[Effect of acidic oligosaccharides on P-selectin of pulmonary hypertensive rats induced by monocrotaline].

Objective: To observe the effects of acidic oligosaccharides (AOS) on P-selectin levels in the serum and the pulmonary arteries of pulmonary hypertensive rats induced by monocrotaline. Methods: Sixty healthy adult male Sprague-Dawley rats were randomly divided into control group (n=10), model group (n=10), Alprostadil group (n=10), low-dose AOS group (AOS-L, n=10), medium-dose AOS group (AOS-M, n=10) and high-dose AOS group (AOS-H, n=10). The rat model of pulmonary arterial hypertension was made by a single intraperitoneal injection of monocrotaline(60 mg/kg). Five weeks after injection, pulmonary arterial (PA) acceleration time (PAT) and ejection time (ET) were measured by color Doppler ultrasound. Then, the Alprostadil group was treated by Alprostadil 5 µg·kg(-1)·d(-1)intraperitoneally. Acidic oligosaccharides was administered by intraperitoneal injection to rats in the AOS-L group(5 kg(-1)·d(-1)), AOS-M group (10 mg·kg(-1)·d(-1))and AOS-H group (20 mg·kg(-1)·d(-1)). Control group and model group were given normal saline instead. At the end of experiments, the death rate was recorded and PAT/ET was measured. We calculated the right ventricular hypertrophy index (RVHI) of all rats sacrificed under anesthesia. Precision-cut lung slices were stained with HE for observation of the structure of middle and small arteries. The expression level of P-selectin in serum and pulmonary arterial tissues were detected by ELISA and Western blot respectively. Results: AOS significantly increased the level of PAT/ET (P<0.01), and attenuated RVHI (P<0.01). AOS significantly improved intima-media proliferation in small-to medium-sized pulmonary arteries, and attenuated perivascular inflammation. AOS and Alprostadil significantly down-regulated the protein expression of P-selectin in serum and pulmonary arteries (P<0.01). Conclusion: In this rat model of monocrotaline-induced pulmonary hypertension, AOS decreased the expressions of P-selectin in serum and pulmonary arteries in a dose-dependent manner.

Short fungal fractions of ß-1,3 glucans affect platelet activation.

Platelets are capable of binding, aggregating, and internalizing microorganisms, which enhances the elimination of pathogens from the blood. The yeast Candida albicans is a pathobiont causing life-threatening invasive infections. Its cell wall contains ß-1,3 glucans that are known to trigger a wide range of host cell activities and to circulate during infection. We studied the effect of ß-1,3 glucan fractions (BGFs) consisting of diglucosides (Glc2), tetraglucosides (Glc4), and pentaglucosides (Glc5) on human platelets, their mechanisms of action, and their possible impact on host defenses. The effect of BGFs on the coagulation process was determined by measuring thrombin generation. Platelets pretreated with BGFs were analyzed in terms of activation, receptor expression, aggregation, and adhesion to neutrophils and to C. albicans The results show that BGFs affected the endogenous thrombin potential in a concentration-dependent manner. For platelet activation, BGFs at a low concentration (2 µmol/l) reduced ATP release and prevented the phosphorylation of protein kinase C. BGFs diminished the expression of P-selectin and the activation of αIIbß3 BGFs decreased platelet aggregation and the interaction between thrombin-stimulated platelets and neutrophils, fibrinogen, and C. albicans GLc5 decreased ATP release and TGF-ß1 production in response to TLR4 upregulation in thrombin-stimulated platelets, but TLR4 blockage abolished the effect of BGFs on platelets. This study provides evidence that fungal pentaglucosides modulate platelet activity mediated via TLR4 stimulation and reduce platelet-neutrophil interaction.

Cocaine and specific cocaine metabolites induce von Willebrand factor release from endothelial cells in a tissue-specific manner.

