U, Krallsz, B. Kotelba-Witkowska, T. Pietrucha THE STUDY ON THE EFFECT OF DMSO
ON .'THE THROMBIN-INDUCED PLATELET AGGREGATION
Thrombin-induced aggregation have been employed to study the effect of commonly used cryoprotectant, DMSO, on pla-telet function. This cryoprotectant caused pronounced decre-ase In aggregation response of both human and porcine pla-telets, This harmful effect was dependent on the concentra-tion of OMSO and incubaconcentra-tion time. The Inhibitory effect waa nearly fully reversible. Spin-labelling experimenta indicate that DMSO inhibition of platelet aggregation may be mediated by structural changes In membrene proteins.
Introduction
Platelet transfusion has become a common practice. Several fSctors Including availability of donors end preparation delay the prompt provision of platelets. The problem were, In part, resolved when It was shown that platelets could be stored for up to 72 hours at 4°C or 22°C. However, the short shelf-life of platelets makes Inevitable the lost of unused platelets [18], The extreme polymorphism of HLA system presents slso a signifi-cant obstacle in providing compatible platelets [3], Only the long-term cryopressrvatlon of single-donor platalet concentrate would offer a mechanism to prevent immunization aa well as pro-vide compatible platelets to a previously sensitized recipient [7],
According to current cryoblologlcal theory of cell preserva-tion by freezing, there is an optimal concentration for cryopro-
»ctlve agents. Below this concentration the additional effect til be offsst by the agent Inherent toxlty or by osmotic
dama-ge occuring while the cryoprotective adama-gent is being removed [10]. A variety of extracellular cryoprotectant« can enhance the reco-very of cells following freezing. These include sugars, sugar alcohols and polywers ouch os polyvinylpyrrolidone and hydro- xyothyl sterch, However, the*» extracellular cryoprotectants a- re not officient in reducing freezing damage to platslsta [13, 15]. Numerous studies have shown that dimethyl sulfoxids (DMSO), possesses the best cryoprotective properties with respect to platelets 12, 8, 11]. However, DMSO may be harmful by ltsslf [4, 16] and it Is important to determine the amount of damage ccuaed by this cryoprotectant. The aim of thia study was to e- xomine the influence of DMSO on platelet aggregation Induced by thrombin.
Materials and methods
Preparation of platelets. Platelets were isolated from ACD human or porcine blood and they were washed in the modified Ty- rode buffer (135 mM NaCl, 1 mM MgClg, 3 mM KC1, 5 wM glucose and 10 mM Trls-HCl pH 7.4), All preparation stepe were run at room temperature.
Platelet aggregation measurements. Platelet aggregation indu-ced by thrombin was studied photometrically using a conventional aggragometer equiped with stirring device. 0.1 ml of thrombin solution in saline was added to 1.4 ml of platelet suspension. 0.08 NIH of thrombin per ml of platelets suspension was used to
Induce human platelet ag **egation. In the case of porcine plate-lets, thrombin concentration ranged between 0.1« 3 NIH per ml of platelet suspension was used. Platelet count was adjusted to 0.3 x 109 per ml. DMSO was dissolved in Tyrode buffer and it was added to platelets in 1 : 3 ratio. Incubation of platelets with DMSO was carried out at room temperature snd aggregation moasurensnts were performed at 37°C.
Spin-labeling of platelet membrane proteine. Spin label 4» -(N-maIeinido)-2,2,6,6~tetramethylpiperldine«l-oxyl, (M3L), eyn- thetized by Or. K. Gwoidzirtskl, was used in ESR study. The me-thod of &pin*-labeling of membrane proteins was based on the pro-cedure ussd in our laboratory in the case of erythrocyte
membra-nes [1, 5. 6], Platelete were spin-labeled with MSL by adding appropriate amount of the label into the cell suspension. Tho final label concentration amounted to 2 mM. Incubation with MSL was carried out for -ft h at room temperature with a con-stant stirring. . Unbound label was removed by repetitive washing with Tyrode buffer. No ESR signal could be found in supernatant after th« third wash, indicating that all spin label present wao platelet-bound. Spin-labeled platelets were treated with OMSO for 20 win. at room temperature. ESR spectra were recorded us-ing a 8E/X-28 ESR spectrometer (Wroclaw, Technical University).
