Pathophysiology and management of coagulation disorders in critical care medicine - Chapter 10 Decreased Circulating Levels of Von Willebrand Factor after Intravenous Administration of a Rapidly Degradable Hydroxyeth

Pathophysiology and management of coagulation disorders in critical care

medicine

Citation for published version (APA):

de Jonge, E. (2000). Pathophysiology and management of coagulation disorders in critical care medicine.

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ChapterChapter 10

Decreasedd Circulating Levels of Von Willebrand

Factorr after Intravenous Administration of a

Rapidlyy Degradable Hydroxyethyl Starch

(HESS 200/0.5/6) in Healthy Human Subjects

E.. de Jonge1, M. Levi2, RRBüller2, F. Berends3, J.Kesecioglu1

Departmentss of (1) Intensive Care, (2) Vascular Medicine andd (3) Clinical Chemistry. Academic Medical Center, University of

Amsterdam,, The Netherlands

Summary y

Impairmentt of haemostasis has been described with high molecular weightt hydroxyethyl starch (HES) and with slowly degradable medium molecularr weight HES, whereas rapidly degradable medium molecular weight HESS is generally considered to have no important effects on blood coagulation. Thiss study was undertaken to investigate the effects of a rapidly degradable mediumm molecular weight (HES) plasma substitute on primary haemostasis and bloodd coagulation in human subjects.

Ninee healthy men were studied in a randomised, cross-over study to investigatee the effects of a 60-minutes intravenous infusion of either 1 litre HESS 200/0.5/6 (HAES-steril 6%) or 4% albumin (control). The infusion of HES resultedd in decreased circulating levels of von Willebrand factor antigen (from 855 8% to 59 6% after HES vs. from 80 7% to 69 8% after albumin, p < 0.05)) and ristocetin cofactor activity (-28 3% after HES vs. -5 6% after albuminn (p < 0.01). This was associated with an impairment of in vitro platelet functionn as determined with the PFA-100 platelet function analyzer (change of closuree time of+30 6% after HES vs. +9 7% after albumin at 60 min using collagen/epinephrinee cartridges, p<0.05 and +32 10% vs. +4 8% using collagen/ADPP cartridges, p=0.01). Also, a trend for decreased levels of factor VIIIxx was observed (from 83 7% to 63 7% vs. From 88 6% to 74 7% at 600 min, p=0.06). No significant difference in bleeding time was observed. A slightt decrease of circulating levels of fibrinogen, factor V and factor VII was foundd in both study groups, which could be completely explained by dilution. Thrombinn generation, as assessed by F1+2 and TAT concentrations was not changedd after either infusion.

Ourr results demonstrate that infusion of 1 litre of HES 200/0.5/6 results inn decreased plasma levels of von Willebrand factor associated with impairment off platelet function.

Introduction n

Hydroxyethyll starches (HES) are widely used as plasma substitutes becausee they carry no risk of transmitting viral or other transfusion related diseasee and are less expensive than plasma products. HES is a natural polymer off amylopectin. Different types of HES are characterized by the mean

molecularr weight (MW), the degree of substitution of hydroxyethyl for hydroxyll groups and by the C2/C6 hydroxyethylation ratio. In the United States,, the predominantly used HES has an average molecular weight of 480 andd a substitution ratio of 0.7 (HES 480/0.7, Hetastarch). Significant bleeding complicationss have been described after treatment with HES 480/0.71;2 and are associatedd with decreased levels of circulating factor VIII and von Willebrand factorr (vWF).3"6 HES with medium molecular weight is commonly used in Europe.. After repeated use of medium MW HES that is slowly degradable (high substitutionn ratio, high C2/C6 ratio) a marked decrease of plasma levels of vWF wass observed, probably due to accumulation of HES molecules with a high molecularr weight.7 In contrast, highly degradable medium MW HES with a low substitutionn ratio and low C2/C6 ratio (HES 200/0.5/6) is generally considered too have no important influence on coagulation and platelet function.8"10

However,, also with this compound a prolongation of the bleeding time and increasedd blood loss following orthopedic surgery has been reported.11 The effectss of medium molecular weight HES with low substitution and low C2/C6 ratioo on primary haemostasis and blood coagulation has, however, not been characterizedd precisely.

