General mechanisms of coagulation and their physiological inhibition. II. The regulation of coagulation by physiological inhibitors

Inopportune coagulation of blood in vessels is prevented by defense mechanisms, in which plasma inhibitors play an important role. The inhibitors are glycoproteins and belong to two different groups, according to their mechanism of action. The first group consists of the inhibitors of serine proteases, which form inactive complexes with various coagulation enzymes; it includes antithrombin III, heparin cofactor II, alpha 2-macroglobulin, alpha 1-antitrypsin and C1S-inhibitor. The second group includes protein C and its cofactor, protein S. Protein C, activated by thrombin complexed with a protein cofactor present on the endothelial cell surface (thrombomodulin), is responsible for the proteolytic degradation of two coagulation cofactors (Va and VIII: Ca). The clinical importance of both antithrombin III, protein C and protein S is attested by the strong association between recurrent venous thromboembolic manifestations and inherited deficiencies of one or the other of these proteins.     

Effect of heparin cofactor II on the antithrombin III activities measured by thrombin methods or factor Xa methods--fundamental studies and clinical studies using the plasma of pregnant women

We evaluated the effects of heparin cofactor II(HC II) on the antithrombin III(AT III) activities measured by the methods of thrombin or factor Xa. Reagents A and B were using the method of thrombin and reagent C was based on the method of Xa. Purified HC II was directly measured or indirectly measured after the dilution with control plasma. Cross reaction of HC II in AT III assay were negligible in reagent C, but substantial amount of AT III activities were measured in reagent A and B. Plasma AT III activities from full-term pregnant women were significantly higher than those from non pregnant control women in reagent A, but comparable in reagent B or C. These results indicate that AT III activities measured by thrombin methods by thrombin were overestimated in pregnant women due to the cross-reactivities of HC II. It is recommended that AT III activities would be measured by the methods of factor Xa.     

Coagulation under flow: the influence of flow-mediated transport on the initiation and inhibition of coagulation

A mathematical model of intravascular coagulation is presented; it encompasses the biochemistry of the tissue factor pathway, platelet activation and deposition on the subendothelium, and flow- and diffusion-mediated transport of coagulation proteins and platelets. Simulation experiments carried out with the model indicate the predominant role played by the physical processes of platelet deposition and flow-mediated removal of enzymes in inhibiting coagulation in the vicinity of vascular injury. Sufficiently rapid production of factors IXa and Xa by the TF:VIIa complex can overcome this inhibition and lead to formation of significant amounts of the tenase complex on the surface of activated platelets and, as a consequence, to substantial thrombin production. Chemical inhibitors are seen to play almost no (TFPI) or little (AT-III and APC) role in determining whether substantial thrombin production will occur. The role of APC is limited by the necessity for diffusion of thrombin from the site of injury to nearby endothelial cells to form the thrombomodulin-thrombin complex and for diffusion in the reverse direction of the APC made by this complex. TFPI plays an insignificant part in inhibiting the TF:VIIa complex under the conditions studied whether its action involves sequential binding of TFPI to Xa and then TFPI:Xa to TF:VIIa, or direct binding of TFPI to Xa already bound to the TF:VIIa complex.     

