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Pathophysiology and management of coagulation disorders in critical care
medicine
de Jonge, E.
Publication date 2000
Link to publication
Citation for published version (APA):
de Jonge, E. (2000). Pathophysiology and management of coagulation disorders in critical care medicine.
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ChapterChapter 9
Impairedd Haemostasis by Intravenous
Administrationn of a Gelatin-based Plasma Expander
inn Human Subjects
E.. de Jonge1, M. Levi2, F. Berends3, A.E. van den Ende2, J.W.. ten Cate2 and C.P. Stoutenbeek'
Departmentss of (1) Intensive Care, (2) Vascular Medicine andd (3) Clinical Chemistry.
Academicc Medical Center, University of Amsterdam, The Netherlands
Summary y
Thee aim of this study was to investigate the effects of a gelatin-based plasmaa expander on blood coagulation and haemostasis in human subjects. Sixx healthy men were studied in a randomised, controlled cross-over study to investigatee the effects of a 60-minutes intravenous infusion of either 1 litre gelatin-basedd plasma substitute (Gelofusine) or 0.9% NaCl (control). The infusionn of gelatin resulted in a 1.7 fold increase in bleeding time at 60 min and aa 1.4 fold increase at 120 min, while saline had no effect (p<0.05). Aggregation studiess revealed a significant impairment of ristocetin-induced platelet
aggregationn (p<0.05), associated with a substantial decrease of vWF:ag (-32% vs.. -5%, p<0.05) and ristocetin co-factor (-29% vs +1%, p<0.05) and without in
vitrovitro impairment of the platelet glycoprotein lb receptor. Gelatin caused a
decreasee in thrombin-antithrombin complexes (-45% vs. -4%, p<0.05) and F1+22 (-40% vs. +1%, p<0.05). The decrease in circulating levels of vWF:ag, vWFF R:Co, thrombin-antithrombin complexes and F1+2 was more than could bee expected by the calculated plasma-dilution generated by Gelofusine. Ourr results demonstrated that the administration of a gelatin-based plasma substitutee results in a significant impairment of primary haemostasis and thrombinn generation. The defect in primary haemostasis appears to be related to aa gelatin-induced reduction in von Willebrand factor, whereas the decreased thrombinn generation may be due to the dilution of coagulation factors induced byy Gelofusine.
ImpairmentImpairment ofhaemosatsis by a gelatin-based plasma expander
Introduction n
Plasmaa substitutes are currently used for intravascular volume expansion inn various clinical situations. Particularly, during complicated surgical
proceduress or in patients with serious bleeding and hypotension, large volumes off these plasma expanders are administered intravenously. Obviously, in such clinicall situations interference of these compounds with the haemostatic system iss undesirable.
Gelatin-basedd solutions have been considered relatively safe and it has beenn reported that these compounds did not influence peri-operative
coagulationn other than by dilution in a large retrospective and uncontrolled study.11 However, recent in vitro studies indicate that gelatin-based solutions mightt affect platelet adhesion and clot stability, measured by
thromboelastography,2,33 although the clinical relevance of these observations hass been debated.4'5 To investigate the effects of a gelatin-based plasma expander,, we performed a randomised, controlled cross-over study in healthy humann subjects. We used sensitive and specific tests to explore possible effects onn in vivo thrombin generation and to delineate possible adverse effects on the processs of haemostasis.
Materialss and Methods s
Thee study was approved by the institutional research and ethical committeess of the Academic Medical Center, University of Amsterdam and writtenn informed consent was obtained from all subjects.