Too investigate the effects of HES 200/0.5/6 on blood coagulation we performedd a randomised, controlled cross-over study in healthy human subjects. Wee used sensitive and specific tests to delineate any influence on in vivo thrombinn generation and evaluated primary haemostasis using bleeding time andd ex vivo platelet aggregation measurements.

Materialss and Methods

Thee study was approved by the institutional review board of the Academic Medicall Center, University of Amsterdam and written informed consent was obtainedd from all subjects.

StudyStudy design

Thee study was performed as a randomised, controlled, double-blind, cross-overr experiment. Nine healthy men (20 - 39 years of age) participated in thee study. They did not use any medication and had no illness in the two months beforee the study. Blood pressure, plasma creatinine, platelet count, prothrombin timee (PT) and activated partial thromboplastin time (aPTT) were measured beforee the study and were normal. There was no history of increased bleeding tendency.. Each subject was studied on two occasions with a 2-3 week washout period.. Subjects were randomised to receive a one-hour intravenous infusion of eitherr 1000 ml 6% hydroxyethyl starch in NaCl 0.9% (mean MW 200.000, MS=0.5,, C2/C6 ratio=6; HAES-steril 6%, Fresenius BV, 's-Hertogenbosch, the Netherlands)) or 1000 ml Albumin 4% in NaCl 0.9% as control experiment (200 mll Albumine 20% in NaCl 0.9%, Central Laboratory of the Red Cross Bloodtransfusionn Service, Amsterdam, The Netherlands, with simultaneous infusionn of 800 ml NaCl 0.9%). After the washout period the alternative infusion wass administered. All volunteers were closely monitored during the study period,, including repeated physical examination and blood pressure measurements. .

BloodBlood collection and bleeding time determination

Venouss blood samples were obtained by separate venipunctures with use off 21-gauge butterfly needles, directly before the HES or albumin infusion, and 30,, 60, 120, 240 and 360 minutes thereafter. Blood for haemoglobin, haematocritt measurement and platelet count was drawn in vacutainer tubes containingg 0.38 mmol/1 EDTA (final concentration). Blood for clotting tests, functionall assays of clotting factors, platelet aggregation tests, PFA-100 analysis andd markers for thrombin generation (prothrombin fragment F1+2 and thrombin-antithrombinn complexes) was collected in plastic tubes containing 3.2%% (w/v) sodium citrate; the ratio of anticoagulant to blood was 1:9 (v/v). Platelet-richh plasma (for platelet aggregation tests) was obtained by centrifuging thee blood samples at 180 x g for 10 minutes at room temperature. Blood samples forr other assays were immediately centrifuged at 4°C for 30 minutes at 1,500 x g andd plasma was stored at -70°C until assayed. Bleeding time test was performed usingg a fully-automated incision-making device (Surgicutt®, International Technidynee Corporation, Edison, New Jersey, USA), directly before and 60, 120 andd 240 minutes after start of the infusion and according to a standard procedure6. .

Assays Assays

Routinee haematologic tests (platelet count, haemoglobin, haematocrit) weree performed using standard laboratory methods. Platelet aggregation was measuredd according to standard techniques12 at 37°C with ristocetin (1.2 mg/ml, Paesii and Lorei, Hannan, Germany) and collagen (1 jig/ml, Chronolog Corp. Havertown,, PA). Light transmission of the stirred platelet-rich plasma was recordedd relative to the platelet-poor plasma blank (Whole Blood Aggrometer, Chronologg Corp, Havertown, PA). In vitro platelet function was also evaluated usingg the PFA-100 system (Dade Behring, Marburg, Germany) as described previously.133 The analyzer aspirates whole citrated blood through a capillary into aa cup where platelets adhere and aggregate by coming in contact with a membranee coated with collagen and either epinephrine or ADP and by high shearr rates generated under standardized flow conditions. The time required to obtainn full occlusion of an aperture in the membrane is reported as the "closure time"" and is considered to reflect the function of primary haemostasis.

