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ASSESSMENT OF PLATELET HYPERREACTIVITY R.E. Scharf

PLATELET FUNCTION IN PATIENTS ~TTH TRANSIENT ISCHEMIC ATTACKS AND STROKE Thrombozyksn-Reaktivit~t

Clinical and pathological studies indicate that platelet hyperreactivity occurs in a number of vascular disorders, including unstable angina, stroke, peripheral vascular disease, and following angioplasty or coronary thrombolysis. To detect activated platelets in vivo, sensitive and specific methods are required that may permit the identification of a prethrombotic or thrombotic state. Demonstration of circulating platelet aggregates has been used for this purpose but this test lacks both specificity and sensitivity. At the present time, the most reliable markers of platelet activation in vivo are constituents, such as Bthromboglobulin and platelet factor 4 that are released from activated platelets and measured in the plasma or urine, and metabolites of thromboxane Av However, widespread clinical application of these markers is limited by a number of technical requirements including sample collection, processing, and analysis. Another little used test for activated platelets is the determination of platelet density distribution since activated degranulated platelets are lighter than normal. Recent advances in flow cytometry have made it possible to study platelets rapidly with respect to size and qualitative or quantitative changes of membrane giycoproteins. Using murine monoclonal antibodies that bind specifically to "activation-dependent" epitopes at the pintelet surface, it is now feasible to identify individual activated circulating platelets. This review will summarize recent developments in this area and discuss advantages and limitations of different techniques that are being used for the detection of activated platelets in clinical disorders. Institut f'tir Exp. Hiimatologie und Transfusionsmedizin, Univ. Bonn, Sigmuod-Freud-Str. 25, D-5300 Bonn 1

M. G. Hennsrici. Heidelberg, FRG

bei TIA und zerebralem Insult

Klinlknm Nannhslm. University

TIA and stroke are frequently due to thromboembolic mechanisms a~ising either from cardiac sources or as arterial embolism from atherosclerotic plaques of the vascular system. Among ether mechanisms, platelet aggregation plays a major role in the formation of structural vascular lesions as well as of its embolJc capacity. As a result of this concept in the pathogenesls of cerebra] vascu]ar events, the prevention of TIA and stroke is based on drugs interfering with platelet function. In contrast to the considerable amount of clinical trials to be reviewed, which supports the reduction of stroke risk in patients with generalized atharosclerosis, only limited data exists on the incidence of cerebrovascular events in patients with the defined atherosclerotic lesions of the brain's supplying arteries and the role of platelets involved. Among these is a five year prospective follow-up study investigating patients with carotid plaque lesions for progression and regression by means of ultrasound in parallel with platelet function analysis (Cerebrovasc Dis 1991, i:142-148): This study suggests that intervals of progression of stherosclerosls are closely associated with hyperactlvJty of platelet function whereas stable phases and in particular regresslve periods are more like'ly to be linked to normal status of platelet funcltion.

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PLATELET HYPZP~EACTIVITYIN CORONARY HEART DISEASE (CHD) AND MYOCARDIAL I~ARCTION (HI) J. ~en~st Several lines of experinestaland clinical evidence indicate that platslets are involvedin the initiationof coronary atherosclerosis(platslet-derived niteqe~) end contribute to %.heprecipitationof acute coronary evests such as unstable angina, acute HI and sudden cardiac death. Acute coronary syn~oles ~y be triggered by platelet-relensedv~aetive mediators (serotonin, throlboxaun A2) leading to transient flow reductions in diseased coronary arteries and/er by a~elate formation ultimately resulting in thrombotic vens~l obstruction. Various indices of platelet activation end hyperreactivity have bees detected in patients with manifest CaD, These include augmented platelet aggregability, circulating platetet a~jregates, reduced platelet survival, elevated plasma levels of beta-thro~oqlobulin and platelet factor 4, and increasedt~o~oxene formation. Furtherevidence say be awaited from studies esinq sonoclunal antibodies to detect activation-dependent~a=brene epitopes and platelet-derivednicroparticles. I key question with respect to both the sequence of pathophysiologic events and the rationale for therapeutic intervention is: Does platslet activation in (~]) =erely result fro= the interaction of functionally nor=m], platelets with the atheroeclerotic vessel wall or do abnormally reactive platelets predispose to acute coronary events? Recent longitudinal studies suggest that elevated platelet counts, changes is platelet volu=e and density end finally eahenced platelet aqq~equbilitypredict an increased risk of first occurrence or recurrence of acute coronary events. Hormonal and dietary factors but also intrinsic platelet almor=alitiessuch as abnormal calcium bandiinq or sarotossrqicdysfunctioncould accountfor platelethyperreactivity. ADP end thromboxa~=-depend~t hut also thro=bin-induced=echunism of platelet activation appear to be involved. These findings open the perspective for new strategies in a,tiplatelettherapy in the various settings of

Mad. Klinik end P01iklinikder Westf. Wilbells-Universit&t,Albert- Schweitzer-Stl. 33, 4400 M~nster

