Stephanie A. Olexa

Fibrin polymerization sites in fibrinogen and fibrin fragments.

The inhibition of blood clotting by degradation products of fibrinogen reflects an interference with fibrin network formation. The original discovery that digestion of fibrinogen by plasmin resulted in the formation of fragments with anticoagulant activity 1. did not recognize the importance of inhibition of fibrin polymerization and was interpreted as an interference with thrombin action. During the past 25 years considerable progress has been achieved in the understanding of fibrin clot formation. The use of fibrinogen and fibrin fragments significantly contributed to the advancement of our knowledge, especially since many biologic functions of fibrinogen were recovered in various fragments. Thrombin releases two molecules of fibrinopeptide A (FPA) and two molecules of fibrinopeptide B (FPB) from the Aa and Bg polypeptide chains of the fibrinogen molecule, respectively. The cleavage of four arginylglycine peptide bonds converts fibrinogen into fibrin monomer, which polymerizes spontaneously forming a fibrin clot. This paper addresses the structure and localization of active contact sites with complementary affinities which are thought to specifically organize fibrin monomers and drive the polymerization reaction. Batroxobin, a protease from Bothrops atrox venom, cleaves FPA from fibrinogen. This event is sufficient to initiate clotting and formation of fibrin I, which lacks FPA but still contains FPB, and provides evidence that the loss of FPA alone is sufficient for fibrin gelation. Thrombin releases FPA at a much faster rate than FPB and the ultimate product, fibrin I1 lacks both fibrinopeptides.

Anticoagulant and fibrinolytic properties of salivary proteins from the leech Haementeria ghilianii.

Abstract The salivary glands of the blood-sucking leech Haementeria ghilianii contain an anticoagulant that not only inhibits the clotting of human and bovine plasma, but also dissolves previously formed fibrin clots. This anticoagulant activity is attributable to an enzyme, for which the name hementin is proposed. Hementin catalyzes the proteolytic degradation of fibrinogen and fibrin, even in the presence of the inhibitors of proteases occurring in human plasma. The enzyme has the same affinity for human fibrinogen and fibrin. In human fibrinogen cleaves the Act chain initially and then the γ chain to yield characteristic fragments of high molecular weight that are different from the fragments resulting from the digestion of fibrinogen by plasmin. The salivary extracts do not contain any appreciable amounts of an activator of human plasminogen or an inhibitor of human or bovine thrombin. Thus, H. ghilianii prevents coagulation of its host blood through a fibrinogenolytic mechanism that is entirely different from that of hirudin, a thrombin-inactivating polypeptide present in the saliva of another leech, Hirudo medicinalis.

Composition of Salivary Gland Extracts from the Leech Haementeria ghilianii

Abstract The giant leech, Haementeria ghilianii, originating from French Guyana, has two pairs of salivary glands. From the anterior and posterior glands, approximately 1.5 and 0.3 mg of protein per leech, respectively, have been extracted. Electrophoretic analysis showed that the protein composition of the anterior and posterior gland extracts is completely different. However, both gland extracts contain fibrinolytic activity associated with only one electrophoretic band. The fractionation of salivary gland homogenates by differential ultracentrifugation showed that the fibrinolytic activity is found entirely in the cytosol fraction.

The interference of plasmic degradation products of human crosslinked fibrin with clot formation

The role of plasmic degradation products of human crosslinked fibrin on polymerization of fibrin monomer and clot formation was studied. Both reactions were inhibited by Fragment DD, which formed a complex with fibrin monomer in a molar ratio 1 : 1. The rate of polymerization was slightly increased by Fragment E but it was not affected by (DD)E complex and Fragment A. Approximately the same amount of fibrin was formed in the presence and absence of Fragments A, E and the complex. It was concluded that of the degradation products of crosslinked fibrin, only Fragment DD is a potent anticoagulant at physiologic pH. The (DD)E complex is inert and Fragments A and E have only marginal effects.

