Erythrocyte interactions – Comparison of the aggregation power of polymer molecules used in medicine – Not only size does matter

1 Introduction

Various polymers are used in medical practice – hydroxyethyl starches (HES), dextran (Dx), polyethylene glycols (PEG), polyvinyl pyrrolidone (PVP) and other [19]. These neutral polymers are important component of blood plasma expander fluids used for the management of hypovolemia in critically ill patients and of preservation solutions applied in organ transplantation [6]. Some of the problems associated with their use are related to altered microcirculatory flow [7, 20]. Aggravation of blood rheological properties is thought to contribute substantially in this respect: increased plasma and blood viscosity [9, 22], altered erythrocyte (ERY) aggregation [21, 30] and deformability; activation of the coagulation system [14]. These colloids are also widely used in hemorheological fundamental studies for assessment of the ERY aggregability: i. e. the cellular property determining the interactions of the erythrocyte with themselves [5]. They play also important role in blood cell adhesion to blood vessel endothelial cells (EC) [23, 29]. Many experimental findings exist concerning the macromolecular properties influencing the aggregation power (AP) of these neutral polymers [5, 11]. There are intense discussions related to the mechanism – how these colloids promote ERY aggregation in vitro and in vivo – the bridging hypothesis [10] and the macromolecular depletion hypothesis [24, 28]. The experimental results show that the concentration and molecular weight (MW) and size of the colloids are the major determinants of the aggregation extend and strength [4, 10]. In the present report we show our results, which demonstrate that for the AP of the neutral polymers the chemical nature of macromolecules plays also an important role, i.e. not only size doesmatter.

2 Materials and methods

Washed human RBC from human whole blood and ERY concentrates (from the National Center for Hematology and Blood Transfusion, Blood Bank Sofia) are suspended in phosphate buffered saline (PBS, pH = 7.40, osmolality = 295 mOsmol/kg) containing the polymers. The final hematocrit value (Hct) in the suspensions is 0.40 (v/v). The zeta sedimentation technique [3, 8] is used for quantification of the extent of cell aggregation as described earlier [1, 16–18]. In brief, RBC suspensions are filled in standard hematocrit tubes and placed in the holder of a home-made Zetafuge [15]. The rotation of the holder (originally 7-8 g) enforces the cells to travel towards the outer wall of the tube and to aggregate. After 45-sec long cycles the tubes are rotated 180 and the process repeated. Four spinning cycles take place over three minutes. At the end of the spinning period the apparent packed cell volume (PCV) is measured and referred to as the zetacrit (Zct). The hematocrit is determined in the same tube after centrifugation at 10000 g, for 5 min in standard hematocrit centrifuge. As aggregation index (AI) the zeta sedimentation ratio (ZSR) is calculated: ZSR = Hct/Zct. A new aggregation index is formulated (ZSR_rel), which involves correction for the difference in the viscosities of the various suspending media containing the macromolecules and relates the ZSR in the polymer solution to the ZSR value in PBS (the non-aggregating state of the ERY) [16].

The hydrodynamic radius (R_h) of the polymer molecules is determined from the intrinsic viscosity [η] using the Einstein’s viscosity relation. R H = ( 3 [ η ] 2.5 M W 4 π N A ) 1 3 , where(1)

[η] is in ml/g, M_W is in g/mol, R_H is in nm, N_A is the Avogadro’s number. The intrinsic viscosity [η] was determined using the Huggins equation: η red = [ η ] + k H [ η ] 2 c ,(2) from the plot of the reduced viscosity η_red as function of concentration and extrapolate to η_red = 0. The intercept is taken as a value for [η].

4 Conclusions

Our results show: The aggregation of ERY rises with concentration of polymers within a defined region, specific for every polymer. New formulated aggregation parameter (ZSR_rel) – with viscosity correction and relating to the value of the parameter in non-aggregating states – gives a good approach for comparing AP of the macromolecules. Within one type of polymer the increase in molecular mass results in larger AP of the colloids. The AP of the polymers is in the order PEG >PVP >Dx >HES. The interrelationship between hydrodynamic radius and AP of the polymers is generally present. The chemical nature of the polymers is of substantial importance for their aggregation power – not only size does matter.

5 Medical relevance

The investigated polymers are used: in fluids for blood plasma replacement therapy, in solutions applied for organ preservation in the transplantation medicine [12, 19]. The hyperaggregating effect [20, 21] of these polymers may disturb blood flow especially in critically ill patients and also be a problem during preservation solution wash out from the transplanted organs. Research groups are looking now for new potential alternative to conventionally used macromolecules (Albumin, PEG, HES etc.) – e.g. hyperbranched polyglycerols (HPG) as plasma expander and in cold preservation solutions [19]. New nanotechnology constructs (PEG-albumin) with novel plasma expansion properties directly interact with the microcirculation. They are used in transfusion medicine and to treat blood losses and concomitant effects on microvascular function due to related acute inflammatory conditions and ischemia [25].

Using the presented experimental approach to determine the AP of the macromolecules in PBS but also in blood plasma would be helpful for preparing a scale for comparing the aggregation properties of the colloids and so for optimization of the solutions produced.