Jules A. Gladner

Studies on the physiological activity of the peptide released during the fibrinogen-fibrin conversion

Abstract The thrombin (EC 3.4.4.13) catalyzed conversion of fibrinogen to fibrin results in the release of several peptides. The peptides have been isolated from the interaction of human thrombin with human fibrinogen. Two of the acidic peptides (α and β) released from human fibrinogen contain 16 amino acids each and differ only in that the α-peptide containis a phosphate group covalently bound to a serine residue. The β-peptide is physiologically active in that it possesses the ability to potentiate the bradykinin-induced contraction of rat-uterus muscle. The α-peptide is inactive when tested in a similar manner. Structural modifications may be performed on the β-peptide without loss of physiological activity. This acitivity is not diminished by the removal of the carboxyl end-group or by a selective enzymic cleavage of the peptide. It is suggested that the β-peptide, which is released during the blood-clotting process, may play a vital role in the regulation of the microcirculation of the blood.

Effects of crosslinking on the rigidity and proteolytic susceptibility of human fibrin clots

Clots formed in reconstituted human plasma or from purified human fibrin were studied in order to assess the effects of subunit crosslinking on clot strength and on resistance to plasmin degradation. The relative amounts of alpha chain and gamma chain ligation were varied by adding factor XIII to the samples. We observe, as have others, that appreciable gamma-gamma crosslinking always precedes detectable formation of alpha dimer or alpha polymer. Non-invasive light scattering measurements of the shear modulus G(t) indicate that ligation of gamma chains and of alpha chains have qualitatively similar effects on clot strength. Since alpha crosslinking occurs very slowly in the clots which are formed from plasma, we infer that under physiological conditions the involvement of alpha chains in the development of clot strength probably is only a secondary function. Light scattering techniques also were used to study the size of particles shed from the surfaces of fibrin clots undergoing fibrinolysis, and no differences could be discerned as resulting from ligation of alpha chains.

Influence of cobalt and cadmium on the peptidase and esterase activities of carboxypeptidase B

1. 1. A 100% increase in the peptidase activity of carboxypeptidase B has been observed as a result of incubation of this enzyme with cobaltous ions. An apparent reduction in esterase activity accompanies this peptidase-activating effect under certain experimental conditions. 2. 2. Incubation of carboxypeptidase B with Cd++, on the other hand, results in as much as 100% increase in esterase activity, attended by a parallel loss in peptidase activity. 3. 3. Incubation with Co++ or Cd++ results in a reduction in the zinc content of the enzyme and a concomitant binding of the metal ion employed in incubation. 4. 4. Treatment of the cobalt and cadmium carboxypeptidases B with zinc essentially restores the peptidase and esterase activities to the levels of those of the untreated purified enzyme. 5. 5. The results support the hypotheses that the mechanism of catalytic action of carboxypeptidases A and B are similar and that the catalytic properties of carboxypeptidase B are a function of the metal bound to the enzyme protein at the level of 1 g atom/mole.

Acceleration of fibrin gel formation by unrelated proteins.

Addition of gamma-globulin, serum albumin, hemoglobin, or ovalbumin in concentrations of 1-10 g/dl to solutions of purified fibrinogen results in a substantial (up to six-fold) decrease in the lag time preceding appearance of a firm fibrin gel following addition of thrombin at 24 degrees C. The effect does not appear to be due to a protein-induced enhancement in the enzymatic activity of thrombin, nor does it appear to be due to the co-condensation of the added protein with fibrin/fibrinogen. It is suggested that the observed effect is primarily due to nonspecific volume exclusion arising from increased fractional occupancy of solution volume by macromolecules.

The fragmentation of actin by thrombin. Isolation and characterization of the split products

Abstract Thrombin, a limited protease, hydrolyzes three bonds in actin at Arg-28, Arg-39, and Lys-113, thereby producing two smaller peptides and two larger fragments. The location of the bonds split was identified in the amino acid sequence of actin by isolating the split products and carrying out amino acid analysis, and N- and C-terminal determinations.

Cross-linking of salmon fibrinogen and fibrin by factor XIII and transglutaminase

Abstract Unlike mammalian species, salmon plasma contains 2 cross-linking enzyme systems: Factor XIII and a transglutaminase which appears to be similar in its action to that described by Folk and Chung. Also, salmon plasma contains an exceedingly active protease which possesses the ability to rapidly destroy fibrinogen clottability even when the plasma is stored at a temperature of −20°C.

The biological properties of peptides derived from fibrinogen

Abstract The action of thrombin on fibrinogen results in the release of two acidic peptides and fibrin monomers. Fibrin monomer, under proper conditions of salt concentration, pH, etc., can then aggregate to an insoluble macromolecule, the fibrin clot. The release of acidic peptides, called A and B, is mandatory prior to clot formation. A study in vivo and in vitro has shown that certain of these peptides have potent biological activities in the stimulation of smooth muscle contraction. The injection of anti-inflammatory drugs into rats induces in the skin an enzyme(s) capable of converting fibrinogen to fibrin via a different mechanism. This enzyme(s) has been shown ‘not’ to be thrombin, and only one peptide is released from the fibrinogen. The physiological activity of this peptide has also been evaluated.