OBJECTIVE: Cocaine use is associated with arterial thrombosis, including myocardial infarction and stroke. Cocaine use results in increased plasma von Willebrand factor (VWF), accelerated atherosclerosis, and platelet-rich arterial thrombi, suggesting that cocaine activates the endothelium, promoting platelet-VWF interactions. APPROACH AND RESULTS: Human umbilical vein endothelial cells, brain microvasculature endothelial cells, or coronary artery endothelial cells were treated with cocaine or metabolites benzoylecgonine, cocaethylene, norcocaine, or ecgonine methylester. Supernatant VWF concentration and multimer structure were measured, and platelet-VWF strings formed on the endothelial surface under flow were quantified. Cocaine, benzoylecgonine, and cocaethylene induced endothelial VWF release, with the 2 metabolites being more potent than the parent molecule. Brain microvasculature endothelial cells were more sensitive to cocaine and metabolites than were human umbilical vein endothelial cells or coronary artery endothelial cells. Coronary artery endothelial cells released VWF into the supernatant but did not form VWF-platelet strings. Intracellular cAMP concentration was not increased after treatment with cocaine or its metabolites. CONCLUSIONS: Both cocaine and metabolites benzoylecgonine and cocaethylene induced endothelial VWF secretion, possibly explaining thrombotic risk after cocaine ingestion. VWF secretion is likely to vary between vascular beds, with brain endothelial cells being particularly sensitive. These results suggest that clinical management of cocaine-induced ischemia may benefit from therapies aimed at disrupting the VWF-platelet interaction.

Dysregulated selectin expression and monocyte recruitment during ischemia-related vascular remodeling in diabetes mellitus.

OBJECTIVE: Diabetes mellitus (DM) is associated with impaired ischemia-related vascular remodeling and also dysregulation of the inflammatory response. We sought to determine whether impaired selectin-mediated monocyte recruitment in ischemic tissues contributes to blunted ischemia-mediated angiogenesis in DM. METHODS AND RESULTS: Contrast-enhanced ultrasound perfusion imaging and molecular imaging of endothelial P-selectin expression in the proximal hindlimb were performed at 1, 3, and 21 days after arterial ligation in wild-type and db/db mice. Ligation reduced muscle blood flow to ≈0.05 mL/minute per gram in both strains. Significant recovery of flow occurred only in wild-type mice (60%-65% of baseline flow). On molecular imaging, baseline P-selectin signal was 4-fold higher in db/db compared with wild-type mice (P<0.01) but increased minimally at day 1 after ischemia, whereas signal increased approximately 10-fold in wild-type mice (P<0.01). Immunohistology of the hindlimb skeletal muscle demonstrated severely reduced monocyte recruitment in db/db mice compared with wild-type mice. Local treatment with monocyte chemotactic protein-1 corrected the deficits in postischemic P-selectin expression and monocyte recruitment in db/db mice and led to greater recovery in blood flow. CONCLUSION: In DM, there is dysregulation of the selectin response to limb ischemia, which leads to impaired monocyte recruitment, which may be mechanistically related to reduced vascular remodeling in limb ischemia.

Identification of linoleic acid as an antithrombotic component of Wenxin Keli via selective inhibition of p-selectin-mediated platelet activation.

Atrial fibrillation significantly increases the risk of thromboembolism and stroke. Wenxin Keli (WXKL) is a widely used Chinese patent medicine against arrhythmia but if it has antithrombotic activity is unknown. Since platelet activation is a critical factor in thrombosis and the key target for many antithrombotic drugs, this study aims to demonstrate the antithrombotic efficacy of WXKL. In vitro platelet activation experiments showed that WXKL significantly inhibited platelet adhesion and aggregation. The potential active monomers in WXKL were screened by in silico prediction and in vitro platelet aggregation/adhesion assays. From WXKL chemical fractions and more than 40 monomers, linoleic acid (LA) was identified as the strongest antiplatelet compound. Oral administration of WXKL (1.2 g/kg/day) and LA (50 mg/kg/day) for 7 days significantly improved FeCl3-induced carotid thrombus formation in ICR mice without prolonging bleeding time. Flow cytometry showed that both WXKL and LA inhibited the release of p-selectin after platelet activation. ELISA showed that WXKL and LA also inhibited the expression of 6-Keto-PGF1α in plasma of mice with thrombus, but had no obvious effect on the expression of TXB2. WXKL inhibited platelet activation by broadly inhibiting the phosphorylation of protein kinase B (Akt), mitogen-activated protein kinases (MAPKs) and phospholipase C (PLC) ß3. In contrast, LA only inhibited the phosphorylation of PLCß3. In conclusion, WXKL and its active component LA showed good antiplatelet and antithrombotic efficacy in vivo and in vitro. Mechanistically, the multicomponent Chinese medicine WXKL acts on multiple targets in the platelet activation pathway whereas its active monomer linoleic acid acts specifically on phospholipase C ß3.

