Impact of vascular thromboxane prostanoid receptor activation on hemostasis, thrombosis, oxidative stress, and inflammation.

The activation of thromboxane prostanoid (TP) receptor on platelets, monocytes/macrophages, endothelial cells, and vascular smooth muscle cells (SMC) plays important roles in regulating platelet activation and vascular tone and in the pathogenesis of thrombosis and vascular inflammation. Oxidative stress and vascular inflammation increase the formation of TP receptor agonists, which promote initiation and progression of atherogenesis and thrombosis. Furthermore, TP receptor activation promotes angiogenesis and vessel wall constriction. Besides thromboxane A2 and its endoperoxide precursors, prostaglandin G2 and H2, isoprostanes, and 20-hydroxyeicosatetraenoic acid also activate TP receptor as autocrine or paracrine ligands. These additional TP activators play a role in pathological conditions such as diabetes, obesity, and hypertension, and their biosynthesis is not inhibited by aspirin, at variance with that of thromboxane A2. The understanding of TP receptor function increased our current knowledge of the pathogenesis of atherosclerosis and thrombosis, highlighting the great impact that this receptor has in cardiovascular disorders.

Single ascending oral dose pharmacokinetics and pharmacodynamics study of EV-077: the specific inhibitor of prostanoid- and isoprostane-induced cellular activation.

PURPOSE: This study was performed to determine the oral pharmacokinetics (PK) of EV-077 and its effects on pharmacodynamic (PD) markers. EV-077 blocks prostanoid-induced and isoprostane-induced cellular activation, and is in development for the treatment of vascular inflammation and associated complications of type-2 diabetes.. METHODS: This single-ascending-dose mono-centre study was randomised, placebo-controlled, and double-blinded within each dose group. Seven EV-077 doses were administered sequentially as an oral solution: 0.0125, 0.125, 0.375, 0.75, 1.25, 1.875 and 2.5 mg/kg body weight. PK, platelet aggregation, bleeding time and safety parameters were measured. Seven to eight healthy male subjects were dosed per group: five to six subjects received EV-077 and two subjects received placebo. RESULTS: Tmax was reached rapidly between 0.5 h and 1.0 h. Both Cmax and AUC increased linearly with the dose. The apparent terminal half-life (t½z) increased with the dose, most likely reflecting the increasing last quantifiable concentration with increasing dose; at 2.5 mg/kg, it was 2.7-6.9 h. Measurement of platelet aggregation showed no effect at 0.0125 mg/kg, and a full and reversible inhibition at doses of 0.125-2.5 mg/kg. The average bleeding time was dose-dependently prolonged, but was always below 9 min. The PK/PD profile showed that at plasma concentrations above 20 ng/ml, EV-077 platelet aggregation was completely inhibited (>90 %). All tested doses were well tolerated. CONCLUSIONS: Orally administered EV-077 was well tolerated, readily absorbed, reached Cmax within 1 h, with a linear PK based on Cmax and AUC. The inhibition of platelet aggregation was complete and reversible at doses of 0.125 mg/kg and higher, and average bleeding time was below 9 min.

Role of 4 platelet membrane glycoprotein polymorphisms on experimental arterial thrombus formation in men.

This study investigates whether the polymorphisms of 3 important platelet receptors affected experimental thrombus formation in men. Forty healthy male volunteers randomly recruited were genotyped for the variable number of tandem repeat (VNTR) of GPIbalpha, the -5T/C polymorphism in the Kozak sequence of GPIbalpha, the 807C/T polymorphism of GPIa, and the PI(A1)/PI(A2) polymorphism of GPIIb/IIIa. Platelet thrombus formation was induced ex vivo by exposing a collagen-coated coverslip in a parallel plate perfusion chamber to native blood for 4 minutes. The shear rates at the collagen surface were 650 and 2600 x s(-1). At 2600 x s(-1) platelet thrombus formation was significantly related only to the 807C/T polymorphism. In contrast, at 650 x s(-1) thrombus formation was significantly altered only by the Kozak sequence polymorphism. The VNTR and the PI(A1)/PI(A2) polymorphisms did not influence thrombus formation. Thus, platelet thrombus formation is significantly influenced by genetic variations of the GPIbalpha and GPIa receptors. The effect of these polymorphisms was dependent on the blood flow rate.

Methods and models to evaluate shear-dependent and surface reactivity-dependent antithrombotic efficacy.

