Heparin‐induced non‐necrotizing skin lesions: rarely associated with heparin‐induced thrombocytopenia

See also Warkentin TE, Linkins L‐A. Non‐necrotizing heparin‐induced skin lesions and the 4T’s score. This issue, pp 1483–. Summary. Background: Recently, there has been an increasing number of reports regarding adverse skin reactions to subcutaneous heparin administration. Case series have implied that heparin‐induced skin lesions are predominantly associated with life‐threatening heparin‐induced thrombocytopenia (HIT) in at least 22% of patients. Skin lesions, therefore, have been included in clinical scores for HIT. Objectives: To determine the association of heparin‐induced skin lesions with HIT. This would have a pivotal impact on further anticoagulatory management in patients with heparin‐induced skin lesions. Patients/Methods: In our observational cohort study, 87 consecutive patients with heparin‐induced skin lesions (85 occurring during low‐molecular‐weight heparin administration) were evaluated using a standardized internal protocol, including HIT diagnostics (heparin‐platelet factor 4‐ELISA, heparin‐induced platelet activation assay), platelet count monitoring, clinical/sonographical screening for thrombosis, skin allergy testing and, if necessary, histology. Results: None of the observed heparin‐induced skin lesions was due to HIT; all lesions were caused by delayed‐type IV‐hypersensitivity reactions (DTH) instead. Even the cutaneous reaction in one patient with concomitant HIT could be classified histologically as DTH reaction, amounting to an association of heparin‐induced skin lesions and HIT in 1.2% (1/87; 95% confidence interval, 0.00–0.06). Conclusion: Heparin‐induced skin lesions associated with use of low‐molecular‐weight heparins do not seem to be strongly associated with a systemic immunologic reaction in terms of HIT and might rather be due to DTH reactions than due to microvascular thrombosis. Hence, we propose refining existing pretest probability scores for HIT, unless underlying causes have been clarified.     

Neuroprotective effects of ultra‐low‐molecular‐weight heparin in vitro and vivo models of ischemic injury

This study was conducted to demonstrate ultra‐low‐molecular‐weight heparin’s neuroprotective effects on ischemic injury both in vivo and in vitro studies. In vitro, the effect of ultra‐low‐molecular‐weight heparin was tested in cultured PC12 cells exposed to Earle’s solution containing sodium dithionite, to identify its neuroprotection to PC12 cells damaged by oxygen‐glucose deprivation (OGD). The cell injury was detected by the tetrazolium salt 3‐(4,5‐dimethyl‐2‐thiazolyl)‐2,5 diphenyl‐2H tetrazolium bromide (MTT) assay. In vivo, male Wistar rats with middle cerebral artery occlusion were evaluated for infarct volume followed by the treatment with ultra‐low‐molecular‐weight heparin. The results in vitro showed that ultra‐low‐molecular‐weight heparin significantly inhibited PC12 cells damage induced by OGD. Results in vivo showed that vein injection of Ultra‐Low‐molecular‐weight heparin at doses of 0.5 and 1.0 mg/kg exerted significant neuroprotective effects on rats with focal cerebral ischemic injury by significantly reducing the infarct volume compared with the injury group. All the findings suggest that ultra‐low‐molecular‐weight heparin might act as a neuroprotective agent useful in the treatment of cerebral ischemia.     

Fibrinogen γ‐chain peptide‐coated,ADP‐encapsulated liposomes rescue thrombocytopenic rabbits from non‐compressible liver hemorrhage

