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Thrombospondin fragmentation by alpha-thrombin and resistance to gamma-thrombin.

ABSTRACT: In the presence of 2mM-Ca2+, alpha-thrombin slowly cleaved thrombospondin (Mr 180 000) into 150 000-Mr and 30 000-Mr fragments. In the absence of Ca2+, the platelet glycoprotein was progressively and completely hydrolysed by 3 units of the enzyme/ml to 130 000-Mr, 95 000-Mr and 65 000-Mr fragments. In contrast, the nonclotting enzyme form, gamma-thrombin, did not hydrolyse the platelet protein either in the presence or in the absence of Ca2+, even at 10-fold higher concentrations of enzyme. Protein-interacting regions removed from the catalytic site, like those required for fibrinogen recognition, are necessary for thrombin proteolysis of thrombospondin.

SUBMITTER: Takahashi K

PROVIDER: S-EPMC1144480 | biostudies-other | 1984 Dec

REPOSITORIES: biostudies-other

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Project description:We have studied the tumor genomic evolution in a patient with metastatic breast cancer bearing an activating PIK3CA mutation. The patient was treated with the PI3Kα inhibitor BYL719 and achieved a lasting clinical response on BYL719, but eventually progressed to treatment and died shortly thereafter. A rapid autopsy was performed and a total of 14 metastatic lesions were collected for further analysis. In order to identify possible genetic determinants of acquired resistance to PI3Kα inhibition, we took a three-step approach. First, we examined both the primary tumor (before BYL719 treatment) and the new lung metastasis by whole genome sequencing (DNA from the spleen was used as a normal control). Then, we analyzed the primary tumor, lung metastasis, and the peri-aortic lesion that remained stable (responding) at the time of progression to BYL719 therapy by whole exome sequencing. Finally, to confirm and expand our findings, we sequenced the primary tumor and all the metastatic lesions to >500-fold coverage using a custom targeted deep-sequencing assay, MSK-IMPACT. Using this 3-step approach, we have demonstrated that patient’s lesion underwent parallel genetic evolution at multiple metastatic sites with different PTEN genomic alterations leading to a convergent PTEN- null phenotype resistant to PI3Kα inhibition. In order to expand our observations, we analyzed paired samples (pre-treatment and at progression) from six additional patients enrolled in the BYL719 trial. Acquired bi-allelic loss of PTEN was found in one additional patient treated with BYL719 whereas in two patients PIK3CA mutations present in the primary tumor were no longer detected at the time of progression. To functionally characterize our findings, inducible PTEN knockdown in sensitive cells resulted in resistance to BYL719, while simultaneous PI3K-p110β blockade reverted this resistance phenotype, both in cell lines and in PTEN-null xenografts derived from our patient. We conclude that parallel genetic evolution of separate sites with different PTEN genomic alterations leads to a convergent PTEN-null phenotype resistant to PI3Kα inhibition.

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Thrombospondin fragmentation by alpha-thrombin and resistance to gamma-thrombin.

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