Immunochemical studies of human factor XIII.

Plasma factor XIII circulates as a noncovalently associated, tetrameric zymogen (a2b2). The b subunit may act as a carrier protein for the a subunit, which possesses the potential catalytic function. In order to define interactions that occur between the two subunits, a sensitive and specific RIA for the b subunit has been developed. Purified plasma factor XIII was incubated with thrombin and chromatographed on organomercurial agarose to separate the subunits. Pure b chain eluted in buffer containing CaCl2. This material was used as the standard b preparation, both for preparing a monospecific antiserum and for establishing the assay. The linear range of the assay is 7 to 700 ng/ml (Ca. 0.1 to 10 nM b subunit), with a minimum detectable dose of l. Data were analyzed by use of the logit-log transformation of antigen-binding curves. The dose-response slope for standard b is -2.76 + or - 0.20, with a potency (ED50) of 74.9 + or - 6.5 ng/ml. This RIA is also valid for determining b subunit concentration of plasma and serum. The dose-response slope for the plasma system is -2.69 + or - 0.20 with an ED50 identical to that of the purified system. By this method the mean b subunit concentration in normal human plasma (corrected for anticoagulant) is 13.8 micro g/ml (ca. 0.15 micro M). The concentration in serum is 13.6 micro g/ml, which shows that b subunit is quantitatively recovered after coagulation has occurred.

In vivo radiolabeling of platelet proteins: a new method with identification of platelet factor XIII.

A method for radiolabeling platelets in vivo was developed in which 3H-arginine was injected into the bone marrow of normal dogs. On the third day after injection, a maximum of 6%--7% of the radioactivity had been incorporated into the total platelet mass. This method of isotope administration resulted in a 50--60-fold increase in maximum uptake of radiolabel by platelets, as compared to values obtained by others using intravenous injections of various radioactive compounds. Tritium-labeled platelets were harvested from the animals and then were washed to remove unbound 3H-arginine. On polyacrylamide gel electrophoresis 7 labeled protein bands, with molecular weights ranging from 29,000to 132,000, were obtained from the platelet-soluble fraction. One 3H-containing protein with a molecular weight of 81,000 was identified immunologically and enzymatically as platelet factor XIII.

Fibrinogen Chapel Hill: hypodysfibrinogenemia with a tertiary polymerization defect.

A patient with a functionally defective fibrinogen (fibrinogen Chapel Hill) has been investigated. Fibrinogen Chapel Hill is characterized by hypofibrinogenemia, with a plasma concentration about one third of normal, as measured both functionally and immunochemically. Fibrinogen survival is normal; so also is fibrinopeptide release. A polymerization defect in this fibrinogen results in the delay of fibrin fibrils in solution to form a normal three-dimensional gel. This defect is not associated with end-to-end aggregation or with lateral associations in solution. Delayed gelation results from an abnormality in a tertiary contact site involved in network branching, which is associated with the hydrophilic, carboxy-terminal segment of the alpha chain. Fibrinogen Chapel Hill exhibits two additional abnormal responses, which are also associated with the same region. The early plasmin cleavages of fibrinogen and fragment X are delayed, and there is a concomitant delay in the appearance of fragments Y, D, and E. This fibrinogen also has an unusual sensitivity to Ancrod proteolysis, whereby Ancrod cleaves a large carboxy-terminal segment of the alpha chain more rapidly than in normal fibrinogen. The abnormalities in fibrinogen Chapel Hill can be explained by a structural abnormality which is functionally related to an alpha chain associated polymerization domain.

Difference between type I autoimmune inhibitors of fibrin stabilization in two patients with severe hemorrhagic disorder.

