Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.69 (APC)
 16,337 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The membrane glycoprotein thrombomodulin (TM) is an essential endothelial cell (EC) cofactor, which forms a 1:1 stoichiometric complex with thrombin. Binding of thrombin to the high affinity TM receptor transforms its procoagulant activity into an anticoagulant potential, by activating protein C. The fate of TM in the presence of thrombin is still unclear: some authors claim that the thrombin-TM complex is internalized in EC, while others find this complex to be stable for at least 2 h at 37 degrees C on the EC surface. In the present study, we investigated the interactions of thrombin and Fab-fragments of anti-TM antibodies, coupled to 5 or 15 nm gold particles with saphenous vein endothelial cells. Our results demonstrate that TM can be observed both on the plasma membrane and in coated structures only in the presence of anti-TM antibodies. Addition of thrombin decreased the extent of this labeling, while in double labeling experiments, where cells were incubated with 5 nm gold coupled thrombin and 15 nm gold coupled Fab fragments of anti-TM antibodies, thrombin was cointernalized only when anti-TM antibodies were present. These results show that thrombin-TM complex is not significantly internalized in EC. The internalization of this complex induced by anti-TM antibodies could play an important role in the thrombotic complications induced by anti-EC autoantibodies.
 ...
PMID:Antibodies to thrombomodulin induce receptor-mediated endocytosis in human saphenous vein endothelial cells. 133 32

Thrombomodulin, a membrane glycoprotein present on normal vascular endothelium, binds circulating thrombin and is important in protein C activation. These functions contribute to the nonthrombogenic nature of endothelium. Damage during harvest and ex vivo storage of vein grafts may result in dysfunction of this endothelial anticoagulant barrier and possibly contribute to early graft thrombosis. We studied the functional activity and antigenic expression of thrombomodulin on saphenous veins before (initial) and after (harvested) harvest and storage for coronary artery bypass grafting in 15 patients. Also, fresh saphenous vein was studied after mechanical endothelial stripping. After storage for 2.7 +/- 0.6 hours at room temperature in heparinized saline, thrombomodulin functional activity in harvested vein segments was 28% less than initial segments (p = 0.08). Endothelial stripping resulted in a 79% reduction in thrombomodulin activity compared with initial segments (p = 0.04). Immunohistochemical staining confirmed thrombomodulin antigen on vein grafts after harvest and storage, but not on segments stripped of endothelium. Thrombomodulin functional activity and antigenic expression on human saphenous vein grafts is not significantly changed by harvest and relatively short periods of storage at room temperature in heparinized saline.
 ...
PMID:Thrombomodulin activity on human saphenous vein grafts prepared for coronary artery bypass. 165 Apr 5

The activation of protein C by thrombin is thought to occur at the endothelial cell surface in the presence of an essential membrane glycoprotein cofactor, thrombomodulin. In the present study it is demonstrated that, in the presence of hirudin, the most potent known inhibitor of thrombin, human protein C can be activated by human factor Xa (20 nM), but by a thrombomodulin-independent mechanism requiring only the presence of Ca2+ and phospholipid vesicles bearing a high proportion of negative charges (30-75% phosphatidylserine, depending on the conditions). At an optimal concentration of phosphatidylserine/phosphatidylcholine (1:1, w/w) of 75 microM, the apparent Km was 1 microM with a kcat. of 1 min-1. At 25 microM-phospholipid the Km was unchanged and the kcat. was 0.67 min-1. At either lipid concentration, increasing the density of negative charges by the adjunction of sulphated polysaccharides, like pentosan polysulphate or standard heparin at optimal concentrations of 2-5 micrograms/ml and 5-10 micrograms/ml respectively, resulted in a 4-fold increase of the kcat. without affecting the Km. Sulphated polysaccharides alone were poor promoters of protein C activation by factor Xa. In any case the presence of Ca2+ was essential, the dependence being sigmoidal with Hill coefficients ranging from 1.4 to 2.0. No significant activation of 4-carboxyglutamic acid-domainless protein C, a chymotrypic derivative lacking the phospholipid-binding domain, could be detected in the presence of phospholipids and Ca2+, with or without pentosan polysulphate. In a large molar excess, other phospholipid-binding entities like prothrombin fragments F1 or F1+2 could inhibit protein C activation by factor Xa, but pentosan polysulphate exerted a clear protective effect. Factor Xa irreversibly inhibited at its active centre, but not di-isopropyl phosphoro-thrombin, behaved as an inhibitor but in a more complex manner than simple Michaelis-Menten kinetics. Among several derivatives of pentosan polysulphate or of heparin which were tested, those having the higher degree of sulphation and/or molecular mass were the most efficient in enhancing the rate of activation of protein C by factor Xa in the presence of phospholipids. These results suggest that human factor Xa, at physiological concentrations, could activate human protein C in the presence of anionic phospholipids and that this activation could be potentiated by therapeutic concentrations of sulphated polysaccharides.
 ...
PMID:Activation of human protein C by blood coagulation factor Xa in the presence of anionic phospholipids. Enhancement by sulphated polysaccharides. 247 15