PLATELET HYPERREACTIVI'I'Y IN DIABETES MEUJTUS D.Tschoepe Vascular accidents are the main cause of death in patients with diabetes of beth types (I, IDDM and II, NIDDM). So far, improved management of the metabolic derangement was not paralleled by a satisfactory reduction of the cardiovascular morbidity and mortality. Platelet hyperrsactivity is well known in diabetes and is considered to be a key signature of the prethrombetic state in these patients contributing to the vascular excess mortality. We showed recently that the increased functional properties of diabetic platelets result from the primary release of larger platelets with enhanced thromboxane formation capacity and increased numbers of functional glycoprotein receptors GPIB and GPIIB/IIIA which are synthesized in the megakaryocytes: "The megakarycyte-I~atelet system is turned on in diabetes mellitus'. It was however a matter of controversy whether the increased functional potential of diabetic platelets becomes operative in vivo: "Circulate platelets in an activated state in diabetes mellitus?" The Duesseldorf III method of single platelet flowcytometry (SPFC) for the activation dependent molecular markers CD62 and CD63 allowed the direct answer: "Large p~atsfets circulate in an activated state in diabetes msflitus'. At present, metabolic normalization is the main therapeutic option in the treatment of patients with diabetes. It is however questionable whether this normalizes the underlying primary platelet hyperreactivity. Antiplatelet therapy, e.g: ASA, calcium-channel-blockers or ticlopidin, has not yet become established in diabetics as a beneficial therapeutic adjunct despite even more rational indication compared to the general population. For the first time the platelet membrane activation marker test will be able to identify single patients with a discrete activated platalet system as present at prethrombotic states and this allows an active selection of patients with diabetes who will possibly benefit from additional antiplatelet therapy. "Cellular Haemostasis Group', Diabetes Research Institute at the Heinrich-Heine University, Duesseidorf, Auf'm Hennelamp 65, D-4000 Duesseidorf 1.

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NEW ASPECTS OF THROMBOLYTIC THERAPY IN ACUTE MYOCARDIAL INFARCTION C. Bode and W. Ktibler

Hirudin in the treatment of acute coronary events: a new perspective? H.R. Biiller, M.T. Nurmohamed, J.W. ten Cate.

Whereas the beneficial effects of thrombolytic therapy in acute myocardial infarction have been shown beyond doubt, the results of the GISSI-2 and ISIS-3 studies keep the question open which therapeutic agent should be prefered. New application schemes of known plasminogen activators, new possibilities of adjunctive therapy and the development of new plasminogen activators have been undertaken in an effort to overcome present limitations of this form of therapy. Among the limitations are a primary failure rate to achieve reperfusion of 20-40%, an early reocclusion rate of 5-20% and an increased risk of bleeding that makes many patients ineligible for thrombolytic therapy. "Front-loading", the rapid infusion of large amounts of activator has been successfully tested for rt-PA. A number of combinations of plasminogen activators have been evaluated. A combination of prourokinase and rt-PA appears more promising in terms of efficacy and specificity as combinations of urokinase and prourokinase, rt-PA and urokinase or rt-PA and streptokinase. Early, preclinical application of thrombolyfic agents is also being evaluated in an effort to reduce the ischemic period. In an effort to improve lysis of platelet-rich, arterial clots and to prevent early reocclusion, a number of agents beyond aspirin and heparin have been evaluated as adjunctive therapy among them hirudin, argatrobane, activated protein C and GPIIb/IIIa inhibitors on the basis of peptides or antibodies. Among the new plasminogen activators, a t-PA mutant with altered pharmacokinetics has yielded encouraging results in clinical trials. Experimental in rive data make bat-t-PA and antibody-activator fusion proteins promising candidates for the future.

The first description of the anticoagulant action of a watersoluble, heatresistant substance derived from the salivary glands of leeches (Hirudo Medicinales) dates back to 1884. This substance was later called hirudin in 1904. Almost no research was done with this compound until the 1960's. More recendy, after hirudin or fractions of it have been produced by recombinant techniques as well as by synthetic methods, there has been a fast growing interest in the clinical potential of this specific thrombin inhibitor. Hirudin is in particular effective in the inhibition of the feedback action of the coagulation system by thrombin. In addition Hirudin is a potent inhibitor of the thrombin mediated platelet aggregation. At present this compound is investigated both in the treatment and prevention of venous and arterial thromboembolic diseases in experimental models as well as in man. With respect to acute coronary events Hirudin is evaluated in models and patients with unstable angina, or with acute myocardial infarction treated with thrombolytic therapy, and also in patients undergoing PTCA in order to prevent restenosis. The evidence of the potential effectiveness and the preliminary clinical data in these indications will be reviewed. Centre for llemostasis, Thrombosis, Atherosclerosis and Inflanunation Research. Academic lVledicai Centre, Amsterdam, The Netherlands.

Medical Clinic III (Cardiology.), University of Heidelberg, Bergheimerstrage 58, 6900 He,delberg

6 THE P R O B L E M OF LYSIS ~ PTCA

8 REOCCLUSIOM FOLLOWING THROMBO-

H.J. Rupprecht, J. Meyer Reocclusion is seen in 6-18% of the patients with acute myocardial infarction within 24 hours following successful thrombolyaia and in 11 to 24% of the patients within the next days. Reocclusion is usually associated with further deterioration of left ventricular function and increased mortality. Post-mortem studies have illucidated that ruptered arteriosclerotic plaques with a high grade residual lesion and thrombi are found in most patients with reocclusion. As there is ongolng thrombosis inspite of thrombolysis, antithrombotic treatment with heparin should be performed simultaneously with the initiation of thrombolysis. Concomitant treatment with vasodilating agents should be considered to avoid coronary artery spasm. Moreover aspirin may be valuable in the prevention of reocclusion in the longterm run. Patients with postinfarction-angina or incipient left heart failure should undergo urgent coronary angiography, and in case of need P T C A or coronary artery bypass grafting. Within the first days following successful PTCA about 4-5% of the patients develop reocclusion with the clinical picture of recurrent angina or acute myocardial infarction. In most of these patients, a major dissection or intracoronary thrombi can be seen immediately following the procedure. Thus, a thrombotic process in combination with elastic recoil and early restenosis can lead to early reocclusion following PTCA. II. Medizinische Klinik, Johannes GutenbergUniverait~t, Langenbeckstr. I, 6500 Mainz