Summary. Background: We developed a fibrinogen γ‐chain (dodecapeptide HHLGGAKQAGDV [H12])‐coated, ADP‐encapsulated liposome (H12‐[ADP]‐liposome) that accumulates at bleeding sites via interaction with activated platelets via glycoprotein IIb–IIIa and augments platelet aggregation by releasing ADP. Objective: To evaluate the efficacy of H12‐(ADP)‐liposomes for treating liver hemorrhage in rabbits with acute thrombocytopenia. Methods: Thrombocytopenia (platelets < 50 000 μL^?1) was induced in rabbits by repeated blood withdrawal (100 mL kg^?1 in total) and isovolemic transfusion of autologous washed red blood cells. H12‐(ADP)‐liposomes with platelet‐poor plasma (PPP), platelet‐rich plasma (PRP), PPP, ADP liposomes with PPP or H12‐(PBS)‐liposomes/PPP, were administered to the thrombocytopenic rabbits, and liver hemorrhage was induced by penetrating liver injury. Results: Administration of H12‐(ADP)‐liposomes and of PRP rescued all thrombocytopenic rabbits from liver hemorrhage as a result of potent hemostasis at the liver bleeding site, although rabbits receiving PPP or ADP liposomes showed 20% survival in the first 24 h. Administration of H12‐(ADP)‐liposomes and of PRP suppressed both bleeding volume and time from the site of liver injury. H12‐(phosphate‐buffered saline)‐liposomes lacking ADP also improved rabbit survival after liver hemorrhage, although their hemostatic effect was weaker. In rabbits with severe thrombocytopenia (25 000 platelets μL^?1), the hemostatic effects of H12‐(ADP)‐liposomes tended to be attenuated as compared with those of PRP treatment. Histologic examination revealed that H12‐(ADP)‐liposomes accumulated at the bleeding site in the liver. Notably, neither macrothombi nor microthrombi were detected in the lung, kidney or liver in rabbits treated with H12‐(ADP)‐liposomes. Conclusions: H12‐(ADP)‐liposomes appear to be a safe and effective therapeutic tool for acute thrombocytopenic trauma patients with massive bleeding.     

Angiostatin K1‐3 induces E‐selectin via AP1 and Ets1: a mediator for anti‐angiogenic action of K1‐3

Summary. Background: Angiostatin, a circulating angiogenic inhibitor, is an internal fragment of plasminogen and consists of several isoforms, K1‐3 included. We previously showed that K1‐3 was the most potent angiostatin to induce E‐selectin mRNA expression. The purpose of this study was to identify the mechanism responsible for K1‐3‐induced E‐selectin expression and investigate the role of E‐selectin in the anti‐angiogenic action of K1‐3. Methods and results: Quantitative real time RT‐PCR and Western blotting analyses confirmed a time‐dependent increase of E‐selectin mRNA and protein induced by K1‐3. Subcellular fractionation and immunofluorescence microscopy showed the co‐localization of K1‐3‐induced E‐selectin with caveolin 1 (Cav1) in lipid rafts in which E‐selectin may behave as a signaling receptor. Promoter‐driven reporter assays and site‐directed mutagenesis showed that K1‐3 induced E‐selectin expression via promoter activation and AP1 and Ets‐1 binding sites in the proximal E‐selectin promoter were required for E‐selectin induction. The in vivo binding of both protein complexes to the proximal promoter was confirmed by chromatin immunoprecipitation (ChIP). Although K1‐3 induced the activation of ERK1/2 and JNK, only repression of JNK activation attenuated the induction of E‐selectin by K1‐3. A modulatory role of E‐selectin in the anti‐angiogenic action of K1‐3 was manifested by both overexpression and knockdown of E‐selectin followed by cell proliferation assay. Conclusions: We show that K1‐3 induced E‐selectin expression via AP1 and Ets‐1 binding to the proximal E‐selectin promoter (?356/+1), which was positively mediated by JNK activation. Our findings also demonstrate E‐selectin as a novel target for the anti‐angiogenic therapy.     

Herbal medicine Shakuyaku‐kanzo‐to reduces paclitaxel‐induced painful peripheral neuropathy in mice

Objectives: Paclitaxel is widely used in cancer chemotherapy for the treatment of solid tumors such as breast, ovarian and lung cancer. However, it sometimes induces moderate to severe muscle pain, and impairs the patients’ quality of life. An appropriate method for relieving this pain is not well established. Shakuyaku‐kanzo‐to, a herbal medicine, is known to relieve menstrual pain, muscle spasm, and muscle pain, and its effectiveness is expected. To ascertain the effectiveness of Shakuyaku‐kanzo‐to on paclitaxel‐induced pain, we investigated the effects of Shakuyaku‐kanzo‐to and its constituent herbal medicines in a mouse model. Methods: Seven‐week‐old male ddY mice were used. To make a mouse model of paclitaxel‐induced pain, different single, intraperitoneally injected doses of this drug were tested in various groups of mice, and the optimal dose was determined. To estimate the effects of Shakuyaku‐kanzo‐to, the constituent herbal medicines Shakuyaku and Kanzo, and loxoprofen sodium as a non‐steroidal anti‐inflammatory drug on paclitaxel‐induced pain, mechanical allodynia and hyperalgesia of the hind paw were assessed. Results: Paclitaxel administered at a dose of 10mg/kg or more produced allodynia and hyperalgesia; the time courses were similar to those of pain after paclitaxel administration in cancer patients. Shakuyaku‐kanzo‐to significantly relieved the allodynia and hyperalgesia induced by paclitaxel (10mg/kg). Shakuyaku and Kanzo inhibited the allodynia and hyperalgesia to some extent, but not significantly, while loxoprofen sodium was without effects. Conclusions: A single administration of paclitaxel (10mg/kg) produced allodynia and hyperalgesia in mice, suggesting that it could be used as an animal model resembling the painful conditions observed in humans medicated with this drug. Using this model, Shakuyaku‐kanzo‐to was shown to relieve paclitaxel‐induced painful peripheral neuropathy.     

