Sphingomyelinase and cell-permeable ceramide analogs increase the release of plasminogen activator inhibitor-1 from cultured endothelial cells

We investigated the effect of exogenous staphylococcal sphingomyelinase (SMase) on the release of tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) from cultured human umbilical vein endothelial cells (HUVEC). Addition of SMase (2 units/ml) to the culture medium induced an approx. 15-fold increase in the extracellular level of PAI-1 antigen at 3 h. No significant increase in the level of t-PA antigen was detected. Treatment of HUVEC with SMASE (2 units/ml) for 3 h resulted in a significant decrease in the cellular sphingomyelin (SM) level, accompanied by a corresponding increase in the ceramide level. Cell-permeable ceramide analogs also enhanced the release of PAI-1 from cultured HUVEC in concentration- and time-dependent manners. A 6-fold increase in PAI-1 antigen level was observed after incubation for 3 h with 10 microM N-acetylsphingosine. Similar effect was noted as early as 2 h with 10 microM N-hexyanoylsphingosine. Addition of sphingosine failed to affect the release of PAI-1 from cultured HUVEC, indicating that the effects of ceramide analogs were independent of sphingosine generation. Pretreatment with cycloheximide or actinomycin D abated the response of HUVEC to N-acetylsphingosine in the increased levels of both extracellular and intracellular PAI-1. These results suggest that ceramide, generated via "SM cycle", acts as a lipid mediator of PAI-1 release from vascular endothelial cells, and may contribute to a better understanding of the pathogenesis of the PAI-1-associated thrombotic disorders.     

ADP-induced platelet activation

Platelet activation is central to the pathogenesis of hemostasis and arterial thrombosis. Platelet aggregation plays a major role in acute coronary artery diseases, myocardial infarction, unstable angina, and stroke. ADP is the first known and an important agonist for platelet aggregation. ADP not only causes primary aggregation of platelets but is also responsible for the secondary aggregation induced by ADP and other agonists. ADP also induces platelet shape change, secretion from storage granules, influx and intracellular mobilization of Ca2+, and inhibition of stimulated adenylyl cyclase activity. The ADP-receptor protein mediating ADP-induced platelet responses has neither been purified nor cloned. Therefore, signal transduction mechanisms underlying ADP-induced platelet responses either remain uncertain or less well understood. Recent contributions from chemists, biochemists, cell biologists, pharmacologists, molecular biologists, and clinical investigators have added considerably to and enhanced our knowledge of ADP-induced platelet responses. Although considerable efforts have been directed toward identifying and cloning the ADP-receptor, these have not been completely successful or without controversy. Considerable progress has been made toward understanding the mechanisms of ADP-induced platelet responses but disagreements persist. New drugs that do not mimic ADP have been found to inhibit fairly selectively ADP-induced platelet activation ex vivo. Drugs that mimic ADP and selectively act at the platelet ADP-receptor have been designed, synthesized, and evaluated for their therapeutic efficacy to block selectively ADP-induced platelet responses. This review examines in detail the developments that have taken place to identify the ADP-receptor protein and to better understand mechanisms underlying ADP-induced platelet responses to develop strategies for designing innovative drugs that block ADP-induced platelet responses by acting selectively at the ADP-receptor and/or by selectively interfering with components of ADP-induced platelet activation mechanisms.     

Effect of biodegradable polyrotaxanes on platelet activation

Flip angle dependence of the localized single-voxel 1H NMR spectroscopic sequences STEAM and PRESS using numerically optimized Shinnar-Le Roux (SLR) and conventional sinc RF pulses has been evaluated. Phantom experiments were used to evaluate voxel profiles from MR images of the selected voxels. Information on the total excited volume was recorded from the integrated area under the water peak in the localized spectrum at different flip angles (theta = 0 degrees-180 degrees). The voxel profiles for both the STEAM and PRESS sequences using the SLR RF pulses were found to be identical, unlike the case for the sinc RF pulses. The SLR RF pulses in the PRESS sequence were found to be more sensitive to flip angle variations. Localized, water-suppressed 1H NMR spectra recorded from the frontal gray matter in healthy volunteers (n = 3) showed less lipid contamination using the SLR RF pulses compared with the sinc RF pulses.     