The purpose of the present communication is to evaluate the importance of blood flow and surface reactivity for measurement of antithrombotic drug activity or efficacy in selected model systems of thrombus formation. Such information is essential for proper evaluation of antithrombotic drug profiles. The continuous development of flow-dependent thrombosis models for in vitro (anticoagulated blood) and ex vivo (native blood) studies and their application in in vivo animal models from the early 1970s and onwards are briefly considered. Central to this process was the development of various types of perfusion chambers in which a thrombogenic surface is exposed to flowing blood. Such perfusion chambers have been inserted into arteriovenous (AV) shunts in baboon, pig, dog, and rabbit. These approaches have allowed reproducible testing of traditional and novel experimental antithrombotic drugs, and studies on novel drug strategies under well-defined shear conditions and surface reactivity. Shear-dependent antithrombotic efficacy in these models is observed with anticoagulants such as unfractionated heparin, low-molecular weight heparins, or selective inhibitors of thrombin, Factor Xa, or Factor VIIa. However, the degree of shear dependency depends on the nature of the thrombogenic surface, e.g., the inhibition is more pronounced on a tissue factor (TF)-rich surface than on a collagen-rich surface, particularly at venous or low arterial shear. Platelet antagonists such as the COX-1 inhibitor aspirin, inhibitors of thromboxane A2 (TxA2) synthetase, the TxA2 platelet receptor, and of von Willebrand factor (vWf) are shear dependent also, being more efficient at high arterial shear. In contrast, the platelet ADP antagonist clopidogrel, or antagonists to the active platelet membrane glycoprotein IIb-IIIa complex (GPIIb-IIIa) are shear independent. At extremely high arterial shear, which activates platelets and elicit aggregates of circulating platelets, aspirin looses its antithrombotic effect, whereas ADP and GPIIb-IIIa antagonists still interrupt thrombus formation. In general, results obtained with these models mimic and predict antithrombotic efficacy in man when comparison is possible. Information on antithrombotic efficacy in flow devices with various thrombogenic surfaces is now sufficiently available to suggest recommendations for experimental conditions, particularly with regard to blood flow and reactive surfaces.

The effect of platelet PlA polymorphism on experimental thrombus formation in man depends on blood flow and thrombogenic substrate.

A number of studies have reported conflicting data on the association of the PlA1/PlA2 polymorphism of the GPIIIa gene and coronary syndromes. We have investigated the effect of this polymorphism on experimental platelet thrombus formation in man. Forty healthy male volunteers were genotyped for the PlA1/PlA2 polymorphism. Thrombus formation was induced ex vivo by exposing a tissue factor (TF) or a collagen-coated coverslip in a parallel plate perfusion chamber to native blood for 2 and 4 min. The shear rates at these surfaces were 650 and 2,600 s(-1). Platelet and fibrin deposition was quantified by immunoenzymatic methods. The frequencies of PlA1/PlA1 and PlA1/PlA2 genotypes were 52.5% and 47.5%, respectively. Ex vivo deposition of fibrin on TF was not affected by the PlA1/PlA2 polymorphism. However, the ex vivo platelet deposition at 650 s(-1) was higher in blood from PlA1/PlA1 individuals than in PlA1/PlA2 individuals (P= 0.008 at 4 min). On collagen, neither fibrin nor platelet deposition was significantly affected by the PlA1/PlA2 polymorphism. Platelet thrombus formation is significantly influenced by genetic variations in the GPIIIa platelet receptor. This effect depends on the blood flow properties and the nature of the thrombogenic stimulus.

Synthesis, biological activity, and solution structures of a cyclic dodecapeptide from the EGF-2 domain of blood coagulation factor VII.

The cyclic dodecapeptide, disulfide-cyclo-[H-Cys-Val-Asn-Glu-Asn-Gly-Gly-Cys(Acm)-Glu-Gln-Tyr-Cys-OH], which corresponds to the 91-102 sequence of the second epidermal growth factor domain of human blood coagulation factor VII, was synthesized using solid-phase procedures. It was shown to be an inhibitor at the key step in the induction of coagulation by the extrinsic pathway, i.e. the factor VII/tissue factor-catalyzed activation of coagulation factor X. The solution structure of this peptide was investigated by NMR spectroscopy and was computer-modeled via molecular mechanics. Structures were calculated based on 112 distance and nine dihedral angle constraints. The resulting backbone structures were classified into two structural subsets: one which exhibited a twisted '8'-shaped folding and another describing an open, circular 'O' outline. The local backbone structures of segments Asn3-Glu4-Asn5, Gly7-Cys8 and Gln10-Tyr11 were well preserved among the two subsets. Apart from the unrestrained N- and C-termini, Gly6 and Glu9 sites exhibited marked local disorder between the two subsets, suggesting localized flexible hinges likely to govern tertiary structure interconversion between the two subsets. Two transient hydrogen bonds were identified from pH chemical shift titrations by matching the pKa values of NH and carboxylate groups, which supported the occurrence of the '8' structure, and agreed with temperature coefficients of peptidyl NH resonances. Structure-function relationships of the peptide were discussed in terms of the likely physiological function of the disulfide-bonded loop in factor VII which the peptide represents.

