Adverse Effects of Synthetic Colloids on Coagulation

Synthetic colloids are popular drugs because of their overall therapeutic safety and efficacy, their easy availability and low price. In the past, some synthetic colloids have been shown to have adverse effects on coagulation that have led in individual cases to serious hemorrhagic complications, some of them lethal. This has resulted in a reluctance of some physicians, particularly in the United States, to use synthetic colloids. To investigate the safety of synthetic colloids and their effects on coagulation in particular, we carried out a nine-to-ten-day hemodilution in patients with cerebral perfusion disorders, infusing 500 ml to 1,500 ml of synthetic colloid daily. We studied the rheological, hemostasiological and pharmacokinetic properties of Dextran 40 (n = 12), HES 200/0.62 (n = 20), hydroxyethyl starch (HES) 70/0.5 (n = 10) as well as three types of HES 200/0.5 with different C2/C6-ratios (n = 32). According to our studies, high molecular weight HES 450/0.7 and highly sustituted medium molecular weight HES 200/0.62, have adverse effects on coagulation. Both synthetic colloids are difficult to break down, resulting in an accumulation of large molecules that impair the coagulation system through a significant drop in factor VIII/von Willebrand factor complex, which can lead to hemorrhagic complications. They also result in an increase in plasma viscosity and unfavorable effects on erythrocyte aggregation. Dextran 40 can have adverse effects on coagulation because it interferes with platelet aggregation. Low-substituted, medium molecular weight HES 200/0.5 with favorable substitution pattern, HES 130/0.4 and low molecular weight starch HES 70/0.5 are more easily metabolized and do not accumulate in clinically relevant quantities. Because it is the in-vivo, not the in-vitro molecular weight that determines the effects of a synthetic colloid, these substances have no relevant adverse effects on the coagulation system and improve rheological parameters.



INTRODUCTION



Plasma substitutes are used in the form of synthetic colloids and crystalloid solutions in the prophylaxis and treatment of hypovolemia, to treat the loss of blood and plasma, as well as after surgery, trauma, burns or infections <|[1-3]|>. Another area of use volume therapy, also called hemodilution therapy, carried out in cerebral, retinal, otogenic and peripheral perfusion disorders <|[4-7]|>. Placenta insufficiency and coronary heart disease are additional indications <|[8-10]|>.



Among the plasma substitutes, the most popular infusions are crystalloid solutions <|[2]|>. They are cheap and can be used without the danger of anaphylactic reactions. Crystalloids are used for the treatment of dehydration, increase urinary output and interstitial fluid. Albumin, Fresh Frozen Plasma (FFP), Plasma Protein Fraction (PPF) and non-protein colloids such as hydroxyethylstarch (HES), Dextran and Gelatin are used alternatively instead of a high-dose infusion of crystalloids. This is particularly the case if the main goal is the increase of intravascular volume, for example in the therapy of acute blood loss or during hemodilution. Compared to crystalloids, synthetic colloids cause only a small increase of interstitial fluid and result therefore in a smaller post-ischemic brain edema <|[2, 11]|>.



Synthetic colloids are used more in hemodilution therapy, because the infusion of crystalloids alone does not lower hematocrit effectively nor increase cardiac output <|[12, 13]|>.



Synthetic colloids such as HES, Dextran and Gelatin have shown to be as safe and efficacious as albumin solutions for cardiac operations, burns, sepsis and larger surgery <|[14, 15]|>. Several groups observed a larger increase in CO after infusion of 10% medium molecular weight HES than after infusion of 5% albumin solution, which can be attributed to a greater plasma-expanding effect of starch solution <|[1, 16]|>. Therefore, the use of albumin as a plasma expander only is not advisable anymore. Because of occasionally occurring albumin shortages and for reasons of cost-effectiveness and infectious risk, synthetic colloids (such as medium or low-molecular weight starches or Gelatin) are a better choice in volume therapy than albumin, as long as protein concentration in the blood is above 3-4 g/dl <|[17]|>. The indications for the use of albumin should be reconsidered under new aspects, such as substance-specific effects of albumin itself that go beyond the osmotic effects. Very little data is available about this and more studies need to be conducted in the future to establish these albumin-specific indications.



As long as the volume effect is comparable, the different synthetic colloids are very similar regarding their effects on hemodynamics and oxygen transport <|[18, 19]|>. HES and Dextran are the most frequently used synthetic colloidal volume substitutes because of their great therapeutic safety after a one-time infusion of 500 ml to 1,000 ml. HES and Dextran differ in their chemical structure, metabolization and elimination. This is of decisive importance for their biological effects, because the actual in-vivo effect of a synthetic colloid is determined by the composition and nature of the molecules that are generated through the in-vivo metabolism.



