Clinical features of a new disease concept, IgG4-related thyroiditis.

OBJECTIVES: Immunoglobulin (Ig)G4-related disease is a recently proposed systemic disorder that includes autoimmune pancreatitis (AIP), Mikulicz's disease, and various other organ lesions. In the present retrospective study, we examined whether thyroid lesions should also be included in IgG4-related disease (Ig4-RD) under the new term IgG4-related thyroiditis. METHOD: We enrolled 114 patients with Ig4-RD, including 92 patients with AIP, 15 patients with Mikulicz's disease, and seven patients with IgG4-related cholangitis, and analysed clinical findings, function, serum values of activity markers, computed tomography (CT) images, and histology of the thyroid gland. RESULTS: Among the 22 patients (19%) in our cohort who were found to have hypothyroidism [thyroid stimulating hormone (TSH) > 4 mIU/L], 11 patients had clinical hypothyroidism [free thyroxine (FT4) < 1 ng/dL] and 11 patients had subclinical hypothyroidism (FT4 ≥ 1 ng/dL). Serum concentrations of IgG, IgG4, circulating immune complex (CIC), and ß2-microglobulin (ß2-MG) were significantly higher in the hypothyroidism group compared with the remaining 92 euthyroid patients, and serum C3 concentration was significantly lower. After prednisolone treatment, TSH values had decreased significantly (p = 0.005) in this group and FT4 values had increased significantly (p = 0.047). CT images showed that the thyroid glands of patients with clinical hypothyroidism had a significantly greater volume than those of the euthyroid and other groups. Pathological analysis of one resected thyroid gland disclosed a focused lesion with infiltration of lymphocytes and IgG4-bearing plasma cells and loss of thyroid follicles. CONCLUSIONS: Thyroid lesions associated with hypothyroidism can be considered as a new disease termed IgG4-related thyroiditis. Awareness of this condition should lead to appropriate corticosteroid treatment that may prevent progression to a fibrous state.

A lipid A analog ONO-4007 induces tolerance to plasma leakage in mice.

OBJECTIVE: The effects of pretreatment with ONO-4007, a lipid A analog, on cutaneous plasma leakage induced by ONO-4007, lipopolysaccharide (LPS) and inflammatory mediators were investigated. MATERIAL: Male ddY strain mice. TREATMENT: Mice were pretreated with ONO-4007 (up to 6 mg/kg i.p.), 0-24 h prior to plasma leakage study. METHODS: Plasma extravasation was determined by dye leakage. RESULTS: Systemic ONO-4007 (6 mg/kg i. p.) pretreatment for 2 to 12 h inhibited plasma extravasation in the mouse skin elicited by ONO-4007 and LPS. The inhibition was dose-dependent. Plasma leakage induced by platelet-activating factor (PAF), histamine and 5-hydroxytryptamine (5-HT) was also inhibited by ONO-4007 pretreatment. Plasma corticosterone levels increased 2 and 4 h after systemic ONO-4007 (6 mg/kg) administration and returned to the control level 24 h later. Adrenalectomy and metyrapone but not propranolol reversed the inhibition by ONO-4007 pretreatment of LPS-induced plasma leakage. CONCLUSIONS: A single injection of ONO-4007 in mice induced transient tolerance to plasma leakage elicited by LPS, ONO-4007 and inflammatory mediators. Endogenous corticosterone, at least in part, plays a role in the development of tolerance.

Inhibitory effects of cyclic AMP elevating agents on lipopolysaccharide (LPS)-induced microvascular permeability change in mouse skin.

Anti-inflammatory effects of cyclic AMP elevating agents were examined in a mouse model of lipopolysaccharide (LPS)-induced microvascular permeability change. Vascular permeability on the back skin was measured by the local accumulation of Pontamine sky blue (PSB) after subcutaneous injection of LPS (400 microg site-1) from Salmonella typhimurium. Dye leakage in the skin was significantly increased 2 h after injection of LPS. This LPS-induced dye leakage was suppressed by phosphodiesterase inhibitors, including pentoxifylline (160 mg kg-1), milrinone (5 - 10 mg kg-1), rolipram (0.5 - 10 mg kg-1) and zaprinast (5 - 10 mg kg-1). The dye leakage was also inhibited by beta-adrenoceptor agonists, including isoproterenol (0.5 - 5 mg kg-1) and salbutamol (0.05 - 5 mg kg-1), an adenylate cyclase activator, forskolin (5 mg kg-1), and a cell permeable cyclic AMP analogue, 8-bromo-cyclic AMP (8-Br-cAMP, 10 mg kg-1). LPS caused a transient increase in serum TNF-alpha level peaking at 1 h after the injection. This increase in serum TNF-alpha was completely blocked by a pretreatment with pentoxifylline (160 mg kg-1), milrinone (5 mg kg-1), rolipram (1 mg kg-1), zaprinast (10 mg kg-1), salbutamol (0.5 mg kg-1), forskolin (1 mg kg-1) and 8-Br-cAMP (10 mg kg-1). LPS caused an increase in serum IL-1alpha level peaking at 3 h after injection. This increase in serum IL-1alpha was not significantly suppressed by the cyclic AMP elevating agents. Our study suggests that cyclic AMP elevating agents attenuate LPS-induced microvascular permeability change by suppressing TNF-alpha up regulation.