Effect of radiologic contrast media on cell volume regulation in rabbit proximal renal tubules.

RATIONALE AND OBJECTIVES: Most radiographic contrast media are hyperosmotic and able to shrink cells with which they are in contact. The authors studied cell volume control in rabbit proximal renal tubules after incubation with three contrast media: iohexol, ioxaglate, and iodixanol. MATERIALS AND METHODS: Proximal renal tubules were isolated from rabbit kidneys. The tubules were exposed to Ringer solutions containing 5% vol/vol iohexol (final osmolality, 330 mOsm), ioxaglate (323 mOsm), iodixanol (305 mOsm), or mannitol (control solutions with identical osmolalities), and tubule volumes were monitored. After 2 hours of incubation, the tubules were stimulated with a hyposmotic Ringer solution (165 mOsm). Three groups of 10 experiments were performed. RESULTS: All solutions induced cell shrinkage (8.3%+/-3.8 [standard error] to 15.4%+/-0.5), which was completely or partly reversible in most experiments (volume increase, 44.8%+/-14.7 to 149.9%+/-107.3) but not those with iohexol and iodixanol. With exposure to the hyposmotic solution, the cells swelled by 11.0%+/-1.8 to 39.7%+/-4.8. In general, the tubules that had been exposed to the most hyperosmotic solution swelled the most. Those exposed to contrast media showed less swelling than the mannitol-exposed controls. In all control experiments, the cells exhibited a gradual shrinkage (43.6%+/-28.5 to 87.0%+/-13). This regulatory response was partly inhibited in tubules exposed to iohexol (39.9%+/-15.8 shrinkage) or iodixanol (8.9%+/-15.8) and completely inhibited in those exposed to ioxaglate. Iohexol and ioxaglate exposure also led to a decrease in water permeability. CONCLUSION: Exposure to hyperosmotic contrast medium tends to induce prolonged cell shrinkage, decrease the water permeability of the cellular plasma membranes, and compromise the ability to regulate cellular volume. These changes seem to reflect both the hyperosmolality of the solutions and their inherent chemical properties.

5-aminoimidazole-4-carboxy-amide-1-beta-D-ribofuranoside treatment ameliorates hyperglycaemia and hyperinsulinaemia but not dyslipidaemia in KKAy-CETP mice.

AIM/HYPOTHESIS: 5-aminoimidazole-4-carboxy-amide-1-beta-d-ribofuranoside increases 5'-AMP-activated kinase activity in insulin-sensitive tissues known to control glucose homeostasis. We hypothesised that 5-aminoimidazole-4-carboxy-amide-1-beta-d-ribofuranoside treatment could have a beneficial effect on glucose homeostasis in KKAy-CETP mice, a model of Type II (non-insulin-dependent) diabetes mellitus. Our aim was to examine potential effects of acute and chronic (7-day) 5-aminoimidazole-4-carboxy-amide-1-beta-d-ribofuranoside treatment on glucose homeostasis in KKAy-CETP diabetic mice. METHODS: Female KKAy-CETP mice were treated with 5-aminoimidazole-4-carboxy-amide-1-beta-d-ribofuranoside by a single daily injection for 7 days (100, 300, or 500 mg. kg-1. day-1). RESULTS: After 7 days of treatment with 500 mg. kg-1. day-1 5-aminoimidazole-4-carboxy-amide-1-beta-d-ribofuranoside, blood glucose and plasma insulin concentrations were reduced (p < 0.01). Body weight and food intake were also reduced after treatment (p < 0.01 and p < 0.05, respectively). Glucose and insulin tolerance were improved (p < 0.05), whereas endogenous glucose production was suppressed (p < 0.05). The beneficial effect of 5-aminoimidazole-4-carboxy-amide-1-beta-d-ribofuranoside on hyperglycaemia and hyperinsulinaemia was due to an inhibition of endogenous glucose production, since in vivo and in vitro basal and insulin-stimulated glucose uptake in skeletal muscle was not affected by 5-aminoimidazole-4-carboxy-amide-1-beta-d-ribofuranoside. Other features of the treatment included increased plasma of free fatty acid concentration (1.9-fold, p < 0.01) and triglycerides (1.3-fold, p < 0.05). CONCLUSION/INTERPRETATION: 5-aminoimidazole-4-carboxy-amide-1-beta-d-ribofuranoside treatment attenuated hyperglycaemia and hyperinsulinaemia but not dyslipidaemia in KKAy-CETP mice, a model of Type II diabetes. The blood glucose lowering effects of 5-aminoimidazole-4-carboxy-amide-1-beta-d-ribofuranoside occurs mainly as a consequence of reduced endogenous glucose production because insulin-stimulated skeletal muscle glucose uptake has not been altered.

