Synergistic inhibition of thrombin-induced platelet aggregation by the novel nitric oxide-donor GEA 3175 and adenosine

1. The influence of the novel nitric oxide-donor GEA 3175 on thrombin- and ionomycin-stimulated human platelets was investigated. The effect of GEA 3175 was compared with that of adenosine, an activator of platelet adenylyl cyclase. 2. GEA 3175 inhibited thrombin-induced secretion of ATP but did not affect aggregation; similar results were obtained with adenosine. 3. Thrombin-stimulated rises in the cytosolic free Ca2+ concentration, [Ca2+]i, were dose-dependently inhibited by GEA 3175 and adenosine. GEA 3175 and adenosine maximally reduced the initial rise in [Ca2+]i by 41% and 35%, respectively. 4. Simultaneous exposure to GEA 3175 and adenosine nearly abolished both the functional responses (i.e. aggregation and degranulation) and the rises in [Ca2+]i in thrombin-stimulated platelets. 5. Aggregation and increases in [Ca2+]i triggered in platelets by the Ca(2+)-ionophore ionomycin were only marginally affected by a combination of GEA 3175 and adenosine. 6. GEA 3175 potently increased the guanosine 3':5'-cyclic monophosphate (cyclic GMP) content in platelets but did not affect adenosine 3':5'-cyclic monophosphate (cyclic AMP) levels. Adenosine did not increase either the cyclic AMP or the cyclic GMP levels in platelets. However, adenosine and GEA 3175 combined significantly elevated the platelet cyclic AMP content. 7. The results show that simultaneous exposure to GEA 3175 and adenosine promotes potent anti-aggregatory properties in platelets in vitro. The findings suggest that blockage of the cytosolic Ca(2+)-signal, which is probably mediated by an amplified cyclic nucleotide response, is an important event during the synergistic inhibition of thrombin-induced aggregation.     

The role of histamine in platelet aggregation by physiological and immunological stimuli

This paper reviews the current state of the debate on the relationship between mental disorder and violent behaviour. Starting from the discussion of methodological approaches to assessing a possible association, the most important studies carried out on the issue in recent years are discussed. Their results concur in supporting the assumption that there is a moderate but reliable association between mental disorder and violence. However, this does not imply that people with mental illness are generally more likely to commit violent acts than members of the general population. An elevated risk of violent behaviour is only evident for specific psychiatric diagnoses and for particular symptom constellations. For schizophrenia and other psychotic disorders, a significant increase in the likelihood to commit violent acts is reported. Substance use disorder and antisocial personality disorder, however, represent a markedly higher risk for violent behaviour. The article further discusses possible determinant of violent behaviour such as psychotic symptoms and comorbidity with substance abuse, and considers who is at particular risk of becoming a target of violent acts.     

High shear stress induced platelet aggregation (h-SIPA) and effects of antiplatelet therapy

A physiologic time averaged mean shear stress in stenosed coronary artery reach more than 350 dyne/cm2. Pathologic stenosis can directly lead to shear-induced aggregation of platelets. Platelet aggregation in response to pathologically elevated shear stress is depend on the presence of plasma von Willebrand factor (vWF) and platelet receptor glycoprotein (GP) Ib/IX and GPIIb/IIIa. Fibrinogen bridging thrombus play as key factor at low shear rate, however, vWF is most important factor at high shear rate. When high shear stress are applied to vWF, vWF change the shape round to linear, and bind to extracellular matrix such as collagen type I or III exposed to blood by rupture of atheromatous plaque. Consequently vWF interact with GP Ib/IX for initial adhesion without agonist stimulation, which is followed by activation of GPIIb/IIIa receptor and co-binding with GPIIb/IIIa and vWF. The binding of platelets via vWF is strengthen to sustain the opposing effect of high shear forces in coronary artery. In our study, significant increases of h-SIPA and plasma vWF levels were observed in patients with acute coronary syndrome compared with patients with chronic coronary artery disease. The additional application of ticlopidine or cilostazol to aspirin therapy significantly inhibition of h-SIPA in patient with acute coronary syndrome, however, less effective than patients with chronic coronary artery disease.     

