Blur

Blut (1990) 60:105-162

© Springer-Verlag 1990

6th Congress of the GTH (Gesellschaft fiir Thrombose- und H~imostaseforschung) Kiel, February 2 1 - 2 4 ,

1990

Abstracts

105 - 158

Author index

1 5 9 - 162

Springer International

106 I

3

ACTIN DETERMINATION IN PLATELETS ADHERED TO PLASTIC ANO IN CULTUREO ENOOTHELIAL CELLS P. Spangenberg, A.V. Mazurov, M.E. Lukashev, U. Till

ACTIN POLYMERIZATION DURING AGONIST-INDUCED ACIIVATION OF HUMAN BLOOD PLATELETS P. Spangenberg, U. Till, g. Heptinstall, C.M. Kirchmaier, A. Schirmer, M. Meyer, K. Breddin

Actin determination in cells requires lysis of cells which is carried out more easily in cell suspensions compared to lysis of cells attached to artificial surfaces. Here we report our data of actin analysis in platelets adhered to plastic and of cultured endothelial cells (ec). Analysis of the actin pools in platelets and ec was performed using the ONase I inhibition assay. Ouri~g adhesion of platelets to plastic wells (Nunclon , Denmark) a shift in the G-/F-actin equilibrium occured. An increase of 22.0 + 6.7~ (n = 5) F-actin was found compared to control washed platelets. This increase during adhesion is moderate in comparison to that found during thrombin-induced platelet aggregation. Ec from human umbilical veins were cultured on gelatin-coated p~astic dishes. Ihey contain 12.9 + 2.3 /ug actin/lO ec (n : iO), 57.4 + 9.1% were found to be G-actin and 42.6 + 9.1% F-actin. For one single determination ~ne petri dish (90 mm diameter) with about 4.5 x lO ec was necessary. Whereas lysed ec were scraped off for actin determination from the dish and transferred into Eppendorf tubes, actin analysis in platelets was quantitatively only after depolymerization of the filaments on the surface, indicating that filaments stick tightly to the plastic. Institut for Pathologische Biochemie, Medizinische Akademie, Nordh~user Str. 74, Erfurt, OOR, 5020

Aotin exists in platelets in two main forms, the monomer or G-aotin and the filamentous polymeric form, F-actin. The equilibrium of G- to F-astin in resting platelets is maintained at a constant ratio, but upon stimulation G-actin can readily polymerize to form filaments. After irreversible aggregation induced by AOP, collagen, adrenaline, arachidonic acid, PAF or thrombin, a significant increase in F-actin was found (30-50~ more cellular F-actin compared to controls). Actin polymerization is a very fast process reaching a significant level already with shape change and well before aggregation commenced or release of serotonio was signilicant. During reversible aggregation the degree of actin polymerization is smaller and at the end of deaggregation the F-actin level nearly returned to values found in unstimulated platelets. A 23187 or ionomycin also induce actin polymerization, but the degree is smaller (12-25~ more cellular F-a~$in compared to controls). The increase in cytosolic Ca ~ induced by the ionophores precedes the actin polymerization. EDTA inhibits partially ADP- or collagen-induced actin polymerization. From both results and the finding that phorbol ester (O.B/uM PMA) leads to an increase of about l ~ F-actin in pl@telets we conclude tentatively that Ca Z + and proteinkinase C seem to be involved at least in part into the initial actin assembly process in platelets. Institut fur Pathologische Biochemie, Medizinische Akademie, Nordh~user Str. 74, Erfurt, OOR, 5020

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ACQUIRED THROMBOPATHY ASSOCIATEO WITH AUTOANTIBOOIES AGAINST MEMBRANE GLYCOPROTEIN IIb-IIIa COMPLEX. 2. AGGREGATION ANO ALTERATIONS IN THE MICROFILAMENTAL SYSTEM P. Spangenberg, C.M. Kirehmaier, A. Schirmer, M. Meyer, K. Breddin

CONGENITAL OISOROERS OF PLATELET FUNCTION: D~TECTION OF STRUCTURAL ANOMALIES OF PLATELET MEMBRANE GLYCOPROTEINS M. Meyer, C~M. Kirchmaier, A. Schirmer, I. Schellenberg, Ch. Str~hl, P. Spangenberg, K. Breddin

Platelet functions in a 63 years old woman with autoantibodies against the GP IIb-IIIa complex (see A. Schirmer et al., part l) were severely impaired. Aggregation could not be induced by AOP and collagen, thrombin caused only a weak aggregation. Agglutination by ristocetin as well as adhesion were also defective. Patient's serum (i-5~ final concentration) inhibits thrombin- or collageninduced aggregation of normal platelets, q Patient's platelets contain only 473/ug actin/lO ~ platelets and an extremely low F-actin value (3Q of total platelet actin). Stimulation of these platelets with 0.i U/ml thrombin for 3 min and measurement of the G-/Factin equilibrium resulted in an increase of only 5% F-actin, whereas ADP and collagen did not induce any actin polymerization. Addition of patient's serum to normal platelets leads to an inhibition of thrombin-induced actin polymerization. Both effects of patient's serum on normal platelets (inhibition of aggregation and actin polymerization) points to receptor blockade by platelet autoantibodies found in th%patient's serum. Ca ~ movement in the patient's platelets is severely impaired after AOP or collagen stimulation whereas a normal Ca movement was induced by O.l U/ml thrombin. However, normal Ca Z + movement by thrombin but inhibited actin polymerization by this agonist does not necessarily+point to an independence of actin polymerization from Ca ~ ions, but emphasizes the receptor-mediation for actin polymerization. Institut for Pathologische Biochemie, Medizinische Akademie, Nordh~user Str. 74, Erfurt, OOR, 5020

Classical hereditary thrombocytopathies (Glanzmann's thrombasthenia, Bernard-Sculler syndrome) are caused by deficiencies of certain platelet membrane glycoproteins (GPs). He report on patients from three families with excessive bleeding. Bleeding time was prolonged or at the upper normal limit. Patients exhibited variable degrees of defective platelet aggregation: either a complete lack of response with ADP and collagen or a decreased aggregation with desaggregation. Platelet GPs were analysed by one- and two-dimensional electrophoresis, lectin- and immunoblotting, and immunoelectrophoretic techniques. In one family the patients' platelets showing decreased aggregability in response to AOP contain only 25 - 50 of GP IIb-IIIa complex. In addition, the complex migrated with an altered mobility in crossed immunoelectrophoresis. In a second family a structural abnormality of the GP IIb-IIIa complex is suggested dy the finning that platelets although containing 40 - 60 ~ of the normai amount of GP IIb-IIIa complex show a complete lack of aggregation. In the third family the patient's platelets contain an abnormal GP idemtifieo as a variant of GP Ib. This variant is different from the polymorphic GP Ib forms reported so far. In conclusion, hereditary thrombopathies with similar functional defects may be caused by a variety of structural abnormalities of platelet GPs. Abteihng hedizinische Genetik der Medizinischen Akademie Erfurt, Klement-Gottwald-Strage 34, OOR-5O~2 Erfurt

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~Da~e tyse Del LuoQenemDoile E. SCnmldt. R. Zac~orsKy. e. Melsener. W. ~roll, 8, Wllle. K.H. Zurborn. R. Slson Ole Indlkatlon zur thrombolytlsc~en lheraple bel Lungenembolie (LE) Wlrd blsner auf des akute Stadium beschranKt, de ,ach Truheren Studlen yon e l n e r Lyse lm chronlscRen StadlU~ keine Verbesserung erwarZet wlr~. Wlr b e r l c h t e n uber einen Pat.~ bel de~ elne spate Thro~bolyse zu einer Rekanalisation der Lungenstrombahn nach rezldzv. L£ fuhrte. Der 35 Jahre alte NaDn eRtwlcMelte 5 Tage oach einem Kozetrauma elne Phlebothrombose mlt konsekutlver LE. Wegen elnes Ha~arthroswurde io elne~ auswar%igen Krankenhaus auf ezne Lyse verzloh%et u. ezne Heparzn-TheraDle mlt 30 000 E/24 h elngeleltet. Ezne Woche sparer wurde er nach elner erneuten, ~assiven E~bolie mit Schocksy~ptomatlk in unsere Kllnik verlegt. Im Perfuelonsszintlgramm fanden s i c h h u s f a l l e in beiden Lungen. ~uch 3 e t z t war elne Lyse wegen elner Throabozytopenle (17xlO ~ /i) nlcht m~llch. Der iPatzent Konnte s t a b l l Z s l e r t werden. Unter Fragmzn-Therapie inor~alieierten s i c h d i e Thro~bozytenzahlen. 3 Wochen ~ p a t e r , b e r e l t s nach Narcu~arlslerung t r a t elne erneute Rezldlv-LE auf. [m Per~uslonSszln~l~ra~l fanden sled entsprechend ver~ehrte e u s f a l l e . £1ne ~n~lc~raphle d e r ILun~enarZerleo Zelqte Throsben 1B l l n k e n H a u p t s t a ~ ~1~ ~er~chluB ~ehrerer SeGmentar%erlen Sowie ~ U l t l p l e ~ p e r i p h e Fen GeTaBabbruchen rechZs. OaraufDln wurde - 6 W~hen nach ~er e r s t e n LE - aloe Lysetheraple e i n g e l e i t e t . £e wurde ~ber 6 Tage Bit 2,4 Mill E, UroKinase/24 h u. 30 000 E Heparln/24 h behande%t. H i e r u n t e r kam es zu e l n e r p r ~ r e d . R l l n i s c h e n Besserung. Elne erneute S z l n t i - U. ~ g l c ~ r a p h l e Uer LungeRarterien z e l g t e elne naRezu volls%andl~e ReKanat i s a t z o ~ der Lungenstro~bahn a i r our nc(:h geringen Kontur~rregularltaten der Perlpherle. Alia zentralen ~rterlen Waren offen. Der Patient konnte beschwerdefrei entlassen Warden. er Fall zelgt, dab bei LE auch im postaKuten Stadiu~ elne ysetherapie erfolgneich seio kaon u. bei Pat. mit ausgedehnten VerleGungen der Lungenarterlen durchaus erwogen Werden sollte.

NEL~RALIZATZC~q OF T~E A N T Z ~ A C T M T Y ( ~ %~E NEW M ~ W E I G ~ HEPAR/~ LU 47311 (REVIPAR~N) IN MAN BY ~ - ' ~ CHLORIDE K. Andrassy, E. Weber, J, Koderiach, M. SuJatta, V. Eschenfelder

The low nDlecular weight he~garin ([/~) i~ 47311 is chazacterised by ~ favorable p ~ c c d ~ c and p ~ o o k ~ o properties ~ m ~oti0nated he~ (UFH). In anlmal models f.he a n t i - - t i c e f f e c t i s oor~ar~hle to UFH %~uLle the risk of bleeding is ~ l l e r .

m ~

i n d u ~ by UF~ =ay be stop~,:t by ~ = o ~ .