Spectroscopic,radiochemical, and theoretical studies of the Ga3+‐N‐2‐hydroxyethyl piperazine‐N′‐2‐ethanesulfonic acid (HEPES buffer) system: evidence for the formation of Ga3+‐ HEPES complexes in 68 Ga labeling reactions

Recent reports have claimed a superior performance of HEPES buffer in comparison to alternative buffer systems for ^67/68 Ga labeling in aqueous media. In this paper we report spectroscopic (¹H and ⁷¹ Ga NMR), radiochemical, mass spectrometry and theoretical modeling studies on the Ga³⁺/HEPES system (HEPES = N‐2‐hydroxyethylpiperazine‐N′‐2‐ethanesulfonic acid) performed with the aim of elucidating a potential contribution of HEPES in the ^68/67 Ga radiolabeling process. Our results demonstrate that HEPES acts as a weakly but competitive chelator of Ga³⁺ and that this interaction depends on the relative Ga³⁺: HEPES concentration. A by‐product formed in the labeling mixture has been identified as a [⁶⁸ Ga]Ga(HEPES) complex via chromatographic comparison with the nonradioactive analog. The formation of this complex was verified to compete with [⁶⁸ Ga]Ga(NOTA) complexation at low NOTA concentration. Putative chelation of Ga³⁺ by the hydroxyl and adjacent ring nitrogen of HEPES is proposed on the basis of ¹H NMR shifts induced by Ga³⁺ and theoretical modeling studies. Copyright © 2013 John Wiley & Sons, Ltd.     

Protective effects of PF‐4708671 against N‐methyl‐d‐aspartic acid‐induced retinal damage in rats

We previously demonstrated that rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR), protects against N‐methyl‐d ‐aspartic acid (NMDA)‐induced retinal damage in rats. Rapamycin inhibits mTOR activity, thereby preventing the phosphorylation of ribosomal protein S6, which is a downstream target of S6 kinase. Therefore, we aimed to determine whether PF‐4708671, an inhibitor of S6 kinase, protects against NMDA‐induced retinal injury. Intravitreal injection of NMDA (200 nmol/eye) caused cell loss in the ganglion cell layer and neuroinflammatory responses, such as an increase in the number of CD45‐positive leukocytes and Iba1‐positive microglia. Surprisingly, simultaneous injection of PF‐4708671 (50 nmol/eye) with NMDA significantly attenuated these responses without affecting phosphorylated S6 levels. These results suggest that PF‐4708671 and rapamycin likely protect against NMDA‐induced retinal damage via distinct pathways. The neuroprotective effect of PF‐4708671 is unlikely to be associated with inhibition of the S6 kinase, even though PF‐4708671 is reported to be a S6 kinase inhibitor.     

Prognostic value of combination of heart‐type fatty acid‐binding protein and ischemia‐modified albumin in patients with acute coronary syndromes and normal troponin T values