Coagulationn times (aPTT and PT) were performed on a fully-automated coagulometerr (Electra 1600C, Medical Automated Inc, Pleasantville, NY), using Actinn FS and Thromborel S (Dade-Behring, Marburg, Germany) respectively. Thee functional assays of coagulation factors were performed by one stage clottingg assays with Recombiplastin (Ortho-Diagnostics Systems Inc, Rariton, NY)) and Actin FS (Dade-Behring, Marburg, Germany). Antigenic levels of von Willebrandd factor (vWF:ag) were measured using an ELISA.14 Ristocetin cofactorr activity (vWF:RCo) was measured as described previously.15 To assess thrombinn generation in vivo measurements of prothrombin activation peptide (Fl+2)) and thrombin-antithrombin complexes (TATc) were performed with ELISA'ss (Dade-Behring, Marburg, Germany).

PlasmaPlasma dilution

Plasmaa dilution by HES or albumin was calculated using the following equation:: D = Hb/Hb0x (l-Hto)/(l-Ht) in which D is the dilutionfactor, Hb0 and

Htoo are the haemoglobin concentration and haematocrit before infusion and Hb andd Ht are the haemoglobin and haematocrit after infusion.16 The calculated concentrationn of a plasma substance after dilution is C = C0 x D, in which C0 is

thee concentration before dilution, C is the concentration after dilution and D is thee dilutionfactor.

StatisticalStatistical Analysis

Valuess are given as means SEM. Differences in results between the HES andd albumin experiments were tested by repeated measurements analysis of variance.. A p-value < 0.05 was considered to represent a significant difference.

Figuree 1. Mean SEM

bleedingg time after intravenouss infusion of HESS (circles) or albumin (triangles).. P values representt difference betweenn HES and albumin experimentss by multiple measurementss analysis of variance. .

Results s

BleedingBleeding time and platelet function

Base-linee bleeding time was normal in all subjects. Infusion of both HES andd albumin resulted in a slight prolongation of bleeding time at 60 min (from 3.77 0.5 to 5.3 0.6 min after HES and from 3.9 0,6 to 4.9 0.7 min after albumin,, fig 1). Whereas bleeding time returned to base-line at 120 min after HES,, a prolongation was still observed up to 240 min after albumin. However, thee difference between the HES and albumin experiments did not reach

statisticall significance (p=0.08 by repeated measurements analysis of variance). Thee platelet count slightly decreased during infusion of either HES or albumin (-133 2% vs. -11 2%, p=NS). Platelet aggregation induced by ristocetin or collagenn was normal in all experiments and not influenced by either infusion (dataa not shown). Results of the platelet function analyzer PFA-100 are shown inn fig 2. Closure time with collagen-epinephrine test cartridges increased 30 6%% after infusion of HES and 9 7% after infusion of albumin (p<0.05). Closuree time with collagen-ADP cartridges increased 32 10 % after HES and 44 8% after albumin (p=0.01). Bleedingg time p=NS S 66 EE 4 -00 -1 ~ i — r r 300 60 120 0 240 0

1,66 - I 1.4 4 | |

I I

Figuree 2. In vitro platelet

functionn measured with PFA-1000 analyzer. Mean

SEM closure time (relativee to baseline) was measuredd with

collagen/ADPP (top) and collagen/epinephrine e cartridgess (bottom) after intravenouss infusion of HESS (circles) or albumin (triangles).. P values representt difference betweenn HES and albumin experimentsexperiments by multiple measurementss analysis of variance. . 44 33 22 11 00 11 0,88 0,66 0,44 0,22 -TT aP-TT 11 p=0.09 II 1 1 00 30 60 F1+2 2 p=NS S II I 1200 2' timee (min) 10 0