Inhibitors of fibrin stabilization of apparently autoimmune origin, found in two severely bleeding unrelated patients (W. G. and G. A.), were compared with regard to their biological target specificities, potencies and immunological characteristics. Both interfered only with the activation of fibrin stabilizing factor (coagulation Factor XIII) and, while totally preventing the conversion of this zymogen to the functional transamidating enzyme, fibrinoligase (Factor XIII(a)), they showed very little inhibition toward the enzyme itself. Thus, according to the classification of Lorand concerning biological specificities, both can be characterized as Type I inhibitors of fibrin stabilization. Potencies of the two inhibitors were quite similar when measured in conjunction with the plasma zymogen, but they differed remarkably in tests with platelet Factor 13. The inhibitor of patient W. G. prevented the activation of the zymogen from platelets, but that of G. A. had no effect on the platelet factor. It may therefore be concluded that the inhibitor of W. G. is directed exclusively against the a subunit which is a common constituent of plasma as well as platelet factors. The inhibitor of G. A., however, must be targeted against determinants uniquely characteristic for the ab ensemble of the plasma zymogen including the b subunit. On the basis of this difference in target specificity, the inhibitor of W. G. is designated as Type I-1 and that of G. A. as Type I-2. The inhibitors of both patients were isolated as immunoglobulins, and neutralization tests revealed that the antibody of W. G. comprised mainly heavy chains of the IgG1 and light chains of the kappa class. The antibody of G. A. proved to be considerably more heterogeneous and contained IgG1 and IgG3 heavy chains as well as kappa- and lambda-light chains. The finding that the antibody of W. G. inhibited conversion of platelet Factor 13 and also its thrombinmodified form, but had no effect on the thrombin and Ca(2+)-activated factor, is an indication that antigenic determinants existing both on the native zymogen and on its hydrolytically modified form become buried in the Ca(2+)-dependent step of activation. This is clear evidence for the occurrence of a significant conformational change in the protein structure attendant to the process of unmasking of its enzymic activity.

Role of fibrinopeptide B release: comparison of fibrins produced by thrombin and Ancrod.

The gelation time, opacity, light scattering, and elastic moduli of human fibrin gels clotted in the presence of thrombin, Ancrod, and Reptilase have been compared. At low ionic strength lateral association to thick fibers is observed in all cases. At all ionic strengths thrombin fibrin forms thicker fibers than does Ancrod fibrin. We have demonstrated that an increase in the extent of lateral association is linked to an increase in its velocity and to a decrease in the gelation time. One may consider the removal of fibrinopeptide B to act as a switch: after it is removed fibrin assembles rapidly to thick fibers and gelation is fast; but when this peptide is still attached, there is a slow assembly of thin fibers, and gelation, especially of dilute fibrin, is delayed. We believe that this delay is critical for the complete digestion by plasmin of fibrin formed during in vivo defibrination with Ancrod and of fibrin produced by very small amounts of thrombin (which would still contain fibrinopeptide B), and that slow release of fibrinopeptide B is part of a control mechanism for the regulation of fibrin formation and the prevention of intravascular coagulation.

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+.

Purification and immunochemical characterization of human plasma factor XIII.

A detailed study of the conditions for purification of plasma factor XIII was undertaken and an optimal procedure developed. This procedure involves adsorption with aluminum hydroxide, repeated fractionation with ammonium sulfate, heat denaturation of fibrinogen, precipitation with polyethylene glycol, cryoprecipitation, and chromatography on DEAE-cellulose with gradient elution. The specific activity of the final product, as measured by the fluorescent amine incorporation assay for factor XIII, averages about 3,000-4,000 mumol dansylcadaverine incorporation/mg protein. The yield is about 20%. In SDS electrophoresis the final product has two bands under nonreducing conditions, characteristic of the a and b subunits.

Alternative pathways for the activation of factor XIII.

Factor XIII is present in plasma as a proenzyme, which when activated catalyses the formation of epsilon(gamma-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 serin 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.

Isolation and characterization of the S-carboxymethyl derivatives of crosslinked and noncrosslinked human fibrin.

The S-carboxymethyl derivative chains of crosslinked and noncrosslinked fibrins were prepared from purified human fibrinogen. For crosslinked fibrin, fibrinogen was clotted with thrombin in the presence of calcium and purified human factor XIII. For noncrosslinked fibrin, ethylenediaminetetraacetate was substituted for factor XIII and calcium. After reduction with dithiothrcitol and alkylation with tritiated iodoacetic acid, the derivative chains were separated on carboxymethyl cellulose in a sodium acetate-pH gradient that contained 8 M urea. Purity of the separated chains was determined by polyacrylamide gel electrophoresis at acid and at neutral pH. The derivative chains of noncrosslinked fibrin were eluted from carboxymethyl cellulose in the order: gamma-chain, beta-chain, and alpha-chain. Each of the purified derivative chains was characterized and identified by amino-terminal aminoacid analysis, aminoacid composition, tryptic peptide mapping, and molecular weight estimation by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. In like manner, the derivative components of crosslinked fibrin were eluted from carboxymethyl cellulose in the order: gamma-gamma-dimer, beta-chain, and alpha-polymer. Application of the same analytical criteria and comparision with the derivatives of noncrosslinked fibrin confirmed the identity of these components. These data provide conclusive evidence that crosslinking of human fibrin involves formation of peptide bonds between two gamma-chains to form gamma-gamma-dimer and between multiple alpha-chains to form high molecular weight polymers of alpha-chains.