Formation and regulation of platelet and fibrin hemostatic plug

Formation of a hemostatic plug represents one of the earliest responses to vessel wall injury. Platelets react to any discontinuity in the vascular endothelium through initial contact, spreading, and formation of a thrombus (or aggregate). This development of a primary hemostatic plug requires platelet membrane receptors through which the adhesive macromolecules, von Willebrand factor (vWF) and fibrinogen, anchor platelets to the vessel wall and link them to each other. There are two receptor pathways--classic and alternative--for the binding of vWF to platelets; the latter induced by thrombin, and adenosine diphosphate (ADP) is shared with fibrinogen. Synthetic peptides, patterned after known binding domains of adhesive molecules, have been designed to inhibit their interactions with platelet receptors. A secondary hemostatic plug, composed of platelets enmeshed in fibrin, results from the action of thrombin, which is not only essential for formation of fibrin but also for exposure of platelet receptors for adhesive molecules and for "activation" of factors V and VIII. Thrombin generation is greatly enhanced through the activity of the prothrombinase complex formed on the surface of platelets, perturbed endothelial cells, and leukocytes. A pivotal event is activation of factor X through the intrinsic and extrinsic coagulation pathways. Binding of factors IXa and VIIa to the vascular endothelium represents a localized mechanism for factor Xa generation. Formation of a platelet and fibrin thrombus is controlled by regulatory mechanism: prostacyclin, endogenous heparin-antithrombin III complex, thrombomodulin-protein C-protein S system, and the fibrinolytic system. The balance of all components--vessel wall, platelets, adhesive and coagulation proteins, regulatory mechanisms--determines the effectiveness of the hemostatic plug in maintaining the structural and functional integrity of the circulatory system. An approach to detection of hemostatic derangements in patients at risk evolves from a full understanding of inherited and acquired deficiencies affecting each step of hemostatic plug formation and from selective use of laboratory tests.     

Abnormal formation of the prothrombinase complex: factor V deficiency and related disorders

A membrane-bound, Ca2-dependent complex of the cofactor factor Va and the enzyme factor Xa comprises the prothrombinase coagulation complex, which catalyzes the proteolytic conversion of prothrombin to thrombin. In normal hemostasis, the platelet is presumed to supply the surface membrane and thus constitutes the site at which an enzymatically functional complex assembles and thrombin generation occurs. Factor Va, the two subunit protein produced by thrombin activation of factor V, is an essential, nonenzymatic cofactor of the prothrombinase complex. Factor Va performs its cofactor role in part by binding to the platelet membrane and functioning as the membrane receptor for factor Xa in a 1:1 stoichiometric complex of high affinity (Kd = 10(-10) M). Factor Va also appears to participate in the binding of prothrombin to the enzymatic complex. Because deletion of factor Va from the prothrombinase complex decreases the rate of thrombin generation by four orders of magnitude, the essential role it plays is easily understood. Therefore, in the evaluation of factor Va function in the prothrombinase complex, the ability of factor Va to support various binding interactions with the platelet, factor Xa, and prothrombin must be considered. Factor Va can be made available from two potential blood compartments: the plasma and platelets. Approximately 80 per cent of the total blood factor V circulates in plasma whereas the remaining 20 per cent is contained within platelet granules. The relative contribution of plasma versus platelet factor V to factor Va binding interactions in the prothrombinase complex are not clearly defined. However, data from our laboratory and several others suggest that factor V stored and released from platelets is of utmost importance in maintaining normal hemostasis. A discussion of these data relative to congenital and acquired deficiencies of both plasma and platelet factor V is the subject of this report.     

Multifunctional roles of thrombin.

Thrombin is an unique molecule that functions both as a procoagulant and anticoagulant. In its procoagulant role it activates platelets through its receptor on the platelets. It regulates its own generation by activating coagulation factors V, VIII and even XI resulting in a burst of thrombin formation. It activates factor XI, thus preventing fibrin clots from undergoing fibrinolysis. Thrombin not only cleaves fibrinogen to fibrin, but also through the activation of factor XIII effects the cross-linking of fibrin monomers to produce a firm fibrin clot. Thrombin's role as an anticoagulant is mediated through binding to thrombomodulin, a receptor protein on the endothelial membrane of the blood vessel, initiating a series of reactions that leads to fibrinolysis. Thrombin has chemotactic properties enabling it to exert its effects during inflammation and vascular injury. It has a mitogenic effect stimulating growth of mammalian cells, fibroblasts and macrophage-like tumor cell lines. It has also been implicated in brain development. A molecule with multifunctional roles such as thrombin has its activity in vivo modulated by only a few endogenous inhibitors.     

Antithrombin III. Theory and clinical applications. H. P. Smith Memorial Lecture.