Human protein C is the precursor of a serine proteinase in plasma which contains nine 4-carboxyglutamic acid residues and functions as a potent anticoagulant. It is activated by thrombin in the presence of an essential endothelial-cell-membrane glycoprotein cofactor, thrombomodulin. In a purified human system, vitamin K-dependent proteins such as factor X, prothrombin and prothrombin fragment 1 were able to inhibit protein C activation by the thrombin-thrombomodulin complex, using either detergent-solubilized thrombomodulin or thrombomodulin reconstituted into vesicles consisting of phosphatidylcholine and phosphatidylserine (1:1, w/w). Factors VII and IX and protein S were much less efficient. Prothrombin fragment 1 behaved as a non-competitive inhibitor with apparent Ki values of 4 microM in the absence, and of 2-2.5 microM in the presence, of phospholipids. Heat decarboxylation of fragment 1 abolished its ability to interfere in protein C activation, and high phospholipid concentrations could attenuate its inhibitory effect and were responsible for a gradual loss of the non-competitive character. Fragment 1 also inhibited the activation of 4-carboxyglutamic acid-domainless protein C, a proteolytic derivative of protein C lacking the 4-carboxyglutamic acid residues, without any influence from phospholipids. At high thrombin concentrations, with respect to thrombomodulin, the inhibitory effect of fragment 1 was diminished. Fragment 1, at 3.8 microM, inhibited by 50% the activation of protein C (0.1 or 0.3 microM) by thrombin. These results suggest that the 4-carboxyglutamic acid domain of vitamin K-dependent proteins can act as a modulator of the protein C anticoagulant pathway through two distinct types of interaction. The functional 4-carboxyglutamic acid domain would be necessary to allow the enhancement of protein C activation in the presence of anionic phospholipids and it could recognize a phospholipid-independent binding site on the thrombin-thrombomodulin complex.
 ...
PMID:Interference of blood-coagulation vitamin K-dependent proteins in the activation of human protein C. Involvement of the 4-carboxyglutamic acid domain in two distinct interactions with the thrombin-thrombomodulin complex and with phospholipids. 285 94

Human thrombomodulin, an endothelial-cell-membrane glycoprotein, has been purified from placenta by Triton X-100 extraction and by affinity chromatography on concanavalin A-Sepharose and thrombin-Sepharose. It has been characterized by its ability to promote the activation of human protein C by human alpha-thrombin in the presence of Ca2+ and fulfilled the requirements of a cofactor. Reconstitution of thrombomodulin into phospholipid vesicles containing anionic phospholipids resulted in an increased rate of activation of protein C. Cardiolipin and vesicles containing phosphatidylcholine/phosphatidylserine (1:1, w/w) were the most effective. The apparent Km of the thrombin-thrombomodulin complex for protein C was 2 microM. It was not changed in the presence of phospholipid, whereas the Vmax. could be apparently increased up to 3.2-fold depending on the phospholipid and on its concentration, the catalytic-centre activity reaching 15.7 mol of activated protein C formed/min per mol of thrombin. Above their optimal concentrations, phospholipids inhibited the amidolytic activity of activated protein C. Phospholipids had no effect on the activation of 4-carboxyglutamic acid-domainless protein C, a proteolytic derivative of protein C lacking the 4-carboxyglutamic acid residues. These results show that the positive effect of anionic phospholipids in the activation of protein C by the thrombin-thrombomodulin complex involves a Ca2+-dependent interaction between protein C and phospholipids. They suggest that the enhancement of thrombomodulin activity by such phospholipids may be of functional significance.
 ...
PMID:The effect of phospholipids on the activation of protein C by the human thrombin-thrombomodulin complex. 302 38

