alpha-chymotrypsin and arginyl-glycyl-aspartyl-serine

Activation of the platelet integrin alpha(IIb)beta3, an essential step in platelet aggregation, is regulated by intracellular signal pathways (inside-out signaling). In this study, we characterize a 35-year-old Japanese female, HM, with a life-long history of mucocutaneous bleeding. HM showed a Glanzmann thrombasthenia-like phenotype with normal expression of alpha(IIb)beta3, and failure of platelet aggregation induced by various agonists. An activation-independent ligand mimic monoclonal antibody (mAb), OP-G2, and RGDS peptides bound normally to the patient's alpha(IIb)beta3, while an activating anti-beta3 mAb, AP5, induced normal aggregation of HM platelets. The nucleotide sequence of the entire coding region of the patient's alphaIIb and beta3, including the cytoplasmic domains of each subunit, revealed no abnormalities. Agonist-induced phosphorylation of platelet pleckstrin and myosin light chain was not impaired. Recently, we proposed that a Na+/Ca2+ exchanger is involved in inside-out signaling, especially in the case of chymotrypsin-induced alpha(IIb)beta3 activation (Blood 88: 2594, 1996). However, chymotrypsin-induced platelet aggregation occurred normally in patient HM. Measurement of changes in cytosolic free calcium concentration ([Ca2+]i) revealed that the plateau level of [Ca2+]i after thrombin stimulation was significantly inhibited in patient HM. Our data suggest that patient HM exhibits a Glanzmann thrombasthenia-like phenotype associated with an abnormality in inside-out signaling which would otherwise activate alpha(IIb)beta3.

Topics: Adult; Antibodies, Monoclonal; Blood Platelets; Blood Proteins; Chymotrypsin; DNA, Complementary; Female; Fibrinogen; Hemorrhagic Disorders; Humans; Myosin Light Chains; Oligopeptides; Phenotype; Phosphoproteins; Phosphorylation; Platelet Aggregation; Platelet Glycoprotein GPIIb-IIIa Complex; Protein Processing, Post-Translational; Signal Transduction; Sodium-Calcium Exchanger; Thrombasthenia; Thrombin

Topics: Batroxobin; Blood Platelets; Chymotrypsin; Fibrin; Fibrinogen; Humans; Inositol; Inositol Phosphates; Oligopeptides; Phosphates; Phosphatidylinositol Phosphates; Phosphorus Radioisotopes; Platelet Aggregation; Platelet Aggregation Inhibitors; Radioisotope Dilution Technique; Tritium

Tetrafibricin; a nonpeptidic fibrinogen inhibitor from microbial origin, showed potent antiaggregation activities on human platelet aggregation induced by either ADP, thrombin or collagen (IC50s = 5.6, 7.6 and 11 microM, respectively) in platelet rich plasma. The ability to inhibit aggregation in platelets treated with chymotrypsin confirmed the GPIIb/IIIa blockage of tetrafibricin. Tetrafibricin blocked the release of serotonin induced by ADP but it did not block the release reaction induced by thrombin. When added to platelets formerly aggregated with ADP, tetrafibricin caused rapid and complete deaggregation. As for the selectivity among other Arg-Gly-Asp -dependent integrins, tetrafibricin seems to be more specific for glycoprotein (GP) IIb/IIIa than RGDS is. This is because it had no effect on adhesion of bovine aortic endothelial cells to RGD-containing proteins. Tetrafibricin is the first nonpeptidic fibrinogen receptor inhibitor that may be valuable for the study on platelet aggregation inhibitors.

Topics: Adenosine Diphosphate; Amino Acid Sequence; Animals; Anti-Bacterial Agents; Blood Platelets; Cattle; Cell Degranulation; Chymotrypsin; Fibrinogen; Humans; In Vitro Techniques; Macrolides; Molecular Sequence Data; Oligopeptides; Platelet Aggregation; Platelet Aggregation Inhibitors; Platelet Membrane Glycoproteins; Serotonin; Streptomyces