Glycoprotein V (GPV) is a membrane-associated, 82 Kd platelet glycoprotein that is hydrolyzed during thrombin activation to yield 69 Kd fragment. We have developed a rapid and simple method for isolation of the protein from platelet extracts using a combination of gel permeation, anion-exchange, and lectin affinity chromatography. The partial amino acid sequence was determined by analysis of peptides generated by digestion of the S-carboxyamido-methylated protein with Achromobacter protease I or cyanogen bromide. The sequence shows a remarkable periodicity of leucine residues, which is homologous to the consensus sequence of a highly diversified protein super-family with a common repetitive module. Thrombin cleavage site was determined to be located at the C-terminal region of GPV by analysis of the products separated by sizing and reversed-phase high performance liquid chromatography. By lectin blot analysis, the existence of mucin-type carbohydrate chains was indicated, as well as the existence of asparagine-linked carbohydrate chains shown by the amino acid sequence analysis. From these data, we report a structural model of GPV that is analogous to glycoprotein Ib.
A case of chronic myelogenous leukemia with Klinefelter's syndrome mosaicism in a 27‐yr‐old male is reported. Cytogenetic analysis provided evidence that the Philadelphia chromosome occurred monoclonally in the XXY cells but not in the XY cells.
SummaryTime course change of the platelet cytoskeletal protein component in the Triton X-100 insoluble fraction after stimulation was analyzed in Hermansky-Pudlak syndrome and thrombasthenia.In Hermansky-Pudlak syndrome (HPS), a 31 kDa protein, myosin, actin, and a 100 kDa protein assembled as in the normal platelets at the shape change and release reaction phases after ADP or collagen stimulation, suggesting that, the deficient dense granule content do not lead to an abnormal platelet cytoskeletal protein assembly. In thrombasthenia (Type I), myosin increased at the shape change and release reaction phases as it does in normal platelets, but actin and the 100 kDa protein increased only at the initial activation phase, and then subsequently decreased to the level of the resting phase. The actin-binding protein (ABP) and the 31 kDa protein increased a little following stimulation. Similar cytoskeletal protein change after stimulation were found in normal platelets which were prevented from the aggregation process by chelating the external Ca2+ or by using synthetic decapeptide of fibrinogen γ-chain of carboxyl terminus. The decreased platelet cytoskeletal protein assembly in thrombasthenia or in platelets stimulated without aggregation, was derived from a loss of the platelet aggregation process due to the defect of GP Ilb-IIIa complex or an interaction failure between GP IIb-IIIa complex and fibrinogen. The interaction between platelets and either fibrinogen or fibrin can induce a more stable platelet cytoskeletal protein assembly, however, agonistic stimulation without these interactions cannot do it directly.
Keyword: platelet membrane glycoprotein V, purification, anti-GPV antibody GPV has a very important role of activating platelets in the presence of thrombin. We now have established a new rapid method for the purification of GPV. The purification procedure consists of (1 extraction of GPV 2 ammonium sulfate precipitation 3 gel permeation HPLC 4 anion exchange HPLC 5 WGA-affinity chromatography. We recently purified GPV which showed a single silver and PAS stained band on the SDS-PAGE and the apparent molecular weight was 80 K dalton. The GPV was hydrolysed by thrombin but not by endoglycosidase F. IEP was estimated as 5.6-6.6. Anti-GPV antibody was raisd by immunizing a rabbit and its reactive protein was analysed on a nitrocellurose membrane by using a Western blotting method.Anti-GPV IgG reacted only with GPV and its fragment, but it did not inhibit the thrombin-induced platelet aggregation. The anti-GPV antibody reactive material in plasma was quantified by using Dot-ELISA method. Two patients showed relatively high concentrations of the antibody reactive material before their thrombotic attacks.We conclude that GPV purified by our rapid technique has the same characteristics as reported before and the detection of anti-GPV antibody reactive materials in patient's plasma is useful for the prediction and/or diagnosis of thrombosis.
309ChemInform Abstract The peptide (I), prepared by the title method, has different effects on molluscan muscles. Depending on the concentration relaxation or contraction is achieved.
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