In clotting tests a noznmlizaticn of thrombi~ time and activated partial thrcmboplastin t ~ (al~) can be observed. In this study it Was investigated ix) what degree protamlne chloride antagonizes the effect of LU 47311 on plasma clotting (aPTT, bleeding t/ms a~Z ~ time) and on factor Xa. ~he investigation8 performed in 12 healthy subjects showed that the anticoagulant effects of It/ 47311 on the a l ~ , thrombin time (%~f) and bleeding t ~ are immediately and cxmpZstely antegonised by intrav~mou~ injection of p r o t s ~ chloride. The anti-factor Xa activity is neutralized h~ about 40 %, A rebound phencmsnmn of the ~Lnticc~CJu_lant effect of heparin was not observed. The results are o 3 m ~ l e to other trials with Lv~H and Bhow that protsmine chloride can be applied as an antidote for the anticoagulant effects of the I ~ molecular weight hel:arL~ LU 47311. Unlvaz~ity of Heidelberg, Medical D e ~ , Bergheimer Strafe 58, D-6900 Heidelberg (FRG) Research and Dewlopnent, Knoll AG, Ludwigshafen, I~ollst/. 50, D-6700 Ludwigehnfsn (FRG)

~

6

8

FLOW CYTOMETRIC ANALYSIS OF PLATELETS FLUORESCENTLY LABELED WITH THIAZOLE ORANGE IN PATIENTS WITH QUANTITATIVE PLATELET DISORDERS

TWO POINT MUTATIONS IN THE FACTOR X CODING SEQUENCE (61a+14~-~,Lys and G l u + 1 0 2 - . ) Lys) IN A FAMILY Wl'[~-I CRM-~' FACTOR X DEFICIENCY( FX " V o r a r l b e r g " ) : i 191-1'9/~tzke ,K Lechner ,G M~hr ,KA High. 2nd D e p a r t m e n t of Internal Medci ne and 1st Department of Internal Medicine, U n i v e r s i t y of Vienna, A u s t r i a Departments of Medicine and Pathology,University of North Carolina at Chapel Hi I I ; and M uni ci pal Hospi t al Fel dki r ch, Aust r i a Factor X ( F X ) " V o r a r l b e r g " is a c o n g e n i t a l CRM+ FX d e f i c i e n c y c h a r a c t e r i z e d c l i n i c a l l y by a m i l d b l e e d i n g ten den cy. Horn ozy gou s i n di v i du als hav e a FX- act i v i t y of-less than 1 0 % in the e x t r i n s i c system and 3 2 % in the i n t r i n s i c system. F X a n t i g e n is 2 0 % . To e l u c i d a t e the genetic defect in F X " V o r a r l b e r g " genomic D N A w a s p r e p a r e d f r o m t h e leucocytes of an a f f e c t e d f a m i l y m e m b e r . S o u t h e r n blot a n a l y s i s of t h e genomio DNA cut w i t h EcoRI and p r o b e d w i t h a FX cDNA gave t h e same r e s t r i c t i o n p a t t e r n as n o r m a l DNA. The enzy m at i c am p l i f i cat i on t echni que was used t o i sol at e theexonscodingfor t h e FX p r o t e i n . T h e s e w e r e subcloned in M 1 3 and sequenced. Two point m u t a t i o n s were detected: a G 4 A a t bp 1 6 0 in exon II resulting In a change from Gla + 1 4 (GAA) to Lys(AAA) ; a G-~A at bp 4 2 4 in exon V r e s u l t i n g in a change f r o m Glu + f 02(GAG) to Lys( AAG). The m u t a t i o n s abol i shed a Taql r est r i ction s i t e i n exon II anda Mnll s i t e i n exon V. T o d e t e r m i n e wether these m u t a t i o n w e r e p r e s e n t on one or on both a l l e l s , r e s t r i c t i o n a n a l y s i s of a m p l i f i e d e x o n l l and exon V f r a g m e n t s w a s p e r f o r m e d . In three m e m b e r s of the kindred w i t h b l e e d i n g episodes and a FX a c t i v i t y of less than 1 0 % , a h o m o z y g o y u s s t a t e f o r t h e defect in exon II was detected. These i n d i v i d u a l s w e r e heterozygous for t h e defect in exon V. Six i n d i v i d u a l s w i t h no b l e e d i n g tendency and o n l y a s l i g h t l y reduced F X a c t i v i t y e x h i b i t e d a h e t e r o z y g o u s s l a t e for both m u t a t i o n s . We t h e r e f o r e conclude thai the mutation in exon II, which alters a G l a r e s i d u e , i s r e s p o n s i b l e for the f u n c t i o n a l defect i n F X " V o r a r l b e r g ' .

J. Kienast* and G. Schmitz § The f l u o r e s c e n t dye t h i a z o l e orange (TO) i s character i z e d by a l a r g e fluorescence enhancement and high quantum y i e l d upon binding t o n u c l e i c acids, p a r t i c u l a r l y RNA. In a d d i t i o n , the dye r e a d i l y permeates l i v e c e l l membranes. Whole blood samples from h e m a t o l o g i c a l l y normal subjects and from p a t i e n t s w i t h q u a n t i t a t i v e p l a t e l e t disorders were s t a i n e d w i t h t h i a z o l e orange and the p l a t e l e t populations were analyzed by f l o w eytometry. The mean percentage (+ SD) o f T O - p o s i t i v e p l a t e l e t s i n 50 c o n t r o l subjects was 8.6 + 2.8 %. In 21 thrombocytopenic p a t i e n t s whose bone marrow contained normal or increased numbers o f megekaryooytes, the percentage o f T O - p o s i t i v e p l a t e l e t s was s i g n i f i c a n t l y elevated to 26.9 + 10.9 % (p < 0.0001). In c o n t r a s t , the percentage o f p o s i t i v e l y s t a i n e d p l a t e l e t s i n 23 p a t i e n t s w i t h thrombocytopenza due to underproduction o f p l a t e l e t s did not s i g n i f i c a n t l y d i f f e r from c o n t r o l s (7.1 ~ 3,2 %). Both the s e n s i t i v i t y and the s p e c i f i c i t y o f the assay i n d i f f e r e n t i a t i n g between these two c a t e g o r i e s o f thrombocytopenia was > 95 %. In p a t i e n t s w i t h thrombocythemza/thrombocytosis (n = 13), the percentage o f T O - p o s i t i v e p l a t e l e t a averaged 7.2 + 2.9 %. We conclude, t h a t f l o w cytometrlc a n a l y s i s o f p l a t e l e t s a f t e r s t a i n i n g w i t h TO i s a s e n s i t i v e and spec±fic t e s t t h a t r a p i d l y prov].des i n f o r m a t i o n on the adequacy o f p l a t e l e t production and, thus, on the mechanism o f thrombocytopenza. * M e d . K h n z k u. P o l i k l l n z k und § I n s t . f o r K l i n . Chemie und laboratorlumsmedzzin der Westf. W l l h e l m s - U n l v e r s l t ~ t , A l b e r t - S c h w e z t z e r - S t r . 33, D-4400 MOnster

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EXPERIMENTALLY INDUCED THROMBOSIS AT ARTERIOLAR BRANCHING. D. Seiffge; E. Kremer; V. Laux; P. Reifert

CTS-NEOTHROMTIN DETERMINATION

Blood flow at vessel segments with complicated geometry are the objects of fluid-dynamically orientated investigations concerning atherogenesis and thrombosis. The development of atherosclerosis in large arteries is determined by two pathogenetic factors: the arterial wall and the contents of the vessel. Both factors should by considered as patho~genetic equals. The areas of predilection for atheroscleros~s and thrombosis in the vascular system are characterized by curvature, branding, bifurcation or embanking. We have investigated induced thrombus formation at arteriolar bifurcations with migrating stagnation points and local vortex flow. Thrombogenic endothelial lesions were produced by argon laser injury or by photochemical reactions (using FITC-dextran 70). The following items have been evaluated: 1. local red blood cell velocity 2. geometr)/of the vessel 3. localisatmn of the first thrombus growth 4. estimation of the extent of thrombogenic stimulus in relation to the extent of thrombus area or volume (video densitometry). The obtained results of increased thrombus formation at arteriolar vessel segments with complicated geometry are in accordance with findings in artifical tubes or h u m a n arteries. We could demonstrate that the onset of thrombus formation will not appear at stagnation points, but at areas of complicated flow within the branching vessel segments (80 to 90 %). Whether those areas are reattachment points or areas of recirculation and local vortex flow remains to be open. Specific three dimensional flow dynamic calculations to determine those points were just carried out. Hoechst A G W e r k Kalle-Albert, Pharma-Forschung, Postfach 3540, 6200 Wiesbaden 1, F R G

C. Wagner,

R

-

A

NEW TWO STEP

REAGENT FOR

K. Fickenscher, U. Konheiser

A new aPTT reagent using chromogenic s u b s t r a t e (Behringwerke AG, Marburg) was developed f o r the ChromoTimeSystem (Behringwerke AG, Marburg), in order to obtain a s e n s i t i v e reagent showing a s i m i l a r referance range as the coaguLometric methods. CTS-Neothromtin c o n s i s t s of two components: the a c t i v a t o r containing etlagic acid and v e g e t a b l e phospho[ipids f o r contact a c t i v a t i o n , and the sub ° strate reagent containing CaCt and a thrombin s p e c i f i c chromogenic s u b s t r a t e . A f t e r a 3 min. incubation with the a c t i v a t o r at 370C the r e a c t i o n is s t a r t e d by adding the s u b s t r a t e r e a g e n t . For CTS-Neothromtin a p r e l i m i n a r y r e f e r a n c e range of 28-40 sec. was c a L c u l a t e d , i n t r a and i n t e r assay CVIs were found to be b ~ w e e n 1,5 and 3 % in normal plasma and 3 - 4 % in c o n t r o l plasma with prolonged aPTT. The chromogenic aPTT assay shows a good heparin s e n s i t i v i t y of 0 . 0 8 IU/ml h e p a r i n . The f a c t o r V I I I s e n s i t i v i t y of CtS-Neothromtin is comparable with the e s t a b t i s t e d coagu[ometric reagents PathromR R tin and Neothromtin . The chromogenic aPTT method, especiatLg developed f o r the Chromot i m e r s c~n also be perfomed on the c l i n i c a l chemistry analyzer Cobas Fara . In conclusion, CTS-Neothromtin o f f e r s f o l l o w i n g f e a t u r e s : 1) f i b r i n o g e n - i n p e n d e n t r e s u l t s , 2) smaLL sample volumes 3) easy performance, 4) good c o m p a r a b i l i t y to coagutometric methods. Behringwerke

AG, P.O.Box 1140, 3550 Marburg,

routine

FRG

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16

PREVENTION OF DEEP VENOUS THROMBOSIS AFTER MA3OR GYNECOLOGIC OPERATIONS L. Heilmann The efficacy and safety of Low Molecular Weight Heparin (LMWH, Sandoz, FRG, -i500 aPTT units once daily + 2 placebo injections daily) and a convential Unfractionated Heparin (UFH-3xS000 I.U. daily) were compared. In a prospective double blind investigation of the prophylaxis of deep vein thrombosis undergoing major gynecologic surgery, 300 patients were randomized into two groups. Treatment was initiated 2h preoperatively in both groups and continued for 7 days. Screening for the Deep Vein Thrombosis (DVT) was performed by impedance plethysmography. The following parameters were investigated: Anti Xa-activity, aPTT, fibrinogen, D-Dimer, F VIII, AT III, Protein C, Erythrocyte aggregation and plasma visosity. Results: Two patients (1.2%) treated with LMWH and 6 patients (4%) receiving UFH developed a DVT. Haemorrhagic complications occured more olten in the UFH-group: Intraoperative high blood loss g% vs. 7.3%, blood transfusion 24~1% vs. 2 ! . 3 % drainage '31eedins. ?0.t5% vs. 27.t~% and wound haematoma lgg" vs. 12.6%. ""ne a c c e e t a n c e of 9-e '.MV7H-regimen was decisivly better. Concerning the results of the laboratory tests, the most striking effect of the LMWH was a significantly 2 3 times higher Anti Xa-activity in the postoperative period in comparison to the UFH. The aPTT was only minimally affected by both groups. There results show that once daily prophylaxis is equally as effective and as safe as the three daily regimen using UFH in patients undergoing elective, major gynecologic surgery.

Antithrombin III-Heoantigene als sensitiver A k t i v i e r u n g des Gerinnungssystems

Abteilung for Gyn~kologie und Geburtshilfe, Akademisches Lehrkrankenhaus der Universit~t Mainz, August-Bebel-Str. 5% D-6090 R ~sselsheim

CHROMOGENIC APTT

Marker e i n e r

E. Zimmer und E. Spanuth

Boehringer Mannheim GmbH, 6800 Mannheim 31 Antithrombin III-Neoantigene sind Komplexverbindungenzwischen Antithrombin I l l (AT I l l ) und aktivierten Gerinnungsfaktoren (Proteasen wie z.B. F IIa, F Xa, F IXa etc.) Sie treten als Folge der Regulation eines Aktivierungsprozesses des H~mostasesystemsdurch AT I l l in der Zirkulation auf. Die Bestimmung dieser AT III-Protease-Komplexe erfolgt mit Hilfe yon monoklonalen Antik~rpern, die spezifisch das im Komplex vorliegende, modifizierte AT I l l erfassen. Eine mit F (ab')2-Fragmenten von monoklonalen Antik6rpern beschichtete Mikroelisaplatte wird mit Citratplasmaprobe inkubiert. Die AT III-Neoantigene (AT-N) werden durch diese Antik6rper gebunden. Die Menge der Antigen-AntikSrper-Komplexe wird mit Hilfe peroxidase markierter polyclonaler AT III-Antik6rper, die an noch freien antigenen Determinanten binden (Sandwhich-Komplexe) bestimmt. Mit dem ELISA AT-N werden im Gegensatz zu TAT-Bestimmungen sEmtliche Komplexe erfaBt, die im Zuge einer Aktivierung der plasmatischen Gerinnung von AT I l l mit den proteolytischen Faktoren gebildet werden. D.h. intravasale Aktivierungsvorg~nge sollten frQher mit hEherer Spezifit~t und Sensitivit~t erfaSt werden. Ein entsprechender Methodenvergleich zwischen ELISA AT-N und der TAT-Bestimmung an Patienten mit unterschiedlichen Grunderkrankungenwird vorgestellt.