Modification of high molecular weight plasmic degradation products of human crosslinked fibrin

The predominant high molecular weight products of plasmic digestion of human crosslinked fibrin Fragments DD, E and (DD)E complex were purified by column gel filtration in a non-dissociating buffer or by ion-exchange chromatography on DEAE-cellulose. The structure of the degradation products was studied by proteolytic degradation, polyacrylamide gel electrophoresis immunodiffusion and sucrose density gradient centrifugation. Unaltered derivatives were very resistant to proteolytic degradation by plasmin. In the the presence of 10 mM EDTA the (DD)E complex did not dissociate, but similar to Fragment DD, became susceptible to plasmic degradation forming Fragment D derivatives. The (DD)E complex dissociated in 3 M urea at pH 5.5, had an altered conformation as evidenced by its aggregability and by its increased susceptibility to degradation by plasmin resulting in the formation of Fragment d. The gammagamma chain remnants of Fragment DD were attacked first, followed by cleavage of the beta chain remnants. It is concluded that plasmin resistance is a function of the intact structure and it is not directly dependent on the presence of the crosslink bonds or calcium ions.

Specific uptake of radioiodinated fragment E1 by venous thrombi in pigs.

Fragment E1, a product of plasmic digestion of cross-linked fibrin, binds specifically in vitro to polymerized fibrin but not to fibrinogen. Purified human Fragment E1 was radiolabeled with 125I or 131I by the Iodogen technique. The uptake of radioiodinated Fragment E1 in vitro into forming or preformed clots was demonstrated. Animal biodistribution studies of radioiodinated Fragment E1 showed its rapid removal from the circulation; radioactive catabolites did not reside long in any organ and were excreted in the urine. The uptake in vivo was evaluated in pigs with preexisting venous thrombi of various ages from 1 h up to 5 d at the time of intravenous systemic injection of the tracer. Radioiodinated fibrinogen was also injected into the same animals to compare the uptake of the two tracers. Thrombus-to-blood ratios for Fragment E1 averaged 43:1 (range 10-108) and 29:1 (range 8-107) in thrombi 1-6 h and 1-5 d old, respectively. In contrast, mean thrombus-to-blood ratios for fibrinogen were, in the same time intervals, 26:1 (range 17-41) and 2:1 (range 0.5-3.9), respectively. It is concluded that radioiodinated Fragment E1 is a specific marker of thrombi in vivo: its uptake by fresh thrombi is better than that of labeled fibrinogen and, in contrast to radioiodinated fibrinogen, this fragment is incorporated into old thrombi as well.

Macromolecular complexes of aminoacyl-tRNA synthetases in Drosophila

Abstract Aminoacyl-tRNA synthetases in Drosophila are present in large macromolecular aggregates having mol. wt in excess of 10 6 Daltons. After cellular fractionation these enzymes are found in the post-microsomal hard pellet, the post-microsomal soft pellet, and the post-microsomal supernatant fractions. The distribution of specific enzyme activities within these fractions varies and is dependent upon the specific tRNA synthetase being studied.

Unique degradation of human fibrinogen by proteases from western diamondback rattlesnake (Crotalus atrox) venom.

Thrombin-coagulability of both human fibrinogen and plasma was rapidly lost upon incubation with western diamondback rattlesnake (Crotalus atrox) venom. The dose- and time-dependent effect was due to direct proteolytic degradation of fibrinogen (Mr 340,000) by venom enzymes. Using purified fibrinogen as the substrate it was demonstrated that the venom degraded the A alpha chain first and then the B beta chain. The degradation pattern of fibrinogen in plasma was different to that of purified fibrinogen, since only the B beta chain was cleaved. A fibrinogen derivative isolated from venom-treated plasma had impaired thrombin-coagulability, Mr 325,000 +/- 10,000, its A alpha and gamma chains appeared intact and only the B beta chain was degraded to a species of Mr 52,000 +/- 1,500. The venom contained three proteolytic enzyme fractions as revealed by gel filtration chromatography. All abolished coagulability of purified fibrinogen, however, only one enzyme fraction rendered plasma incoagulable. The proteolytic enzyme with anticoagulant activity against plasma degraded only the B beta chain of purified fibrinogen, generating a derivative of Mr 325,000, which was identical to that obtained upon incubation of the crude venom with plasma. The polypeptide chain structure of the derivative indicates that the intact B beta chain of fibrinogen plays an important role in the formation of fibrin clots.