StudyStudy design
Thee study was performed as a randomised, controlled, double-blind, cross-overr experiment. Six healthy men (31-37 years of age) volunteered to participatee in the study. They did not use any medication and had no illness in thee two months before the study. Blood pressure, plasma creatinine, platelet count,, prothrombin time (PT) and activated partial thromboplastin time (aPTT) weree measured before the study and were normal. There was no history of increasedd bleeding tendency. Each subject was studied on two occasions with a
1-- 3 week wash-out period. Subjects were randomised to receive either a one-hourr intravenous infusion of 1000 ml 4% gelatin in 0.9% NaCl (Gelofusine, Viforr Medical SA, Switzerland) or 1000 ml NaCl 0.9% (control). After the
wash-outt period the alternative infusion was administered. All volunteers were closelyy monitored during the study period, including repeated physical
examinationn 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 gelatin or isotonic saline infusion,, and 30, 60, 120 and 240 minutes thereafter. Blood for haematocrit measurementt and platelet count was drawn in vacutainer tubes containing 0.38 mmol/11 EDTA (final concentration). Blood for clotting tests, functional assays off clotting factors, platelet aggregation tests, and markers for thrombin
generationn (prothrombin fragment F1+2 and thrombin-antithrombin complexes) wass collected in plastic tubes containing 3.2% (w/v) sodium citrate; the ratio of anticoagulantt to blood was 1:9 (v/v). Platelet-rich plasma (for platelet
aggregationn tests) was obtained by centrifuging the blood samples at 180 x g for 100 minutes at room temperature. Blood samples for other assays were
immediatelyy centrifuged at 4oC for 30 minutes at 1,600 x g and plasma was storedd at -70oC until assayed. Bleeding time test was performed using a fully-automatedd incision-making device (Surgicutt, International Technidyne Corporation,, Edison, New Jersey, USA), directly before and 60 and 120 minutess after start of the infusion and according to a standard procedure.6
Assays Assays
Routinee haematologic tests (platelet count, haematocrit) were performed using standardd laboratory methods. Platelet aggregation was measured according to standardd techniques? at 37oC with ristocetin (1.2 mg/ml, Paesi and Lorei, Hannan,, Germany), collagen (0.4 ug/ml, Organon Technika, Turnhout, Belgium)) and ADP (1.0 Mg/ml). Light transmission of the stirred platelet-rich plasmaa was recorded relative to the platelet-poor plasma blank (Whole Blood Aggrometer,, Chronology Corp, Haverton, PA). Data are given as the maximal increasee in light transmission within 5 minutes (amplitude) or as the slope of thee aggregation curve.
Coagulationn times (aPTT and PT) and functional assays of coagulation factorss were performed by one-stage clotting assays with thromboplastin IS (Drade,, Diidingen, Switzerland) on a semi-automated coagulometer (Electra
1600C,, Medical Laboratory Automated Inc, Pleasantville, NY). Antigenic levelss of von Willebrand factor (vWF:ag) were measured using an ELISA.8 Ristocetinn cofactor activity (vWF R:Co) was measured as describedd previously9
ImpairmentImpairment ofhaemosatsis by a gelatin-based plasma expander
(normall values 80-120%). Multimeric analysis of von Willebrand factor was donee in plasma samples before and 60 min after the start of the infusion with gelofusinee or saline. Multimeric analysis was performed, essentially as describedd previously10, by electropheresis on a 1.7% agarose gel using a PhastGell system (Pharmacia Biotech, Uppsala, Sweden) and subsequent immunoblottingg using a horseradish peroxidase labelled anti-human von Willebrandd antibody (Dako, Glostrup, Denmark). The plasma concentrations of F1+22 and TAT complexes were measured with ELISA's (Beringwerke AG, Marburg,, Germany).
PlasmaPlasma dilution
Plasmaa dilution by gelatin or saline was calculated using the following equation:: D = (1-Ht*)/[(1-Ht) x (Ht'/Ht)], in which D is the dilutionfactor, Ht is thee haematocrit before infusion and Ht' is the haematocrit after infusion.11 The calculatedd concentration of a plasma substance after dilution is C' = C / D, in whichh C is the concentration before dilution, C' is the concentration after dilutionn and D is the dilutionfactor.