Recent studies have suggested that heart‐type fatty acid‐binding protein (H‐FABP) may detect ongoing myocardial damage involved in the progression of acute coronary syndromes (ACS). This study was prospectively designed to examine whether the combination of H‐FABP, a marker for ongoing myocardial damage, and ischemia‐modified albumin (IMA), a marker for myocardial ischemia, would effectively diagnose patients with ACS. H‐FABP values above 1.5 µg/l can be correctly measured via an ELISA and 6 µg/l is the currently used cut‐off value (1–3). We measured serum H‐FABP and IMA of 108 patients on admission within 12 hr after onset of chestpain and normal troponin T. serum samplesfrom ACS group (n=82) had decreased capacity of ACB [64 (61–67) U/ml] compared with non‐ACS ischemic chest pain group (n=26) samples [75 (71–78) U/ml] (P<0.05). The combination of IMA and H‐FABP usually had better sensitivity [96.3% (92.2–100%)] (P<0.05) and accuracy [92.6 (87.7–97.5%)] (P<0.05) than when individually used. Thus, the combination of H‐FABP and IMA measurements after initiation of chest pain may be highly effective for risk stratification in patients with ACS and normal cardiac troponin T. J. Clin. Lab. Anal. 23:14–18, 2009. © 2009 Wiley‐Liss, Inc.     

Hepatic effects of flunixin‐meglumin in LPS‐induced sepsis

The aim of this study was to evaluate the actions of the non‐steroidal anti‐inflammatory drug flunixin‐meglumin (FM) on the changes caused by lipopolysaccharide (LPS)‐induced sepsis in the rat liver. Eight groups of five adult male Wistar rats were analysed: (1) saline injected (controls), (2) FM treated with 1.1 mg/kg, (3) FM treated with 2.2 mg/kg, (4) LPS‐injected (10 mg/kg), (5) LPS‐injected with 1.1 mg/kg FM pretreatment, (6) LPS‐injected with 2.2 mg/kg FM pretreatment, (7) LPS‐injected with 1.1 mg/kg FM post‐treatment and (8) LPS‐injected with 2.2 mg/kg FM post‐treatment. All drugs were intraperitoneally injected. The following parameters were evaluated: plasma levels of hepatic enzymes and urea, hepatic histological characteristics, antioxidant enzymes and several metabolic fluxes. The latter comprised gluconeogenesis, ureagenesis and oxygen consumption. Liver damage in LPS‐induced sepsis was characterized by histological changes, increased plasma levels of alanine aminotransferase and aspartate aminotransferase (P < 0.001) and diminished gluconeogenesis (P < 0.001) and ureagenesis (P < 0.01). LPS also induced oxidative stress as evidenced by increased catalase (P < 0.05) and superoxide dismutase activities and enhanced lipid peroxidation (P < 0.001). Pretreatment of the animals with FM minimized the histological changes and normalized, in part, all enzymatic activities. Pretreatment of the animals with FM also normalized gluconeogenesis and partly restored ureagenesis (P < 0.05). These and other results show that LPS‐induced sepsis may lead to severe liver damage, affecting both structure and function. Treatment with FM can be used to avoid this damage. The antioxidant properties of FM can be, partly at least, responsible for this protective action.     

Peripheral N‐methyl‐d‐aspartate receptors contribute to mechanical hypersensitivity in a rat model of inflammatory temporomandibular joint pain

The aim of this study was to determine whether peripheral N‐methyl‐d ‐aspartate (NMDA) receptors are involved in inflammation‐induced mechanical hypersensitivity of the temporomandibular joint (TMJ) region. We developed a rat model of mechanical sensitivity to Complete FreunD's Adjuvant (CFA; 2μl containing 1μg Mycobacterium tuberculosis)‐induced inflammation of the TMJ and examined changes in sensitivity following injection of NMDA receptor antagonists (dl ‐2‐amino‐5‐phosphonovaleric acid (AP5) or Ifenprodil) with CFA. CFA injected into the TMJ resulted in an increase in mechanical sensitivity relative to pre‐injection that peaked at day 1 and lasted for up to 3 days (n =8, P <0.05). There was no change in mechanical sensitivity in vehicle‐injected rats at any time‐point (n =9). At day 1, there was a significant increase in mechanical sensitivity in animals injected with CFA+vehicle (n =7) relative to those injected with vehicle alone (n =7, P <0.05), and co‐injection of AP5 (n =6) or Ifenprodil (n =7) with CFA blocked this hypersensitivity. Subcutaneous injection of AP5 (n =7) and Ifenprodil (n =5) instead of into the TMJ had no significant effect on CFA‐induced hypersensitivity of the TMJ region. Western blot analysis revealed constitutive expression of the NR1 and NR2B subunits in trigeminal ganglion lysates. Immunohistochemical studies showed that 99% and 28% of trigeminal ganglion neurons that innervated the TMJ contained the NR1 and NR2B subunits respectively. Our findings suggest a role for peripheral NMDA receptors in inflammation‐induced pain of the TMJ region. Targeting peripheral NMDA receptors with peripheral application of NMDA receptor antagonists could provide therapeutic benefit and avoid side effects associated with blockade of NMDA receptors in the central nervous system.     

