Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.5 (thrombin)
33,306 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Glycoprotein IIb-IIIa (alpha IIb beta 3) and the vitronectin receptor (alpha v beta 3), two integrins that share the common beta 3 subunit, have been reported to function as promiscuous receptors for the RGD-containing adhesive proteins fibrinogen, vitronectin, fibronectin, von Willebrand factor, and thrombospondin. The present study was designed to establish a cell system for the expression of either GP IIb-IIIa or the vitronectin receptor in an otherwise identical cellular environment and to compare the adhesive properties of these two integrins with those of native GP IIb-IIIa and the vitronectin receptor constitutively expressed in HEL cells or platelets. M21 human melanoma cells lack GP IIb-IIIa and use the vitronectin receptor to attach to vitronectin, fibrinogen, fibronectin, and von Willebrand factor. To study the functional properties of GP IIb-IIIa in these cells, we transfected GP IIb into M21-L cells, a variant of M21 cells (Cheresh, D.A., and R.C. Spiro. 1987. J. Biol. Chem. 262:17703-17711), which lack the expression of functional alpha v and are therefore unable to attach to vitronectin, fibrinogen, and von Willebrand factor. Transfectants expressing GP IIb were isolated by immunomagnetic beads and surface expression of the GP IIb-IIIa complex was documented by FACS analysis and immunoprecipitation experiments performed with 125I-labeled M21-L/GP IIb cells. Comparative functional studies demonstrated that GP IIb-IIIa expressed in M21-L/GPIIb cells as well as native GP IIb-IIIa constitutively expressed in HEL-5J20 cells (an HEL variant lacking alpha v beta 3) mediated cell attachment to immobilized fibrinogen, but not to vitronectin or von Willebrand factor, whereas the vitronectin receptor expressed in M21 cells and HEL-AD1 cells (an HEL variant expressing alpha v beta 3) mediated cell attachment to fibrinogen, vitronectin, and von Willebrand factor. Similarly, PGl2-treated resting platelets attached to immobilized fibrinogen but not to vitronectin or von Willebrand factor, and this attachment could be inhibited by mAb A2A9 (directed against a functional site on the GP IIb-IIIa complex). However, in contrast to platelets, which adhered to vitronectin and von Willebrand factor after stimulation by thrombin or PMA, activation of the protein kinase C pathway in M21-L/GP IIb or HEL cells did not induce cell adhesion to vitronectin or von Willebrand factor.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Adhesive properties of the beta 3 integrins: comparison of GP IIb-IIIa and the vitronectin receptor individually expressed in human melanoma cells. 170 57

Vitronectin is one of the glycoproteins that mediate cell adhesion and spreading of a variety of cells through the RGD(S) sequence. Vitronectin is demonstrated to bind to glycoprotein IIb-IIIa and play a role in platelet aggregation. Synthetic peptides containing the RGD(S) sequence can inhibit vitronectin binding to platelets, but the affinity of these peptides is less than 1/100th that of native vitronectin. The present study thus examined the ability of modified RGD(S)-containing peptide to inhibit vitronectin binding to thrombin-stimulated platelets. The cyclicization of GRGDSPA peptide was done by the linkage of NH2-terminal glycin and the COOH-terminal alanin. The circular dichroism spectrum of cyclic GRGDSPA peptide only showed negative minimum at approximately 220 nm, but those of other linear peptides such as GRGDSPA and GRGESPA had no effect. This result indicated that only the cyclic GRGDSPA peptide retained some conformational structure to restrict its flexibility. Inhibition experiments revealed that the affinities of the ligands for the receptor decreased in the order of vitronectin = fibronectin = fibrinogen = von Willebrand factor (vWF) greater than cyclic GRGDSPA peptide greater than GRGDSPA peptide. GRGESPA peptide had no effect. These results demonstrate that the conformational structure of the RGD(S) sequence plays the important role for the affinity of vitronectin binding to activated platelets and the increased affinity of the modified peptide is a prerequisite for the potential antithrombotic use.
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PMID:Novel effect of cyclicization of the Arg-Gly-Asp-containing peptide on vitronectin binding to platelets. 170 43

Previous work has shown that adhesion of anchorage-dependent cells to fibronectin via integrin alpha 5 beta 1 leads to activation of the Na-H antiporter and a rise in intracellular pH (pHi). We now show that adhesion of bovine capillary endothelial cells (BCE) to fibrinogen; collagens type III, IV, and V; laminin; and vitronectin; ligands that bind other members of the integrin family, resulted in significant elevations in pHi. Other ligands (basic fibroblast growth factor, concanavalin A, and thrombin), which bind cells when immobilized on plastic, but that do not bind integrins and do not support cell growth, do not elevate pHi. Adhesion to an antibody against integrin alpha v beta 3 also elevates pHi. Adhesion of peripheral human T lymphocytes to an antibody against the integrin LFA-1 induced a rise in pHi. Antibodies to CD2 or ICAM-2 had only slight effects on pHi, whereas an antibody to the T cell receptor complex that strongly activates T cells induced a large increase in pHi. We conclude that elevation of pHi by integrins is specific and is a property shared by many members of the integrin family.
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PMID:Multiple integrins share the ability to induce elevation of intracellular pH. 171 34