FlowcytometryFlowcytometry experiments
Too study the effect of gelatin on the pivotal platelet receptor responsible forr platelet adhesion mediated by ristocetin and von Willebrand factor (i.e. glycoproteinn lb), a separate in vitro experiment was performed. Blood samples off volunteers (n=2) were incubated with various amounts of gelofusine or 0.9% NaCll (control), resulting in 0,20 and 50% dilution. Aliquots of these mixtures weree fixed in 1 ml PBS/EDTA (140 mM NaCL, 9.2 mM Na2HP04, 1.3 mM NaH2P04,, 5 mM EDTA, pH 7.4) with 2% paraformaldehyde (w/v) for 1 hour, thenn washed with PBS/EDTA (without formaldehyde). The washed cells were centrifugedd at 1,000 x g for 10 minutes and the pellet was incubated with 20 ul off FITC-conjugated antibodies to glycoprotein lb (CD42, Immunotech, Vienna, Austria).. After a next washing step in PBS/EDTA, the platelets were analysed withh a FAC-scan flow cytometer (Becton Dickinson Immunocytometry Systems,, San Jose, CA, USA). Platelet discrimination was performed using forwardd and side light scatter properties.
InIn vitro dilution experiments
Inn a separate experiment we studied the effects of in vitro incubation of plasmaa with gelofusine on collagen-binding of vWF and on vWF:ag. Plasma sampless of volunteers (n=2) were incubated during 60 minutes at 37°C with
increasingg amounts of gelofusine or NaCl 0.9% (control), resulting in 0, 10, 25, 333 and 50% dilution. After incubation collagen-binding and vWF:ag were measured.. Collagen-binding of vWF was measured as previously described 12 andd using a commercially available ELISA (Gradipore, North Ryde, Australia). Resultss are expressed as a percentage of values obtained in plasma that was not dilutedd with gelofusine (100%).
StatisticalStatistical analysis
Valuess are given as means SEM. Differences in results between the gelatinn and saline experiments were tested by analysis of variance and Newman-Keul'ss test for multiple comparison, as indicated. A p-value < 0.05 wass considered to represent a significant difference.
Bleedingg time 200 0 150 0 «100 0 50 0 * * 00 60 120 Timee (min)
Figuree 1. Prolongation of bleeding time (as percentage of
baseline,, mean SEM) after intravenous infusion of gelatin (open bars)) or saline (hatched bars). Normal values 2-6 min. Asterisk indicatee statistical significance for the comparison of gelatin with salinee (p < 0.05 by Newman-Keul's test)
ImpairmentImpairment of haemosatsis by a gelatin-based plasma expander
Results s
BleedingBleeding time and platelet aggregation aggregation studies
Resultss of bleeding time measurements are given in fig 1. Base-line bleedingg time was normal in all experiments. Infusion of gelatin resulted in a
1.77 fold prolongation of bleeding time at 60 min and a 1.4 fold prolongation at 1200 min, whereas the bleeding time after the infusion of saline remained unchangedd (p < 0.001 gelatin vs. saline at t=60 and t=120 min). In two subjects bleedingg time was outside the normal range after gelatin infusion (10 and 10,5 minn respectively, normal values <6 min)
ristocetin-induced d aggregationn (amplitude) 800 600 400 200 -ristocetin-induced d aggregationn (slope) Figuree 2. Platelet aggregationn induced by ristocetinristocetin and collagen coiiagen-mducedd after intravenous infusion aggregationn (amplitude) o f g e l a t i n ( s q u a r e s ) Or
TT saline (triangles). Asterisk indicate
statistical significance for thee comparison of gelatin ,, with saline (p < 0.05 by oo 60 120 180 240 Ne wman-Keul' s test)
Timee (min)
Thee platelet count slightly decreased during infusion of either gelatin or saline (-8%% vs -4%). However, these changes were not significant. Platelet
aggregationn induced by ristocetin was significantly impaired after infusion of gelatin,, as compared with the infusion of saline which effect lasted for the entiree observation period, (fig 2). There was no influence of gelatin on
collagen-inducedd aggregation (fig 2). Platelet aggregation induced by ADP was nott different between the two groups (data not shown).