Reduced thrombin generation and soluble P-selectin after intravenous enoxaparin during PCI.

PURPOSE: The objective of our study was to identify changes in the coagulation and serum concentration of soluble P-selectin (sP-sel) after i.v. bolus of 0.75 mg/kg enoxaparin in a group of 33 patients during PCI. METHODS AND RESULTS: As compared to baseline, i.v. enoxaparin increased anti -Xa activity and FIIa inhibition together with APTT and thrombin time tests within 20 min, that persisted for 60 min. At 6 h, the results of all tests had returned to baseline. In contrast, the level of prothrombin fragments (F1 + 2) decreased persistingly for a period of 6 h (baseline 1.19 ± 0.42 nmol/l, after 20 min 1.03 ± 0.46 nmol/l, after 60 min 1.06 ± 0.43 nmol/l, after 6 h 0.95 ± 0.40 nmol/l, p < 0.001 vs. baseline for all values). In addition, i.v. enoxaparin decreased serum sP-sel level (baseline 111.80 ± 37.05 ng/ml, after 20 min 87.80 ± 33.17 ng/ml, after 60 min 86.45 ± 29.15 ng/ml, after 6 h 92.24 ± 31.34 ng/ml, p < 0.001 vs. baseline value for all). sP-sel level mildly correlated with both F Xa inhibition (r = -0.275, p < 0.05) and F1 + 2 level (r = 0.274, p < 0.05). CONCLUSION: Intravenous enoxaparin induced target F Xa inhibition (>0.6 IU/ml) for 60 min in 82% of study patients. During the 6 h of monitoring, a decrease of thrombin generation (F1 + 2) and sP-selectin levels were observed.

Anti-platelet effects of olive oil extract: in vitro functional and proteomic studies.

PURPOSE: Platelets play a key role in haemostasis and wound healing, contributing to formation of vascular plugs. They are also involved in formation of atherosclerosic plaques. Some traditional diets, like the Mediterranean diet, are associated with a lower risk of cardiovascular disease. Components in these diets may have anti-platelet functions contributing to their health benefits. METHODS: We studied the effects of alperujo extract, an olive oil production waste product containing the majority of polyphenols found in olive fruits, through measurement of effects on platelet aggregation and activation in isolated human platelets, and through identification of changes in the platelet proteome. RESULTS: Alperujo extract (40 mg/L) significantly decreased in vitro ADP- (p = 0.002) and TRAP- (p = 0.02) induced platelet activation as measured by the flow cytometry using the antibody for p-selectin (CD62p), but it did not affect the conformation of the fibrinogen receptor as measured by flow cytometry using the antibodies for anti-fibrinogen, CD42a and CD42b. Alperujo extract (100 mg/L) inhibited both collagen- and TRAP-induced platelet aggregation by 5% (p < 0.05), and a combination of hydroxytyrosol and 3,4-dihydroxyphenylglycol were, at least partly, responsible for this effect. Proteomic analysis identified nine proteins that were differentially regulated by the alperujo extract upon ADP-induced platelet aggregation. These proteins represent important mechanisms that may underlie the anti-platelet effects of this extract: regulation of platelet structure and aggregation, coagulation and apoptosis, and signalling by integrin αIIb/ß3. CONCLUSIONS: Alperujo extract may protect against platelet activation, platelet adhesion and possibly have anti-inflammatory properties.

[Effect of benzo(a)pyrene on platelet aggregation and expression of P-selectin].

