Robert C. Wohl

Comparative activation kinetics of mammalian plasminogens

Five native mammalian plasminogen species, namely, cat, dog, bovine, rabbit and horse, were studied and compared to native human plasminogen with respect to their substrate and enzymatic properties in various activated forms. These studies are an extension of previous work and were designed to confirm our previously proposed mechanism of plasminogen activation, using a series of native, but different, plasminogen substrates. The plasminogen activator species used were high molecular weight urokinase, streptokinase, human Glu-plasminogen-streptokinase complex, human plasmin-derived light(B)-chain-streptokinase complex, and the equimolar streptokinase activator complexes prepared from cat and dog plasmins. The peptidase parameters of the plasmins, plasmin-streptokinase and plasminogen-streptokinase complexes were determined with H-D-valyl-L-leucyl-L-lysyl-p-nitroanilide and Tos-glycyl-L-prolyl-L-lysyl-p-nitroanilide. Activation kinetics were measured with the same substrates. The peptidase parameters of all plasmin species were found to be similar, but with minor variations. The equimolar streptokinase mixtures of bovine, rabbit and horse plasminogens and plasmins did not form complexes and did not form active sites with plasminogen, under the conditions used. The second-order rate constants of activation revealed great differences (as much as 1400-fold), presumably expressing differences in the tertiary structure of the various plasminogen scissile bonds. The catalytic rate constants of activation, kplg, varied by as much as a 100-fold, while differences in Kplg were relatively small. The results of this study confirm the activation mechanism we have postulated previously, namely, that rapid-equilibrium rather than steady-state conditions prevail and that k2 (acylation) is the catalytic rate constant and the rate-determining step, while KS is a true dissociation constant. Calculations of the free energy of interaction of the peptidase and plasminogen activation reactions showed -4.4 to -5.6 kcal/mol for peptidase and -6.5 to -10 kcal/mol for the activation reaction. These values indicate 1-3 subsite binding interactions for the peptidase activity and 3-5 subsite binding interactions for the activation catalytic event. Streptokinase activator complexes have at least one more interacting subsite than the urokinase active site.

[30] Human plasmin

Publisher Summary Various forms of human plasmin are described in this chapter, prepared, and isolated—namely, Glul-plasmin, Lys-plasmin, Val-plasmin. Glu-plasmin can be prepared from the native Glu-zymogen in the presence of plasmin inhibitors, Lys-plasmin can be prepared from either the native zymogen or the plasmin-degraded Lys-zymogen, and Val-plasmin can be prepared from the elastase-degraded Val-zymogen. Val-plasmin, the light (B) chain of Glu-plasmin, Lys-plasmin, and Val-plasmin, can be prepared by partial reduction of the two interchain-disulfide bonds connecting the heavy (A) chain and light (B) chain of the larger plasmins. Also, the afffinity-chromatography forms of each of the plasmins containing the heavy (A) chains with lysinebinding sites can be prepared. Recombinant plasmin can be prepared from the isolated sulfhydryl forms of the heavy (A) chain and light (B) chain.

Identification of the active-site serine in human lecithin: Cholesterol acyltransferase☆

Purified human lecithin:cholesterol acyltransferase (LCAT) was covalently labeled by [3H]diisopropylflourophosphate with concomitant loss of enzymatic activity (M. Jauhiainen and P.J. Dolphin (1986) J. Biol. Chem. 261, 7023-7043). Some 60% of the enzyme was labeled in 1 h. Cyanogen bromide (CNBr) cleavage of the labeled, reduced, and carboxymethylated protein, followed by gel permeation chromatography yielded a 5- to 6-kDa peptide (LCAT CNBr-III) containing at least 60-70% of the incorporated label. Comparison of the amino acid composition of LCAT CNBr-III with that of the CNBr peptides predicted from the LCAT sequence (J. McLean et al. (1986) Proc. Natl. Acad. Sci. USA 83, 2335-2339) indicates that LCAT CNBr-III is peptide 168-220. In 22 cycles of automated Edman degradation of CNBr-III a radioactive derivative was only observed at cycle 14, and of the predicted CNBr fragments only peptide 168-220 contains a serine at position 14 from the amino terminus. Tryptic peptides predicted from the sequence should contain Ser181 at positions 22 and 23 from the N-terminus of fragments 160-199 and 159-199, respectively. On the other hand, Ser216 should be in position 15 from the N-terminus in fragment 202-238. Radiolabel sequencing of the tryptic digest of [3H]diisopropylphosphate-LCAT resulted in recovery of radioactivity in cycles 22 and 23, whereas cycle 15 yielded negligible radioactivity. These results establish that Ser181 is the major active site serine in human LCAT.

