© 1991 S. Karger AG, Basel 0301 -0147/91/0217-0156S2.75/0

Haemostasis 1991;21(suppl 1): 156—160

Hirudin - A Potential Stabilizing Factor for Platelet Preservation in Transfusion G. Pindur, E. Wenzel, M. Koehler, M. Heiden, B. Braun Department of Clinical Haemostaseology and Transfusion Medicine, University of the Saarland, Homburg/Saar, FRG

Key Words. Hirudin • Platelet concentrates • Thrombin inhibition • Platelet factor 4 • p-Thromboglobulin • Platelet volume

Introduction Citrate is commonly used as an anticoag­ ulant in the preservation of blood compo­ nents for transfusion-related purposes. At­ tempts have also been made to use heparin for anticoagulation. The results however, have not been satisfactory due to druginduced hemolysis, rapid damage to platelet function and generation of microaggregates [1]. Hirudin, a potent thrombin inhibitor, seems to be an alternative approach to im­

prove the preservation of blood products since there is evidence of thrombin genera­ tion being involved in storage lesions, in par­ ticular of platelets. Its general anticoagulant activities in vitro and in vivo have already been well demonstrated [2], Hirudin was shown to establish a long-lasting anticoagu­ lant effect and was particularly advanta­ geous for processing blood into plasma frac­ tions [3]. Bode and Miller [4, 5] demon­ strated that platelet concentrates (PC) con­ taining hirudin had significantly better mor­

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Abstract. The influence of additional hirudin in low doses on platelet and coagulation parameters was studied in platelet concentrates (PC) prepared by cytapheresis using citratedextrose solution formula A. The in vitro release of platelet factor 4 and P-thromboglobulin was markedly lowered and morphological platelet alterations, as assessed by platelet size studies were considerably less accentuated in the presence of hirudin over a 5-day storage period; this suggests a possibly protecting effect on platelet function. No clear activation of the coagulation and fibrinolysis system was evident during PC storage with and without hirudin since thrombin-antithrombin III complexes and Z)-dimers did not markedly change.

Hirudin in Platelet Preservation

Material and Methods Recombinant hirudin (r-hirudin) was kindly pro­ vided by Prof. F. Markwardt, Erfurt (FRG); its bio­ chemical characteristics were described previously [ 6],

PC for replacement therapy were harvested from healthy volunteers by cytapheresis using a Fenwal CS3000 blood cell separator (flow rate: 45 ml/min, pro­ cessing volume: 3.5 liters). 250 ml of PC was collected in a closed-system apheresis kit for extended platelet storage and plasma collection (Travenol Laborato­ ries, Inc., Fenwal Division, Deerfield, 111., USA) using citrate-dextrose solution, USP formula A anticoagu­ lant (ACD). 50 ml of the PC were transferred into a sampling pack for mere study purposes. Hirudin was dissolved in 0.9% saline to a final concentration of 10 antithrombin units/ml and added (ex vivo) to the samples immediately after collection. PC were stored for 5 days at 20-24 °C with continuous gentle agita­ tion. Platelets were counted electronically using an au­ tomated hematologic analyzer: Sysmex CC-780, Toa Medical Electronics, Kobe, Japan. Thrombin-anti­ thrombin III complex in platelet-poor plasma ob­ tained by centrifugation (3,000 g, 15 min, 22 °C) of the PC samples was examined by ELISA (Behringwerke, Marburg, FRG), D-dimers by ELISA (MAB; Boehringer, Mannheim, FRG). Platelet factor 4 (PF 4) and p-thromboglobulin (|3-TG) were determined by ELISA (Boehringer), in platelet-poor plasma obtained by centrifugation of the original PC samples (2,000 g, 15 min, 4 °C) using CTAD-tubes (containing citrate, theophyllin, adenosine and dipyridamol) according to the manufacturer’s instructions. Platelet volume dis­ tribution was measured using a Coulter Counter Channelizer as previously described [7]. The Wilcoxon signed rank test and Student’s t test were used for statistical analysis.

