Crocetin, a carotenoid from Gardenia jasminoides Ellis, protects against hypertension and cerebral thrombogenesis in stroke-prone spontaneously hypertensive rats.

Crocetin is a natural carotenoid dicarboxylic acid that is found in the fruit of Gardenia jasminoides Ellis (Cape Jasmine) and in the stamen and pistil of Crocus sativus L. (saffron). It is used worldwide as an important spice, food colorant, and herbal medicine. In the current investigation, we have examined the cardiovascular effects of crocetin using stroke-prone spontaneously hypertensive rats (SHRSPs). Male SHRSPs (6 weeks old) were classified into three groups: a control group and two crocetin groups (25 and 50 mg/kg/day). The animals were given crocetin for 3 weeks. Body weights in each group were not significantly different during the treatment period, but the increase in systolic blood pressures observed with age was significantly moderated by crocetin. Thrombogenesis, assessed using a He-Ne laser technique in pial vessels, was significantly decreased. Antioxidant activity, assessed by measuring urinary 8-hydroxy-2'-deoxyguanosine levels, together with urinary nitric oxide (NO) metabolite levels, was increased significantly after treatment. Acetylcholine-induced vasodilation was measured using the aorta and indicated that endothelial function was significantly improved by crocetin. These results strongly suggest that the antihypertensive and antithrombotic effects of crocetin were related to an increase in bioavailable NO, possibly mediated by decreased inactivation of NO by reactive oxygen species.

Preventive effects of hesperidin, glucosyl hesperidin and naringin on hypertension and cerebral thrombosis in stroke-prone spontaneously hypertensive rats.

The effects of hesperidin, glucosyl hesperidin (G-hesperidin), a water-soluble derivative of hesperidin, and naringin on blood pressure and cerebral thrombosis were investigated using stroke-prone spontaneously hypertensive rats (SHRSP). Hesperidin, G-hesperidin and naringin were mixed with diet and fed to the animals for 4 weeks. No effect was evident on body weight, but the supplements significantly suppressed the age related increase in blood pressure. Thrombotic tendency, as assessed using a He-Ne laser technique in the cerebral blood vessels, was significantly decreased in the treated animals compared with the control animals. Measurements of 8-hydroxy-2'-deoxyguanosine (8-OHdG) demonstrated that the supplements had strong antioxidant activity. Furthermore, these supplements significantly increased the production of nitric oxide (NO) metabolites in urine measured with Griess reagent. Vasodilation induced by acetylcholine-mediated NO production in the endothelium was assessed using thoracic aortic ring preparations and indicated that endothelial function was significantly improved by the administration of these supplements. These findings suggest that the strong antioxidant properties of hesperidin, G-hesperidin and naringin could modulate the inactivation of NO and protect endothelial function from reactive oxygen species (ROS). In this manner, the flavonoids could contribute beneficial effects on the mechanisms of hypertension and thrombosis by increasing the bioavailability of NO.

Identification of a thrombin-interactive site within the FVIII A2 domain that is responsible for the cleavage at Arg372.

FVIII is activated by cleavage at Arg(372), Arg(740), and Arg(1689) by thrombin. This study showed that an anti-A2 monoclonal antibody, with a specific epitope for residues 484-509, and anti-FVIII inhibitor alloantibodies with similar A2 epitopes, inhibited thrombin-catalyzed FVIII activation. Sodium dodecyl sulphate polyacrylamide gel electrophoresis analysis showed that cleavage at Arg(372) but not at Arg(740) occurred at approximately fourfold decreased rate in the presence of anti-A2 antibody. Peptide 484-509 also inhibited co-factor activation, consistent with inhibition of cleavage at Arg(372). Direct binding studies using active-site modified thrombin showed that a 484-509 peptide as well as the anti-A2 antibodies blocked the A2-thrombin binding. Furthermore, covalent cross-linking was observed between the 484-509 peptide and thrombin following reaction with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide. Mutant A2 molecules in which the clustered basic residues in this sequence were converted to alanine were used to assess the binding reactions in a surface plasmon resonance-based assay. Mutants R484A, R489A, R490A, H497A and K499A possessed two to fivefold lower affinity than wild-type A2. These findings demonstrate that clustered basic residues within the 484-509 region of the A2 domain play a part of key role in thrombin-binding, which is responsible for thrombin-catalyzed FVIII activation by cleavage at Arg(372).