Very late activation antigens (VLAs) are glycoproteins (GPs) that play a major role in platelet adhesion to extracellular matrix. These GPs, members of the integrin family, are heterodimer complexes with different alpha subunits noncovalently associated with a common beta 1 subunit known as GPIIa. GPIa-IIa (also known as VLA2), GPIc-IIa (VLA5), and GPIc*-IIa (VLA6) are involved, respectively, in platelet adhesion to collagen, fibronectin, and laminin. At this stage, very little is known about the role of GPIIa in platelet adhesive functions. In this study, we have generated a monoclonal antibody (MoAb) (LYP22) directed against GPIIa. Immunoaffinity chromatography using LYP22 combined with two-dimensional nonreduced-reduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows that the antibody brings down all VLA subunits. Western blots indicate that the binding site of LYP22 on GPIIa is disulfide bridge-dependent. The number of LYP22 binding sites is not increased on stimulation with thrombin and is in the range of what is observed with another anti-GPIIa MoAb (A-1A5). LYP22 is the first anti-GPIIa MoAb to inhibit aggregation and secretion of washed platelets stimulated with collagen, thrombin, or arachidonic acid. Moreover, the lag-phase usually observed on collagen stimulation is significantly prolonged (by 60 seconds) in the presence of LYP22. This lag-phase, mediated by LYP22, is also observed in the presence of plasma proteins and is coupled with a reduced effect on collagen-induced platelet aggregation. In addition, LYP22 affects the adhesion of resting platelets to type III collagen, but not to fibronectin, laminin, or type I collagen. These results strongly indicate that the site on GPIIa, bearing the LYP22 epitope, is an active participant in signal transduction controlling platelet functions.
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PMID:Role of glycoprotein IIa (beta 1 subunit of very late activation antigens) in platelet functions. 171 78

Vitronectin, which is present in both plasma and extracellular matrix, inhibits the complement cascade and promotes the growth and attachment of cells in vitro. Like other adhesive proteins such as fibrinogen, von Willebrand factor, and fibronectin, vitronectin contains the sequence Arg-Gly-Asp and binds to some members of the family of receptors called integrins. Platelet membrane glycoprotein IIb-IIIa (GPIIb-IIIa) is well known as a member of integrins that bind to vitronectin as well as to fibrinogen, von Willebrand factor, and fibronectin. The interaction of vitronectin with GPIIb-IIIa was studied. Vitronectin bound to thrombin-stimulated platelets in a calcium-dependent, specific, and saturable manner with a molecular weight of 290 nmol/L and 9,100 sites per platelet. The binding was inhibited by the other adhesive proteins with IC50s of 0.078-0.15 mumol/L. The binding also was inhibited by the tetrapeptide Arg-Gly-Asp-Ser and the monoclonal antibody to GPIIb-IIIa (LJ-CP8). Vitronectin inhibited thrombin-induced platelet aggregation in a dose-dependent manner and fibrinogen enhanced platelet aggregation. These results suggest that vitronectin might modulate platelet aggregates by interfering with the interaction of fibrinogen with thrombin-activated GPIIb-IIIa.
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PMID:How vitronectin binds to activated glycoprotein IIb-IIIa complex and its function in platelet aggregation. 171 97

The pathophysiology of peripheral circulatory disturbance in patients presenting with vibration syndrome was studied from the viewpoint of blood coagulation. Plasma levels of fibronectin (FN), vitronectin (VN), thrombin-antithrombin III complex (TAT), and alpha 2-plasmin inhibitor-plasmin complex (PIC) were measured in 23 subjects who showed no evidence of vibration-induced white finger [VWF(-) group] and in 24 patients who presented with VWF [VWF(+) group]. In the VWF(-) group, plasma FN concentrations were elevated but plasma TAT and PIC levels were within the normal ranges. In the VWF(+) group, plasma FN concentrations were normal but plasma TAT and PIC levels were significantly elevated. In both groups, plasma VN concentrations were similar to those in normal controls. For purposes of comparison, 32 patients presenting with diabetes mellitus were also studied. They were divided into 2 groups, 13 subjects who showed no evidence of angiopathy [complication(-) group] and 19 patients who presented with angiopathy [complication(+) group]. In the complication(+) group, plasma TAT and PIC concentrations were significantly elevated, as in the VWF(+) group. These results suggest that in vibration syndrome, vibration, cold stimulus, or other factors first injure the vascular endothelium, resulting in a rise in plasma FN, and that in the VWF(+) group, augmentation of coagulation and fibrinolysis induces a state of compensated disseminated intravascular coagulation (DIC).
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PMID:Activation of blood coagulation and fibrinolysis in vibration syndrome. 172 Jul 65

