Influence of acetylsalicylic acid (1.0 g/day) on platelet survival time, β-thromboglobulin and platelet factor 4 in patients with peripheral arterial occlusive disease

In this study we investigated the influence of acetylsalicylic acid (ASA) 1.0 g/day on 111-In-platelet survival time (PST) and on plasma levels of β-thromboglobulin (β-TG) and platelet factor 4 (PF 4) in 37 patients (median age: 63.4 years) with arteriographically proven peripheral arterial occlusive disease (PAOD) in a chronic stable phase. We found a slight but significant increase of PST during therapy with ASA (weighted mean (WM): 184.3 → 193.2 [median]hours, p < 0.05; multiple hit (MH): 182.4 → 192.8 hours, p<0.005) for the total group of patients. Concerning the influence of risk factors of PAOD on PST during ASA-therapy, there was a significant increase of PST only in the nondiabetics (WM: 180.3 → 204.6 hours p<0.01; MH: 176.8 → 195.3 hours, p<0.01). There was a negative correlation between the baseline values of PST and their increase following ASA therapy (WM: R = - 0.63; p<0.0001; MH: R = - 0.61, p<0.0001). The pretreatment levels of β-TG - but not PF 4 - were significantly (p<0.001) elevated compared to healthy controls. Therapy with ASA caused a significant decrease in the plasma levels of β-TG (median: 30.4 → 26.6 ng/ml, p<0.001) and PF 4 (2.95 2.2 ng/ml, p<0.01).     

Low molecular weight heparin and the heparin mobilisable pool of platelet factor 4 are reduced in young survivors of myocardial infarction

The platelet factor 4 (PF4) mobilisation properties of low molecular weight heparin (Fraxiparine, Sanofi Winthrop, France) in young survivors of myocardial infarction (YSMI) and healthy volunteers have been investigated. The study group consisted of 42 YSMI less than 44 years old, all of them with angiographically proven occlusive coronary artery disease, studied 6 to 24 months after the acute event. The control group was composed of 30 healthy men of similar age. Subjects from the study and control groups were allocated to the following subgroups: those receiving 60 or 120 IU/kg b.w. of standard heparin (Polfa Kutno, Poland) and those receiving 60, 120 or 180 IC anti-Xa U/kg b. w. of low molecular weight heparin (Fraxiparine, Sanofi Winthrop, France) as a single intravenous injection. Additionally, in five YSMI patients the influence of prolonged aspirin administration (0.3g daily for more than 30 days) on the Fraxiparine mobilisable pool of PF4 and β-thromboglobulin (β-TG) concentration in the plasma was determined after injection of 180 IC anti-Xa U/kg b.w. of the drug. The PF 4 and β-TG concentration in the plasma was evaluated using enzyme immunoassay methods before heparin or Fraxiparine intravenous injection and 2, 5, 10, 20, 30 and 60 min after. In both, the control and YSMI groups baseline PF4 levels were found to be normal. Moreover, similar marked dose-dependent increases of PF4 concentration in the plasma measured after 60 and 120 IU/kg b.w. of heparin as well as after 60 and 120 IC anti-Xa U/kg b.w. of Fraxiparine was found. The administration of 120 IU/kg b.w. of heparin resulted in a reduced rise in plasma PF 4 in YSMI as compared to healthy controls. The same phenomenon was observed when 180 IC anti-Xa U/kg b. w. of Fraxiparine was injected intravenously. In YSMI treatment with aspirin had no influence on the Fraxiparine mobilisable pool of PF 4 or the β-TG concentration in the plasma. These results suggest that mobilisable pool of platelet factor 4 in young survivors of myocardial infarction derives from the “nonplatelet pool” and that reduction of heparin- or Fraxiparine-releasable pool of PF4 may reflect an impaired endothelium function, probably due to atherosclerosis.     

Experimental evidence for renal catabolism of fibrin fragment β15–42

The elimination of labelled human fibrin fragment β15–42 from the circulation has been studied in an experimental rat model. When ¹²⁵I β15–42 was injected as a bolus into a control group of rats, its elimination from the circulation could be fitted to a two compartment exponential model. A fast initial elimination had a t$\text{1}\text{2}$ of less than 2 min and this was followed by a slower component with a calculated t$\text{1}\text{2}$ of 135 min. The fast component was caused by equilibration of peptide throughout the intra and extra vascular spaces and the slow component reflected the action of catabolic processes once equilibration had been attained. Rats that had undergone bilateral nephrectomy eliminated significantly less of the labelled peptide than the control animals at a given time and the t$\text{1}\text{2}$ of the slow component was prolonged, but not significantly , to 159 min. Rats with ligated ureters had statistically indistinguishable elimination curves from control rats. Examination of the heterogeneity of labelled peptide in plasma samples taken during the experiments by immunoprecipitation and by gel filtration revealed progressive extensive degradation in the control and ureteral ligated rats, but less degradation in nephrectomized rats. These results suggest that β15–42 is eliminated,in part, from the circulation by uptake and catabolism by the kidney. It is concluded that impaired renal function may result in elevated plasma levels of β15–42 antigen in human renal failure without the need for an increased rate of production of the peptide.     