Cytokine induction of platelet activation

A three-dimensional, two-part model of the foot, for use in a simulation of human gait, is presented. Previous simulations of gait have not included the foot segment (e.g. Siegler et al., 1982, J. Biomechanics 15, 415-425) or have fastened it to the ground (e.g. Onyshko and Winter, 1980, J. Biomechanics 13, 361-368). A foot model based on viscoelastic elements (e.g. Meglan, 1991, Ph.D. thesis, Ohio State Univ.), allows more freedom of movement and thus models the physical system more closely. The current model was developed by running simulations of the foot in isolation from just before heel contact to just after toe-off. The driving inputs to the simulation were the resultant ankle joint forces and moments taken from a gait analysis. Nine linear, vertically oriented spring/damper systems, positioned along the midline of the foot were used to model the combined viscoelastic behaviour of the foot, shoe and floor. Associated with each vertical spring/damper system were two orthogonally placed, linear, horizontal dampers used to provide the shear components of the ground reaction force. Torques at the metatarsal-phalangeal joint were supplied by a linear, torsional spring and damper. Control about the vertical axis and the long axis of the foot was achieved by the use of linear, torsional dampers. The predicted kinetic and kinematic values are very similar to those taken from the gait analysis. The model represents an improvement over previous work because the transition from swing to stance was smooth and continuous without the foot being constrained to any specific trajectory.     

Effects of sodium nitroprusside on hemodialysis-induced platelet activation

Plasminogen activator inhibitor-2 (PAI-2), a member of the serpin gene family, is thought to serve as a primary regulator of plasminogen activation in the extravascular compartment. High levels of PAI-2 are found in keratinocytes, monocytes, and the human trophoblast, the latter suggesting a role in placental maintenance or embryo development. The primarily intracellular distribution of PAI-2 also may indicate a unique regulatory role in a protease-dependent cellular process such as apoptosis. To examine the potential functions of PAI-2 in vivo, we generated PAI-2-deficient mice by gene targeting in embryonic stem cells. Homozygous PAI-2-deficient mice exhibited normal development, survival, and fertility and were also indistinguishable from normal controls in response to a bacterial infectious challenge or endotoxin infusion. No differences in monocyte recruitment into the peritoneum were observed after thioglycollate injection. Epidermal wound healing was equivalent among PAI-2 -/- null and control mice. Finally, crossing PAI-2 -/- with PAI-1 -/- mice to generate animals deficient in both plasminogen activator inhibitors failed to uncover an overlap in function between these two related proteins.     

Evidence of platelet activation in hypertension

To test the hypothesis that platelet activation is present in hypertension, we measured plasma markers beta thromboglobulin and soluble P-selectin in hypertensive patients and normotensive controls. Both markers were raised in the patients (P < 0.05), and in a subgroup of patients, beta thromboglobulin was reduced with successful treatment of hypertension with the ACE inhibitor quinapril. We suggest that reversible platelet activation is present in hypertension. This may be a contributing factor to the link between this risk factor and the development of thrombotic disease such as stroke.     

