Jan McDonagh

Identification of intracellular factor XIII in human monocytes and macrophages.

Factor XIII is a blood protransglutaminase that is distributed in plasma and platelets. The extracellular and intracellular zymogenic forms differ in that the plasma zymogen contains A and B subunits, while the platelet zymogen has A subunits only. Both zymogens form the same enzyme. Erythrocytes, in contrast, contain a tissue transglutaminase that is distinct from Factor XIII. In this study other bone marrow-derived cells were examined for transglutaminase activity. Criteria that were used to differentiate Factor XIII proteins from erythrocyte transglutaminase included: (a) immunochemical and immunohistochemical identification with monospecific polyclonal and monoclonal antibodies to Factor XIII proteins, (b) requirement for thrombin cleavage to express activity, (c) pattern of fibrin cross-linking catalyzed by the enzyme, and (d) different electrophoretic mobilities in nondenaturing gel systems. By these criteria human peripheral blood monocytes, peritoneal macrophages, and monocytes maintained in culture contain an intracellular protransglutaminase that is the same as platelet Factor XIII. The monocyte-macrophage protein is thrombin-sensitive, and under appropriate conditions there is no enzyme expression without activation of the zymogen. Both the monocyte-macrophage zymogen and enzyme have the same electrophoretic mobilities as platelet Factor XIII zymogen and enzyme. Antibody to A protein reacts with the monocyte-macrophage protein. B protein is not associated with this intracellular zymogen. By immunoperoxidase staining monocyte-macrophage protein seems to be localized in the cytoplasm, similar to the known cytoplasmic distribution of platelet and megakaryocyte Factor XIII. These procedures were also used to study populations of human granulocytes and lymphocytes, and protransglutaminase activity was not observed in these cells.

Factor XIII in human plasma and platelets

Plasma and platelet factor XIII levels were measured in normal human donors and in a patient congenitally deficient in factor XIII. The purpose of these experiments was to study the role of platelet factor XIII in blood coagulation. On polyacrylamide disc electrophoresis, factor XIII activity in extracts of washed normal platelets appeared as a single peak. This peak was missing or very low when factor XIII-deficient platelet extract was used. The patient was also studied before and after transfusion of fresh frozen plasma. Before transfusion, factor XIII activity could not be detected in the patients plasma or platelet extracts. 24 hr after transfusion the plasma factor XIII level was still at the anticipated value, but factor XIII activity could not be detected in the platelet extracts. These experiments imply that plasma factor XIII was not transported across the platelet membrane in measurable quantities in vivo. This suggests that platelet factor XIII is a true platelet component and originates in the platelet precursor, the megakaryocyte. Thrombelastographic studies suggest that platelet factor XIII participates in the coagulation process. Thrombelastograms of factor XIII-deficient samples had decreased maximum amplitude and clot elasticity values. The abnormalities were fully corrected by the addition of washed normal platelets to factor XIII-deficient plasma; preincubation of the normal platelets in the deficient plasma had no additional effect. This indicates that platelet factor XIII is immediately available during clot formation.

Effects of calcium ion and covalent crosslinking on formation and elasticity of fibrin gels

Abstract The elastic properties of fibrin gels prepared under a variety of conditions were studied using a Couette elastometer. Fibrin gels formed either by addition of thrombin to fibrinogen or by dilution of previously prepared fibrin monomer were seen to be dependent on calcium ion for the expression of maximum elasticity. By dialyzing a fibrinogen solution it was shown that two calcium ions per mole are tightly bound. Factor XIIIa was shown to have an effect on the elastic modulus by means of its crosslinking the α-chains to form α-polymer. γ-γ crosslinking caused no additional increase over calcium in elasticity of fibrin gels. The relative effects of calcium concentration on the rates of crosslinking of α-chains and γ-chains to form α-polymers and γ-γ dimers were determined by means of SDS gel electrophoresis in the presence of dithiothreitol. At calcium concentrations below 2×10 −4 M α- and γ-chain crosslinking proceed at the same rate which depends strongly on the calcium concentration, while at higher concentrations the rate of α-crosslinking remains constant and the γ-chain crosslinking rate is dependent on calcium concentration. Factor XIIIa was shown also to lower the gel point of dilute solutions of fibrinogen clotted with thrombin in the presence of calcium. This demonstrates that Factor XIIIa promotes gelation in very dilute fibrin solution, probably by irreversibly crosslinking intermediate polymers of fibrin.