Fibrinogen receptor (GPIIb-IIIa) antagonists derived from 5,6-bicyclic templates. Amidinoindoles, amidinoindazoles, and amidinobenzofurans containing the N-alpha-sulfonamide carboxylic acid function as potent platelet aggregation inhibitors

A series of highly potent and specific fibrinogen receptor antagonists have been discovered and optimized through structural modification of the novel amidinoindole and benzofuran compounds, I and II. Systematic linker optimization afforded the amidinobenzofuran-containing inhibitor 29, which displayed an IC50 value of 250 nM in platelet aggregation assays. Attempts to enhance activity by modification of the beta-position of the beta-alanyl carboxylate group of 29 had only a modest effect on inhibitory activity in aggregation assays. Analogues prepared to enhance the activity by conformational restriction were also found to be equally or less potent. In contrast, modification at the alpha-position of the beta-alanyl carboxylate group resulted in the identification of extremely potent and novel amidinobenzofuran-containing derivatives 46-49. Reexamination of 5,6-bicyclic aromatic nucleus led to the further identification of amidinoindole- and amidinoindazole-containing derivatives 53-55. These analogues, 46-49 and 53-55, exhibited potent in vitro activity with IC50 values of 25-65 nM in platelet aggregation assays and an IC50 value of 2 nM in fibrinogen binding assays and demonstrated a selectivity of > 50,000-fold for GPIIb-IIIa versus the most closely related integrin, the vitronectin receptor, alpha v beta 3.     

Role of chondroitin 4-sulphate as a receptor for polycation induced human platelet aggregation

1. Proteoglycans provide negatively charged sites on the surface of platelets, leukocytes and endothelial cells. Since chondroitin 4-sulphate is the main proteoglycan present on the platelet surface, the role of this molecule in mediating the activation of human platelets by polylysine was studied. 2. Platelets were desensitized with phorbol 12-myristate 13-acetate (PMA, 10 nM) 5 min before the addition of polylysine to platelet-rich plasma (PRP). Changes in the intracellular Ca2+ concentration were measured in fura2-am (2 microM) loaded platelets and protein phosphorylation was assessed by autoradiography of the electrophoretic profile obtained from [32P]-phosphate labelled platelets. The release of dense granule contents was measured in [14C]-5-hydroxytryptamine loaded platelets and the synthesis of thromboxane (TXA2) was assessed by radioimmunoassay. Surface chondroitin 4-sulphate proteoglycan was degraded by incubating platelets with different concentrations of chondroitinase AC (3 min, 37 degrees C). The amount of chondroitin 4-sulphate remaining in the platelets was then quantified after proteolysis and agarose gel electrophoresis. 3. The addition of PMA to PRP before polylysine inhibited the aggregation by 88 +/- 18% (n = 3). Staurosporine (1 microM, 5 min) prevented the PMA-induced inhibition. Chondroitinase AC (4 pu ml-1 to 400 muu ml-1, 3 min) abolished the polylysine-induced aggregation in PRP but caused only a discrete inhibition of ADP-induced aggregation. The concentration of chrondroitin 4-sulphate in PRP (0.96 +/- 0.2 microgram/10(8) platelets, n = 3) and in washed platelets (WP; 0.35 +/- 0.1 microgram/10(8) platelets, n = 3) was significantly reduced following incubation with chondroitinase AC (PRP = 0.63 +/- 0.1 microgram/10(8) platelets and WP = 0.08 +/- 0.06 microgram/10(8) platelets). 4. Washed platelets had a significantly lower concentration of chondroitin 4-sulphate than platelets in PRP. The addition of polylysine to WP induced a rapid increase in light transmission which was not accompanied by TXA2 synthesis or the release of dense granule contents. This effect was not inhibited by sodium nitroprusside (SNP), iloprost, EDTA or the peptide RGDS. This event was accompanied by the discrete phosphorylation of plekstrin and myosin light chain, which were inhibited by staurosporine (10 microM, 10 min). The hydrolysis of platelet surface chondroitin 4-sulphate strongly reduced the polylysine-induced phosphorylation. 5. Our results indicate that polylysine activates platelets through a specific receptor which could be the proteoglycan chondroitin 4-sulphate present on the platelet membrane.     

