Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.5 (thrombin)
 33,306 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Investigations were conducted to determine whether rabbit aortic smooth muscle cells (SMC) reproduce their essential in situ features in culture. Enzymatically isolated cells in culture were compared with their in situ state in terms of myosin and caldesmon isoform expression, sensitivity to Ca(2+)-mobilizing agonists, and contractility. Protein marker expression was assessed by electrophoresis and quantitative immunoblotting, and intracellular free Ca2+ ([Ca2+]i) measurements were accomplished using indo-1, a Ca(2+)-sensitive fluorescent dye. Contraction of SMC grown on deformable silicone films was monitored optically. Before the onset of cell division (3 to 6 days in culture), SMC still contained significant although decreasing amounts of smooth muscle myosin (SM1 and SM2 isoforms) and they started to express nonmuscle-type myosin. The relative content of 150-kDa caldesmon decreased, whereas the expression of 77-kDa caldesmon increased during this period. In the confluent primary culture (11 days), SM1 was expressed, but 150-kDa caldesmon was hardly detectable. Histamine (10(-5) mol/L), serotonin (10(-6) mol/L), and thrombin (1.5 units/mL) contracted deendothelialized rings of rabbit aorta, but only histamine was able to elevate [Ca2+]i 2.5- to 3-fold and induce reversible contraction of primary nondividing cells. [Ca2+]i elevation in response to histamine was due both to Ca2+ mobilization from intracellular stores and Ca2+ flux across the plasma membrane. After the onset of proliferation, SMC regained the ability to elevate [Ca2+]i in response to serotonin and thrombin but lost the ability to contract. Thus, primary cultured quiescent rabbit aortic SMC (3 to 6 days in culture) retain the essential features of vascular SMC in situ (eg, smooth muscle specific contractile and regulatory proteins, vasoactive hormone sensitivity, and contractility).
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PMID:Contractile rabbit aortic smooth muscle cells in culture. Preparation and characterization. 163 34

Recombinant human single-chain urokinase-type plasminogen activator (suc-PA) (SM0: wild type) and its variants resistant to plasmin and/or thrombin (SM1: Lys135 to Gln; SM3: Phe157 to Asp; and SM4: Lys135 to Gln and Phe157 to Asp) have been constructed by site-directed mutagenesis with the aim of producing more efficient thrombolytic agents [Miyake, T. et al. (1988) J. Biochem. 104, 643-647]. In the present study, we characterized the recombinant variant scu-PAs expressed in Escherichia coli. They appeared to have structural integrity because their heat-stabilities, immunological reactivities, and circular dichroism spectra were essentially identical to those of each other and of native scu-PA (nscu-PA). In the presence of thrombin, SM3 and SM4 showed efficient clot lysis by all of the assays used, compared with SM0, SM1, and nscu-PA. While in the absence of thrombin, when measured by a fibrin plate method in a purified system, SM3 and SM4 had lower specific activities than SM0, SM1, and nscu-PA, because of their catalytic constants for conversion to the two-chain form (tcu-PA) by plasmin are lower. However, SM4 lysed clots as efficiently as SM0 in plasma by retaining the single-chain form, whereas SM0 was partly converted to the two-chain form.
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PMID:Characterization of thrombin- and plasmin-resistant mutants of recombinant human single chain urokinase-type plasminogen activator. 214 58

A major step in the pathogenesis of atherosclerosis is the vectorial migration of smooth muscle cells (SMCs) from the arterial media into the intima. Although subcultured SMCs usually show synthetic phenotype, the behaviour of contractile SMCs may be crucial for the subsequent migration of the cells. In the present study, we utilized an in vitro assay system to evaluate the effects of fibrin gels on the migration of SMCs from explants taken from rabbit aorta. After cultured for 5-7 days in a serum-free condition, SMCs appeared from explants covered with fibrin gel. The cells were positive on immunostaining for SMC specific alpha-actin. No migration of SMCs from the control explants without fibrin gel was observed. Then the percentage of explants showing cell migration and the number of migrating cells increased with time. The migration of SMCs into fibrin gels was not dependent on the concentration of fibrinogen used for the preparation of fibrin gel in the range of 1.5-3 mg/ml. Variations of thrombin concentration in the range of 0.25-1.25 U/ml had no significant effect. However, there was less migration of SMCs with higher concentrations of thrombin. Thrombin inhibitors, hirudin and PPACK had no significant effect on the migration of SMCs. An RGD-containing peptide, GRGDS inhibited the migration of SMCs although a control peptide GRGES at the same concentration had no significant effect. A monoclonal antibody to alphavbeta3, LM609, completely inhibited the migration of SMCs from the explants, suggesting that alphavbeta3 integrin is involved in the migration of SMCs into fibrin gels. SMCs which migrated from the explants showed the positive staining with the monoclonal antibodies against SMC myosin heavy chain isoforms, SMemb, SM1 and SM2, suggesting that they are in an intermediate state changing from contractile to synthetic state. In conclusion, the present study showed that fibrin gel induces the migration of SMCs from explants into itself and the process may not need other growth factors or cytokines.
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PMID:Fibrin gel induces the migration of smooth muscle cells from rabbit aortic explants. 1054 26