The influence of fibrin crosslinking on the kinetics of urokinase-induced clot lysis.

Summary. The effect of fibrin crosslinking on the lysis of plasma clots was investigated with plasma from a patient congenitally deficient in plasma factor XIII (fibrin stabilizing factor). The thrombin‐activated plasma factor XIII was found to render clots more resistant to fibrinolysis when urokinase (UK) was used to induce plasminogen activation. Incorporation of UK in the clot by addition to plasma immediately before clotting resulted in a log‐log relationship when lysis time was plotted against UK concentration, with greater differences between normal and factor‐XIII deficient clots at lower UK concentrations. Addition of UK to the clot externally, after preincubation of the clot, gave linear plots on rectangular coordinates when either plasma or euglobulin fraction was used; and the difference in lysis time between factor‐XIII deficient and normal clots was nearly constant over the range of UK concentrations tested. When the fluorescent amine, dansylcadaverine, was used to measure factor‐XIII activity quantitatively, plasma clot lysis times were found to be directly proportional to amine‐incorporating activity over a wide range of activities at low UK concentrations. The range of proportionality was reduced when UK concentration was increased. Addition of purified factor XIII to the patients plasma restored the resistance of these clots to within the normal range.

Subcellular distribution of fibrinogen and factor XIII in human blood platelets.

Abstract Fresh human platelets were disrupted by nitrogen decompression, and homogenates were fractionated by centrifugation in a linear gradient of sodium diatrizoate (18–33%). Five distinct fractions were obtained. Electron microscopy showed that the membrane fraction was free of contaminating subcellular organelles. The granule fraction contained primarily intact granules, with some mitochondria and some swollen granules. A portion of the granules was also distributed in the fractions above and below the primary granule fraction. Total protein was found to be 1.95 mg/10 9 platelets, of which 0.18 mg was identified as fibrinogen. The ratio of platelet to plasma fibrinogen in whole blood was determined to be 1:30. Total factor XIII activity was 69 μmole dansylcadaverine incorporation/10 9 platelets, and factor XIII activity was found to be equally distributed between plasma and platelets. 70% of the platelet fibrinogen was found in association with granules, with the remainder appearing primarily in the soluble fraction. Platelet factor XIII was localized in the cytoplasm, with 94% appearing in the soluble fraction. Platelet fibrinogen was released during the platelet release reaction induced by collagen, whereas factor XIII was not.

Fibrin gel structure: influence of calcium and covalent cross-linking on the elasticity.

Abstract Calcium ion causes the development of a higher elastic modulus in fibrinogen solutions clotted by thrombin. By also measuring the development of covalent crosslinks introduced by activated Factor XIII, we find that (a) calcium ion causes an overall increase of the modulus, (b) covalent crosslinking of α-chains causes an increase of the modulus, while (c) covalent crosslinking of γ-chains does not.