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EXPERIMENTALLY INDUCED THROMBOSIS AT ARTERIOLAR BRANCHING. D. Seiffge; E. Kremer; V. Laux; P. Reifert

CTS-NEOTHROMTIN DETERMINATION

Blood flow at vessel segments with complicated geometry are the objects of fluid-dynamically orientated investigations concerning atherogenesis and thrombosis. The development of atherosclerosis in large arteries is determined by two pathogenetic factors: the arterial wall and the contents of the vessel. Both factors should by considered as patho~genetic equals. The areas of predilection for atheroscleros~s and thrombosis in the vascular system are characterized by curvature, branding, bifurcation or embanking. We have investigated induced thrombus formation at arteriolar bifurcations with migrating stagnation points and local vortex flow. Thrombogenic endothelial lesions were produced by argon laser injury or by photochemical reactions (using FITC-dextran 70). The following items have been evaluated: 1. local red blood cell velocity 2. geometr)/of the vessel 3. localisatmn of the first thrombus growth 4. estimation of the extent of thrombogenic stimulus in relation to the extent of thrombus area or volume (video densitometry). The obtained results of increased thrombus formation at arteriolar vessel segments with complicated geometry are in accordance with findings in artifical tubes or h u m a n arteries. We could demonstrate that the onset of thrombus formation will not appear at stagnation points, but at areas of complicated flow within the branching vessel segments (80 to 90 %). Whether those areas are reattachment points or areas of recirculation and local vortex flow remains to be open. Specific three dimensional flow dynamic calculations to determine those points were just carried out. Hoechst A G W e r k Kalle-Albert, Pharma-Forschung, Postfach 3540, 6200 Wiesbaden 1, F R G

C. Wagner,

R

-

A

NEW TWO STEP

REAGENT FOR

K. Fickenscher, U. Konheiser

A new aPTT reagent using chromogenic s u b s t r a t e (Behringwerke AG, Marburg) was developed f o r the ChromoTimeSystem (Behringwerke AG, Marburg), in order to obtain a s e n s i t i v e reagent showing a s i m i l a r referance range as the coaguLometric methods. CTS-Neothromtin c o n s i s t s of two components: the a c t i v a t o r containing etlagic acid and v e g e t a b l e phospho[ipids f o r contact a c t i v a t i o n , and the sub ° strate reagent containing CaCt and a thrombin s p e c i f i c chromogenic s u b s t r a t e . A f t e r a 3 min. incubation with the a c t i v a t o r at 370C the r e a c t i o n is s t a r t e d by adding the s u b s t r a t e r e a g e n t . For CTS-Neothromtin a p r e l i m i n a r y r e f e r a n c e range of 28-40 sec. was c a L c u l a t e d , i n t r a and i n t e r assay CVIs were found to be b ~ w e e n 1,5 and 3 % in normal plasma and 3 - 4 % in c o n t r o l plasma with prolonged aPTT. The chromogenic aPTT assay shows a good heparin s e n s i t i v i t y of 0 . 0 8 IU/ml h e p a r i n . The f a c t o r V I I I s e n s i t i v i t y of CtS-Neothromtin is comparable with the e s t a b t i s t e d coagu[ometric reagents PathromR R tin and Neothromtin . The chromogenic aPTT method, especiatLg developed f o r the Chromot i m e r s c~n also be perfomed on the c l i n i c a l chemistry analyzer Cobas Fara . In conclusion, CTS-Neothromtin o f f e r s f o l l o w i n g f e a t u r e s : 1) f i b r i n o g e n - i n p e n d e n t r e s u l t s , 2) smaLL sample volumes 3) easy performance, 4) good c o m p a r a b i l i t y to coagutometric methods. Behringwerke

AG, P.O.Box 1140, 3550 Marburg,

routine

FRG

14

16

PREVENTION OF DEEP VENOUS THROMBOSIS AFTER MA3OR GYNECOLOGIC OPERATIONS L. Heilmann The efficacy and safety of Low Molecular Weight Heparin (LMWH, Sandoz, FRG, -i500 aPTT units once daily + 2 placebo injections daily) and a convential Unfractionated Heparin (UFH-3xS000 I.U. daily) were compared. In a prospective double blind investigation of the prophylaxis of deep vein thrombosis undergoing major gynecologic surgery, 300 patients were randomized into two groups. Treatment was initiated 2h preoperatively in both groups and continued for 7 days. Screening for the Deep Vein Thrombosis (DVT) was performed by impedance plethysmography. The following parameters were investigated: Anti Xa-activity, aPTT, fibrinogen, D-Dimer, F VIII, AT III, Protein C, Erythrocyte aggregation and plasma visosity. Results: Two patients (1.2%) treated with LMWH and 6 patients (4%) receiving UFH developed a DVT. Haemorrhagic complications occured more olten in the UFH-group: Intraoperative high blood loss g% vs. 7.3%, blood transfusion 24~1% vs. 2 ! . 3 % drainage '31eedins. ?0.t5% vs. 27.t~% and wound haematoma lgg" vs. 12.6%. ""ne a c c e e t a n c e of 9-e '.MV7H-regimen was decisivly better. Concerning the results of the laboratory tests, the most striking effect of the LMWH was a significantly 2 3 times higher Anti Xa-activity in the postoperative period in comparison to the UFH. The aPTT was only minimally affected by both groups. There results show that once daily prophylaxis is equally as effective and as safe as the three daily regimen using UFH in patients undergoing elective, major gynecologic surgery.

Antithrombin III-Heoantigene als sensitiver A k t i v i e r u n g des Gerinnungssystems

Abteilung for Gyn~kologie und Geburtshilfe, Akademisches Lehrkrankenhaus der Universit~t Mainz, August-Bebel-Str. 5% D-6090 R ~sselsheim

CHROMOGENIC APTT

Marker e i n e r

E. Zimmer und E. Spanuth

Boehringer Mannheim GmbH, 6800 Mannheim 31 Antithrombin III-Neoantigene sind Komplexverbindungenzwischen Antithrombin I l l (AT I l l ) und aktivierten Gerinnungsfaktoren (Proteasen wie z.B. F IIa, F Xa, F IXa etc.) Sie treten als Folge der Regulation eines Aktivierungsprozesses des H~mostasesystemsdurch AT I l l in der Zirkulation auf. Die Bestimmung dieser AT III-Protease-Komplexe erfolgt mit Hilfe yon monoklonalen Antik~rpern, die spezifisch das im Komplex vorliegende, modifizierte AT I l l erfassen. Eine mit F (ab')2-Fragmenten von monoklonalen Antik6rpern beschichtete Mikroelisaplatte wird mit Citratplasmaprobe inkubiert. Die AT III-Neoantigene (AT-N) werden durch diese Antik6rper gebunden. Die Menge der Antigen-AntikSrper-Komplexe wird mit Hilfe peroxidase markierter polyclonaler AT III-Antik6rper, die an noch freien antigenen Determinanten binden (Sandwhich-Komplexe) bestimmt. Mit dem ELISA AT-N werden im Gegensatz zu TAT-Bestimmungen sEmtliche Komplexe erfaBt, die im Zuge einer Aktivierung der plasmatischen Gerinnung von AT I l l mit den proteolytischen Faktoren gebildet werden. D.h. intravasale Aktivierungsvorg~nge sollten frQher mit hEherer Spezifit~t und Sensitivit~t erfaSt werden. Ein entsprechender Methodenvergleich zwischen ELISA AT-N und der TAT-Bestimmung an Patienten mit unterschiedlichen Grunderkrankungenwird vorgestellt.

110 17

19

CHANGES OF HEMOSTATIC POTENTIAL IN COMPARISON OF 4 METHODS FOR PLASMAPHERESIS. H. Neumeyer, B. Schuite, S. Quentin and J.U. Wiedlng

COMPARATIVE STUDIES ON THE ANTICOAGULANT AND ANTITHROMBOTIC EFFECTS OF HEPARIN, LMWH, SYNTHETIC HEPARIN-PENTASACCHARIDE AND DERMATAN SULFATE. K. Krupinski, H.K. Breddin, J.M. Walenga, J. Fareed, B. Casu and J. Choay

In the course of this study, separation of plasma using the systems 'Ptasmapur Monitor' (Fa. Organon), 'Autopheresis-C' (Fa. Baxter) and 'PCS' (Fa. Haemonetios) was compared with the conventional blood bag cantrifugatlon. In 16 apherasls per method, parameters mainly reflecting the blood coagulation were examined In addition to other criteria. Blood/plasma samples were drawn from the donor before, during and after the separation run as well as from the used device during the separation and from the collected plasma. Thrombin-antithrombin-oomplexes and the fibdn(ogen)-derivatNes, soluble fibrin and flbrinopeptidA, proved to be sensitive indicators of a slight activation of the coagulation system. This activation was less pronounced by separation through cantrifugation than through filtration; however, even the filtration devices activated the coagulation system tess than common bag centrifugatlon. Furthermore, a decrease in blood coagulation factors and inhibitorswere most pronounced using bag centrifugation. The number of cells remaining in the collected plasma was highest after separation by centrifugationalone, whereas less than 1/10 as many were seen usingfiltration methods. Considering the fact that presently most of the dally transfused fresh frozen plasma Is collected by centrlfugatlon of blood bags, the comparatively slight activation of blood coagulation by plasma separation machines seems to be negligible. However, the number of remaining cells in the collected plasma may be of greater interest: a total elimination of cells from transfused plasma should be the goal to avoid activation of blood coagulation in recipients. Abtlg.f. Transfusionsmedizinder Universit&t, 3400 G6ttingen / FRG

Seven glycosaminoglycans (GAGs) were studied for t h e i r a n t i coagulant effects in v i t r o , p l a t e l e t adhesion to bovine e x t r a c e l l u l a r matrix (ECM) and antithrombotic effects in a r a t thrombosis model. Unfractionated heparin (UFH), low molecular weight heparin (CY 216), a synthetic heparin pentasaccharide (PS) and dermatan sulfate IDS) were obtained from I n s t i t u t e Choay, Paris, France and a supersulfated low molecular weight heparin (GAG 869) and heparin in which the binding site f o r antithrombin I I I had been inactivated (GAG 262) were obtained from Prof. Casu ( I s t . S c i e n t i f i c o di Chimica "G. Ronzoni", Milano, I t a l y ) . A series of in v i t r o assays including aPTT, TT, heptest, anti Xa and anti l l a assays using chromogenic substrates were performed in both human and r a t p l a t e l e t poor plasma. All agents i n h i b i t e d thrombus formation in venules at minimal e f f e c t i v e doses from 0.01 to 5 mg/kg 30 min a f t e r a single i . v . i n j e c t i o n . The dose-dependent antithrombotic e f f e c t was not p o s i t i v e l y correlated with the i n h i b i t i o n of F. l l a or F. Xa in human or r a t plasma. These data suggest that i n h i b i t i o n of thrombosis at the endothelial surface does not d i r e c t l y correlate with current ex vivo coagulation assays. Higher doses were required to observe antithrombotic e f f e c t s a f t e r subcutaneous injections and t h e i r duration lasted f a r longer a f t e r s.c. administration than a f t e r i . v . i n j e c t i o n . The antithrombotic effects persisted longer than detectable ex vivo anticoagulant a c t i v i t i e s . The low molecular weight heparins, pentasaccharide and dermatan sulfate showed in v i t r o i n h i b i t o r y effects on p l a t e l e t adhesion to bovine ECM. This i n h i b i t o r y e f f e c t was not observed with two unfractionated heparins (GAG 262 and Liquemin). Further studies are needed to c l a r i f y a possible correlation between inhibition of platelet adhesion and antithrombotic effects of GAGs. Center of Internal Medicine, Division of Angiology, J.W. Goethe-University, Theodor-Stern-Kai 7, 6000 Frankfurt/M., FRG

18

2O

U ROKINASE-TYPE PLASMINOGEN ACTIVATOR GENE: TRANSCRIPTIONAL CONTROL OF PLASMINOGEN ACTIVATOR SYNTHESIS AND REGULATION OF EXPRESSED ACTIVITY. D. vonder Ahe, Y. Nagamine--and G. MOIler-Berghaus The genes coding for the two plasminogen activators urokinasetype (uPA) and tissue-type (tPA) have been identified. They are located on different chromosomes. Both genes are organized with separate exons. The homology between the two PAs at the amino acid level is only 40%, but the enzymes are highly similar in their basic structures, which demonstrates a close evolutionary relationship, uPA is implicated in various aspects of cellular functions such as fibrinolysis, cell migration, tissue reorganization during morphogenesis and invasive growth in both normal and pathological conditions linked to increased plasmin activity, uPA synthesis is modulated by a variety of effector molecules such as phorbol esters, growth factors, peptide and steroid hormones retinoids and others. The regulation of uPA synthesis occurs mainly at the level of gene transcription. To elucidate the mechanisms of regulation, we studied the human and pig uPA gene including their putative regulatory 5'flanking regions. For this purpose, we constructed a series of hybrid genes with uPA-5'-flanking deletion mutants and the E. coil chloramphenicol acetyl-transferase as a reporter gene. Using gene transfer techniques, this hybrid gene was stably integrated in the host cell genome and the activity of the reporter gene measured. This analysis revealed that sequences proximal to the transcription start site as well as sequences far upstream play an important rol e in hormonal regulation through the protein kinaee A pathway and by phorbol esters using the protein kinase C pathway. Furthermore, we identified the protein-binding sequences within the regulatory regions by DNAase I footprint and gel band shift analysis. A comparison of regulatory sequences of human and pig uPA genes will be discussed.