Effect of radiologic contrast material on cell volume regulation in proximal renal tubules from trout (Salmo trutta).

RATIONALE AND OBJECTIVES: Most radiographic contrast media (CM) are hyperosmotic and pose an osmotic threat to cells they are in contact with. To study these effects at the cellular level, cell volume regulatory mechanisms were observed in proximal renal tubules following exposure to the CM iohexol, ioxaglate, and iodixanol. MATERIALS AND METHODS: Isolated renal tubules from trout (Salmo trutta) were exposed to 5% vol/vol iohexol (326 mOsm), ioxaglate (314 mOsm), or iodixanol (300 mOsm) or mannitol (to achieve the same osmolalities), and cell volume changes were observed videometrically. RESULTS: Iohexol and ioxaglate solutions induced a rapid shrinkage (12%-13%) not followed by cell volume regulation. Without CM (same osmolality), the cells shrank 11% but then showed a 77%-88% volume recovery. This reswelling was inhibited by 55% with the Na+, K+, Cl- symporter inhibitor bumetanide (50 micromol/L). Iodixanol did not significantly affect cell volume. Tubules preincubated with CM or mannitol were then stimulated with a hypoosmotic Ringer solution (160 mOsm) resulting in a 26%-36% cellular volume increase. Compared with results of experiments without mannitol and CM, preexposure to iohexol or ioxaglate almost completely inhibited the expected regulatory shrinkage phase, while previous exposure to hyperosmotic solutions with mannitol reduced the shrinkage response by 40%-53%. CONCLUSION: In this system, the hyperosmotic iohexol and ioxaglate cause cell shrinkage followed by an impaired cell volume regulatory response. Exposure to these two CM also inhibits cell volume regulation on hypoosmotic stimulation. The isosmotic iodixanol has no such effects. These changes appear to some extent to be a result of the CM's degree of hyperosmolality, but this property alone does not explain these findings.

Early potent antithrombotic effect with combined aspirin and a loading dose of clopidogrel on experimental arterial thrombogenesis in humans.

BACKGROUND: We conducted a double-blind, randomized, crossover study to assess the antithrombotic effects of the combination of aspirin (acetylsalicylic acid, ASA) and clopidogrel, with or without a loading dose, versus ASA alone in a model of arterial thrombosis in humans. METHODS AND RESULTS: Eighteen male volunteers received the following 3 regimens for 10 days separated by a 1-month period: (1) 325 mg ASA daily, (2) 325 mg ASA+75 mg clopidogrel daily, (3) 325 mg ASA daily+300-mg clopidogrel loading dose on day 1 and +75 mg clopidogrel per day on days 2 to 10. The antithrombotic effect was measured 1.5, 6, and 24 hours after drug intake on day 1 and 6 hours after drug intake on day 10. Arterial thrombus formation was induced ex vivo by exposing a collagen-coated coverslip in a parallel-plate perfusion chamber to native blood for 3 minutes at an arterial wall shear rate. Without a loading dose, clopidogrel+ASA developed an antithrombotic effect within 6 hours after the first intake. It was superior to that produced by ASA, but it was moderate (P

Sequestrated thrombolysis: comparative evaluation in vivo.