Alpha chain crosslinking of human fibrin: Purification and radioimmunoassay development for two Aα chain regions involved in crosslinking

Rapid and efficient purification methods that include hydrophobic chromatography on Phenyl Sepharose CL-4B have been developed for the peptides that span residues 241–476 (CNBr VIII), and 518–584 (CNBr X) in the Aα chain of human fibrinogen. Amino acid analysis, NH₂-terminal sequence determination, and SDS-PAGE indicated that greater than 95% purity of each peptide was achieved. Sensitive and specific radioimmunoassays capable of detecting antigen in the range of 0.1–10.0 pmol/ml have also been developed for CNBr VIII and CNBr X. These assays have been characterized and successfully applied in studies designed to localize the two COOH-terminal Aα chain regions in column effluents of CNBr-digested fibrinogen and crosslinked fibrin. When these immunoassays were used to study purified preparations of a high molecular weight, crosslink-containing CNBr derivative of α polymer, the data provided immunologic confirmation for the involvement of CNBr VIII and CNBr X in α chain crosslinking.     

Inhibitory effect of vitamin E (α -tocopherol) on spontaneous platelet aggregation in whole blood

Vitamin E (D-α-tocopherol) inhibited spontaneous human platelet aggregation in whole blood in the 20–200 μ g/ml range. When α -tocopherol (20 μ g/ml) and aspirin (0.5 mM), or α -tocopherol and the mixture of phosphocreatine (1.5 mM) and creatine phosphokinase (50 U/ml) (CP/CPK) were added to this reaction system, a synergic inhibitory effect on aggregation was observed. On the other hand, when both α -tocopherol and the specific inhibitor of platelet activating factor (CV-3988 : 0.38 mM) were added to this system, the inhibition was the same as that caused by the addition of CV-3988 alone, suggesting there was no synergism, i.e., that the effect of α -tocopherol is related to the inhibition of platelet activating factor (PAF)-induced platelet aggregation in whole blood. However, α -tocopherol (20 or 50 μ g/ml) did not inhibit PAF (10 nM) induced platelet aggregation in platelet rich plasma (PRP). These results suggest that the inhibition of platelet aggregation in whole blood by α -tocopherol is due to the inhibition of PAF synthesis, and is unrelated to adenosine diphosphate (ADP) or thromboxane A2.     

Effect of edta and PGE1 on β-thromboglobulin liberation from platelets

The aim of this study was to evaluate the effect of PGE₁ and EDTA on liberation of β-thromboglobulin (βTG) from platelets in vitro. Liberation of BTG was followed in citrated blood at room temperature for 120 minutes after venesection. PGE₁ reduced βTG liberation, and maximal inhibition was attained by concentrations greater than 2 × 10⁻⁶M. EDTA induced the efflux of βTG. This EDTA-induced efflux was delayed but not prevented by PGE₁ and by citrate; it was not found at 0–4°C. Therefore the use of EDTA to prevent βTG liberation during sampling for in vitro or in vivo studies depends heavily on modifying factors such as PGE₁ and low temperature, and on the time taken to process samples. Its effectiveness must be in some doubt where the platelets may be sufficiently stimulated to overcome these modifying influences, or where handling of samples is less than optimal.     

Inhibition of the availability of platelet factor 3 by increased concentration of lecithin in platelets caused by gonadal steroids

Platelet factor 3 (PF-3) activity and the phosphatide composition of plasma and platelets were studied in a group of oophorectomized women on estrogen-gestagen treatment. Based on the results the following hypothesis is put forward. An increased concentration of lecithin in the platelets stabilizes the platelet membrane and inhibits the conformational changes of the membrane which is supposed to precede the “availability” of PF-3. Estrogen treatment induces a decrease of plasma lysolecithin resulting in a decreased formation of platelet lecithin. The increased PF-3 activity observed during the estrogen dominated phase may thus not be due to an increase of lipids promoting clotting but to a decrease of platelet lecithin which normally prevents the “release” of clot promoting activities.     