Because they were thought to have favorable rheological properties, hemodilution therapy was initially mostly carried out with low molecular weight Dextrans <|[20]|>. After Haa et al. <|[21, 22]|> and Kroemer et al. <|[23]|> were able to show that Dextran 40 loses its favorable rheological properties after repeated administration because of the accumulation of macromolecules, starch solutions gained popularity.



High molecular weight starch solutions, such as Hetastarch with a Mw of 450,000 D, were more popular in the beginning, particularly in the United States, because of their long-lasting volume effect. However, after repeated infusion or the infusion of larger volumes, hemorrhagic complications were observed in individual cases <|[24-29]|>. Recently, Trumble et al. <|[30]|> reported the increased occurrence of hemorrhagic complications after infusion of Hetastarch during vasospasm treatment in patients with subarachnoid hemorrhage. Because of this, Trumble et al. advised against infusions of HES and recommended instead the use of Plasma Protein Fraction (PPF).



The hemorrhages observed under Hetastarch were caused by an acquired von Willebrand syndrome <|[31, 32]|>. Strauss and colleagues <|[33]|> observed that medium molecular weight starch (Pentastarch, Mw 264 kD) has fewer unfavorable effects on coagulation than high molecular weight starch. In addition, medium molecular weight starch has better rheological properties and remains for a shorter time in the human body <|[21-23, 34-37]|>. This increased the popularity of medium molecular weight starches.



The effects of a one-time administration of the different synthetic colloids on rheology and coagulation have been studied repeatedly <|[38-44]|>. The effects of a repeated infusion of larger volumes, such as during a hemodynamically oriented volume therapy, have not been studied sufficiently, although more pronounced effects are to be expected. A long-term volume therapy is a good model to judge therapeutic safety of synthetic colloids, because possible side effects will show more clearly due to the much higher doses.



We carried out a hypervolemic volume therapy in 74 patients with cerebral perfusion disorders after obtaining informed consent from the patients <|[45-61]|>. No patient suffered from a manifest cardiac or renal insufficiency. Infused synthetic colloids included 10% Dextran 40 (NaCl-free), 6% and 10% HES 200/0.62 (Elohst, manufactured by Hormon-Chemie, Munich) and 6% HES 70/0.5 (Expafusion, manufactured by Pfrimmer Kabi). In addition, we infused three derivatives of a 10% HES 200/0.5 solution: HES 200/0.5 (HES-steril, manufactured by Fresenius) and two HES solutions that were identical except for their pattern of substitution, both manufactured by Pfrimmer, in the following called 10% HES 200/0.5 (1) and (2). The C2/C6 ratio for 10% HES 200/0.5 (1) was 13.4 and for (2) it was 5.7 (for details, see original manuscripts <|[45-50]|>).



All patients received initially a rapid infusion of a “loading dose” of 500 ml of synthetic colloids and 500 ml electrolyte solution. The subsequent protocol showed only minor differences for the individual substances. Patients received 1,000 ml of a synthetic colloid and 1,000 ml of a crystalloid on days 1-4 and 500 ml of a synthetic colloid and 500 ml of a crystalloid on days 5-10. For HES 200/0.62 a smaller total dose was chosen, because this starch has a longer-lasting volume effect due to its higher degree of substitution.



EFFECT OF SYNTHETIC COLLOIDS ON RHEOLOGICAL PARAMETERS



Effect of synthetic colloids on hematocrt, erythrocyte aggregation and plasma viscosity



All HES solutions administered by us lowered hematocrit by approximately the same amount. The volume effect was shorter for a HES with a low in vivo molecular weight than for HES that is difficult to metabolize and has a high in-vivo molecular weight.



Dextran and HES affected erythrocyte aggregation differently. The initial values for HES 200/0.5 and Dextran 40 were 18.3 and 21.7, respectively. For Dextran 40, erythrocyte aggregation reached 29.5 at the end of the therapy. The individual infusions of HES 200/0.5 lowered erythrocyte aggregation more than Dextran 40 and the tendency to aggregate was lowered continuously. The loading dose of the highly substituted HES (200/0.62) caused a significant increase from 15.9 to 18.3. The loading dose of HES 70/0.5 decreased the tendency to aggregate significantly from 17.7 to 12.9.



The infusion of both Dextran 40 and HES 200/0.62 caused an overall increase in plasma viscosity. Plasma viscosity in the Dextran group was 1.38 mPas at the beginning of therapy, reaching 1.78 mPas on day 10 (+28%). HES 200/0.62 caused a continuous, significant increase from 1.30 mPas to 1.54 mPas (+18.5%). Individual infusions of HES 200/0.5 and HES 70 lowered plasma viscosity. This trend continued during the therapy. The reduction was more long-lasting for medium molecular weight starch than for the low-molecular weight starch, which showed its maximum effect on day 3.



