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T B Tschopp

Publications and source records attributed to T B Tschopp.

At least 19 recordsLinked to original sources
PubMed1995

Role of collagen-adherent platelets in mediating fibrin formation in flowing whole blood.

Activated platelets provide assembly sites for coagulation enzyme complexes and in this way can mediate coagulation during hemostasis and thrombosis. In this study, we examined the procoagulant activity of platelets adhering directly to fibrillar collagen, a main thrombogenic constituent of subendothelium. For this purpose, we used a human ex-vivo thrombosis model in which collagen-coated coverslips were exposed to flowing nonanticoagulated blood (shear rate, 65/s) for 5.5 minutes, which led to the deposition of adherent platelets, platelet thrombi, and fibrin. To examine the procoagulant activity of adherent platelets only, a selective antagonist of the platelet GPIIb-IIIa complex, Ro 44-9883, was infused via a mixing device, resulting in a complete abrogation of platelet thrombus formation but leaving the collagen-adherent platelet layer intact. This platelet layer generated increased postchamber fibrinopeptide A (FPA) levels (203 +/- 33 ng/mL) as compared with control experiments without infusion of inhibitor (95 +/- 13 ng/mL). Concomitantly, fibrin deposition measured by morphometric analysis of cross-sections was also increased, as was the platelet adhesion to collagen. An immunochemical staining of fibrin fibers further showed that the adherent platelets formed the nuclei for fibrin fiber formation. This increase in fibrin deposition was mediated by the intrinsic factor X (F.X) activation complex on adherent single platelets, because almost complete inhibition of FPA generation (9 ng/mL) and fibrin deposition (0.4% +/- 0.2% coverage) was achieved upon coinfusion of the GP IIb-IIIa antagonist and active site-inhibited F.IXa. The large platelet thrombi that were deposited in control experiments contained no significant amounts of immunodetectable fibrin except at the thrombus base, where adherent platelets anchored the thrombi to the collagen surface. These results suggest that the collagen-adherent platelets are important promoters of coagulation during the initial phase of thrombogenesis by providing assembly sites for the F.X activation complex.

Acetates↗
PubMed1995

Activated clotting time as an appropriate test to compare heparin and direct thrombin inhibitors such as hirudin or Ro 46-6240 in experimental arterial thrombosis.

BACKGROUND: Specific thrombin inhibitors are considered to be more potent antithrombotics than heparin. However, the relation between the systemic anticoagulation generated by thrombin inhibitors and their antithrombotic effect is not well defined. In a guinea pig carotid thrombosis model, the activated clotting time (ACT), the activated partial thromboplastin time (aPTT), and thrombin-generation tests were evaluated for their ability to predict the arterial antithrombotic effect of direct thrombin inhibitors such as hirudin and Ro 46-6240 compared with heparin. METHODS AND RESULTS: Thrombosis of the carotid artery was induced by subendothelial damage in guinea pigs, and the subsequent cyclic flow variations were monitored. The effects of pretreatment with intravenous heparin, hirudin, and Ro 46-6240 were tested. After doubling the baseline aPTT, 1 IU.kg-1.min-1 heparin was inactive, whereas either hirudin or Ro 46-6240 (30 micrograms.kg-1.min-1) prevented thrombus formation by 80%. Heparin (10 IU.kg-1.min-1) induced the same antithrombotic effect but with indefinite aPTT prolongation. However, for similar prolongation of the ACT, the three compounds had equivalent antithrombotic effects. Thrombin generation was predictive of the antithrombotic effect of the thrombin inhibitors but not of heparin. CONCLUSIONS: The arterial antithrombotic effect of direct thrombin inhibitors, when compared with those of heparin, should be evaluated by the ACT and not the aPTT or thrombin-generation assays. For a "therapeutic" aPTT prolongation, thrombin inhibitors induce higher systemic anticoagulation than does heparin and thus might unduly have higher bleeding liability.

Animals↗
PubMed1995

Inhibition of extrinsic and intrinsic thrombin generation by a novel synthetic thrombin inhibitor (Ro 46-6240), recombinant hirudin and heparin in human plasma.