Results and discuslon
Typical tlme-couree of thrombin-induced aggregation response of porcine platelet Is presented in Fig. 1. With control calls aggregation reached its maximum about 9 minutes after the ad-dition of thrombin, as reflected in light tran9mitance changes. Platelets Incubated for 20 min. with DMSO exhibited markedly smaller aggregation response. 35% inhibition was observed for platelets treated with 2.5% OMSO, At 5% DMSO inhibition of platelet aggregation reached about 61%. Besides porcine plate-lets, the effect of DMSO on aggregation response to thrombin has been also studied with human platelets. The general pattern of DMSO effect on human platelets closely reeeroble3 that observ-ed for porcine platelets. Figure 2 shows typical aggregation curves obtained for human platelets preincubated for 15 min.with increasing concentration of DMSO. Like with porcine platelets there is a pronounced decrease in aggregation response upon ad-dition of DMSO.
The inhibitory effect of OMSO on platelet eggr&gation res-ponse to thrombin depends on the incubation time. Figure 3 •hows typical data obtrined for human platelets incubated with l% DM30, The inhibitory effect of the cryoprotectant increases mar-kedly with increasing incubation time. The data obtained for hu- •an platelets are summarized in Fig, 4. This Figure sho?.-3 both the effect of OMSO concentration and the effect of incubation time on thrombin-induced platelet aggregation. The inhibitory effect of DMSO increases with increasing cryoprotectant
concen-1 min
10 %
LIGHT TRANSMISSION
Fig. 1. Time-course of thrombin-induced aggregation rasponae of porcine platelets. Platelets wer* Incubated for 20 «in. with DMSO
e) control (without DMSO); b) 2.5% DMSO; c) 5% DMSO Agregacja krwinek płytkowych świni domowej. Krwinki inkubowano 20
min, z DMSO
TIME / mm j
Fig. 2. Aggregation response of humen platelets Incubated for 15 *ln. with OMSO. 1 - control (without OMSO); 2 - 1 % OMSO; 3
2.5% DMSO; 4 - 5 % DMSO
Agregacja kminek płytkowych człowieka. Krwinki lnkubowano 15 •In. * OMSO. 1 - kontrola (be* DMSO)j 2 - 1 % OMSOi 3 - 2,5% OMSO
tratlon. For a given OMSO concentration the affect Increases with lncreaalng incubation time. OMSO induced Inhibition of the pla-telet aggregation rasponee appears to be almost fully rever-sible. Figure 5 shows that aftsr washing of 5% OMSO, aggre-gation response to thrombin was nearly the sane as In the ca-
of untrestedplatelete. On washing off OMSO aggregation re-ached about 94% of Its control value in the case of porcine
latelsts and about 90% In tha case of human platelets. The btalnad in this study results are compatible with those of c h l f f e r at al. [17]. Our data confirm the observa- lon that OMSO in tha concentration range of 1-5% perturbe the
TIME /min./
Fig. 3. Aggregation response of human platelete lncubateu with 1% OMSO. 1 control (without DMSO); 2 1 5 »In.j 3 3 0 mln.; 4
-45 mln.
Agregacja krwinek płytkowych człowieka. Krwinki inkubowano z 1% OMSO. 1 - kontrola (bez DMSO)t 2 1 5 min.j 3 3 0 aln.j 4
-45 min. >
functional propartles of platelete and they point to the re-versibility of thle effect.