Effect of EDTA and PGE1 on beta-thromboglobulin liberation from platelets

The aim of this study was to evaluate the effect of PGE₁ and EDTA on liberation of β-thromboglobulin (βTG) from platelets in vitro. Liberation of BTG was followed in citrated blood at room temperature for 120 minutes after venesection. PGE₁ reduced βTG liberation, and maximal inhibition was attained by concentrations greater than 2 × 10⁻⁶M. EDTA induced the efflux of βTG. This EDTA-induced efflux was delayed but not prevented by PGE₁ and by citrate; it was not found at 0–4°C. Therefore the use of EDTA to prevent βTG liberation during sampling for in vitro or in vivo studies depends heavily on modifying factors such as PGE₁ and low temperature, and on the time taken to process samples. Its effectiveness must be in some doubt where the platelets may be sufficiently stimulated to overcome these modifying influences, or where handling of samples is less than optimal.     

Genetic variation in the transforming growth factor β1 gene in multiple sclerosis

Transforming growth factor β1 (TGFβ1) is a Th2 cytokine encoded on chromosome 19q13, a region possibly linked to multiple sclerosis (MS). TGFβ1 exerts favorable effects on experimental allergic encephalomyelitis. We performed a comprehensive search for genetic variants in this gene in 122 population-based sporadic cases of MS. We detected six variants, including three missense variants. We tested for association of the variants with susceptibility and course of MS and for linkage and transmission disequilibrium in a family series consisting of 395 samples in 59 pedigrees. Genetic variation in TGFB1 does not appear to contribute in a major way to susceptibility to MS.     

β-endorphin and β-lipotropin plasma levels in chronic schizophrenia, primary affective disorders and secondary affective disorders

Plasma levels of β-endorphin (β-EP), β-lipotropin (β-LPH) and ACTH were assayed in 15 chronic schizophrenics, nine patients with primary affective disorders (PAD) and seven patients with secondary affective disorders (SAD). Patients received no therapy for 10 days prior to study. All subjects were studied once; eight schizophrenics were studied again after 10 days of Haloperidol therapy, at a dose of 0.1 mg/kg body weight. β-LPH levels were significantly higher in the schizophrenics without hallucinations and in the PAD and SAD patients in comparison to the controls; β-EP levels were higher in the schizophrenics and SAD patients compared to the controls; and ACTH concentrations were significantly lower in the SAD than in the PAD patients. Haloperidol therapy failed to induce significant changes in β-LPH, β-EP or ACTH plasma levels.

Statistical evaluation by multiple linear regression confirmed the significant positive correlations among β-LPH, β-EP and ACTH in the controls, schizophrenics with hallucinations, and PAD and SAD patients, while inverse correlations between β-LPH and the other two peptides were found in the schizophrenics without hallucinations. The same analysis revealed that, while in the PAD patients equimolar amounts of the three peptides occurred, the SAD patients were characterized by ACTH/β-LPH and ACTH/β-EP molar ratios of 0.5.

Although these results are preliminary, they seem to indicate that β-LPH, β-EP and ACTH secretion patterns in chronic schizophrenia, PAD and SAD may help in revealing specific groups of patients with different biochemical substrates.     

Tissue factor pathway inhibitor (TFPI) activity in uremic patients during hemodialysis

We studied tissue factor pathway inhibitor (TFPI) activity during hemodialysis in 10 uremic patients who were not receiving anticoagulant for at least 120 minutes. TFPI activity before dialysis was normal (patients 107 ± 5.8%, controls 104 ± 4.5%). During extracorporeal circuit it rose progressively with a statistically significant difference, reaching a plateau between 60 and 120 minutes. Since thrombin induces a marked redistribution and release of TFPI from stimulated endothelial cells and platelets contain about 10% of TFPI activity that is secreted following activation it is possible that thrombin-induced release of TFPI by endothelium and platelets could account for the increased TFPI we found during hemodialysis. To investigate this possibility we measured during dialysis β-thromboglobulin (β-TG), thrombinantithrombin complex (TAT) and prothrombin fragment 1.2 (F 1.2). The increased levels of β-TG, TAT and F1.2 we noted during extracorporeal circuit are in keeping with this concept.

One hundred eighty minutes after initiation of dialysis, by which time all patients were receiving heparin there was a further increase in TFPI (to more than 200% of baseline), due to the presence of the glycosaminoglycan. This was due the previously reported displacement by heparin of the major intravascular pool of TFPI, from endothelial cell surfaces.     