II s.v 100 0 80 0 60 0 4 0 0 90 0 80 0 70 0 en n
2.8 8 2.4 4 , 2 . 0 0 1.6 6 1.2 2 1.2 2 1.0 0 0.8 8 0.6 6 0.4 4 T a TT c o m p l e x e s 600 120 180 240 Timee (min)
Figuree 3. Mean SEM
plasmaa concentrations of thrombin-antithrombin n complexess (normal: 2-7 ug/1)) and prothrombin fragmentt F 1+2 (normal: 0.8-1.33 nmol/1) after intravenouss infusion of gelatinn (squares) or saline (triangles).. Asterisk indicatee statistical significancee for the comparisonn of gelatin withh saline (p < 0.05 by Newman-Keul'ss test)
CoagulationCoagulation studies
Base-linee values of coagulation times and functional assays of coagulationn factors were similar in both study groups. In the gelatin group theree was a small increase in aPTT, whereas the aPTT remained unchanged in thee saline group (+1.8 sec vs. +0.4 sec at t=60 min, p < 0.005). Also the PT showedd a small prolongation in the gelatin group as compared to the saline group,, but this difference did not reach statistical significance (+0.7 sec vs. +0.1 sec,, p=0.06).
Inn both groups there was a decrease in fibrinogen, factor V, VII, VIIIx, XII and XII. At 240 min virtually all values had returned to base-line. This decreasee could be explained completely by dilution by the infused fluid (data nott shown). However, this gelatin-induced decrease in function coagulation factorss resulted in lower thrombin generation, as reflected by plasma levels of prothrombinn activation peptide F1+2 and complexes of thrombin with
antithrombinn (TAT) (fig 3). The difference in F1+2 and TAT between the two studyy groups was evident up to 4 hours after start of the infusion (p<0.0001 by analysiss of variance) After correction for dilution, these differences were still significantt (p<0.05).
VonVon Willebrandfactor
Afterr infusion of gelofusine we found a decrease in vWF:ag which was significantt even after correction for dilution (p<0.05 by analysis of variance). Parallell to the decrease in antigenic levels of vWF we also observed a
significantt decrease in ristocetin cofactor activity (p<0.05 by analysis of variance)) (fig 4). There were no essential changes in multimeric pattern after
ImpairmentImpairment of haemosatsis by a gelatin-based plasma expander
thee infusion of gelofusine and there were no differences between the gelofusine andd the saline treatment experiments (data not given).
2.8 8 2.44 -, 2 . 0 0 1.6 6 1.2 2 1.2 2 1.0 0 0.8 8 0.6 6 0.44 J T a TT c o m p l e x e s FF 1 + 2 600 120 180 240 Timee (min)
Figuree 4. Mean SEM
plasmaa concentrations of thrombin-antii thrombin complexess (normal: 2-7 ug/1)) and prothrombin fragmentt F 1+2 (normal: 0.8-1.33 nmol/1) after intravenouss infusion of gelatinn (squares) or saline (triangles).. Asterisk indicatee statistical significancee for the comparisonn of gelatin withh saline (p < 0.05 by Newman-Keul'ss test)
Ass shown in figure 5, collagen-binding of vWF was markedly decreased after in
vitrovitro incubation of plasma with gelofusine as compared to incubation with
saline.. There was no change in vWF:ag after incubation with gelofusine as comparedd to saline (data not given).
vWFF c o l l a g e n - b i n d i n g oo > i -o -o 200 30 40 dilutionn [%) Figuree 5. Mean vWF collagen-bindingg (as percentagee of base-line, n=2)) after in vitro dilution withh gelofusine (squares) orr saline (triangles).
PlasmaPlasma dilution
Infusionn of gelofusine resulted in dilution factors of 1.15, 1.25, 1.14 and 1.111 at t = 30, 60, 120 and 240 minutes. Dilution factors after saline were 1.03, 1.03,, 1.04 and 1.03 respectively.
FlowFlow cytometry experiments
Glycoproteinn lb expression on platelets and its ability to bind to the antibodyy were unchanged after incubation of blood with both gelatin and saline too a final dilution of 20 and 50% as compared with no incubation (data not shown). .
