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)

This report identifies a component of normal human fibroblasts that forms a covalent linkage with thrombin and urokinase (urinary plasmingoen activator) and mediates most of the specific cellular binding of these proteases. This component, here named protease-nexin (PN), is both associated with the cell surface and released into the culture medium. In several ways PN resembles antithrombin III (AT3), a prominent inhibitor of thrombin in serum: PN links thrombin, probably via an ester bond; PN does not link thrombin blocked at its catalytic site serine; PN has a high-affinity heparin-binding site; and heparin greatly accelerates the rate of linkage between soluble PN and thrombin. Despite these similarities, PN and AT3 are distinct; they differ in size and are not immunologically cross-reactive. Whereas AT3 regulates the proteolytic activity of thrombin in serum, PN may regulates the activity of serine proteases at and near the cell surface.
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PMID:Protease-nexin: a cellular component that links thrombin and plasminogen activator and mediates their binding to cells. 615 79

Human alpha-thrombin, the procoagulant activation product of prothrombin, elicits chemotaxis in several macrophage-like continuous cell lines, most notably J774. Effective alpha-thrombin concentrations eliciting cell movement range from 10(-10) to 10(-6) M, with the optimal response occurring at about 10(-8) M. At the latter concentration, the response is equivalent, on a molar basis, to that observed with f-Met-Leu-Phe-OH, the positive control used in these experiments. Blockage of alpha-thrombin's active center with diisopropylfluorophosphate or by tryptic proteolysis of the procoagulant exosite (i.e., gamma-thrombin) does not decrease chemotactic activity. However, formation of enzymatically inactive complexes with AT3 or hirudin eliminates chemotactic potency. Competition chemotaxis assays, carried out by comparing the abilities of varying concentrations of test substances placed in the upper compartment of Boyden chambers to inhibit gradient-oriented movement of cells toward a fixed concentration of thrombin in the lower compartment, demonstrate that, although the modified forms of thrombin cross-compete, formylated peptide will not inhibit thrombin's chemotactic effects. Binding studies carried out using 125I-alpha-thrombin on paraformaldehyde-fixed J774 cells show a Kd of approximately 7.5 nM with an estimated 14,100-binding sites/cell. This Kd is in agreement with the optimal chemotactic thrombin dose (approximately 10 nM). On the basis of the competition chemotaxis assays and the radiolabeled thrombin-binding data, it is proposed that unique thrombin-specific chemotactic receptors exist on J774 cell membranes, and that these receptors are distinct from those mediating chemotaxis by agents such as formylated peptides.
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PMID:Receptor-mediated chemotactic response of a macrophage cell line (J774) to thrombin. 631 83

Human alpha-thrombin, the procoagulant activation product of prothrombin, elicits chemotaxis in human peripheral blood monocytes and several macrophagelike continuous cell lines, most notably J-774.2, but not in human peripheral blood granulocytes. alpha-Thrombin is effective in stimulating cell movement at concentrations ranging from 10(-10) to 10(-6) M but is optimally active at 10(-8) M. At the latter concentration, the degree of response is equivalent, on a molar basis, to that observed with the peptide formylmethionylleucylphenylalanine, (FMP). In contrast to thrombin, prothrombin produces a minimal chemotactic response in monocytes and J-774.2. Blockade of alpha-thrombin's active center with diisopropylfluorophosphate (DIP-F) or tryptic proteolysis of the procoagulant exosite (i.e., gamma-thrombin) fails to alter chemotactic activity. On the other hand, addition of equimolar amounts of antithrombin III (AT3) to alpha-thrombin reduces thrombin-mediated chemotaxis by 60%, and increased ratios of AT3 to enzyme completely suppress chemotaxis. We conclude that thrombin is a potent monocyte chemotaxin and that the domains in thrombin involved in stimulating cell movement are distinct from the catalytic site and the fibrin recognition exosite. These chemotactic domains appear to be sequestered in prothrombin and in the thrombin-AT3 complex and, as such, are unavailable to the chemotactic receptor on the monocyte cell membrane.
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PMID:Chemotactic response of monocytes to thrombin. 682 48