Effect of platelet activation on coronary collateral blood flow

BACKGROUND: The platelet products thromboxane A2 and serotonin have been shown to cause constriction of well-developed coronary collateral vessels. This study was performed to determine whether intravascular platelet activation produced with platelet activating factor (PAF) can cause a decrease in coronary collateral blood flow. METHODS and RESULTS: Collateral vessel growth was induced by embolization of a hollow stainless steel plug into the left anterior descending coronary artery (LAD) of adult dogs. The animals were returned to the laboratory 3 to 6 weeks later for surgical instrumentation and measurement of collateral blood flow. Collateral flow was assessed by measuring retrograde blood flow from the cannulated collateral-dependent artery. PAF (10 nmol) was injected into the left main coronary artery to allow products of platelet activation to reach collateral vessels arising from the left coronary system. PAF caused a vasoconstrictor response, which became maximal 3 minutes after injection and resulted in a 40.3+/-7.4% decrease in retrograde blood flow (32.1+/-2.1 to 19.6+/-3.2 mL/min; P<0.05). By 15 minutes after the PAF injection, both retrograde blood flow and transcollateral resistance had returned to normal. After pretreatment with the thromboxane A2 receptor antagonist SQ30, 741, the vasoconstrictor response to PAF was abolished and, in contrast to the decrease in retrograde blood flow from PAF alone, a weak vasodilator effect was unmasked. CONCLUSIONS: PAF caused a decrease in coronary collateral blood flow. This vasoconstrictor response required the participation of thromboxane A2.     

Gender differences in platelet GPIIb-IIIa activation

Gender differences in the development of thrombotic diseases have been described in numerous clinical settings. Enhanced platelet reactivity in both sexes is associated with the development of vascular thromboses. Because activation of platelet GPIIb-IIIa receptors is a central event in thrombus formation, we examined GPIIb-IIIa function in normal male and female volunteers. Using flow cytometry, we quantitated gender differences in the number of binding sites for FITC-labeled fibrinogen (FITC-FGN) and FITC-labeled PAC-1 antibody (FITC-PAC-1). Washed platelets were incubated with either FITC-FGN or FITC-PAC-1 and activated with either ADP or thrombin receptor activating peptide (TRAP) prior to cytometric acquisition of data. The dissociation constant for FITC-FGN was the same in both sexes (approx. 1.6 x 10(-7)M), however, the number of GPIIb-IIIa receptors per platelet capable of binding fibrinogen was significantly greater in women than men in response to 2 microM ADP (16,319 +/- 1871 vs 9669 +/- 1994, p = 0.02), 20 microM ADP (39,951 +/- 4711 vs 25,948 +/- 4953, p = 0.05) and 50 microM TRAP (39,236 +/- 3965 vs 21,848 +/- 4159, p = 0.007). Similarly, the number of GPIIb-IIIa receptors capable of binding PAC-1 in response to ADP and TRAP was 50% to 80% greater in women than men. Binding experiments using specific anti-GPIIb-IIIa monoclonal antibodies (P2 and 10E5), as well as quantitative Western blotting experiments, showed no gender difference in the total number of GPIIb-IIIa molecules expressed. Analysis of data from female subgroups demonstrated an association of GPIIb-IIIa reactivity with menstrual phase. We conclude that GPIIb-IIIa receptors on platelets from premenopausal women are more "activatable" than those on platelets from young men. Variations in the serum concentrations of estrogens and/or progestins may modulate GPIIb-IIIa function.     

Influence of purified apoprotein E on platelet activation induced by serotonin

In what ways can a premenopausal woman be pregnant after a breast carcinoma's treatment? A survey organized in 1985 by the French Gynecologic Association gave us the opinion of 316 gynecologists: 50% of them disagreed with this pregnancy because of an accrued risk of relapse. 68 observations were collected including 41 of full term pregnancies; the survival curves were, in each group, similar to those of the controls at the same stage. The interruption doesn't improve the prognosis. This confirms the whole of the published series: a subsequent pregnancy doesn't seem to affect the prognosis of a breast carcinoma; it is important to take the contraception into account so that this pregnancy shall be really desired.     