Results Hirudin was added to samples of PC ob­ tained by cytapheresis from healthy donors using ACD. Platelet parameters were studied and compared in samples prepared with (ACD + hirudin; n = 3) and without (ACD only; n = 3) hirudin over a storage period of 5 days. The mean platelet count of the PCs was 1.019 vs. 1.013 X 106/ml initially in the samples with and without hirudin. Platelets showed a slight decreasing tendency (ta­ ble 1); however, there was no difference be­ tween hirudin-treated and nontreated PC. Platelet size distribution was similar in both groups at days 1 and 3 of storage. At day 5 there was a marked shift towards smallersized platelets in PCs without hirudin (ta­ ble 1). Hereby a second population of plate­ lets with low volumes appeared (fig. 1). There was only a slight shift in mean platelet size in the hirudin-treated PC. The marked increase of PF 4 and (3-TG immediately after collection of the PC at day 1 results from platelet activation during cy­ tapheresis (table 1). The initial range of val­ ues for PF 4 was 150-653 ng/ml and 658— 2,525/ml for (3-TG. During storage, PF 4 increased more than 10-fold compared to initial amounts and (3-TG more than 8-fold when ACD was used alone. The values of PF 4 and (3-TG did not exceed 1.8-fold over the storage period in PC containing hirudin. This difference is significant ( p < 0.05). The mean ratios of (3-TG to PF 4 were markedly decreased at days 3 and 5 in PC containing solely ACD-A whereas in those with hirudin the ratios remained constant (table 1). Thrombin-antithrombin III complex was not markedly elevated following cytaphere­ sis and decreased gently during storage in

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phological scores and functions than con­ trols even after 15 days of storage. In this study, the protective effect of low concentrations of hirudin on the function of platelets obtained by cytapheresis was inves­ tigated.

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Pindur/Wenzel/Koehler/Heiden/Braun

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Table 1. Summary of means of in vitro markers during storage of PC Day 1

Day 2

Day 5

Formula

Platelet count, %

100 100

88.9 105

86.9 88.9

ACD ACD + hirudin

Mean platelet volume, fl

7.0 6.1

7.0 6.1

3.7 5.3

ACD ACD + hirudin

Maximal frequent platelet size, fl

3.7 3.7

3.7 3.7

2.0 4.5

ACD ACD + hirudin

P-TG, %

100 100

465 122

835 176

ACD ACD + hirudin

PF 4, %

100 100

1,218 124

1,346 164

ACD ACD + hirudin

P-TG/PF 4 ratio

4.4 3.9

1.7 3.8

2.7 4.1

ACD ACD + hirudin

TAT, ng/ml

7.8 24.5

8.1 18.4

3.3 14.2

ACD ACD + hirudin

D-dimer (ng/ml)

44.1 59.9

60.9 73.7

65.3 86.3

ACD ACD + hirudin

pH

7.13 7.13

7.29 7.18

7.31 7.27

ACD ACD + hirudin

both PC. Z)-dimers were within the normal range and increased slightly over the storage period, which was not clearly influenced by hirudin.

Discussion Thrombin is known to be a potent platelet-aggregating agent, and is suggested, even in the presence of trace amounts, to account for platelet lesion during storage when ci­ trate is used solely as a conventional antico­ agulant in blood processing for transfusionrelated purposes [4, 8], Emphasizing on mor­

phological characteristics and metabolic pa­ rameters, Bode and Miller [4, 5] demon­ strated using various agents that hirudin is more efficient in maintaining platelet re­ sponsiveness during storage than anticoagu­ lants containing citrate alone. In this trial, the influence of additional hirudin in ACDanticoagulated PC on platelet parameters and markers of the activation of the hemo­ static and fibrinolytic system was studied. The concentrations of PF 4 and (3-TG in the PC obtained by cytapheresis were consider­ ably elevated, which suggests enhanced re­ lease due to mechanical trauma to the plate­ lets during extracorporal circulation. In con-

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Hirudin was added to a final concentration of 10 antithrombin units/ml (n = 3 for each preparation). TAT = Thrombin-antithrombin III.