Relationship between the binding sites for von Willebrand factor, phospholipid, and human factor VIII C2 inhibitor alloantibodies within the factor VIII C2 domain.

Some factor VIII (FVIII) inhibitor alloantibodies block FVIII binding to von Willebrand factor (VWF) and phospholipid (PL) and recognize a C2 domain epitope that overlaps both binding sites. We previously showed that FVIII peptide 2315-2330 neutralized FVIII inhibitors and that Cys2326 and Glu2327 contributed to the maximum neutralizing effect. In the present study, we investigated the relationship between the essential binding sites for VWF, PL, and anti-C2 inhibitors by means of competitive-inhibition assays with overlapping synthetic peptides that span the C terminus of the C2 domain (residues 2288-2332). We identified 2 peptides (residues 2303-2317 and 2315-2330) that specifically blocked FVIII binding to VWF or PL by approximately 80% (50%-inhibitory concentration [IC50], 9.0 microM) and 95% (IC50, 0.12 microM), respectively. To examine in detail the residues responsible for PL binding, we prepared mutants of peptide 2315-2330 in which we sequentially substituted each residue with Gly. Two residues, Ile2317 and Met2321, were shown to be essential for PL binding. Their substitution with Gly reduced the inhibitory effect by >90%. The data suggest that the binding sites for VWF, PL, and anti-C2 inhibitors in the C2 domain are in very close proximity but are not identical.

Heparin-induced inhibitory effects of a prothrombin complex concentrate on global tests of haemostasis.

Prothrombin complex concentrates (PCC) have been used as bypassing agents for the treatment of haemophilia A patients with inhibitor as well as for replacement therapy in congenital and acquired deficiencies of vitamin-K-dependent clotting factors. The efficacy of PCC is variable, however, especially during long-term and high-dose use, and all currently available products of this nature contain heparin. We have examined the haemostatic properties of PCC using reconstituted whole blood made by mixing coagulation-factor-deficient plasma and washed blood cells. In rotation thromboelastometry (ROTEM), the recommended therapeutic dose of Proplex ST corrected the abnormal patterns. At higher concentrations, however, the ROTEM patterns regressed. In addition, specific assays of coagulation factors appeared unreliable in the presence of 2.5 U/ml Proplex ST; the abnormalities were corrected when protamine sulfate was added. The findings suggest that the presence of heparin in PCC might have a greater effect on global haemostasis. Careful attention to the anticoagulant effect as well as thrombogenicity of PCC is required. Monitoring therapy using such as ROTEM analysis could be highly informative.

C-reactive protein-induced in vitro endothelial cell activation is an artefact caused by azide and lipopolysaccharide.

OBJECTIVE: C-reactive protein (CRP) has been proposed to be an independent risk factor for cardiovascular disease. In vitro studies investigating the mechanism behind this have used purified commercial CRP (cCRP) and endothelial cells. We investigated the role of contaminants in cCRP preparations. METHODS AND RESULTS: Human umbilical vein endothelial cells and the human endothelial cell line EA.hy926 were incubated with Escherichia coli-derived cCRP, in-house-generated azide-free recombinant, and ascites-purified CRP, azide, or lipopolysaccharide (LPS) equivalent to the concentration present in cCRP preparations. Cells were investigated for change in cell proliferation, morphology, apoptosis, and expression of endothelial NO synthase and intercellular adhesion molecule-1. Cell supernatants were assessed for monocyte chemoattractant protein-1 (MCP-1), interleukin-8, von Willebrand factor secretion, and pH change. Only cCRP was able to induce all activation events analyzed; however, this ability was lost on extensive dialysis, suggesting that low molecular weight contaminants were responsible for these events. Indeed, the effects of cCRP were mirrored by azide or LPS. CONCLUSIONS: We investigated a wide range of effects on endothelial cells ascribed to CRP; however, azide and LPS, but never CRP itself, were responsible for the cell activation events. We conclude that CRP, per se, does not activate endothelial cells.