Antithrombin III is one of the main inhibitors in the blood coagulation mechanisms. Thrombin and factor Xa are slowly inactivated by it, as well as other serine proteinases of the coagulation mechanisms. Heparin tremendously accelerates the inhibitory function of antithrombin III. In the process antithrombin III activity is also reduced. Heparin retards the thrombin-fibrinogen reaction, but otherwise the effectiveness of heparin as an anticoagulant depends on antithrombin III in laboratory experiments, as well as in therapeutics. The activation of prothrombin is inhibited, and any thrombin or other vulnerable protease that might generate becomes inactivated. The measurement of antithrombin III concentration in blood is now achieved by research methods, as well as by methods that are practical for routine use. The tests require either thrombin or factor Xa as substrate, and could be specific for antithrombin III. There are congenital as well as acquired deficiencies of antithrombin III. The inhibitor is also found in tissues.     

A mathematical model of thrombin production in blood coagulation,Part I: The sparsely covered membrane case

This paper presents the first attempt to model the blood coagulation reactions in flowing blood. The model focuses on the common pathway and includes activation of factor X and prothrombin, including feedback activation of cofactors VIII and V by thrombin, and plasma inhibition of factor Xa and thrombin. In this paper, the first of two, the sparsely covered membrane (SCM) case is presented. This considers the limiting situation where platelet membrane binding sites are in excess, such that no membrane saturation or binding competition occurs. Under these conditions, the model predicts that the two positive feedback loops lead to multiple steady-state behavior in the range of intermediate mass transfer rates. It will be shown that this results in three parameter regions exhibiting very different thrombin production patterns. The model predicts the effect of flow on steady-state and dynamic thrombin production and attempts to explain the difference between venous and arterial thrombi. The reliance of thrombin production on precursor procoagulant protein concentrations is also assessed.     

Mechanism of factor VIII inactivation by human antibodies. III. Proteolytic cleavage of factor VIII:C and C antigen by thrombin

Thrombin activation of factor VIII results in a marked, but transient, increase in factor VIII procoagulant activity. This proteolytic process has been examined by immobilizing factor VIII in a solid phase system using mouse monoclonal antibody specific for the factor VIII related antigen. These studies demonstrate that thrombin activation is the result of proteolytic cleavage from the factor VIII:C protein of a peptide fragment which contains both the factor VIII:C procoagulant and VIII:C antigen sites recognized by human antibodies.     

Effect of heparin on chronically induced intravascular coagulation in dogs.

When intermediate-strength thromboplastin was continuously infused into dogs for 10 days or more, platelet counts decreased sharply and factor VIII concentrations decreased by more than 50%. There was little change in plasma fibrinogen, prothrombin, factor V, antithrombin III, plasminogen, prothrombin time, and thrombin time values. When heparin was infused (25-50 U/kg per h) along with the same thromboplastin, there was no change in onset or degree of thrombocytopenia. However, the decrease in factor VIII was abolished and there were significant increases in fibrinogen, prothrombin, and factor V. The absolute concentrations of the various clotting factors seemed to give no indication of their turnover rates. Unexplained is the remarkable heparin tolerance that developed in these dogs.     

What is new in antithrombotic treatment?

Real progresses have been made during the past years in the comprehension of hemostasis mechanisms, along with rising of new antithrombotic drugs. The later include: 1) direct inhibitors of thrombin such as hirudin, bivalirudin, argatroban, melagatran and ximelagatran; 2) inhibitors of factor Xa such as the synthetic pentasaccharid and DX-9065a; 3) inhibitors of factor IXa; 4) inhibitors of tissue factor-factor VIIa complex such as tissue factor pathway inhibitor (TFPI) or NAPc2 (nematode anticoagulant peptide); 5) drugs enhancing endogenous anticoagulant activity, such as protein C or activated protein C; 6) drugs modulating endogenous fibrinolytic activity. These new drugs are promising a real decrease in mortality and morbidity due to venous thrombo-embolic disease, which is considered as a public health issue. Both physicians and biologists are concerned by these new antithrombotic agents, the former to think about new treatment strategies, the later to monitore, if necessary, the effects of such new drugs. Our review does not include antiplatelet agents which are indicated only in arterial thrombosis.     

