Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.4 (trypsin)
 42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Platelet glycocalicin has been purified to homogeneity by a two step procedure involving affinity chromatography on WGA-Sepharose and then on thrombin-Sepharose using selective elution with heparin. The procedure is more rapid (3-4 days), more reproducible and gives about twice the yield (10 mg/40 units platelets) of the previous method (Okumura et al. J. Biol. Chem. 251, 5950-5955, 1976). The two preparations showed identical inhibition of aggregation of gel filtered platelets induced by thrombin and by ristocetin. Glycocalicin was cleaved in a controlled fashion by trypsin-Sepharose over 3hr to yield the macroglycopeptide and peptide "tail" fragments and there was no apparent further degradation with 18 hrs digestion.
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PMID:Isolation of platelet glycocalicin by affinity chromatography on thrombin-sepharose. 715 25

The levels of glycoprotein (GP) Ib and GPV and phospholipase C activity were measured in platelets from three Bernard-Soulier syndrome patients. The patients' platelets had 46%, 46%, and 24% of control levels of GPIb alpha and 43%, trace, and 13% of control levels of GPV as determined by immunoblot analysis. Stimulation by thrombin, trypsin, the thromboxane analogue U46619, and the combination of U46619 and trypsin caused the formation of [32P]phosphatidic acid, an index of phospholipase C activity, in [32P]orthophosphate-prelabeled platelets. With all agonists, however, the formation of [32P]phosphatidic acid was markedly reduced in Bernard-Soulier syndrome platelets compared with control platelets. These data indicated a postreceptor defect in phospholipase C activation in Bernard-Soulier syndrome platelets and confirmed earlier observations of potential proteolytic and nonproteolytic mechanisms of platelet activation.
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PMID:Phospholipase C activity in platelets from Bernard-Soulier syndrome patients. 821 96

Platelet glycocalicin (GC) is the extramembranous portion of GPIb alpha that can be rapidly cleaved by enzymes such as calpain, plasmin, trypsin, elastase, etc. Quantitative cleavage will ultimately result in an acquired Bernard-Soulier-like bleeding disorder, and circulating GC may act as a potential inhibitor of platelet adhesion. We have developed and standardized a new enzyme-linked immunosorbent assay (ELISA), which uses two monoclonal antibodies (mAbs), both of which bind to the amino-terminal 45-kD fragment of GC and inhibit platelet-von Willebrand interactions and the streptavidin-biotin system. First, the methodology was evaluated and standardized with special emphasis on the anticoagulant and the inhibitors (EDTA, prostaglandin E1 [PGE1], aprotinin, N-ethyl-maleimide), the mode of high-speed centrifugation (to avoid platelet microparticles), and the standards used (purified GPIb and GC). This assay was then used to analyze the GC levels of healthy subjects (2.04 +/- 0.46 micrograms/mL) and of patients with selected diseases. The results of patients with aplastic anemia and thrombocytosis confirmed that GC levels are clearly dependent on the platelet count, which was the basis for the introduction of the GC index, the standardization of GC for a platelet count of 250 x 10(9)/L. The GC index discriminates reliably patients with active immune thrombocytopenic purpura from those in remission. GC levels are elevated in patients on hemodialysis (3.62 +/- 0.75 micrograms/mL, P < .001). The high GC index (6.93 +/- 4.21, P < .001) in cirrhosis patients suggests an increased platelet turnover and/or abnormal proteolysis. In contrast to other groups, we have not found that recombinant tissue plasminogen activator (rtPA) treatment of patients with myocardial infarction increases GC levels. However, concentrations are elevated in leukemia and the highest levels found are approximately 40 micrograms/mL. These studies suggest that GC is a useful platelet marker in certain diseases, which directly reflects platelet damage and possibly platelet dysfunction.
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PMID:Glycocalicin: a new assay--the normal plasma levels and its potential usefulness in selected diseases. 829 32

Jararaca GPIb-BP, a snake venom protein composed of alpha and beta subunits purified from Bothrops jararaca, binds to platelet glycoprotein (GP)Ib and functions as a receptor blocker for von Willebrand factor binding to GPIb (Fujimura, Y., Ikeda, Y., Miura, S., Yoshida, E., Shima, H., Nishida, S., Suzuki, M., Titani, K., Taniuchi, Y., and Kawasaki, T. (1995) Thromb. Haemostasis 74, 743-750). We present here the entire 142- and 123-residue amino acid sequence of the respective alpha and beta subunits and also demonstrate that the platelet GPIb-binding site resides on the beta and not on the alpha subunit based on an enzyme-linked immunosorbent assay using biotin-labeled jararaca GPIb-BP and competing ligands. Sequences of the alpha and beta subunits were determined by analysis of the intact S-pyridylethylated proteins and their peptides generated by digestion with Achromobacter protease I, Staphyloccocus aureus V8 protease, pepsin, endoproteinase Asp-N, or L-1-tosylamino-2-phenylethyl chloromethyl ketone-trypsin. A 38-39% identity of amino acid sequence between the alpha and beta subunits of jararaca GPIb-BP was observed, as well as a high degree of sequence identities (38-64%) with the respective subunits of botrocetin (Usami, Y., Fujimura, Y., Suzuki, M., Ozeki, Y., Nishio, K., Fukui, H., and Titani, K (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 928-932) and the beta-chain of echicetin (Peng, M., Holt, J. C., and Niewiarowski, S. (1994) Biochem. Biophys. Res. Commun. 205, 68-72).
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PMID:Complete amino acid sequence and identification of the platelet glycoprotein Ib-binding site of jararaca GPIb-BP, a snake venom protein isolated from Bothrops jararaca. 863 68