Identification of two novel splicing variants of human type II iodothyronine deiodinase mRNA.

Human type II iodothyronine deiodinase (hDII) belongs to a family of selenoproteins. It catalyzes 5'-deiodination of thyroxine to generate an active thyroid hormone, 3,3',5-triiodothyronine. Two novel splice variants of hDII gene transcript; namely hDII-b and hDII-c, were identified. Three distinct DNA fragments (hDII-a-c) were amplified by a reverse transcription-polymerase chain reaction (RT-PCR) with hDII intron-spanning primers using total-RNA from human umbilical vein endothelial cell line, ECV304. The sequence of hDII-a was identical to that of previously reported hDII. hDII-b and hDII-c had an additional insertion of 108 and 242 bp, respectively. The insertion sequences were found in the intron of hDII gene, therefore, two novel exons exist between exons 1 and 2 of hDII gene. The splice sites of new exons (b and c) conserved the consensus sequences of splice acceptor and donor sites. hDII-b contains exon b with an in-frame TGA codon that may encode an extra selenocysteine. hDII-c contained exons b and c and the predicted open reading frame is interrupted by a stop codon (TAA) produced by a frame shift. RT-PCR analysis showed that hDII-a and hDII-b mRNAs are expressed in human tissues including brain, kidney, lung and trachea. The mRNA abundance of hDII-c was lower than that of hDII-a or hDII-b. Thus, the new variants of hDII transcripts suggest the presence of two short exons between exons 1 and 2 of hDII gene, and of functional variant of hDII.

Molecular and functional characterization of HSC54, a novel variant of human heat-shock cognate protein 70.

A novel variant of human heat-shock cognate protein 70 (HSC70) transcript, named heat-shock cognate protein 54 (HSC54), was identified and characterized. The transcript encodes the protein lacking 153 amino acid residues of HSC70 in a part of the protein-binding and variable domains, resulting in a calculated molecular mass of 53.5 kDa. HSC54 mRNA was detected in all human cells and tissues examined. The protein was also detected in peripheral mononuclear cells and U937 human histiocytic lymphoma cells. Heat treatment of U937 cells up-regulated the expression of HSC54. The chaperoning activity of HSC54 was examined by luciferase renaturation assay. HSC70 recovered the luciferase activity in the presence of reticulocyte lysate as a source of cochaperones. However, HSC54 did not facilitate the recovery of denatured luciferase; besides, HSC54 significantly inhibited the HSC70-mediated chaperoning activity. In pull-down experiments, HSC54 interacted with cochaperones, p60, HSP40, and p48, as HSC70 did. The resonant mirror detection analysis showed that p60 binds to HSC54 with a higher association rate constant than HSC70 with a similar affinity constant. These results suggest that HSC54 is constitutively expressed and also inducible by stress and may function as an endogenous inhibitory regulator of HSC70 by competing the cochaperones.

A novel degradative pathway of 2-nitrobenzoate via 3-hydroxyanthranilate in Pseudomonas fluorescens strain KU-7.

