Blood stasis, thrombosis and fibrinolysis.

Blood stasis is a very common occurrence associated with many of our daily activities. Prophylaxis against development of thrombosis in high-risk conditions is well established. In this issue of Hematology/Oncology Clinics of North America, international experts present new and provocative information on the basic mechanisms, diagnosis, and management of blood stasis, thrombosis, and fibrinolysis. Reviews of pertinent topics by three physicians in training also are included. I hope that the information provided will expand our understanding of this interesting subject.

Hypercoagulable states.

The hypercoagulable state has been defined as the potential to develop thrombosis in association with hereditary and noninherited genetic mutations and acquired disorders. It is a condition that places an individual at risk for, but does not in itself inevitably lead to, thrombosis. The focus of this article is understanding mechanisms in the hypercoagulable state that enhance and maintain the production of thrombin in circulating blood while preventing its progression to thrombosis. These mechanisms include reactions that produce thrombin from prothrombin, feedback loop mechanisms that affect the rate of thrombin production from prothrombin and the inactivation of thrombin in blood. The fibrinolytic system is involved in clot lysis but not in thrombin production and inactivation.

Mechanisms of thrombosis in spinal cord injury.

Many studies failed to identify a hypercoagulable imbalance in the blood factors or decreased anticoagulant activity. On the other hand, fibrinolysis, a process unrelated to hypercoagulability but closely related to endothelial cell integrity, is predictably altered and contributes to the persistence of venous occlusion by thrombosis. There is considerable evidence that interruption of neurologic impulses and the ensuing paralysis cause metabolic changes in blood vessels and that blood vessel changes are accountable for venous thrombosis. Altered venous competence with complete spinal cord injury manifests by a decrease in venous distensibility and capacity and an increase in venous flow resistance. Vascular adaptations to inactivity and muscle atrophy, rather than the effect of a nonworking leg-muscle pump and sympathetic denervation, seem to lead to the thrombosis; indicating that thrombosis resulting from venous incompetence cannot be reversed by anticoagulation alone.

Statin drugs and dietary isoprenoids as antithrombotic agents.

Statin drugs and various isoprenoids from plant origins inhibit mevalonic acids, cholesterol, and other isoprenoid products. Among these, reduction of farnesyl and geranylgeranyl prenylated proteins impedes signal transduction at the cellular level. The authors envision that limiting such prenylated proteins downregulates thrombin-stimulated events, including decreasing the expression and availability of protease-activated receptor-1 mitigating thrombin stimulation of cells, tissue factor preventing additional thrombin generation, and plasminogen activator inhibitor-1 allowing thrombosis. Additional processes may enhance nitric oxide production and induce other processes. Downregulation of thrombin-stimulated events should promote hypothrombotic or quiescent conditions that reduce cardiovascular disease, thus contributing to longevity.

Thrombin and antithrombotics.

From injury through healing, thrombin has several important functions in blood clotting, subsequent clot lysis, and tissue repair. These include edema, inflammation, cell recruitment, cellular releases, transformations, mitogenesis, and angiogenesis. Thrombin also participates in disease states, such as venous thrombosis, coronary thrombosis, stroke, and pulmonary emboli, among others and is implicated in atherosclerosis, the growth and metastasis of certain cancers, Alzheimer's disease, and perhaps other conditions. Thrombin must be continually generated to sustain normal and pathogenic processes. This is because of a variety of consumptive mechanisms. Unlike other activated factors in thrombotic and fibrinolytic pathways, and because thrombin promotes its own generation (feedback and cellular activation), thrombin is a primary target for therapeutics. Besides recombinant hirudins, Argatroban (Novastan) and Bivalirudin (Hirulog) are promising thrombin-directed inhibitors for antithrombotic intervention.

Understanding thrombin and hemostasis.

Unlike other factors in blood coagulation and fibrinolysis, thrombin has several functions in hemostasis from injury to recovery. Because of continual consumption, thrombin generation controls prethrombotic thrombin functions and may be prevented by inactivation of its precursors or by inhibition of thrombin-mediated amplification steps. The direct activation product of prothrombin, alpha-thrombin, not only converts fibrinogen into clottable fibrin but also is actively incorporated into the forming thrombus, where it is protected and transformed into other or inactive forms with thrombus maturation. Larger protein inhibitors, such as antithrombin III, cannot penetrate the thrombus, whereas hirudin and small thrombin inhibitors can. Unique structural features of thrombin allow the design and synthesis of a variety of small inhibitors. Such small inhibitors may prevent rethrombosis upon lysis of immature thrombi. On the other hand, such intervention must be used with caution, because low levels of thrombin appear to promote wound healing. In this regard, the scars of healing are but manifestations of the many functions of thrombin.

Laboratory evaluation of hemostatic disorders.

This article sketches a historical overview of milestones in diagnostic technologies to put into perspective a proposed role for laboratories in the diagnosis of coagulation disorders. Divided into five sections, the information presented in tables, illustrations, and appropriate vignettes for easy reference. The first section deals with the roles of coagulation testing in the management of bleeding and thrombotic disorders. Limitations of coagulation testing in defining the hemostatic state are discussed in the second section, and coagulation testing is reviewed in the third. In the fourth section interpretation of abnormal coagulation test results and the possible relationship to excessive bleeding and thrombosis are thoroughly discussed. The final section examines the contribution of gene analysis in establishing a diagnosis.