PURPOSE: Lysis of a thrombus is a function of the local concentration of thrombolytic enzymes. This study was designed to determine in a porcine model of acute deep vein thrombosis (DVT) whether perithrombic sequestration of small volumes of a concentrated enzyme solution can accelerate the process of thrombolysis. METHODS: DVT was induced in both hind limbs using a previously described technique (n = 32). Thirty minutes later the animal was heparinized and unilateral thrombolysis was attempted using 8 mg recombinant tissue plasminogen activator (rt-PA); saline was administered in the opposite leg. For conventional high-volume infusion (CI) (n = 5) rt-PA (0.067 mg/ml) was infused at 1 ml/min. For sequestrated thrombolysis the external iliac vein was endoluminally occluded, and rt-PA (0.25 mg/ml) administered either for proximal injection (ST-P) (n = 5), as a bolus every 3 min through a microcatheter placed via the balloon catheter, or for transthrombic injection (ST-T) (n = 5), as a bolus every 3 min through a Katzen wire in the balloon catheter. At autopsy, the thrombus mass in the iliofemoral veins was measured, and the extent of residual thrombosis in the venous tributaries graded at four sites. From these data a thrombolysis score was calculated. RESULTS: One pig died before thrombolysis could be performed. Only with ST-T was residual thrombus mass in the test limb normalized to control, residual thrombus index (RTI), consistently less than unity. The median RTI of this group was 0.50 (range 0.39-0.97) compared with 1.22 (0.64-1.38) for ST-P and 0.88 (0.37-1.13) for CI. Compared with contralateral controls, a lower grade of residual thrombosis in tributaries was observed in test limbs at more venous sites with ST-T (8/20; 95% confidence interval 5-13) and ST-P (9/20; confidence interval 5-13) than with CI (2/20; confidence interval 0-5) (p = 0.04). A trend toward lower thrombolysis scores was observed with ST-T (p = 0.08). Systemic fibrinogenolysis was not observed in any of the groups. Changes in coagulation parameters during thrombolysis were similar irrespective of treatment protocol. CONCLUSIONS: "Transthrombic" sequestrated thrombolysis may offer some advantages over conventional selective infusion for the treatment of acute DVT. However further refinements will be necessary before it can be considered an alternative to the latter.

Protamine sulphate inhibits platelet membrane glycoprotein Ib-von Willebrand factor activity.

Platelet adhesion to the injured vessel wall is essential in haemostasis and thrombosis. This process involves the interaction of the platelet glycoprotein Ib (GPIb) with surface bound von Willebrand factor (vWF). Since synthetic polycationic peptides of the general formula (Arg)n, (Lys)n or (Arg-Lys)n inhibit GPIb-vWF interaction, they were suggested as potential antithrombotics. Protamine sulphate is a highly cationic polypeptide, arginine accounting for approximately 60% of the primary sequence, utilized to neutralize the anticoagulant effect of heparin after cardiac surgery. We have investigated potential effects of protamine sulphate on the function of GPIb-vWF. Addition of protamine sulphate to platelet-rich plasma (PRP), reduced significantly the GPIb-vWF activity as assessed by ristocetin-induced platelet agglutination. When protamine sulphate was added to PRP containing heparin, even at clinically relevant neutralizing doses the GPIb-vWF activity was reduced by 20-30% (p < 0.001). Protamine sulphate in excess of heparin nearly abolished the activity. Furthermore, the direct effect of protamine sulphate on collagen-induced platelet thrombus formation in non-anticoagulated human blood was investigated by employing an ex-vivo parallel-plate perfusion chamber device. Protamine sulphate (200 microg/mL) reduced platelet-collagen adhesion at shear rates of 650 and 2600 sec(-1) by 40% (p< 0.004) and 45% (p < 0.0001), respectively. The corresponding platelet thrombus volumes were concomitantly reduced by 90% (p < 0.006) and 84% (p < 0.05). Our data are questioning the rationale for empirical repetitive protamine sulphate administration when so-called "heparin rebound" after cardiac surgery is suspected, since protamine sulphate in excess of heparin may impair the platelet GPIb-vWF interaction necessary for normal haemostasis.

Selective thrombolysis in acute deep vein thrombosis: evaluation of adjuvant therapy in vivo.

