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)

There are three different aspects in the etiological diagnosis of hemostasis disorders: (1) primary hemostasis evaluation; (2) coagulation evaluation; and, (3) thrombosis evaluation. In every case, a look at the medical history is an essential step before proceeding to biological investigations. Bleeding time is the first step of the primary hemostasis evaluation; prolonged drug use (particularly salicylates) and thrombocytopenia must be first considered; only then will the Von Willebrand factor and platelet functions be studied. Coagulation evaluation first requires the study of the overall coagulation tests (Quicks test, partial thromboplastin time test and thrombin time test); determination of the different plasma coagulation factors and search for a circulating anticoagulant will be performed secondarily, allowing differentiation between the different acquired or constitutional coagulation disorders. Thrombosis evaluation must first consider a local or general inciting factor before looking for anti-phospholipid antibodies, an acquired protein C or S deficiency or a constitutional hemostasis disorder.
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PMID:[Exploration of hemostasis disorders in children (excluding the neonatal period)]. 1054 86

The aim of this study was to design a simple laboratory method that can screen the overall haemostatic potential in plasma (OHPP) when a hyper- or hypocoagulable state is present. A fibrin time curve was made via spectrophotometric registration of fibrin generation and lysis in plasma, to which exogenous thrombin and tissue type plasminogen activator was added. The area under the curve, calculated by the sum of absorbance (ABS-sum), varied in correlation to the concentrations of platelets or purified pro-/anticoagulants: tissue factor, von Willebrand factor, fibrinogen, antithrombin, plasminogen, or plasminogen activator inhibitor type 1. The ABS-sums also changed in positive relation to the haemostatic function investigated in 16 menopausal women and 14 young healthy nonpregnant women (controls). The findings imply that the ABS-sums not only offer a general information about fibrin generation and lysis in vitro, but also reflect the OHPP (i.e., final combined effects of platelet activity, coagulation, and fibrinolysis in vivo). Preliminary results were satisfactory; the levels of OHPP, expressed as the ABS-sums, were higher in normal pregnant women than in the controls, and even higher in preeclamptic patients than in pregnant women with no complications, which corresponds to the different grades of hypercoagulability in the three groups. Moreover, the level of OHPP was considerably lower in an untreated infant with von Willebrand's disease type 3 and in factor VIII- or factor IX-deficient plasma samples.
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PMID:A laboratory method for determination of overall haemostatic potential in plasma. I. Method design and preliminary results. 1057 92

Recent studies emphasize the role of blood constituents in Staphylococcus aureus (S. aureus) adherence to subendothelial extracellular matrix. In the present study, the adherence of two strains of S. aureus (ATCC 29213 and RN 6390) grown to a postexponential phase to cultured human umbilical vein endothelial cells (EC-304) was examined. Under flow conditions (600 s(-1)), pretreatment of endothelial cells (ECs) with human alpha-thrombin (2 U/mL) significantly (2- to 4-fold) increased bacterial adherence to ECs. Adherence of both S. aureus strains to thrombin-treated ECs was similarly higher in the presence of whole blood, platelet-rich plasma, or platelet-poor plasma when compared with Tris-buffered saline solution (TBS). Platelet inactivation in whole blood by prostaglandin E1 did not reduce the adherence rate. When ATCC 29213 bacteria were suspended in TBS containing increasing concentrations of fibrinogen at near-physiologic ranges (0.25 to 2 mg/mL), a dose-dependent increase in S. aureus adherence to thrombin-activated ECs was observed that reached a maximum level of about 12-fold. Fibronectin used at the above physiologic concentrations (12.5 to 100 microg/mL) enhanced bacterial adherence up to 2-fold. Von Willebrand factor (1 IU/mL) did not support bacterial adherence to ECs, either alone or in combination with fibrinogen. Inhibition of fibrin formation either by the Gly-Pro-Arg-Pro peptide or by hirudin increased bacterial adherence by 50% and 90%, respectively. Blockage of either ICAM-I, alpha5beta1, or alphavbeta3 receptors on ECs by appropriate monoclonal antibodies resulted in substantial inhibition of bacterial adherence (by 42%, 65%, and 72%, respectively). Preincubation of S. aureus with a fibrinogen gamma-chain binding domain peptide led to 65% inhibition of adherence to ECs in the presence of fibrinogen. In contrast, preincubation of bacteria with the Arg-Gly-Asp-Ser peptide failed to affect their adherence. The data suggest that S. aureus adherence to the EC surface was (1) significantly enhanced by thrombin treatment of ECs, (2) not mediated by platelets, and (3) mediated by plasma fibrinogen, which binds to the bacteria via the C-terminus gamma-chain binding domain but not via the Arg-Gly-Asp sequence.
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PMID:Staphylococcus aureus adherence to thrombin-treated endothelial cells is mediated by fibrinogen but not by platelets. 1063 93