Hirudin in Platelet Preservation

159

Mean platelet volume, fl

trast, less platelet activation is observed when PC are processed from blood obtained by conventional nonmechanical techniques [9]. Ongoing release during storage reflected by marked increases in PF 4 and (3-TG was observed in this study when PC were pre­ pared with ACD alone. Only a minor in­ crease was recognized under the influence of hirudin, indicating its protective effect on platelet function obviously due to thrombin inhibition. The lowering of the ratios of (3TG to PF 4 over the storage period in the PC without hirudin might also be attributed to an enhanced in vitro release reaction accord­ ing to Kaplan et al. [10]. Platelet size, ex­ pressed as mean platelet volume, was stud­ ied as a morphological criterion possibly cor­ relating with functional properties. Larger

platelets appear to be more active in particu­ lar concerning aggregability [11]. In hirudintreated PC, the mean platelet size only showed a slight shift without aleration of the basic distribution patterns. In contrast, in the absence of hirudin PC displayed a marked shift towards lowered platelet vol­ umes accompanied by a dissociation of the size distribution at day 5, thus indicating a loss of possibly superior platelets during pro­ longed storage. No marked activation of the coagulation system following cytapheresis was observed since the values of thrombin-antithrombin III were close to the upper normal range immediately after PC collection and during the storage period. This is ascribed to the anticoagulant effect of citrate. An additional

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Fig. 1. Frequency distribution of mean platelet volume at day 5 of storage in a PC prepared with ACD-A alone and with additional hirudin (final concentration: 10 antithrombin units/ml. ■ = ACD; + = ACD + hiru­ din.

Pindur/Wenzel/Koehler/Heiden/Braun

influence of hirudin could not be recognized. D-dimers as a marker of activation of the fibrinolytic system were not elevated but showed a gently increasing tendency. How­ ever, no clear difference was observed in the presence or absence of hirudin. In conclusion, according to our in vitro studies of PCs obtained by cytapheresis us­ ing ACD, the addition of hirudin seems to be advantageous for the protection of platelet function during storage. However, currently it cannot yet be evaluated if these findings will become important for the processing of PC in the support of thrombocytopenic pa­ tients unless hirudin is introduced into clini­ cal use.

References 1 Swank RL: Alteration of blood on storage: Mea­ surement of adhesiveness of aging platelets and leukocytes and their removal by filtration. N Engl J Med 1961;265:728-733. 2 Markwardt F: Development of hirudin as an anti­ thrombotic agent. Semin Thromb Hemost 1989; 25:269-282. 3 Drawert J, Stein P: Untersuchungen zur Stabili­ sierung von Spenderblut mit Thrombininhibito­ ren. Folia Haematol (Leipz) 1988; 118:560-565. 4 Bode AP, Miller DT: Preservation of in vitro function of platelets stored in the presence of inhibitors of platelet activation and a specific inhibitor of thrombin. J Lab Clin Med 1988; 111: 118-124.

5 Bode AP, Miller DT: The use of thrombin inhibi­ tors and aprotinin in the preservation of platelets stored for transfusion. J Lab Clin Med 1989; 113: 753-758. 6 Markwardt F, Fink G, Kaiser B, Kloecking HP, Nowak G, Richter M, Stuerzenbecher J: Pharma­ cological survey of recombinant hirudin. Pharma­ zie 1988;43:202-207. 7 von Blohn G, Jaeger H, Brill G, Wenzel E, Hell­ stem P: Methodik und klinische Bedeutung der Thrombozyten-Volumenhäufigkeitsverteilung. Lab Med 1984;8:5-9. 8 Martin BM, Feinman RD, Detwiler TC: Platelet Stimulation by thrombin and other proteases. Bio­ chemistry 1975;14:1308-1314. 9 Levine SP, Suarez AJ, Sorenson RR, Knieriem LK, Raymond NM: The importance of blood col­ lection methods for assessment of platelet activa­ tion. Thromb Res 1981;24:433-443. 10 Kaplan K, Owen J: Plasma levels of ß-thromboglobulin and platelet factor 4 as indices of platelet activation in vivo. Blood 1981;57:199-201. 11 Karpatkin S: Heterogeneity of human platelets. VI. Correlation of platelet function with platelet volume. Blood 1978;51:307-316.

Dr. G. Pindur Abteilung für Klinische Hämostaseologie und Transfusionsmedizin der Universität des Saarlandes D-W-6650 Homburg/Saar (FRG)

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Hirudin--a potential stabilizing factor for platelet preservation in transfusion.

The influence of additional hirudin in low doses on platelet and coagulation parameters was studied in platelet concentrates (PC) prepared by cytapher...
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