To further define the anticoagulant activity of Ro 46-6240, a novel, synthetic, thrombin inhibitor, we compared its effect on extrinsic and intrinsic thrombin generation in human platelet-poor plasma with that of recombinant hirudin and standard heparin. The time course of thrombin generation was followed with a chromogenic substrate assay. The total amount of active thrombin formed was quantified by calculating the area under the thrombin generation curve. Ro 46-6240 and r-hirudin delayed thrombin formation in a concentration-dependent manner in both activation systems whereas heparin showed this effect only in the intrinsic system. Heparin was the most potent inhibitor of extrinsic and intrinsic thrombin generation with IC50 values of 20 and 27 nM, respectively. Ro 46-6240 was nearly as potent as r-hirudin for inhibiting extrinsic thrombin generation (IC50 418 vs 229 nM) and intrinsic thrombin generation (IC50 463 vs 343 nM) despite a much lower affinity of Ro 46-6240 for thrombin (Ki apparent: 0.3 nM) in a purified buffer system. The similar potency of the small active-site thrombin inhibitor compared to the larger hirudin may be explained by different kinetic mechanisms for inhibition of thrombin and by a higher accessibility to the phospholipid surface where thrombin generation takes place. In conclusion, our results show that a specific small thrombin inhibitor efficiently inhibits and delays thrombin generation in human coagulating plasma. This reduced thrombin generation might be caused by inhibition of thrombin-mediated feedback reactions during blood coagulation.

Antithrombins↗
PubMed1995

Active site-blocked factors VIIa and IXa differentially inhibit fibrin formation in a human ex vivo thrombosis model.

The role of tissue factor/factor VIIa (FVIIa) and factor VIIIa/factor IXa (FVIIIa/FIXa) complexes in thrombus formation was examined in a human ex vivo blood flow system by use of active site-blocked FVIIa (FVIIai) and FIXa (FIXai) as selective inhibitors. Blood was drawn directly from the veins of volunteers into a mixing device where FVIIai and FIXai were mixed with flowing blood. The blood then entered parallel-plate chambers containing coverslips coated with human fibrillar collagen or tissue factor-expressing cell layers of tumor necrosis factor-alpha-stimulated human endothelial cells, human smooth muscle cells, and J82 cells. Exposure of stimulated endothelial cells to blood flowing at a venous shear rate of 65/s led to fibrin deposition, which was inhibited by infusion of FVIIai (IC50, 3 nmol/L), as quantified by micro-densitometry of fibrin-stained coverslips. Whereas FIXai (600 nmol/L) was only a weak inhibitor, FVIIai (60 nmol/L) reduced fibrinopeptide A (FPA) plasma levels from 504 +/- 79 to 171 +/- 27 ng/mL and concomitantly inhibited platelet thrombus deposition. Similarly, experiments with smooth muscle cells and J82 cells showed that FVIIai but not FIXai efficiently reduced FPA levels. Conversely, with tissue factor-free collagen, ,hich induces platelet-dependent fibrin formation, infusion of FIXai but not of FVIIai inhibited fibrin deposition (IC50, 8 nmol/L) and reduced FPA levels from 55 +/- 8 to 9 +/- 5 ng/mL. However, FIXai did not affect the number of platelet thrombi deposited on collagen.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

Design and synthesis of potent and highly selective thrombin inhibitors.

Thrombin, a serine protease, plays a central role in the initiation and propagation of thrombotic events. An extensive search for new thrombin inhibitors was performed, using an unconventional approach. Screening of small basic molecules for binding in the recognition pocket of thrombin led to the discovery of (aminoiminomethyl)piperidine (amidinopiperidine) as a weak, but intrinsically selective, thrombin inhibitor. Elaboration of this molecule provided compounds which inhibit thrombin with Ki's in the range of 20-50 nM and with selectivities of 1000-4000 against trypsin. These inhibitor compounds show a new and unexpected binding mode to thrombin. Modification of the central building block and then of one of the hydrophobic substituents led to the discovery of a new family of thrombin inhibitors which has reverted to the former binding mode to thrombin. This last class of compounds shows inhibitory activities in the picomolar range, low toxicity, and a short plasma half life which favors its use for an intravenous application. From this series of thrombin inhibitors, 19f(Ro 46-6240) was selected for clinical development as an antithrombotic agent for intravenous administration.