EVIDENCE FOR SYNERGISTIC EFFECTS OF CATHEPSIN G AND PAF IN THE ACTIVATION OF PLATELETS BY STIMULATED NEUTROPHILS A. Maras, A. Ruf, R. Schlenk and H. Patscheke Cathepsin G seems to be the major stimulus involved in neutrophfl (PMNL) induced platelet aggregation (Evangelista V., 1991, Blood 77:2379). An additional role of platelet activating factor (PAF) and other mediators is still controversially discussed. We used eglin C (a serine protease inhibitor) and Web 2086 (a PAF receptor antagonist) in order to investigate whether cathepsin G and PAF are effective in mixed suspensions of platelets and PMNL. Aggregation and luminolenhanced chemiluminescence (CL) were simultaneously measured with a lumiaggregometer as parameters for platelet and PMNL activation, respectively. The chemotactic peptide FMLP induced both CL and platelet aggregation in the presence of cytochalasin D. The prostacylin mimetic iloprost and EDTA inhibited the aggregation but did not affect the CL of PMNL. When cytochalasin D was omitted, CL decreased and no platelet aggregation oceured. Both eglin C and Web 2086 inhibited the aggregation induced by FMLP in the presence of cytochalasin D. At a high concentration of FMLP 2x10 -6 M, eglin C 50,ug/ml and Web 2086 2x10 -5 M produced a maximal inhibition of no more than 54% and 36%, respectively. Both inhibitors used in combination showed an inhibition of 91%. At a Lower FMLP concentration 2x10-7 M the inhibition of platelet aggregation by eglin C was 75% and by Web 2086 74%. Both inhibitors added together produced an inhibition of 92%. In the controls, neither eglin C nor Web 2086 inhibited the FMLP-induced CL of PMNL. These results suggest that neutrophil derived cathepsin G and PAF may act synergistically in platelet activation in mixed suspensions of platelets and PMNL. (Supported by the DFG, Pa-263) Medizinisch-Diagnostisches Institut, Klinikum Kerlsruhe, 7500 Karlsruhe 1

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THE INFLUENCE OF NEUTROPHIL PREPARATION ON THEIR ACTIVATION INDUCED BY PLATELETS

THE CALPAIN-ACTIVATING DRUG DIBUCAIN INDUCES ALPHA-GRANULE RELEASE IN HUMANELATEL~TS E. Morgenstern, H. Patscheke - j and A. Ruf *) The release of proagulant-rich microvesicles (MV) from the p l a t e l e t surface was a t t r i b ~ e d to drugs which dissociate the submembranous cytoskeleton (SMC) from i t s membrane attachment sites (~E.B.Fox et a l . , J Cell Biol 111: 483, 1990). Under conditions comparable to those of these investigators we use the dissociating calpain act i v a t o r dibucain (0.5 mM) in order to observe d i r e c t l y this phenomenon ("membrane shedding"). Suspensions of washed p l a t e l e t s (6x10~ were incubated (I-3 min at 37~ rapidly frozen and freeze substituted or fixed with glutardialdehyde (GA) and embedded after dehydration For electron microscopy (see E. Morgenstern, Scanning Micr Suppl 5: in press, 1991). With dibucain GMP140 (an indicator of alpha-granule release, determined by flow cytometry) was expressed on the p l a t e l e t surface. After GA, the drug induced MV and myelin figures (MF) as described in thrombin-stimulated pTatele~ (see Morgenstern 1991). Furthermore, dibucain induced destructive p l a t e l e t alterations (rounding without pseudopodia f o r mation, rippled plasmalemma, inhomogeneous hyaloplasm). Frozen and freeze substituted platelets did not show vesiculation or MF after dibucain. Fusion of secretory organelles, swollen and compound granules were observed as characteristics f o r exocytosis. Different from GA, the spheric p l a t e l e t s after dibucain treatment revealed a smooth outline and a homogeneous appearance of the hyaloplasm. I t is suggested that the phenomenon (shedding of MV) results from fusion of alpha-granules which is f a c i l i t a t e d by the alteration of the fusion-preventing SMC. GA-fixation induces vesiculation on fusion sites (see Morgenstern 1991) and may cause also membrane rippling and a l t e r a t i o n of the hyaloplasm.

R. Schlenk, A.Ruf, A.Maras and H.Patscheke The activation of neutrophils (PMNL) by platelets requires mutal contacts and fibrinogen as a cofactor (Ruf et al. 1991 Thromb. Haemostas. 65:189). We examined how the procedure of PMNL preparation influences platelet induced activation of PMNL. Luminol-enhanced chemiluminescence (CL) of PMNL was measured in platelet PMNL suspensions or human whole blood with a lumiaggregometer. Preparation 1 (PMNL1): PMNL were isolated by dextran sedimentation from blood anticoagulated with ACD, subsequent centrifugation through FicolI-Hypaque and hypotone red cell lysis. All steps were carried out at room temperature. Preparation 2 (PMNL2) comprised sedimentation with hydroxyethyl starch, hypotone lysis and centrifugation through PercollR at 4~ An elutriation centrifuge was used for PMNL isolation at room temperature in preparation 3 (PMNL3). Unstimulated PMNL1 showed a bipolar shape with surface ruffling and a basal CL, whereas PMNL2 and PMNL3 were spherical with a smooth surface and did not show CL. The extent of the CL induced by the chemotactic peptide (FMLP) was: CLpMNL1 > CLpMNL2 = CLpMNL3, Addition of unstimulated platelets to the PMNL induced a CL of PMNL1 and PMNL2 but not of PMNL3. The extent of CL induced by platelets stimulated with U 46619 was also different in the three PMNL preparations: CLpMNL 1 > CLpMNL2 > CLpMNL 3. These observations suggest different degrees of preactivation and priming in PMNL1 > PMNL2. Both PMNL3 and whole blood required stimulation by FMLP or U 46619 to show CL. This indicates the absence of preactivated or primed PMNL. We conclude that response of PMNL1 and PMNL2 to unstimulated platelets are due to preactivation and priming of neutrophils during their preparation. (Supported by the DFG, Pa=263) Medizinisch-Diagnostisches Institut, Klinikum Karlsruhe, 7500 Karlsruhe 1