- 4 4

Figuree 3. Mean SEM

aPTTT (top), given as changee from base-line and prothrombinn fragment F1+22 (bottom) after intravenouss infusion of HESS (circles) or albumin (triangles).. P values representt difference betweenn HES and albumin experimentsexperiments by multiple measurementss analysis of

CoagulationCoagulation studies

Base-linee values of PT, aPTT and functional assays of coagulation factors weree similar in both study groups. In the HES group, there was a trend to

prolongedd aPTT values, whereas the aPTT remained unchanged in the albumin groupp (p = 0.09), fig 3). A small increase in PT was found after infusion of bothh HES and albumin without difference between groups (+1.1 0.3 vs. 0.7 0.11 sec, p=NS). In both study groups, theree was a decrease in fibrinogen, factor VV and VII. This decrease could be completely explained by the dilution effect (dataa not shown). No difference was observed between groups. Thrombin generationn as measured by prothrombin activation peptide F1+2 (fig 3) and thrombin-antithrombinn complexes (TATc, data not shown) was not influenced byy either infusion.

vWF:ag g

p<0.01 1

I I - I — ? ?

vWF:ag g

correctedd for dilution

p<0.05 5

- i — II

r-ii i i 1 1

-00 30 60 120 240 timee (min)

Figuree 4. Mean SEM plasmaa concentrations of vWF:agg (top) and factor VIIIxx (bottom) after intravenouss infusion of HESS (circles) or albumin (triangles).. The middle panell shows vWF:ag after correctionn for dilution by multiplyingg by the dilutionfactorr D. P values representt difference betweenn HES and albumin experimentss by multiple measurementss analysis of variance. .

VonVon Willebrandfactor

Resultss of vWF:ag and factor VIII:c are presented in fig 4. Plasma vWF levelss decreased in both groups. This decrease was more pronounced after HES comparedd to albumin (from 85 8% at base-line to 59 6% at 60 min after HESS and from 80 7% to 69 8% after albumin, p < 0.05). In the albumin group,, the decrease in vWF could be explained by dilution by the infused fluid, whereass in the HES group vWF decreased more than by dilution only (p O.01). Circulatingg levels of factor VIIIx decreased after HES from 83 7% to 63 7%% and after albumin from 88 6% to 74 7% at 60 min with a trend towards statisticall significance for the difference between HES and albumin (p=0.06). Parallell to the decrease in antigenic levels of vWF we also observed a 28 3% decreasee in ristocetin cofactor activity after HES and 5 6% decrease after albuminn (fig 5, pO.01).

vWRRco o p<0.01 1

Figuree 5. Mean SEM

ristocetinristocetin cofactor activity (vWF:Rco)) after

intravenouss infusion of HESS (dark bars) or albuminn (open bars). Valuess are given relative too base-line. P value representss difference betweenn HES and albumin experimentss by multiple measurementss analysis of variance. .

Timee (rrin)

PlasmaPlasma dilution

Noo difference was observed in plasma dilution between both study groups.. Dilution factors at 30, 60, 120, 240 and 360 min were 0.89, 0.81, 0.84, 0.877 and 0.93 after HES infusion and 0.93, 0.87, 0.91, 0.89 and 0.91 after albuminn (p = NS). C C ~T ~T 0) ) CO O Co o -Q Q 1--0 1--0 | 0 , 8 8 0,6 6 -30 0 60 0 120 0

Discussion n

Inn this study we found a HES-induced decrease of circulating levels of factorr VIIIx and von Willebrand factor. After correction for dilution of plasma byy the infused fluid, vWF:ag levels did not change in the albumin experiments, whereass a 26% decrease was found after infusion of HES. Associated with the reductionn in vWF antigen, we found a similar decrease in ristocetin cofactor activity.. Although platelet aggregation studies were normal in all experiments, wee did find an impairment of in vitro platelet function as determined with the PFA-1000 platelet analyzer. Indeed, this PFA-100 analyzer has been shown to be veryy sensitive and specific in detecting von Willebrand disease.17 Although a slightt prolongation of bleeding time was observed after both HES and albumin administration,, this difference was not statistically significant. HES

administrationn resulted in only a small prolongation of aPTT of borderline significance.. This was probably caused by the decrease in factor VIII/vWF in combinationn with some dilution of coagulation factors. Thrombin generation as measuredd by prothrombin fragment F1+2 was unchanged after either infusion.