Binding of Ulex europaeus lectin to microvessels was used to isolate endothelial cells from cycling human endometrium. Cultured human endometrial endothelial cells (HEECs) exhibited endothelial cell-specific characteristics such as tube formation on a basement membrane matrix and sequestration of acetylated low-density lipoprotein. Markers for potentially contaminating epithelial, stromal, smooth muscle, and bone marrow-derived cells were not detected in the HEEC cultures. Basal and proinflammatory-stimulated immunostaining profiles for endothelial cell-specific adhesion markers, as exemplified by Von Willebrand's factor and E-selectin, were similar for cultured HEECs and human umbilical venous cord endothelial cells (HUVECs). However, HUVECs expressed several extracellular matrix proteins that were absent from cultured HEECs. In the latter, the protein kinase C agonist phorbol myristate acetate transiently enhanced tissue factor (TF) mRNA levels and elicited a more prolonged elevation in TF protein levels, but did not affect plasminogen activator inhibitor-1 (PAI-1) mRNA and protein levels. Inappropriate expression of TF, which initiates hemostasis by generating thrombin, and of PAI-1, which regulates hemostasis by acting as the primary inhibitor of fibrinolysis, can each lead to thrombosis. The differential regulation of TF and PAI-1 expression revealed in the current study emphasizes the importance of using HEECs to evaluate mechanisms regulating the hemostatic/thrombotic balance in human endometrium.
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PMID:Human endometrial endothelial cells: isolation, characterization, and inflammatory-mediated expression of tissue factor and type 1 plasminogen activator inhibitor. 1068 11

The role of the platelet glycoprotein (GP) Ib-V-IX receptor in thrombin activation of platelets has remained controversial although good evidence suggests that blocking this receptor affects platelet responses to this agonist. The mechanism of expression of procoagulant activity in response to platelet agonists is also still obscure. Here, the binding site for thrombin on GPIb is shown to have a key role in the exposure of negatively charged phospholipids on the platelet surface and thrombin generation, in response to thrombin, which also requires protease-activated receptor-1, GPIIb-IIIa, and platelet-platelet contact. Von Willebrand factor binding to GPIb is not essential to initiate development of platelet procoagulant activity. Inhibition of fibrinogen binding to GPIIb-IIIa also failed to block platelet procoagulant activity. Both heparin and low molecular weight heparin block thrombin-induced platelet procoagulant activity, which may account for part of their clinical efficacy. This study demonstrates a new, critical role for platelet GPIb in hemostasis, showing that platelet activation and coagulation are tightly interwoven, which may have implications for alternative therapies for thrombotic diseases.
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PMID:The GPIb thrombin-binding site is essential for thrombin-induced platelet procoagulant activity. 1100 99