The polymerase chain reaction and direct sequencing were used to determine the nature of the mutations in the antithrombin III (AT3) gene in seven unrelated patients with familial antithrombin III (ATIII) deficiency and recurrent venous thrombosis. Three novel mutations were found, two associated with a type I deficiency state (Pro80-->Thr and His120-->Tyr) manifesting reduced synthesis of ATIII. The other novel lesion (Met251-->Ile) was associated with a dysfunctional ATIII protein (type II ATIII deficiency) and is predicted to interfere either with a heparin-induced conformational change in the ATIII molecule or with docking to thrombin. A novel polymorphism (Tyr158-->Cys) was also found to occur in several individuals of Scandinavian origin.
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PMID:Three novel missense mutations in the antithrombin III (AT3) gene causing recurrent venous thrombosis. 795 85

Human antithrombin is the major plasma inhibitor of thrombin both in the presence and absence of heparin. Its physiological importance is emphasised by the recurrent thromboses that individuals with a deficient or functionally abnormal protein are prone to develop. Such deficiencies are estimated to affect as many as 1:630 of the general population and between 3% and 5% of patients with thrombotic disease. The gene for antithrombin (AT3) has been cloned and shown to map to the long arm of chromosome 1 at 1q23-25. The gene consists of seven exons and six introns and spans 13,477bp of DNA. Advances in molecular genetic techniques have facilitated identification of the underlying DNA mutation(s) in > 80 families with antithrombin deficiency. Such work has proved invaluable in structure-function studies and in helping to provide informed genetic counselling to "at-risk" individuals based upon the natural history of similar variants.
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PMID:Molecular genetics of human antithrombin deficiency. 866 6

Selective adhesion of plasma proteins to immobilized heparin is considered to be beneficial regarding hemocompatibility of foreign materials in contact with blood. Prothrombin, thrombin, antithrombin III (AT3), and fibrinogen were selected for analysis in an experimental model. Biomolecular interaction analysis employing surface plasmon resonance was utilized to record and analyze their binding properties in real time. Biotinylated heparin, heparin-albumin conjugate, and albumin, respectively, were immobilized onto streptavidin-coated sensors as ligands. Prothrombin did not bind to any of the ligand surfaces and no specific binding of any of the plasma proteins to albumin was observed. Binding kinetics of thrombin to heparin and to heparin-albumin conjugate were calculated using two different methods. For heparin, identical K(D)(equilibrium dissociation constant) values of 61 x 10(-9) M were obtained with both methods. For the conjugate, only slightly different K(D) values of 111 x 10(-9) and 104 x 10(-9) M, respectively, were calculated. The affinity of thrombin toward the heparin-coated surface proved to be higher than its affinity toward the heparin conjugate. The binding pattern of AT3 to both heparin and heparin-albumin conjugate, although specific, was biphasic, possibly due to a conformational change during the binding process. Steady-state kinetic analysis revealed a K(D) value of 281 +/- 24 x 10(-9) M for the heparin surface. For the conjugate surface, a K(D) of 53 +/- 5 x 10(-9) M was calculated, indicating a higher affinity toward heparin-albumin conjugate. A high-affinity binding of fibrinogen to high-density surfaces of both heparin and the conjugate was observed. However, as binding to low-density surfaces was considerably reduced, specificity remained uncertain.
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PMID:Plasma protein binding properties to immobilized heparin and heparin-albumin conjugate. 1753 59