Amino Acids↗
PubMed1994

Inhibition of clot-bound and free (fluid-phase thrombin) by a novel synthetic thrombin inhibitor (Ro 46-6240), recombinant hirudin and heparin in human plasma.

Clot-bound thrombin remains active and is less accessible to heparin-antithrombin III than fluid-phase thrombin. To determine whether clot-bound human thrombin is more susceptible to inactivation by direct thrombin inhibitors, the activity of a novel synthetic competitive thrombin inhibitor Ro 46-6240, recombinant hirudin and unfractionated heparin were compared with fluid-phase thrombin and clot-bound thrombin. Fibrinopeptide A generated in human plasma was used as an index of thrombin activity. Hirudin was the most potent inhibitor of fluid-phase and clot-bound thrombin. However, Ro 46-6240 inhibited clot-bound thrombin three times more potently than fluid-phase thrombin (IC50 19 vs 56 ng/ml) while hirudin was two times (IC50 8 vs 3 ng/ml) and heparin six times (IC50 1,205 vs 200 ng/ml) less active against clot-bound thrombin compared with fluid-phase thrombin. The relative selectivity for clot-bound thrombin is not a unique property of Ro 46-6240 since two other synthetic thrombin inhibitors tested inhibited clot-bound thrombin more effectively than fluid-phase thrombin and a third was equally active against both forms of thrombin. In contrast, the affinities of two chromogenic substrates were similar for both forms of thrombin. This study shows that direct thrombin inhibitors inhibit clot-bound thrombin more potently than heparin and suggests that an apparent selectivity for clot-bound thrombin can be achieved with some synthetic thrombin inhibitors. Further studies have to show whether the high potency of these direct thrombin inhibitor translates into antithrombotic efficacy in clinical situations with pre-existing clots.

Binding, Competitive↗
PubMed1994

Thrombin plays a key role in late platelet thrombus growth and/or stability. Effect of a specific thrombin inhibitor on thrombogenesis induced by aortic subendothelium exposed to flowing rabbit blood.

Thrombin is involved in the pathogenesis of venous and arterial thrombosis. This study addressed the question of the relative importance of thrombin in the early and late phases of thrombogenesis. The effect of Ro 46-6240 (1.43 mg/kg bolus and 0.1 mg/kg per minute i.v.), a novel, selective thrombin inhibitor on thrombogenesis induced by rabbit aorta subendothelium, was measured ex vivo in a perfusion chamber model after a short (5-minute) and long (30-minute) exposure time to rabbit native blood. The role of the perfusion time was assessed at shear rates of 100/s, 650/s, and 2600/s. These shear rates mimic blood flow conditions found in veins, arteries, and small or stenosed arteries, respectively. Fibrin deposition and platelet thrombus formation on subendothelium were evaluated by microscopic morphometry. In the presence of Ro 46-6240, fibrin deposition was abolished at both perfusion times and at all shear rates. In the 5-minute experiments, thrombus height was reduced by Ro 46-6240 at shear rates of 100/s (85%) and 650/s (35%) but not at a shear rate of 2600/s, whereas thrombus area was not affected at any shear rate. In contrast, both thrombus height and thrombus area were reduced (60% to 90%) by Ro 46-6240 in the 30-minute perfusion groups at all wall shear rates. The antithrombotic effect of Ro 46-6240 after 30-minute perfusion was confirmed by the minimal increase in the pressure difference between the entrance and the exit of the perfusion chamber compared with the control groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Endothelial cells stimulated with tumor necrosis factor-alpha express varying amounts of tissue factor resulting in inhomogenous fibrin deposition in a native blood flow system. Effects of thrombin inhibitors.