OBJECTIVE: To investigate the effect of benzo(a)pyrene (BaP) on platelet aggregation and expression of P-selectin. METHODS: Blood samples were collected from healthy volunteers and the platelets was washed. Platelet aggregation was monitored by aggregometer and the expression of P-seletin was detected by whole blood flow cytometry. RESULT: BaP (10 µmol/L, 1 µmol/L and 0.1 µmol/L) did not induce platelet aggregation; however, preincubation with BaP (10 µmol/L) significantly enhanced ADP-induced platelet aggregation (P < 0.01) and platelet aggregation was (80 ± 10)%, while BaP-preincubation failed to enhance platelet aggregation under collagen and thrombin stimulation. Flow cytometry showed that preincubation with BaP increased ADP-induced, but not thrombin-induced P-selectin expression (P < 0.01). CONCLUSION: BaP can stimulate ADP-induced platelet aggregation and P-selectin expression, probably through the interaction with ADP-mediated signal pathway.

Activation of cannabinoid CB2 receptor ameliorates atherosclerosis associated with suppression of adhesion molecules.

OBJECTIVE: Adhesion molecules have been implicated in the development and progression of atherosclerosis. Cannabinoids have been reported to modulate the migration and adhesion molecules expression of various cell types. Here we examined the effects of WIN55212-2, a cannabinoid receptor 1 (CB1-R)/cannabinoid receptor 2 (CB2-R) agonist on the development of atherosclerotic lesions in apolipoprotein E-deficient (ApoE-/-) mice, which are vulnerable because of their high plasma cholesterol and triacylglycerol levels, focusing on the expression of endothelial adhesion molecules. METHODS AND RESULTS: In the aorta of ApoE-/- mice, WIN55212-2 significantly reduced aortic root plaque area. The mechanism for this seemed to be reduced infiltration of macrophages into the atherosclerotic plaque which was also associated with reduced expression of vascular cellular adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and P-selectin in the aorta. In vitro studies revealed reduced cell adhesion of a monocytic cell line (U937) to human umbilical vein endothelial cells after incubation with WIN55212-2. The reduction in macrophage adhesion also correlated with significant reductions in the expression of VCAM-1, ICAM-1, and P-selectin, indicating that reduced infiltration of macrophages in atherosclerotic plaques may occur as a result of the direct effect of WIN55212-2 on adhesion molecules in macrophages and endothelial cells. CONCLUSION: In conclusion, WIN55212-2 seems to have direct anti-atherosclerotic effects in an animal model of atherosclerosis. These effects were at least partly due to effects on the expression of VCAM-1, ICAM-1, and P-selectin, which led to reduced macrophage adhesion and infiltration. Furthermore, the protective effects completely blocked by the highly selective CB2 receptor antagonist AM630 suggest that these beneficial effects of WIN55212-2 may be mediated through the CB2 receptor.

Effect of erythropoietin on angiogenesis with the increased adhesion of platelets to the microvessels in the hind-limb ischemia model in mice.

Erythropoietin (EPO) has been shown to enhance angiogenesis, but its precise mechanisms of enhancement during ischemia are not fully elucidated. We examined the effect of EPO on blood flow recovery from acute hind-limb ischemia induced by ligation of the femoral artery in male C57Bl/6 mice. The density of microvessels with platelet adhesion in ischemic tissues was assessed by intravital microscopy. Treatment with EPO (100 and 1000 IU/kg, i.p.) restored blood flow in a dose-dependent manner and increased plasma levels of soluble-P-selectin, vascular endothelial growth factor (VEGF), and stromal cell-derived factor (SDF-1). Flow cytometric analysis revealed increased P-selectin expression on platelets in EPO-treated mice compared to PBS-treated mice. Intravital microscopic studies showed that EPO increased density of microvessels with platelet adhesion selectively in the ischemic tissues. Neutralizing antibody against P-selectin reduced the density of microvessels with platelet adhesion enhanced with EPO and impaired blood flow recovery with reductions in VEGF and SDF-1 levels. These results suggest that EPO administration enhances recovery from hind-limb ischemia, and platelet adhesion to the microvessels is a key event to enhance the angiogenesis in the ischemic tissues.

Influence of treatment with ultralow-dose aspirin on platelet aggregation as measured by whole blood impedance aggregometry and platelet P-selectin expression in clinically normal dogs.