Discussion n
Intravenouss administration of the gelatin-based solution gelofusine in healthyy male volunteers impaired primary haemostasis and in vivo thrombin generation. .
Thee defect in primary haemostasis, as revealed by the prolongation of the bleedingg time, appeared to be associated with impaired ristocetin-induced platelett agregation. The underlying mechanism for this impairment could either bee a defect in the platelet receptor glycoprotein lb or interference with the ligandd of this receptor, i.e. von Willebrand factor. Flow cytometry experiments showedd no changes in platelet glycoprotein lb receptor expression after incubationn of whole blood with high concentrations of gelofusine, hence an effectt on von Willebrand factor might explain the observations. Indeed, plasma levelss of vWF:ag as well as ristocetin cofactor activity were significantly reducedd after infusion of gelofusine. This reduction was more than might be expectedd from plasma dilution only (the observed decline of vWF:ag after 120 minutess was 33% compared to base-line, whereas the anticipated decline by dilutionn would be 12%). The mechanism by which gelatin interacts with vWF is nott completely clear. Gelatin is composed of bovine collagen, and may bind to thee collagen binding site on the von Willebrand molecule.13 Indeed, we found in ourr in in vitro experiment that collagen-binding of vWF was decreased after incubationn of plasma with gelofusine, whereas vWF:ag remained unchanged. Thiss suggests that vWF binds to gelofusine using its collagen binding sites. The loww levels of vWFrag found in our in vivo experiments after gelofusine infusion mightt be explained by rapid clearance of the vWF-gelofusine complexes. Our conclusionss are supported by the findings of Tabuchi et al, who found that, in ann in vitro experiment, gelatin reduced both the velocity and the extent of
ristocetin-inducedristocetin-induced platelet aggregation and the velocity of polybrene-induced aggregation.. They concluded that gelatin interferes with plasma von Willebrand
factor.. Platelet-vWF was not affected by gelatin, thus preserving the extent of polybrene-inducedd aggregation.2
ImpairmentImpairment ofhaemosatsis by a gelatin-based plasma expander Besidess this impairment of primary haemostasis, blood coagulation was
significantlyy impaired as well. Most of this effect could be explained by gelofusine-inducedd plasmadilution, causing lower levels of functional coagulationn factor activity. The overall decrease in coagulation factors might thuss be an aspecific side-effect of plasma expanding. Nevertheless, the resulting effectt was a reduction in thrombin generation, as reflected by decreasing levels off prothrombin fragment F1+2 and thrombin-antithrombin (TAT) complexes. Thee effects of gelatin on platelet function and coagulation in healthy volunteers weree relatively small. However, they might be relevant in clinical practice in patientss with pre-existent abnormal haemostasis, e.g. after cardio-pulmonary bypass.. Indeed a positive correlation was found between blood loss and gelatin usee after cardiac surgery.2 Furthermore, the effects of gelatin may be more pronouncedd in patients with already low circulating levels of factor VIII as in mildd forms of Von Willebrand's disease, and upon infusion of large amounts of thiss plasma expander. Unfortunately, the alternatives for gelatin as plasma expanderr also might be associated with abnormal haemostasis. High-molecular weightt hydroxyethyl starch (HMW-HES) reduces platelet aggregation, prolongs bleedingg time and decreases the levels of circulating factor VIII.14
Low-molecularr weight hydroxyethyl starch is generally considered to have no importantt influence on coagulation and platelet function,15,16 but prolongation off the bleeding time has been reported.17 Dextran infusion lowers factor VIII in plasmaa and prolongs the bleeding time.18 It would be of interest to perform a comparativee study regarding the effects on coagulation and platelet function of differentt plasma substitutes in the future. It seems most important that clinicians aree aware of the adverse effects on the haemostatic system of these plasma expanders.. Furthermore, in some clinical situations (e.g. abnormal haemostasis orr need for massive plasma expansion) the use of plasma or plasma derivatives, albeitt associated with other limitations, should be considered.