In von Willebrand disease (vWD) type 1 and mild haemophilia A patients we studied the effect of an infusion of DDAVP (0.3 microg/kg body weight) on thrombin generation in platelet-rich plasma (PRP) and platelet-poor plasma (PPP). Baseline thrombin generation in PRP was diminished both in the haemophilia A and vWD patients. It was normal in vWD plasma when sufficient procoagulant phospholipids were present, either via adding phospholipid vesicles to PPP or via scrambling of the platelet membrane with ionomycin in PRP. In haemophilia A plasma, thrombin generation did not normalize by providing procoagulant phospholipids. Treatment with DDAVP temporarily restored thrombin generation in PRP to normal in both diseases. To investigate the individual roles of von Willebrand factor (vWF) and factor VIII, we also studied the effect of factor VIII infusion on thrombin generation in a severe haemophilia patient. It appears that at a fixed normal vWF concentration, <25% factor VIII is sufficient for normal thrombin generation in PRP. At a sufficient factor VIII concentration, however, thrombin generation is still lower than normal in vWD patients; approximately 40% of vWF is required for half-normal thrombin generation in PRP. It thus appears that vWF is also a clotting factor, in the sense that it is required for normal thrombin generation. This underlines the importance of the interaction between coagulation and the platelets in normal haemostasis. Thrombin generation in PRP appears to be a suitable test to reflect the combined function.
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PMID:The effect of DDAVP infusion on thrombin generation in platelet-rich plasma of von Willebrand type 1 and in mild haemophilia A patients. 1105 63

The management of unexpected bleeding must be directed at the specific abnormality identified, as there is no universally effective and safe procoagulant product. Where practical, a purely pharmaceutical approach obviates the residual risks of exposure to plasma-derived products. Desmopressin is often effective in bleeding due to mild haemophilia A, Type I von Willebrand's disease and some platelet function disorders. Where replacement therapy is necessary, it should be as specific as possible, preferably using purified components singly or in combination. Recombinant proteins provide the greatest margin of safety, but it must be borne in mind that these are biologicals, and that they may contain human and animal plasma-derived proteins. Where specific replacement is unavailable or impractical, plasma or crudely fractionated plasma derivatives may be used. In the case of inhibitor antibodies to factor VIII, high dose human factor VIII or porcine factor VIII may be used. Where replacement therapy is impossible due to a high inhibitory titre, it may be necessary to bypass the specific haemostatic defect using activated prothrombin complex concentrates or recombinant activated factor VIIa. The latter product is being studied in patients with various disorders of platelet function, and in the more global haemostatic failure that accompanies end-stage liver disease. Ancillary methods are often of great value in securing haemostasis. These may be derived from pharmacological or biological sources, and their sites of action may be systemic or topical. Examples include antifibrinolytic lysine analogues, corticosteroids where inflammation accompanies bleeding, and the topical application of fibrin sealants or thrombin. Simple physical measures such as pressure, ice, or splinting are also valuable adjunctive measures. Finally, it must be emphasized that the ultimate control of bleeding often depends upon effective management of the inciting cause, such as eliminating the trigger for DIC, or suppressing the causative antibody of ITP. These principles will be presented using a practical algorithmic approach. The initial question when considering treatment should be whether or not the patient is acutely unstable. Instability may be due to one of two causes: the volume of blood loss leading to a compromised cardio-vascular status, or the site of the bleed. The relevance of the site of the bleed is independent of the volume of blood loss, so for example, a closed bleed into CNS will cause critical functional compromise even though the volume of bleeding may be minimal. Similarly bleeding into a compartment, such as into a forearm or a calf will cause critical functional compromise irrespective of the volume of bleeding.
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PMID:Unexpected bleeding disorders: Algorithm for approach to therapy. 1125 56

The macromolecular composition of the extracellular matrix (ECM) produced by the human microvascular endothelial cell line (HMEC-1) was determined by ELISA and its thrombogenicity was studied in blood perfusion assays. Results were compared with those obtained with the ECM produced by human umbilical vein endothelial cells (HUVEC). The HMEC-1's ECM contains collagen type IV, fibronectin, laminin and thrombospondin, but no detectable levels of collagen types I, III and VI, or von Willebrand factor (vWF), whereas all these components were found in the ECM synthesized by HUVEC. HMEC-1's ECM was perfused with low-molecular-weight heparin-anticoagulated blood at two wall shear rates (650/s and 2,600/s), representative of moderate and high arterial wall shear rates, in parallel plate flow chambers for 5 min. This resulted in the formation of large platelet aggregates, compared to essentially a monolayer of adherent platelets on HUVEC's ECM. Interestingly, large thrombi were formed at 2,600/s when HMEC-1's ECM was perfused with the blood of a patient with severe type III von Willebrand disease lacking both plasma and platelet vWF, indicating that vWF was not absolutely required for thrombus formation on this matrix. Thrombin generated on the HMEC-1's ECM contributed importantly to the large platelet thrombi formed, shown by performing blood perfusion experiments in the presence of thrombin inhibitors. Our results indicate that 1) platelet adhesion and aggregate formation on a subendothelium may occur at a high shear rate (2600/s) without the participation of collagen types I, III and VI, and vWF; and 2) the HMEC-1 cell line may prove useful for in vitro studies of the thrombogenic properties of microvascular subendothelium which in most cases does not contain fibrillar collagens and vWF.
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PMID:The subendothelium of the HMEC-1 cell line supports thrombus formation in the absence of von Willebrand factor and collagen types I, III and VI. 1130 30