Gold labeling of cell monolayers grown on dextran beads.

Gold labeling of antigenic sites has become an increasingly useful tool in the study of cultured cell monolayers. If these monolayers are grown on flat substrates, major difficulties in both scanning (SEM) and transmission electron microscopy (TEM) specimen preparation and imaging may result. An alternate surface, that of dextran microcarrier beads, eliminates a majority of these difficulties and facilitates correlative TEM and SEM. The SEM procedure for using backscattered electron imaging requires the use of carbon planchets as the cell growth matrix to eliminate background signals. These planchets are expensive and are not an optimal cell-attachment matrix in that they result in loose and abnormally shaped cells. In contrast, the dextran beads were produced specifically for cell culture and, therefore, provide an excellent surface for growth. The beads have an average diameter of 100 microns, allowing attachment directly to aluminum stubs without signal generation from the aluminum to interfere with the gold signal. With TEM preparation, the monolayer poses the major disadvantage. Specimen preparation for thin sectioning is often preceded by extensive manipulation. In the microcarrier bead system, the beads are directly sectionable, and it is possible to cut five to eight full beads per thin section. This increase in cell surface makes quantification of gold labeling easier and also provides a more representative sampling of the monolayer. The ease of preparation, the decrease in reagents used (via cell pooling), and the ability to use one cell preparation for TEM and SEM make this procedure an ideal technique for gold labeling.

Efficacy of antithrombin III in endotoxin-induced disseminated intravascular coagulation.

Antithrombin III (AT III) is a major modulator of the clotting cascade and is decreased in disseminated intravascular coagulation (DIC). AT III was given as a pretreatment to dogs with endotoxin-induced DIC. Significant improvement in clotting parameters (prothrombin time, fibrinogen, fibrin degradation products) was noted. There was no effect on platelets. Mean arterial blood pressure was improved, while there were no other significant changes in other measured hemodynamic, acid-base, or biochemical variables. It was concluded that AT III was effective in ameliorating endotoxin-induced changes in the clotting profile. AT III may prove to be a beneficial therapy in acquired DIC.

Interaction of protamine sulfate with thrombin.

Protamine sulfate (salmine), a basic protein with a molecular weight of 4,626 +/- 109, is a known antiheparin agent which in the absence of heparin demonstrates an anticoagulant activity. To date, much work has been done to elucidate the interaction of heparin with thrombin and its physiologic inhibitor, Antithrombin III (ATIII). Little is known, however, about the mechanism of anticoagulant action of protamine sulfate and its mode of thrombin inactivation. We provide information about the interaction of protamine sulfate with purified, labeled thrombin and ATIII through binding experiments in which protamine is shown to inhibit the inactivation of thrombin by ATIII. Furthermore, we show in clotting assays that protamine sulfate has an inhibitory effect on thrombin in the conversion of fibrinogen to fibrin, and that this inhibition is concentration dependent, partial, and reversible.

Usefulness of new analytical reagents demonstrated with coumadin-modified plasma.

Prothrombin, factor X or factor IX were removed specifically from bovine plasma by immunological techniques. Each depleted plasma is responsive to the respective purified component, and was used to follow the blood changes produced in a steer's blood by Coumadin administration. A factor IX-VII reagent was also used to follow changes in factor IX and factor VII activity. It is suggested that the new reagents may have practical value for qualitative and quantitative work in blood coagulation studies.

Improved procedures for the purification of selected vitamin K-dependent proteins.

Improved methods are described to obtain bovine prothrombin, Factor IX, Protein C, and autoprothrombin III (Factor X, Auto-III) in purified form. The prothrombin had a specific activity of 4,340 Iowa units/mg. Theoretically, a preparation of clean thrombin should have a specific activity of 8,200 U/mg, because 47.08% of the protein in prothrombin is lost when thrombin forms. Such thrombin preparations have been obtained (Arch. Biochem. Biophys. 121, 372 (1967)). The prothrombin concentration of bovine plasma is near 60 mg/liter. Protein C, first isolated by Stenflo (J. Biol. Chem. 251, 355 (1976)), was found to be the precursor of autoprothrombin II-A (Auto-II-A), discovered earlier (Thromb. Diath. Haemorrh. 5, 218 (1960)). Protein C (Factor XIV) was converted to Auto-II-A (Factor XIVa) by thrombin. Digesting purified Auto-III with purified thrombin removed a small glycopeptide from the COOH-terminal end of the heavy chain to yield Auto-IIIm. Auto-III thrombin leads to Auto-IIIm + peptide. Auto-IIIm was not converted to the active enzyme with thromboplastin, and furthermore, inhibited the activation of purified native Auto-III with thromboplastin. Auto-IIIm was also not converted to the active enzymes when the procoagulants consisted of purified Factor VIII, purified Factor IXa, platelet factor 3 and calcium ions. The "activation peptide" released by RVV-X from the NH2-terminal end of the heavy chain and the active enzyme (Auto-Cm) were purified. Auto-III was also activated with purified RVV-X. The same "actid of Auto-Cm. Purified Factor IX developed anticoagulant activity when reacted with an optimum concentration of purified thrombin. A suitable reagent for the assay of Factor IX was prepared by removing prothrombin complex from anticoagulated bovine plasma and restoring the prothrombin and Auto-III concentration with use of the respective purified proenzymes.