Victor J. Marder

Comparative Effects of Fibrinogen Degradation Fragments D and E on Coagulation

The mechanism of anticoagulant action of the fibrinogen degradation products (fragments D and E) has been investigated using purified products in selected esterolytic, proteolytic and coagulation systems. Their modes of action were distinctly different. Fragment D acted primarily by inhibiting fibrin monomer polymerization, while fragment E did not significantly alter this process. The latter product appeared to be a competitive inhibitor of the clotting action of thrombin on fibrinogen. Its comparative effect in thrombin proteolytic and esterolytic systems indicated that fragment E did not irreversibly inactivate the enzyme centre of thrombin. Rather, it appeared to block a thrombin binding site essential for proper alignment with the fibrinogen substrate. This competitive inhibition suggests that fragment E may derive from and share physical structures with the thrombin‐susceptible, N‐terminal portion of the fibrinogen molecule.

Comparison of the physicochemical properties of fragment D derivatives of fibrinogen and fragment D-D of cross-linked fibrin

The molecular weight of Fragment D derivatives obtained from plasmic digests of fibrinogen and cross-linked fibrin was determined by equilibrium sedimentation and compared with the summated molecular weight of their polypeptide chains observed after electrophoresis of reduced protein in sodium dodecyl sulfate polyacrylamide gels. The measured molecular weight of Fragment D (Stage 2) of fibrinogen is 103 500, which is compatible with a molecule containing only one each of the Aalpha (13 000), Bbeta (43 000) and gamma (39 000) chain remnants. Fragment D-D of cross-linked fibrin has a molecular weight of 189 000, compatible with a molecule containing one isopeptide-bound gamma-gamma chain (80 000) and two each of Bbeta (43 000) and Aalpha (13 000) chain remnants. The NH2-terminal amino acid residues of the Fragment D derivatives were measured quantitatively using a thioacetic-thioglycolic acid method, and molar quantities were calculated on the basis of the molecular weights determined by equilibrium sedimentation. Fragment D preparations obtained from Stage 2 and Stage 3 digests of fibrinogen have 3 mol of NH2-terminal amino acids per molecule, while Fragment D-D has seven. These data support the view that two Fragment D molecules, each of three polypeptide chains, are derived by plasmic degradation from each fibrinogen molecule, and that an isopeptide-bound, six chain Fragment D-D molecule is released from cross-linked fibrin by plasmin. Equlibrium sedimentation measurement of the molecular weights of Fragment X (Stage 1 and Stage 2) and Fragment Y are 265 000 and 148 000, respectively. These values are compatible with asymmetric cleavages of Fragment X to Fragments Y and D (Stage 2), and of Fragment Y to Fragments D (Stage 2) and E, and with a fibrinogen model in which the two halves are joined by disulfide bonds only in the amino-terminal regions.

The relationship of platelet coagulant activities to venous thrombosis following hip surgery.

Platelets have recently been shown to trigger intrinsic coagulation by two alternative pathways, protect active clotting factors from inactivation by plasma inhibitors and catalyse intrinsic coagulation reactions on the platelet surface to form fibrin. To determine whether these platelet coagulant activities (PCA) might have a role in the pathogenesis of DVT, 29 patients have been studied before and after arthroplasty or other surgery for fractured hip or degenerative hip disease. The occurrence of DVT was detected by [125I]fibrinogen uptake in the legs and confirmed by venography. In patients who developed DVT, all PCA increased progressively and significantly on day 1 (mean rise, 146% of baseline), day 3 (228%) and day 5 (298%) after surgery before isotopic evidence of DVT appeared (mean 3.27 days postoperatively). In patients without DVT no changes in PCA were observed. Plasma coagulation factor assays were no different in patients with and without DVT. Platelet counts and total platelet antiheparin activity increased during the early postoperative period in DVT patients but not in patients without DVT. It is suggested that progressive increases in PCA concerned with triggering and catalysing intrinsic coagulation reactions may play a pathogenetic role in DVT after hip surgery.

Formation of highly ordered polymers from fibrinogen and fibrin degradation products

