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:3.4.21.6 (thromboplastin)
13,278 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The procoagulant activity (PCA) of four T-lymphoblastoid cell lines (CEM-CCRF, Jurkat, Molt-4 and A3.01) at different stages of differentiation has been characterized and compared with that of a monocytoid cell line (THP-1). Four assay systems were employed; the activated partial thromboplastin time (APTT); prothrombin time/tissue factor (TF) activity; a purified factor (F)Xa generation system and cancer procoagulant. High levels of TF activity were seen only with the monocytic cells. However the more differentiated of the T-lymphoblastoid cells (Molt-4 and A3.01) were more active than monocytic cells in supporting FXa generation. This pattern was not repeated for the APTT assay, which was related to cell-surface TF activity, since it was partially inhibited by antiTF antibody. Annexin V totally inhibited the activity observed in all three assay systems, indicating that the PCA of T-lymphoblastoid cells is primarily due to expression of negatively charged phospholipids. However, antiphosphatidylserine antibody even at a high concentration gave only partial inhibition of the activity observed in the APTT and FXa generation systems for the cells compared with almost total inhibition for the phospholipid standard, suggesting either that cellular phosphatidylserine (PS) is less accessible to the antibody, or that PS is not the sole negatively charged phospholipid responsible for this activity. Flow cytometry studies using propidium iodide and annexin V showed that the PCA, although linked to PS exposure, was not the result of apoptosis.
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PMID:Characterization of the cell-surface procoagulant activity of T-lymphoblastoid cell lines. 1500 64

Autoantibodies to prothrombin are common in patients with systemic lupus erythematosus. Although their presence is a risk factor for thrombosis, neither their origin nor their precise role in inducing the procoagulant state is known. We have developed a phage-display antibody library from patients with systemic lupus erythematosus with antiprothrombin antibodies, and we have selected two single-chain Fv antibody fragments (ScFvs) by panning on a prothrombin-coated surface. In prothrombin activation assays using purified components, these antibodies promoted prothrombin activation. These ScFvs, termed AN78 and AN129, bound to immobilized prothrombin in a concentration-dependent specific manner but not to other anionic phospholipid binding proteins such as beta2-glycoprotein I or annexin V. Phosphatidylserine-bound prothrombin, but not soluble prothrombin, inhibited the binding suggesting that the epitope is available only on immobilized prothrombin. To localize the epitope, prothrombin was treated with thrombin or factor Xa and various prothrombin activation fragments were subsequently isolated and tested in ELISA with the ScFvs. Both AN78 and AN129 bound to prethrombin I (the fragment lacking the Gla domain and the first kringle domain), to fragment 1.2 (containing Gla and the two kringle domains only) and to fragment 2 but not to thrombin, thus localizing the cognate epitope to the kringle 2 domain in prothrombin. Analysis of the cDNA sequences of these antibodies show clustered mutational patterns in the complementarity determining region, suggesting that variable domains are the products of antigen-driven B cell clonal maturation.
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PMID:Lupus-derived antiprothrombin autoantibodies from a V gene phage display library are specific for the kringle 2 domain of prothrombin. 1504 12

The activated platelet surface serves as an integral part of the prothrombinase complex upon activation by potent platelet agonists such as thrombin and collagen. We determined the receptor specificity through which thrombin was enhancing collagen-induced thrombin generation. Whereas SFLLRN or AYPGKF alone produced minimal thrombin generation or phosphatidylserine exposure through protease activated receptor (PAR) stimulation, they caused a leftward shift in the collagen-induced thrombin generation dose-response curve. Although SFLLRN or AYPGKF potentiated collagen-induced thrombin generation, neither of them potentiated to the same extent as thrombin. However, SFLLRN and AYPGKF together potentiated collagen-induced thrombin generation to the same extent as thrombin. We conclude that thrombin mediates its procoagulant activity through activation of both PAR1 and PAR4 receptors. Similarly, neither PAR1 nor PAR4 stimulation alone mimicked the annexin V-binding response caused by thrombin stimulation. The combination of PAR activating peptides caused minimal increases in annexin V binding, but caused significant thrombin generation, suggesting that events other than phosphatidylserine exposure may play a role in platelet prothrombinase complex formation. We also investigated the ability of ADP to potentiate agonist-induced thrombin generation. Whereas P2Y(1) antagonism did not affect collagen or thrombin-induced thrombin generation, P2Y(12) antagonism did decrease both collagen- and thrombin-induced thrombin generation, suggesting that ADP potentiates thrombin generation primarily through the P2Y(12) receptor. Collectively, these results suggest that stimulation of both the PAR1 and PAR4 receptors are necessary for thrombin-induced procoagulant activity, and that the P2Y(12) receptor, but not the P2Y(1) receptor, is responsible for the potentiation of agonist-induced platelet procoagulant activity.
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PMID:Role of protease-activated and ADP receptor subtypes in thrombin generation on human platelets. 1509 88

