Synthesis of novel anti-inflammatory peptides derived from the amino-acid sequence of the bioactive protein SV-IV.
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SV-IV is a basic, thermostable, secretory protein of low Mr (9758) that is synthesized by rat seminal vesicle (SV) epithelium under strict androgen transcriptional control. This protein is of obvious pharmacological interest because it has potent nonspecies-specific immunomodulatory, anti-inflammatory, and pro-coagulant activities. In evaluating the clinical relevance and the possible use in medicine of SV-IV, we became interested in the study of its structure-function relationships and aimed to identify in its polypeptide chain specific peptide fragments possessing the marked anti-inflammatory properties of the protein not associated with other biological activities (pro-coagulation and immunomodulation) typical of this molecule. By using two different experimental approaches (the fragmentation of the protein into peptide derivatives by chemical methods and the organic synthesis on solid phase of selected peptide fragments), data were obtained showing that in this protein: (a) the immunomodulatory activity is related to the structural integrity of the whole molecule; (b) the anti-inflammatory activity is located in the N-terminal region of the molecule, the 8-16 peptide fragment being the most active; (c) the identified anti-inflammatory peptide derivatives do not seem to possess pro-coagulant activity, even though this particular function has been located in the 1-70 segment of the molecule. (+info)
Antibodies from patients with heparin-induced thrombocytopenia stimulate monocytic cells to express tissue factor and secrete interleukin-8.
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Thrombosis is a life-threatening complication that occurs in a subset of patients with heparin-induced thrombocytopenia (HITT). The pathogenic mechanisms underlying the variable occurrence of thrombosis in HITT is poorly understood. It was hypothesized that monocyte activation leading to tissue factor expression may play a role in promoting a thrombogenic state in HITT. This study demonstrates that a human platelet factor 4 (PF4)/heparin-specific murine monoclonal antibody (KKO) binds to peripheral blood-derived human monocytes in a PF4-dependent manner. KKO and antibodies from patients with HITT induce monocytes to synthesize and secrete interleukin-8 and induce cell-surface procoagulant activity, which is abrogated following treatment with antihuman tissue factor antibody. The findings suggest a novel mechanism by which PF4/heparin antibodies may promote a hypercoagulable state in patients with HITT. (Blood. 2001;98:1252-1254) (+info)
Ultrasound guided human thrombin injection. A new modality in the management of femoral artery pseudo-aneurysms.
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OBJECTIVES: We report our initial results of a prospective study of duplex ultrasound-guided injection (UGTI ) of thrombin in the management of femoral artery pseudo-aneurysms. We used human thrombin to avoid the increase in the human antibodies directed against fibrinogen, with the use of bovine thrombus, that preclude further utilisation of the bovine fibrin glue during cardio-thoracic surgery. METHODS: From 1999 to 2001, 19 patients, aged 69 (range 52-85) years presented with 21 femoral pseudo-aneurysms were treated. The mean pseudoaneurysm diameter was 30 (15-55) mm. All but two were secondary to cardiac procedures and the common femoral artery was the injured vessel in all instances. Patients were referred within 2-21 days following their iatrogenic injury. RESULTS: Immediate thrombosis of the sac occurred in 19 (90%) of the 21 pseudo-aneurysms. After a second injection, complete occlusion occurred in the remaining two patients. Two patients (CI 95%; 1-19) with three femoral pseudo-aneurysms developed leg pain. Duplex ultrasound follow-up showed two recurrences (9.5% - CI 95%; 1-19) and both were treated by repeat UGTI. There was no conversion to surgical repair. CONCLUSION: This percutaneous minimally invasive technique is safe and effective in the management of iatrogenic pseudo-aneurysms in this high-risk group of patients. Human thrombin has significant advantages over bovine thrombin. (+info)
Thrombin induces increased expression and secretion of angiopoietin-2 from human umbilical vein endothelial cells.