PURPOSE: To evaluate in a porcine model of acute deep vein thrombosis (DVT) the efficacy of dalteparin and antithrombin with respect to heparin for local adjuvant therapy during selective thrombolysis, and the utility of nitroglycerin and iloprost as heparin supplements. METHODS: DVT was induced in both hind limbs using a previously described technique (n = 20). Thirty minutes later, the animal was heparinized (2500 IU IV), and bilateral sequestrated thrombolysis was performed using 8 mg alteplase: both external iliac veins were endoluminally occluded with Swan-Ganz catheters, and a multi-sideport infusion wire coaxially introduced through each catheter and advanced into the ipsilateral popliteal vein. In the control limbs, tissue plasminogen activator (tPA) 8 mg was injected as 0.8-ml boluses at 3-min intervals for 2 hr as a 0. 25-mg/ml solution containing heparin 50 IU/ml (n = 20). On the contralateral side, heparin was substituted with either dalteparin 50 IU/ml (n = 5) or antithrombin 12.5 IU/ml (n = 5), or supplemented with either nitroglycerin 0.075 mg/ml (n = 5) or iloprost (150 ng/ml) (n = 5). Blood samples were taken at predetermined intervals to measure the activated partial thromboplastin time (aPTT), prothrombin time (PT), and fibrinogen concentration. At autopsy, the thrombus mass in the iliofemoral veins was measured, and the extent of residual thrombosis in the venous tributaries graded at four sites. RESULTS: Bilateral thrombolysis was successfully completed in all animals. The median thrombus mass in the iliofemoral veins after thrombolysis was 0.48 g (range 0.06-1.58 g), 0.95 g (0.59-1.29 g), 0. 74 g (0.52-0.96 g), and 0.29 g (0.0-0.77 g) for dalteparin, antithrombin, iloprost, and nitroglycerin respectively, as compared with 0.53 g (0.18-0.88 g) (p = 0.69), 0.97 g (0.46-1.15 g) (p = 0. 69), 0.53 g (0.48-1.10 g) (p = 0.69), and 0.18 g (0.13-1.04 g) (p = 0.5) for the respective controls. Likewise, the severity of residual thrombosis in the venous tributaries was not affected by the constituents of adjuvant therapy. Nitroglycerin induced a small drop in blood pressure, which was transient. The temporal change in aPTT was similar in all four groups. Invariably PT progressively shortened during thrombolysis (p = 0.0001); this effect was somewhat blunted with antithrombin. Fibrinogen levels demonstrated a time-dependent increase (p = 0.004) that was not influenced by the adjuvant therapy used. CONCLUSIONS: Dalteparin or antithrombin demonstrated no appreciable advantage over heparin as local adjuvant therapy for selective venous thrombolysis. Supplementation of heparin with iloprost or nitroglycerin also had virtually no effect on thrombolytic efficacy.

Antithrombotic efficacy of the vitamin K antagonist fluindione in a human Ex vivo model of arterial thrombosis : effect of anticoagulation level and combination therapy with aspirin.

Thrombin is a main mediator of arterial thrombus formation, and its inhibition is an important antithrombotic strategy. However, the place of vitamin K antagonists among the different therapeutic strategies for preventing arterial thrombus formation is still debated. We studied the antithrombotic efficacy of the vitamin K antagonist fluindione in a human ex vivo model of arterial thrombosis and determined whether aspirin enhances fluindione efficacy. Ten healthy male volunteers were randomly assigned to receive fluindione, alone or in combination with aspirin (325 mg/d). Fluindione was given at increasing doses to give a stable international normalized ratio (INR) between 1.5 and 2.0 and between 2.1 and 3.0. We induced arterial thrombus formation ex vivo by exposing collagen- or tissue factor (TF)-coated coverslips in a parallel-plate perfusion chamber to native blood for 3 minutes at an arterial wall shear rate of 2600 s(-1). Platelet and fibrin deposition were measured by immunoenzymatic methods. Fluindione inhibited thrombus formation on TF-coated coverslips in a dose-dependent manner by 50% and 80% at INR 1.5 to 2.0 and INR 2.1 to 3.0, respectively (P<0.05). Fluindione in combination with aspirin inhibited TF-induced thrombus formation in a comparable manner. Collagen-induced thrombus formation was not reduced in subjects treated by fluindione. It was reduced by 50% to 60% in those treated with fluindione plus aspirin, regardless of the level of anticoagulation (P<0.05). Thus, the effectiveness of fluindione for preventing arterial thrombosis is dependent on the nature of the thrombogenic trigger. Fluindione is very effective in preventing TF- but not collagen-triggered thrombus formation. Aspirin enhances the antithrombotic effectiveness of fluindione, because combined treatment interrupts both TF- and collagen-induced thrombus formation.