Backgrounds and Aim: Intrasinusoidal microthrombosis is considered to be a cause of massive hepatocyte death in fulminant hepatic failure. Generally, apoptotic cells express phosphatidyl serine (PS) outside the plasma membrane, which is also expressed on the surface of activated platelets and accelerates fibrin-thrombus formation. Therefore, the acceleration of blood coagulation on the surface of apoptotic hepatocytes may occur because hepatocytes are in direct contact with plasma that passes through fenestrations of the sinusoidal endothelium. To test this hypothesis, we investigated the coagulation activity of apoptotic hepatocytes. Methods: (1) The apoptosis of Hep G2 cells was induced by staurosporin (STS). PS expression was determined by confocal microscopy with FITC-annexin V, a specific inhibitor of PS activity. (2) One million HepG2 cells treated with STS with or without pretreatment with annexin V were exposed to activated factor X, CaCl(2) and prothrombin. Thrombin generation was determined using a thrombin-specific chromogenic substrate. Results: (1) The percentage of apoptotic cells and PS-expressing cells markedly increased following the STS-treatment. (2) The thrombin generation significantly increased with STS-treatment in a dose dependent manner. (3) The increase in thrombin generation by STS-treatment was abolished following pretreatment with annexin V. Conclusion: Apoptotic hepatocytes accelerate blood coagulation through the expression of a PS-dependent pro-coagulant surface.
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PMID:Apoptotic hepatocellular carcinoma HepG2 cells accelerate blood coagulation. 1520 81

Circulating procoagulant microparticles (MP) were measured as markers of vascular damage and prothrombotic risk in patients undergoing ST-segment myocardial infarction (STEMI) treated by primary percutaneous transluminal coronary angioplasty (PTCA) and additional GPIIb-IIIa antagonists. Cells possibly more responsive to GPIIb-IIIa (alpha(IIb)beta(3)) antagonists were evidenced through MP phenotypes by comparison with healthy volunteers (HV) and STEMI patients treated by PTCA without GPIIb-IIIa antagonist (CP). In 50 STEMI patients, blood samples were collected at day 1 and day 6. Circulating procoagulant MP were captured on annexin V and quantified by prothrombinase assay as nanomolar phosphatidylserine equivalents (nm PhtdSer). Platelet activation by thrombin was confirmed through independent measurement of soluble GPV (sGPV). With respect to HV, procoagulant MP levels were high in patients with STEMI or unstable angina, platelet-derived MP and elevated sGPV testifying to significant platelet activation. A substantial release of endothelial-derived MP was evidenced simultaneously. In abciximab-treated patients, procoagulant MP, mainly of platelet origin, decreased precociously at day 1 (4.2 +/- 0.6 vs. CP 15.5 +/- 2.1 nm PhtdSer; P = 0.001) together with sGPV (36 +/- 3 vs. CP 58 +/- 8 ng mL(-1); P = 0.02). Leukocyte-derived MP decreased at day 6 (0.12 +/- 0.04 vs. CP 0.56 +/- 0.12 nm PhtdSer; P = 0.01) suggesting a possible effect on underlying inflammatory status. In patients presenting cardiovascular events at 6-month follow-up, procoagulant MP levels at day 1 could be indicative of a worsened outcome. MP could constitute a relevant parameter for the follow-up of STEMI patients treated by GPIIb-IIIa antagonists.
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PMID:Circulating procoagulant microparticles and soluble GPV in myocardial infarction treated by primary percutaneous transluminal coronary angioplasty. A possible role for GPIIb-IIIa antagonists. 1521 95

