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)

Present methods for assay of platelet aggregating agents use freshly prepared platelets. Much time is spent in daily preparation of platelets and standardization presents problems. The preparation of fixed washed platelets (FWP) and their use in two bioassays are described in this report. Washed human platelets were fixed for 48 hours with 4 per cent paraformaldehyde, washed twice in phosphate buffer, pH 6.4, and stored at 4 degrees C. Aggregation of FWP was studied with a macroscopic test and a light absorbance measurement. FWP did not aggregate with adenosine diphosphate, collagen, adrenalin, and thrombin. FWP aggregated with bovine or porcine plasma, poly-L-lysine, and ristocetin with normal human plasma but not with von Willebrand's disease plasma. These observations confirm the direct aggregating effect of these agents. Macroscopic aggregation times were dependent on the amount of aggregating agent (bovine plasma, normal human plasma). A quantitative assay for bovine platelet aggregating factor (PAF) and von Willebrand factor (vWF) with FWP was developed. The ability of FWP to aggregate remained unchanged after 1 month of storage at 4 degrees C. Ristocetin alone caused a decrease in light transmission of FWP suspensions, depending upon the concentration of ristocetin, but did not cause aggregation. FWP constitute a stable reagent suitable for quantitative measurement of PAF and vWF.
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PMID:Platelets fixed with paraformaldehyde: a new reagent for assay of von Willebrand factor and platelet aggregating factor. 23 99

Cyanate reacts with unchanged amino groups of various proteins in a specific irreversible carbamylation reaction. The effect of this molecule on the clotting process and the effects of carbamylation on the clotting proteins and platelet functions were investigated in vitro. An immediate effect on the clotting proteins, not related to pH, was seen in the screening tests prothrombin time, partial thromboplastin time and thrombin time at the highest concentration (100 mM), to a lesser degree at the lower concentration (10 mM). These changes reflected decreases of 19 and 36% respectively in Factor V and X activity, an inhibition of 63-75% of Factors VII, IX, X and XI activity, and 80% inhibition of thrombin activity. The inhibitory changes of carbamylation increased with time. No changes were seen in the activity of Factors I and VIII. Platelet function studies revealed no inhibition of Factor III release; aggregation was abnormal only at high concentrations with epinephrine and collagen induction and partially reversible by resuspension in normal plasma.
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PMID:The effect of cyanate on the clotting proteins and platelet function. 23 14

The ubiquitous connective tissues contain a wide range of cells including fibroblasts, osteoblasts and chondroblasts. Recently it has been demonstrated that another principal cell of the connective tissue is the smooth muscle cell in several organ systems. These have been shown to be responsible for the synthesis of the connective tissue matrix components of the uterine myometrium and of the arterial system, including collagen, both elastic fibre proteins and glycosaminoglycan. Microtubule inhibitors such as colchicine and vinblastine, and iron chelators such as alpha,alpha -dipyridyl have been used to study their morphologic and chemical effects on collagen synthesis and secretion. Colchicine produces an increase in large Golgi-associated vacuoles, which sometimes contain material reminiscent of aggregates of collagen macromolecules. Vinblastine produces alterations in the endoplasmic reticulum cisternae similar to alterations seen in ascorbic acid deficiency, and alpha,alpha-dipyridyl increases the frequency of regions in cells, interpretable as potential sites of communication of rough endoplasmic reticulum cisternae with the cell surface. Ferritin conjugated anti-procallagen sera were used to localize procollagen in cells and demonstrated procollagen not only in the cisternae of rough endoplasmic reticulum but in all of the elements of the Golgi complex as well. The studies reported in this review have shown that in cell culture arterial smooth muscle will produce not only the microfibrillar protein of the elastic fibre but soluble and/or insoluble elastin as well. Recent studies on serum factors responsible for the proliferation of connective tissue cells have demonstrated that at least one of the principal factors responsible for fibroblast and/or smooth muscle cell proliferation in culture is derived from thrombocytes. Medium containing serum derived from cell-free plasma lacks most of this proliferative effect which can be reinstated when platelets are present during recalcification to form serum. This effect is due to the platelet release reaction as shown by combining supernatant factors derived from platelets exposed to purified thrombin to cell-free, plasma derived serum. Studies with macrophages have also suggested that phagocytic macrophages release factor(s) into a cell culture medium that may also participate in stimulating fibroblasts to proliferate in vitro. The means by which these factors stimulate fibroblast proliferation and connective tissue synthesis remains to be elucidated.
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PMID:Connective tissue cells, cell proliferation and synthesis of extracellular matrix-a review. 23 19