Measuring the serum concentration of the infused synthetic colloids showed an increase for Dextran 40 and the highly substituted HES 200/0.62 during the individual infusions as well as during the whole therapy that paralleled plasma viscosity. Both substances accumulated and reached at the end of therapy a concentration of approximately 20 mg/ml. For HES 200/0.5 and HES 70/0.5 no such accumulation was observed. The measured concentrations for medium molecular weight starch were approximately 10 mg/ml. For the low molecular weight starch which was only infused as 6% solution, the values ranged from 2.6 to 5.4 mg/ml.



EFFECTS OF SYNTHETIC COLLOIDS ON COAGULATION PARAMETERS



Effects on platelet count, platelet volume and platelet aggregation



The initial platelet count was very similar for all groups at the beginning, ranging from 237,000 to 246,000/mm3. No group showed changes beyond the dilution effect, which can be judged from the change in hematocrit. The initial values of the patients treated with HES 200/0.62 was 240,700 55.600/mm3. Before the last day of infusion, the lowest value was 194,000/mm3. In the Dextran 40 group, the platelet count dropped on day 1 from 244,000/mm3 to 206,000/mm3. On day 10, the value was 202,000/mm3. HES 200/0.5 lowered the platelet count during the 10-day therapy from 246,000/mm3 to 243,000/mm3. In the group treated with low molecular weight starch, platelet count was before therapy 237,000/mm3. During the infusion therapy, platelet number increased again and was on day 10 higher (261,000/mm3) than the initial value.



During the infusion therapy with HES 200/0.5 (1) a slight decrease of mean platelet volume from 10.29 m3 to 9.8 m3 was observed. Infusion of HES 70/0.5 resulted in a decrease from 10.36 1.25 m3 to 9.88 m3. The decrease was more pronounced for the 0.62 substituted starch. Initial volume was 9.67 m3, the lowest value was reached on day 7 at 8.43 m3.



Dextrans and HES have different effects on platelet aggregation. Whereas Dextrans inhibit the spontaneous and induced platelet aggregation, HES 200/0.62 is the only starch that causes a slight decrease in platelet aggregation. For HES 200/0.5 and HES 70/0.5 no significant effect on platelet aggregation could be observed.



Effects quick, PTT, thrombin time and fibrinogen



The infusion of the loading dose of HES 200/0.5 resulted in a small decline of the quick value from 87.5% to 86.7%. During the remaining therapy, the quick fluctuated little and was at the end of therapy at 89.0% slightly above the initial value. The first Dextran infusion lead to a significant decrease. The quick on the last day was 72%. The reduction in quick was therefore most pronounced in the Dextran group, followed by the 0.62 substituted starch, which caused a 20% drop from the initial value. HES 70/0.5 and HES 200/0.5 caused no significant changes in quick.



HES 200/0.5 caused a 10.8% increase in PTT. Dextran raised the initial value by 23.9%. The higher substituted starch HES 200/0.62 increased PTT by 42.8%. The initial value was 29.9 0.9 s and was increased by the loading dose significantly to 31.7% and increased further during therapy to 42.7 3.3 s. In the low molecular weight starch group PTT was initially 32.1 1.2 s. This particular synthetic colloid had no effect on PTT.



At the beginning of therapy, the rapid infusion of HES 200/0.5 shortened thrombin time from 19.6 s to 17.7 s. During the remaining therapy, thrombin time hardly changed, final value on day 10 was 18.4 1.2 s. The reduction in thrombin time was more pronounced for Dextran. The loading dose caused an 24.7% drop. At the end of therapy, the rapid infusion of HES 200/0.62 led to a 28.9% decrease from the initial value of 22.8 11.1 s, and a further significant decline to 13.7% 1.1 s during the remaining therapy. Low molecular weight starch caused only small changes in thrombin time, which dropped at the end of therapy from 17.3 s to 16.4 s.



All synthetic colloids lowered the concentration of fibrinogen at the beginning of the therapy.A loading dose of HES 200/0.5 lowered fibrinogen concentration by 19.9%. During the remainder of therapy, only small changes were observed. Under therapy with Dextran, a continuous, 34.7% decrease was observed. The first infusion of the highly substituted starch caused a decrease in fibrinogen from 278 mg% to 217 mg%. Later infusions caused no notable changes. The low molecular weight HES 70/0.5 caused the greatest drop from the initial value after the loading dose, 18.2% below the initial concentration. The highest value was measured on day 10.