Heparin-induced thrombocytopenia (HIT) is a complication of heparin therapy caused by antibodies against a complex of platelet factor 4 and heparin. Fondaparinux (Arixtra) is a new synthetic selective factor Xa inhibitor. We performed a serologic study to determine the cross-reactivity of HIT sera with fondaparinux. Using a prospective, blinded study design, 39 clinically and serologically confirmed sera from patients with HIT and 15 control sera were sent to 3 different laboratories, each of which specialized in a particular HIT assay. These include the serotonin release assay, heparin-induced platelet agglutination assay, and platelet aggregation assay. Two of 82 assays (2.4%) performed in the presence of control sera were positive, both with unfractionated heparin. In the presence of HIT sera, 75 of 94 (79.8%) evaluable assays were positive with unfractionated heparin; fondaparinux was significantly (P < .001) less reactive than unfractionated heparin, only 3 of 91 evaluable assays (3.3%) being positive. Using flow cytometry, unlike unfractionated heparin, fondaparinux did not induce the binding of PAC1 and anti-CD62 monoclonal antibodies or of annexin V to platelets with HIT sera. Together, these results suggest that fondaparinux is nonreactive to HIT sera and raise the possibility that the drug may be used for prophylaxis and treatment of thrombosis in patients with a history of HIT.
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PMID:Effect of fondaparinux on platelet activation in the presence of heparin-dependent antibodies: a blinded comparative multicenter study with unfractionated heparin. 1538 75

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

Autoimmune factors are involved in some of the cases of reproductive failure. These factors entail several autoantibodies, especially in patients having systemic lupus erythematosus (SLE) or the antiphospholipid syndrome (APS). These autoantibodies include mainly antibodies directed to phospholipid such as cardiolipin, phosphatidylserine, phosphatidylethanolamine or phospholipids binding glycoproteins such as beta2glycoprotein-I, annexin V, prothrombin and protein-Z. There are also some other autoantibodies directed to laminin-I, thromboplastin, mitochondrial antibodies of the M5 type, corpus luteum, prolactin, poly (ADP-ribose), thyroglobulin and more, which were also found in SLE or APS patients with reproductive failure. Moreover, the presence of additional autoantibodies directed to actin, enolase, cubilin and others, needs further investigation to support a firm association to reproductive failure in women. Future studies are likely to help to determine and expand the number of autoantibodies screened in these patients, as well as by the use of proteomics technology, to determine peptides resembling the epitope specificities associated with the specific clinical manifestations.
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PMID:Autoantibodies associated with reproductive failure. 1548 94

Some cases of reproductive failure with autoimmune background are characterized by the involvement of autoantibodies. This occurs mainly in patients having systemic lupus erythematosus or antiphospholipid syndrome. The autoantibodies associated with reproductive failure include: a) antibodies which directly bind phospholipid (e.g., cardiolipin, phosphatidylserine, phosphatidylethanolamine); b) antiphospholipid Abs which bind the phospholipid via phospholipid-binding glycoproteins such as beta2glycoprotein-I, annexin V and prothrombin; c) autoantibodies directed to laminin-I, actin, thromboplastin, the corpus luteum, prolactin, poly (ADP-ribose), thyroglobulin and mitochondrial antibodies of the M5 type. This paper will focus on the association of antiphosphatidylserine autoantibodies and reproductive failure. Future studies are likely to help to identify peptides resembling the epitope specificities associated with the specific clinical manifestations.
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PMID:Antiphosphatidylserine antibodies and reproductive failure. 1548 98

The anionic phospholipid, phosphatidyl-L-serine (PS), is sequestered in the inner layer of the plasma membrane in normal cells. Upon injury, activation, and apoptosis, PS becomes exposed on the surfaces of cells and sheds microparticles, which are procoagulant. Coagulation is initiated by formation of a tissue factor/factor VIIa complex on PS-exposed membranes and propagated through the assembly of intrinsic tenase (factor VIIIa/factor IXa), prothrombinase (factor Va/factor Xa), and factor XIa complexes on PS-exposed activated platelets. We constructed a novel series of recombinant anticoagulant fusion proteins by linking annexin V (ANV), a PS-binding protein, to the Kunitz-type protease inhibitor (KPI) domain of tick anticoagulant protein, an aprotinin mutant (6L15), amyloid beta-protein precursor, or tissue factor pathway inhibitor. The resulting ANV-KPI fusion proteins were 6- to 86-fold more active than recombinant tissue factor pathway inhibitor and tick anticoagulant protein in an in vitro tissue factor-initiated clotting assay. The in vivo antithrombotic activities of the most active constructs were 3- to 10-fold higher than that of ANV in a mouse arterial thrombosis model. ANV-KPI fusion proteins represent a new class of anticoagulants that specifically target the anionic membrane-associated coagulation enzyme complexes present at sites of thrombogenesis and are potentially useful as antithrombotic agents.
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PMID:Fusion proteins comprising annexin V and Kunitz protease inhibitors are highly potent thrombogenic site-directed anticoagulants. 1567 61


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