Thrombomodulin (TM) binds thrombin to form a complex that activates the plasma anticoagulant zymogen protein C. TM is an integral membrane glycoprotein that contains a chondroitin sulfate moiety. Interaction with thrombin involves both the protein component of TM, specifically the growth factor-like repeats 4-6 (TM 4-6), and chondroitin sulfate. Removal of chondroitin sulfate decreases the affinity for thrombin approximately 10-fold and shifts the Ca2+ dependence of protein C activation from simple saturation at > or = 500 microM Ca2+ to a distinct optimum at approximately 100 microM Ca2+. Thrombin possesses two regions of high positive charge, anion binding exosites 1 and 2. Anion binding exosite 1 interacts with the growth factor region of TM while exosite 2 is involved in binding prothrombin activation fragment 2 or heparin. We demonstrate that recombinant TM, truncated at the membrane-spanning domain, or TM 4-6 can bind thrombin when fragment 2 is present either covalently attached (meizothrombin des-fragment 1) or in reversible association. With meizothrombin des-fragment 1, the Ca2+ dependence of protein C activation is independent of the presence of the chondroitin sulfate on TM. At 0.27 mM Ca2+, TM containing chondroitin sulfate binds thrombin (Kd(app) = 0.3 nM) approximately 45 times tighter than meizothrombin des-fragment 1 (Kd(app) = 14 nM). However, the chondroitin-free form binds thrombin (Kd(app) = 2.4 nM) only approximately 4 times tighter than meizothrombin des-fragment 1 (Kd(app) = 9.4 nM). These studies suggest that occupancy of anion binding exosite 2 by either chondroitin sulfate or fragment 2 alters thrombin conformation resulting in the altered Ca2+ dependence of protein C activation.
 ...
PMID:Occupancy of anion binding exosite 2 on thrombin determines Ca2+ dependence of protein C activation. 816 79

Thrombomodulin (TM) is an endothelial cell membrane glycoprotein which neutralizes thrombin procoagulant activity and accelerates the thrombin-catalyzed activation of protein C. We expressed recombinant human soluble TM (rhs-TM) in Chinese hamster ovary cells and compared the effects of rhs-TM and heparin on endotoxin-induced experimental disseminated intravascular coagulation (DIC) in rats. Experimental DIC was induced by a continuous intravenous infusion of endotoxin for four hours. rhs-TM or heparin was infused simultaneously with endotoxin. Treatment with rhs-TM significantly reversed the endotoxin-induced changes in significantly reversed the endotoxin-induced changes in following parameters: platelet count, fibrinogen level and fibrinogen and fibrin degradation products. Furthermore, glomerular fibrin deposits elevated by endotoxin treatment were reduced by the rhs-TM administration. Heparin showed the similar effects to rhs-TM. Activated partial thromboplastin time (APTT) in rats receiving rhs-TM were slightly longer than APTT in endotoxin-treated rats, but rats receiving heparin had much more prolonged APTT. From these results, we concluded that rhs-TM may be useful for the clinical treatment of DIC while having only minor adverse effects on APTT.
 ...
PMID:Antithrombotic effect of recombinant human soluble thrombomodulin on endotoxin-induced disseminated intravascular coagulation in rats. 823 61

Activation of the coagulation system is postulated to play an important role in the pathogenesis of endotoxin-induced tissue injury. Thrombomodulin (TM) is an endothelial cell membrane glycoprotein receptor for thrombin. Once bound to TM, thrombin loses its procoagulant activity, which results in decreased clotting. In addition, the binding of thrombin to TM activates the endogenous anticoagulant pathway through protein C. We studied the effect of recombinant human TM (rh-TM) on endotoxin-induced multiple-system organ failure (MSOF) in Sprague-Dawley rats weighing 400 to 450 g: 2 mg/kg of rh-TM was injected (T1/2 = 4.5 h) 30 min prior to intravenous injection of 20 mg/kg of Escherichia coli endotoxin. The study presented here consisted of three separate experiments. Experiment 1: 24-h survival study. Experiment 2: multiple-system organ microthrombi study in which 125I-human fibrinogen was injected 30 min prior to an endotoxin or saline injection and tissue microthrombi formation was assessed by measuring the percentage of organ radioactivity (lung, heart, liver, and kidney) against total injected radioactivity (microthrombi index, MI) 2.25 h after an endotoxin or saline injection. Experiment 3: endotoxin-induced MSOF study in which 125I-rat albumin was injected 5 h after an endotoxin or saline injection, and endotoxin-induced organ injury was evaluated by measuring tissue wet-to-dry ratios (W/D) and tissue-to-plasma 125I-rat albumin concentration ratios (T/P) 8 h after the endotoxin or saline injection. Blood contamination in samples from Experiments 2 and 3 was corrected by using 131I-rat albumin measurements. Pretreatment with rh-TM improved the survival from 12 h through 23 h as compared with that of the endotoxin control group (p < 0.05). However, at 24 h, after essentially all injected rh-TM had been eliminated, there was no difference in survival. Significant reductions in MI, W/D, and T/P in the organs sampled were observed in the rh-TM pretreated groups (p < 0.05). In conclusion, rh-TM improved short-term but not overall survival and decreased MSOF in endotoxemic rats.
 ...
PMID:The effects of recombinant human thrombomodulin on endotoxin-induced multiple-system organ failure in rats. 866 42