EFFECT OF VWP CONTENT OF THE CULTURE MEDIUM ON THE VWF PRODUCTION OF CULTURED ENDOTHELIAL CELLS. I . A l t o r j a y , C.M. Kirchmaier, Zs. Vigh, K. Krupinski, I . Scharrer, H.K. Breddin

MPG-Clinical Research Group for Blood Coagulation and Thrombosis, D-6300 Giessen (West Germany) and * Fnednch " " Miescher Institute, CH-4002 Basel, Switzerland

The exact regulation of synthesis of yon Willebrand factor by endothelial cells is s t i l l unclear. The effect of the vWF content of the culture medium on the production of vWF by cultured human ECs was studied. The amount of vWF produced and released by ECs in the presence of culture medium containing normal human serum was significantly higher than that produced in the presence of medium containing serum from patients with severe Willebrand syndrome. The l a t t e r correlated well with the amount of vWF produced in the presence of a serum-free culture medium. The multimeric structure of vWF released by ECs was the same in a l l cases. I f human ECs were cultivated in a medium containing normal serum the adhesion of platelets to the matrix produced by these cells was higher than the adhesion to matrix produced by ECs which had been cultivated in a mediumcontaining serum from a patient with severe vWS. This finding may be correlated with the higher vWF content of ECM. A stimulating effect of yon Willebrand factor contained in the culture medium on the vWF synthesis of cultured ECs is supposed.

Center of Internal Medicine, Division of Angiology, J.W. Goethe-University, Theodor-Stern-Kai 7, 6000 Frankfurt/M., FRG

111 23

21 IN VITRO PREVENTION

CHANGES OF THE FIBRINOLYTIC EARLY PHASE OF POLYTRAUMA : A PROSPECTIVE STUDY

EFFECTS OF UROKINASE AND THEIR BY DIFFERENT INHIBITORS •

E. Seifried, K.H. Bock

M.D. Oethinger and E. Seifried We investigated dose-, time- and temperaturedependent effects of urokinase (UK) on normal citrated plasma in vitro. When 5 ~g/ml UK were added to pooled normal plasma and incubated for 30 minutes at an ambient temperature (25 ° C), ~ 2 - a n t i p l a s m i n decreased to 8% of the control value. Incubation on ice yielded a decrease to 45% of control, whereas ~2-antiplasmin was fully consumed at 37 ° C. Fibrinogen and p l a s m i n o g e n fell to 46% and 39%, respectively, after a 30 minutes incubation at 25 ° C. Thrombin time prolonged to 182% of control. Various inhibitors were studied with respect to their suitability and efficacy to prevent these in vitro effects. Aprotinin exhibited a good protective effect on fibrinogen at concentrations exceeding 500 KIU/ml plasma. Its use however was limited due to interferences with some haemostatic assays. We could d e m o n s t r a t e that L-Glutamyl-L-Glycyl-L-Arginyl c h l o r o m e t h y l ketone (GGACK) and a specific polyclonal antiUK-antibody effectively inhibited urokinaseinduced plasmin generation without interfering with haemostatic assays. The a n t i - U K - a n t i b o d y afforded full protection of ~ 2 - a n t i p l a s m i n at therapeutic levels of UK. It is concluded that UK in plasma samples from patients during thrombolytic therapy may induce in vitro effects which should be p r e v e n t e d by the use of a suitable inhibitor such as GGACK or specific anti-UK-antibody. Medizinische Klinik und Poliklinik, U n i v e r s i t y of Ulm, Robert-Koch-Str. 8, D - 7900 Ulm, F.R.G.

22

IN

THE

L. Lampl, M. Tisch,

Changes of the fibrinolytic system were investigated in twenty adult polytrauma p a t i e n t s with a mean injury severity score (ISS) of 37 (20 - 50) and a NACA-score between IV and VI. Citrated blood samples were taken w i t h o u t delay at the site of the accident with a m e d i a n time interval between trauma and sampling of 18 m i n (range: i0 - 29 min). No patient had r e c e i v e d more than 500 ml of cristalloid or colloidal fluid before sampling. Blood samples were centrifuged and snap frozen immediately using a portable centrifuge and cool aggregate. A second blood sample was taken in the emergency room at hospital. The following parameters were determined in both blood samples (mean results are given, sample taken in hospital in brackets): Plasminogen 93% (72); A n t i p l a s m i n 68 %(48); t-PA-antigen 14,5 ng/ml (3,3); t-PA-activity 21,6 ng/ml (4,7); PAI 8,0 AU/ml (19,1); D-dimer 13,7 ~g/l (15,4); FgDP 8,9 ~g/ml (6,7); FbDP 15,4 ~g/ml (19,2); TDP 27,4 ~g/ml (28,1). The meaning of these changes of the fibrinolytic system are unclear. Longitudinal studies of the fibrinolytic system after polytrauma have to be performed to answer the question whether therapeutic use of inhibitors of fibrinolysis i.e. aprotinin have a positive effect on the outcome of these patients, especially with regard to the frequency of acute r e s p i r a t o r y distress syndrom (ARDS). M e d i z i n i s c h e Klinik und Poliklinik, U n i v e r s i t y of Ulm, Robert-Koch-Str. 8, D - 7900 Ulm, F.R.G.

24

ALTERATIONS OF COAGULATION PARAMETERS THE EARLY PHASE OF POLYTRAUMA : A PROSPECTIVE STUDY

E. Seifried, K.H. Bock

D.Ellbr~ck,

SYSTEM

L.

Lampl,

D.

Ellbr~ck,

M.

IN

tHE CLINICAL SUFFERING FROM

COURSE COUMARIN

OF 3 NECROSIS

D. Ellbr~ck, H. Wankm~ller, Gabelmann, E. Seifried

M.D.

PATIENTS

Oethinger,

A.

Tisch,

Changes of the blood coagulation system were investigated in twenty adult polytrauma patients with a mean injury severity score (ISS) of 37 (20 - 50) and a NACA-score between IV and VI. Citrated blood samples were taken w i t h o u t delay at the site of the accident with a m e d i a n time interval between trauma and sampling of 18 min (range: i0 - 29 min). No patient had r e c e i v e d more than 500 ml of cristalloid or colloidal fluid before sampling. Blood samples were centrifuged and snap frozen immediately using a portable centrifuge and cool aggregate. A second blood sample was taken upon admission to hospital. The following parameters were determined in both blood samples (median results are given, sample taken in hospital in brackets): Quick 91% (64); aPTT 31 s (38); TT ii s (12); fibrinogen 1,6 g/l (0,95); AT III 81% (52); TATcomplex 138,7 ~g/l (132,5); protein C activity 60% (21); protein S antigen 70% (50); F.V 137% (86); F.VIII:C 225% (121); hematocrit 39% (28); platelets 269 000/~i (166 000). We conclude that distinct alterations in the coagulation system occur including a decrease of inhibitors of coagulation. Prospective studies need to be done in order to evaluate the clinical importance of these effects and the benefit of early therapeutic substitution. M e d i z i n i s c h e Klinik und Poliklinik, U n i v e r s i t y of Ulm, Robert-Koch-Str. 8, D - 7900 Ulm, F.R.G.

Coumarin necrosis is a rare but clinical important complication of treatment with oral anticoagulants. We report three cases which we observed over the last years. First case: A 35 year old woman was a d m i t t e d with a deep vein thrombosis (DVT) of the femoral and iliacal veins. After heparin therapy oral anticoagulation with phenprocoumon Was initiated. The patient developed a coumarin necrosis at the right breast. Second case: A 21 year old obese woman (weight: i00 kg, height: 160 cm) presented with an acute DVT which was c o m p l i c a t e d by a subsequent p u l m o n a r y e m b o l i s m of grade III. The patient had a longstanding history of severe allergic bronchial asthma. She was a heavy smoker and took the o./c. pill. I.v. h e p a r i n was started, followed by M a r c u m a r R. After a few days, the patient developed a large necrosis at the anterior abdominal wall which finally had to be treated with skin grafting. Third case: A 25 year old obese p a t i e n t was admitted with PE. He had a i0 years h i s t o r y of hay fever and allergic bronchial asthma and smoked 40-60 cigarettes/day. The patient r e c e i v e d h e p a r i n followed by M a r c u m a r R. On the 5th day a large coumarin necrosis d e v e l o p e d on the right hip with involvement of the muscles; the patient had to undergo five operations. No abnormalities in the haemostatic system were detected in any of the patients, in p a r t i c u l a r no protein C-deficiency. It is concluded that an allergic diathesis may play an important role in the pathogenesis of coumarin necrosis. M e d i z i n i s c h e Klinik und Poliklinik, U n i v e r s i t y of Ulm, Robert-Koch-Str. 8, D - 7900 Ulm, F.R.G.

112

25

27

THROMBOPROPHYLAXIS IN GENERAL SURGERY WITH A LOW MOLECULAR WEIGHT H E P A R I N (LMW 21-23). A PROSPECTIVE RANDOMIZED CLINICAL STUDY E. Seifried, D. Ellbr~ck, J. Limmer, A. Schwarz, H. MUller, E. Eisele, H.G. B e g e r

SYSTEMIC EFFECTS OF INTRAVENOUS RT-PA UROKINASE IN ACUTE MYOCARDIAL INFARCTION A. Eii~rs~ 0, Claws; U. Tebbe; H, K6sterino Dept. of internal Medicine, University Clinic, 3400GOttingen~ FR6

S t u d y desiqn: A p r o s p e c t i v e r a n d o m i z e d c l i n i c a l s t u d y was p e r f o r m e d t o c o m p a r e the e f f i c a c y of low m o l e c u l a r w e i g h t h e p a r i n (LMW 21-23, Braun) and u n f r a c t i o n a t e d h e p a r i n (UFH, H e p a r i n Braun) in 203 p a t i e n t s u n d e r g o i n g g e n e r a l surgery. 103 p a t i e n t s r e c e i v e d a s i n g l e d a i l y i n j e c t i o n of 2500 a n t i - f a c t o r Xa u n i t s of LMW 21-23 and i00 p a t i e n t s r e c e i v e d 3 x 5000 IU UFH/day. A r a d i o active fibrinogen uptake test was used for s c r e e n i n g of d e e p v e i n t h r o m b o s i s (DVT). If DVT w a s s u s p e c t e d e i t h e r c l i n i c a l l y or by leg s c a n n i n g the d i a g n o s i s was v e r i f i e d by p h l e b o g r a p h y . R e s u l t s : No p a t i e n t s of e i t h e r g r o u p p r e s e n t e d clinical signs of DVT. In the L M W H g r o u p 4 p a t i e n t s (4,1%) and in the UFH g r o u p 5 p a t i e n t s (5%) d e v e l o p e d DVT (leg s c a n n i n g ) . As far as p h l e b o g r a p h y was c a r r i e d out it was n e g a t i v e . One p u l m o n a r y e m b o l i s m o c c u r e d in the U F H - g r o u p (clinical diagnosis). In the LMWH group we observed a tendency to less haemorrhagic c o m p l i c a t i o n s (n.s.). T h e r e was a s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e b e t w e e n a n t i - X a l e v e l s in both groups (median r e s u l t s ± SEM are given, v a l u e s of the UFH g r o u p in b r a c k e t s ; s a m p l e s w e r e t a k e n on the 3rd day p o s t - o p ) : 0.i0 ± 0.01 anti-Xa U/ml (0.02 ± 0.008). V a l u e s for PT, PTT, TT, f i b r i n o g e n , AT III, p r o t e i n C, p l a s minogen, a n t i p l a s m i n and t - P A a n t i g e n d i d not differ significantly. C o n c l u s i o n s : The r e s u l t s i n d i c a t e t h a t a s i n g l e i n j e c t i o n of 2500 a n t i f a c t o r Xa u n i t s of LMW 21-23 is as e f f e c t i v e and at least as safe as 5000 IU of UFH g i v e n daily. M e d i z i n i s c h e K l i n i k und P o l i k l i n i k , U n i v e r s i t y of Ulm, R o b e r t - K o c h - S t r . 8, D - 7900 Ulm, F.R.G.