Different effects of indomethacin and nabumetone on prostaglandin-mediated gastric responses to central vagal activation in rats

Intracisternal injection of a stable thyrotropin-releasing hormone (TRH) analog increases gastric prostaglandins release and mucosal resistance to injury through central vagal pathways. The effects of two nonsteroidal anti-inflammatory drugs, indomethacin (INDO) and nabumetone on intracisternal injection of various doses of TRH-induced gastric acid secretion and changes in mucosal resistance were investigated in urethane-anesthetized rats. Doses of INDO (5 mg/kg) and nabumetone (13.75 mg/kg) producing similar acute anti-inflammatory response in the carrageenin-induced paw edema were injected i.p. in all studies. INDO potentiated the acid secretion induced by intracisternal injection of TRH at 25, 50 and 200 ng by 5.1-, 1.9- and 1.4-fold, respectively, whereas nabumetone did not modify the secretory response to TRH. Moderate erosions were observed in 100% of rats treated with the combination of INDO and TRH (200 ng) whereas no erosions were observed when TRH or INDO were given alone or TRH in combination with nabumetone. TRH at 7 ng reduced mucosal damage induced by intragastric administration of ethanol (60%, 1 ml/kg) by 63%. The mucosal protective action of TRH was abolished by INDO but not altered by nabumetone pretreatment. These data indicate that at comparable anti-inflammatory doses, nabumetone, unlike INDO, neither blocks the protection against ethanol injury induced by low doses of TRH injected intracisternally nor potentiates the gastric acid secretion or lesions induced by higher dose of TRH. We speculate that these differences reflect reduced inhibition of gastric prostaglandins by nabumetone.     

Aprotinin inhibits plasmin-induced platelet activation during cardiopulmonary bypass

PURPOSE: We correlated the expression of bcl-2 with accumulation of p53 protein in bone marrow metastases from patients with androgen independent prostate cancer and a history of hormonal ablation therapy. These results were correlated with clinical parameters, including the extent of bone marrow metastases and patient survival. MATERIALS AND METHODS: All 43 patients studied had evidence of prostate cancer progression following androgen deprivation therapy and histologically confirmed bone marrow metastases. Decalcified tissue sections were used for immunohistochemical evaluation of bcl-2 protein and p53 protein accumulation. RESULTS: We previously established that p53 protein accumulation as detected by immunohistochemistry is a reliable indicator of p53 gene mutation in prostate cancer. Immunoreactivity was demonstrated for p53 protein in 22 of 43 cases and for bcl-2 protein in 14. A total of 28 cases (65%) exhibited immunohistochemical evidence of p53 and/or bcl-2 expression, and 15 (35%) were negative for p53 and bcl-2. The expression of bcl-2 and accumulation of p53 were independent events (p < 0.01). The expression of bcl-2 or accumulation of p53 protein in prostate cancer metastases did not significantly influence patient survival or the extent of metastatic disease. CONCLUSIONS: The presence or absence of p53 protein accumulation and/or bcl-2 expression did not correlate with tumor burden or patient survival in stage D androgen independent prostate cancer bone marrow metastases. The expression of bcl-2 protein occurs independently of and is inversely correlated with p53 mutations in advanced prostate cancer.     

A dual thrombin receptor system for platelet activation

Platelet-dependent arterial thrombosis triggers most heart attacks and strokes. Because the coagulation protease thrombin is the most potent activator of platelets, identification of the platelet receptors for thrombin is critical for understanding thrombosis and haemostasis. Protease-activated receptor-1 (PAR1) is important for activation of human platelets by thrombin, but plays no apparent role in mouse platelet activation. PAR3 is a thrombin receptor that is expressed in mouse megakaryocytes. Here we report that thrombin responses in platelets from PAR3-deficient mice were markedly delayed and diminished but not absent. We have also identified PAR4, a new thrombin-activated receptor. PAR4 messenger RNA was detected in mouse megakaryocytes and a PAR4-activating peptide caused secretion and aggregation of PAR3-deficient mouse platelets. Thus PAR3 is necessary for normal thrombin responses in mouse platelets, but a second PAR4-mediated mechanism for thrombin signalling exists. Studies with PAR-activating peptides suggest that PAR4 also functions in human platelets, which implies that an analogous dual-receptor system also operates in humans. The identification of a two-receptor system for platelet activation by thrombin has important implications for the development of antithrombotic therapies.     