The P2Y1 receptor is necessary for adenosine 5''-diphosphate-induced platelet aggregation

Population control has become a major problem in many wildlife species. Fertility control through immunocontraception has been proposed as a method for reducing population size. We have tested the concept that immunocontraception can be achieved with a recombinant ectromelia virus expressing an ovary-specific antigen, the mouse zona pellucida 3 (ZP3) glycoprotein. Female mice infected with the recombinant virus produced autoimmune antibodies against ZP3 and were infertile for 5-9 mo after infection. For almost half the infertile mice, immunity to ZP3 was associated with a disruption of ovarian follicular development and the depletion of mature follicles without observable oophoritis. Mice returned to fertility as the anti-ZP3 antibody level in the serum decreased. Reinfection of the mice with the recombinant virus boosted the anti-ZP3 response and restored infertility.     

Phospholipase A2: its role in ADP- and thrombin-induced platelet activation mechanisms

ADP and thrombin are two of the most important agonists of platelet aggregation--a cellular response that is critical for maintaining normal hemostasis. However, aberrant platelet aggregation induced by these agonists plays a central role in the pathogenesis of cardiovascular and cerebrovascular diseases. Agonist-induced primary or secondary activation of phospholipases leads to generation of the second messengers that participate in biochemical reactions essential to a number of platelet responses elicited by ADP and thrombin. Phospholipase A2 (PLA2) has been linked to cardiovascular diseases. However, the mechanism(s) of activation of PLA2 in platelets stimulated by ADP and thrombin has remained less well defined and much less appreciated. The purpose of this review is to examine and compare the molecular mechanisms of activation of PLA2 in platelets stimulated by ADP and thrombin.     

Plastic instability and flow localization in shear at high rates of deformation

The occurrence of adiabatic shear bands in metals is analyzed using models based on

• 1.(i) load instability and
• 2.(ii) flow localization. In the former case, shear strain concentration is identified with the occurrence of a maximum in the shear stress-shear strain curve.

By contrast, the latter model treats localization as a process which begins to develop at a material imperfection at the onset of deformation. In both cases, the flow softening arising from adiabatic heating is taken to be the driving force behind the process. The predictive capabilities of the two types of model are compared using data on shear band formation during the explosive expansion of 4340 steel cylinders. It is shown that the flow localization model forecasts the occurrence of adiabatic shear bands more accurately and is thus more useful for the prediction of nonuniform flow at high rates of strain such as those which occur during impact loading and metalcutting.     

HDL3-mediated inhibition of thrombin-induced platelet aggregation and fibrinogen binding occurs via decreased production of phosphoinositide-derived second messengers 1,2-diacylglycerol and inositol 1,4,5-tris-phosphate

We demonstrate that physiological concentrations of HDL3 inhibit the thrombin-induced platelet fibrinogen binding and aggregation in a time- and concentration-dependent fashion. The underlying mechanism includes HDL3-mediated inhibition of phosphatidylinositol 4,5-bis-phosphate turnover, 1,2-diacylglycerol and inositol 1,4,5-tris-phosphate formation, and intracellular calcium mobilization. The inhibitory effects of HDL3 on inositol 1,4,5-tris-phosphate formation and intracellular calcium mobilization were abolished after covalent modification of HDL3 with dimethylsuberimidate. Furthermore, they could be blocked by calphostin C and bis-indolylmaleimide, 2 highly selective and structurally unrelated protein kinase C inhibitors. However, the inhibitory effects of HDL3 were not blocked by H89, a protein kinase A inhibitor. In addition, HDL3 failed to induce cAMP formation but stimulated the phosphorylation of the protein kinase C 40- to 47-kD major protein substrate. We observed a close temporal relationship between the HDL3-mediated inhibition of thrombin-induced inositol 1,4,5-tris-phosphate formation, intracellular calcium mobilization, and fibrinogen binding and the phosphorylation of the protein kinase C 40- to 47-kD major protein substrate. Taken together, these findings indicate that the HDL3-mediated inhibition of thrombin-induced fibrinogen binding and aggregation occurs via inhibition of phosphatidylinositol 4,5-bis-phosphate turnover and formation of 1,2-diacylglycerol and inositol 1,4,5-tris-phosphate. Protein kinase C may be involved in this process.     