LAMA-84, a human leucocytic cell line, which upon establishment was described as having megakaryocytic, erythroid and granulocytic characteristics, was analysed for expression of various differentiation markers. In addition to some of the previously described phenotypic characteristics, this cell line was found to express mRNA for several proteins characteristic for basophilic leucocytes and mast cells. The authors show that LAMA-84 cells express mRNA for the mast cell tryptase, the proteoglycan core protein, carboxypeptidase A and the alpha and beta chains of the high affinity IgE receptor (Fc epsilon RI). The authors examined the potential of LAMA-84 to differentiate in serum-free medium or after DMSO or PMA treatment. Depending on the inducing factor, surface expression of the Fc epsilon RI alpha-chain was increased from 20% to 35-50% of the cells and mRNA levels for tryptase were increased in serum-free medium and after DMSO treatment. LAMA-84 was found to express CD13, CDw17, CD29, CD33, CD40, CD45 and CD117. Furthermore, mRNA for the eosinophil/basophil markers Charcot-Leyden crystal (CLC) protein and the major basic protein (MBP), as well as the erythrocyte differentiation marker alpha-globin, was detected. However, the authors observed only trace amounts of mRNA for another erythroid differentiation marker (glycophorin), trace amounts of the megakaryocytic marker GPIIIa, and no detectable level of GPIb alpha. By comparing the expression pattern of a panel of differentiation markers in LAMA-84, and a second human cell line (KU812) expressing a basophil phenotype, it is evident that these cell lines, which presently are the only two cell lines identified with basophilic characteristics, share a large number of phenotypic characteristics.
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PMID:Characterization of a human basophil-like cell line (LAMA-84). 869 92

A platelet glycoprotein Ib-binding protein, termed TSV-GPIb-BP, was isolated from the venom of Trimeresurus stejnegeri. On SDS-polyacrylamide gel electrophoresis, TSV-GPIb-BP showed a single band with an apparent molecular weight of 28,000 and two distinct bands with apparent molecular weights of 16,000 and 15,000 under non-reducing and reducing conditions, respectively. cDNA clones containing the coding sequences for both TSV-GPIb-BP subunits were isolated and sequenced. The deduced amino acid sequences of TSV-GPIb-BP subunits were confirmed by N-terminal protein sequencing and trypsin-digested peptide mass fingerprinting. Interestingly, the alpha subunit of TSV-GPIb-BP is identical to that of alboaggregin-B, and the sequence identity of their beta subunits is 94.3%. TSV-GPIb-BP inhibited ristocetin-induced human platelet agglutination in platelet-rich plasma under lower dosages (<5 microg/ml). On the other hand, it directly aggregated washed human platelets in the absence of additional Ca2+ or any other cofactors under higher dosages (>5 microg/ml). This platelet aggregation activity was dose-dependently inhibited by specific GPIbalpha antibodies, but not by those antibodies against platelet GPIa, GPIIa, GPIIb and GPIIIa.
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PMID:Molecular cloning and characterization of a platelet glycoprotein Ib-binding protein from the venom of Trimeresurus stejnegeri. 1278 89

The endangered sea turtles are living "fossils" that afford us an opportunity to study the hemostatic process as it likely existed millions of years ago. There are essentially no data about turtle thrombocyte aggregation prior to our studies. Thrombocytes are nucleated cells that serve the same hemostatic functions as the anucleated mammalian platelet. Sea turtle thrombocytes aggregate in response to collagen and beta-thrombin. Ristocetin induces an agglutination/aggregation response indicating the presence of a von Willebrand-like receptor, GPIb, found in all mammalian platelets. Samples treated with alpha-thrombin plus gamma-thrombin followed by ristocetin results in a rapid, stronger response than ristocetin alone. These responses are inhibited by the RGDS peptide that blocks fibrinogen cross-linking of mammalian platelets via the fibrinogen receptor, GPIIb/IIIa. Three platelet-like proteins, GPIb, GPIIb/IIIa and P-selection are detected in sea turtle thrombocytes by fluorescence activated cell sorting. Turtle thrombocytes do not respond to ADP, epinephrine, serotonin, thromboxane A2 mimetic, U46619, trypsin, or alpha-thrombin and gamma-thrombin added alone. Comparison of hemostasis in sea turtles to other vertebrates could provide a framework for understanding the structure/function and evolution of these pathways and their individual components.
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PMID:Comparison of sea turtle thrombocyte aggregation to human platelet aggregation in whole blood. 1618 11


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