Medizinische Biologie, Universit~t des Saarlandes D-W6650 Homburg/Saar and ) Medizinlsch-Dzagnostlsches Ins t i t u t am Klinikum, D-W-7500 Karlsruhe, FRG, .

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BILIRUBIN=INDUCED PLATELETAGGREGATION,EXVIVO.

INHIBITION OF PLATELETAGGREGATIONBY GLUTATHIONE, ASCORBIC ACID AND OTHER FREE RADICAL SCAVENGERS.

A.Evangel ou,S .Karkabounas ,K.Liveris,G .Sofis, K.Charalambopoulos. Technical Assistance, A.Beca. Activation of p l a t e l e t aggregation is induced by a variety of endogenous agonists. B i l i r u b i n , a metabolic product of heme degradation, is not known as p l a t e l e t aggregating agonist so far. On rabbit washed plateletes, action of non-conjugated bil i r u b i n was tested by a photoaggregometer. 9 i l i r u b i n induced a potent i r r e v e r s i b l e aggregation at zonc.of 10-6M, which became maximun at concentration of IO-5M. B i l i r u b i n f a i l e d to induce aggregation on platelets suspended in rabbit plasma,even at conc. of IO-3M, in cont r a s t to platelets suspended in deproteinated serum where b i l i r u b i n provoked aggregation at conc. of 10-5M. Platelets treated by CP/CPK, aspirin,and gingka biloba extract, in order to prevent aggregation by activating ADP, Aracbidonic Acid and p l a t e l e t activating Factor biochemical proccess,exhibited a strong aggregatory effect to bil i r u b i n at conc. of IO-5M. Hematoporphyrin,arelative to b i l i r u b i n substance, induced as w e l l , p l a t e l e t aggrefation at similar to b i l i r u b i n conc., whi~'e hemine,cyanocobalamin and b i l i v e r d i n had no effect. Data indicate that b i l i r u b i n and r e l a t i v e substances,induce an i r r e v e r s i b l e p l a t e l e t aggregation ex vivo,which seems to be unrelated to ADP,AA and Paf biochemical proocess. This aggregation is probably inhibited in vivo by plasma proteine. Although the physiological importance of this phenomenon can not be evaluated so far,the aggregatory effect of b i l i r u b i n on platelets,seems of great value for understanding the mechanistic effects,taking place during p l a t e l e t aggregation. L a b o r a t o r y of Exp. P h y s i o l o g y , F a c u l t y of Medic i n e , U n i v e r s i t y of I o a n n i n a , 4 5 1 1 0 - I o a n n i n a , Greece

S.Karkabounas,G.Sofis,D.Galaris*,K.Liveris,K.Charalambo poulos and A.Evangelou. Technical Assistance,A.Beca. Platelets aggregate,in v i t r o , b y a variety of agonists which activate some of the three d i f f e r e n t biochemical patways,i.e the ADP,Arachidonic Acid (AA) and p l a t e l e t Act i v a t i n g Factor (Paf) one. On rabbit washed platelets maximun aggregation induced by ADP,AA and Paf was determined by a photoaggregometer. Pretreatment of platelets by CP/CPK,Aspirin and ginkgo b i loba extract was used in order each of the agonists to act i v a t e i t s own pathw~j of aggregation proccess. Complete i n h i b i t i o n of p l a t e l e t aggregation induced byADP, AA and Paf was attained in presence of ~educed Glutathione(GSH) Ascorbic Acid, a-Mercaptopropionyl glycine (MPG), Butyl Hydroxy Toluene (BHT) and the spin tra~per Phen~l Butyl Nitrone (PBN) at concentrations of 10-4M to IO-JM. Vitamin C oxidation curve during i n h i b i t i o n of Paf and AA aggregation was estimated spectrophotometrically. A~Sition of GSH, Ascorbic Acid and MPG in the aggregometer cuvettes during the irreversible phase of aggregation induced by Paf and AA, reversed the phenomenon. The above mentioned data, strongly suggest that free radicals of some kjnd are involved to the proccess of p l a t e l e t aggregation induced by ADP,AA and Paf and free radicals are probably a common step of the biochemical patways activated by the above agonists. The ~pecies of free radicals involved,haye not been i d e n t i f i e d so far, but are currently under investigation by our laboratories.