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Angiogenesis is required for tumor growth and metastasis. It has recently been suggested that thrombin is a potent promoter of angiogenesis. We therefore examined the possibility that thrombin could be inducing the expression of angiopoietin-2 (Ang-2), necessary for remodeling. Human umbilical vein endothelial cells were incubated with or without thrombin (1 U/mL) for 1 to 24 hours and then examined for messenger RNA (mRNA) by Northern analysis. Enhanced mRNA expression (about 4-fold over baseline) was noted at 4 hours. Enhanced expression of Ang-2 mRNA was secondary to enhanced transcription (about 4-fold), with no effect on stabilization. Enhanced Ang-2 mRNA transcription was inhibited by H7 and PD98059, indicating the requirement of serine/threonine kinases as well as the mitogen-activated protein kinase pathway. Up-regulation of mRNA was associated with enhanced Ang-2 protein synthesis and secretion as assayed by immunoblot. Thrombin-induced secreted Ang-2 inhibited the binding of recombinant (35)S-Ang-1 to its Tie-2-Fc receptor, demonstrating functionality. Hirudin reversed this effect, demonstrating thrombin specificity. Thus, thrombin-induced tumorigenesis and metastasis is associated with enhanced Ang-2 protein synthesis and secretion via enhanced transcription of Ang-2. This could help explain how thrombin promotes angiogenesis. (+info)
NADPH oxidase mediates tissue factor-dependent surface procoagulant activity by thrombin in human vascular smooth muscle cells.
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BACKGROUND: Tissue factor (TF) initiates the extrinsic coagulation cascade leading to thrombin formation. Thrombin induces TF mRNA in vascular smooth muscle cells (VSMCs), thereby contributing to the prolonged procoagulant activity and enhanced thrombogenicity at sites of vascular injury. However, the signaling mechanisms mediating this thrombogenic cycle are unclear. Characteristically, vascular injury promotes the generation of reactive oxygen species (ROS). Because ROS exert signaling functions, we investigated whether the NADPH oxidase, an important source of ROS in VSMCs, contributes to upregulation of TF by thrombin. METHODS AND RESULTS: Thrombin not only stimulated TF mRNA expression, but also TF-dependent surface procoagulant activity in cultured human VSMCs. This response was attenuated by antioxidants; the flavin inhibitor diphenylene-iodonium, Clostridium difficile toxin B, which inhibits Rho GTPases, p22phox antisense oligonucleotides, or the dominant-negative RacT17N mutant. Inhibitors of p38 MAP kinase and phosphatidylinositol (PI) 3-kinase also prevented thrombin-stimulated TF mRNA expression. Furthermore, thrombin stimulated the phosphorylation of the PI 3-kinase target protein kinase B/Akt in a redox-sensitive and NADPH oxidase-dependent manner. CONCLUSION: These findings indicate that the NADPH oxidase is essentially involved in the redox-sensitive induction of TF mRNA expression and surface procoagulant activity by thrombin. This response is mediated by NADPH oxidase-dependent activation of p38 MAP kinase and the PI 3-kinase/protein kinase B/Akt pathway. Given that active TF promotes thrombin formation, the NADPH oxidase may play a crucial role in perpetuating the thrombogenic cycle in the injured vessel wall. (+info)
Peptidoglycan from Staphylococcus aureus induces tissue factor expression and procoagulant activity in human monocytes.
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Staphylococcus aureus is one of the most significant pathogens in human sepsis and endocarditis. S. aureus can initiate blood coagulation, leading to the formation of microthrombi and multiorgan dysfunction in sepsis, whereas in endocarditis the bacterium induces fibrin clots on the inner surface of the heart, so-called endocardial vegetations. In the present study, we show that live and heat-killed S. aureus bacteria are potent inducers of procoagulant activity in human peripheral blood mononuclear cells. Furthermore, purified peptidoglycan, the main cell wall component of S. aureus, induced procoagulant activity in mononuclear cells in a concentration-dependent fashion. The procoagulant activity in these cells was dependent on expression of tissue factor, since antibodies to tissue factor inhibited the effect of peptidoglycan. In mononuclear cells stimulated with peptidoglycan, reverse transcription-PCR showed tissue factor gene expression, and the gene product was detected by enzyme-linked immunosorbent assay. Finally, flow cytometry identified tissue factor at the surface of CD14-positive monocytes. Peptidoglycan is known to induce proinflammatory cytokine production in monocytes. The present investigation shows that peptidoglycan also activates the extrinsic pathway of coagulation by inducing the expression of tissue factor in these cells. This mechanism helps to explain the procoagulant activity, which plays such an important role in the pathogenicity of severe S. aureus infections. (+info)
A clottable protein (coagulogen) from amoebocyte lysate of Japanese horseshoe crab (Tachypleus tridentatus). Its isolation and biochemical properties.
