Andrei Z. Budzynski

A prospective study of platelets and plasma proteolytic systems during the early stages of Rocky Mountain spotted fever.

We prospectively examined early changes in platelets and plasma proteolytic systems in 12 vaccinated and 6 unvaccinated volunteers in whom Rocky Mountain spotted fever developed after challenge with Rickettsia rickettsii. The platelet counts declined while the plasma concentration of beta-thromboglobulin and the ratio of beta-thromboglobulin to platelet factor 4 increased, indicating in vivo activation of platelets. Plasma levels of antithrombin III decreased and levels of fibrinopeptide A increased, indicating in vivo activation of the coagulation system. Plasma fibrinogen levels peaked at 24 hours and gradually declined; this is consistent with the behavior of fibrinogen as an acute-phase reactant. Prolongation of the prothrombin time and a decrease in plasma levels of factor VII in the absence of evidence of liver injury suggested possible activation of the extrinsic pathway of coagulation. A decline in plasma prekallikrein levels with an increase in plasma C1-inhibitor-kallikrein complexes suggested activation of kallikrein, probably through the intrinsic coagulation system. Elevations in levels of plasma fibrin-degradation products and alpha 2-antiplasmin-plasmin complexes with declines in plasminogen and alpha 2-antiplasmin levels provided evidence of activation of the fibrinolytic system. Elevated plasma levels of tissue plasminogen activator and von Willebrand factor reflected endothelial stimulation. Thus, even early in the course of Rocky Mountain spotted fever that is treated promptly, there is activation of platelets, coagulation pathways, and the fibrinolytic system. These changes may be related to endothelial perturbation, a major pathogenetic mechanism in the disorder.

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.

High-dose, brief-duration intravenous infusion of streptokinase in acute myocardial infarction: description of effects in the circulation

The effects in the circulating blood of a 1-hour intravenous infusion of 1.5 X 10(6) units of streptokinase (SK) were measured during the subsequent 24-hour period in 7 patients with acute myocardial infarction. At the end of the infusion, the activator activity, expressed in SK units, averaged 65 U/ml, all of the plasminogen had disappeared, only a small amount of free plasmin was still present and functionally active alpha 2 antiplasmin had been reduced to 21% of the preinfusion level. All of the native fibrinogen had been degraded and the thrombin-coagulable protein was composed entirely of fragment X species, but the circulating plasma also contained significant amounts of the more extensively degraded fragments Y, D and E. The biologic half-life of the SK-induced activator activity was 23 minutes and that of the fibrinogen degradation products was 6.3 hours. The lytic effects persisted for 4 hours before any signs of recovery from the hemostatic defect were evident; considerable recovery was present at 25 hours.

FIBRINOGEN INTERACTION WITH PLATELET RECEPTORS

In summary: Incubation of platelets with ADP or proteolytic enzymes (chymotrypsin or pronase) results in an exposure of two classes of specific binding sites on platelet surface: low and high affinity fibrinogen receptors. Fibrinogen interaction with these receptors results in platelet aggregation. High affinity fibrinogen receptors are not exposed on thrombasthenic platelets stimulated by ADP but are rendered available on chymotrypsin-treated thrombasthenic platelets; low affinity receptors cannot be exposed by ADP or chymotrypsin on these platelets. Availability of high affinity fibrinogen receptors on thrombasthenic platelets may depend on the residual glycoprotein IIIa. Fibrinogen receptors appear to be associated with glycoproteins IIb, IIIa and a 66,000 Mr platelet membrane component that is exposed during proteolysis of platelet membranes. Some of the platelet-binding sites on the fibrinogen molecule appear to be associated with the COOH-terminal portion of the gamma chain (gamma 374-411). Additional binding sites may also be located in the COOH-terminal portion of the A alpha chain. The conformation of the fibrinogen molecule may be important in its interaction with platelets. Platelet aggregation may result from bridging platelets by fibrinogen molecule in the presence of bivalent cations. In conclusion, platelet interaction with fibrinogen is a complex process involving different binding sites of the fibrinogen molecule. Our own data and review of literature suggest that platelet-interaction with fibrinogen is of major significance in hemostasis.

Fibrinogen and fibrin: biochemistry and pathophysiology.

Fibrinogen is a thrombin-coagulable glycoprotein occurring in the blood of vertebrates. The primary structure of the alpha, beta, and gamma polypeptide chains of human fibrinogen is known from amino acid and nucleic acid sequencing. The intact molecule has a trinodular, dimeric structure and is functionally bivalent. Thrombin cleaves short peptides from the amino termini of the alpha and beta chains exposing polymerization sites that are responsible for the formation of fibrin fibers and appearance of a clot. The major physiological function of fibrinogen is the formation of fibrin that binds together platelets and some plasma proteins in a hemostatic plug. In pathological situations, the network entraps large numbers of erythrocytes and leukocytes forming a thrombus that may occlude a blood vessel. Fibrinogen and fibrin are multifunctional proteins. Fibrinogen is indispensable for platelet aggregation; it also binds to several plasma proteins, however, the biological function of this interaction is not completely understood. Fibrin is an essential matrix for regulation of fibrinolysis and for facilitation of cell attachment in wound healing.

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.

Purification and characterization of hementin, a fibrinogenolytic protease from the leech Haementeria ghilianii

The fibrinogenolytic enzyme hementin, present in extracts of the posterior salivary glands of the giant leech Haementeria ghilianii, was isolated by ultrafiltration, high-performance ion-exchange chromatography and subsequent reversed-phase liquid chromatography. Approximately 100 micrograms (1 nmol) of hementin, present at less than 0.5% in the crude leech salivary extract, was brought to about 90% purity in three steps. Hementin migrated at an Mr of about 73,000 on non-reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and at 82,000 on reducing SDS-PAGE. The amino terminal sequence was determined to be TTLTE-PEPDL. The amino terminal sequences of two inactive proteins that partially coeluted with hementin in the first chromatographic step were also determined.

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.