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)

Desmopressin (DDAVP) action on platelets is associated with the development of procoagulant response but the underlying mechanism of this phenomenon is not known. We investigated whether this effect of DDAVP might be due to activation of plasma membrane Na+/H+ exchanger. The DDAVP-induced platelet procoagulant response, measured as phospholipid-dependent thrombin generation, was dose dependent and significantly weaker than that produced by collagen or monensin (mimics Na+/H+ antiport). Both the DDAVP- and collagen-produced procoagulant responses were less pronounced in the presence of EIPA, an Na+/H+ exchanger inhibitor. Flow cytometry studies revealed that in vitro treatment of platelets with DDAVP or collagen was associated with the appearance of both degranulated (and fragmented) and swollen cells. The DDAVP-evoked rise in size and granularity heterogeneity was similar to that produced by collagen or monensin and was not observed in the presence of EIPA. Using flow cytometry and annexin V-FITC as a probe for phosphatidylserine (PS) we demonstrated increased and uniform binding of this marker to all subsets of DDAVP-treated platelet population. The DDAVP-evoked PS expression was dose dependent, strongly reduced by EIPA and weaker than that caused by monensin or collagen. As judged by optical swelling assay, DDAVP in a dose dependent manner produced a rise in platelet volume. The swelling was inhibited by EIPA and its kinetics was similar to that observed in the presence of monensin. Electronic cell-sizing measurements showed an increase in mean platelet volume and a decrease in platelet count and platelet crit upon treatment with DDAVP. DDAVP elicited a slow (much slower than collagen) alkalinization of platelet cytosol. Altogether the data indicate an involvement of Na+/H+ exchanger in the generation of procoagulant activity in DDAVP-treated platelets.
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PMID:Involvement of Na+/H+ exchanger in desmopressin-induced platelet procoagulant response. 1544 38

Circulating platelets play a pivotal role in hemostasis. The platelet hemostatic function involves the direct interaction with damaged vessel walls, and circulating coagulation factors, primarily thrombin resulting in platelet activation, aggregation and formation of hemostatic plug. Flow cytometry is a useful technique for the study of platelet activation in circulating blood. Platelet activation markers for ex vivo analysis may include a) activation-dependent epitopes of the membrane glycoprotein (GP) IIb/IIIa (CD41a) receptor, as demonstrated by the binding of activation-specific monoclonal antibodies (MoAbs) PAC1, anti-LIBS1 and anti-RIBS); b) the expression of P-selectin (CD62p), the alpha-granule GP translocated to the platelet surface following release reaction; and c) platelet procoagulant activity, as demonstrated by the binding of i) annexin V protein to the prothrombinase-complex (prothrombin, activated factor X (Xa) and V (Va)) binding sites on the surface of activated platelets, and of ii) MoAbs against activated coagulation factors V and X bound to the surface of activated platelets. Using this method, platelet activation as a marker for in vivo prothrombotic activity can be demonstrated in various clinical conditions including coronary angioplasty, orthostatic challenge in primary depression, sickle cell disease in clinical remission and during pain episode, and in pregnancy-related hypertension with marked increase during preeclampsia. The finding of platelet procoagulant activity is corroborated by increased levels of plasma markers for thrombin generation and fibrinolytic activity.
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PMID:Platelet activation as a marker for in vivo prothrombotic activity: detection by flow cytometry. 1547 Dec 23

In normal pregnancy, there is a marked increase in the procoagulant activity in maternal blood characterized by elevation of factors VII, X, VIII, fibrinogen and von Willebrand factor, which is maximal around term. This is associated with an increase in prothrombin fragments (PF1+2) and thrombin-antithrombin complexes. There is a decrease in physiological anticoagulants manifested by a significant reduction in protein S activity and by acquired activated protein C (APC) resistance. The overall fibrinolytic activity is impaired during pregnancy, but returns rapidly to normal following delivery. This is largely due to placental derived plasminogen activator inhibitor type 2 (PAI-2), which is present in substantial quantities during pregnancy. D-dimer, a specific marker of fibrinolysis resulting from breakdown of cross-linked fibrin polymer by plasmin, increases as pregnancy progresses. Overall, there is a 4- to 10-fold increased thrombotic risk throughout gestation and the postpartum period. Local haemostasis at the placental throphoblast level is characterized by increased tissue factor (TF) expression and low expression of the inhibitor TFPI. Microparticles derived from maternal endothelial cells and platelets, and from placental throphoblasts may contribute to the procoagulant effect. Local anticoagulant mechanisms on placental throphoblasts are important for counterbalance of the procoagulant milieu. Disruption of anticoagulant mechanisms, for example, autoantibodies, to annexin V may increase pregnancy complications in patients with antiphospholipid antibodies (APLA).
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PMID:Haemostatic changes in pregnancy. 1550 71