Effects of factor II, V, VIII: C, von Willebrand Ristocetin co-factor and von Willebrand factor antigen



Long-term infusion of HES 200/0.5 (1) (degree of substitution 13.4) caused a significant (p



Initial factor V concentration in the HES 200/0.5 (1) group was 59.9%. The lowest value was reached on day 3 at 41.5%. In the group treated with low molecular weight starch, lowest factor V activity was observed at the end of day one, 20.3% below the initial value.



HES 200/0.5 (1) lowered factor VII during the rapid infusion from 83.5% to 56.7%. During therapy, the concentration of factor V increased reached 69.4% on day 10. HES 70/0.5 also caused the greatest drop directly after the rapid infusion. This was a significant 21.6% decrease from the initial activity.



Factor VIII:C was affected very differently by the different synthetic colloids. The loading dose of 0.5 substituted HES with an initial molecular weight of 200,000 D caused only a small decrease in factor VIII:C activity from 120.4% to 109.6%. During the remaining therapy, only small changes occurred, the final value was 110.9%. The more highly substituted starch on the other hand caused a significant decrease to 28.3% (-70.5%). Low molecular HES caused a significant decrease in factor VIII:C activity only at the end of day 1. Factor VIII:C was not lowered significantly during the remaining time.



Measuring von Willebrand Ristocetin cofactor yielded comparable results. HES 200/0.5 with a more favorable substitution pattern (C2/C6 ratio 5.7) and HES 70/0.5 did not significantly affect von Willebrand Ristocetin cofactor. Activities measured during infusion therapy changed a maximum of 12% from the initial values and were at the end of therapy even higher than the initial values. However, similar to factor VIII:C, the more highly (0.62) substituted HES caused a 70% decrease in activity.



Similar to von Willebrand Ristocetin cofactor, von Willebrand factor antigen was affected little by a therapy with HES 200/0.5 and HES 70/0.5. The more highly substituted HES 200/0.62 led directly after the initial infusion to a significant decrease from 127.5 31.9% to 88.3%. Von Willebrand factor antigen dropped continuously until the last day of infusions by 84.4%.



Because all multimers in the complete multimer spectrum of von Willebrand factor were affected similarly, the observed decrease was most likely a purely quantitative effect. This corresponds to an acquired (type 1) von Willebrand syndrome <|[62, 63]|>.



Effects of factor IX, X, XI and XII



Factor IX was not lowered significantly by HES 200/0.5 (1) or HES 70/0.5. Factor X (Stuart-Prower factor) was lowered significantly by HES 200/0.5 (-13%). HES 70/0.5 caused only a 17.8% drop



Activity of factor XI was reduced by 49.0% through infusion of HES 200/0.62. The effect of HES 200/0.5 (1) and HES 70/0.5 on factor XI was smaller.



For the HES 200/0.62, the reduction in factor XII activity was similar to factor XI. HES 200/0.5 decreased activity of factor XII significantly (p



Influence of the HES Substitution Pattern



We studied the effect of the substitution pattern with two 10% HES 200/0.5 solutions that were identical except for their C2/C6 substitution pattern. In the following, they are called 10% HES 200/0.5 (1) and (2). HES 200/0.5 (1) has a C2/C6 ratio of 13.4 and HES (2) a C2/C6 ratio of 5.7.



Initial hematocrit in the HES (1) group was higher than in the HES (2) group (47.2% vs. 42.9%, respectively). Bloodletting and loading dose caused an 18% decrease in both groups. The lowest value of 32.8% was reached in group 1 on day 7 and in group 2 on day 5. This corresponds to a 30.5% and 23.5% drop, respectively. HES 200/0.5 (1) has a stronger dilution effect, which occurs however with a delay after repeated infusions.



Erythrocyte aggregation was before therapy (11.4) in group 1 and 14.2 in group 2. In both groups, a lower tendency to aggregate was observed.



The starch with a higher C2/C6 ratio caused a continuous, significant 10% increase in plasma viscosity. HES (2) on the other hand caused a smaller increase



The measurements of serum concentration in group 1 showed in the high-dose phase a continuous increase (from 9.3 mg/ml to 14.9mg/ml). In the following low-dose phase, concentration decreased again to 12.0 mg/ml. The starch solution with the low C2/C6 ratio of 5.7 showed no accumulation of HES in the serum. The concentration of 9.0 mg/ml reached during the loading dose, remained stable during the high-dose infusion therapy and dropped towards the end of therapy to 5.8 mg/ml.



The effect on PTT showed a clear dependency on the substitution pattern. Whereas the HES with a high C2/C6 ratio caused a significant, 30% increase in PTT, the PTT increase was much smaller in the other group (13%).



