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)

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.
 ...
PMID:Platelets fixed with paraformaldehyde: a new reagent for assay of von Willebrand factor and platelet aggregating factor. 23 99

A platelet-aggregating activity was found in many snake venoms, predominantly those of the genus Bothrops, that is apparent only in the presence of the platelet-aggregating von Willebrand factor of plasma. It is designated "venom coagglutinin." The coagglutinin can be largely separated from the thrombin-like enzyme of the venoms by ion-exchange chromatography. The venom factor acts on formaldehyde-fixed platelets and is effective with decalcified, heparinized, and afibrinogenemic plasmas but not with severe von Willebrand disease plasmas or with normal plasmas in which the von Willebrand factor has been neutralized by specific antibodies. Use of this coagglutinin permits the assay of von Willebrand factor without the many disadvantages of the ristocetin test. The coagglutinin is active with human, dog, pig, and bovine plasmas and with platelets of any one of these species. This broad-spectrum activity without regard to species contrasts with the ristocetin-resistance of many combinations of plasma and platelets from various species. The assay provides a procedure for studying human, porcine, and canine von Willebrand disease. The lack of species specificity of the coagglutinin suggests that it may be a universal activator of the von Willebrand factor-platelet reaction.
 ...
PMID:Venom coagglutinin: an activator of platelet aggregation dependent on von Willebrand factor. 30 34

Recent observations suggest that plasma F VIII consists of a series of molecules with different molecular weights. The data described in this paper suggest that sup F VIII represents the molecules with relatively low molecular weights whereas the molecules with the highest molecular weights appear in cryo F VIII. Sup F VIII was associated with VIII:C and VIIIR:Ag, but ristocetin cofactor activity was lacking. Although the immunoprecipitation characteristics of sup F VIII with rabbit antifactor VIII were different from those of cryo F VIII, immunological identity was observed in immunodiffusion and crossed immunoelectrophoresis. In 0.8M NaCl sup F VIII dissociated into VIIIR:Ag of relatively high molecular weight and VIII:C of low molecular weight. No indications were obtained that the presence of sup F VIII was the result of proteolytic degradation of factor VIII. VIII:C of sup F VIII was more labile in vitro than VIII:C in plasma. It could be activated by traces of thrombin in a way similar to plasma F VIII. In patients with classic von Willebrand's disease relatively more VIII:C remained in the supernatant after cryoprecipitation of plasma.
 ...
PMID:Heterogeneity of human factor VIII. I. Characterization of factor VIII present in the supernatant of cryoprecipitate. 61 89

The maximal rate of change in optical density (Vmax-deltaOD)of plasma clots forming in the activated partial thromboplastin time test (APTT) may be significantly influenced by reductions in factor VIII activity insufficient to also cause a distinctly abnormal timed clotting endpoint. Analysis of relationships between Vmax-deltaOD of clotting plasma in the APTT, prothrombin time, and thrombin time tests provides a basis for increasing the screening value of the APTT in suggesting intrinsic system abnormalities. Three illustrative case reports support the added benefit of thrombokinetics in the detection of mild factor VIII deficiency in hemophilia A and in von Willebrand's disease.
 ...
PMID:Detection of mild factor VIII Deficiency by thrombokinetics. 115 79

avWD is a rare entity that is primarily associated with lymphoproliferative disorders, most commonly with multiple myeloma, lymphoma, and the myeloproliferative diseases. Various pathogenetic mechanisms have been postulated. The most commonly seen is antibodies directed against the FVIII complex, resulting in either its accelerated destruction or its accelerated clearance by the reticuloendothelial system. There may be immunoadsorption of the FVIII complexes onto the clones of malignant cells, as has been reported in several cases, or proteolysis may be causing the peripheral destruction of the FVIII complex. Lastly, as seen in hypothyroidism, global decrease in production of the multimers also results in avWD. The treatment, in general, should be aimed at controlling the underlying disorder and at stopping any life-threatening hemorrhage. The treatment includes any or all of the following: DDAVP, cryoprecipitate, FVIII concentrates, extracorporeal immunoadsorption, and chemotherapy as needed to control the underlying disorders. The screening tests that will allow for the detection of the avWD include measurement of the bleeding time, the FVIII:C, FVIII:vWF, and the FVIII:RCoF. FVIII:C inhibitors can be demonstrated by mixing the patient plasma with normal plasma. A normal prothrombin time (PT), activated partial thromboplastin time (APTT), and thrombin time (TT) are expected. Clinically, these patients present with mucosal bleeding, and in avWD tend to have an association with lymphoproliferative malignancies. They tend to be elderly patients with no prior history of bleeding diathesis and to have negative family histories for coagulopathies. Further study of these patients is warranted, because this disorder appears to have a multifactorial etiology. Increasing our understanding of avWD may increase our understanding of congenital vWD, thus allowing us to more effectively treat all patients with von Willebrand's disease.
 ...
PMID:Acquired von Willebrand's disease. 145 20