Fibrin gel structure has been shown to be dependent on the thrombin concentration as well as the rate of thrombin generation. Accordingly, factor VIII (FVIII)- and FIX-deficient plasma (hemophilia A and B) form loose fibrin clots with high permeability constants. By adding rFVIIa in vitro to FVIII-deficient plasma containing platelets (frozen and thawed), the fibrin gel permeability constant normalized, indicating that extra rFVIIa (1.2 microg mL(-1) or higher) induced a tight fibrin structure. Thrombin generation is highly dependent on the number of platelets, and in this study it was demonstrated that the addition of rFVIIa (5 microg mL(-1)) normalizes the fibrin gel permeability in samples containing platelets (frozen-thawed) in numbers of at least down to 20 x 10(6) mL(-1). The effect of rFVIIa was not observed when unfrozen platelets instead of frozen-thawed platelets were added. Neither was any effect on the fibrin permeability seen, in the presence of annexin V, known to block the effect of phospholipids on the platelet surface. This indicates an important role of platelet phospholipids for the effect of rFVIIa. A similar effect on the fibrin permeability of rFVIIa was observed when added to platelet-rich plasma from a patient with Glanzmann thrombasthenia. Recombinant FVIIa has been found to induce hemostasis in patients with hemophilia and inhibitors against FVIII/FIX as well as in patients with Glanzmann thrombasthenia, indicating the importance of the formation of a tight fibrin gel structure, more resistant against premature proteolysis, for maintaining hemostasis. In conclusion, the addition of rFVIIa (5 microg mL(-1)) also substantially decreased the permeability constant of fibrin gels formed in FVIII-deficient plasma in the presence of low numbers of frozen-thawed platelets (down to 20 x 10(6) mL(-1)). A similar pattern was obtained in plasma from a Glanzmann patient. No effect was found in the presence of unfrozen instead of frozen-thawed platelets. Annexin V blocked any effect of rFVIIa. A normalization of the overall fibrinolysis potential (OFP) during the same condition supports the effect of rFVIIa on the fibrin permeability in the presence of a limited number of platelets.
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PMID:The effect of platelets on fibrin gel structure formed in the presence of recombinant factor VIIa in hemophilia plasma and in plasma from a patient with Glanzmann thrombasthenia. 1567 32

Exposure of tissue factor (TF)-bearing cells to blood is the initial event in coagulation and intravascular thrombus formation. However, the mechanisms which determine thrombus growth remain poorly understood. To explore whether the procoagulant activity of vessel wall-bound cells regulates thrombus expansion, we studied in vitro spatial clot growth initiated by cultured human cells of different types in contact pathway-inhibited, non-flowing human plasma. Human aortic endothelial cells, smooth muscle cells, macrophages and lung fibroblasts differed in their ability to support thrombin generation in microplate assay with peaks of generated thrombin of 60 +/- 53 nmol L(-1), 135 +/- 57 nmol L(-1), 218 +/- 55 nmol L(-1) and 407 +/- 59 nmol L(-1) (mean +/- SD), respectively. Real-time videomicroscopy revealed the initiation and spatial growth phases of clot formation. Different procoagulant activity of cell monolayers was manifested as up to 4-fold difference in the lag times of clot formation. In contrast, the clot growth rate, which characterized propagation of clotting from the cell surface to plasma, was largely independent of cell type (< or = 30% difference). Experiments with factor VII (FVII)-, FVIII-, FX- or FXI-deficient plasmas and annexin V revealed that (i) cell surface-associated extrinsic Xase was critical for initiation of clotting; (ii) intrinsic Xase regulated only the growth phase; and (iii) the contribution of plasma phospholipid surfaces in the growth phase was predominant. We conclude that the role of TF-bearing initiator cells is limited to the initial stage of clot formation. The functioning of intrinsic Xase in plasma provides the primary mechanism of sustained and far-ranging propagation of coagulation leading to the physical expansion of a fibrin clot.
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PMID:Initiation and propagation of coagulation from tissue factor-bearing cell monolayers to plasma: initiator cells do not regulate spatial growth rate. 1567 39

Long-term storage of platelets is infeasible due to platelet activation at low temperatures. In an effort to address this problem, we evaluated the effectiveness of a formulation combining trehalose and phosphate in protecting platelet structure and function following cryopreservation. An annexin V binding assay was used to quantify the efficacy of the trehalose and phosphate formulation in suppressing platelet activation during cryopreservation. Of the platelets cryopreserved with the trehalose plus phosphate formulation, 23% +/- 1.2% were nonactivated, compared with 9.8% +/- 0.26% nonactivated following cryopreservation with only trehalose. The presence of both trehalose and phosphate in the cryopreservation medium is critical for cell survival and preincubation in trehalose plus phosphate solutions further enhances viability. The effectiveness of trehalose plus phosphate in preserving platelets in a nonactivated state is comparable to 6% dimethyl sulfoxide (Me(2)SO). Measurements of platelet metabolic activity using an alamarBlue assay also established that trehalose plus phosphate is superior to trehalose alone. Finally, platelets protected by the trehalose plus phosphate formulation exhibit similar aggregation response upon thrombin addition as fresh platelets, but an increase of cytosolic calcium concentration upon thrombin addition was not observed in the cryopreserved platelets. These results suggest that trehalose and phosphate protect several aspects of platelet structure and function during cryopreservation, including an intact plasma membrane, metabolic activity, and aggregation in response to thrombin, but not intracellular calcium release in response to thrombin.
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PMID:Platelet cryopreservation using a trehalose and phosphate formulation. 1593 43

