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Quantitative evaluation of tissue-type plasminogen activator (tPA) was done to evaluate substrate structure-efficiency correlations and to assess the specificity of tPA in hydrolyzing peptidyl fluorogenic substrates. Synthetic substrates used in the experiment contained 6-amino-1-naphthalenesulfonamide (ANSN) leaving group, Arg in the P1 position, various amino acids in the P2 and P3 positions, and different substituents in the sulfonamide moiety in the leaving group. The results showed that the factors that affect the efficiency and affinity of the substrates for tPA include hydrophobicity of the substrates, the volume of amino acids in the peptide core and the structure of the leaving group.

The membrane bound factor Xa appears to be the major activator of the vitamin K dependent zymogen, human factor VII, in vivo. The activation by Xa is anionic phospholipid (PCPS) dependent. The VIIa-tissue factor complex (factor IXa) and thrombin activate factor VII in vitro, but in vivo activation is uncertain. In the presence of PCPS, factor VIIIa has no affect on the activation by factor IXa, while factor Va decreases the activation by Xa. Factor XIa has no observable effect on factor VII activation rates.

The amidolytic activity and cooperative interactions during factor VIIa-tissue factor (TF) complex formation was studied in the presence of EDTA or divalent cations such as Me2+. The amidolytic activity of the factor VII-a TF complex increased in the presence of Me2+ while cooperative interactions occurred between Me2+ factor VIIa, tissue factor and substrate.

Factor VIIa, a two chain serine protease hydrolyzes tripeptide fluorogenic substrates depending on the hydrophobicity of the P1 structure of the substrate, and its reactivity is enhanced by complex formation between factor VIIa and its initiation of the mechanism of coagulation. The configurational modifications in factor VIIa caused by TF leads to acceleration of the degradation process of the cofactor-substrate complex, and magnifies the catalytic constant of substrate hydrolysis. The volume of the components in the P structure determines substrate efficiency for factor VIIa.

A study was conducted on the use of aminonaphthalenesulfonamide (ANSN) compounds as modifiable fluorescent detecting groups. These compounds possess a high quantum fluorescent yield and their sulfonyl moiety allows for numerous chemical modifications. ANSN was found to be capable of elevating the specificity of substrate hydrolysis by thrombin more than 30 times. Alterations on the peptidyl and detecting group side of the resultant substrate were also suggested to optimize the substrate's specificity for use in an assay system with a mixture of proteases.