Affinity chromatography of human plasma and platelet factor XIII on organomercurial agarose

A method for affinity chromatography of plasma and platelet factor XIII has been developed, based on known structural characteristics of these molecules. Plasma factor XIII is composed of a and b subunits which are held together by noncovalent interactions; platelet factor XIII has only a subunits. a subunit contains free sulfhydryl groups, while in b subunit all the cystines form disulfide bonds. The affinity gel is an organomercurial agarose with p-chloromercuribenzoate as the reactive group. Both the zymogen and activated forms of a subunit reversibly bind to the ligand by forming covalent mercaptide bonds and are eluted by reducing agents. b subunit does not bind to the affinity gel and is held to it only through interaction with a subunit. Affinity chromatography can be used to purify plasma and platelet factor XIII and to study interactions of the subunits. Experiments on the affinity chromatography of purified plasma factor XIII in several stages of activation agree with earlier observations that activation is a two-step procedure in which b subunit is not quantitatively released from the complex until the final stage of activation by Ca2+.

Alternative Pathways for the Activation of Factor XIII

Summary. Factor XIII is present in plasma as a proenzyme, which when activated catalyses the formation of ε(γ‐glutamyl)lysyl bonds in fibrin. In this study the activation of purified plasma factor XIII was examined quantitatively with the fluorescent amine incorporation assay. Activation products were examined by polyacrylamide gel electrophoresis. The serine proteases, thrombin, trypsin, chymotrypsin, and factor Xa, and also Reptilase were tested for their ability to activate factor XIII. Highly purified thrombins activated purified factor XIII; this reaction was not calcium dependent. Trypsin was also a potent activator, but no transglutaminase activity was found with chymotrypsin. The most highly purified preparations of Reptilase had no effect on factor XIII activity. Less purified Reptilase preparations activated factor XIII, which suggests the presence of another enzyme in these Reptilase preparations. Highly purified factor Xa was found to be an effective activator of purified factor XIII, In contrast to thrombin activation, this reaction required calcium. It may be that under certain circumstances factor XIIIa could be formed in vivo directly by the alternative pathway of factor Xa. Factor XIIIa could then crosslink fibrinogen, which would also provide an alternative pathway for thrombus formation. Also, the activation of factor XIII by both factor Xa and thrombin provides a further point of control in the blood coagulation process.

Comparative Studies on Blood Coagulation Factor XIII

Summary Factor XIII activity and fibrinogen concentrations were determined in multiple, fresh, platelet-poor plasma samples from 15 species of vertebrates. Factor XIII and fibrinogen levels varied independently. In monkey, rat, cat, horse, pig, sheep, goat, goose, chicken, and turkey plasmas, factor XIII activity ranged from 56 to 96% of that found in human plasma. For guinea pig, rabbit, canine, and bovine plasmas the values ranged from 173 to 480%. Fibrinogen concentration in all species ranged from 2.16 to 4.24 mg/ml. Factor XIII of all species was neutralized to varying degrees by two human antibodies to plasma factor XIII and, except for rabbit and avian plasmas, by rabbit anti-serum to human a subunit. Rabbit antiserum to human b subunit formed a precipitin line with plasma of all species except rabbit and avian.

Factor XIII from human platelets: Effect on fibrin crosslinking

Abstract Human platelets were separated from plasma proteins by gel filtration on Sepharose in order to study the fibrin crosslinking activity of platelet factor XIII and the release of factor XIII during platelet aggregation. It was found that the platelet soluble fraction prepared from sonicated, gel-filtered platelets contained two enzymes which had activity in the fluorescent amine incorporation assay for factor XIII. One of these was heat stable and required thrombin activation and was identified as platelet factor XIII. The crosslinking activity of this enzyme was examined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. When platelet soluble fraction was the only source of factor XIII, electrophoretic analysis showed the typical pattern of crosslinked fibrin, consisting of β-chain, γ-γ-dimer and α-polymer. When platelets were aggregated by adenosine diphosphate sufficient platelet factor XIII was released to form γ-γ-dimer crosslinks. However plasminogen was also activated during aggregation which degraded α-chains of fibrin. The crosslinking pattern of this plasmin degraded fibrin suggested that an α-chain fragment of molecular weight 46,000 to 49,000 consisting of two crosslinked α-chain fragments was formed. α-chain fragments of molecular weight 17,000 and 26,000 were also found.