HIV‐1 Tat‐induced platelet activation and release of CD154 contribute to HIV‐1‐associated autoimmune thrombocytopenia

Summary. Background: Enhanced platelet activation in human immunodeficiency virus (HIV)‐1‐infected patients has been reported and shown to strongly correlate with plasma viral load. Activated platelets are known to express and to release a variety of proteins that can modulate the immune system. Specifically, platelet‐derived CD154 has been shown to be directly involved in the development of autoimmune thrombocytopenia (ITP). The mechanism by which HIV‐1 infection leads to platelet activation and the effect of this activation on the development of HIV‐1 ITP, however, is not fully understood. Objective: We have investigated the effect of HIV‐1 Trans activating factor (Tat) on platelet activation. Results: We report that HIV‐1 Tat directly interacts with platelets and induces platelet activation resulting in platelet micro‐particle release. This activation by Tat requires the chemokine receptor CCR3 and β3‐integrin expression on platelets, as well as calcium flux. Tat‐induced activation of platelets releases platelet CD154, an immune modulator. Enhanced B‐cell activity is found in mouse spleen B cells co‐cultured with platelets treated with Tat in vitro. An early antibody response against adenovirus is found in Tat‐injected mouse immunized with adenovirus, suggesting an enhanced immune response in vivo. Conclusions: We have described a role of Tat‐induced platelet activation in the modulation of the immune system, with implications for the development of HIV‐1‐associated thrombocytopenia.     

Mesenchymal stem cell expression of SDF‐1β synergizes with BMP‐2 to augment cell‐mediated healing of critical‐sized mouse calvarial defects

Bone has the potential for spontaneous healing. This process, however, often fails in patients with comorbidities. Tissue engineering combining functional cells, biomaterials and osteoinductive cues may provide alternative treatment strategies. We have recently demonstrated that stromal cell‐derived factor‐1β (SDF‐1β) works in concert with bone morphogenetic protein‐2 (BMP‐2) to potentiate osteogenic differentiation of bone marrow‐derived mesenchymal stem/stromal cells (BMSCs). Here, we test the hypothesis that SDF‐1β overexpressed in Tet‐Off‐SDF‐1β BMSCs, delivered on acellular dermal matrix (ADM), synergistically augments BMP‐2‐induced healing of critical‐sized mouse calvarial defects. BMSC therapies alone showed limited bone healing, which was increased with co‐delivery of BMP‐2. This was further enhanced in Tet‐Off‐SDF‐1β BMSCs + BMP‐2. Only limited BMSC retention on ADM constructs was observed after 4 weeks in vivo, which was increased with BMP‐2 co‐delivery. In vitro cell proliferation studies showed that supplementing BMP‐2 to Tet‐Off BMSCs significantly increased the cell number during the first 24 h. Consequently, the increased cell numbers decreased the detectable BMP‐2 levels in the medium, but increased cell‐associated BMP‐2. The data suggest that SDF‐1β provides synergistic effects supporting BMP‐2‐induced, BMSC‐mediated bone formation and appears suitable for optimization of bone augmentation in combination therapy protocols. Copyright © 2015 John Wiley & Sons, Ltd.     

Induction of anti‐β2‐glycoprotein I autoantibodies in mice by protein H of Streptococcus pyogenes

