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)

An enzyme-linked immunosorbent assay (ELISA) for quantitation of natural and recombinant plasminogen activators containing the serine protease domain (B-chain) of urokinase-type plasminogen activator (u-PA) was developed, based on two murine monoclonal antibodies, MA-4D1E8 and MA-2L3, raised against u-PA and reacting with non-overlapping epitopes in the B-chain. MA-4D1E8 was coated on microtiter plates and bound antigen was quantitated with MA-2L3 conjugated with horseradish peroxidase. The intra-assay, inter-assay and inter-dilution coefficients of variation of the assay were 6%, 15% and 9%, respectively. Using recombinant single-chain u-PA (rscu-PA) as a standard, the u-PA-related antigen level in normal human plasma was 1.4 +/- 0.6 ng/ml (mean +/- SD, n = 27). The ELISA recognized the following compounds with comparable sensitivity: intact scu-PA (amino acids, AA, 1 to 411), scu-PA-32k (AA 144 to 411), a truncated (thrombin-derived) scu-PA comprising AA 157 to 411, and chimeric t-PA/u-PA molecules including t-PA(AA1-263)/scu-PA(AA144-411), t-PA(AA1-274)/scu-PA(AA138-411) and t-PA(AA87-274)/scu-PA(AA138-411). Conversion of single-chain to two-chain forms of u-PA or inhibition of active two-chain forms with plasminogen activator inhibitor-1 or with the active site serine inhibitor phenyl-methyl-sulfonyl fluoride, did not alter the reactivity in the assay. In contrast, inactivation with alpha 2-antiplasmin or with the active site histidine inhibitor Glu-Gly-Arg-CH2Cl resulted in a 3- to 5-fold reduction of the reactivity.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:An enzyme-linked immunosorbent assay for urokinase-type plasminogen activator (u-PA) and mutants and chimeras containing the serine protease domain of u-PA. 137 17

The platelet glycoprotein (GP) Ib-IX-V receptor complex has a central role in primary haemostasis and possesses binding sites for the plasmatic adhesive protein von Willebrand Factor (VWF) and thrombin. Several snake venom components have been identified in recent years that target this receptor complex and modulate its functionality. Among them, agkicetin-C is from Deinagkistrodon acutus and proved to be a potent antagonist of GPIb-IX-V. We further studied the structure-activity relationships of agkicetin-C in order to reveal the molecular mechanisms of its antagonistic effect. Agkicetin-C concentration-dependently inhibited botrocetin-, ristocetin- and low dose thrombin- (0.32-0.4nM) induced platelet aggregation. Moreover, it abolished platelet adhesion to collagen under high shear conditions (2600/s), while having only minor effects at low shear rate (650/s), which suggested it targets mainly GPIbalpha instead of other platelet glycoproteins. The interaction site of agkicetin-C was further refined: it recognizes a linear sequence in a recombinant GPIbalpha (AA1-289) fragment and inhibited completely the ristocetin-induced VWF binding to this fragment. Using cross-blocking studies with epitope-mapped anti-GPIbalpha monoclonal antibodies, the binding region of agkicetin-C was refined to the AA201-282 region. In conclusiong the C-type lectin agkicetin-C is a potent GPIb-IX-V antagonist, inhibiting both VWF and thrombin interaction through binding to the AA201-282 region in GPIbalpha. Another thing of interest is that, although agkicetin-C did not agglutinate platelets in all conditions tested in vitro, it caused a severe thrombocytopenia in rats, suggesting a different mechanism than with flavocetin-A or echicetin.
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PMID:How does agkicetin-C bind on platelet glycoprotein Ibalpha and achieve its platelet effects? 1577 51