Spontaneous platelet aggregation in arterial insufficiency: mechanisms and implications

To investigate the clinical implications and mechanisms of spontaneous platelet aggregation (SPA) in man, 150 normal subjects, 22 patient controls and 130 patients with vascular insufficiency were studied. SPA was negative in normal subjects and patient controls whereas it was positive in 36 of 66 (54%) patients with transient ischemic attacks, 6 of 32 (19%) patients with stable angina, 7 of 10 (70%) patients with acute myocardial infarction and 11 of 14 (80%) patients with acute peripheral arterial insufficiency. The SPA was inhibited with aspirin in vivo, and inhibited competitively in vitro by low concentrations of aspirin, 2-chloroadenosine, prostaglandin E1 or apyrase but only by high concentrations of heparin or hirudin. Addition of platelet-poor plasma from patients with positive SPA did not cause normal platelets to aggregate. Treatment of patients who had acute peripheral arterial insufficiency with aspirin and dipyridamole prevented SPA with notable clinical improvement of the ischemic changes.     

Shear-induced platelet aggregation (SIPA) monitoring of hemostatic effect during platelet transfusion in a patient with Glanzmann's thrombasthenia: a comparison between SIPA and conventional platelet aggregation

Shear-induced platelet aggregation (SIPA) in a patient with Glanzmann's thrombasthenia was examined during platelet infusion therapy. Prior to platelet infusion, SIPA measured with the modified cone-and-plate type viscometer, as well as ADP-and collagen-induced platelet aggregation measured with the conventional platelet aggregometer, were absent. The patient's SIPA after multiple infusions of platelet, in parallel with the bleeding time and the clinical hemostatic effect, improved to the normal level, while ADP-and collagen-induced platelet aggregation remained abnormal. These observations imply that SIPA is physiologically more relevant than the conventional agonist-induced platelet aggregometry in this type of the disease.     

Chronic electrical stimulation of the auditory nerve at high stimulus rates: a physiological and histopathological study

The ubiquitous distribution of peroxisomes and the identification of a number of inherited diseases associated with peroxisomal dysfunction indicate that peroxisomes play an essential part in cellular metabolism. Some of the most important metabolic functions of peroxisomes include the synthesis of plasmalogens, bile acids, cholesterol and dolichol, and the oxidation of fatty acids (very long chain fatty acids > C22, branched chain fatty acids (e.g. phytanic acid), dicarboxylic acids, unsaturated fatty acids, prostaglandins, pipecolic acid and glutaric acid). Peroxisomes are also responsible for the metabolism of purines, polyamines, amino acids, glyoxylate and reactive oxygen species (e.g. O-2 and H2O2). Peroxisomal diseases result from the dysfunction of one or more peroxisomal metabolic functions, the majority of which manifest as neurological abnormalities. The quantitation of peroxisomal metabolic functions (e.g. levels of specific metabolites and/or enzyme activity) has become the basis of clinical diagnosis of diseases associated with the organelle. The study of peroxisomal diseases has also contributed towards the further elucidation of a number of metabolic functions of peroxisomes.     

Amylin: physiological roles in the kidney and a hypothesis for its role in hypertension

1. There are high-affinity binding sites for amylin in the renal cortex associated with proximal tubules. These appear to represent seven transmembrane (heptatopic) receptors that are known to form ternary complexes with G-proteins and activate second messenger systems. 2. Amylin stimulates sodium/water reabsorption from the basolateral side of the proximal tubules and plays a role in sodium homeostasis. 3. The transient expression of amylin-like mRNA has been detected perinatally, using in situ hybridization, in the subnephrogenic zone of the metanephros and is associated with proximal tubules of the developing nephron. There it is thought to play a role as a growth factor for brush border epithelial cells in the developing kidney and in renal regrowth in the adult kidney. 4. In two models of hypertension, the spontaneously hypertensive rat (SHR) and one created surgically by subtotal nephrectomy, renal amylin receptors are activated. In the SHR, activation precedes the rise in blood pressure and suggests that activation of the amylin system may be an important event in the development of hypertension.     