Platelets from patients with myeloid leukaemia showed reduced aggregation with collagen or thrombin. These platelets also had a lower capacity to bind thrombin. This lower thrombin binding is due to a decrease in the total quantity of receptors available and not because of a change in the affinity. In the presence of the patients' plasma, the aggregation behaviour of normal platelets induced by thrombin as well as the clotting time of fibrinogen remained unchanged. The results suggest that the platelet dysfunction in myeloid leukaemia is partially due to a membrane defect involving the thrombin receptors which leads to an impaired induction of the initial stimulation.
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PMID:Defective binding of thrombin to platelets in myeloid leukaemia. 27 56

Fibronectin is a major glycoprotein component of normal fibroblasts in culture. External fibronectin is predominantly present in a pericellular fibrillar matrix that mediates distant cell-cell and cell-substratum contacts. A small proportion of external fibronectin is closely associated with the plasma membrane. In the matrix, fibronectin is partially disulfide bonded into complexes. Plasma transglutaminase, activated by thrombin, also cross-links external fibronectin into high-molecular-weight covalent complexes. In cultures of normal fibroblasts, pericellular matrix fibronectin displays extensive codistribution with (pro)collagens types I and III. Transformed adherent cells show decreased formation of the fibronectin-collagen matrix. The deficient synthesis of fibronectin and other matrix components and abnormal interactions with the matrix may account for several phenotypic characteristics of transformed cells. The pericellular matrix structure has been prepared by use of deoxycholate and hypotonic medium to solubilize the cells. The matrix contains glycosaminoglycans, procollagens, and fibronectin. The fibronectin codistributes with the procollagens. The matrix may be considered to be an in vitro equivalent of the connective tissue matrix and basal laminae found in vivo. Human sarcoma cells spread rapidly on the prepared matrix and assume an elongated morphology characteristic of normal fibroblasts. The prepared matrix may provide a general tool to study the effects of matrix on cellular behavior and differentiation.
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PMID:Fibronectin and the pericellular matrix of normal and transformed adherent cells. 29 68

Platelet Factor VIII-related antigen (VIIIR:Ag) represents a significant proportion of the total circulating VIIIR:Ag pool. However, its participation in the events of primary hemostasis has not been shown. We now report that platelet-contained VIIIR:Ag is released from platelets by collagen, ADP and thrombin. The concentrations of these agonists, required for VIIIR:Ag release, are the same or lower than those required for release of serotonin, lysosomal enzymes, or fibrinogen. This release has the features of an energy-dependent secretory response because it is blocked by the metabolic inhibitors, antimycin A and 2-deoxy-D-glucose. The electrophoretic characteristics of the VIIIR:Ag released by collagen and ADP are similar to those of plasma VIIIR:Ag. However, thrombin-released platelet VIIIR:Ag differs from that of plasma in that the less anodal forms are relatively depleted. These differences do not appear to be the result of proteolytic degradation of platelet-derived VIIIR:Ag, but may reflect interactions between specific molecular forms of VIIIR:Ag and the platelet membrane. These studies suggest mechanisms by which platelet-contained VIIIR:Ag may contribute to the primary events of hemostasis.
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PMID:Active release of human platelet factor VIII-related antigen by adenosine diphosphate, collagen, and thrombin. 31 83

Excessive reactivity of blood platelets may contribute to atherosclerotic vascular disease. Hence drugs which alter platelet function may be protective. Prompted by findings that propranolol therapy normalized hyperactive platelet aggregation in patients with coronary artery disease, we studied propranolol in vitro to assess its action on platelets. At concentrations similar to those achieved in vivo (0.1-1 muM), propranolol raised the thresholds for aggregation of some normal paltelets by adenosine diphosphate (ADP). At higher concentrations (10-50 muM), propranolol abolished the second wave of platelet aggregation induced by ADP and epinephrine, and inhibited aggregation induced by collagen, thrombin, and the ionophore A23187. Propanolol blocked the release of 14C-serotonin from platelets, inhibited platelet adhesion to collagen, and interfered with clot retraction. Propranolol blocked ionophore-induced uptake of 45Ca by platelets. Inhibition appeared unrelated to beta-adrenergic blockage, as d(+) propranolol (which lacks beta-blocking activity) was equipotent with 1(-) propranolol. Moreover, practolol, a beta-blockading drug which is nonlipophilic, did not inhibit platelet function. These studies suggested that propranolol, like local anesthetics, decreased platelet responsiveness by a direct action on the platelet membrane, possibly by interfering with calcium availability. Modulation of platelet function by propranolol may occur at concentrations achieved at usual clinical doses of the drug.
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PMID:Effect of propranolol on platelet function. 31 74