Thromboplastin time (quick) was not considerably affected by either substance. Reduction in quick was 11.9% in group 1 and 6.1% in group 2.



The shortening of thrombin time was also more pronounced in group 1 than in group 2 Fibrinogen concentration decreased by 29.0% (p



The most pronounced difference between the two starch solutions was observed in their effect on factor VIII/von Willebrand factor complex. HES 200/0.5 (1) reduced factor VIII:C strongly (from 171.5% to 61.2%). The starch with the lower C2/C6 ratio of 5.7 caused a much smaller decline of 19.3%.



Under therapy with HES 200/0.5 (1) von Willebrand Ristocetin Cofactor decreased by 45.6% The decrease in group 2 was only 7%. HES 200/0.5 (2) lowered von Willebrand Ristocetin Cofactor at the end of day 1 from 123.0% to 114.3%. During the remaining therapy, von Willebrand Ristocetin co-factor increased again and the final value was higher then the initial activity (134.4%).



The decrease in von Willebrand Factor Antigen was also larger in group 1 (55.8%) than in group 2 (42.2%).



Distributions of molecular weight of different starches



The examination of the distribution of the molecular weight explains the different attributes of the studied HES-solutions.



The mean molecular weight (Mw) of the different synthetic colloids during the volume therapy varied greatly. The in-vitro Mw of the HES 200/0.62 infusion solution was 270,000 D. Through rapid enzymatic breakdown, the Mw was 120,000 at the end of therapy. HES (1) and (2) had an in-vitro Mw of 214,000 and 180,000 respectively. The Mw reached 95,000 and 84,000 on the final day. The low initial Mw of HES 70/0.5 (60,000 D) changed little in vivo.



Due to the more difficult elimination, Dextran 40 leads to an accumulation of larger molecules. The distribution of molecular weight was shifted to the right and a continuous increase from the in-vitro Mw of 52,000 to an in-vivo Mw on the last day of 102,000 was observed.



The analysis of the average molar masses (Mn) showed different trends for different starch solutions. With HES 200/0.62, a slight increase from 66,000 to 85,000 occurred. HES 200/0.5 (1) showed a decrease from 93,000 to 65,000, whereas HES 200/0.5 (2) caused an 11.5% increase of the in-vitro value of 52,000 D. The in-vivo Mn of low molecular weight starch increased considerably from 24,000 to 51,000 D.



The distribution width of the molecular weight, indicated by the quotient Mw/Mn narrowed for all starch solution through elimination of the smaller and hydrolysis of the larger molecules.



DISCUSSION



A variety of synthetic colloids are available to carry out a volume therapy. They have in part significantly different, substance-specific properties. Studies regarding these substances are often limited to a one-time infusion of 500 to 1,000 ml and are of limited value for a long-term volume therapy. Important criteria for the choice of a volume substitute are rheological effectiveness and therapeutic safety.



RHEOLOGICAL PROPERTIES



Plasma viscosity, erythrcyte aggregation



It is important to differentiate between the effects on micro- and macrocirculation when judging the rheological effectiveness of a synthetic colloid. For the macrocirculation, the hematocrit and the full blood viscosity, which depends on the hematocrit, are of decisive importance. For the microcirculation on the other hand, the hematocrit, which is physiologically lower, is of lesser importance <|[8, 64]|>. Under impaired flow conditions, as they exist in the ischemic penumbra, erythrocyte aggregation and plasma viscosity are less relevant because of reduced perfusion pressure and frictional forces <|[65, 66]|>. Due to the Fahraeus-Lindqvist effect, the effective viscosity of blood in the capillaries approaches the viscosity of plasma <|[67]|>. Plasma viscosity depends strongly on macromolecules in the plasma, such as fibrinogen. Large molecules increase erythrocyte aggregation when they bridge the physiological distance of approximately 30 nm between the erythrocytes <|[68, 69]|>.



It is not well understood what role rheological factors play in cerebral perfusion disorders <|[70]|>. In patients with subcortical arteriosclerotic encephalopathy Ringelstein et al. <|[71]|> were able to demonstrate the clinical relevance of fibrinogen concentration and plasma viscosity. By reducing the fibrinogen concentration from 326 mg/dl to 152 mg/dl, plasma viscosity decreased from 1.38 to 1.31 mPas. This led to a normalization of the cerebral perfusion reserve and an improvement of microcirculation, manifested by a 30% faster arterio-venous passage time in the retina. An acute improvement of cerebral competence or a long-lasting decline in the number of lacunar reinfarctions was not observed, so that the therapeutic effect of a fibrinogen and plasma-viscosity-lowering therapy was questioned.