Antibody assay for adenoviruses and mycoplasma pneumoniae by the platelet aggregation test

The practicability of the platelet aggregation test (PAT) in routine antibody assays for adenoviruses and Mycoplasma pneumoniae is demonstrated. An increase in reliability and sensitivity was achieved by employment of EDTA for washing the platelet and by addition of albumin to the test medium. Various lots of platelets yielded reproducible PAT titres which attained 1 : 156,250 in positive patient's sera. Commercially available antigens assigned for complement-fixation (CF) tests proved to be appropriate for the PAT. Checkerboard titrations revealed that the platelet reactivity of immune complexes formed was maintained in a range of up to 625-fold alterations of the antibody: antigen ratio. The fact that PAT and CF titres did not always correlate is interpreted as being due to the differences between complement-fixing and platelet reactivities of various immunoglobulin classes.     

Intracerebral hemorrhage and treatment with platelet aggregation inhibitors: study of 21 cases

INTRODUCTION: The place of platelet antiaggregants in the aetiology of intracerebral hemorrhage (IH) has not been extensively studied. OBJECTIVE: To analyze the characteristics of IH in patients treated with platelet antiaggregants and the possible clinical and prognostic differences from other primary IH. PATIENTS AND METHODS: A retrospective study of patients admitted to hospital with primary IH from 1985 to 1997. The cases were IH patients while being treated with platelet antiaggregants. For each case we selected two controls with IH and similar age and IH risk factors. The following data was analyzed: start of clinical condition; type, dose, indication and duration of treatment with antiaggregants; mortality, localization, volume and extension of IH to the ventricles. The last four variables were compared with the control group using the ji squared test (chi 2) and the t student test. RESULTS: 21one patients had a primary IH while being treated with antiaggregants: 20 with salicylates (17 aspirin and 3 riflusal) and one with ticlopidine. The dose of aspirin was 500 mg or less in 90% of the cases. In the group treated with salicylates, this was given for more than 20 months in 90% of the cases. Initially there was no clinical progression in any case. No significant differences were observed between the variable compared, although there was a tendency to greater volume, extension to the ventricles and mortality in the group treated with antiaggregants. CONCLUSIONS: More studies with larger numbers of patients are necessary to be able to confirm the tendencies observed.     

2-Alkynyl derivatives of adenosine-5'-N-ethyluronamide: selective A2 adenosine receptor agonists with potent inhibitory activity on platelet aggregation

A series of new 2-alkynyl and 2-cycloalkynyl derivatives of adenosine-5'-N-ethyluronamide (NECA) and of N-ethyl-1'-deoxy-1'-(6-amino-2-hexynyl-9H-purin-9-yl)-beta-D- ribofuranuronamide (1, HE-NECA), bearing hydroxy, amino, chloro, and cyano groups in the side chain, were synthesized. The compounds were studied in binding and functional assays to assess their potency for the A2 compared to A1 adenosine receptor. The presence of an alpha-hydroxyl group in the alkynyl chain of NECA derivatives accounts for the A2 agonist potency, leading to compounds endowed with sub-nanomolar affinity in binding studies. However, these analogues also possess good A1 receptor affinity resulting in low A2 selectivity. From functional experiments the 4-hydroxy-1-butynyl (6) and the 4-(2-tetrahydro-2H-pyranyloxy)-1-butynyl (16) derivatives appear to be very potent in inducing vasorelaxation without appreciable effect on heart rate. The new compounds were also tested as inhibitors of platelet aggregation induced by ADP. Introduction of an alpha-hydroxyl group in the alkynyl side chain caused a greater increase in antiaggregatory activity than either NECA or HE-NECA, resulting in the most potent inhibitors of platelet aggregation so far known in the nucleoside series. The presence of an alpha-quaternary carbon such as the 3-hydroxy-3,5-dimethyl-1-hexynyl (12) and the 3-hydroxy-3-phenyl-1-butynyl (15) derivatives markedly reduced the antiaggregatory potency without affecting the A2 affinity. The hydrophobicity index (k') of the new nucleosides barely correlated with the binding data, whereas high k' values were associated with increased A2 vs A1 selectivity but with reduced activity in all functional assays. Some of the compounds synthesized possess interesting pharmacological properties. Compounds having an appropriate balance between vasorelaxation and antiplatelet activity, if confirmed in vivo, deserve further development for the treatments of cardiovascular disorders.     