A bacterial strain KU-7, identified as a Pseudomonas fluorescens by 16S rDNA sequencing, was one of the 12 new isolates that are able to grow on 2-nitrobenzoate as a sole source of carbon, nitrogen, and energy. Resting cells of KU-7 were found to accumulate ammonia in the medium indicating that degradation of 2-NBA proceeds through a reductive route. Metabolite analyses by thin layer chromatography and high pressure liquid chromatography indicated that 3-hydroxyanthranilate is an intermediate of 2-nitrobenzoate metabolism in KU-7 cells. This offers an alternative route to 2-nitrobenzoate metabolism since anthranilate (2-aminobenzoate) or catechol were detected as intermediates in other bacteria. Crude extracts of KU-7 cells converted 2-nitrobenzoate to 3-hydroxyanthranilate with oxidation of 2 mol of NADPH. Ring cleavage of 3-hydroxyanthranilate produced a transient yellow product, identified as 2-amino-3-carboxymuconic 6-semialdehyde, that has a maximum absorbance at 360 nm. The initial enzymes of the 2-nitrobenzoate degradation pathway were found to be inducible since succinate-grown cells produced very low enzyme activities. A pathway for 2-nitrobenzoate degradation in KU-7 was proposed.

A Ca(2+) channel blocker-like effect of dehydrocurdione on rodent intestinal and vascular smooth muscle.

Effects of dehydrocurdione, a zedoary-derived sesquiterpene, on smooth muscle were investigated by recording the mechanical activity of intestines and aorta from guinea pigs and rats. Dehydrocurdione (0.1-3 mM) induced a sustained relaxation of rat duodenum and inhibited spontaneous motility. Dehydrocurdione (0.1-1 mM) inhibited the contractile response of guinea pig ileum induced by acetylcholine (0.01-10 microM), histamine (0.03-10 microM) and substance P (0.1-30 nM) in a non-competitive manner. Acetylcholine (0.5 microM) elicited a transient contraction followed by a sustained contraction of guinea pig ileum, and dehydrocurdione pretreatment inhibited the sustained component, which depends on Ca(2+) entry from the extracellular space. The high K(+)-induced contraction of rat aortic ring is reported to be blocked by Ca(2+) channel blockers, while the norepinephrine-induced contraction includes a Ca(2+) channel blocker-resistant component. Dehydrocurdione (1 mM) blocked the high K(+) (60 mM)-induced contraction of rat aortic ring by 81%, while it inhibited the norepinephrine (1 microM)-induced contraction by only 28%. Dehydrocurdione (1 mM) significantly reduced the high K(+)-stimulated increase in cytosolic Ca(2+) level of Fura-2-loaded mesenteric artery from rats. These results suggest that the inhibitory effects of dehydrocurdione on intestinal and vascular smooth muscle are mediated by blockade of Ca(2+) entry from the extracellular space.

Iodophosphoryloxylation of carbon-carbon multibonds and its application to glycals

Iodophosphoryloxylation of carbon-carbon multibonds was attempted. Alkynes and cyclohexene were converted to the corresponding 1,2-iodophosphoryloxylated compounds in moderate to good yields with a trivalent iodine compound/iodine system, while glucal gave mainly the corresponding iodohydrin compound in this system. However, 2-deoxy-2-iodoglycosyl diphenylphosphinates were obtained from the corresponding glycals with a diphenylphosphinic acid/iodine/potassium carbonate system in good yields. Moreover, triethylborane smoothly reduced 2-deoxy-2-iodoglycosyl diphenylphosphinates to 2-deoxyglycosyl diphenylphosphinates in a 1,4-cyclohexadiene solvent.

Role of inflammatory mediators in lipid A analogue (ONO-4007)-induced vascular permeability change in mouse skin.

1. Endotoxin shock is accompanied by an increase in peripheral vascular permeability. It has been postulated that most biological activities of LPS are derived from lipid A moiety. Here we examined the effect of lipid A analogue ONO-4007 in increasing vascular permeability and the possible mediators in mouse skin by a dye leakage method. 2. Subcutaneous injection of ONO-4007 (1 - 2 mg site(-1)) induced a dose-dependent increase in vascular permeability which was evident after 120 min. 3. ONO-4007-induced dye leakage was significantly attenuated by pretreatments with anti-tumour necrosis factor-alpha (TNF-alpha) and anti-interleukin-1alpha (IL-1alpha) antibodies, but not with indomethacin (5 mg kg(-1)) or diphenhydramine (10 mg kg(-1)). ONO-4007-induced dye leakage was significantly inhibited by a pretreatment with N(G)-nitro-L-arginine methyl ester (L-NAME) (10 mg kg(-1)) but not with aminoguanidine (50 mg kg(-1)). In inducible nitric oxide synthase (iNOS)-deficient mice, ONO-4007 significantly increased the dye leakage, while ONO-4007 dilated rat thoracic aortic rings pre-contracted with phenylephrine, and the L-NAME pretreatment inhibited the dilation. 4. Thus, TNF-alpha, IL-1alpha and constitutive NOSs-derived nitric oxide but not prostaglandins or histamine play a role in ONO-4007-induced increase in vascular permeability. Although ONO-4007 mimics LPS in increasing vascular permeability, mechanisms of permeability change elicited by ONO-4007 were not identical to those of LPS.