Methods for studying fibrinolytic pathway components in human plasma

Methods have been developed to quantitatively measure the major plasma components of the human fibrinolytic system. Plasminogen is measured functionally with a 9M excess of streptokinase and immunochemically by rocket immunoelectrophoresis; the normal range was found to be 16.7-23.8 mg/dl and 17.4-21.6 mg/dl, respectively. Alpha 2-plasmin inhibitor is measured functionally and immunochemically; the normal range for the major plasma plasmin inhibitor was found to be 5.30-6.60 mg/dl by both methods. Plasminogen activator concentrations, as well as, free, and complexed, protease activities are measured along with plasmin generation rates by spectrophotometric assays with chromogenic substrates. Both activator and free protease activities are zero in plasma samples from normal human subjects. Plasmin generation rates are 0.25-0.47% with urokinase and 5.30-9.70% with streptokinase; these values are the percentages of the respective initial velocities of activation in purified systems.

PLASMJN AND PLASMINOGEN ACTIVATORS: KINETICS, AND KINETICS OF PLASMINOGEN ACTIVATION

The human blood fibrinolytic system in vivo is regulated primarily by activation of plasminogen, a circulating plasma zymogen, to plasmin by one or more activators found in the plasma and the vascular endothelium. Plasmin, a proteolytic enzyme, is regulated in the blood by its interaction with the plasma a,-plasmin inhibitor to form enzymatically inactive, irreversible, complexes that are rapidly eliminated. Fibrin and thrombus formation probably accelerate plasminogen activation. The naturally occurring plasminogen activators found in plasma and tissues appear to be serine proteases and will activate plasminogen to plasmin by peptide-bond cleavage. A single molecular event is involved in the activation of plasminogen to plasmin, and, in isolated systems, it is the primary cleavage of the Arg,,,-Val peptide bond in the enzymatically inactive zymogen monomer to form an active two-chain enzyme with two interchain disulfide bonds. Since enzymatic reactions in plasma operate by kinetic principles, it is essential that we understand the kinetic behavior of plasmin and plasminogen activators, and the kinetic parameters of plasminogen activation both in isolated systems and in the plasma milieu. Since native plasma and vascular endothelial cell plasminogen activators are not readily available, we have used highly purified human urinary urokinase( s) and streptokinase, obtained from culture filtrates of hemolytic streptococci, to develop the methodology to study plasminogen activation. Other types of activators were also studied to develop the concept of physiological activation in soluble systems.

Congenital Abnormal Plasminogen, Frankfurt I, a Cause for Recurrent Venous Thrombosis

A new abnormal plasminogen, Frankfurt I, has been identified in the plasma of a 42 year-old male patients who had recurring thromboses, thrombophlebitis and pulmonary embolism since his age of 29. Reduced functional and also slightly reduced antigen plasminogen concentrations were found in both the proposituts and his mother. Plasmin generation rates carried out by Streptokinase and Urokinase were also abnormal. The plasmin generated was very unstable in the absence of stabilizing ligands and/or substrates. Crossed immunoelectrophoresis of the purified Frankfurt I revealed a peak with normal size and shape, but displaced with respect to normal Glu-plasminogen toward the anode. Isoelectric focusing followed by zymography on an agarose-fibrin plate proved this observation but did not indicate a separation of the normal from the abnormal plasminogen molecular species, also, fewer bands were found in the abnormal plasminogen isozyme pattern. Kinetic studies of Frankfurt I Glu-plasminogen and plasmin showed that most of the functional abnormality is related to absence of active sites in half of the molecules.