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PHARMACOLOGY OF HIGH MOLECULAR WEIGHT, POLYVALENT DERIVATIVES OF HYDROXYETHYLSTARCH WITH THE TXAJPGH: RECEPTOR ANTAGONISTSULOTROBAN M. Miiller and H. Patscheke

EFFECTS OF ALPHA-TOCOPHEROL PLATELETS. U. Maschke and R. Distichs

Under certain pathophysiological conditions TXA, and PGH2reach very high local concentrations, that may overcome the inhibitory effects of competitive receptor antagonists. Therefore, non-competitveantagonists may provide considerable advantages in TXA, pharmacology. In order to develope non-competitive TXAJPGH: receptor antagonists, we synthesized polyvalent ligands with the capability of binding simultaneously to two or several TXA2/PGH2 receptor sites. Such a multiple binding can be anticipated to induce a cooperative binding and a non-competitivetype of inhibition associated with an increase in inhibitory potency. The synthesis of such polyvalent TXAJPGH~ receptor antagonists was accomplishedby linking sulotroban, a competitive TXA.JPGH2 receptor antagonist, to hydroxyethylstarchwith molecular weights of 450000, 200000 and 40000 (Sulo-HES450, 200 and 40). Sulo-HESderivatives inhibited the shape change in aspirin-treated platelets stimulated by the TXA2mimetic U 46619, but showed no effect on the platelet activation induced by PAF, ADP, serotonin, collagen or thrombin. In aspirin-untreated platelets, the polyvalent antagonists prevented the stimulation by exogenous arachidonic acid. They displaced [3Hl-Daltrobanfrom the specific binding to the platelet TXA2/PGH,receptors. In various Sulo-HES preparations with a comparable degree of derivatization, 2 % of the free hydroxyl groups, the 1C50for the inhibition of the U 46619 inducedshapechangedecreased withincreasingmolecularweight: 0.99 /~M for Sulo-HES40, 0.24 ~M for Sulo-HES200 and 0.066 ~M for Sulo-HES 450. On the other hand, the IC~decreased with increasingdegree of derivatization at same molecularweights. Dose-effect-curvesof Sulo-HES40, 200 and 450 for the U 46619 induced serotonin release and platelet aggregation revealed a non-competitive type of inhibition. These results show, that a linkage of the competitive antagonistsulotrobanwith hydroxyethylstarchretains the receptor specificity and generates a polyvalent TXAJPGH2 receptor antagonist with a non-competitive inhibitoryeffect. Its inhibitorypotencydepends on the molecular weight of the polymer matrix and on the degree of derivatization with sulotroban. (Supportedby the Deutsche Forschungsgemeinschaft,PA 263) Institutefor Clinical Chemistry,KlmikumMannheimof the Universityof Heidelberg, 6800 Mannheim, and Institute of Medical Diagnostics, Klinikura Karlsruhe,7500 Karlsruhe

ON

HUMAN

Alpha-tocopberol was administered to washed human platelets or PRP in concentrations varying from 0.001 mM to 10 raM. In concentrations of 0.1 mM or higher the substance leads to activation washed platelets. Electron micrographs give evidence that a considerable amount of platelets have been destroyed by tocopherol. The contents secretory granules of the destructed platelets might have initiated the activation of the remaining cells. The tocopherol-induced activation can be inhibited by PgE1. High concentrations of tocopherol can overcome the inhibiting effect of PgE1. In PRP similar observations cannot be made even with the highest concentration of tocopherol, suggesting that a significant amount of the vitamin is bound to plasma constituents and has no effect on the platelets. In concentrations up to 0.075 mM tocopherol inhibits platelet activation by thrombin, in a dose-dependent manner. A similar inhibitory effecct has been found when platelets treated with less than 0.03 mM alpha-tocopberol were activated using the polycation Cationized Ferdtin (CF). In the range of 0.1 m_M tocopberol acts synergistically on platelet activation by CF, whereas in higher concentrations no effect of CF can be found and the activating effect of tocopherol predominates. Electron micrographs prove that the polycation binds to cellular fragments and does no longer come into contact with the surface of undestroyed cells. These experiments give evidence that tocopherol interacts with the platelet membrane and that high concentrations of the substance strongly destabilizes the cell membrane. Institut ftir Anatomie der Westfiilischen Wilhelms-Universit~t Mtinster, Vesaliusweg 2-4, D-4400 Mtinster

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COMPARISON OF THE EFFECT OF ALPHA-TOCOPHEROL (VITAMIN E) ON THE ADHESION AND AGGREGATION OF PLATELETS IN PATIENTS WITH THROMBOCYTOSIS AND IN HEALTHY VOLUNTEERS J. Pieper, M. Sosada and H. Poliwoda

DECREASED RESPONSIVENESSOF PLATELETS TO ILOPROST IN PATIENTS WITH CROHN'S DISEASEAND ITS REVERSALBY POLYUNSATURATED FATTY ACIDS (n-3 PUFA).

i0 healthy volunteers (m/f=6/4, age=24,9~8,3) and 16 patients (m/f=10/6, age=44,3~14,8)with thrombocytosis (>BOO 000/~I blood) suffering of myeloproliferative diseases were treated during 14 days with alpha-tocopherol (3 x 300 mg per day orally). Measurement of platelet aggregation (Born test) did not show any significant (student t-test) changes in the collagen and ADP-induced aggregation before and after 14 days of alpha-tocopherol treatment in either group. The thrombometer time (time in which a collagen channel is blocked by platelet adhesion and aggregation) was not effected by alpha-tocopherol in the healthy volunteers (28~2s = normal range of the used charge, n=10). Pathological decreased thrombometer times were obtained before alpha-tocopherol treatment in the patient group indicating increased platelet adhesion and aggregation (25,5• n=16, p, D-Dimer, platelet count and WBC count. All coagulation parameters were significantly increased at all 4 hour draws, hut no accumulative or desensitization effect was observed during the time of investigatlon. The WBG and the platelet count remained unchanged and in the normal range throughoutthe study. The flbrinolytic parameters also remained unchanged. No adverse reactions or signs of allergic reactlon~ were observed. In conclusion, over a treatment period of 6 weeks the LMW-heparin tested was safe and no accumulation or desensitization effects were noticed. Further studies need to be performed to confirm these results and to suggest the long-term use of LMWH's in humans. Hemostasis Research Lab., Loyola University Medical Center, 2160 S. First Aven~e, Maywood, IL 60153, U.S.A.