Plasma viscosity was clearly increased through the infusion of Dextran 40 and HES 200/0.62. This increase paralleled serum concentration and depended on the concentration of the infusion solution and infusion velocity. Despite comparable serum concentrations, HES 200/0.62 led to a smaller increase in plasma viscosity, because from a rheological perspective, the more spherically shaped starch molecules are more favorable than the chain-like configuration of Dextrans <|[8]|>. HES 200/0.5 and HES 70/0.5 lower plasma viscosity, if sufficient amounts of fluid are infused.



Erythrocyte aggregation is also affected differently by Dextrans and HES. Erythrocyte aggregation is caused by reversible bridge-binding between erythrocytes which can approach each other only up to a distance of 30 nm, because of repulsive Coloumb forces. Erythrocytes can only attach to each other when larger molecules, such as fibrinogen, form “bridges” between the erythrocyte membranes <|[68, 69]|>. Smaller molecules on the other hand can crowd out the aggregation-supporting macromolecules and therefore have an aggregation-inhibiting effect.



Among other factors, different molecular shapes are probably responsible for the varying effects of different synthetic colloids. While Dextran, due to its chain-like shape can increase erythrocyte aggregation already at molecule sizes above 60,000 D, starch with its more spherical shape exerts this effect only at molecule sizes of several hundred thousand Dalton <|[72]|>. Dextran, which causes a decrease in erythrocyte aggregation after a single infusion, leads to a steady increase in aggregation after repeated infusion over several days. HES on the other hand improves this parameter after repeated infusion. Only after infusion of high doses of the more highly substituted starch, aggregation increases slightly.



The in-vivo molecular weight helps explaining this differing behavior. In a single, short infusion of Dextran, the smaller molecules, which lower the tendency to aggregate, dominate. After repeated, slow infusion, larger molecules that are difficult to eliminate accumulate, leading to an increase in erythrocyte aggregation.



Erythrocyte aggregation is lowered by HES because HES molecules are metabolized in-vivo and the resulting small molecules inhibit erythrocyte aggregation. A loading dose of 10% HES 200/0.62 leads through the massive presence of large molecules to an increase in aggregation. With continuing therapy, however, the large molecules are broken down enzymatically and smaller, aggregation-inhibiting molecules dominate. Through a loading dose of HES 70/0.5 many small molecules appear in the blood, slowing aggregation. After long-term infusion therapy, smaller molecules are eliminated and aggregation increases again. HES 200/0.5 has the strongest aggregation-lowering effect, which reaches its maximum effect at the end of therapy.



The different distribution of the molecular weight causes the different rheological properties of the substances studied. After infusing Dextran 40, with an in vitro molecular weight of 52,000 D, larger molecules cumulate in vivo and Mw reaches twice the initial value. HES, on the other hand, is metabolized rapidly in vivo through alpha-amylases.



The in-vitro Mw of the HES 200/0.5 infusion solution is 200,000 D. In vivo, this substance is broken down quickly. Highly substituted starch (HES 200/0.62) is broken down slowly, the average Mw in-vivo is much higher at 120,000 D. Because elimination is more difficult, macromolecules accumulate. Low molecular weight starch hardly changes its initial Mw (60,000 D) and has favorable rheologic properties, similar to HES 200/0.5. Both substances do not lead to an increase in serum concentration.



A pronounced increase in plasma viscosity after a hemodilution therapy with HES 450/0.7 was observed by Boldt et al. <|[73]|>. Because of the high initial Mw and the high degree of substitution, this starch solution has a high intravascular Mw, which explains the increase in plasma viscosity.



Platelet count and function



Platelet count was not affected beyond the dilution effect by any of the solutions studied. After an initial dilution-induced decrease in platelet count, the number of platelets increased during the therapy, possibly the result of a reactive release of platelets. For the starch solutions, platelet volume was also measured. All the starch solution caused a small, yet significant decrease in platelet volume.



Platelet volume decreases in a dose-dependent effect. The more highly substituted HES 200/0.5 caused a larger decrease in platelet volume than HES 200/0.5 and HES 70/0.5. Our analysis of the MW-distribution showed that the decline in platelet volume was more pronounced, if the in-vivo Mw of the starch was higher <|[47]|>.



The infusion of synthetic colloids causes an increase in colloid-osmotic pressure, resulting in a shrinkage of the platelets and a decline in platelet volume. This hypothesis is supported by the fact that platelet count at the maximum value and distribution width of the platelets remained constant <|[47]|>. An increased breakdown or phagocytosis of platelets after attaching to HES can also not be excluded, because platelets are a part of the unspecific immune defenses of the body and play an important role in the phagocytosis and elimination of foreign particles <|[74]|>. It is also possible that platelet microaggregates are dissolved through hemodynamic and rheological effects. The dissolution of these reversible platelet aggregates would increase the number of platelets counted and seemingly lower the mean platelet volume, explaining the increasing platelet count and decreasing platelet volume.