Effect of lipoteichoic acid on dermal vascular permeability in mice.

Lipoteichoic acid (LTA), the cell wall component of Gram-positive bacteria, has been shown to cause inflammatory responses comparable to lipopolysaccharide (LPS) of Gram-negative bacteria. This study examined the activity of LTA to induce dermal microvascular permeability changes in mice. Vascular permeability was assessed by extravasation of Pontamine sky blue. Subcutaneous injection of LTA (200-400 microg/site) in mice that were preinjected i.v. with the dye increased local dye leakage in the skin at 1 to 3 h. The LTA-induced dye leakage was inhibited by indomethacin, valeryl salicylate, diphenhydramine, and a platelet-activating factor antagonist but not by inhibitors of nitric-oxide synthase, cyclooxygenase-2, or guanylate cyclase or by antibodies against tumor necrosis factor-alpha or interleukin-1alpha. LTA induced comparable increases in dye leakage in inducible nitric-oxide synthase-deficient mice and wild-type controls. Pretreatment of normal mice with i.v. LTA did not confer tolerance to LTA- or LPS-induced dye leakage. In contrast, systemic LPS administration induced tolerance against subsequent challenge with LPS but not LTA. Serum corticosterone levels, which were suggested to induce tolerance, were not increased by LTA pretreatment but were increased by LPS. Thus, LTA increases dermal microvascular permeability in mice. Among the inflammatory mediators, eicosanoids, platelet-activating factor, and histamine mediate the effect of both LTA and LPS, whereas nitric oxide, tumor necrosis factor-alpha, and interleukin-1alpha may not play a major role in LTA-induced dye leakage. The difference between LTA and LPS to stimulate corticosterone may partially explain the failure of LTA to induce tolerance against vascular dye leakage.

Evaluation of iNOS-dependent and independent mechanisms of the microvascular permeability change induced by lipopolysaccharide.

1. Subcutaneous injection of lipopolysaccharide (LPS) increases plasma leakage in mouse skin. Pretreatment with LPS conditions mice tolerant to the LPS-induced plasma leakage. Nitric oxide (NO) has been suggested to be involved in these LPS effects. A specific role of inducible NO synthase (iNOS) was investigated in the LPS-induced plasma leakage using iNOS deficient mice. 2. Plasma leakage in mouse skin was measured by the local accumulation of Pontamine sky blue at the site of subcutaneous injection of LPS (Sal. typhimurium). LPS (100 - 400 microg site(-1)) produced a dose-related increase in dye leakage in both iNOS deficient and wild-type mice with about 40% less dye leakage in iNOS deficient mice. 3. Indomethacin (5 mg kg(-1)), N-[-2-cyclohexyloxy]-4-nitrophenyl methanesulphonamide (NS-398) (1 mg kg(-1)), diphenhydramine (10 mg kg(-1)) and anti-TNF-alpha antibody (dilution 1 : 400, 10 ml kg(-1)) inhibited the LPS-induced dye leakage in both iNOS deficient and wild-type mice, whereas N(G)-nitro-L-arginine methyl ester (L-NAME) (10 mg kg(-1)) or aminoguanidine (10 mg kg(-1)) inhibited that in wild-type but not in iNOS deficient mice. 4. Pretreatment with LPS (0.15 mg kg(-1) i.p.) 4 h before decreased the LPS-induced dye leakage in wild-type but not in iNOS deficient mice. LPS pretreatment increased serum corticosterone levels in both mice, while it increased the serum nitrate/nitrite levels in wild-type but not in iNOS deficient mice. 5. These studies indicate that an increase in vascular permeability induced by LPS is mediated by NO produced by iNOS, eicosanoids, histamine and TNF-alpha. The tolerance against LPS-induced vascular permeability change may be mediated by iNOS induction but not by an increased release of endogenous corticosteroids.

Anti-NO action of carvedilol in cell-free system and in vascular endothelial cells.