Thrombomodulin is an endothelial cell membrane glycoprotein that promotes protein C activation. It has been clearly demonstrated that the anticoagulant functions of the protein C system are important in the prevention of thromboembolic disease. Patients with protein C or protein S deficiency and/or resistance to activated protein C (APC resistance) are at higher risk for developing thromboembolic disease. The first mutation in the thrombomodulin gene was discovered in an American patient suffering from pulmonary embolism at the age of 45 (Ohlin and Marlar 1995). Here we report a case of sagittal sinus thrombosis in a 42-year-old Swedish woman. She was found to carry a heterozygous point mutation changing G127 to A, predicting an Ala25 to a Thr change in the mature thrombomodulin protein. This mutation was also found in her 16-year-old daughter, who so far has not suffered from any thrombotic events. The patient had no other detectable prothrombotic genetic defects associated with the coagulation system. This case supports the hypothesis of an association between mutations in the thrombomodulin gene and venous thrombosis.
 ...
PMID:A novel thrombomodulin gene mutation in a patient suffering from sagittal sinus thrombosis. 936 78

Blood clotting involves a multitude of proteins that act in concert in response to vascular injury to produce the procoagulant enzyme alpha-thrombin, which in turn is responsible for the generation of the fibrin plug. However, while generation of the fibrin plug is required for the arrest of excessive bleeding, unregulated clotting will result in the occlusion of the blood vessels and thrombosis. Thus, the regulation of the delicate balance between the procoagulant and anticoagulant mechanisms is of extreme importance for survival. While the majority of proteins involved in blood coagulation circulate as inactive zymogens that require proteolytic activation in order to function, approximately 1% of the circulating factor VII molecules are active. Factor VIIa, possess a serine protease active site, has poor catalytic activity, and is not inhibited by the circulating stoichiometric protease inhibitors. Following injury to the vasculature and subsequent exposure of the integral membrane glycoprotein, tissue factor (TF), the circulating factor VIIa molecules can bind to the exposed TF forming the extrinsic tenase complex (TF/factor VIIa) and initiate the blood coagulation process. Formation of the TF/factor VIIa complex increases the catalytic efficiency of the enzyme by four orders of magnitude when compared with factor VIIa alone. This cell-associated enzymatic complex initiates a series of enzymatic reactions, leading to the generation of alpha-thrombin and ultimately to the formation of the fibrin plug. The procoagulant enzymatic complexes (i.e., prothrombinase, intrinsic tenase, and extrinsic tenase) are similar in structure and composed of an enzyme, a cofactor, and the substrate associated on a cell surface in the presence of divalent metal ions. While the activity of the extrinsic tenase complex is limited by the availability (exposure) of its cell-associated cofactor (TF) it is remarkable that the activities of both the prothrombinase complex (factor Va/factor Xa) as well as the intrinsic tenase complex (factor VIIIa/factor IXa) are limited by the presence of the two soluble, nonenzymatic cofactors, factor Va and factor VIIIa. Factor Va and factor VIIIa, which are very similar in structure and function, are required for prothrombinase and intrinsic tenase activities, respectively, because both cofactors express a dual function in their respective complexes, acting as an enzyme receptor and catalytic effector on the cell surface. The cofactors derive from inactive plasma precursors by regulatory proteolytic events that involve alpha-thrombin. In general, bleeding tendencies are usually associated with defects in the activation of one of the zymogens or the cofactors of the procoagulant complexes. However, the activity of all of the complexes is also limited by the availability of an adequate membrane surface provided by endothelial cells, platelets, and monocytes. The cell surface provides a site for the recruitment of the appropriate proteins and allows for fast and efficient clot formation. In the absence of an appropriate membrane surface, the procoagulant complexes have limited catalytic efficiency. Thus, timely exposure of the adequate membrane surface is an additional step in the regulation of alpha-thrombin formation. alpha-Thrombin participates in its own down-regulation by binding to the endothelial cell receptor thrombomodulin, initiating the protein C pathway, which in turn leads to the formation of activated protein C (APC). APC is required for efficient neutralization of factor Va cofactor activity, which results in the inactivation of the prothrombin-activating complex. This inactivation can only occur in the presence of the appropriate membrane surface. Thus, while following alpha-thrombin activation, factor VIIIa is rapidly and spontaneously inactivated by dissociation of the A2 domain from the rest of the cofactor, APC is required for down-regulation of alpha-thrombin formation by prothrombinase. (ABSTRACT
 ...
PMID:The regulation of clotting factors. 939 73


1 2 Next >>