20] Quantifying thrombin-catalyzed release of fibrinopeptides from fibrinogen using high-performance liquid chromatography

Abstract

Publisher Summary This chapter focuses on the quantifying thrombin-catalyzed release of fibrinopeptides from fibrinogen using high-performance liquid chromatography (HPLC). It presents several methods in which the HPLC analysis of fibrinopeptides can yield (1) initial rates of thrombin-catalyzed release of fibrinopeptides from fibrinogen, (2) the Miehaelis–Menten parameters catalytic constant( k CAT ) and (Michaelis constant) K M for a-thrombin-catalyzed release of fibrinopeptide A (FPA) and fibilnopeptide B (FPB) from fibrinogen and fibrin I, (3) the specificity constant k CAT / K M for both FPA and FPB at concentrations of fibrinogen below K M , and (4) the inhibition constant for hirugen, an exosite inhibitor of α-thrombin. The chapter also discusses the HPLC profile of coeluting fibrinopeptides and activation peptides from factor XIII. The chapter describes a method for determining the concentration of α-thrombin that is catalytically competent to process fibrinogen.