Hydrogen peroxide is involved in collagen-induced platelet activation

Leptin is a 16-kDa protein secreted by adipocytes that has been proposed to regulate feed intake in mice, rats, and humans. The present study was designed to characterize porcine leptin structure and expression. Successful RT-PCR resulted in development of a cDNA clone to the full length coding region of porcine leptin. Sequence data demonstrate 85% base homology to rodent, 88% to human, and a 92% homology to the bovine sequence. For assessment of porcine leptin gene expression, total RNA was extracted from the subcutaneous adipose tissue of genetically selected high backfat pigs and from contemporary crossbred swine. Total RNA derived from genetically selected high fat pigs contained 113% higher (P < .05) concentrations of porcine leptin mRNA than total RNA derived from contemporary crossbred pigs. Western blotting was used to evaluate serum levels of porcine leptin in genetically selected high backfat and contemporary, crossbred pigs. Relative levels of porcine leptin in sera from obese swine were approximately 306% higher (P < .05) than levels present in sera from contemporary, crossbred swine. These data indicate that leptin is expressed in pigs, the expressed protein is secreted into the bloodstream, and obese swine express higher levels of leptin mRNA and protein than nonobese swine at similar body weight.     

Inhibition of collagen-induced platelet activation by arachidonyl trifluoromethyl ketone

Collagen-induced platelet activation is associated with, and markedly potentiated by, the release of arachidonic acid and its subsequent conversion to thromboxane A2. The precise mechanism of arachidonic acid release is unknown. An inhibitor of isolated cytosolic phospholipase A2 (cPLA2), arachidonyl trifluoromethyl ketone (AACOCF3), was used to examine the role that cPLA2 plays in this process. AACOCF3 inhibited platelet aggregation in response to collagen and arachidonic acid but not to thrombin, calcium ionophore, phorbol ester, or a thromboxane mimetic. Thromboxane formation stimulated by thrombin or collagen was inhibited by AACOCF3. However, AACOCF3 did not inhibit collagen-induced [14C]arachidonic acid release. These data are consistent with the inhibitory effects of AACOCF3 on collagen-induced aggregation involving an action on the conversion of arachidonic acid to thromboxane.     

The effects of ceramide and its analogues on the secretion of the mucocyst content of Tetrahymena

Monoclonal antibody to Geodia lectin is bound by the mucocyst content of Tetrahymena. By using this (fluorescent-labelled) antibody and confocal microscopy, the actual state of the mucocyst (position, resting, filling or extruding) can be studied. Treatment with C2 ceramide and non-hydroxy fatty acid caused a rapid depletion of mucocyst material. Another ceramide analogue, psychosine caused fusion of mucocysts. In these cases--in contrast to the controls--the contractile vacuole was filled with mucocyst material and this was seen in the tubules in contact with the contractile vacuoles. Hydroxy fatty acid ceramide, sphingomyelin and sphingosine-1-phosphate were ineffective. As the former materials influence also the cytoskeleton, while the latter do not, the cytoskeleton is presumed to have a mediatory effect. Neither the connection of contractile vacuoles with tubular structures nor mucocyst fusion have been described before.     

Comparative effects of recombinant staphylokinase and streptokinase on platelet aggregation