OBJECTIVE: To evaluate the influence of treatment with ultralow-dose aspirin (ULDAsp) on platelet aggregation, P-selectin (CD62P) expression, and formation of platelet-leukocyte aggregates in clinically normal dogs. ANIMALS: 18 clinically normal dogs. PROCEDURES: Studies were conducted before and 24 hours after ULDAsp administration (0.5 mg/kg, PO, q 24 h, for 2 days). Whole blood impedance aggregometry for the assessment of platelet function was performed with sodium citrate-anticoagulated blood and aggregation agonists (ADP at 20, 10, and 5 µmol/L; collagen at 10, 5, and 2 µg/mL). Onset, maximum response, and rate of platelet aggregation were recorded. Flow cytometric assays were configured to detect thrombin-induced CD62P expression and platelet-leukocyte aggregates in EDTA-anticoagulated whole blood. Externalized platelet CD62P and constitutive CD61 (GPIIIa) were labeled with antibodies conjugated to phycoerythrin (PE) and fluorescein isothiocyanate (FITC), respectively. Red blood cell-lysed paraformaldehyde-fixed EDTA-anticoagulated whole blood was dual labeled with CD61-FITC and a panleukocyte antibody (CD18-PE) to characterize platelet-leukocyte aggregates. RESULTS: ULDAsp significantly delayed platelet aggregation onset with ADP at 20 µmol/L by 54% to 104%, attenuated maximum aggregation with various concentrations of ADP and collagen by ≥ 41%, and slowed aggregation rate with the highest ADP and collagen concentrations by ≥ 39%. Depending on the parameter tested, up to 30% of dogs failed to have an ULDAsp effect. Thrombin stimulation significantly increased CD62P expression in platelets and platelet-leukocyte aggregates, but ULDAsp did not alter basal or thrombin-stimulated CD62P expression. CONCLUSIONS AND CLINICAL RELEVANCE: ULDAsp treatment of clinically normal dogs impaired platelet aggregation in most dogs, but did not influence CD62P platelet membrane expression.

The effect of ellagic acid on platelet activation as measured by the quantification of P-selectin using flow cytometry.

The purpose of this study was to investigate the effects of ellagic acid on platelet expression via the cyclooxygenase (COX) pathway by examining its effects on platelet activation and comparing them with known COX inhibitors in male Sprague-Dawley rats. Ellagic acid is a major compound found in certain fruits and nuts. It has been attributed as having anti-inflammatory, free radical scavenging, and coagulation properties as well as effects on tumor genesis in multiple forms of cancer. We assessed the similarities of ellagic acid to known COX-2 specific and nonspecific COX inhibitors by examining their effects on platelet activation via use of P-selectin flow cytometry. Compared with the vehicle group, both the ellagic acid (P = .035) and the ketorolac (P = .038) groups demonstrated a significant decrease in platelet activation (P = .026). Furthermore, compared with all other groups, ellagic acid plus ketorolac group showed a significant decrease in platelet activation (P = .01). Our findings suggest that ellagic acid is likely a nonspecific COX inhibitor. It also suggests that combining ellagic acid with a known nonspecific COX inhibitor such as ketorolac may cause a significant decrease in platelet activity and an increase in blood loss.

Prophylactic P-selectin inhibition with PSI-421 promotes resolution of venous thrombosis without anticoagulation.

P-selectin inhibition has been evaluated as a therapeutic for prevention and treatment of venous thrombosis. In this study, a novel oral small-molecule inhibitor of P-selectin, PSI-421, was evaluated in a baboon model of stasis induced deep vein thrombosis (DVT). Experimental groups included i) primates receiving a single oral dose of 1 mg/kg PSI-421 two days prior and continued six days after thrombosis (n = 3); ii) primates receiving a single daily subcutaneous dose of 0.57 mg/kg enoxaparin sodium two days prior and continued six days post thrombosis (n = 3); and iii) primates receiving no treatment (n = 3). PSI-421 treated primates had greater percent vein reopening and less vein wall inflammation than the enoxaparin and controls at day 6. Microparticle tissue factor activity (MPTFA) was significantly lower in the animals receiving PSI-421 immediately after thrombosis (T+6 hours day 0) suggesting lower potential for thrombogenesis in these animals. PSI-421 also reduced soluble P-selectin levels versus controls at T+6 hours day 0, day 2 and 6. Experimental animals in any group showed no adverse effects on coagulation. This study is the first to demonstrate a reduction in MPTFA associated with vein reopening and reduced vein inflammation due to oral P-selectin inhibition in a baboon model of DVT.