We have shown previously that washed human platelets resuspended in Tyrode solution containing albumin and apyrase maintain their disc shape and their ability to aggregate upon the addition of low concentration of ADP, providing fibrinogen is added to the suspending medium. We have now examined their responses to other aggregating and release-inducing agents. Collagen, arachidonate, thrombin, immune serum globulin, the ionophore A23, 187 and phytohaemagglutinin from Phaseolus vulgaris caused aggregation and release of granule contents. The response to adrenaline was variable. Serotonin caused the platelets to change shape but no aggregation or release occurred. Addition of a small amount of plasma was necessary for ristocetin-induced aggregation. Polylysine caused immediate platelet-to-platelet adherence with little or no release of granule contents. Responses to collagen or thrombin were greater in a modified medium containing magnesium but no calcium; in this medium, aggregation caused by ADP or polylysine was followed by the release of granule contents whereas these agents caused aggregation without release in a medium with both calcium and magnesium. When protein was omitted from the suspending medium, platelet aggregation in response to ADP was variable. In this medium, collagen and thrombin caused more extensive release than in the albumin-containing medium. Aggregation by polylysine was accompanied by release and extensive lysis in the protein-free medium. Thus, the composition of the final resuspending medium has a major effect on the responses of washed human platelets to aggregating agents.
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PMID:Properties of washed human platelets. 32 7

The mechanism of stimulus-response coupling in human platelets was investigated with a new instrument that simultaneously monitors aggregation and secretion in the same sample of plateletrich plasma. When platelets were stimulated by high concentrations of ADP, secretion began only after aggregation was almost complete. With lower concentrations of ADP or with epinephrine, biphasic aggregation was observed, and secretion began simultaneously with, or slightly after, the second phase of aggregation. When platelets were stimulated with high concentrations of gamma-thrombin or A23187, secretion and aggregation began essentially together. With very low concentrations of gamma-thrombin or A23187, biphasic aggregation was observed with secretion paralleling the second phase. At every concentration of collagen, secretion and aggregation appeared to be parallel events. Under every condition where the beginning of secretion lagged behind aggregation, secretion was dependent upon aggregation and was inhibited by indomethacin; this is referred to as aggregation-mediated platelet activation. When secretion began at the same time as aggregation, it also occurred in the absence of aggregation and was not blocked by indomethacin; this is referred to as directly induced platelet activation. These observations are consistent with a simple model of platelet stimulus-response coupling that includes two mechanisms for activation; aggregation-mediated activation is inhibited by indomethacin, while direct activation does not depend upon aggregation and is not inhibited by indomethacin. Secretion and second wave aggregation appear to be parallel events, with little evidence for second wave aggregation being a consequence of secretion as usually described.
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PMID:Interrelations of platelet aggregation and secretion. 33 May 68

The interaction of platelets with damaged vessel walls leads to the formation of platelet-fibrin thrombi and may also contribute to the development of atherosclerotic lesions because platelets adherent to exposed collagen release a mitogen that stimulates smooth muscle cell proliferation. The first step in thrombus formation, platelet adherence to an injured vessel wall, can be studied quantitatively by the use of platelets labeled with 51chromium. In these investigations, rabbit aortas were damaged by passage of a balloon catheter and segments of the aortas were everted on probes that were rotated in platelet suspensions. Collagen-coated glass cylinders were also used. Adherence was measured in a medium containing approximately physiologic concentrations of calcium, magnesium, protein and red blood cells. Conditions of testing influence the effect of non-steroidal anti-inflammatory drugs, sulfinpyrazone, and dipyridamole on platelet adherence. Aspirin and sulfinpyrazone were not inhibitory when tested in a medium with a 40% hematocrit; this indicates that products formed by platelets from arachidonate probably do not play a major part in the adherence of the first layer of platelets to the surface, although they may be involved in thrombus formation. Indomethacin, dipyridamole, prostaglandin E1, methylprednisolone and penicillin G and related antibiotics did inhibit platelet adherence although the concentrations required were higher than would likely be achieved in vivo upon administration to human patients. None of the non-steroidal anti-inflammatory drugs inhibited the release of granule contents from adherent platelets. Pretreatment of the damaged vessel wall with aspirin increased platelet adherence, presumably because it prevented the formation of PGI2 by the vessel wall. Platelet adherence to undamaged or damaged vessel walls was enhanced by prior exposure of the wall to thrombin. Platelet reactions with aggregating agents and platelet survival can be modified by changes in dietary lipids but there is very little evidence concerning the effects of lipids on platelet adherence. If some forms of dietary fat damage the endothelium, platelet interaction with the damaged area and release of the mitogen for smooth muscle cells would contribute to the development of atherosclerotic lesions.
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PMID:Drug effects on platelet adherence to collagen and damaged vessel walls. 36 49


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