Enhancement of endogenous plasminogen activator-induced thrombolysis by argatroban and APC and its control by TAFI, measured in an arterial thrombolysis model in vivo using rat mesenteric arterioles.

Recent in vitro studies have demonstrated that thrombin inhibits fibrinolysis through thrombin-activatable fibrinolysis inhibitor (TAFI, plasma procarboxypeptidase B). We have recently shown that endogenous fibrinolysis in vivo is enhanced by activated protein C (APC) and the selective thrombin inhibitor, argatroban. The aim of the present study was to examine the role of TAFI in these fibrinolytic mechanisms in vivo using purified porcine pancreatic carboxypeptidase B (PPCPB) and a specific TAFIa inhibitor, potato tuber carboxypeptidase B inhibitor (PTCI) in a newly established arterial thrombolysis model. Non-occlusive, mural, platelet-rich thrombi were formed by helium-neon laser irradiation in rat mesenteric arterioles and thrombus size was measured by computerised image analysis. We confirmed that endogenous thrombolysis was enhanced by argatroban (2.0 mg/4 ml/kg/h) or APC (1.62 mg/ 2.31 ml/kg). PTCI (5.0 mg/2 ml/kg) also accelerated endogenous thrombolysis. PPCPB (3.5 mg/2 ml/kg) inhibited thrombolysis in the absence and presence of argatroban or APC. PTCI tended to further promote APC-induced thrombolysis but the differences did not reach statistical significance. The present findings were in keeping with the results of earlier studies and demonstrated that arterial, platelet-rich thrombi in vivo are degraded by naturally generated plasminogen activators. TAFI may play a significant role in the control of these mechanisms.

Factor VIIa induced release of von Willebrand factor from human umbilical vein endothelial cells by a tyrosine kinase dependent pathway.

The interaction of FVIIa with surface-bound tissue factor (TF) induces various cellular changes including cytosolic Ca2+ signals. The release of von Willebrand factor (VWF) from endothelial cell stores may be triggered by an elevation in cytosolic free Ca2+, therefore we investigated the effect of rFVIIa on the release of VWF from human umbilical vein endothelial cells (HUVEC). We show here that rFVIIa induces the release of VWF from HUVEC with or without prestimulation with lipopolysaccharide (LPS). The effect of rFVIIa was dose dependent. However, the release of VWF by HUVEC in response to rFVIIa was significantly greater with LPS prestimulation (3.18 times control) than without LPS prestimulation (1.45 times control) (p < 0.001). Cytosolic Ca2+ signals were detectable only after LPS prestimulation of HUVEC and these were small compared to those elicited by thrombin. No effect on rFVIIa induced release of VWF was seen in the presence of hirudin, site inactivated rFVIIa or the protein kinase C (PKC) inhibitor staurosporine. However, a tyrosine kinase inhibitor genistein, inhibited the rFVIIa induced release of VWF. These data show that release of VWF can occur without involvement of the cytosolic Ca2+/ PKC pathway. FVIIa induced VWF release from endothelial cells may have in vivo significance at sites of TF expression.

Store-mediated calcium entry in the regulation of phosphatidylserine exposure in blood cells from Scott patients.