AND

Recombinant tissue-type p l asminogen activator (rt-PA) was d e s c r i b e d as e f f e c t i n g a more s p e cific thrombolysis avoiding a systemic f i b r i n o genolysis as w e l l as s e r i o u s bleeding complications. The systemic activation of the

fibrinolytic s y s t e m by i.v. rt-PA and u r o k i n a s e was studied in 69 patients presenting with symptoms of acute myocardial infarction of less than 6 h d u r a t i o n . 34 p a t i e n t s randomized t o t h e rt-PA group r e c e i v e d a bolus i n j e c t i o n o f i 0 mg

rt-PA followed by 60 mg rt-PA over 90 min. 35 patients randomized to the urokinase group received a bolus injection of l. SxlO ~ IU urokinase and l. SxlO~ IU u r 0 k i n a s e over 90 min. Blood samples were obtained before therapy, after 90 min, 4, 12 and 24 h. Both groups showed a significant systemic reaction: after 90 min f i b r i n o g e n (Clauss), p l a s m i n o g e n and a~antiplasmin decreased considerably, while fibrin(ogen) d e g r a d a t i o n products, r e p t i l a s e time and plasmin i n c r e a s e d a c c o r d i n g l y . Even though the a v e r a g e s y s t e m i c e f f e c t s of rt-PA were lower than those of u r o k i n a s e . In later blood s a m p l e s a significantly more rapid return to normal coagulation s t a t u s was e s t a b l i s h e d in the rt-PA group. However, d i s p e r s i o n of p a r a m e t e r s in the rt-PA group were quite remarkable: There were some cases of considerable fibrinogenolysis in the rt-PA group which may lead to s e r i o u s hemorrhagic c o m p l i c a t i o n s . So far, these m a r k e d individual r e a c t i o n s have not been s u f f i c i e n t l y explained. T h e r e f o r e , as in any other t h r o m b o lytic therapy, a careful m o n o t o r i n g of p a t i e n t s treated with rt-PA is s t r o n g l y a d v i s a b l e . G e r i n n u n g s l a b o r der U n i v e r s i t a t G O t t i n g e n , R o b e r t - K o c h - S t r . 40, D - 3400 S o t t i n g e n

26

28

LEVELS OF PIBRIN(OGEN) DEGRADATION PRODUCTS AND OF INTACT FIBRINOGEN IN PATIENTS RECEIVING THROMBOLYTIC THERAPY WITH rt-PA E. Seifried, E. H o e g e e - d e Nobel, D. E l l b r ~ e k , W. Haerer, P. T a n s w e l l , W. N i e u w e n h u i z e n

ROLE OF FIBRINOGEN-MEDIATED CONTACTS FOR THE ACTIVATION OF POLYMORPHONUCLEAR LEUKOCYTES (PMNLs) BY STIMULATED PLATELETS. A. Ruf, R. Schlenk, A. Maras, E. Mor,qenstern* and H. Patscheke. Cell suspensions containing human washed platelets (2 x 108/ml) and human PMNLs (2 x 106/ml) were stimulated with the platelet agonists thrombin 0.06 IU/ml, U 46619 1 pM, or ADP 10 pM. Platelet aggregation and the luminolenhanced chemiluminescence (CL) of the PMNLs were simultaneously monitored in a Lumiaggregometer. Thrombin and U 46619 were able to t r i g g e r the platelet aggregation and the CL in the absence of exogenous fibrinogen, whereas ADP required the presence of added fibrinogen to aggregate aspirin-treated platelets and to induce the CL. EDTA and no s t i r r i n g suppressed the platelet aggregation and the CL in the mixed suspensions. Interference and electron microscopy revealed mixed plateletPMNL aggregates. Cell-bound fibrinogen was localized by immunofluorescence and immunoelectron microscopy. It was found in the contacts between platelets, between platelets and PMNLs, at the free surfaces of both cell species and in the platelet C-granules. Neither thrombin, U 46619 and ADP nor the supernatant or a f i l t r a t e of thrombin-stimulated platelets induced any CL in pure PMNL suspensions. In asplrin-treated whole blood, U 46619 4 IJM or ADP 10 pM also led to an aggregation as measured by the impedance method and to a strong CL. The CL and the aggregation were again suppressed by EDTA, no s t i r r i n g and RGD peptides. In contrast, ristocetin-induced platelet agglutination was not associated with CL. It is concluded that the platelet-dependent activation of PMNLs requires a close intercellular contact that is mediated by fibrinogen-binding to the surfaces of both cell species. (Supported by the DFG, Pa 263 and Mo 124)

In 12 M I - p a t i e n t s t r e a t e d w i t h i00 mg r t - P A / 3 h o u r s and in 18 h e a l t h y s u b j e c t s (0.25 mg/kg, 0.50 m g / k g r t - P A and p l a c e b o over 30 min, n = 6 e a c h group) levels of f i b r i n o g e n (fbg) and its derivatives were measured in serial plasma samples containing citrate and the t-PA inhibitor PPACK. Fbg was measured with 2 c l o t t i n g rate a s s a y s and a new E L I S A for i n t a c t fbg (Thromb H a e m o s t 60, 415, 1988). D - d i m e r , degradation products of fbg (FgDP), fibrin (FbDP) and total DP (TDP) in plasma were a s s e s s e d by s p e c i f i c ELISA. FDP in s e r u m w e r e also measured. In M I - p a t i e n t s D-dimer, FgDP, FbDP, TDP and FDP in s e r u m i n c r e a s e d from 0.i, 0.4, 0.4, 0.8 and 6.0 to 2.3, 20, 8, 30 and 120 ~ g / m l at the end of the infusion, r e s p e c t i v e l y . In n o r m a l subjects FbDP and D - d i m e r i n c r e a s e d f r o m 0.5 to 0.9 and f r o m 0.i to 0.5 ~g/ml. M e a n fbg l e v e l s w e r e 51%, 61% (Clauss and p h o t o m e t r i c assay) a n d 81% (ELISA) of the p r e i n f u s i o n values. A f t e r 3 d a y s all f i b r i n ( o g e n ) d e g r a d a t i o n p r o d u c t v a l u e s w e r e n o r m a l w h e r e a s fbg levels w e r e 120 - 140% (Clauss, p h o t o m e t r i c ) and 105% (ELISA). The discrepancies between the functional and ELISA l e v e l s of fbg s u g g e s t t h a t a b o u t 25% of fbg is c o n v e r t e d to (slowly clotting, i.e. less functional) L M W ' f b g and that r t - P A i n d u c e s less fbg d e g r a d a t i o n t h a n e x p e c t e d from f u n c t i o n a l a s s a y s The levels of FbDP and D - d i m e r are too h i g h to be d e r i v e d e x c l u s i v e l y from a c o r o n a r y t h r o m b u s and p r o b a b l y o r i g i n a t e from a n o t h e r f i b r i n pool. M e d i z i n i s c h e K l i n i k und P o l i k l i n i k , University of Ulm, R o b e r t - K o c h - S t r . 8, D - 7900 Ulm, F.R.G.

Inst. f . Kiln. Chem., Klinikum Mannheim der Uoiv. Heidelberg, P.O.B. 100023, D-6800 Mannheim and "'Medizin. Biol., Univ. des Saarlandes, D-6650 Homburg.

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29 INBORN PROTEIN C DEFICIENCY IN A 15-YEAR-OLD GIRL WITH DISSIMINATED INTRAVASCULR COAGULOPATHY (DIC) DUE TO FULMINANT MENINGOCOCCEMIA A . M . M i n g e r s , H . B a r t e l s , H . B . v . Stockhausen A 1 5 - y e a r - o l d g i r l was a d m i t t e d t o h o s p i t a l w i t h fulminant meningococcemia. Besides a c i r c u l a t o r y breakdown accompanied by r e n a l i n s u f f i c i e n c y the p a t i e n t had acute DIC w i t h numerous l a r g e c u t a neous n e c r o s e s . D u r i n g the a c u t e d i s e a s e as w e l l as a f t e r she had c o m p l e t e l y r e c o v e r e d 10 weeks l a t e r the p a t i e n t showed s i g n i f i c a n t protein C deficiency. In the mother who had a l r e a d y gone t h r o u g h a t h r o m b o s i s p r o t e i n C was a l s o found deficient. Thus the g i r l i s most l i k e l y t o have an i n b o r n p r o t e i n C d e f i c i e n c y . I t i s suggested t h a t DIC and the e x t r e m e l y s e v e r e and f u l m i n a n t c o u r s e o f the d i s e a s e were i n i t i a t e d by p r o t e i n C deficiency. To date t h e r e i s no c l e a r e v i d e n c e why in an o n l y small number o f p a t i e n t s mening o c o c c o s i s t a k e s such a f u l m i n a n t , m o s t l y f e t a l c o u r s e . The q u e s t i o n a r i s e s w e t h e r , more f r e q u e n t l y than known so f a r DIC in t h i s cases must be a t t r i b u t e d t o an i n b o r n i p c l i n a t i o n to t h r o m b o s i s due f o r example t o p r o t e i n C or AT I l l deficiency. Universit~ts-Kinderklinik, D-8700 WUrzburg

Josef-Schneider-Str.2

IS THROMBOLYSIS IM F I V O M O R E EFFECTIVE WHEN rtPA IS GIVEN BY INFUSION THAN AS A BOLUS ? K.S. Herrmann, H. Kreuzer und U.Tebbe Acute mycardial infarction is treated by parenteral application of thrombolytic compounds. Different drugs are available, mode of application and dosages are still under discussion. Clinical studies to standardize therapy are time consuming and expensive; animal experiments can shorten the way to find a therapeutical standard. We developped a method to induce local endothelial trauma Jm rive by employing a contactless, photochemical process. Thus, a reproducible damage can be initiated and stable arterial thrombi are produced in a highly reproducible way. As thrombogenesis and thromholysis take place under direct visual control, direct information about blood flow can be obtained and quantified. After occlusive thrombi were formed, thrombolysis was induced by (A) bolus (B) or infusion of rtPA at different dosages or (C) solvent alone. Patencies were compared consecutively and for dosages of 0,5 and 1.4 mg/kg rtPA. 1.4 mg/kg rtPA induced reperfusion to 80% (A) or 60% (B) (SEM=I6 or 14%; n.s.). 0.5 mg/kg rtPA was more effective when given as an infusion than when applied as a bolus (49 or 19%, SEM = 12 or 3%; p < 0.01). (C) was without efficacy. We conclude, that similar dosages of rtPA obtain better patencies when the drug is applied as infusion rather than as a bolus. Med. KlinJk u. Poliklinik, Zentrum Innere Medizin, Robert-Koch-Str. 40, 3400 Gbttingen

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30 BINDING AND INTERNALIZATION BY T}IROMBOCYTES H. H ~ r m a n n , V. J e ] i n i 6 and

OF H.

SOLUBLE

FIBRIN

Richter

Gelfiltered human thrombocytes f a i l to h i n d soluble 125-I-fibrin unless they had been supplemented with activated plasma transamidase (coagulation factor XIIIa). In addition, a free 30 kDa-domain from the N-terminus of the fibron e c t i n s u b u n i t c h a i n s is r e q u i r e d as a e o f a c tot. F i b r i n b i n d i n g is i n h i b i t e d by the transamidase c o m l ) e t i t o r s l,u t r e s c i n e and histamine. T h e r e is e v i d e n c e t h a t the f r e e f i b r o n e c t J n d o m a l n is c i r c u l a t i n G in p l a s m a , a l t h o u g h its quantitative determination meets some difficulties . CentrifuGed platelets still containin G their halo of adsorbed plasma proteins bind ]25-If i b r i n to a v a r i a b l e e x t e n t . B i n d i n G is i m p r o v e d by t h e 30 k D a - d o m a i n at l o w e r c o n c e n t r ations than required for fibrin binding to 6 e l filtered platelets suGGestinG that some free fibronectin d o m a i n is a m o n G tile a d s o r b e d prot e i n s . O n t h e o t h e r h ~ n d , b i n d i n G is i n c o m pletely inhibited by p u t r e s e i n e i n d i c a t i n G that still a second i)athway f o r f i b r i n b i n d i n g is possible . Platelets in a thrombocyte concentrate even internalize ]25-1-fibrim within few hours. The trapped activity Js o n l y d e t e c t a b l e for about 6 hours indieatin 6 intracellLllar de{iradation a n d r e l e a s e o f the fra/Dllents° I n t e r n a l i z a t i o n is promoted by the f r e e f i b r o n e c t i m domain and is i m p r o v e d by f a c t o r X l l I a . l{owever, o t h e r unknown plasma compounds are still essential. M a x i ' l a n c k - l n s t i t u t f~ir B i o c h e m i e , Am Klopfers p i t z , D - 8 0 3 3 5 ? a r t i n s r i e d n e a r 5;umich.