The role of complex formation and epidermal growth factor-like domains in the regulation of blood coagulation by the thrombomodulin-protein C system

The explosive nature of the coagulation cascade led many scientists to investigate how it is regulated. Proteinase inhibitors such as antithrombin III inhibit active proteases of the coagulation cascade. Cofactors such as factor Va and factor VIIIa are proteolytically inactivated by activated protein C. Protein C is activated by the thrombin-thrombomodulin complex on the endothelial cell surface. Thus, the independent actions of the proteinase inhibitor system and the thrombomodulin-protein C system complement each other to maintain regulation of blood coagulation. The thrombin binding site of thrombomodulin was identified to be the fifth and sixth repeats of the epidermal growth factor-like domain. The same binding template contains sufficient information to block the functions of thrombin as a procoagulant. However, additional repeats are required for the activation of protein C. Thrombomodulin is the first example which illustrates that the epidermal growth factor-like domain functions as a binding template for thrombin and as a switch to turn off the procoagulant activity of thrombin as well as to trigger the protein C anticoagulant pathway. Epidermal growth factor-like structures are found in many of the coagulation factors. Complex formation is a repeated theme not only in the blood coagulation cascade but also in the thrombomodulin-protein C anticoagulant pathway.     

The role of endothelium in the homeostatic balance of haemostasis

As the cells forming the luminal vascular surface, endothelial cells are strategically positioned to play an important role in the regulation of coagulation. They cannot be regarded as an inert surface lining the vessel wall since they possess multiple activities. Anticoagulant properties include provision of a cell surface with heparin-like molecules (which can serve as binding sites for antithrombin III), synthesis of thrombomodulin (which alters the substrate specificity of thrombin), maintenance of a low level of tissue factor and generation of prostacyclin. In addition to these anticoagulant properties, endothelial cells can play a role in procoagulant reactions. Studies which have examined mechanisms underlying the localization of thrombotic processes have suggested the possible involvement of endothelial cells in procoagulant events. Endothelial cells have been found to propagate factor X and prothrombin activation once factor IXa and factor Xa have been formed. Factors regulating the balance of plasminogen activator and inhibitor synthesis are also under study. Perturbation of endothelial cells with induction of tissue factor and production of platelet-activating factor and thromboxane provides a model of the thrombotic state in which endothelium can promote coagulation. The multiple properties of endothelial cells indicate that a continuous blood flow in an unperturbed region of the vessel wall results from a complex interplay of anticoagulant and procoagulant activities. In a perturbed region, endothelial cells might initiate coagulation and the thrombin formed on the surface of endothelial cells might then lead to recruitment of platelets.     

Anticoagulant mechanism of sulfonated polyisoprenes.

The influence of sulfonated polyisoprene (SPIP) on coagulation factors and human blood cells was investigated to elucidate and compare its anticoagulant mechanism with that of heparin. While the number of red cells was unaffected, the number of platelets decreased dramatically in the presence of SPIP due to aggregation. Using a synthetic peptide substrate to assay thrombin activity in the presence of its natural inhibitor, antithrombin (AT), we observed no stimulation by SPIP of AT-mediated inhibition. Nevertheless, thrombin cleavage of its natural substrate fibrinogen to fibrin peptide A was slightly inhibited. SPIP altered the electrophoretic mobility of fibrinogen and completely inhibited fibrinogen from clotting. We detected no significant influence of SPIP on factors II, VII, IX, and X, while factor XI and factors V and VIII were only slightly affected. Therefore, the main mechanism of SPIP's anticoagulant activity appears to be a strong interaction with fibrinogen and fibrin monomer, first, to prevent proteolytic conversion of the former to the latter and second, to inhibit polymerization of the fibrin monomer, once formed.     