Monoclonal antibodies to bovine platelet factor 4: Species interactions to platelets and megakaryocytes using indirect immunocytofluorescence

Murine monoclonal antibodies (mAb) were raised to a purified product of bovine PF-4, a 9,500 dalton protein with heparin neutralization activity comparable to that of human PF-4. Using a non-radioactive slide immunoenzymatic assay, four major classes of mAb could be identified when comparisons were made between purified antigens of PF-4 and β-TG-like protein from both bovine and human species. Type 1 cross-reacted with all four antigens; type 2 reacted with PF-4s; type 3 reacted with only bovine PF-4 and β-TG-like protein; and type 4 reacted only with bovine PF-4. Differences in immunoreactivities of types 1, 2 and 3 were retained throughout the growth of succeeding clones and in ascitic fluids. Using a modified factor Xa, S-2222 chromogenic substrate-heparin inhibition assay, no mAb was found to block PF-4's ability to neutralize heparin. mAbs representative of types 1, 2 and 3 were successfully raised in stable cell lines from at least second generation clones. These were purified with protein A agarose and found to be IgG₁. By indirect immunocytofluorescence a purified type 2 mAb, 2E7, was found to specifically stain granules of human platelets and megakaryocytes, as well as masses (putative platelets within late stage megakaryocytes) without staining other cellular types in either bone marrow or peripheral blood. Species comparisons displayed positive staining for human, rat, and rabbit platelets and megakaryocytes, and negative staining for mouse, guinea pig and dog platelets and megakaryocytes. It seems likely that mAb, 2E7, is directed against an epitope, common to PF-4 of bovine, human, rabbit and rat.     

δ‐Catenin/NPRAP: A new member of the glycogen synthase kinase‐3β signaling complex that promotes β‐catenin turnover in neurons

Through a multiprotein complex, glycogen synthase kinase‐3β (GSK‐3β) phosphorylates and destabilizes β‐catenin, an important signaling event for neuronal growth and proper synaptic function. δ‐Catenin, or NPRAP (CTNND2), is a neural enriched member of the β‐catenin superfamily and is also known to modulate neurite outgrowth and synaptic activity. In this study, we investigated the possibility that δ‐catenin expression is also affected by GSK‐3β signaling and participates in the molecular complex regulating β‐catenin turnover in neurons. Immunofluorescent light microscopy revealed colocalization of δ‐catenin with members of the molecular destruction complex: GSK‐3β, β‐catenin, and adenomatous polyposis coli proteins in rat primary neurons. GSK‐3β formed a complex with δ‐catenin, and its inhibition resulted in increased δ‐catenin and β‐catenin expression levels. LY294002 and amyloid peptide, known activators of GSK‐3β signaling, reduced δ‐catenin expression levels. Furthermore, δ‐catenin immunoreactivity increased and protein turnover decreased when neurons were treated with proteasome inhibitors, suggesting that the stability of δ‐catenin, like that of β‐catenin, is regulated by proteasome‐mediated degradation. Coimmunoprecipitation experiments showed that δ‐catenin overexpression promoted GSK‐3β and β‐catenin interactions. Primary cortical neurons and PC12 cells expressing δ‐catenin treated with proteasome inhibitors showed increased ubiquitinated β‐catenin forms. Consistent with the hypothesis that δ‐catenin promotes the interaction of the destruction complex molecules, cycloheximide treatment of cells overexpressing δ‐catenin showed enhanced β‐catenin turnover. These studies identify δ‐catenin as a new member of the GSK‐3β signaling pathway and further suggest that δ‐catenin is potentially involved in facilitating the interaction, ubiquitination, and subsequent turnover of β‐catenin in neuronal cells. © 2010 Wiley‐Liss, Inc.     

Platelet factor 3 in plasma fractions: Its relation to microparticle size and thromboses

Platelet factor 3 (PF3) was assayed by Russell's viper venom (RVV) in three plasma fractions, platelet-rich plasma (PRP), platelet poor plasma (PPP), and 0.1 μm particlefiltered plasma (PFP), in 42 healthy controls, 34 patients with recent cerebrovascular accidents (CVA) and 28 with recent ischemic events from coronary artery disease (CAD). Platelet microparticles (PMP) were assayed in PPP by flow cytometry. Relative to controls, the RVV clotting times were shortened in all three plasma fractions in both patient groups, p<0.001. PMP were also elevated in both patient groups, p<0.001. Linear regression analysis showed that the RVV times of PPP are inversely correlated with PMP, p<0.005, in patient groups but not in controls. There was no correlation of RVV time with PT, APTT or FIB. After converting RVV times to units of PF3 activity, it could be shown that only about 1/4 of the total PF3 activity was contributed by platelets. The major contribution to the PF3 activity in controls was from microparticles <0.1 μm but in patients was due mainly to microparticles >0.1 μm. The RVV time was superior to routine coagulation tests in discriminating thrombotic patients from healthy controls.     