The transfusion of fresh-frozen plasma (FFP) is suggested to minimize dilution coagulopathy when applied instead of colloids during paediatric craniofacial surgery (pCFS). We prospectively compared plasmatic haemostaseologic function between volume replacement with FFPs versus human albumin (HA) in a pilot study. Thirty infants with primary craniosynostosis were scheduled for pCFS. In 15 of those, FFPs were available from the identical donor as for packed red blood cells (pRBC), and were thus employed for intraoperative volume replacement. The remaining 15 infants were infused with HA-5% instead. Haemoglobin(Hb)-values, global coagulation parameters (activated partial thromboplastin time-aPTT; prothrombin time-PT), selected clotting factors (F) (VIII, XI, XIII), antithrombin-AT, fibrinolytic factors (fibrinogen; plasminogen; alpha2-antiplasmin-alpha2A), and activation parameters (thrombin-antithrombin-complex-TAT; plasmin-antiplasmin-complex-PAP; D-dimers) were assessed and compared between both groups after induction of anaesthesia, before transfusion of pRBC, and at the end of surgery. Patients and treatment characteristics were balanced between both groups. Prolongation of aPTT and decreases of PT, FXI, FXIII, AT3, and fibrinolytic factors were more pronounced in the HA-group. Increases in F VIII activity, activation parameters, and the course of Hb-values were similar among both groups. There was no difference regarding clinical endpoints (peri-/postoperative pRBC-transfusions, postoperative blood loss). In conclusion, the application of HA was associated with a more distinct dilution of procoagulant factors, AT3, and fibrinolytic factors than the use of FFPs. However, the course of activation markers suggested a similar extent of clotting and fibrinolytic activation with the use of both transfusion regimens, and there were no differences with regard to clinical endpoints.
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PMID:Intraoperative fresh-frozen plasma versus human albumin in craniofacial surgery--a pilot study comparing coagulation profiles in infants younger than 12 months. 1759 10

Pathologic blood clotting is a leading cause of morbidity and mortality in the developed world, underlying deep vein thrombosis, myocardial infarction, and stroke. Genetic predisposition to thrombosis is still poorly understood, and we hypothesize that there are many additional risk alleles and modifying factors remaining to be discovered. Mammalian models have contributed to our understanding of thrombosis, but are low throughput and costly. We have turned to the zebrafish, a tool for high-throughput genetic analysis. Using zinc finger nucleases, we show that disruption of the zebrafish antithrombin III (at3) locus results in spontaneous venous thrombosis in larvae. Although homozygous mutants survive into early adulthood, they eventually succumb to massive intracardiac thrombosis. Characterization of null fish revealed disseminated intravascular coagulation in larvae secondary to unopposed thrombin activity and fibrinogen consumption, which could be rescued by both human and zebrafish at3 complementary DNAs. Mutation of the human AT3-reactive center loop abolished the ability to rescue, but the heparin-binding site was dispensable. These results demonstrate overall conservation of AT3 function in zebrafish, but reveal developmental variances in the ability to tolerate excessive clot formation. The accessibility of early zebrafish development will provide unique methods for dissection of the underlying mechanisms of thrombosis.
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PMID:Targeted mutagenesis of zebrafish antithrombin III triggers disseminated intravascular coagulation and thrombosis, revealing insight into function. 2499 78

Hemophilia A and B are inherited bleeding disorders characterized by deficiencies in procoagulant factor VIII (FVIII) or factor IX (FIX), respectively. There remains a substantial unmet medical need in hemophilia, especially in patients with inhibitory antibodies against replacement factor therapy, for novel and improved therapeutic agents that can be used prophylactically to provide effective hemostasis. Guided by reports suggesting that co-inheritance of prothrombotic mutations may ameliorate the clinical phenotype in hemophilia, we developed an RNA interference (RNAi) therapeutic (ALN-AT3) targeting antithrombin (AT) as a means to promote hemostasis in hemophilia. When administered subcutaneously, ALN-AT3 showed potent, dose-dependent, and durable reduction of AT levels in wild-type mice, mice with hemophilia A, and nonhuman primates (NHPs). In NHPs, a 50% reduction in AT levels was achieved with weekly dosing at approximately 0.125 mg/kg, and a near-complete reduction in AT levels was achieved with weekly dosing at 1.5 mg/kg. Treatment with ALN-AT3 promoted hemostasis in mouse models of hemophilia and led to improved thrombin generation in an NHP model of hemophilia A with anti-factor VIII inhibitors. This investigational compound is currently in phase 1 clinical testing in subjects with hemophilia A or B.
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PMID:An RNAi therapeutic targeting antithrombin to rebalance the coagulation system and promote hemostasis in hemophilia. 2584 32