Estrogen receptor beta (betaER) is the predominant estrogen receptor in platelets. Experiments were designed to define phenotypic changes in platelets with aging following deletion of betaER (betaERKO). Blood was collected from wild-type and betaERKO female mice at 4-7 (young) and 24-25 (aged) months of age. In young animals, total number of platelets, number of platelets containing RNA (reticulated platelets), aggregation, dense body adenosine triphosphate secretion, and alpha granular secretion were the same in both groups. With aging, total number of platelets decreased but reticulated platelets increased in betaERKO mice; aggregation and dense granule adenosine triphosphate secretion decreased whereas basal expression of fibrinogen receptors increased with age in wild-type and betaERKO mice. Basal expression of P-selectin and annexin V binding increased with aging only in betaERKO mice; thrombin did not increase expression in these mice. Therefore, deletion of betaER is associated with specific platelet functions, which are expressed only with age-associated reproductive senescence.
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PMID:Platelet characteristics change with aging: role of estrogen receptor beta. 1607 2

Platelet activation in response to dual stimulation with collagen and thrombin results in the formation of a subpopulation of activated platelets known as coated platelets. Coated platelets are characterized by high surface levels of alpha-granule proteins and phosphatidylserine, which support the assembly of procoagulant protein complexes. Using murine models, we tested the hypothesis that the collagen receptor-associated molecule FcRgamma and the transglutaminase factor XIIIA are required for the formation of coated platelets. Following dual stimulation with the collagen receptor agonist convulxin and thrombin, 68% of platelets from C57BL/6 mice acquired the coated platelet phenotype, defined by high surface levels of fibrinogen and von Willebrand factor and decreased binding of the alphaIIbbeta3 activation-dependent antibody PE-JON/A. In FcRgamma-/- mice, only 10% of platelets became "coated" after dual stimulation with convulxin plus thrombin (P < .05 vs C57BL/6 platelets). Decreased coated platelet formation in FcRgamma-/- platelets was accompanied by decreased annexin V binding (P < .01) and decreased platelet procoagulant activity (P < .05). Platelets from FXIIIA-/- mice did not differ from control platelets in coated platelet formation or annexin V binding. We conclude that FcRgamma, but not factor XIIIA, is essential for formation of highly procoagulant coated platelets following dual stimulation with collagen and thrombin.
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PMID:Role of FcRgamma and factor XIIIA in coated platelet formation. 1610 83

Thrombin derived from bovine sources commonly is used to arrest bleeding during surgical procedures. However, complications such as postoperative hemorrhage can occur because of the development of cross-reactive anti-bovine antibodies that inhibit human coagulation factor V. It would thus be advantageous to develop techniques to generate human thrombin. This study evaluated thrombin produced from human plasma using a new Thrombin-Processing Device (TPD). Plasma was introduced into the TPD, mixed with an ethanol/ CaCl2 reagent, incubated for 1 h, and the harvested thrombin was assayed for activity and the ability to activate platelets by in vitro assays. TPD-produced thrombin activity was found to be 51.8 +/- 12.4 IU/mL (n = 145). TPD-produced thrombin also stimulated P-selectin (CD62) expression (83 +/- 13% of the platelet population) and Annexin V binding (10.3 +/- 2% of the platelet population) on platelets in a similar fashion to commercial thrombin (P-selectin expression: 88 +/- 3%; Annexin-V binding: 11.4 +/- 3%). Compared with CaCl2 and batroxobin, TPD-produced thrombin had a significantly greater ability to activate platelets. TPD-produced thrombin from human plasma has consistent activity and significantly activates platelets and, thus, may have attractive applications such as the production of autologous thrombin for surgical patients.
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PMID:Quality of thrombin produced from the patient's own plasma using the TPD, a new Thrombin-processing Device. 1611 59

The response of human platelets to low temperature (below 15 degrees C) requires that they are stored at elevated temperatures and limits their storage time to 5 days for use in transfusion. Prolonged storage at room temperature leads to loss of platelet function and risk of septic conditions. The need for improved platelet storage is an important issue, and finding a key component allowing platelets to be maintained at low temperatures would have significant practical benefit. Developing such a component is challenging, because the process of cold-activation resembles that of a physiological agonist-mediated activation, but without a specific receptor that can be inhibited. A component preventing platelets' low temperature response will potentially inhibit their physiological function, making them less useful after transfusion. In the present study, we report that pretreatment of platelets with flavonoids before chilling prevents an increase in cytosolic calcium concentration, actin polymerization and platelet shape change. After warming, platelets that were chilled in the presence of flavonoids retain a normal shape change and aggregation response after stimulation by thrombin. Additionally, cold platelet activation does not increase platelet procoagulant activity evaluated by annexin V-FITC binding in the presence and absence of flavonoids. These data confirm the important links that exist between agonist- and cold-mediated platelet activation, suggesting a possible advantage of incorporating the use of flavonoids to allow platelet hypothermic-storage.
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PMID:Flavonoid-mediated inhibition of actin polymerization in cold-activated platelets. 1619 66


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