Sodium/hydrogen exchange inhibition with cariporide reduces leukocyte adhesion via P-selectin suppression during inflammation.

BACKGROUND AND PURPOSE: The Na(+)/H(+) exchange (NHE) inhibitor cariporide is known to ameliorate ischaemia/reperfusion (I/R) injury by reduction of cytosolic Ca(2+) overload. Leukocyte activation and infiltration also mediates I/R injury but whether cariporide reduces I/R injury by affecting leukocyte activation is unknown. We studied the effect of cariporide on thrombin and I/R induced leukocyte activation and infiltration models and examined P-selectin expression as a potential mechanism for any identified effects. EXPERIMENTAL APPROACH: An in vivo rat mesenteric microcirculation microscopy model was used with stimulation by thrombin (0.5 micro ml(-1)) superfusion or ischaemia (by haemorrhagic shock for 60 min) and reperfusion (90 min). KEY RESULTS: Treatment with cariporide (10 mg kg(-1) i.v.) significantly reduced leukocyte rolling, adhesion and extravasation after thrombin exposure. Similarly, cariporide reduced leukocyte rolling (54+/-6.2 to 2.4+/-1.0 cells min(-1), P<0.01), adherence (6.3+/-1.9 to 1.2+/-0.4 cells 100 microm(-1), P<0.01) and extravasation (9.1+/-2.1 to 2.4+/-1.1 cells per 20 x 100 microm perivascular space, P<0.05), following haemorrhagic shock induced systemic ischaemia and reperfusion. The cell adhesion molecule P-selectin showed a profound decrease in endothelial expression following cariporide administration in both thrombin and I/R stimulated groups (35.4+/-3.2 vs 14.2+/-4.1% P-selectin positive cells per tissue section, P<0.01). CONCLUSIONS AND IMPLICATIONS: The NHE inhibitor cariporide is known to limit reperfusion injury by controlling Ca(2+) overload but these data are novel evidence for a vasculoprotective effect of NHE inhibition at all levels of leukocyte activation, an effect which is likely to be mediated at least in part by a reduction of P-selectin expression.

CD41-YFP mice allow in vivo labeling of megakaryocytic cells and reveal a subset of platelets hyperreactive to thrombin stimulation.

OBJECTIVE: Development of a mouse line permitting live imaging of cells expressing CD41/GpIIb as a means to study megakaryopoiesis. MATERIALS AND METHODS: The gene encoding yellow fluorescent protein (eyfp) was inserted by homologous recombination into embryonic stem cells at the start site of the gpIIb locus. A knockin mouse line, designated CD41-yellow fluorescent protein (YFP), was developed and was characterized by fluorescence microscopy and flow cytometry. Activity of YFP(+) platelets was determined by induction of P-selectin expression in response to thrombin stimulation. RESULTS: CD41-YFP mice contained YFP-labeled megakaryocytes and platelets, the proportions of which varied, depending on the genotype and individual animal, while lymphoid, myelomonocytic, and erythroid lineages were negative. In addition, a fraction of hematopoietic stem cells and intermediate progenitors expressed YFP at low levels. Crossing CD41-YFP mice with lysozyme green fluorescent protein and globin cyan fluorescent protein mice, followed by in vivo imaging of fetal liver, revealed megakaryocytic cells as a subset distinct from myeloid and erythroid cells. This experiment is also the first to show the distribution of three hematopoietic lineages in a minimally perturbed organ. Surprisingly, analysis of CD41-YFP platelets showed that the YFP(+) subset is more responsive to thrombin stimulation than the YFP(-) subset. Experiments aimed at determining the stability of the YFP(+) platelets showed that after lethal irradiation of CD41-YFP mice, the proportion of labeled platelets in the blood declines more rapidly than the bulk of the platelets. CONCLUSION: The newly developed mouse line should become useful not only for in vivo imaging experiments of megakaryocytes and platelets, but also for studies on platelet aging and function. Our irradiation experiments suggest that the YFP(+) platelets are enriched for newly made cells because YFP has a shorter half-life than platelets. Therefore, the finding that YFP(+) platelets are more responsive to thrombin stimulation raises the possibility that platelet activity decreases rapidly during physiological aging.