Savor8l observations Indicate that OMSO aay aot at a mem-brane level. This interaction aay occur either by changing the Interfaclel water structure at cell interface or by forming hydrogen bonds between OMSO aoleculee end verloue membrane com-ponents. Both types of Interactions mentioned are likely to in-duce diverse perturbation in membrane etructure [9, 19], Structu-ral changes induced In platelet membrane by OMSO have been ob-served by spln-lebollng method. A typical ESR spectrum of
ma-77m * [ m m . ]
Pig. 4. Effect of OMSO concentration end the effect of Incu-bation tine on human platelet aggregation. 1 - 1 % OMSO; 2 - 2.5% OMSOj 3 - 5 % DMSO. Each value repreeente the mean
tstandard deviation of eeven experiments
Wpływ stężenia OMSO 1 czasu inkubacji na agregację krwinek płytkowych człowieka. Każdy punkt przedstawia wartość średnlę
¿odchylenie standardowe z 7 pomiarów
lelmide spin-labeled platelets membrane proteins is shown in Fig. 6, Similar epectra have been deecribod ae reflective of at leaet two classes of spln-label binding elt^st one strongly immobilized (S) and one weakly immobilized (vv). The hyt/h8 ratio of signal height of MSL attached to weakly immobilized sites to those attached to strongly immobilized sites may be used ee a convenient and sensitive monitor of protein organizatio-nal changee in the membrane [1. 11, 13]. Figure 7 shows the effect of OMSO on the above defined structural parameter. The concentration-dependent decreese in h ^ h g ratio in DMSO trea-ted semples indicate that this cryoprotectant produce signifi-cant structural perturbation in platelet membrane proteins. The- ®e perturbation manifest itself in a conversion of weakly
im-TIME /min/
Fig. 5. Reversibility of DMSO-lnhlbltlon of human platelets ag-gregation. 1 - control (without DMSO)» 2 - after washing
Of DMSOs 3 - 5& DHSO
Odwracelność hamowania agregacji krwinek płytkowych człowieka. 1 -kontrola (bez OMSO); 2 - po przemyciu krwinek 1 usunlęolu
DMSO
mobilized spln-label binding sites to strongly Immobilized ones. In addition, the etructural changes produced by OMSO appear to be fully reversible. On washing off OMSO the origi-nal spectra have been reatored.
The data presented in this paper indicate that OMSO In the concentration range routinelly employed at platelet cryo- preservatlon exerts the Inhibitory effect on platelet aggrega-tion. This effect is almost fully reversible and it la probab-ly mediated by structural changee In membrane protelna. Xt may be suggested that the decreaaa eusceptlblllty of thaw-ed and washthaw-ed platelets to aggregation la not due to the toxic effect of the cryoprotectant. The depressed response to aggregation stimuli of thawed platalete should be at-tributed rather to the cell injury during the freezlng- -thawing cycle.
Fig. 6. ESR spectra of naleimide (M SL) spin lebelled humen pla-telet menbrane proteina
Widno EPR znacznika nsleinidowego przyłączonego do biełek błony krwinek płytkowych człowieka
Fig. 7, Effect of DMSO on the etructurel parameter hw/hs. Deto are expressed aa neons 51 stendard error# of the neen of five
experi-ments
Wpływ DMSO 17» perametr strukturalny h*/hc. Każdy punkt przedstawia wartoic średnis i odchylenie standerdowa z 5 pomiarów
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U, Kralisz, B. Kotelba-Wltkowske, T. Pietrucha WPŁYW DMSO NA AGREGACCJĘ KRWINEK PŁYTKOWYCH
STYMULOWANYCH TROMBINĄ
W pracy wykazano. Ze DMSO hamuje egregację kminek płytko-wych w sposób zależny od jego stężenia i czasu działanie* Stwier-dzono« Ze hamowanie agregacji ma charakter odwracalny. Wyniki uzyskane dzięki metodzie znakowanie spinowego pozwalają przypu-szczać. że zmiany strukturalne białek błony krwinek płytkowych zachodzące pod wpływem DMSO s q odpowiedzialne za antyagregacyj-