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237

T~OMBIN-ANTITHROMBIN HI COMPLEX FORMATION IN PATIENTS WITH DEEP-VEIN THROMBOSIS RECEIVINGEITHER LOW MOLECULAR WEIGHT HEPARIN OR IfNFRACTIONATEDHEPARIN W, D 9 1 r E, B~laun. E~ Gatterere. G. Bonner*. J. Slan~* and P. HODmeier It is still a topic of discussionwhether low molecular weight heparin (LMWH) can be recommendedfor the treatment of deep-veinthrombosis. In this connection, we studied thrombln formation as reflected by the measurement of thrombln-antltlwombinHI complexes(TAT) in patients with pldebographlcallyrecorded acute venous thrombosis, who were treated either with LMWH (group I, N = 6, age: 6L7 • 14.6 years) or with tmfractionated heparin (UFH) (group EL,N ffi 6, age: 62.7 • 5.2 years). Dos~e: Patients in group I were given LMWH suheutaneously(Fragmin, Kabi Vitrum, Swedeif~(if < 80 kg b.w.: 2 x 10.000 IU/d, if ~ 80 kg b.w,: 2 x 12.000 IU/d)~the plasma levelw ~ kept between0~5aF XaU/ml (bottom value) and 1.0 aF XaU/ml (peak value), Patients in group H receivedUFH (Heparin Immtmo, lmmtmo, Austria) intravenously(initially 100 IU/kg b.w. followed by an infusion of L000 lU/h). TAT were measured by immunoassay(EnzygnostTAT, Behring, Germany).

MODULATION OF HEPARD4 COFACTOR II OICH) ACTIVITY BY GLYCOSAMINOGLYCANS (GAGs) A N D A D H E S I V E GLYCOPROTE[NS E. Petzelbauer, D, Seiffert, R. Beckma_rm, M. Geiger and B. R, Binder HCII is a Very specific thrombin inhibitor. Its inhibitory a c t i v i t y is s t i m u l a t e d by he pa ri n (Hop) and d e r m a t a n s u l f a t e (DS). V i t r o n e c t i n (VN), a he pa ri n binding a d h e s i v e glycoprotein present in plasma and extracellular matrix, has been shown t o d e c r e a s e t he s t i m u l a t o r y e f f e c t of Hop but not of DS (Preissner and Si%, 1988). We were i n t e r e s t e d to analyze t h e e f f e c t of GAGs and GAG-binding proteins on t he HCH/thrombin interaction in more detail. HCII was purified from human plasma by barium chloride adsorption and polyethylene glycol precipitation followed by heparin-affinity- and ion-exchange chromatography. Inhibition of thrombin (0.03U/mi} by HCII (8-0.4/Lg/ml) was studied in t he absence and presence of GAGs (heparin 0.03-300/zg/ml, DS 0.05-50#g/ml, heparan s ul fa t e (HS) 0.05-50#g/ml, chondroi t i n s u I f a t e A a n d C 0.05-500/zg/ml) in a c h r o m o g e n i c s u b s t r a t e assay using S-2366 us a thrombin s ubs t ra t e . The e f f e c t s of VN and fibronectin (FN) on HCII s t i m u l a t i o n by GAGs were determined. In a ddi t i on to Hep and DS t h e i n h i b i t o r y e f f e c t of HCII was s t i m u l a t e d , by c hondroi t i n s u l f a t e A a n d C and Ha, however, to a lesser extent. This stimulatory e f f e c t was completely inhibited when Hep (s 0.3/tg/ml) was preincubated with VN (60pg/mt) and decreased to less than 50% when HS (50pg/ml) or chondroitin sulfate A or C (50pg/rnt each) were preineubated w i t h VN. VN had no e f f e c t on DS as already described previously. FN (75/zg/mt) ~ b i t e d the stimulatery e f f e c t of Hep on t h e H C I i / t h r o m b i n i n t e r a c t i o n l e s s t h a n 50% and had no e f f e c t on t h e s t i m u l a t o r y e f f e c t of DS. Thus, FN a p p e a r s t o be a w e a k m o d u l a t o r of t h e GAG/HCll/thrombin system in comparison to ON. In conclusion, adhesive glycoproteias m i g h t modul a te t he coagulation system by reducing the stimulatory e f f e c t of GAGs on t he HCII]thrombin interaction.

Results:

time after the onset of therapy l~mrs

0

2

4

12

. 18

24 .....36

48

~,roua I

T A T (ng/ml) 33.3

29.5

II.0

24.8

15.5

27.3

11.5

7.0

+ 1S

23.2

I0,9

23.7

8.8

24.4

14.6

4.2

21,6

~r0uD H

TAT (ag/ml) 23,3

9,2

T I ~

4,0

7.3 1,6

8.7 3.7

7.3 1.8

2.4

r30-35% are to be stated but seldom. In case suchlike will be found out, the patient will get about 30 minutes after blood-taking a subcutaneous injection of heparin. Krankenhaus Friedrichshain, Abt.B~mostaseologie-Angiologie, Leninallee 49, O-1017 Berlin

263 [131a]

U. Bohn,

D.C.