Thrombus formation on collagen fibrils was quantified at venous (100/s) and arterial (650/s and 2,600/s) wall shear rates in blood from patients with various subtypes of von Willebrand disease (vWD) and with hemophilia A (HA). Nonanticoagulated blood was drawn directly from an antecubital vein over purified type III collagen fibrils exposed in parallel-plate perfusion chambers. Blood-collagen interactions were differentiated and quantified by morphometry as platelet adhesion, thrombus height, thrombus volume, and deposition of fibrin strands. Sixteen patients with vWD, including four type III, six type I, four type IIA, and two type IIB, were compared with 26 normal subjects and nine patients with HA, including six severe HA and three mild HA. Platelet adhesion and thrombus formation at 2,600/s were significantly decreased in blood from patients with vWD type III, IIA, and IIB, but not in blood from patients with type I and in HA. The abnormal thrombus formation was apparently not related to the decreased levels of factor VIII (F.VIII), because thrombus height and volume were normal in severe and mild HA. Thrombus formation at 650/s was also significantly decreased in patients with vWD type III, IIA, and IIB and slightly reduced in type I. Significant reduction in thrombus volume and height was also observed in blood from patients with severe HA, but not in mild HA. Thrombus dimensions were not affected at 100/s in the vWD subtypes. However, significantly decreased thrombus height and virtually absent fibrin deposition were observed in blood from patients with severe HA. Apparently, F.VIII supports thrombus formation at low and intermediate shear conditions, presumably through the generation of thrombin. In contrast, von Willebrand factor (vWF) mediates not only platelet adhesion, but also thrombus formation at intermediate and high shear rates. Thus, the relative contribution of coagulation (F.VIII) and platelet function (vWF) in thrombus formation appears to be shear rate dependent, but having optimal effects at different shear conditions.
 ...
PMID:Shear rate-dependent impairment of thrombus growth on collagen in nonanticoagulated blood from patients with von Willebrand disease and hemophilia A. 149 39

We recently reported that after activation of human platelets by thrombin, glycoprotein (GP) Ib-IX complexes are translocated to the surface-connected canalicular system (SCCS) (Blood 76:1503, 1990). As GPIb is a major receptor for von Willebrand factor (vWF) in platelet adhesion, we have now examined the consequences of thrombin activation on the organization of vWF bound to GPIb on the platelet surface. Studies were performed using monoclonal or polyclonal antibodies in either immunogold staining and electron microscopy (Au-EM) or in flow cytometry. When unstirred platelet-rich plasma was incubated with ristocetin, bound vWF was located by Au-EM as discrete masses regularly distributed over the cell surface. Platelets from a patient with Glanzmann's thrombasthenia, lacking GPIIb-IIIa complexes, gave a similar pattern, confirming that this represented binding to GPIb. That ristocetin was not precipitating vWF before their binding to the platelets was shown by the detection of similar masses on the surface of platelets of a patient with type IIB von Willebrand disease. Experiments were continued using washed normal platelets incubated in Tyrode-EDTA, the purpose of the EDTA being to limit the surface expression of endogenous vWF after platelet stimulation. Under these conditions, platelets were treated with ristocetin for 5 minutes at 37 degrees C in the presence of increasing amounts of purified vWF. This was followed by incubation with thrombin (0.5 U/mL) for periods of up to 10 minutes. Flow cytometry showed a time-dependent loss in the surface expression of vWF bound to GPIb and these changes were confirmed by Au-EM. In particular, immunogold staining performed on ultrathin sections showed that the bulk of the vWF was being cleared to internal membrane systems. Surface clearance of vWF during thrombin-induced platelet activation is a potential mechanism for regulating platelet adhesivity.
 ...
PMID:von Willebrand factor bound to glycoprotein Ib is cleared from the platelet surface after platelet activation by thrombin. 156 27