Evaluation of thrombin in urine as a real-time indicator of clotting activation in glomerulonephritis

When tissues are injured and bleeding occurs, blood clotting is immediately activated and fibrin clots are formed by thrombin. Afterwards, antithrombin III promptly inactivates thrombin, which restricts the clotting to the bleeding site. In inflamed sites, tissue factor is expressed on cells in the lesion by stimulation from cytokines, and produces thrombin. In this case, thrombin may survive longer because of inefficient inactivation by antithrombin III due to dilution and less perturbation in the interstitial fluid, and therefore, has a greater chance to activate thrombin receptors (protease-activated receptors: PARs) on the cells, which induces various cellular events including proliferation, migration, and shape change. Recent studies have suggested a pathophysiological association of the PAR pathway with crescentic glomerulonephritis. However, the role of thrombin in human diseases has not been fully studied, probably because of a lack of simple and reliable methods for measuring thrombin in clinical samples. To solve this problem, we developed an ELISA system for human alpha-thrombin and applied it to the measurement of thrombin in the urine of patients with glomerulonephritis. Thrombin in urine was detected in glomerulonephritic patients but not in healthy volunteers or disseminated intravascular coagulation patients, which suggests that thrombin in urine may reflect thrombin generation by clotting activation in the glomerular lesion.     

Factor V Leiden: association with venous thromboembolism in pregnancy and screening issues

Disturbances of the natural balance between procoagulant and anticoagulant mechanisms can result in bleeding or thrombotic tendencies. Factor V, on activation by thrombin to factor Va, forms an essential component of the prothrombinase complex, in which it demonstrates its cofactor activity for factor Xa. Down-regulation of factor Va by activated protein C (APC) occurs through cleavage of specific peptide bonds in the heavy chain of the molecule. Factor V Leiden (FV Leiden) is a mutation of factor V that renders factor Va resistant to APC, due to loss of one of these cleavage sites. This mutation predisposes the patient to thrombosis. Prevalence of FV Leiden varies; however, heterozygosity for the FV Leiden mutation is recognised as the most common heritable thrombophilic defect in Caucasian populations. The association this inherited thrombophilia has with venous thromboembolism (VTE) is well established. Pregnancy is notably an acquired hypercoagulable state, due in part to physiological changes that occur in the coagulation system. This seems to have potential for interaction with FV Leiden to cause adverse experiences. A role has been suggested for FV Leiden in VTE events during pregnancy. At present only selected women are screened for FV Leiden. Pregnant women with a history of VTE or with a family history of the mutation are investigated. Whether or not the introduction of a routine screening plan for this mutation is justified remains a matter for debate.     

Sulfated Sericin is a Novel Anticoagulant Influencing the Blood Coagulation Cascade

Sulfated sericin's influence on factors in the blood coagulation cascade was investigated to elucidate its anticoagulant mechanism. Prolongation of the prothrombin time (PT) and activated partial thromboplastin time (APTT) were observed in the presence of sulfated sericin. Fluorogenic peptide substrates on thrombin (factor IIa) and factor Xa were used to study the influence of sulfate sericin on their respective activities. Sulfated sericin inhibited neither thrombin nor factor Xa in the presence of antithrombin III (AT III). Gel electrophoresis was used to examine fibrinogen–fibrin conversion by thrombin in the presence of sulfated sericin. FPA and FPB release from fibrinogen by thrombin proceeded in the presence of sulfated sericin. The behavior of polymerization of fibrin monomer (FM) was affected by the presence of sulfated sericin. No initial lag time in the polymerization process was observed by addition of sulfated sericin to FM. This result means that sulfated sericin will interfere in the build-up of normal double-strand fibrils of FM during formation of fibrin fiber. Scanning electron microscopy (SEM) observations supported this inference. The anticoagulant mechanism of sulfated sericin is inferred to interfere with the initial polymerization process of FM.     