The transition of fibrinogen to fibrin and to their degradation products within the arterial wall has been reported to be accompanied by atherosclerotic progression. A major step in the pathogenesis of atherosclerosis is the vectorial migration of vascular smooth muscle cells (SMCs) from the arterial media through the internal elastic lamina into the intima and their subsequent proliferation in the intima. I have been studying the effects of fibrinogen, fibrin and their degradation products on the behaviour, particularly migration, of SMCs. Fibrinogen/fibrin stimulates the adhesion and migration of SMCs and their effects are mediated by both the RGD-containing region of the alpha chain of fibrinogen/fibrin and integrin alpha v beta 3 on the cell surface. SMCs migrate into fibrin gel even with no other chemotactic stimuli. SMCs displayed two-fold increase in migration into crosslinked fibrin gels compared to non-crosslinked gels, suggesting the importance of fibrin crosslinking by factor XIIIa on its three-dimensional structure for the migration of SMCs. Fibrin gels prepared with batroxobin, which cleaves only fibrinopeptide A, with ACTE, which cleaves only fibrinopeptide B, and with protamine sulfate, which cleaves nothing, but forms a fibrin-like gel, induce migration of SMCs in a manner similar to the gel prepared with thrombin, suggesting that the cleavage of fibrinopeptides is not involved in the migration of SMCs. Both anti-fibrinogen fragment D and E antibodies inhibit the migration of SMCs into fibrin gel, suggesting that both D and E regions of fibrin are involved in the migration of SMCs into fibrin gel. The migration of SMCs into fibrin gel also depends on the RGD-containing region and integrin alpha v beta 3. Both fibrinogen fragments D and E inhibit the migration of SMCs into fibrin gels, suggesting that these fragments may be involved in the regulation of SMC migration into fibrin gel as the result of fibrinolysis. Although subcultured SMCs usually show a synthetic phenotype, the behaviour of contractile SMCs may be crucial for the subsequent migration of the cells. We employed an in vitro assay system to evaluate the effects of fibrin gels on the migration of SMCs from explants taken from rabbit aorta. alpha v beta 3 integrin and the RGD-containing region are involved in the migration of SMCs into the fibrin gels. SMCs which migrated from the explants showed positive staining with monoclonal antibodies against SMC myosin heavy chain isoforms, SMemb, SM1 and SM2, suggesting that they are in an intermediate state changing from a contractile to synthetic state. These findings show that fibrin (ogen) itself induces adhesion and migration of SMCs without other chemotactic or chemokinetic substances, suggesting a crucial role for fibrin (ogen) in the development and progression of such vascular diseases as atherosclerosis, thrombosis and restenosis following balloon angioplasty.
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PMID:[Effects of fibrinogen, fibrin and their degradation products on the behaviour of vascular smooth muscle cells]. 1099 26

To investigate whether bound thrombin can induce modulation of SMemb expression in vascular smooth muscle (VSM) cells, messenger RNA (mRNA) expression was measured by in situ hybridization (ISH) and reverse transcription-polymerase chain reaction (RT-PCR) in cultured rabbit aortic VSM cells. To test the concentration- and time-dependent effect of bound thrombin on the expression of SMemb, confluent VSM cells were incubated for 48 h in 10% FBS-DMEM containing 0, 3, 10 and 30 units/ml of bound thrombin. In addition, the confluent VSM cells were incubated for 6, 12, 24 and 48 h in 10% FBS-DMEM containing 10 units/ml of bound thrombin. Consequently, bound thrombin significantly increased SMemb mRNA in a concentration- and time-dependent manner. When compared with the effect of rabbit fibrinogen (10 microg/ml) and native thrombin (10 units/ml), SMemb mRNA was significantly increased by bound thrombin and was slightly increased by native thrombin, but not by fibrinogen. Other myosin heavy chain (MHC) isoform (SM1 and SM2) mRNA expressions were not changed by fibrinogen, native thrombin or bound thrombin. ISH revealed that there was no significant difference in the expression of MHC mRNAs among fibrinogen, native thrombin or bound thrombin. Western blot analysis demonstrated that the SMemb protein level was significantly increased by 2.5-fold by bound thrombin. When the clot-forming activities in cultured medium containing native thrombin or bound thrombin were measured from 0.5 to 48 h, the activity of bound thrombin declined more slowly than that of native thrombin. In conclusion, bound thrombin could upregulate the expression of SMemb mRNA and protein in cultured VSM cells and the activity of bound thrombin was maintained for longer than that of native thrombin in culture medium.
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PMID:Bound thrombin-induced upregulation of myosin heavy chain isoform, SMemb messenger RNA expression in cultured rabbit vascular smooth muscle cells. 1524 19