Scott syndrome is a bleeding disorder, characterized by impaired surface exposure of procoagulant phosphatidylserine (PS) on platelets and other blood cells, following activation with Ca(2+)-elevating agents. Since store-mediated Ca(2+) entry (SMCE) forms an important part of the Ca(2+) response in various blood cells, it has been proposed that deficiencies in Ca(2+) entry may relate to the impaired PS exposure in the Scott syndrome. Here, we have tested this hypothesis by investigating the relationship between Ca(2+) fluxes and PS exposure in platelets as well as B-lymphoblasts derived from the original Scott patient (M.S.), a newly identified Welsh patient (V.W.) with similar bleeding symptoms, and two control subjects. Procoagulant activity of V.W. platelets in suspension, measured after stimulation with collagen/thrombin or Ca(2+)-ionophore, ionomycin, resulted in 52% or 17%, respectively, compared to that of correspondingly activated control platelets. Procoagulant activity of V.W. erythrocytes treated with Ca(2+)-ionophore resulted in less than 6% of the activity of control erythrocytes. Single-cell Ca(2+) responses of M.S. and V.W. platelets, adhering to collagen, were similar to those of platelets from control subjects, while PS exposure was reduced to 7% and 15%, respectively, compared to controls. Stimulation of non-apoptotic B-lymphoblasts derived from both patients and controls with Ca(2+)-ionophore or agents causing Ca(2+) mobilization and SMCE, resulted in similar Ca(2+) responses. However, in lymphoblasts from M.S. and V.W. Ca(2+)-induced PS exposure was reduced to 7% and 13% of the control lymphoblasts, respectively. We conclude that i. patient V.W. is a new case of Scott syndrome, ii. Ca(2+) entry in the platelets and lymphoblasts from both Scott patients is normal, and iii. elevated [Ca(2+)](i) as caused by SMCE is not sufficient to trigger PS exposure.

Suppression of argatroban-induced endogenous thrombolysis by PKSI-527, and antibodies to TPA and UPA, evaluated in a rat arterial thrombolysis model.

We have previously confirmed, using a rat mesenteric arteriole thrombolysis model, that thrombin inhibition induces endogenous thrombolysis in vivo. In addition, we have shown that thrombin-activatable fibrinolysis inhibitor (TAFI) plays a role in the down regulation of endogenous thrombolysis. However, the mechanism of endogenous thrombolysis or spontaneous plasmin generation in vivo remains unclear. It has been shown in an in vitro system that plasma kallikrein activates pro-urokinase (pro uPA) and/or plasminogen, resulting in plasmin generation. These findings suggest that spontaneous fibrinolysis might be mediated by tPA and plasma kallikrein-dependent uPA. The aim of the present study was to examine whether these mechanisms play a dominant role in endogenous thrombolysis in vivo, using our rat mesenteric arterial thrombolysis model. Argatroban infusion enhanced endogenous thrombolysis. PKSI-527, anti uPA and anti tPA IgGs suppressed argatroban-induced thrombolysis. Also, the antibody IgG preparations suppressed endogenous thrombolysis in the absence of argatroban. In the presence of PKSI-527, anti tPA IgG was more effective than anti uPA IgG in suppressing argatroban-induced thrombolysis. The results suggested that both tPA and plasma kallikrein-mediated uPA activation and tPA release contribute to endogenous fibrinolytic or thrombolytic mechanisms.

Human factor VIII inhibitor alloantibodies with a C2 epitope inhibit factor Xa-catalyzed factor VIII activation: a new anti-factor VIII inhibitory mechanism.

Factor VIII (FVIII) inhibitor alloantibodies react with the A2, C2, or A3-CI domains of FVIII and inactivate FVIII activity. We recently demonstrated that an anti-C2 monoclonal antibody with a Val2248-Gly2285 epitope, inhibited factor Xa (FXa)-catalyzed FVIII activation, and that a FXa binding site for FVIII was located within residues Thr2253-Gln2270. In this study, we investigated whether anti-C2 alloantibodies inhibit FXa-catalyzed FVIII activation. Anti-C2 alloantibodies from four patients inhibited FVIII activation by FXa in one-stage clotting assay. Furthermore, analysis by SDS-PAGE showed that all alloantibodies inhibited FVIII proteolytic cleavage by FXa independently of phospholipid. To confirm direct inhibition of FVIII and FXa interaction, we examined the effect of alloantibodies on FVIII binding to anhydro-FXa, a catalytically inactive FXa, in ELISA. All alloantibodies and C2-affinity purified F(ab)'2 preparations inhibited FVIII binding to anhydro-FXa dose-dependently. Our results revealed a new inhibitory mechanism of FVIII, mediated by inhibition of FXa in the presence of anti-C2 alloantibodies.