Blockade of p-selectin reduces neutrophil infiltration into the murine testis after ischemia-reperfusion-injury.

Germ cell specific apoptosis after ischemia-reperfusion (I/R) induced testicular injury is dependent on neutrophil recruitment to the testis. Intravascular adhesion molecules like the P- and E- selectins play an important role in this recruitment.The purpose of this study was to inhibit neutrophil recruitment in I/R induced testicular injury by using a function-blocking monoclonal anti-mouse P-selectin antibody. Adult mice were subjected to a 2 h period of testicular torsion (ischemia) followed by detorsion (reperfusion).Ten minutes after the onset of reperfusion, mice received either 100 microg of a function-blocking monoclonal P-selectin antibody (FBMAB group) or isotype-matched control antibody (IMCA group). Separate groups of mice underwent sham-operation (SO group) or received 500 ng of TNFalpha (IF group) to induce inflammation. Mice were sacrificed 24 h after reperfusion and testiscular interstitial cells were isolated and analyzed for the presence of neutrophils by means of flow cytometry. The function-blocking monoclonal P-selectin antibody reduced neutrophil recruitment in I/R induced testicular injury significantly (FBMAB group as compared to the IMCA group 26 +/- 4 vs. 52 +/- 10% Gr-1 +CD11 b+ of total leucocytes; P < 0.001). Therefore, blocking P-selectin may be therapeutically beneficial to protect postischemic testis.

Correlations of inhaled NO with the cTnI levels and the plasma clotting factor in rabbits with acute massive pulmonary embolism.

PURPOSE: To investigate the correlation of inhaled nitric oxide (NO) on plasma levels of cardiac troponin I (cTnI) and von Willebrand factor (vWF), glycoprotein (GP) IIb/IIIa, granule membrane protein 140 (GMP-140) in rabbits with acute massive pulmonary embolism (PE). METHODS: Thirty apanese white rabbits were divided into 3 groups, thrombus were injected in model group (n = 10), NO were inhalated for 24 h after massive PE in NO group (n = 10), saline were injected in control group (n = 10). The concentrations of vWF, GP IIb/IIIa, GMP-140 and cTnI were tested at 4, 8, 12, 16, 20, and 24 h, Correlation analyses were conducted between cTnI and vWF, GP IIb/IIIa, and GMP-140 by Pearson's correlation. RESULTS: The concentration of cTnI and vWF, GP IIb/IIIa, and GMP-140 was increased in the model group, compared to control group. In the inhaled group, the concentrations of cTnI, vWF, GP IIb/IIIa, and GMP-140 were reduced compared to model group. There was a positive correlation between cTnI and vWF, GP IIb/IIIa, and GMP-140. CONCLUSION: Inhaled nitric oxide can lead to a decrease in levels of cardiac troponin I, von Willebrand factor, glycoprotein, and granule membrane protein 140, after an established myocardial damage, provoked by acute massive pulmonary embolism.

Resolution of venous thrombosis using a novel oral small-molecule inhibitor of P-selectin (PSI-697) without anticoagulation.