The diagnostic importance of decreasing platelet volume is unclear <|[75-79]|>. Several studies showed that a positive correlation exists between platelet volume, function and bleeding time <|[78, 80-82]|>. Therefore, one can suspect that platelet function is impaired during a volume therapy with HES. The significant decline of platelet volume observed in this study seems to have only a small effect on platelet volume, because platelet aggregation is slightly impaired only through the infusion of 10% starch.



Platelet aggregation is impaired more by Dextran 40 solution than by HES 200/0.5 or 70/0.5 <|[22, 83-85]|>. They are suited less for the acute therapy of a stroke, because intracerebral hemorrhage has to be excluded with cranial CT before therapy can be initiated <|[86]|>.



Plasmatic coagulation system



The clinical relevance of coagulation disorders induced through synthetic colloids is underlined by the severe hemorrhagic complications observed after repeated infusion of highly substituted, high molecular weight starch <|[24-30, 32]|>.



The reduction of thromboplastin time (quick) was most pronounced for Dextran 40 (23%), followed by HES 200/0.62. After infusion of HES 200/0.5 or low molecular weight HES, no relevant changes in thromboplastin time were observed.



PTT is affected differently, depending on the substance used, the dose and duration of therapy. Infusion of low molecular weight HES and HES 200/0.5 resulted in no notable effect on PTT beyond the dilution effect. Infusion of Dextran 40 increased PTT by 24%, infusion of 10% HES 200/0.62 by 43%.



The impairment of the intrinsic system of coagulation, mostly due to an impairment of factor VIII/vWF complex is shown by the prolonged PTTtime. The importance of the von Willebrand factor lies in the attachment of platelets to the damaged vascular endothelium. A shortage of von Willebrand factor rarely leads to spontaneous bleeding, but after bleedings even after minor injury can be considerably prolonged. However, after repeated infusion of HES 200/0.62 factor VIII/vWF complex dropped regularly below the hemostasiological limit of 30%. Yet, none of our patients suffered from clinically relevant hemorrhagic complications that needed treatment. Only one patient had spontaneous gum bleeding.



vWF has a stabilizing function as a carrier protein for factor VIII:C. The lowering of factor VIII:C in von Willebrand syndrome is therefore probably a secondary consequence of the lowering in vWF <|[87]|>.



It is not well understood how HES affects factor VIII/vWF complex. After the addition of HES to human serum in in-vitro studies, Batlle et al. <|[88]|> and Stump et al. <|[31]|> did not observe a decrease in factor VIII/vWF complex that went beyond the dilution effect. The authors therefore suspected the occurrence of an in-vivo precipitation or an inhibition of synthesis or release. Because patients with monoclonal gammopathy suffer from an acquired von Willebrand syndrome <|[89-91]|>, an accelerated elimination of factor VIII/vWF complex after attachment of starch molecules is another possible explanation. The finding of a factor VIII-IgG-paraprotein complex by Siostrzonek et al. <|[89]|> supports this hypothesis. The authors explained the low factor VIII levels and the rapid drop after administration of cryoprecipitate or desamino-D-arginine vasopressin (DDAVP) with an accelerated elimination of the complex. The successful treatment of an acquired von Willebrand syndrome with immuno globulins lends further credence to the view of immune-system modulated mechanism <|[91]|>.



In our study, we were able to show for the first time through vWF multimeric analysis that all multimer components are reduced to the same extent by the infusion therapy. The coagulation disorder caused by HES is therefore a purely quantitative defect, corresponding to type I von Willebrand syndrome <|[50, 90, 92]|>. Because the coagulation disorder caused by HES was classified for the first time, it was possible to consider possible therapies, since particularly type I of the von Willebrand syndrome can be treated with the vasopressin derivative DDAVP <|[62]|>.



The use of highly substituted starch could be therapeutically useful in patients with an increased risk of thrombosis or reinfarction, because of the observed coagulation disorder.



Different starches have different effects on coagulation parameters. According to our studies, the drop in factor VIII/vWF complex correlates with the dose, the initial molecular weight, the share of C2-hydroxyethylated starch molecules and particularly the degree of substitution of the starch. This is the case because the larger molecules that are difficult to eliminate are responsible for the coagulation disorder. Therefore, hemorrhagic complications can be avoided through the choice of a suitable starch with a low in-vivo Mw <|[45,49]|>.