Abstract Products of limited plasmin digestion of fibrinogen (FDP) and of fibrin (fdp) retained the ability to form highly organized polymers with protamine sulphate while products of extensive digestion lost this ability. Electron microscope studies showed that highly organized polymers were formed only in samples of FDP or fdp which contained Fragment X. Strong evidence indicated that there are two varieties of Fragment X, one with fibrinopeptides (Xfp) and one devoid of fibrinopeptides (Xo). The significance of fibrinopeptides on the behavior of the fragment was indicated by the difference in mechanisms by which polymers seem to be formed. Fragment Xfp (contained in FDP or purified) reacted with protamine sulphate but not ethanol to form rather small lancet-shaped polymers with a periodicity of 225–240 A without branching. Fragment Xo (contained in fdp) reacted with protamine sulphate and with ethanol to form an extensive net which was indistinguishable from thrombin induced fibrin in extent, branching and periodicity. This likely resulted from dissociation of complexes of Xo with other degradation products Y or D, followed by the spontaneous polymerization of Xo. Purified Fragment Xfp formed a similar net after conversion to Xo by thrombin. After pretreatment with thrombin (to remove fibrinopeptides from Xfp), FDP formed an extensive net with both protamine sulphate and ethanol, probably by a mechanism similar to that described for dissociation of complexes in fdp. Maximal protein precipitation from fibrinogen and FDP required 5–10 times as much protamine sulphate as was required for similar precipitation from fdp and FDP pretreated with thrombin. As digestion progressed, the amount of precipitable protein decreased until only a small amount remained. The formation of highly ordered fibrin-like polymers in fibrin or fibrinogen digests appeared to result from (a) the direct action of protamine sulphate on fibrinogen and Fragment Xfp; (b) the dissociation by ethanol or protamine sulphate of complexes of Fragments Xo with Y or D followed by the subsequent polymerization of Fragment Xo. The latter mechanism is probably responsible for the paracoagulation reaction.

A Quantitative Evaluation of the Inhibition of Platelet Aggregation by Low Molecular Weight Degradation Products of Fibrinogen

Summary. The inhibitory effect of low molecular weight degradation products of fibrinogen (peptides) on in vitro platelet aggregation has been studied quantitatively. In our test system (0.3 μm ADP at 25°C) only concentrations higher than 0.65 mg/ml showed significant inhibitory activity. The higher concentrations of peptides also inhibited platelet aggregation induced by adrenaline, collagen and thrombin. Ultrafiltrates of plasma from patients receiving streptokinase showed no increase in inhibition of platelet aggregation as compared with pretreatment samples. This supports the idea that the peptides produced by streptokinase‐induced fibrinogcnolysis exert a quantitatively weak effect. Peptides produced by digestion of serum with trypsin showed inhibitory activity similar to that of the peptides produced by digestion of fibrinogen with plasmin, indicating that the inhibitory effect is unspccific as far as the protein source and the acting enzyme are concerned. Some specificity may exist, however, related to size and/or charge of the peptides, as suggested by the present experiments. These data do not support the view that the low molecular weight degradation products of fibrinogen significantly impair platelet aggregation in the fibrinolytic states.

Thrombolysis of acute or subacute nonembolic arterial thrombosis.

The cause of arterial occlusion in patients with peripheral arterial disease is usually atherosclerosis. However, arterial thrombosis is at times associated with the intrinsic arterial disease. Progressive arterial stenosis by atherosclerosis may cause decreased arterial flow and eventual intra-arterial thrombosis. This is manifested clinically as an acute or subacute exacerbation of chronic arterial insufficiency. The objective of this study was to evaluate the capability of thrombolysis by Streptokinase in acute or subacute exacerbations of chronic arterial occlusive disease by randomized single-blind comparison to standard anticoagulant therapy.

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.

Electron microscopic studies of plasmic degradation products of fibrinogen: Implications for the disulfide structure of fibrinogen

Fibrinogen, coagulable plasmic derivatives (Fragments X) and Fragments Y, D and E were studied by negative staining electron microscopy. Fragment X obtained from Stage 1 digests and fibrinogen were both globular, while Fragment X of Stage 2 digests appeared as a nodular filament. The Stage 1 and Stage 2 Fragment X preparations had approximately the same molecular weight, but could be differentiated by several subtle differences in polypeptide chain structure. Fragments Y and D were also filamentous, although shorter than Fragment X (Stage 2), and Fragment E appeared as a small, compact or folded filament. These results agree with the concept that fibrinogen consists of a strand of nodules connected by thin strands, folded into a compact, spherical shape. The molecule opens up when stabilizing bonds are disrupted or liberated by plasmin. The data are compatible with a fibrinogen molecule in which the two halves are linked by a single locus of disulfide bonds at the amino terminus and with the asymmetric hypothesis of plasmic degradation to Fragments X, Y, D and E.

Creation of the recombinant tissue plasminogen activator (rt-PA) image and its influence on practice habits

American physicians have commonly practiced thrombolytic therapy for acute myocardial infarction with the recombinant form of tissue plasminogen activator (rt-PA), although its cost is much higher than that of streptokinase. The greater popularity of rt-PA is based on the belief that it is a more effective and a safer drug for achieving myocardial salvage and mortality reduction. However, a series of studies testing this assumption have not substantiated its greater efficacy or safety with respect to not only streptokinase but also urokinase and anisoylated plasminogen-streptokinase activator complex (APSAC). This editorial reviews the sequence of events that led to the creation of the rt-PA image, the mistaken premises on which it was based and the questions that need to be addressed if we are to strengthen the scientific method for evaluating similar types of drugs and its influence on practice habits including the costs to the health system.