THE EFFECT OF CISAPRIDE AND METOCLOPRAMIDE ON THE BIOAVAILABILITY OF PHENPROCOUMON D.Wesemeyer, H.M~ig, T.Gaska, S.Masuch, K.U.Seiler*, H.D.Bruhn i. Med. Universititsklinik Kiel/ * J ~ Research Foundation, Neuss To e v a l u a t e t h e p o s s i b l e i n t e r a c t i o n s b e t w e e n p h e n p r o coumon and e i t h e r m e t o c l o p r a m i d e or c i s a p r i d e t h e f o l l o w i n g open c r o s s o v e r t r i a l was c a r r i e d out: 24 h e a l t h y v o l u n t e e r s were g i v e n 0.22 m g / k g p h e n p r o coumon as a s i n g l e oral dose on d a y 4 of a 10 day l a s t i n g metoclopramide or c i s a p r i d e a d m i n i s t r a t i o n and, a f t e r a w a s h o u t period of 4 weeks, a l o n e w i t h o u t t h o s e drugs. S e v e r a l c o a g u l a t i o n p a r a m e t e r s ( p r o t h r o m b i n tlme, f a c t o r X - and f a c t o r VII- a c t i v i t y , p r o t e i n C a c t i v i t y and p r o t e i n C a n t i g e n ) a n d t h e plasma l e v e l s of p h e n p r o coumon were d e t e r m i n e d a t c e r t a i n i n t e r v a l s up to 7 days after administration. Metoclopramide d e c r e a s e d t h e a r e a u n d e r t h e c u r v e (AUC) ( a b o u t I6%) and t h e h a l f l i f e of p h e n p r o c o u m o n (from 132 to 111 hours), b u t did n o t a l t e r peak c o n c e n t r a t i o n or t h e time to p e a k c o n c e n t r a t i o n . C i s a p r i d e h a d no e f f e c t on t h e p h a r m a c o c i n e t i c s of phenprocoumon. These f i n d i n g s f i t for an i n c r e a s e in t h e e l i m i n a t i o n of p h e n p r o c o u m o n t h r o u g h metoclopramide. Except for a s i g n i f i c a n t d e c r e a s e i n f a c t o r X a c t i v i t y b e t w e e n 2 and 36 h o u r s a f t e r phenprocoumon i n t a k e d u r i n g m e t o c l o p r a m i d e c o a d m i n i s t r a t i o n t h e r e were no s i g n i f i c a n t changes in the coagulation parameters through c o a d m i n i s t r a t i o n of e i s a p r i d e or m e t o c l o p r a m i d e . As t h e p h a r m a c o k i n e t l c and t h e c o a g u l a t i o n p a r a m e t e r s v a r i e d to a l a r g e e x t e n t i n t r a - and i n t e r i n d i v i d u a l l y we c o n c l u d e t h a t b o t h d r u g s s h o u l d be used w i t h c a u t i o n in c o m b i n a t i o n w i t h phenprocoumon.

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M O N I T O R I N G OF INTRAVENOUS "HEPARIN THERAPY BY A M O D I F I E D CHROMOGENIC A N T I - F A C T O R - X a - A S S A Y P. Hellstern, A. Seeliger, E. Wenzel .............................................. A commercial chromoqenic heparin assay (Coatest Heparin) was m o d i f i e d in order co allow m e a s u r e m e n t of anti-Xa-activities between 0.05 and 0.7 U/ml without changing the reaction conditions. The most important m o d i f i c a t i o n was replacement of the chromogenic S-2222 by S-Xa-I ~resenting a greater sensltivity against factor AN-A n t i c o a g u l a n t activities w e r e m e a s u r @ d in 50 patients under intermedlate or hlgh dose h e p a r i n treatment using the m o d i f i e d anti-Xaassay, Coatest heparin, an a n t i - I I a - a s s a y , and activated partial thromboplastin time (APTT). The closest correlation between dose of administered heparin and anticoagulant a c t i v i t y was observed with the m o d i f i e d anti-Xa-assay. whereas only weak or no siqnificant correlations were received by Coatest, antiIIa-assay, and APTT. There was also a close correlation between plasma anti-Xa-activities measured by the modified test and the corresponding values obtained by Coatest. Correlations between anti-Xa- and a n t i - I I a - a s s a y resu*its were weak. Both anti-Xa-assavs utilize excess antithrombin III which is adaed to the reaction mixture, while no antithrombin III is is added in the anti-IIa-assay. This may be the cause of weak correlation. The value of the different assays available for m e a s u r i n g anticoagulant heparin activities in preventing underdosage and overdosaqe remains ~o be established in further clinical studies. Abt. f. Klin. Haemostaseologie onsmedizin, D-6650 Homburg-Saar

u.

Transfusi-

PHARMACOLOGICAL IN VIVO-PROPERTIES OF RECOMBINANT TISSUE-TYPE PLASMINOGEN ACTIVATOR PRODUCED IN ESCHERICHIA COLl (BM 06.021) U. Martin m

g. Fischer, U. Kohnert, R. Rudolph, G. Sponer, A. Stern and K. Strein Tissue-type plasminogen activator (t-PA) produced by recombinant DNA technology in eucaryotic ceils is well known as a t h r o m b o l y t i c agent. Recombinant t-PA produced in E. coli and renatured tacks c a r b o h y d r a t e moieties in contrast to t-PA produced in eucaryotic cells. The aim of

our studies was to investigate i f there were differences between recombinant tissue-type piasmlnogen activator (rt-PA) derived from eucaryot i c cells (Aiteplase, Thomae GmbH, glberach, FRG) and rt-PA from E.coli (BM 06.021) regarding their pharmacoklnetic and pharmacodynamic characteristics. Rabbits were infused with 200 OOO lU/kg b.w. of DM 06.021 or Altepiase for 30 minutes. Plasma samples were assayed for t-PA-activity by a spectrophotometric method with ChromozymRpL. In comparison with AIteplase (n=6), BM 06.021 (n=5) showed an about three-fold longer i n i t i a l h a l f - l i f e (2.1+_0.6vs.5.6±2.6 mln, mean+-So) a three-fold slower plasma clearance (22.2±7.6vs. 7.5+1.7 ml • min -1 • kg-1) and a three-fold increased area under the curve (133+_44vs. 452+_105 IU • m1-1 " min). In a rabbit model of jugular vein thrombosis, BM 06.021 or A t t e p t a s e was infused for 4 hours at at least 3 dose levels. The specific activity of RM 06.021 was comparable with that of Alteplase. The analysis of the dose-response curves showed that BM 06.021 has a two-fold increase in thrombotytlc potency (ED~ 0.26 mg/kg b.w. vs. 0.55 mg/kg b.w.). At equieffective doses there were no s t a t i s t i c a l l y significant differences either in the plasma levels of t-PA-activity, fibrinogen, plasminogen or alpha~antiplasmln between BM 06.021 and Alteplase at the end of the experiments or in the hemodynamics during the infusion of the two agents.

I t is concluded that unglycosyLated rt-PA produced in E. coli is biologically active. Furthermore, BM 06.021 shows improved pharmacokinetic properties and an increase in thrombolytic potency. At equieffective thrombolytic doses BM 06.021 is comparable with ALteplase in its effect on the hemostatic and hemodynamic systems. Therefore, one might anticipate that non-gtycosyLated recombinant t-PA from E. coti exhibits superior pharmacological properties. Department of Pharmacology, Boehringer Mannheim GmbR, Sandhofer Str. 116, D-6800 Mannheim ]1, FRG

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34

PERFORMANCE OF A CHROMOGENIC AT III TEST ON A HITACHI 717 A N A L Y Z E R A.Hubbuch, G.Tensierowski and H.Lill We have applied the manual AT III reagent (Boehringer Mannheim,Cat.No.759376) using TosG l y - P r o - A r g - p N A (Chromozym ® TH) as substrate on a Hitachi 717 analyzer. At 25 and 30°C reaction t e m p e r a t u r e i0~i sample (diluted 1+50) and 250BI of a buffered (Tris/HCl,100 mmol/l, pH 8.1) thrombin solution (0.024 U/l) are incubated for 5 minutes. Then the chromogenic substrate solution is added (50BI) and the increase of absorbance is m e a s u r e d kinetically d u r i n g a time intervall of 2 minutes after another minute. At 37°C reaction temperature half of the sample volume and half of the thrombin c o n c e n t r a t i o n are used. R e s u l t s ( 2 5 ° C ) : W i t h i n run CVs (n=20) ranged from 1% to 3.3% between 14 and 5.6 IU/ml (112-45% AT III), day to day CVs (n=10) from 1.2% to 3.7% using the same specimens (control plasma and frozen human plasma). No deviation from linearity was observed between 3 and 25 IU/ml (25-200% AT III) using +/- 5% as acceptance criteria. The recovery of assigned values in two controls (PreciChrom ® I and II) was 98% and 101%. M e t h o d comparison in 50 deep frozen plasma specimens with AT III activities from 2 to 25 IU/ml showed an excellent agreement with the manual method: y= -0.2 + 1.00 x. The m e d i a n AT III activity in 15 fresh frozen plasma samples from h e a l t h y v o l u n t e e r s was 12.8 IU/ml (102%).Using the same reagent in the analyzer the calibration factor was found to be stable for at least 3 weeks. Results of the same quality were obtained at 30 and 37°C. We conclude, that the described p r o c e d u r e is well suited for routine analysis. Boehringer M a n n h e i m GmbH, Sandhofer Str. D-6800 M a n n h e i m

116,

CHANGES IN PLATELET PARAMETERS AFTER CORONARY-ARTERY BYPASS SURGERY MAY HELP DETECT THROMBOTIC AND SEPTIC COMPLICATIONS. A.A. yon Ruecker, R. Dickerhoff, P. Hufnagel, D. Murday, and F. Bidlingmaier Patients with c o r o n a r y heart disease u n d e r g o i n g c o r o n a r y - a r t e r y b y p a s s s u r g e r y r e p r e s e n t a well-studied g r o u p in w h i c h c h a n g e s of a n u m b e r of hematological, mostly platelet p a r a m e t e r s have b e e n described. N e w electronic cell c o u n t i n g a n d sizing t e c h n i q u e s in h e m a t o l o g y enable us t o analyze m o s t of t h e s e partially unc o m m o n parameters s u c h as m e a n platelet v o l u m e (MPV), platelet v o l u m e distribution width (PDW) and RBC-distribution width (RDW) o n a fast scale. It is i m p o r t a n t to be a w a r e of t h e e x t e n t of normally o c c u r r i n g c h a n g e s s o that misinterpretation of findings can be avoided. In this study, o u r first aim w a s to g e n e r a t e d i a g r a m s s h o w i n g t h e a v e r a g e r e s p o n s e (and 9 9 % c o n f i d e n c e intervals) of t h o s e h e m a t o logical p a r a m e t e r s t h a t s h o w e d characteristic p o s t o p e r a t i v e c h a n g e s in patients u n d e r g o i n g c o r o n a r y - a r t e r y b y p a s s grafting w i t h o u t c o m p l i c a t i o n s (n = 50). S e c o n d l y , w e e x a m i n e d if b y m e a n s of h e m a t o l o g i c a l p a r a m e t e r s p o s t o p e r a t i v e d i s e a s e states s u c h as t h r o m b o s i s , reinfarction o r infections could be differentiated f r o m c h a n g e s that normally o c c u r during t h e p o s t o p e r a t i v e period. O u r investigation s h o w s that t h e monitoring o f plate!et c o u n t and M P V during t h e postoperative p h a s e with t h e aid of t h e m e n t i o n e d d i a g r a m s helped identify t h r o m b o t i c d i s e a s e and infections in all patients with t h e s e postoperative c o m p l i c a t i o n s (7 o u t of a total of 83) up to 48 h before clinical signs w e r e apparent. T h e monitoring of platetet p a r a m e t e r s w a s clearly superior to l e u c o c y t e monitoring. L e u c o c y t e p a r a m e t e r s w e r e misleading in 16 c a s e s (5 false negative, 11 false positive). Furthermore, this s t u d y w a s able t o confirm r e c e n t findings t h a t an a b n o r m a l increase in PDW and low platelet c o u n t s f o u n d preoperatively in patients with c o r o n a r y heart d i s e a s e m a y serve as g o o d indicators for t h e p r e t h r o m b o t i c state and t h e risk of m y o c a r d i a l infarction. Institut f0r Klinische Biochemie der Universit&t Bonn, Sigmund-Freud-Strasse 25, D-5300 Bonn 1