Modulation of thrombin-induced platelet aggregation by inhibition of calpain by a synthetic peptide derived from the thiol-protease inhibitory sequence of kininogens and S-(3-nitro-2-pyridinesulfenyl)-cysteine

Thrombin-induced platelet aggregation has been suggested to play an important role in reocclusion following thrombolytic therapy of angioplasty for treatment of myocardial infarction. We previously demonstrated that aggregation of washed platelets by thrombin is accompanied by cleavage of aggregin, a putative ADP receptor, and that these events are indirectly mediated by calpain, expressed on the surface of the external membrane. High-molecular-mass kininogen (HK) contains, in its heavy chain, domain 2, which is responsible for its action as a potent inhibitor of platelet calpain. Domain 3 of the heavy chain of HK directly inhibits binding of thrombin to platelets, confounding mechanistic studies using the entire molecule. Moreover, HK, a protease of 120 kDa, is unsuitable as a potential pharmacological agent. The highly conserved sequence Gln-Val-Val-Ala-Gly, present in HK and its evolutionary precursors, the cystatins, is thought to be involved in the binding of cysteine proteases but is, itself, not inhibitory. An affinity analog, Phe-Gln-Val-Val-Cys(Npys)-Gly-NH2(Npys, 3-nitro-2-sulfenylpyridine), P1, corresponding to the thiol-protease-binding sequence in HK and containing a ligand, Npys, that can react with the free sulfhydryl group in the active site of calpain, was synthesized. P1 was an irreversible inhibitor of platelet calpain. P1 selectively inhibited thrombin-induced aggregation of washed platelets and platelets in plasma, but did not inhibit the aggregatory effects of other platelet agonists. P1 did not inhibit the amidolytic activity and coagulant activity of thrombin. Unlike HK, P1 did not inhibit binding of thrombin to washed platelets. P1 did not inhibit thrombin-induced platelet-shape change. P1 neither raised intracellular levels of cAMP nor did it interfere with the ability of thrombin to antagonize the rise in intracellular levels of cAMP induced by iloprost, an analog of prostaglandin I2. The design and synthesis of P1 could leave to the development of a new class of inhibitors that selectively block thrombin-induced platelet aggregation while sparing other functions of this pathophysiological protease and without inhibiting the action of other platelet agonists.     

Blood viscosity and triglyceridemia. Findings using a co-axial cylindrical viscosimeter at low "shear rates"

Examination of blood viscosity at low shear rates using a co-axial cylinder viscometer showed a significant difference between the means of values observed in hypertrigliceridemic patients compared with that of control subjects. This result differs from what has been reported by most workers although generally greater shear rates have been used. Calculation of the "r" coefficient and plotting of the regression line for each shear rate showed that there is no linear correlation between blood viscosity and triglyceridaemia, whose variations occur quite independently. It is suggested that the absence of a correlation between the two parameters examined may depend on various factors, of which the most important are those pertaining to the rheological properties of red blood cells and to the structure and chemical and physical characteristics of the triglyceride molecule and of the lipoproteins and chylomicrons which transport them.     

Evidence for a third component in neutrophil aggregation: potential roles of O-linked glycoproteins as L-selectin counter-structures

The homotypic aggregation of neutrophils requires the participation of L-selectin and the beta 2-integrins, but it has not been clear whether the two receptors recognize one another as counter-structures or whether other adhesion molecules are involved. We have examined aggregation of live neutrophils with target populations, manipulated to alter expression of adhesive epitopes, using flow cytometry. A target population depleted of both L-selectin and activatable beta 2-integrin displayed an ability to aggregate with live neutrophils, suggesting that these two molecules are not counter-structures. We also found that an O-sialoglycoprotease (GCP) from Pasteurella haemolytica is capable of inhibiting homotypic aggregation. Neutrophils treated with GCP lose O-glycosylated proteins but retain L-selectin and activatable beta 2-integrin. One or more of the GCP substrates appears to function in L-selectin-dependent binding but not in beta 2-integrin-dependent binding. Together the data suggest a mechanism of aggregation that is analogous to leukocyte-endothelial cell adhesion in which a low-affinity carbohydrate-dependent interaction precedes a high-affinity integrin-dependent adhesion.