Wee conclude that gelatin infusion to normal healthy volunteers leads to a decreasee in plasma levels of coagulation factors and a decrease in thrombin generation.. Furthermore, it prolongs the bleeding time probably due to decreasedd circulating levels of plasma-vWF. This decrease in plasma-vWF is moree than can be explained by dilution alone. Gelatin-based plasma substitutes shouldd be given cautiously to patients with pre-existent abnormal platelet functionn like patients after cardio-pulmonary bypass surgery or in patients with mildd forms of von Willebrand's disease. Furthermore, massive transfusion with gelatinn should be avoided.
References s
1.. Lundsgaard-Hansen P, Tschirren B. Clinical experience with 120,000 units of modifiedd gelatin. Dev Biol Stand 1980;48:251-6
2.. Tabuchi N, de Haan J, Gallandat Huet RCG, Boonstra PW, van Oeveren W. Gelatin usee impairs platelet adhesion during cardiac surgery. Thromb Haemost 1995; 74(6):
1447-51. .
3.. Mardel SN, Saunders F, Ollerenshaw L, Edwards C and Baddeley D. Reduced quality off in vitro clot formation with gelatin-based plasma solutions. Lancet 1996; 347: 825. 4.. Heath ML. Clot formation and gelatin-based plasma substitutes. Lancet
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5.. Baddeley DT, Saunders FM, Ollerenshaw L, Edwards CM. Clot formation and gelatin-basedd plasma substitutes. Lancet 1996;347:1763.
6.. Mielke CH, KaneshiroMM, Maher IA et al. The standardized normal Ivy bleeding timee and its prolongation by aspirin. Blood 1969;34:204-215.
7.. Born GVR. Aggregation of blood platelets by adenosine diphosphate and its reversals. Naturee 1962; 194:927.
8.. Cejka J. Enzyme immunoassay for factor Vlll-related antigen. Clin Chem 1982;28:1356-1358. .
9.. Weiss HJ, Hoyer LW, Rickles FR et al. Quantitative assay of a plasma factor deficient inn von Willebrand's disease that is necessary for platelet aggregation. J Clin Invest 1973;; 52: 2708-12.
10.. Meyer D, Obert B, Pietu G et al. Mulimeric structure of factor VIII/vWF in von Willebrandss disease. J Lab Clin Med 1980; 95: 950.
11.. Flordal PA. The plasma dilution factor: predicting how concentrations in plasma and serumm are affected by blood volume variations and blood loss. J Lab Clin Med 1995;
126:: 353-357
12.. Brown JE, Bosak JO. An ELIS A test for the binding of von Willebrand antigen to collagen.. Thromb Res 1986; 43: 303-11.
13.. Meyer D, Girma JP. Von Willebrand factor: structure and function. Thromb Haemost 1993;; 70: 99-104
14.. Stump DC, Strauss RG, Henriksen RA et al. Effects of hydroxyethyl starch on blood coagulation,, particularly factor VIII. Transfusion 1985: 25: 349-354.
15.. London MJ, Ho JS, Triedman JK et al. A randomized clinical trial of 10% pentastarch (loww molecular weight hydroxyethyl starch) versus 5% albumin for plasma volume expansionn after cardiac operations. J Thorac Cardiovasc Surg. 1989; 97: 785-97. 16.. Tigchelaar I, Gallandat Huet RCG, Korsten J et al. Hemostatic effects of three colloid
plasmaa substitutes for priming solution in cardiopulmonary bypass. Eur J Cardio-Thoracc Surg 1997;11:626-632.
17.. Mortelmans YJ, Vermaut G, Verbruggen AM et al. Effects of 6% hydroxytehyl starch andd 3% modified fluid gelatin on intravascular volume and coagulation during intraoperativee haemodilution. Anesth Analg 1995;81:1235-42.
18.. Strauss RG. Volume replacement and coagulation: a comparative review. J of Cardiothoracc Anaesth 1988; (2)6, suppl 1: 24-32.