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F I B e I N O L Y S I S IN P A T I E N T S W I T H N E P H R O T I C S Y N D R O M E U.Vertolli, K.Andrassv. J.Koderisch, E. Ritz ................................................ H y p e r c o a g u l a b i l i t v a n d t h r o m b o t i c e p i s o d e s are w e l l - k n o w n E e a t u r e s oE n e p h r o ~ i c s v n d r o m e ( N S ) , b u t the r e l a t i v e c o n t r i b u t i o n o~ - a l l e d g e d l v impaired~ G i b r i n o l y s i s ( F I > has not been e l u c i d i c a t e d . W e . t h e r e f o r e e x a m i n e d FI in NS in more detail under basal c o n d i t i o n s and f o l l o w i n g two s t i m u l a t o r y m a n o e v e r s ( v e m o u s o c c l u s i o n ( V O ) ~ i n ~ l a t i n g c u f ~ ~or 15 min b e t w e e n systol, and diastol DP; i n g u s i o n o4 D D V A P o,4 ug/kg over 30 m i n ; b l o o d s a m p l i n g 3o min t h e r e a f t e r ) . 13 pat with NS( p r o t e i n u r i a > 3,5g/ d/1,73 m =, normai GFR, prior to any m e d i c a t i o n ) and 8 c o n t r o l s w e r e analysed. RESULTS: N e p h r o t i c s s h o w e d the e × p ~ c t e d e l e v a t i o n of 9 i b r i n o g e n , G V I I I ( A g , C ) and a = - m a c r o g l o b u l i n ; no FDP: no s i g n c h a n g e s oF TPA, UPA( u r o k i n a s e type P A ) , a , a n t i t r y p s i m , a = - a n t i p l a s m i n , p l a s m i n o g e n (protein and chromogen, substr.), TAT( t h r o m P l n / a n t i t h r o m b i n 3) complexes, p r e k a l l i krein and F ×II levels. PAI-I was decreased. VO sign s h o r t e n e d e u g l o b u l i n ivsis time, h e m a t o c r i t rose less, TPA, PAI 1 and FDP rose s i g n . , w h e r e a s UPA and TAT r e m a i n e d u n c h a n g e d in NS in c o m p a r i s o n to controls. AFter DDAVP f VIII rose less in NS than in controls. Other f i b r i n o l v t i c p a r a m e t e r s were not diFFerent. C O N C L U S I O N : In NS p r o v o c a t i o n manoeuvers demonstrate unexpected h y p e r r e s p o n s i v e n e s m i . e . a u g m e n t e d r e l e a s e oF FIc o n t r o l l i n g p r o t e i n s 9tom e n d o t h e l i a l cells. S t u d i e s are u n d e r w a y to c l a r i g y w h e t h e r this is due to an i n c r e a s p d pool oE s t o r e d proteins.

AGE-DEPENDENCY OF THE FIBRINOLYTIC SYSTEM. K. Hager, S. N e u f a n g e r , T. V o g l und D. P l a t t .

D e p a r t m e n t int Medicine, U n i v e r s i t y Hospital, H e i d e l b e r o , F R G , B e r g h e i m e r s t r . 56 a and Div.di Ne#roloqia, O s e p d a l e C i v i l e di Padova, Italy

Aging i n f l u e n c e s plasma p r o t e i n c o n c e n t r a t i o n s and may t h e r e f o r e change t h e f u n c t i o n a l state of p r o t e i n s y s t e m s . An age d e p e n d e n t i n c r e a s e o f tissue plasminogen activator ( t - P A ) , and p l a s m i nogen a c t i v a t o r inhibitor ( P A l ) , as w e l l as a slight d e c r e a s e o f w h o l e b l o o d l y s i s t i m e have been r e p o r t e d . In o r d e r t o i n v e s t i g a t e age i n f l u e n c e s on f i b r i nolysis, s e v e r a l p a r a m e t e r s were d e t e r m i n e d w i t h tests commercially available. 48 h e a l t h y s u b jects of different age and 23 g e r i a t r i c patients participated. Comparing t h e young ( 2 0 - 3 5 y e a r s , n=18) w i t h t h e o l d e s t p a r t i c i p a n t s (75 y e a r s and more, n = 2 0 ) , p l a s m i n o g e n , ~ - 2 - a n t i p l a s m i n and Cl-inhibitor d i d n o t change. A n t i t h r o m b i n III (AT I I I ) sank f r o m 107 + 11 t o 94 + 12 % (p 500 ml

45 18

44 16

As shown above were observed no significant differences in the frequencies of thromboembolic events or postoperative bleeding complications. Thus, the single application of LMWH/DHE in a dosage of 1500 aPTT-U/0.5 mg a day was foundto be a safe and effective therapy regimen in perioperative prophylaxis of deep venous thrombosis in high risk patients. Abteilung fEJrklinische H&mostaseologie und Transfusionsmedizin, Universit&tskliniken, D-6650 Homburg/Saar

70

178

180 Congenital Protein-C-Deficiency-Symptoms and therapie in two cases K. Auberger 1 C. BrGckmannI, Th. Vukovich 2, H.-S. HadornI 1Children's Hospital, University of Munich, Lindwurmstr. 4, 28000 M~nchen 2 Institute of Phys. Medicine, University of Vienna, A-1090 Wien

Two patients with protein-C-deficlency are being followed up at our institution. Until 17 cases of this disorder have been described in the literature. A 5-year-old girl presented at the age or 8 months with ecchymosls and necrosis of the glutea[ area. Blood coagulation profile revealed protein-Cdeficiency (type I). Therapy with fresh-frozen plasma (FFP) and prothrombincomplex-concentrate (PPSB) was instituted which resulted in resolution of the necrosis. Transient skin bleedings remained unaffected. Upon administration of protein-C-concentrate every other day the child was free of symptoms for two years. Therapy had to be dlsconHnued because of severe anaphylactic reaction and administration of FFP (two times per week) was reinstituted. Again bleeding or skin and in additon severe retroocul~r hemorrhage was observed. Changing therapy to phenprocoumon-administration resulted in complete resolution of symptoms. A 6-year-old boy had had an intracranlal hemorrhage of two weeks, resulting in severe brain damage. Suspecting a diagnosis or dysfribrinogenemla FFP and cryoprecipitate was given, which prevented the child from further bleeding for two years. Unexpected profound necrosis of the skin with severe disseminated intravasal coagulation resolved again upon FFP administration but transient skin bleeding raised the suspicion of protein-C-deficiency (type I]]. e~eding was reduced by continuing this therapy. When FFP was replaced by PPSB after one year no further bleeding was observed. Nevertheless therapy had to be discontinued after two months because of deep venous thrombosis which lead to institution of phenprocoum-administration. Again complete resolution of symptoms could be achieved by this therapy. We conclude that phenprocoumon-administration was safe and effective in these two patients who had no further thromboembolic complications and no bleeding throughout treat,..ment period.

COMPARISON OF LOW MOLECULAR WEIGHT HEPARIN AND UNFRACTIONATED HEPARIN IN HAEMODIALYSIS PATIENTS: A CROSS OVER STUDY M KOhler, S. ROcker*, R. Bambauer*, M Heiden, U. Weber*, S. M0rsdorf, B. Braun, G. Pindur, G.A. Jutzler*.

The use of low molecular weight heparin (LMWH, Fragmin, KabWitrum) in comparison with unfractionatad beparin (UFH, Braun, Melsungen) was investigated in a cross over study in 27 haemodialysis (HD) patients over 12 month. The patients (15 female, 12 male) had a mean age of 65 years and were on HD for 5,7 years. 5 patients died during the study and one was transplanted. LMWH was given initially at a 60% dose of previous UFH requirement and subsequently adjusted according to AP'I-r and thrombin time (l-r) values. Several assays were performed prior to, 30 rain and after HD including: AP13-, "IT, anti-Xa, anti-Ila (Boehringer, Mannbeim), t-PA and PAl-1 (Biopool, Umea), von Willebrand factor antigen (Boehringer, Mannheim) and factor VIII:C anti-

gen. A mean heparin dose of 4958 U (UFH) vs. 4216 U (LMWH) was given per dialysis. Mean APTT (Boehdnger, Mannbeim) values before HD were 33.8 s and 33.9 s, and after HD were 49.9 and 46.3 s for UFH and LMWH, respectively. Significant correlations between chromogenic substrata anti-Xa assay (KabWitrum) and Heptest (Haemachem, St.Louis) were observed during UFH (r=0.71) and LMWH (r=0.72). The correlation between APTT and anti-Xawas r= 0.62 for UFH, but only r= 0.27 for LMWH. Factor vIn:CAg decreased during LMWH treatment (425_+151 to 317_+ 260 U/dl), but remained constant during UFH (313_+122 and 292_+144 U/dl). Mean platelet count showed no differences during LMWH (228_+84x 1000/ul) or UFH (220+89 x 1000/ul) treatment. Transfusion requirements were 1.59 _+2.62 U and 1.6 -+ 1.44 U red cells during UFH and LMWH treatment, respectively. Shunt thromboses were observed in 1 (UFH) and 3 (LMWH) patients. Our data confirm earlier findings that LMWH may be used as an anticoagulant for HD, however, dose finding, drug monitoring and possible advantages have to be investigated and confirmed in further studies. Abteilung for Klinische H~mostaseologie und Transfusionsmedizin und Abteilung for Dialyse und Nephrologie der Universitt=t des Saarlandes, D-6650 Homburg/Saar, F.R.G.

151 181

183

THE INFLUENCE OF RECOMBINANT HUMAN ERYTHROPOIETIN ON MACRO- AND MICROCIRCULATION, COAGULATION AND FIBRINOLYSlS IN HAEMODIALYSlS PATIENTS.

THE EFFECT OF LOW MOLECULAR WEIGHT HEPARINS (LMWH) AND UNFRACTIONATED HEPARIN (UFH) DURING SUCCESSIVE ADMINISTRATION IN HEALTHY SUBJECTS: (2) CUMULATION OF ANTICOAGULANT ACTIVITY.

B. Braun, M. K~hler, S. M6rsdorf, F. Jung, E. Wenzel, U. Weber*, R. Boalet*, J. Liamann*, H. Tarrach*, R. Bambauer*, G.A. Jutz!er*

12 months therapy

Low molecular weight heparins (LMWH) are more and more substituting uofractlonationatod heparin (UFH) as prophylactic agents against deep venous thrombosis. The use of LMWH appears to be more convenient, since due to an Increased haif-life and bioavailabilify only one injection per day is required. This, however, bears the risk of cumulation of anticoagulant effects during long term administration. We therefore investigated the effects of different LMWHs and UFH, when injected over a 5 day period in a randomized cross over study in 12 healthy subjects. Fragmin (KabWitrum, Munich), Fraxiparin (Sanofi, Munich), Embolex (without dihydroergotamine, Sandoz, Nemberg) and Liquemin (Roche, GrenzachWyhlen) were Injected subcutaneously using the recommended dosage at 8:00 for 5 days. Between the different drugs was a washout period of 14 d. Investigations on heemostasis were performed using standard methods and commercially available test systems. The anticoagulant effects (LMWHs calibrated against the WHO standard for LMWH) 3 h after the 5th injection (3 h after the I st injection in parentheses) are shown in the following table (mean values): APT[ (s) anti-Ila (U/ml) anti-Xa (U/ml) HepTest (U/ml)

3,69 (0,34) 260 (71,8) 1,44 (0,16) 465 (118) 198 1,55 (66,7) (0,62)

Fragmln Fraxiparin Embolex Liquemin

Recombinant human erythropoietin (rh-EPO) has been shown to be effective in the treatment of renal anaemia. Additionally rh-EPO improves the haemostatic defect of uraemia. On the other hand, a hypertensinogen effect and an increased risk for thrombosis have been reported in haemodialysis HD) patients. 20 HD patients in Hombor~l were recruited for a multicenter, cebo-controlled study (MF 3981), aiming to assess the risk of rh-EPO. e investigated the effect of rh-EPO on micro- and macrocirculation, heemostasls and haemorheological paramsters. Initially, 10 patients received rh-EPO at a dose of 3 x 80 U/kg body weight per week which was subsequently ad usted according to the haematocrit, After 6 months the 10 patients receiving placebo were changed to rh-EPO therapy. Clinical and laboratory Investigations were done before, 1, 3, 6 and 12 months after the beginning of treatment. The following table demonstrates the influence of rh-EPO on some of the parameters (mean values and standard deviation - corrected for haematokrit - in parentheses) studied: Parameter erythrocytes (mio/ul) I thrombocytes (1000/ul) I plasma viscosity (mpas) J fibrinogen (mg/dl) I I A, dors. pedis (mmHg) I | arterial flow (ml/100ml tis- ~ I sue) I

prior to treatment 2,67 (0,33) 234 (103) 1,39 (0,16) 458 (129) 204 (64,1) 2,14 (1,20)

3 months therapy 3,52 (0,42] 268 (101) 1,46 (0,121 536 (152) I 2,o6 195 (62,3 (1,16

M Heiden, G.Weishaupt, G. Pindur, B. Wagner, M. K6hler

Erythrocyte and platelet counts increased significantly during rh-EPO. When the other results, obtained in the placebo group, were taken into consideration, no significant changes, attributable 1o rh-EPO, were observed 12 months after treatment. Blood pressure and arterial flow remained constant, presumably due to an increased antlhypertensive treatment regimen. /,%Abteilung fQr Klinlsche H~mostaseologle und Transfusionsmedizln und bteilung for Dialyse und Nephrologie der Unlversit~tskliniken des Saarlandes, D-6650 Homburg/Saar, F.R.G.