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A clottable protein, named coagulogen, was highly purified from the amoebocyte lysate of Japanese horseshoe crab (Tachypleus tridentatus) by a method similar to that used for the lysate of Limulus polyphemus amoebocytes. The isolated material gave a single protein band on analytical gel electrophoresis at pH 3.2, gel electrofocusing, and sodium dodecyl sulfate (SDS) gel electrophoresis with or without 2-mercaptoethanol. It was 90 percent coagulable, and the total yield from 10 ml of the amoebocyte lysate was about 40 mg. The sedimentation coefficient of purified coagulogen was 2.6 S and its molecular weight was estimated to be about 15,300 by sedimentation equilibrium analysis. The molecular weight estimated by SDS-gel electrophoretic analysis was 19,500 +/- 1,000. This discrepancy was apparently due to abnormal mobility arising from the basic nature of this protein on electrophoresis. The protein had a high isoelectric point of pH 10.0 +/- 0.2, as measured by the isoelectric focusing technique. It consisted of a total of 132 to 135 amino acid residues and contained high levels of basic amino acids, which accounted for more than 16 per cent of the total amino acid residues. No methionine was detected. High contents of valine, half-cystine, glutamic acid (glutamine), and phenylalanine were found. The N-terminal sequence of the first three residues of the coagulogen was Ala-Asx-Thr, and its C-terminal residues was identified as phenylalanine, indicating that it consists of a single polypeptide chain. It is of interest that the first three N-terminal residues are homologous with those of the Aalpha-chain of non-human primate fibrinogen. (+info)
Role of the adapter protein SLP-76 in GPVI-dependent platelet procoagulant responses to collagen.
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The adapter protein SLP-76 is a critical mediator of signal transduction via the platelet collagen receptor glycoprotein VI (GPVI) and its coreceptor FcRgamma. We tested the hypothesis that SLP-76 is required for collagen-induced procoagulant responses in murine platelets. Platelets from SLP-76 null (SLP-76(-/-)) or heterozygous (SLP-76(+/-)) mice were activated with the GPVI agonist convulxin, and surface expression of P-selectin (a marker of granule release) and annexin V binding (a marker of procoagulant phospholipid) were determined by flow cytometry. Convulxin induced surface expression of P-selectin in SLP-76(+/-) platelets, but not SLP-76(-/-) platelets (P <.01), and failed to stimulate annexin V binding to either SLP-76(+/-) or SLP-76(-/-) platelets. Platelet procoagulant activity was measured in a prothrombinase assay. Convulxin did not stimulate procoagulant activity in either SLP-76(+/-) or SLP-76(-/-) platelets, but fibrillar collagen produced a 1.9-fold increase in procoagulant activity in both SLP-76(+/-) and SLP-76(-/-) platelets (P <.001 versus unstimulated platelets). Similar results were obtained with platelets from FcRgamma null mice, for which collagen, but not convulxin, induced procoagulant activity (P <.01). Costimulation with thrombin and collagen produced a further (2.3-fold) increase in procoagulant activity in SLP-76(+/-) platelets (P <.05), but not in SLP-76(-/-) platelets. SLP-76(-/-) platelets also exhibited less annexin V binding than SLP-76(+/-) platelets after costimulation with thrombin and convulxin (P <.05). These findings demonstrate that an intact GPVI/FcRgamma/SLP-76 signal transduction pathway is not essential for platelet procoagulant activity induced by collagen but is necessary for maximal procoagulant response to costimulation with thrombin plus collagen. Thus, both GPVI-dependent and GPVI-independent pathways contribute to collagen-induced platelet procoagulant activity. (+info)