Penclomedine [3,5-dichloro-4,6-dimethoxy-2-(trichloromethyl)pyridine], an antitumor agent, is currently in Phase I clinical trials and is believed to be a prodrug. In these studies, cerebellar effects have been dose limiting. Previous studies identified 4-demethylpenclomedine (4-DM-PEN) as the major plasma metabolite in rodents and humans. 4-DM-PEN was demonstrated to be an antitumor-active metabolite of penclomedine in vivo when evaluated against the penclomedine-sensitive MX-1 human breast tumor xenograft implanted either s.c. or intracerebrally and is believed to be on the metabolic activation pathway of penclomedine. Because earlier studies revealed an absence of neurotoxic cerebellar effects for 4-DM-PEN in contrast to penclomedine in a rat model, this metabolite may be a candidate for an alternative to penclomedine in the clinic for treatment of breast cancer or brain tumors, if the cerebellar effects of penclomedine preclude its further clinical development. Because neither penclomedine nor 4-DM-PEN were very active in vitro, the metabolism of penclomedine was also investigated using rat liver microsomes in an attempt to identify the ultimate active form of the drug. Metabolites and putative metabolites were prepared by chemical synthesis for antitumor evaluation in vitro and in vivo. A reductive metabolite, alpha,alpha-didechloro-PEN, was observed to be much more cytotoxic than penclomedine or 4-DM-PEN in vitro, but evaluation of this and the other metabolites and putative metabolites in vivo against the MX-1 tumor failed to identify any active metabolite among the structures evaluated other than 4-DM-PEN. The limited activity of 4-DM-PEN in vitro indicates that it, like penclomedine, is also a prodrug, demonstrating a need for additional studies on the metabolic activation of penclomedine to identify the ultimate active form of the drug.     

The effects of heparinase 1 and protamine on platelet reactivity

BACKGROUND: Protamine is currently the most widely used drug for the reversal of heparin anticoagulation. Heparinase 1 (heparinase) is being evaluated as a possible alternative to protamine for the reversal of heparin anticoagulation. The authors evaluated the effects of equivalent doses of heparinase and protamine on platelet reactivity by measuring agonist-induced P-selectin expression. METHODS: After Institutional Review Board (IRB) approval, informed consent was obtained from 12 healthy volunteers and 8 patients undergoing surgery requiring cardiopulmonary bypass (CPB). Twenty-four ml of blood was obtained from each volunteer; 10 ml of blood was obtained from each patient before the CPB, and another 10 ml was obtained after CPB. Heparin was neutralized using heparinase or protamine. Platelet reactivity was assessed by measuring the expression of P-selectin after stimulation of platelets with increasing concentrations of a thrombin receptor agonist peptide (TRAP). Data were analyzed using analysis of variance. P < 0.05 was considered significant. RESULTS: For the healthy volunteers, the activated coagulation times (ACTs) of the heparinized samples returned to baseline values with heparinase (12.5 U/ml) or protamine (32.5 microg/ml). For the 8 patients, the ACTs returned to baseline with heparinase (20 U/ml) or protamine (50 microg/ml). The authors found no difference in the expression of P-selectin in samples neutralized with heparinase, but samples neutralized with protamine showed a significant decrease in the expression of P-selectin when compared with heparinized samples. CONCLUSIONS: At dosages that reverse the anticoagulant effects of heparin, heparinase has minimal effects on platelets, whereas platelet reactivity was markedly inhibited by protamine.     

Conformational and topological requirements of cell-permeable peptide function

BACKGROUND: Magnetic resonance imaging and positron emission tomography were used to study the size and metabolic rate of the caudate and the putamen in 18 patients with schizophrenia (n=16) or schizo-affective disorder (n=2) and 24 age- and sex-matched control subjects. METHODS: The patients were either never medicated (n=7) or drug free (n=11) for a median of 3 weeks. During uptake of fludeoxyglucose F 18, all patients performed a serial verbal learning test. Positron emission tomographic and magnetic resonance imaging scans were coregistered, and the caudate and the putamen were traced on the magnetic resonance image. RESULTS: The striatum had a significantly lower relative metabolic rate in schizophrenics than in controls. Never-medicated patients had lower metabolic rates in the right putamen (ventral part of the dorsal striatum) than previously medicated patients. The caudate was significantly smaller in never-medicated patients than in controls and largest in previously medicated patients. Patients with higher relative metabolic rates in the putamen scored higher on the Abnormal Involuntary Movements Scale. CONCLUSIONS: The findings are consistent with reports of striatal enlargement in previously medicated patients and size increases after neuroleptic treatment. Never-medicated patients, in contrast, had ventral striatal structures that were smaller and less active than those observed in controls and previously medicated patients.