Effect of tumor necrosis factor α and β on human oligodendrocytes and neurons in culture

Cytokines produced by infiltrating hematogenous cells or by glial cells activated during the course of central nervous system disease or trauma are implicated as mediators of tissue injury. In this study, we have assessed the extent and mechanism of injury of human-derived CNS oligodendrocytes and neurons in vitro mediated by the cytokines tumor necrosis factor α and β and compared these with the tumor necrosis factor independent effects mediated by activated CD4⁺ T-cells. We found that activated CD4⁺ T-cells, but not tumor necrosis factor α or β, could induce significant release of lactate dehydrogenase, a measure of cell membrane lysis, from oligodendrocytes within 24 hr. Neither induced DNA fragmentation as measured using a fluorescence nick-end labelling technique. After a more prolonged time period (96 hr), tumor necrosis factor α did induce nuclear fragmentation changes in a significant proportion of oligodendrocytes without increased lactate dehydrogenase release. The extent of DNA fragmentation was comparable to that induced by serum deprivation. Tumor necrosis factor β effects were even more pronounced. In contrast to oligodendrocytes, the extent of DNA fragmentation, assessed by propidium iodide staining, induced in neurons by tumor necrosis factor α was less than that induced by serum deprivation. In-situ hybridization studies of human adult glial cells in culture indicated that astrocytes, as well as microglia, can express tumor necrosis factor α mRNA.     

Tumor necrosis factor α and transforming growth factor β upregulate astrocyte expression of monocyte chemoattractant protein-1

Astrocytes participate in the pathophysiology of central nervous system (CNS) inflammatory disease. Astrocyte expression of adhesion molecules, cytokines, and major histocompatibility complex antigens may contribute to these inflammatory processes. In addition, recent data suggested that astrocytes may be a source of monocyte chemoattractant protein-1 (MCP-1). MCP-1 is a member of the chemokine family of small cytokines and functions both as a chemoattractant as well as a stimulator of monocytes. To further characterize the role of astrocytes in CNS inflammation, we examined the effect of inflammatory cytokines on MCP-1 expression by astrocytes. Results of these studies demonstrate that the pro-inflammatory cytokine tumor necrosis factor alpha (TNFa) upregulates MCP-1 message and protein expression. The pleiotropic cytokine transforming growth factor beta (TGFβ) also stimulated MCP-1 expression. When astrocytes were exposed to both cytokines simultaneously, an additive effect on MCP-1 message, but not MCP-1 protein expression, was observed. These data suggest that TNFa and TGFβ, each present during CNS inflammatory disease, may upregulate the expression of MCP-1 which, in turn, may function to both recruit monocytes to the site of inflammation as well as to activate those monocytes already present in an inflammatory lesion.     

The influence of heparin and indol on the catalytic properties of α- and β/δ -thrombins

Heparin was shown to form an equimolar complex with α- and β/δ -forms of thrombin. The formation of the complex resulted in inhibition of the TAME esterase activity of thrombin ( by 40% form α- and 17% for β/δ-form ) and in stimulation of its BAME esterase activity ( by 50% for α- and 64% for β/δ-form ). Heparin caused the 70% inhibition of the activity of both forms of the enzyme towards the synthetic amid substrate Bz-Phe-Val-Arg-pNA; at the same time it had little if any effect on the enzyme activity towards Tos-Gly-Pro-Arg-pNA and stimulated the α- and β/δ-thrombins activities towards H-D-Phe-Pip-Arg-pNA by 16% and 57% respectively. In the case of both ester and amid substrates heparin exerted its effect on kcat, but had no effect on Km(app).Indol was shown to activate the TAME hydrolysis catalyzed by α- and β/δ-thrombins. The identity of the binding site for indol and for the additional TAME molecule in the effect of substrate activation was demonstrated. Heparin did not prevent the effects of indol and substrate activation of the thrombin-catalyzed hydrolysis of ester substrates. Moreover the kinetic parameters of indol activation are similar for the free enzyme and its complex with heparin indicating the different localization of the binding sites for indol and heparin in the molecule of thrombin.