Gulba*,

A stable thrombin-antithrombin III-complex (TAT) was formed of thrombin (4 IU/ml) and antithrombin III (0,05 IU/ml) in the presence of unfractionated heparin (0.025 IU/ml). After in vitro addition of plasmin (0.i -i CU/ml) to the TAT, the TAT-level decreases about 60% within a few seconds, and thrombin is released from the TAT-complex in the presence of heparin. There exists a linear correlation between initial plesmin- and released thrombin concentration. This thrombin from the TAT is able to split fibrinopeptide A from fibrinogen despite the presence of heparin (0.025 - 0.i IU/ml). After 30 s incubation of 1 CU/ml p l a s m i n with the TAT, an amount of thrombin is released which has the equivalent activity of 0.012 IU/ml purified thrombin detected by the release of FPA from fibrinogen under the same conditions. However, the hydrolytic activity of the released thrombin detected with the chromogenic substrate S-2238 is 10 times higher. The explanation of this effect could be an altered thrombin molecule. The activity of this thrombin is inhibited completely by hirudin (no FPA generation), but not by another addition of antithrombin III and heparin (I0 fold concentration). These in vitro several causes thrombolysis.

observations could be for thrombin activity

one of during

Division of Haematology and Oncology, *Division of Cardiology, Medizinische Hochschule Hannover, Konstanty-Gutschow-Str.8, 3000 Hannover 61

265

INHERITED SEVERE PROTEIN S DEFICIENCY BOY WITH CONSUMPTIVE COAGULOPATHY F,Bergmann,

M. Barthels,

P.F.Hoyer,

IN A

YOUNG

C.Osterreich * , M.Barthels*

A previously healthy eight year old boy with a short history of sore throat, cervical lymph node enlargement and fever, treated with oephaclor was hospitalized one week later with purple discoloration and painful induration on the lower legs. Laboratory data showed signs of infection (CRP 150 mg/l, leucocytosis, elevated antistrep~olysin ~iter). Coagulation parameters suggested a DIC inspire a normal antithrombin Ill of 117%: fibrinogen 0.5 g//l, platelets 33000/~i, fibrin degradation products 14623 pg/l, t h r o m b i n - a n t i t h r o m b i n III complex >70 pg/l. A mild toxic shock syndrom was suspected and treatment was started with antibiotics, heparin and FFP. He improved shortly but consequently developed h e m o r r h a g i c blisters in his face and on the lower limbs and fibrinogen became undetectable despite aprotinin infusions Zo prevent hyperfibrinolysis. He developed massive thrombosis of superficial veines and despite heparinization and replacement therepy with FFP pulmonary emboli were suspected. Further investigation revealed a severe deficiency of total Protein S (5~). Despite continous replacement therapy with FFP an increase of free Protein S was not detected'even when measured 15 min post infusion. Total Protein S was kept at 40-60%. replacement therapy with FFP was continued for about 4 weeks. During this time ~he boy recovered. The last level of total Protein S was about 35% and free Protein S about 6%, Phenprocoumon therapy was started and tolerated without complications. Kinderklinik, ~Division Kons~anty-Gutschow-Str.8,

of Haematology, 3000 Hannover 61

M-HH

MOLECULAR GENETIC ANALYSIS TECHNIQUES: THEIR A P P L I C A T I O N FOR THE D I A G N O S I S OF C O A G U L A T I O N DEFECTS. C. M a n n h a l t e r , G, M i t t e r b a u e r D u r i n g the lest few years m o l e c u l a r g e n e t i c a n a l y s i s t e c h n i q u e s b e c a m e i n c r e a s i n g l y important for the d i a g n o s i s of m o n o g e n i c disorders. ~or c o a g u l a t i o n defects, a m u c h more a c c u r a t e d i a g n o s i s of c a r r l e r s of the d i s o r d e r can be obtained, and p r e n a t a l d i a g n o s i s can be c a r r i e d out at an early stage of p r e g n a n c y . D i f f e r e n t technical a p p r o a c h e s are c u r r e n t l y available. The goal is, of course, the d i r e c t i d e n t i f i c a t i o n of the genetic defect (deletion, point m u t a t i o n ) . However, some d i s e a s e s , eg. h e m o p h i l i a A are caused by a large n u m b e r of d i f f e r e n t m u t a t i o n s . Thus, direct i d e n t i f i c a t ion of the m u t a t i o n is l a b o r i o u s and i m p r a c t i c a l for d i a g n o s t i c l a b o r a t o r i e s . An a l t e r n a t i v e approach, gene tracking, by w h i c h a d e f e c t i v e allele can be f o l l o w e d w i t h i n a given family, has p r o v e n v e r ~ useful. S o u t h e r n blot t e c h n i q u e s and a m p l i f i c a t i o n of p o l y m o r p h i c regions by the p o l y m e r a s e chain r e a c t i o n ( r e s t r i c t i o n f r a g m e n t length p o l y m o r p h i s m s and other length polym o r p h i s m s , eg. v a r i a b l e n u m b e r t a n d e m repeats) can be applied. Very r e c e n t l y , the use of single strand c o n f o r m a t i o n p o l y m o r p h i s m a n a l y s i s was e s t a b l i s h e d for the l o c a l i z a t i o n of point m u t a t i o n s in larger groups of patients. Once the area c a r r y i n g the m u t a t i o n is localized, it can be s e q u e n c e d and the m u t a t i o n can be identified. E x a m p l e s for each m e t h o d will be p r e s e n t e d . K l i n i s e h e s I n s t i t u t fHr m e d i z i n i s c h e nnd ehemische L a b o r d i a g n o s t i k , U n i v e r s i t ~ t Wien, W g h r i n g e r GHrtel 18-20, A-I090 Wien.

