similar after addition of each agonist utilized, (Figures 1, 2, 3). Furthermore, the mean values of maximal aggregation showed no difference between the groups of WB-XHb and WB-saline mixtures following addition of each agonist. The mean differences in impedance observed were as follows: ADP - 17 and 19 ohms at three minutes with the WB-XHb and WB-saline mixtures respectively; collagen - 23 ohms at 75 seconds with both mixtures: and ristocetin -18 ohms at 90 seconds with both mixtures. In addition, no spontaneous aggregation occurred during incubation and stirring of both mixtures for six minutes in aggregometer cuvettes
.
DISCUSSION: Although experimental data suggest that the infusion of stroma free and modified hemoglogin solutions in rats and some other animals is without adverse effects on hemostasis, these conclusions may not be applicable to all species. Infusion of stroma free hemoglobin in dogs was associated with DIC,(6). Furthermore, little experience has been reported with other larger mammals and laboratory primates. In-vivo experience in humans is limited to early safety studies in which relatively small amounts of hemoglobin solutions were administered. In one study, the infusion of an only partially purified hemoglobin solution was not accompanied by clinical or laboratory evidence of DIC,(11). On the other hand, the infusion of a stroma free preparation has been associated with a disproportionate prolongation of the partial thromboplastin time,(l2). In the recent clinical trials of pyridoxylated polymerized hemoglobin, none of the subjects infused with this material demonstrated changes in coagulation screening tests, platelet count, or bleeding time,(l3). Given the incompleteness of our current knowledge, it remains possible that the infusion of larger quantities of these solutions to humans during resuscitation might be associated with significant hemostatic abnormalities other than hemodilution. For this reason, i t is important that their effects on hemostatic mechanisms be more completely understood. In this study, we have demonstrated that human platelets maintain normal whole blood aggregation responses to ADP, collagen, and ristocetin following exposure to concentrations of an a - Q cross-linked hemoglobin solution which would be reached during massive transfusion.
REFERENCES:
1. Rabiner SF, Friedman LH: The role of intravascular hemolysis and the reticuloendothelial system in the production of a hypercoagulable state. Brit J Haematol 14: 105, 1968. 2. Spector J I , Lang E, Crosby WH: Coagulation changes in baboons during acute experimental hemoglobinemia and dextran infusion. Am J Pathol 78: 469, 1975. 3 . Rabiner SF, Helpert JR, Lopas HL, Friedman LH: Evaluation of stroma free hemoglobin as a plasma expander. J Exp Med 126: 1127, 1967. 4 . Feola M, Simoni J , Tran R, Canizaro PC: Mechanism of toxicity of hemoglobin solutions. Biomat Art Cells Art Org 16: 217, 1988. 5 . White CT, Murray AJ, Smith DJ, Qreene JR, Bolin RB: Synergistic toxicity of endotoxin and hemoglobin. J Lab Clin Med 108: 132, 1986. 6. Bolin R, Smith D, Moore G, Boewell a, DeVenuto F: Hematologic effects of hemoglobin solutions in animals, in Bolin RB, Geyer RP,
Downloaded by [New York University] at 19:32 17 March 2016
EFFECTS OF X-LINKED HEMOGLOBIN ON PLATELET FUNCTION
655
Nemo 0 , (ads.): Advances in Blood Substitute Research. New York, Liss, 1983, pg. 117. 7. DeVenuto F, Moores WY, Zegna AI, Zuck TF: Total and partial blood exchange in the rat with hemoglobin prepared by crystallization. Transfusion 17: 555, 1971. 8. Ning J, Chang TMS : Effects of stroma-free hemoglobin and polyhemoglobin on complement activation, blood cells and coagulation factors in rats. Biomat Art Cells Art Org 16: 651, 1988. 9. Ning J, Chang TMS: In vivo effects of stroma-free hemoglobin and polyhemoglobin on coagulation factors in rats. Int J Art Organs 13: 509, 1990. 10.Lalla FR, Ning J, Chang TMS: Effects of pyridoxylated polyhemoglobin and stroma free hemoglobin on ADP induced platelet aggregation. Biomat Art Cells Art Org 17: 363, 1989. 11.Spector J I , Crosby WM: Coagulation studies during experimental hemoglobinemia in humans. J Appl Physiol 38: 195, 1975. 12.Savitsky JP, Doczi J, Black J, Arnold JD: A clinical safety trial of stroma free hemoglobin. Clin Pharmacol Ther 23: 73, 1978. 13.Moss GS, Gould SA, Rosen AL, et al.: Results of the first clinical trial with a polymerized hemoglobin solution (abs). Biomat Art Cells Art Org 17: 633, 1989.