TNF-alpha induces changes in endothelial cell functions, such as upregulation of tissue factor, resulting in endothelial procoagulant activity which may play a role in disseminated intravascular coagulation. The procoagulant activity of TNF-alpha-stimulated endothelial cell monolayers was studied in a human ex vivo native (nonanticoagulated) blood flow system using the three thrombin inhibitors recombinant hirudin, Ro 46-6240, and heparin. Under venous blood flow conditions (shear rate 65 s-1) recombinant hirudin, Ro 46-6240, and heparin inhibited fibrin deposition on the endothelial cells by 50% at concentrations of 14, 28, and 412 ng/ml, respectively. The highest tested concentrations of the thrombin inhibitors reduced the postchamber fibrinopeptide A levels from 713 +/- 69 to < 70 ng/ml. Surprisingly, even at relatively high inhibitor concentrations, some local fibrin deposits were found on TNF-alpha-stimulated cells, suggesting that some endothelial cells possess higher procoagulant activity than others. Therefore, the surface expression pattern of tissue factor, the primary initiator of coagulation in this system, was examined by immunogold-silver staining. The results showed that the tissue factor density on the cell surface varied strongly among TNF-alpha-stimulated endothelial cells. Using TNF receptor-selective agonistic mutants of TNF-alpha, it was demonstrated further that the heterogenous surface expression of tissue factor was mediated entirely by the 55-kD TNF receptor and did not involve the 75-kD TNF receptor. We conclude that in this system TNF-alpha induces heterogenous tissue factor expression which may lead to a high local thrombin concentration, such that even in the presence of thrombin inhibitors focal fibrin deposition occurs.

Blood Circulation↗
PubMed1993

Relationship between tissue factor expression and deposition of fibrin, platelets, and leukocytes on cultured endothelial cells under venous blood flow conditions.

Endothelial cell-mediated coagulation and leukocyte adhesion are processes that might be connected by the generation of thrombin. To examine the interaction of procoagulant and proadhesive activity, cultures of endothelial cells were stimulated with tumor necrosis factor-alpha, which resulted in the surface expression of tissue factor. Subsequent exposure to human nonanticoagulated blood at a shear rate of 100 s-1 in a parallel plate perfusion device led to the deposition of polymerized fibrin, which covered 63% of the endothelial surface. In addition, numerous platelet aggregates (71 per 10 mm cross-section) and leukocytes (53 +/- 6/mm2) were deposited on stimulated endothelial cells, whereas no fibrin and only a few platelet aggregates (4 +/- 1 per 10 mm cross-section) and leukocytes (6 +/- 1/mm2) were detected on control cells. A significant portion of the adherent leukocytes bound to fibrin and platelets. However, when the deposition of fibrin and platelet aggregates was inhibited with the anti-tissue factor antibody HTFI-7B8 by 100% and 86%, respectively, leukocyte adherence remained unchanged (68 +/- 6/mm2). This indicated that leukocytes could efficiently adhere to endothelial cells through direct cell-cell contact independent of both thrombin and deposited fibrin. Moreover, this direct adhesion of leukocytes to the endothelial surface was reduced twofold to threefold when fibrin deposition occurred. These data suggest a relationship between endothelial procoagulant and proadhesive properties in that tissue factor-initiated coagulation may contribute to leukocyte adhesion through the formation of an adhesive fibrin/platelet meshwork but concurrently prevents the adhesive endothelial surface to bind leukocytes at its full capacity.

Antigens, CD↗
PubMed1990

Heparin, carboxyl-reduced sulfated heparin, and Trestatin A sulfate. Antiproliferative and anticoagulant activities.

Human smooth muscle cells were used to investigate the antiproliferative activities of sulfated carbohydrates. The antiproliferative potencies of coarse heparin fractions prepared by ultrafiltration increased with the mean molecular-weight, whereas the anticoagulant activities of a high-molecular-weight fraction had submaximal values. Furthermore, the dependence of antiproliferative activity on sulfate content is discussed. Carboxyl-reduction of heparin abolished both antiproliferative and anticoagulant activities. Sulfation of this compound yielded CRS-heparin with restored antiproliferative potency but devoid of antithrombin III-mediated anticoagulant activity. Sulfation of the pseudo-nonasaccharide, Trestatin A, yielded a compound having the highest antiproliferative activity, so far observed for a low-molecular-weight compound, and having only weak anticoagulant properties.