Summary. Background: The antiphospholipid syndrome (APS) is characterized by the persistent presence of anti‐β₂‐glycoprotein I (β₂‐GPI) autoantibodies. β₂‐GPI can exist in two conformations. In plasma it is a circular protein, whereas it adopts a fish‐hook conformation after binding to phospholipids. Only the latter conformation is recognized by patient antibodies. β₂‐GPI has been shown to interact with Streptococcus pyogenes. Objective: To evaluate the potential of S. pyogenes‐derived proteins to induce anti‐β₂‐GPI autoantibodies. Methods and results: Four S. pyogenes surface proteins (M1 protein, protein H, streptococcal collagen‐like protein A [SclA], and streptococcal collagen‐like protein B [SclB]) were found to interact with β₂‐GPI. Only binding to protein H induces a conformational change in β₂‐GPI, thereby exposing a cryptic epitope for APS‐related autoantibodies. Mice were injected with the four proteins. Only mice injected with protein H developed antibodies against the patient antibody‐related epitope in domain I of β₂‐GPI. Patients with pharyngotonsillitis caused by S. pyogenes who developed anti‐protein H antibodies also generated anti‐β₂‐GPI antibodies. Conclusions: Our study has demonstrated that a bacterial protein can induce a conformational change in β₂‐GPI, resulting in the formation of antiβ₂‐GPI autoantibodies. This constitutes a novel mechanism for the formation of anti‐β₂‐GPI autoantibodies.     

PKC inhibition markedly enhances Ca2+ signaling and phosphatidylserine exposure downstream of protease‐activated receptor‐1 but not protease‐activated receptor‐4 in human platelets

To cite this article: Harper MT, Poole AW. PKC inhibition markedly enhances Ca²⁺ signaling and phosphatidylserine exposure downstream of protease‐activated receptor‐1 but not protease‐activated receptor‐4 in human platelets. J Thromb Haemost 2011; 9 : 1599–607. Summary. Background: Cytosolic calcium concentration is a critical regulator of platelet activation, and so platelet Ca²⁺ signaling must be tightly controlled. Thrombin‐induced Ca²⁺ signaling is enhanced by inhibitors of protein kinase C (PKC), suggesting that PKC negatively regulates the Ca²⁺signal, although the mechanisms by which this occurs and its physiological relevance are still unclear. Objectives: To investigate the mechanisms by which PKC inhibitors enhance thrombin‐induced Ca²⁺ signaling, and to determine the importance of this pathway in platelet activation. Methods: Cytosolic Ca²⁺ signaling was monitored in fura‐2‐loaded human platelets. Phosphatidylserine (PS) exposure, a marker of platelet procoagulant activity, was measured by annexin V binding and flow cytometry. Results: PKC inhibition by bisindolylmaleimide‐I (BIM‐I) enhanced α‐thrombin‐induced Ca²⁺ signaling in a concentration‐dependent manner. PAR1 signaling, activated by SFLLRN, was enhanced much more strongly than PAR4, activated by AYPGKF or γ‐thrombin, which is a potent PAR4 agonist but a poor activator of PAR1. BIM‐I had little effect on α‐thrombin‐induced signaling following treatment with the PAR1 antagonist, SCH‐79797. BIM‐I enhanced Ca²⁺ release from intracellular stores and Ca²⁺ entry, as assessed by Mn²⁺ quench. However, the plasma membrane Ca²⁺ ATPase inhibitor, 5(6)‐carboxyeosin, did not prevent the effect of BIM‐I. PKC inhibition strongly enhanced α‐thrombin‐induced PS exposure, which was reversed by blockade of PAR1. Conclusions: Together, these data show that when PAR1 is stimulated, PKC negatively regulates Ca²⁺ release and Ca²⁺ entry, which leads to reduced platelet PS exposure.     

Release of sphingosine‐1‐phosphate from human platelets is dependent on thromboxane formation

Summary. Background: Platelets release the immune‐modulating lipid sphingosine‐1‐phosphate (S1P). However, the mechanisms of platelet S1P secretion are not fully understood. Objectives: The present study investigates the function of thromboxane (TX) for platelet S1P secretion during platelet activation and the consequences for monocyte chemotaxis. Methods: S1P was detected using thin‐layer chromatography in [³H]sphingosine‐labeled platelets and by mass spectrometry. Monocyte migration was measured in modified Boyden chamber chemotaxis assays. Results: Release of S1P from platelets was stimulated with protease‐activated receptor‐1‐activating peptide (PAR‐1‐AP, 100 μm ). Acetylsalicylic acid (ASA) and two structurally unrelated reversible cyclooxygenase inhibitors diclofenac and ibuprofen suppressed S1P release. Oral ASA (500‐mg single dose or 100 mg over 3 days) attenuated S1P release from platelets in healthy human volunteers ex vivo. This was paralleled by inhibition of TX formation. S1P release was increased by the TX receptor (TP) agonist U‐46619, and inhibited by the TP antagonist ramatroban and by inhibitors of ABC‐transport. Furthermore, thrombin‐induced release of S1P was attenuated in platelets from TP‐deficient mice. Supernatants from PAR‐1‐AP‐stimulated human platelets increased the chemotactic capacity of human peripheral monocytes in a S1P‐dependent manner via S1P receptors‐1 and ‐3. These effects were inhibited by ASA‐pretreatment of platelets. Conclusions: TX synthesis and TP activation mediate S1P release after thrombin receptor activation. Inhibition of this pathway may contribute to the anti‐inflammatory actions of ASA, for example by affecting activity of monocytes at sites of vascular injury.     