P-selectin inhibition has been shown to decrease thrombogenesis in multiple animal species. In this study, we show that a novel oral small-molecule inhibitor of P-selectin, PSI-697, promotes thrombus resolution and decreases inflammation in a baboon model of venous thrombosis. Experimental groups consisted of the following: 1) primates receiving a single oral dose of PSI-697 (30 mg/kg) daily starting three days pre-iliac vein balloon occlusion, and continued for six days; 2) primates receiving a single treatment dose of a low-molecular-weight-heparin (LMWH) (1.5 mg/kg) daily starting one day pre-iliac balloon occlusion, and continued for six days; and 3) primates receiving a single oral dose of a vehicle control daily starting three days pre-iliac vein balloon occlusion, and continued for six days. Animals receiving PSI-697, although thrombosed after balloon deflation, demonstrated greater than 80% vein lumen opening over time, with no opening (0%) for vehicle control (p < 0.01). LMWH opening evident after balloon deflation slightly deteriorated over time compared to PSI-697. PSI-697 therapy also significantly decreased vein wall inflammation determined by magnetic resonance venography (MRV). Importantly, this beneficial opening occurred without measured anticoagulation. Animals receiving PSI-697 demonstrated significantly increased plasma D-dimer levels versus LMWH and control animals six hours post thrombus induction (p < 0.01). This study is the first to demonstrate the effectiveness of oral P-selectin inhibition to modify venous thrombogenesis, increase vein lumen opening, and decrease inflammation in a large animal model.

Effects of triple antiplatelet therapy (aspirin, clopidogrel, and cilostazol) on platelet aggregation and P-selectin expression in patients undergoing coronary artery stent implantation.

The purpose of this study was to determine the effect of the addition of cilostazol to aspirin plus clopidogrel on platelet aggregation after intracoronary stent implantation. Twenty patients who underwent coronary stent placement were randomly assigned to therapy with aspirin plus clopidogrel (dual-therapy group, n = 10) or aspirin plus clopidogrel plus cilostazol (triple-therapy group, n = 10). A loading dose of clopidogrel (300 mg) and cilostazol (200 mg) was administered immediately after stent placement, and clopidogrel (75 mg/day) and cilostazol (100 mg twice daily) were given for 1 month. Platelet aggregation in response to adenosine diphosphate (ADP; 5 and 20 micromol/L) or collagen and P-selectin (CD-62P) expression was assayed at baseline, 2 hours, 24 hours, 1 week, and 1 month after stent placement. Inhibition of ADP-induced platelet aggregation was significantly higher in patients receiving triple therapy than those receiving dual therapy from 24 hours after stent placement, and inhibition of collagen-induced platelet aggregation was significantly higher in the triple-therapy group beginning 1 week after stent placement. P-Selectin expression was significantly lower in the triple-therapy than dual-therapy group at 1 week and 30 days. In conclusion, compared with dual antiplatelet therapy, triple therapy after coronary stent placement resulted in more potent inhibition of platelet aggregation induced by ADP and collagen. These findings suggest that triple therapy may be used clinically to prevent thrombotic complications after coronary stent placement.

Effect of candesartan and various inflammatory markers on maintenance of sinus rhythm after electrical cardioversion for atrial fibrillation.

Inflammatory markers, their relation to maintenance of sinus rhythm after electrical cardioversion for atrial fibrillation (AF), and the effect of candesartan were investigated in a double-blind placebo-controlled study (CAPRAF). One hundred seventy-one patients with persistent AF were randomly assigned to receive candesartan 8 mg/day or placebo for 3 to 6 weeks before and candesartan 16 mg/day or placebo for 6 months after electrical cardioversion. Serum levels of high-sensitivity C-reactive protein (hs-CRP), tumor necrosis factor-alpha, interleukin-6, P-selectin, E-selectin, CD-40 ligand, and vascular cell adhesion molecule-1 were measured at baseline and end of study. Compared with patients with a relapse of AF, patients still in sinus rhythm at 6 months after cardioversion (n = 40) had lower baseline hs-CRP and E-selectin levels: median 2.36 mg/L (25th, 75th percentiles 1.28, 4.09) versus 3.44 mg/L (25th, 75th percentiles 1.66, 6.05, p = 0.031) and 32 ng/ml (25th, 75th percentiles 23, 42) versus 37 ng/ml (25th, 75th percentiles 28, 51, p = 0.042), respectively. Neither sustained sinus rhythm for 6 months nor treatment with candesartan had an impact on measured concentrations of markers of inflammation. In conclusion, low hs-CRP and E-selectin at baseline were associated with maintenance of sinus rhythm after electrical cardioversion.