Platelet glycoprotein (GP) Ibalpha is a component of the GPIb-IX receptor complex, which is involved in multiple physiological and pathological processes, including platelet adhesion at sites of vascular injury, thrombin binding, Bernard-Soulier syndrome, platelet-type von Willebrand disease, and immune-mediated thrombocytopenias. The amino-terminal domain of approximately 300 residues of GPIbalpha mediates both normal biological function (by providing the sites for direct ligand interaction) and aberrant function (through amino acid substitutions). To investigate the molecular interactions mediated by this region of GPIbalpha, we have developed a recombinant baculovirus to facilitate its expression as a calmodulin fusion protein from insect cells. By employing the calmodulin tag, the fusion protein could be obtained at >90% purity after a single isolation step at yields of 8 mg/L of insect cell medium (purified fusion protein). The recombinant GPIbalpha fragment was shown to be posttranslationally sulfated and glycosylated, although its glycosylation differed from that of the equivalent GPIbalpha fragment isolated from human platelets. The differential glycosylation, however, did not affect the function of the recombinant GPIbalpha fragment in either von Willebrand factor (vWf) or thrombin binding as these were both found to be identical to those of the same-length GPIbalpha fragment derived from human platelets. The calmodulin tag was also exploited in the development of assays to measure directly vWf and thrombin binding, since it did not interfere with either, demonstrating the feasibility for the use of this soluble receptor fusion protein in detailed biophysical assays to investigate the molecular mode of binding of platelet glycoprotein Ibalpha to these ligands.
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PMID:Expression of the amino-terminal domain of platelet glycoprotein Ib alpha: exploitation of a calmodulin tag for determination of its functional activity. 1143 95

Von Willebrand factor (vWF) is synthesized in endothelial cells as pre-provWF and processed intracellularly to propeptide (vWFpp) and mature vWF. Building on previous studies indicating that recombinant provWF when infused into animals can also be processed extracellularly in vivo, we investigated the processing of provWF in vitro. Incubation of a recombinant provWF (rpvWF) preparation with canine and human vWF-deficient plasma induced a time-dependent decrease in provWF antigen and an increase in vWFpp antigen without changing total vWF antigen or collagen-binding activity. Multimer analysis showed the gradual transformation of the provWF multimers to mature vWF multimers and cleaved vWFpp was visualized on autoradiograms of SDS-polyacrylamide electrophoresis gels using 125-labeled provWF. Processing was facilitated by CaCl2 but prevented by a thrombin inhibitor and did not occur in prothrombin-depleted plasma. When recombinant provWF was incubated with increasing amounts of purified thrombin, the extent of provWF processing was dose-dependent. The specific cleavage of vWFpp was confirmed by immunoblots using an anti-vWFpp antibody and by amino terminal amino-acid analysis. Binding of provWF to collagen decreased the thrombin concentration necessary for propeptide removal to a concentration in the range of that found during blood clotting. Meizothrombin, an intermediate of prothrombin activation, was also able to induce dose-dependent removal of the propeptide from rpvWF. Hirudin preconditioning of vWF-deficient mice attenuated processing of infused rpvWF suggesting that thrombin plays a part in the processing events in vivo.
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PMID:Thrombin-mediated in vitro processing of pro-von Willebrand factor. 1177 13


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