Enhanced endogenous thrombolysis induced by a specific factor Xa inhibitor, DX-9065a, evaluated in a rat arterial thrombolysis model in vivo.

We have previously established an animal model to investigate mechanisms of arterial thrombolysis in vivo and have demonstrated that endogenous thrombolysis, mediated by thrombin-activatable fibrinolysis inhibitor, is enhanced by administration of specific thrombin inhibitors. The aim of the present study was to evaluate the effects of a synthetic and specific factor Xa inhibitor, DX-9065a, on endogenous fibrinolysis. Mural thrombi were formed in rat mesenteric arterioles by helium-neon laser irradiation in the presence of Evans blue. Thrombolysis was continuously monitored by video microscopy and was quantified using image analysis software. Oral and intravenous administration of DX-9065a enhanced endogenous thrombolysis in vivo. The mechanisms require additional investigation using other experimental systems, but nevertheless, the present results extended our previous findings and further suggested that the enhanced fibrinolysis might be due to depressed activity thrombin-activatable fibrinolysis inhibitor. The synthetic factor Xa inhibitor could provide the basis for a useful thrombolytic agent.

Short-term high-dose folic acid does not alter markers of endothelial cell damage in patients with coronary heart disease.

BACKGROUND AND OBJECTIVE: Endothelial dysfunction is an early, pre-clinical manifestation of coronary heart disease and is associated with increased plasma levels of von Willebrand factor (vWF), soluble E-selectin, and thrombomodulin, markers of endothelial cell damage/activation and reduced nitric oxide bioavailability. Homocysteine is associated with an increased risk of cardiovascular disease and mortality. High-dose folic acid treatment lowers plasma homocysteine by 25% and improves nitric oxide bioavailability; however, the effects on other indices of endothelial cell activation/damage has not been examined in patients with coronary heart disease and normal renal function. DESIGN AND METHODS: In a randomised, double-blind, cross-over study in 50 patients with coronary heart disease and normal serum creatinine, folic acid (5 mg/daily) was administered for 6 weeks and blood was analysed for von Willebrand factor, soluble E-selectin, and thrombomodulin. Endothelial nitric oxide bioavailability was assessed by flow-mediated dilatation. RESULTS: Plasma folate levels increased (9.1+/-3.4 vs. 310+/-235 microg/l; p<0.001) and nitric oxide bioavailability improved (47+/-35 vs. 110+/-43 microm; p<0.001) following active treatment. However, markers of endothelial cell injury were not significantly influenced (von Willebrand factor 118+/-33 vs. 119+/-34%; E-selectin 52+/-17 vs. 51+/-16 microg/l; thrombomodulin 3.94+/-1.81 vs. 3.94+/-1.51 microg/l; p=NS comparing post-placebo with post-folate). No correlation was observed between improvement in flow-mediated dilatation and change in endothelial marker proteins. INTERPRETATION AND CONCLUSION: These data suggest that endothelial markers are not useful surrogates of endothelial nitric oxide bioavailability in coronary heart disease and may be a less sensitive marker of endothelial function than nitric oxide.

Effects of voluntary exercise on cerebral thrombosis and endothelial function in spontaneously hypertensive rats (SHRSP/Izm).