34.9 (34.0) 36.4 (35.2) 33.8 (34.2) 42.5 (38.2)

0.04 (0.05) 0.04 (0.02) 0.01 (0.01) 0.13 (0.10)

0.39 (0.30) 0.38 (0.179 0.30 (0.18) 0.25 (0.20)

0.19 (0.09) 0.32 (0.12) 0.16 (0.11) 0,19 (0.17)

When using the anti-Xa assay (KabWitrum, Munich), several results were outside the measuring range, especially after Fraxiparin. A significant correlation between anti-Xa assay and HepTest (Heemachem, at.Louis) was found (y= 0.019+ 0.35 * x; n=221; p< 0.00001; r= 0.34). Our data suggest a significant cumulation of anticoagulant activity after successive administration of several LMWHs. Abteilung filr Klinische H~mostaseologie und Transfusionsmedizin Universit=~ltdes Saarlandes, D-6650 Homburg/Saar, F.R.G.

182

184

THE EFFECT OF LOW MOLECULAR WEIGHT HEPARINS (LMWH) AND UNFRACTIONATED HEPARIN (UFH) DURING SUCCESSIVE ADMINISTRATION IN HEALTHY SUBJECTS: (1) EFFECTS ON PRIMARY HAEMOSTASlS, FiBRINOLYSlS AND ADVERSE EFFECTS

Results of the TAT-ELISA in patients undergoing percutaneous transluminal coronary angloplasty (PTCA) and cardiopulmonary bypass surgery

M. K0hler, G.Weishaupt, G, Pindor, B. Wagner, M, Heiden Low molecular weight heparins (LMWH) were introduced as substitutes for uofractionated heparin (UFH) in order to decrease adverse effects of conventional prophylaxis of deep vein thrombosis. There are, however, controversial communications on the effect of UFH and LMWH on the fibrinolytic system. We therefore investigated the effects of different LMWHs and UFH, when injected over a 5 day period in a randomized cross ever study in 12 healthy subjects. Fragmin (KabWitrum, Munich), Fraxiparin (Sanofi, Munich), Embolex (without dihydroergotamine, Sandoz, NOmbcrg) and Uquemin (Roche, Grenzach-Wyhlen) were injected subcutaneously using the recommended daily dosage at 6:00 for 5 days. Between the different drugs was a washout period of 14 d. Investigations on heemostasis were performed using standard methods and commercially available test systems. No significant changes of plasminogen activator antigen and plasminogen activator inhibitor antigen (Biopool, Umea) were observed during successive administration. No significant changes of bleeding time (Duke), thromboxane B2 and 6-ksto-PGF1 (NEN, Dreielch) were found. Factor VIII antigen and von Willebrand factor antigen (Boehringer, Mannheim) remained unchanged. A significant increase of liver enzymes (ALAT > 30 U/I) was observed in 3 (of 12) subjects after Uquemin, and in one after Fraxiparin and Fragmln. Our data suggest no effects of UFH and LMWH on the fibrinolytic system as well as on the primary haemostasis. Elevation of liver enzymes may also occur after LMWH, however, may be less frequent when compared with UFH. Abteilung f0r Klinische H~mostaseologie und Transfusionsmedizin Universfl,~t des Saarlandes, D-6650 Homburg/Seer, F.R.G.

F.Mahlstedt, Medizinische

H.K6stering, G.Claus, J.U.Wieding; Universit~tsklinik, 3400 G6ttingen

The research conducted was focussed on investigating the reliability of the ThrombinAntithrombin III-ELISA. Starting from the observation that the concentration of the TATcomplex in blood taken from an in-dwelling cannula (IDC) increased after 90 min, after a dose of 15 U of heparin per kg body weight had been injected, in vitro tests were carried out to examine the effect of the IDC and the time of passage through it. From this experiment it emerged that the results of the TAT-complex measured in blood taken from the IDC displayed higher readings than in blood obtained through cannulation with a butterfly and that it increased in proportion to the time of passage. In addition two groups of patients were examined. The first group consisted of patients who had to undergo a PTCA, whereas the second one were to undergo cardiopulmonary bypass surgery. It turned out that, after an injection of 15,000 U of heparin and after extracorporeal circulation, the TAT-complex increased, which suggests an increased activation of coagulation. Despite these results the question whether the TAT-test is a suitable one to be carried out routinely, still remains open because it is the method of taking blood samples which is of major importance, and falsely positive results are achieved very frequently.

152

185

187

HYPOFIBRINOGENEMIA CAUSED BY VINCRISTINE/PREDNISOLONE THERAPY OF LYMPHOID BLAST CELL CRISIS OF CHRONIC MYELOID LEUKEMIA W.Speiser, G.Sunder-Plassmann, C.Korninger, P.Bettelheim, K.Lechner

R E S T I N G AND A C T I V A T E D PLATELETS CONSTRUCTION R. Dierichs and E. M o r g e n s t e r n

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Therapy with v i n c r i s t i n e and prednisolone caused a pronounced decrease in fibrinogen levels in 9 patients treated f o r lymphoid blast c r i s i s (LBC) of chronic myeloid leukemia (CML). In two patients the i n i t i a l decrease in white blood c e l l count (WBC) in response to therapy was accompanied by a marked decrease in fibrinogen levels to values below 80 ng/d], within 'he f i r s t three days of trealmu.L, a pronounced rise i,, b-Db,er ieveis~ the occurence of soluble f i b r i n in the c i r c u l a t i o n and a drop in p l a t e l e t count to values below 20.000 c e l l s / u ] . Induct i o n of disseminated intravascular coagulation DIC in these two patients caused profuse bleeding and necessitated s u b s t i t u t i o n therapy with fibrinogen and p l a t e l e t concentrates. In the remaining 7 patients no signs of DIC were detected a f t e r i n i t i a t i o n of therapy, nevertheless 4 of them showed a moderate increase in D-Dimer levels. In these patients a well known side e f f e c t of steroid therapy, namely a decrease in fibrinogen levels accnrding to the h a l f l i f e of 96 hours was observed. Fibrinugen levels did not f a l l below 150 mg/dl and increased a f t e r dose reduction from 100 mg/day to 50 mg/day. We conclude from our results that "two types of disturbances in f i b r i nogen metabolism can be observed during vincristine/prednisolone therapy of LBC in CML: i) a decrease in fibrinogen levels according to i t s biological h a l f l i f e due to a steroid mediated impairment of l i v e r synthesis and 2) a rapid f a l l in fibrinogen levels in the course of DIC most l i k e l y induced by the release of procoagulants from d e t o r i a t i n g blast c e l l s , leading to severe bleeding. Therefore blood coagulation parameters should c a r e f u l l y be monitored in such c l i n i c a l settings. First

Dept.

of Medicine,

University

of

Vienna,

IN T H R E E - D I M E N S I O N A L

RE-

Human and b o v i n e p l a t e l e t s were fixed by impact freezing and f r e e z e - s u b s t i t u t i o n in resting state and after activation. E l e c t r o n m i c r o g r a p h s of serial sections were digit i z e d by a M a c i n t o s h II computer (Apple Inc.), a n d threed i m e n s i o n a l r e c o n s t r u c t i o n was done with r e g a r d to the surface shape, the d i s t r i b u t i o n of internal organelles, the c o n t r a c t i l e sphere, and the d i s t r i b u t i o n of m i c r o t u bules. The p r o g r a m allows p r e s e n t a t i o n of d i f f e r e n t selections of the c o m p o n e n t s of the m o d e l s under various aspects. T r a n s p a r e n t r e c o n s t r u c t i o n of some surfaces give insight into internal o r g a n i z a t i o n s . The c r y o - p r e p a r a t i o n acts very rapid. C o n f o r m a t i o n a l changes of p l a t e l e t s therefore most likely are the result of activation, and not to be r e g a r d e d as artifacts due to the slow p r o c e s s of chemical fixation. Both p l a t e l e t types have a d i s c o i d shape in the resting state. In b o t h types a c t i v a t i o n causes the f o r m a t i o n of a c o n t r a c t i l e sphere. By this way c e l l u l a r c o m p a r t m e n t s are r e d i s t r i b u t e d and centralized. The dense tubular s y s t e m forms a b r a n c h e d m e m b r a n e c o m p l e x with n a r r o w lumina which o c c a s i o n a l l y e n l a r g e into distinct vesicles. AS b o v i n e p l a t e l e t s have no s u r f a c e - c o n n e c t e d c a n a l i c u l a r system, they p e r f o r m no shape c h a n g e in the way human p l a t e l e t s do. Nevertheless, they exhibit m e m b r a n e c o m p a r t m e n t s that have contact to the cell surface a n d result from fusion of g r @ n u l e m e m b r a n e s with the cell surface when s e c r e t i o n has occurred. The c o m p u t e r models give evidence of the c o m p l e x m o r p h o logical r e o r g a n i z a t i o n that occurs when p l a t e l e t s transform from the resting into the a c t i v a t e d state. Institut M~nster,

fur A n a t o m i e der U n i v e r s i t ~ t V e s a l i u s w e g 2-4

MUnster,

D-4400

Austria

186 DYNAMICS OF PLATELET ULTRASTRUCTURE WITHIN THE FIRST SECONDS OF PLATELET ACTIVATION E. iViorgenstern, A. Ruf, and H. Patscheke Shape change (1), internalization of surface-bound iigands (2) and the exocytosJs of secretory organelles (3) characterize stimulated platelets. These reactions involve membrane rearrangements and the action of the contractile cytoskeleton and appear to run off simultaneously within the first seconds of activation. To study the earliest structural dynamic processes during activation the platelets were stimulated for 10 up to 60 seconds with thrombin or collagen and than chemically fixed or - for demonstration of membrane fusion - rapidly frozen (impact freezing) and freezesubstituted. Using electron micregraphs from serial sections the structural alterations were reconstructed. (1) Within the first seconds of piatelet stimulation the membranes of the "surface connected system" were evaginated for the purpose of surface enlargement required to change shape. (2) Simultaneously, the plateJets started to internalize surface areas with bound ligands. The plasmalemmaJ invaginations were observed attached to the contractile get and thus they followed the centripetally moving constriction. This could be demonstrated for several surface bound ligands as cationized ferritin, fibrin as weJI as collagen fibers. The internalization of fibrillar ligands by platelets in this mode caused the retraction of fibers. (3) Within the first seconds after stimulation the membranes of the secretory organel]es form appositions to the plasmalemma or to membranes of the SCS and seemed to maintain this position during shape change and interna] contraction. The exocytosis started after formation of a fusion pore and was then continued by compound exocytosis of secretory granules. The observed membrane rearrangement may explain how piatelets are able to carry out their different actions (shape change, surface-ligand interaction, release reaction and retraction) within a short period of time. Thereby, a retrieval of membranes and integrated components seems to occur. (supported by DFG, Grant Me 124/4-2/3) Medizinische Biologie, Universit~t des Saartandes, D-6650 Homburg/Saar und Institut for KJinische Chemie, Klinikum Mannheim der Universit~Lt Heidelberg, D-680Q Mannheim

188 DAY TO DAY VARIATIONS OF THE FIBRINOLYTJC SYSTEM IN HUMAN PLASMA L. Barthold, J. Grulich-Henn, D. Heinrich and G . M011erBerghaus In recent years the knowdlege on the regulation of the fibrinolytic system has substantially increased. Several clinical conditions have been associated with alterations of the fibrinolytic system in plasma. However, it is difficult to draw definite conclusions from the laboratory data obtained as the plasma levels of several components of the fibrinolytic system, such as tissue-plasminogen activator (tPA) and plasminogen activator inhibitor type 1 (PAl-l), are influenced by diurnal variation, age, exercise, stress, nutrition. In order to study the fibrinolytic system in plasma, it is necessary to standardize influencial factors. We designed a highly standardized procedure for the investigation of the fibrinolytic system in plasma. Ten healthy volunteers were investigated three times, with a minimal interval of 7 days between each examination. Blood was withdrawn under fasting conditions in the morning between 7.30 and 9.00 a.m., after a resting period of 20 minutes, before and after 10 minutes of venous occlusion (VO). In order to evaluate the day to day variation, the coefficient of variation (CV) was estimated for each parameter before and after VO. The mean CV for the eug!obulin lysis time was 9.5% before VO and 9.3% after VO. The CVs for tPA were 14.4% and 27.0% before and after VO, respectively. PAl-1 antigen showed a CV of 10.5% before and 19.0% after VO, and the CV for PAl-capacity were 5.0% before and 33.0% after VO. There was a significant correlation between PAl-1 antigen and PAl.capacity before and after VO (r=0.64, p