Orthotopic liver transplantation is frequently associated with a complex coagulation disorder, influencing the outcome of the procedure. In this respect, disseminated intravascular coagulation (DIC) had been suggested to be of causative importance for bleeding complications after reperfusion of the liver graft. In 10 consecutive patients undergoing orthotopic liver transplantations, we studied the occurrence of two phagocyte proteinases of different origin in the graft liver perfusate and in systemic blood during the operation, as well as their effects on hemostasis. As compared with plasma samples taken at the end of the anhepatic phase, highly significant increases of cathepsin B and thrombin-anti-thrombin III complexes (TAT), as well as highly significant decreases in antithrombin III, protein C, and C1-inhibitor were observed in graft liver perfusate. Von Willebrand factor and fibrinogen were slightly decreased, whereas the elastase-alpha 1 proteinase inhibitor complexes (EPI) were elevated. In plasma the activity of cathepsin B remained unchanged during the prereperfusion phases, but immediately after revascularization of the graft this cysteine proteinase increased. The EPI showed a gradual increase in plasma during the preanhepatic and anhepatic phases but a more pronounced increase in the reperfusion phase. In parallel with the rise in these two proteinases TAT increased and the activities of antithrombin III and C1-inhibitor in plasma decreased after reperfusion. At 12 hr after revascularization plasma levels of TAT, antithrombin III, and C1-inhibitor had returned to the prereperfusion ranges, whereas cathepsin B and EPI were significantly above the baseline levels. These observations are consistent with the hypothesis that extracellularly released lysosomal proteinases may play a role in the development of a DIC-like constellation, including thrombin formation after revascularization of the liver graft. For the first time we could prove the occurrence of phagocyte proteinases in graft liver perfusate and evaluate the importance of these proteinases for the understanding of the pathophysiology leading to bleeding complications in patients undergoing orthotopic liver transplantation.
 ...
PMID:Possible role of extracellularly released phagocyte proteinases in coagulation disorder during liver transplantation. 189 20

A very highly purified von Willebrand factor (vWF) concentrate was analyzed in order to evaluate its suitability for the treatment of von Willebrand's disease (vWD). The functional activity of vWF assessed by its ristocetin cofactor activity (vWF:RCo) correlated with the level of vWF antigen (vWF:Ag), with the vWF:RCo/vWF:Ag ratios ranging from 0.56 to 1.02, and the specific activity being always greater than 50 IU vWF:RCo/mg protein. Electrophoretic analysis showed a normal pattern of high, intermediate and low-molecular-weight multimers of vWF. The biological activity of vWF was also evaluated by studying its ability to bind to collagen and to platelet receptors in the presence of either ristocetin or thrombin. Furthermore, these functional activities of vWF were confirmed by the capacity of this concentrate to induce platelet adhesion to collagen in a perfusion system. Moreover, the vWF present in this preparation was able to bind factor VIII. All these in vitro data suggest that this preparation is likely to be effective in the treatment of vWD patients.
 ...
PMID:In vitro evaluation of a very-high-purity, solvent/detergent-treated, von Willebrand factor concentrate. 194 3

Endothelial cells were cultured from the umbilical veins of two neonates with type I von Willebrand disease (vWD) and compared with cells cultured in parallel from normal control umbilical veins. In both cases, cultured vWD endothelial cells contained less messenger RNA (mRNA) encoding von Willebrand factor (vWF), and constitutively secreted two- to fourfold less vWF protein than their matched controls. Regulated secretion of stored vWF induced by thrombin or phorbol-12-myristate-13-acetate (PMA) was also diminished in vWD cells. Both the mRNA and protein produced by each of these type I vWD cells appeared to be of normal size. However, despite the diminished size of the vWF storage pool, electron microscopy of endothelial cells in situ showed normal appearing vWF storage organelles (Weibel-Palade bodies). These studies show that cultured umbilical vein endothelial cells can be used to explore the molecular defects in type I and perhaps other forms of vWD, and suggest that at least some forms of type I vWD are caused by diminished mRNA transcription or subsequent translation due to a defective vWF allele.
 ...
PMID:Molecular studies of von Willebrand disease: reduced von Willebrand factor biosynthesis, storage, and release in endothelial cells derived from patients with type I von Willebrand disease. 231 58


1 2 3 4 5 6 7 8 9 10 Next >>