Kinetic Characterization of Tissue-Type Plasminogen Activator (t-PA) and t-PA Deletion Mutants

Author:

Vries Carlie de1,Veerman Harry1,Nesheim Michael E2,Pannekoek Hans1

Affiliation:

1. The Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Department of Molecular Biology, Amsterdam, The Netherlands

2. The Queen’s University, Department of Biochemistry, Kingston, Canada

Abstract

SummaryThe binding of t-PA to fibrin is mediated both by its “finger” (F) and its “kringle 2” (K2) domain. In addition, these domains are involved in the stimulation of t-PA activity by fibrin. We analyzed the kinetic characteristics of Glu-plasminogen activation by t-PA and a set of t-PA deletion mutants in the absence and the presence of desA-fibrin. In the absence of desA-fibrin, the activity of t-PA (variants) is determined by the presence of the pro tease domain, irrespective of the composition of the amino-terminal heavy chain. In the presence of the cofactor desA-fibrin, the activity of t-PA (variants) is dependent on the domain composition of the heavy chain. The activity of t-PA is stimulated 2,400 fold by desA-fibrin, whereas the activity of the mutant lacking the K1 domain (del. Kl) increases 936 fold in the presence of this cofactor. Mutants lacking either the K2 domain (del. K2) or the F domain (del. F) exhibit an enhanced activity upon desA-fibrin addition of 200 and 210 fold, respectively. DesA-fibrin has no stimulatory effect on the activity of the mutant containing only the serine-protease domain (del.FE Kl K2) nor on the activity of the variant containing only the Kl domain and the serine-protease domain (del. FE K2). Furthermore, we determined the relative fibrin affinity of each t-PA variant, which is similarly dictated by the composition of the heavy chain.

Publisher

Georg Thieme Verlag KG

Subject

Hematology

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2. Biophysical Mechanisms Mediating Fibrin Fiber Lysis;BioMed Research International;2017

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