Reviewingg the literature on the effects of HES on haemostasis yields conflictingg results. High molecular weight HES can prolong aPTT values, decreasee factor VIIIx and vWF,1;3;18;19 and induce increased post-operative bloodd loss.20 However, this effect of HES on haemostasis seems to depend on itss in vivo molecular weight.7 Unsubstituted starch is degraded in vivo within minutess through action of amylases in the blood. Hydroxyethylation of HES slowss this process. Thus, in vivo molecular weight is determined by the mean molecularr weight of the infused HES and its degree of substitution of hydroxyl byy hydroxyethyl groups. Degradation of HES is also impaired by a high C2/C6 ratio,, i.e. the relative number of hydroxyethyl groups at the C2 atom compared too the C6 atom of the glucose units.9 Accordingly, decreased vWF levels have beenn described after administration of medium weight HES with highh degree of substitution77 or high C2/C6 ratio,21 both leading to in vivo accumulation of HES moleculess with high molecular weight. Medium weight HES with a degree of substitutionn of 0.5 and a C2/C6 ratio of 6 (HES 200/0.5/6) is commonly used as plasmaa substitute in Europe, is rapidly degradable without accumulation of macromolecules,, and is considered safe regarding coagulation disturbances.8*10 Treibb et al. carried out a 10-day haemodilution therapy in neurologic patients andd found no influence on factor VIIIx and vWF after HES 200/0.5/6 administration.9 9

actingg as an acute-phase protein, increases in acutely-ill patients, masking the loweringg effects of HES infusion. Our findings are supported by Kapiotis et al. whoo found a specific lowering effect of HES 200/0.5/6 on factor VIIIx in healthyy volunteers.22 In this study vWF was not measured.

Althoughh statistically significant, the magnitude of the effects of HES in ourr study was only modest. It seems unlikely that HES, when given to patients inn quantities comparable to the amount given in this study (i.e. 1 L), will induce ann important impairment of haemostasis. This is in accordance with clinical studiess that found no increased bleeding tendency after administration of mediumm molecular weight HES.10:20;23 Nevertheless, especially when given in largee quantities, HES could induce a clinically relevant bleeding tendency in particularr in patients with already low circulating levels of vWF or if

haemostasiss is already compromised e.g. after cardio-pulmonary bypass. Unfortunately,, alternatives for HES have also been associated with disturbed haemostasis.. Dextran infusion lowers factor VIII in plasma and prolongs the bleedingg time.24 Gelatin-based plasma substitutes induce a decrease of vWF, prolongg bleeding time and decrease thrombin generation.25 Albumin is

expensivee and its use on intensive care units has been associated with increased mortality.266 Few studies have been published, comparing the effects of different plasmaa substitutes on post-operative blood loss. One study found increased bloodd loss after orthopaedic surgery when HES 200/0.5/6 was given compared too gelatin.11 However, other studies found no difference in post-operative blood losss when medium molecular weight HES was compared with gelatin20;23 or albumin.20;277 Based on the present study and on review of the literature, there is noo reason to restrict the use of medium weight HES as plasma substitute. In situationss when the vWF lowering effects become clinically relevant (e.g. if massivee amounts of plasma substitutes are given in bleeding patients, the use of plasmaa or desmopressin (DDAVP)6 to increase vWF levels, should be

considered. .

Wee conclude that rapidly degradable medium molecular weight HES 200/0.5/66 induces a mild reduction of plasma vWF, associated with impaired platelett function. Caution should be exercised when large amounts of HES are administeredd and when pre-existent disturbances in haemostasis exist.

References s

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