A69 266

268

GENEDIAGNOSIS;ANDGENETHERAPYIN HEMOPHIUA HHWatzke

Chronic idiopathic thrombocytopenic purpura (ITP) treatment results

Molecular biologyand gone technology are currently applied to many fields in medicine Pharmaceuticals engineered by means of gone technology are routinely used by almost every physician. Gone technology is also used for the diagnosisof genetic disorders and represents a major breakthrough in genetic counseling. Gone therapy, in contrast, is currently not applicable on a routine basis but is gaining more and more attention as the first clinicaltrialson yena therapy in humans are underway. Molecular biology has a tremendous impact in blood coagulation. It enhances our knowledge

on structure function relationshipof the proteinsim/olved in hemostasis The analysis of the genetic basisof hereditarycoagulationdefectsallows the identificationof the defect on the amino acid level. The correlation of the

molecular defect with the functional defect elucidatesthe function of the normal protein. Functionallyaltered moleculescan be generatedbg site directed mutagenesisand in vitro expression. This results in specifically altered proteins "...,'ith distinct functional characteristics which again enhancesour knovledgeon the interaction of the coagulationfactors For the diagnosisof hereditarydefects in bloodcoagulationgoneanalysis is performed routinely. Somedefects (Hemophilia B) are diagnosedby sequence analysis of the respective gene. In others (Hemophilia A), the diagnosis is still dependent on linkage analysis of affected genes (restriction fragment length polymorphismus). Hemophilia is an ideal model for gone therapy for many reasons: coagulalion factors are constilutively secreted into the plasma not requiring specific regulation processes; elevationof as littleas l O 9 of circulating F YIIl or F IX would tremendously improve the clinical ou'{come; finally,there ia an animal modeI which perfecflg suitsthe naedsofgenetherapg. Clinicalapplication of gone therapy in hemophilia is ho;,.xeverlimited by the fact that the currently used replacement of coagulation factors is an '~lell established and clinicallyuaeful therapy. Thus, gone therapy is clinicWly only used in genetic disorders without establishedtreatment (ADA deficiency). Universit~tsklinik fur Interne I/H~roatologie,W~hMnger GUrtol 18-20 , A- 1090 Wien

M. Winkelmann, P. Leifeld, W. Schneider We are reporting the treatment results of 161 patients suffering from ITP (115 female, 46 male; ration 2,5:1, age between 17 and 93 years). Complete remission was defined as at least 6 months of platelet counts over 150.O00//ul without any therapeutic intervention. Results:- only 2 patients died i n i t i a l l y of cerebral bleeding, 10 patients (6,2%) achieved complete remission without therapy. 24 patients (14,9%) had platelet counts over 30.O00//ul and no signs of bleeding without therapy. 120 (74,5~) patients were primarily treated with glucocorticoids (in fact prednisolon). 29 of them reached complete remission after one therapy cycle, 9 patients had to be treated with glucocorticoids a second time and then reached complete remission (total remission rate 31,7%). An outstanding result of this regimen is the significant correlation between the ini t i a l l y applied glucocorticoid dose and the i n i t i a l increase of platelet count (p 0,02) and the rate of complete remissions (p 0,001). 34 patients (21%) underwent splenectomy. 22 of them reached complete and 4 partial remission (total success rate 76,5%), only 8 needed further therapy. However, 19 (glucocorticoid treatment failures) out of 31 non-splenectomised patients have to receive permanent treatment. Our study reveals the following aspects about therapy and the cause of ITP: 1. In idividual cases, the risk involved in splenectomy should be weight against the risk of a second cycle of glucocorticoid treatment. 2. The i n i t i a l glucocorticoid dose correlates s i g n i f i cantly with the rate of complete remission. 3. The number of patients having to undergo treatment after glucocorticoid and splenectomy regimen is clearly below the 10% mark. " Department of Hematology, Oncology and Clinical Immunology, Heinrich-Heine-University, Medical Center, Moorenstr. 5, 4000 DUsseldorf i .

267

IMMUNE THROMBOCYTOPENIA:DIAGNOSTIC STRATEGIES V. Kiefel, S. Ssntoso, and C. Mueller-Eckhardt Plstelet specific antibodies bring about thrombocytopenis through accelerated platelet destruction in different conditions. Autoimmune thrombocytopenia (AITP), caused by platelet specific autoantibodies (Bob) occurs as "idiopathic" AITP or "secondary" AITP, accompanying diseases as SLE, malingnant solid tumors, CLL, lymphomas, or HIV infection. Neonatal alloimmune thrombocytopenia (NAIT) is equivalent to hemolytic disease of the newborn. In NAITP, thrombocytopenia of the fetus end the neonate is the consequence of maternal immunization against incompatible platelet alloantigens. Another condition always associated with platelet allosntibodies is post transfusion purpura (PTP), a rare transfusion reaction characterized by severe immune-mediated thrcmbocytopenia. Discrimination between platelet specific auto- and alloantibodies and immunoglobulin reacting with HLA class I antigens, Fc receptors or Ig trapped in platelet alpha granules is possible with glycoproteinspecific immunoassays using monoclonsl antibodies for antigen isolation (MAIPA), immunoblot or radioimmunoprecipitation. Most platelet specific antibodies characterized so far react with monomorphic (aab) or polymcrphic determinants (alloantibodies) on glycoproteins IIb/IIIe, Ib/IX, Ia/IIa or IV of the platelet membrane. Institut for Klinische Immunologie und Transfusionsmedizin der dustus-Liebig-UniversitBt, Langhansstr. 7, D-6300 Giessen