1. Effects of voluntary exercise on blood pressures, oxidative stress, urinary nitric oxide (NO) level and expression of endothelial NO synthase (eNOS) mRNA were studied in stroke-prone spontaneously hypertensive rats (SHRSP/Izm). 2. SHRSP at the age of 6 weeks were divided into four groups: (i) the control group, sedentary group; (ii) the L-NAME group, which was the sedentary control group given L-NAME (5 mg/kg per day) in drinking water; (iii) the exercise group, which was allowed to run voluntarily on running wheel attached to the metal cages; and (iv) the exercise plus L-NAME group which was loaded exercise and given L-NAME solution for 3 weeks. 3. The bodyweight and systolic pressure of rats were increased with age and the bodyweight of the rats in an exercise plus L-NAME group was less than control but systolic pressure in the exercise group were significantly lower than control. 4. Thrombotic tendency assessed by He-Ne laser method in an exercise group was significantly decreased compared with the rest of the groups. 5. Urinary nitrite/nitrate level was significantly increased in the exercise group compared with before (6 weeks) and after exercise (9 weeks), but there were no significant differences in the rest of groups. 6. eNOS mRNA expression of aorta in the exercise group measured after exercise was significantly higher than the other groups. 7. Urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG) level after exercise was significantly decreased in the exercise group compared with before exercise. 8. These results suggested that voluntary exercise decreased thrombotic tendency by increasing NO level through enhanced expression of eNOS mRNA and antioxidative effects.

Hemostatic treatment using factor VIII concentrates for neutralizing high-responding inhibitors prior to CVAD insertion for immune-tolerance induction therapy.

We have utilized high-dose factor VIII (FVIII) concentrates in 4 hemophilia A patients with inhibitors prior to surgery for the insertion of a central venous access device. In total, 3 patients out of 4 had high responding inhibitors. Dosing algorithms for this type of therapy have not been previously validated and established. We devised an effective formula to calculate the initial dose required to neutralize the inhibitors, although some of the patients demonstrated a lower recovery of FVIII than expected. An anamnestic inhibitor response was evident in 3 cases, but overall our strategy provided a reliable hemostatic effect for at least 4 days after surgery. In addition, our protocol appeared to be more cost-effective than FVIII bypass therapy. The financial saving in 1 case for the initial 3 days was estimated to be approximately US$49 122. Our results demonstrated that high-dose FVIII therapy provided clinically effective and economically viable results even in high responders.

Astaxanthin inhibits thrombosis in cerebral vessels of stroke-prone spontaneously hypertensive rats.

It is known that vitamin E and some carotenoids have antioxidant activities that alleviate endothelial dysfunction and play a protective role against cardiovascular disease. The current study was designed to examine the hypothesis that astaxanthin, a red pigment carotenoid found in salmonid and crustacean aquaculture, protects stroke-prone spontaneously hypertensive rats (SHRSP) from vascular oxidative damage, hypertension, and cerebral thrombosis. Male 6-week-old SHRSP were classified into 4 groups: a control group, 2 astaxanthin groups, and a vitamin E group. The treated animals were given either astaxanthin or vitamin E for 3 weeks. Body weights in each group were not significantly different from control group during the treatment period, but the usual increase in systolic blood pressure in SHRSP observed with age was significantly suppressed by treatment. Thrombogenesis, assessed using a helium-neon (He-Ne) laser technique in pial blood vessels, together with antioxidant activity, assessed by measuring urinary 8-OHdG levels, were significantly moderated. Urinary nitric oxide (NO) metabolites were increased after treatment. These results supported our hypothesis and strongly suggested that the antithrombotic and antihypertensive effects of astaxanthin or vitamin E may be related to an increase in bioavailable NO, possibly mediated by decreased inactivation of NO by reactive oxygen species.

Impaired endothelial function may be due to decreased aortic tetrahydrobiopterin, assessed by a new flow-mediated vasodilation in vivo in hypercholesterolemic/atherogenic mice.