Endothelium‐dependent and endothelium‐independent effects of 1‐nitro‐2‐propylbenzene on rat aorta

A group of nitro compounds contains a benzene ring in a short aliphatic chain with the NO₂ group, property that supposedly favors its vasodilator profile. In this study, we evaluated in isolated rat aorta the effects of 1‐nitro‐2‐propylbenzene (NPB), a nitro compound containing the NO₂ in the aromatic ring. In aorta precontracted with KCl, NPB (1‐3000 μm ) induced full endothelium‐independent relaxation. In endothelium‐intact preparations, phenylephrine‐induced contractions were fully relaxed by NPB, effect unaltered by N(ω)‐nitro‐L‐arginine methyl ester (L‐NAME) or 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one (ODQ). In the concentration range of 30–300 μm , NPB slightly but significantly potentiated the phenylephrine‐induced contraction. Such potentiation was unaltered by the thromboxane‐prostanoid receptor antagonist seratrodast, but was abolished by endothelium removal or by preincubation of endothelium‐intact preparations with L‐NAME, ODQ or by ruthenium red and HC‐030031, blockers of subtype 1 of ankyrin transient receptor potential (TRPA₁) channels. Verapamil exacerbated the potentiating effect of NPB. The potentiating effect was undetectable in preparations precontracted by 9,11‐dideoxy‐11α,9α‐epoxymethanoprostaglandin F2α (U‐46619). Relaxation was reduced by ruthenium red while it was enhanced by HC‐030031. In conclusion, NPB has vasodilator properties but with a mechanism of action distinct from its analogues. Contrary to other nitro compounds, its relaxing effects did not involve recruitment of the guanylyl cyclase pathway. NPB has also endothelium‐dependent potentiating properties on phenylephrine‐induced contractions, a phenomenon that putatively required a role of endothelial TRPA₁ channels. The present findings reinforce the notion that the functional group NO₂ in the aliphatic chain of these nitro compounds determines favorably their vasodilator properties.     

Bioeffects of Low‐Intensity Ultrasound In Vitro

Objective. The purpose of this study was to evaluate the potential molecular mechanism of low‐intensity ultrasound‐induced apoptosis by analyzing protein profile alteration in response to ultrasound exposure. Methods. Human hepatocarcinoma SMMC‐7721 cells were used in this study. Cell viability was measured by a trypan blue dye exclusion test. Morphologic changes were examined by light microscopy. Apoptosis was assessed by phosphatidylserine externalization and DNA fragmentation. The pattern of the mitochondrial membrane potential decrease was determined by flow cytometry. Protein profile alteration was analyzed by comparative proteomics based on 2‐dimensional polyacrylamide gel electrophoresis and matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry. Results. Low‐intensity ultrasound (3.0 W/cm², 1 minute, cells incubated for 6 hours after ultrasound exposure) induced early apoptosis (mean ± SD, 26.5% ± 6.2%) significantly (P < .05) with minimal lysis in human hepatocarcinoma cells in vitro. On a molecular level, several proteins, eg, cellular tumor antigen protein 53, BH3‐interacting domain death agonist, apoptosis regulator Bcl‐2, and heme oxygenase 1 were identified as responding to ultrasound irradiation, suggesting that mitochondrial dysfunction and oxidative stresses were involved in ultrasound‐induced apoptosis. It was also assumed that mitofilin‐regulated crista remodeling may be a potential channel of mitochondrial membrane permeabilization pore formation involved in low‐intensity ultrasound‐induced apoptosis. Conclusions. This study suggests that 2 potential molecular signaling pathways are involved in ultrasound‐induced apoptosis. It is a first step toward low‐intensity ultrasound‐induced apoptotic cancer therapy via understanding its relevant molecular signaling and key proteins.