Vascular protease receptors: integrating haemostasis and endothelial cell functions

The endothelium plays a crucial dynamic role as a protective interface between blood and the underlying tissues during the haemostatic process, which maintains blood flow in the circulation and prevents life-threatening blood loss. Following vessel wall injury with initial platelet adhesion and aggregation to exposed subendothelial extracellular matrix, the initiation, amplification, and control of haemostasis depend on structurally unrelated membrane-associated receptors for blood coagulation proteases including tissue factor, G-protein-coupled protease-activatable receptors, thrombomodulin, and protein C receptor, respectively. In addition to their regulatory role in haemostasis, the respective (pro-)enzyme ligands such as Factors VIIa and Xa, thrombin or protein C mediate specific signalling pathways in vascular cells related to migration, proliferation or adhesion. The functional importance of these receptors beyond haemostasis has been manifested by various lethal and pathological phenotypes in knock-out mice. These protease receptors thereby provide important molecular links in the vascular system and serve to integrate haemostasis with endothelial cell functions which are relevant for the (patho-)physiological responses to injury or inflammatory challenges.     

Inherited abnormalities in the protein C activation pathway

The protein C (PC) anticoagulant pathway plays a crucial role in the regulation of fibrin formation via proteolytic degradation of the procoagulant cofactors factor Va and VIIIa by activated PC (APC). PC circulates in plasma as a zymogen, which is activated, on the surface of endothelial cells by the thrombin-thrombomodulin complex. Another endothelial cell-specific protein, the endothelial cell PC/APC receptor (EPCR), binds PC on the endothelial cell surface and further enhances the rate of PC activation. Normal APC generation depends on the precise assemblage, on the surface of endothelial cells, of at least four proteins: thrombin, thrombomodulin (TM), PC and EPCR. Therefore, any change in the efficiency of this assemblage may cause reduced APC generation and an increase in the risk of thrombosis. In the last years, several reports have suggested the association between mutations in TM and EPCR genes and venous and arterial thrombosis. Surprisingly, no studies have been reported linking mutations with levels of circulating APC, the final product of the interaction between thrombin, TM, PC and EPCR. Here, we describe the previously reported mutations in the TM and EPCR genes, and present the design and evaluation of a new strategy to investigate TM, EPCR, PC and prothrombin gene mutations in arterial and venous thrombosis.     

Thrombin Regulates Chemokine Induction during Human Retinal Pigment Epithelial Cell/Monocyte Interaction

Thrombin, an important clotting factor, extravasates at sites of blood-retina barrier breakdown that is often associated with many retinal diseases. Here we investigated the effects of thrombin on human retinal pigment epithelial (HRPE) cells, monocytes, and HRPE cell/monocyte co-cultures. Thrombin induced secretion and mRNA expression of HRPE interleukin (IL)-8 and monocyte chemoattractant protein-1 (MCP-1). Thrombin also enhanced IL-8 and MCP-1 by HRPE cell/monocyte co-cultures, by apparently enhancing cell-cell contact mechanisms. The thrombin effects on IL-6 secretion were similar to those on chemokine secretion. Thrombin-induced chemokines by co-cultures were inhibited by anti-tumor necrosis factor-alpha (TNF-alpha) antibody, but not by anti-IL-1beta antibody. TNF-alpha was detected in cell lysates of monocytes detached from HRPE cells after co-culture stimulation with thrombin. HRPE cells mainly produced these chemokines. However, thrombin generally potentiated exogenous IL-1beta- and TNF-alpha-induced chemokine production by HRPE cells, monocytes, and co-cultures. Interferon-gamma potentiated chemokine secretion by co-cultures with or without thrombin. Our results indicate that thrombin may cause leukocyte recruitment by inducing HRPE cell and monocyte chemokine and by enhancing HRPE cell/monocyte interactions, in part because of monocyte TNF-alpha induction, suggesting important mechanisms for ocular inflammation during blood-retina barrier breakdown and intra-ocular hemorrhage.