Tetrahydrobiopterin (BH4) is an important cofactor for endothelial nitric oxide synthase activity. The relationship between endothelial function in vivo and aortic BH4 level is not fully understood, however. In the present study, we aimed to clarify whether reduction of aortic BH4 levels contributes to endothelial dysfunction in vivo using spontaneously hyperlipidemic mice. To estimate endothelial function in vivo and in real-time state, we developed a flow-mediated vasodilation (FMV) method in mice, which measured changes in the diameter of the femoral artery in response to increased blood flow. C57BL/6 mice and apoE/low-density lipoprotein receptor double knock-out mice were fed a low-fat diet (LFD) or a high-fat diet (HFD) for 12 weeks from 6 weeks of age. HFD feeding impaired FMV in double knock-out mice, but not in C57BL/6 mice. Furthermore, HFD feeding reduced plasma NOx concentration and aortic BH4 level in double knock-out mice. Conversely, exogenous injection of BH4 (2 mg/kg) markedly increased aortic BH4 levels and restored endothelial function. In conclusion, we demonstrated that HFD feeding impaired nitric oxide-mediated endothelial function and reduced BH4 level in vivo, and that acute augmentation of aortic BH4 levels improved endothelial function. These findings indicate that BH4 is a critical determinant of nitric oxide-mediated endothelial function in hypercholesterolemia.

The antithrombotic effects of carrot filtrates in rats and mice.

The prevention of arterial thrombotic disease has a high priority in developed countries. An inappropriate diet is known to enhance the risk for acute thrombotic events and a regular diet with proven antithrombotic effects might be a beneficial way to prevent disease. The present study was undertaken as a part of a series of investigations to examine vegetables and fruits for antithrombotic activity. A shear-induced in vitro platelet reactivity/thrombolysis method [Global Thrombosis Test, formerly Gorog Thrombosis Test] was used to screen 15 different varieties of carrot for antiplatelet and thrombolytic activities. Effective varieties were further investigated using an in vivo, laser-induced thrombosis model in mice. The different carrot varieties demonstrated a variable effect on thrombosis in vitro and in vivo. In particular, a variety designated SAKATA-0421 exerted an antithrombotic effect in vivo independent from heat treatment of the filtrate at 100 degrees C for 10 min. There was no significant correlation between antithrombotic activity and the levels of polyphenolics and any other biochemical parameter, including antioxidant activity, alpha-carotene and beta-carotene, alpha-tocopherol and ascorbic acid. Different varieties of carrot demonstrated a range of antithrombotic and prothrombotic activities. After oral intake, the particular heat resistant variety (SAKATA-0421) showed antithrombotic effect in vivo possibly due to antiplatelet reactivity and/or spontaneous thrombolytic activity. The present study added a new variety to the list of antithrombotic fruits and vegetables.

Reversible inhibition of the platelet procoagulant response through manipulation of the Gardos channel.

The platelet procoagulant response requires a sustained elevation of the intracellular Ca2+ concentration, [Ca2+]i, causing exposure of phosphatidylserine (PS) at the outer surface of the plasma membrane. An increased [Ca2+]i also activates Ca2+-dependent K+ channels. Here, we investigated the contribution of the efflux of K+ ions on the platelet procoagulant response in collagen-thrombin-activated platelets using selective K+ channel blockers. The Gardos channel blockers clotrimazol, charybdotoxin, and quinine caused a similar decrease in prothrombinase activity as well as in the number of PS-exposing platelets detected by fluorescence-conjugated annexin A5. Apamin and iberiotoxin, inhibitors of other K+ channels, were without effect. Only clotrimazol showed a significant inhibition of the collagen-plus-thrombin-induced intracellular calcium response. Clotrimazol and charybdotoxin did not inhibit aggregation and release under the conditions used. Inhibition by Gardos channel blockers was reversed by valinomycin, a selective K+ ionophore. The impaired procoagulant response of platelets from a patient with Scott syndrome was partially restored by pretreatment with valinomycin, suggesting a possible defect of the Gardos channel in this syndrome. Collectively, these results provide evidence for the involvement of efflux of K+ ions through Ca2+-activated K+ channels in the procoagulant response of platelets, opening potential strategies for therapeutic interventions.