Anticoagulants↗
PubMed1990

[Fish oil in thrombosis and arteriosclerosis].

In the past few years interest in the use of fish oil as a dietetic approach in the management of thromboarteriosclerotic diseases has increased. This is based on epidemiological studies which indicate that people living mainly on a fish diet have a low incidence of coronary heart disease (CHD). The beneficial effect is attributed to the high content of polyunsaturated n-3 fatty acids (PUFA), especially eicosapentaenoic acid (C20:5n-3) (EPA), in the marine diet. These fatty acids are built into the phospholipids of various cell membranes, changing their biological reactivity. EPA and docosahexaenoic acid (C22:6n-3) are also metabolized into specific eicosanoids (e.g. prostaglandin I3, thromboxane A3 and leukotriene B5). In contrast to the mediators originating from the n-6 fatty acids, these n-3 autocoids exhibit stronger antiaggregant, vasodilatory and anti-inflammatory activities. When given to volunteers, n-3 PUFAs inhibit platelet aggregation and lower plasma triglycerides. However, first clinical studies in patients with angina pectoris are equivocal. Furthermore, restenosis of coronary arteries in patients after PTCA was not clearly reduced. Therefore, more prospective clinical studies are needed to establish the efficacy of these fish oils before their use in thromboarteriosclerotic CHD can be recommended.

Animals↗
PubMed1985

7-Bromo-1,5-dihydro-3,6-dimethylimidazo[2,1-b]quinazolin-2(3H)- one (Ro 15-2041), a potent antithrombotic agent that selectively inhibits platelet cyclic AMP-phosphodiesterase.

This study with the new analog Ro 15-2041 (7-bromo-1,5-dihydro-3,6-dimethylimidazo[2,1-b]quinazolin-2(3H)-on e) confirms and substantially extends the activity spectrum of imidazoquinazolinones as potent platelet function inhibitors. Ro 15-2041 inhibited platelet aggregation induced by all common platelet agonists in platelet-rich plasma obtained from various species including man (IC50 = 1-3 microM). The compound potentiated platelet inhibition by prostacyclin, the prostacyclin-induced increase of intraplatelet cyclic (c) AMP levels and inhibited the collagen-induced release of serotonin and beta-thromboglobulin. Ro 15-2041 reduced the increase and accelerated the normalization of cytosolic free Ca++ in thrombin-stimulated human platelets. Ro 15-2041 is a potent (IC50 = 70 nM) and selective inhibitor of platelet cAMP-phosphodiesterase activity. Whereas Ro 15-2041 caused complete inhibition of cAMP-phosphodiesterase activity in human platelet supernatants, breakdown of cAMP in cardiac homogenates was depressed to maximally 50%. In human brain and rabbit uterus Ro 15-2041 was at least 1000 times less potent. By comparison, papaverine fully inhibited phosphodiesterase activity in all four tissues with similar IC50 values of about 5 microM. Furthermore, Ro 15-2041 selectively inhibited cAMP-phosphodiesterase activity of a bovine calmodulin-independent but not of a calmodulin-dependent enzyme preparation. The compound exhibited significant p.o. activity in various ex vivo and in vivo platelet function tests.(ABSTRACT TRUNCATED AT 250 WORDS)

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1981

Platelet adhesion and mural platelet thrombus formation on aortic subendothelium of rats, rabbits, and guinea pigs correlate negatively with the vascular PGI2 production.