The binding of fibronectin (Fn) to several integrins involves the Arg-Gly-Asp (RGD) tripeptide sequence. However, linear synthetic RGD peptides do not completely mimic the cell attachment activity of intact Fn or certain large Fn fragments. This suggests that the integrin-Fn interaction involves a more extended surface of Fn than that provided by the RGD sequence. To test this possibility, three novel monoclonal anti-Fn antibodies that inhibit its binding to a purified integrin, alpha IIb beta 3, were developed. The epitopes of these three antibodies mapped to a region at least 55 residues amino-terminal of the RGD sequence. Further, recombinant fragments of Fn containing these epitopes and lacking the RGD site also inhibited the binding of Fn to purified alpha IIb beta 3. These fragments, which spanned Fn residues 1359-1436, bound to alpha IIb beta 3 in a divalent cation-dependent manner. In addition, this region of Fn bound specifically to alpha IIb beta 3 on thrombin-stimulated but not resting platelets. These results demonstrate the presence of additional sequences in Fn that interact with integrin alpha IIb beta 3 and suggest that multiple sites in Fn are involved in its recognition by this integrin.
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PMID:Integrin alpha IIb beta 3 (platelet GPIIb-IIIa) recognizes multiple sites in fibronectin. 172 Jul 79

Extracts of atherosclerotic lesions contain a range of fibrin degradation products (FbDP), similar fragments have been detected in extracts from human and mouse healing skin wounds and from the invasive edge of human breast carcinomas, which are all proliferating systems. We have previously shown that FbDP stimulate cell proliferation including angiogenesis in the chick chorioallantoic membrane (CAM), and sought to characterize further the active components. Fibrin prepared from platelet-rich and platelet-free plasma, and purified Kabi fibrinogen, was treated with plasmin, and the digests were all active. FbDP from platelet-rich plasma clots also increased vascularity of the CAM. Prior removal of fibronectin from plasma by gelatin-Sepharose affinity chromatography did not affect proliferative activity. Current studies showed that long digests of fibrin, in which the only major band detectable is fibrin fragment E are active. Commercial fibrinogen derived fragment E, itself inactive on the CAM, becomes active after exposure to thrombin cleavage of fibrinopeptides. Recently fragment E has been isolated from shorter digests, by simple filtration through a Millipore 0.2 microns centrifuge filter. It displayed similar activity to the fragment E obtained from long digests. Fragment E in plaque extracts has been shown consistently to lack fibrinopeptide A indicating it is of fibrin origin.
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PMID:Factors relevant to stimulatory activity of fibrin degradation products in vivo. 172 7

Platelets contain a pool of endogenous adhesive proteins that can be released and may bind to surface membrane receptors under appropriate conditions. Because the binding of exogenous fibrinogen to platelets was shown previously to be accompanied by a time-dependent decrease in fibrinogen accessibility to antibody and enzymes, studies were performed to evaluate changes in the expression of endogenous fibrinogen released from thrombin-stimulated platelets using monospecific polyclonal and monoclonal antibody F(ab')2 fragments. Parallel studies were performed to compare the expression of released fibronectin and von Willebrand factor (vWF). Binding of polyclonal antibody F(ab')2 fragments directed against individual adhesive proteins was inhibited by EDTA or the 10E5 monoclonal antibody, suggesting that fibrinogen, fibronectin, and vWF expression was mediated, in large part, by divalent cation-dependent interactions with the glycoprotein IIb-IIIa complex. Interestingly, when polyclonal antibody F(ab')2 fragments were added to platelet suspensions at discrete times after thrombin stimulation, antifibrinogen F(ab')2 binding decreased by 72% +/- 15% (mean +/- SD, n = 22) over a 60-minute time course, whereas antifibronectin and anti-vWF antibody F(ab')2 fragment binding changed minimally (6% +/- 23%, n = 22 and 3% +/- 26%, n = 14, respectively). Similar observations were made with monoclonal antibodies. Parallel experiments using 125I-labeled fibrinogen as a marker indicated that the observed decrease in antifibrinogen F(ab')2 binding was not accompanied by fibrinogen dissociation. Moreover, antibody accessibility to platelet-bound fibrinogen could be restored after Triton X-100 platelet lysis. The data suggest that fibrinogen, fibronectin, and vWF are not coordinately expressed on thrombin-stimulated platelets. Rather, fibrinogen expression appears transient compared with the expression of fibronectin and vWF. The ability of platelets to secrete and organize adhesive proteins on their surface is likely to have important implications for hemostasis and thrombosis.
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PMID:Adhesive protein expression on thrombin-stimulated platelets: time-dependent modulation of anti-fibrinogen, -fibronectin, and -von Willebrand factor antibody binding. 173 4

The process of wound healing under the influence of locally applied fibronectin, heparin and thrombin was studied in adult rabbits. Using immunofluorescence microscopy, a higher concentration of fibronectin within the wound area was found on the 1st day after fibronectin treatment. The tensile strength of the wounds was tested on the 6th, 9th and 12th day after operation. On the 9th day the tensile strength of the fibronectin-treated wounds was significantly higher than that of the control wounds on the 12th day after operation. The addition of heparin and thrombin, each in combination with fibronectin, did not show any significant effect on wound healing.
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PMID:Acceleration of wound healing by local application of fibronectin. 174 8


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