1. Carvedilol, an adrenoceptor blocker with antioxidant activity, was studied for its ability to interact with NO in a cell-free condition and in an endothelial cell line (ECV304). 2. In a cell-free system, carvedilol attenuated NO-dependent reduction of carboxy-2-phenyl-4,4, 5,5-tetramethyl-imidazoline-1-oxyl-3-oxide induced by a NO donor, 1-hydroxy-2-oxo-3-(aminopropyl)-3-isopropyl-1-triazene (NOC5), which was determined by electron paramagnetic resonance (EPR) spectrometry. The EPR study also showed that nitrosylhaemoglobin formation in rat red blood cells by the addition of NO-saturated solution was attenuated by prior incubation with 0.1 - 10 microM carvedilol. 3. NO-induced fluorescence in 4,5-diaminofluorescein-2 diacethyl (DAF-2DA)-loaded ECV304 cells was attenuated by carvedilol but not by labetalol. The IC(50) of carvedilol for NOC5 or sodium nitroprusside-induced fluorescence of DAF-2DA in ECV304 cells was 1. 0x10(-7) M, which was similar to the reported IC(50) of carvedilol for the antioxidant effect. 4. Cell toxicity induced by a NO donor determined by the number of viable cells after 24 h treatment with 2-2'(hydroxynitrosohydrazino)bis-ethanamine was significantly attenuated by pretreatment with 1 microM carvedilol. 5. Both free and cell-associated carvedilol quenched NO. Because NO mediates both physiological and pathophysiological processes, NO quenching by the drug may have diverse clinical implications depending upon specific functions of local NO in tissues where carvedilol is distributed.

Acetyl-seryl-aspartyl-lysyl-proline is a novel natural cell cycle regulator of renal cells.

A natural tetrapeptide, acetyl-seryl-aspartyl-lysyl-proline (AcSDKP) is a physiological negative regulator of hematopoiesis. The precursor of AcSDKP, thymocin beta 4, is expressed in many tissues including kidney. The present study examined the antiproliferative effect of AcSDKP in two renal cell lines, namely, renal interstitial fibloblasts cell line (NRK 49F) and renal proximal tubular epitherial cells (LLC-PK1). An addition of AcSDKP for 48 hours in theses cells resulted in a concentration-dependent attenuation in the proliferation rate (significant difference to non-treated cells was observed at 10(-9) to 10(-5) M AcSDKP) determined by a colorimetry of alamer blue oxidation. The cell cycle analysis of NRK 49F cells treated with AcSDKP showed that AcSDKP significantly reduced the ratio of S-phase to G2/M-phases. Thus, physiological concentrations of AcSDKP is capable of altering cell cycle to inhibit the proliferation of renal cells.

Infant leukemia suggestive of natural killer cell precursor origin followed an unusual clinical course.

We report a patient with infant leukemia showing the rare phenotypes of CD2+, CD4+, CD7+, CD56+, CD3-, CD5- who followed an unusual clinical course. The patient was a 3-month-old girl who was admitted because of unusual purpura looking like a black ring. On admission WBC count was 85.8 x 10(9)/l and bone marrow aspiration revealed a nucleated cell count of 112.0 x 10(9)/l with 70.8% atypical lymphocytes. On the 3rd hospital day, the WBC count decreased by about 1/5 without chemotherapy and partial remission was obtained. But about 3 weeks later, the WBC count increased again and she died. Based on surface marker analysis, genotypic analysis and autopsy, we diagnosed infant leukemia suggestive of natural killer (NK) cell precursor origin.

Vasculo-protective effects of insulin sensitizing agent pioglitazone in neointimal thickening and hypertensive vascular hypertrophy.

A novel insulin sensitizing agent, thiazolidine, has been demonstrated to inhibit the growth of cultured vascular smooth muscle cells (VSMC) in vitro. This study was undertaken to examine the in vivo effects of the thiazolidine compound pioglitazone (PIO) on carotid neointimal thickening, after endothelial injury in Wistar rats and vascular hypertrophy in stroke-prone spontaneously hypertensive rats (SHR-SP/Izm). PIO treatment (3 mg/kg/day for 1 week prior to endothelial injury and 2 weeks postendothelial injury) remarkably decreased neointimal cross-sectional areas in treated animals (63.8 +/- 4.9 x 10(3) microm2) versus controls (196 +/- 7.6 x 10(3) microm2, P < 0.05). Bromodeoxyuridine uptake in the neointima, a marker of DNA synthesis, was also decreased after treatment compared with controls. In SHR-SP/Izm but not in Wistar rats, PIO treatment decreased blood pressure and plasma insulin levels. PIO treatment in SHR-SP/Izm (3 mg/kg/day from 4 weeks of age for 7 weeks) significantly decreased the medial wall thickness of the mesenteric artery (10.4 +/- 1.2 x 10(3) microm2 versus control, 21.2 +/- 2.4 x 10(3) microm2, P < 0.05). In addition, PIO treatment significantly decreased the expression of EIIIA fibronectin both in the carotid neointima of Wistar rats and the media of the mesenteric artery in SHR-SP/Izm compared with their respective controls (P < 0.05). These results suggest that PIO has vasculo-protective effects in both acute and chronic vascular injury in vivo through inhibition of VSMC proliferation.