After a single infusion of Dextran or starch, a shortening of thrombin time and a lowering of fibrinogen concentration has been described repeatedly in the literature <|[85, 93-95]|>. These effects are probably due to an accelerated polymerization of fibrin. For hemostasis, they are of secondary importance. The largest decrease can be observed for Dextran 40 and highly substituted starch, whereas HES 200/0.5 and low molecular weight HES 70/0.5lead to no relevant changes in thrombin time and fibrinogen concentration.



Highly substituted HES 200/0.62 reduces Factors XI and XII by approximately one-half. This indicates that the impairment of the intrinsic system is not limited to factor VIII/vWF complex. Significant drops of factor XI and XII can be avoided through the use of low molecular weight starch or HES 200/0.5.



In addition, a small, clinically not relevant reduction in factor II and X was observed. Factors V, VII and IX were not affected beyond the dilution effect.



C1-inactivator, a central inhibitor of coagulation factor XI and XII showed no significant changes during the therapy in concentration or activity. The average drop in activity of factor XI and XII can therefore not be explained through a change in C1-inhibitor <|[96]|>.



Substitution patterns of different hydroxyethylstarches



Two starches that differed in their C2/C6 ratio and were identical in concentration, initial molecular weight and degree of substitution were studied by us with respect to their effects on rheology and coagulation system with <|[45, 49]|>.



Yoshida et al. <|[97, 98]|> showed in animal experiments that 2-hydroxyethylated starch is metabolized more slowly. Our studies confirmed this. Hematocrit was lowered in both groups through the loading dose equally by 18%. During the further therapy however, HES (1) with the lower elimination rate had a longer-lasting volume effect and the maximum decrease in hematocrit was significantly larger than in the other group. Erythrocyte aggregation was affected favorably by both substances. The effect was more pronounced for HES (1). The continuous increase in plasma viscosity measured for HES (1) was higher than for HES (2).



The coagulation system is affect more unfavorably by the starch with the higher C2/C6 ratio. PTT increase was more pronounced for HES (1) than for HES (2). Factor VIII:C was also more reduced by HES (1) than HES (2). The longer-lasting volume effect, the larger increase in plasma viscosity and the greater impairment of the coagulation system caused by HES (1) are due to an accumulation of large molecules that are difficult to eliminate. This is confirmed by the serum concentrations and the distribution of the molecular weight. The higher share of C2-hydroxyethylated starch that is difficult to break down resulted in a continuous increase of serum concentration up to 15 mg/ml through the infusion of HES 200/0.5 (1). Maximum serum concentration in group 2 was 10 mg/ml. The analysis of molecular weight distribution showed that the medium molecular weight of HES (1) immediately after the loading dose was higher than for HES (2). At the end of therapy, the values were 95,000 D for group 1 and 84,000 D for group 2.



The starch solutions studied by us, all of which are labeled 10% HES 200/0.5, differ in their hemostasiological and rheological properties because of their different substitution patterns. HES 200/0.5 (1), with the higher C2/C6 ratio is metabolized slowly and has very similar characteristics to HES 200/0.62. HES 200/0.5 (2) occupies a middle position among the three starches, regarding volume effect and hemostasiological and rheological properties <|[45, 49, 58]|>. In general, it is the in-vivo, and not the in-vitro size of the molecules that determines the pharmacological properties of these synthetic colloids and their effects on coagulation <|[99]|>.



CONCLUSION



Dextran 40, high-molecular weight HES 450/0.7 and highly substituted medium molecular weight HES 200/0.62 have adverse effects on the coagulation system. The two latter synthetic colloids cause a decrease in factor VIII/vWF complex, resulting in increased hemorrhagic risk. In addition, they have unfavorable rheological properties, increasing plasma viscosity and impair erythrocyte aggregation. These synthetic colloids are difficult to break down and eliminate for the organism, resulting in an accumulation of large molecules, which are responsible for the adverse effects on coagulation. Dextran 40 increases the risk of bleeding through an inhibition of platelet aggregation and has the added disadvantage of a higher incidence of anaphylactoid reactions <|[100-102]|>.



Medium molecular weight starch HES 200/0.5 has no relevant adverse effects on coagulation, even after repeated administration. It is easily degradable and broken down rapidly in vivo into rheologically favorable molecule sizes.



Low molecular weight starch (HES 70/0.5) has very favorable rheological and hemostasiological properties. It is a safe synthetic colloid without relevant adverse effects of coagulation and the added benefit of improving rheological parameters.



However, one has to take into account that the availability of different HES preparations varies greatly from country to country <|[103]|>. The newly developped and promising HES 130/0.4 should, judging from the pharmacology, have favorable hemostaseological and rheological properties. However, the results of the latest studies are not yet available.

Appendix: 



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