This study investigates whether PGI2 produced locally by the vessel wall affects the interaction of platelets with subendothelium. Aortic endothelium was removed by a balloon catheter. Subsequent platelet deposition on subendothelium was assessed by morphometry. After 10 min exposure, surface coverage with adherent platelets in rats, rabbits, and guinea pigs amounted to 63% +/- 4, 82% +/- 3, and 97% +/- 1 (means +/- S.E.) (n greater than 5), and with microthrombi to 0 +/- 0, 6% +/- 2 and 15% +/- 4, respectively. Conversely, as measured by bioassay, excised aortic segments of rats, rabbits, and guinea pigs produced 135 +/- 14, 34 +/- 4, and less than 15 pmol of PGI2 per 10 mg of aorta per 30 min incubation in buffer, respectively (n = 4). In a second set of experiments, native blood from the aorta of a rabbit was circulated (30 ml/min) through a rat's de-endothelialized abdominal aorta and back into the vena cava of the rabbit. After a 5 min exposure at an approximate gamma w of 3000 sec-1, adhesion and aggregation of rabbit platelets on subendothelium of untreated rats amounted to 6% +/- 2 and 0 +/- 0, respectively. In contrast, on subendothelium of rats pretreated with 50 mg/kg ASA, which inhibits generation of PGI2 by greater than 80%, platelet adhesion and aggregation amounted to 55% +/- 11 and 19% +/- 7, respectively (n = 6, 2 alpha less than 0.01). The deposited thrombus volume per surface area differed by 3 orders of magnitude between the two groups. We conclude that platelet adhesion and aggregation on subendothelium correlate negatively with the generation of PGI2. Thus the smooth muscle cells of the media can produce sufficient amounts of PGI2 to locally influence the thrombogenicity of a de-endothelialized artery.

Animals↗
PubMed1980

Shear rate dependent inhibition of platelet adhesion and aggregation on collagenous surfaces by antibodies to human factor VIII/von Willebrand factor.

The effect of heterologous and homologous antibodies to factor VIII/von Willebrand factor (F.VIII/VWF) and purified VWF on the interaction of human platelets with subendothelium and with a surface consisting of collagen fibrils was investigated using annular flow chambers. Wall shear rates of 200--5200 s-1 were produced by varying the flow rate of the citrated blood which was circulated through these chambers by a pump. Surface coverage with platelets and with platelet aggregates was measured morphometrically. The antibodies had no effect on platelet adhesion at low shear rates. However, an increasing inhibition of adhesion was observed with increasing shear rates. The antibodies to F.VIII/VWF and to purified VWF virtually abolished adhesion at shear rates which are present in the microvascular (1300--5200 s-1). By contrast, antibodies to factor VIII:C which were isolated from two patients with haemophilia A had no effect on adhesion. In addition antibodies to F.VIII/VWF and to purified VWF inhibited adhesion-induced aggregation. We conclude that F.VIII/VWF plays an essential role as shear rates, i.e. when the time available for the establishment of a stable bond between platelets and collagenous surfaces is short.

Animals↗
PubMed1980

Automated microdensitometry and protein assays as a measure for platelet adhesion and aggregation on collagen-coated slides under controlled flow conditions.

A parallel-plate perfusion cell was developed for the in vitro study of platelet adhesion and aggregation. Two collagen-coated slides were exposed simultaneously to citrated blood under laminar and controlled flow conditions. The extent of the surface covered with platelets (adhesion) and platelet accumulations (aggregation) greater than about 5 micrometers in height was determined en face by automated microdensitometry of the fuchsin-stained collagen slides. The global measurement of deposited platelet protein per unit of surface was measured with a modified Lowry technique. Correlation among different parameters used to estimate the mass density on the slides, i.e., platelet number, amount of protein, and extent of platelet aggregation, is demonstrated. Acetylsalicylic acid at 100 microM decreased platelet aggregation without affecting adhesion. The new parallel-plate perfusion system offers rapid quantitation of platelet adhesion and aggregation after exposure to flowing blood.

Animals↗
PubMed1979

Platelet adhesion and platelet thrombus formation on subendothelium of human arteries and veins exposed to flowing blood in vitro. A comparison with rabbit aorta.

Subendothelium of blood vessel segments from elderly patients was exposed in a perfusion chamber to citrated blood under arterial blood flow conditions. The resulting platelet adhesion and thrombus formation was assessed morphometrically. The results show that the time course and the extent of platelet adhesion and aggregation were similar to the results obtained previously with subendothelium from rabbit arteries. The extent of surface-induced platelet aggregation was furthermore governed by both the properties of the exposed surface as well as those of the blood perfusate. No significant differences with regard to platelet reactivity were found between veins and arteries and the extent of surface-induced platelet aggregation was rather low.

Animals↗