Role of endogenous nitric oxide in the nitric oxide donor-induced plasma extravasation of mouse skin.

The role of endogenous nitric oxide (NO) and prostanoids in the increase in microvascular permeability induced by NO donors was investigated in the mouse skin by a dye leakage method. Subcutaneous (s.c.) injection of 1-hydroxy-2-oxo-3-(3-aminopropyl)-3-isopropyl-1-triazene (NOC 5), 1-hydroxy-2-oxo-3,3-bis(2-aminoethyl)-1-triazene (NOC 18) and sodium nitroprusside dose-dependently increased local dye leakage. While indomethacin inhibited the dye leakage elicited by these NO donors, N(G)-nitro-L-arginine methyl ester (L-NAME) inhibited the effect of NOC 5 and NOC 18 but not of sodium nitroprusside. These results suggest that endogenous NO, in addition to the prostanoid biosynthesis, is involved in the dermal microvascular permeability increase induced by the NOC series NO donors.

Protein kinase C mediates tumor necrosis factor-alpha-induced inhibition of obese gene expression and leptin secretion in brown adipocytes.

Previously we showed that tumor necrosis factor-alpha (TNF-alpha) inhibits lipoprotein lipase (LPL) activity and its gene expression, an early marker of adipocyte differentiation, in cultured brown adipocytes. To know whether TNF-alpha also affects late events in brown adipocyte maturation, we examined the effect of TNF-alpha on obese gene expression and leptin secretion in mouse brown adipocytes differentiated in culture. TNF-alpha caused a concentration-dependent decrease in leptin accumulation in culture medium and leptin mRNA amount in brown adipocytes which constitutively express the ob gene. Time-course study showed that TNF-alpha significantly suppressed leptin secretion during incubation for 16, 24 and 48 h. Since some effect of TNF-alpha is mediated by activation of protein kinase C (PKC), the role of PKC in TNF-alpha-induced downregulation of ob gene expression and leptin secretion was studied. The suppressive effect of TNF-alpha on both ob gene expression and leptin secretion was blocked by PKC inhibitors such as bisindolylmaleimide I (BIM) and 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7). Incubation of brown adipocytes with TNF-alpha (20 ng/ml, 15 min) caused a rapid shift of PKC activity from the cytosolic to the membrane fraction, suggesting an activation of PKC by TNF-alpha in brown adipocytes. This effect of TNF-alpha was blocked by a selective PKC inhibitor, BIM. These results suggest that TNF-alpha promotes dedifferentiation of the brown adipocytes as evidenced by a downregulation in ob gene expression and leptin secretion via PKC-dependent mechanisms.

A shear-induced in vitro platelet function test can assess clinically relevant anti-thrombotic effects.

Morphological features of haemostatic plugs formed in vitro under high shear forces were investigated. Electron microscopy confirmed the relevance of such haemostatic plug to a platelet-rich arterial thrombus, which is formed in vivo . In rat blood samples, the effects of anticoagulants and various antiplatelet agents on platelet reactivity (rate of haemostatic plug formation) and subsequent coagulation of the flowing blood were investigated. Haemostasis did not occur in citrated blood, and heparin greatly inhibited the shear-induced platelet reaction. Aspirin (1 mM), a thromboxane A(2) receptor antagonist (5 microM), a stable prostacyclin (0.55 nM), a stable prostaglandin E(1) (141 nM) and a phosphodiesterase inhibitor (100 microM) were tested. All these agents exerted significant inhibitory effect on shear-induced platelet reaction, including the inhibition of the very first phase of platelet plug formation, due to aggregation of shear-activated platelets. Except for the phosphodiesterase inhibitor, which prolonged clotting time, none of the above agents affected dynamic coagulation. These results suggest that the employed in vitro shear-induced thrombosis/haemostasis test can reveal in vivo the antithrombotic effect of various agents independently of their mechanism of action.