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.     

In vitro blood compatibility of polymeric biomaterials through covalent immobilization of an amidine derivative

We present a surface coating with anticoagulant characteristics showing significantly reduced coagulation activation. The synthesis of a monomeric conjugate containing a benzamidine moiety was carried out and its inhibitory activity against human thrombin, the key enzyme of the blood coagulation cascade, was determined using a chromogenic assay. Based on that, low-thrombogenic interfaces were prepared by covalent attachment of this low-molecular weight thrombin inhibitor on poly(octadecene-alt-maleic anhydride) copolymer thin films and characterized using ellipsometry, XPS and dynamic contact angle measurements. The in vitro hemocompatibility tests using freshly drawn human whole blood showed, in agreement with the SEM images, that a PO-MA film modified with a benzamidine moiety using a PEG spacer decreased the activation of coagulation, platelets and the complement system. The decreased protein adsorption, in addition to the specific inhibition of thrombin, effectively enhanced the short-term hemocompatibility characteristics.     

Thrombin: a multifunctional enzyme

Thrombin is the final enzyme of blood coagulation cascade. It belongs to the trypsin family of serine proteases. Its two primary actions are to cleave fibrinogen to release fibrin and to activate platelets through a limited proteolysis of a specific receptor. In addition, thrombin is the major regulator of blood coagulation. It is both a procoagulant enzyme in the activation of factors V and VIII, and an anticoagulant enzyme through the activation of protein C and TAFI. This multi-functionality of thrombin depends upon the conformation of its active site: depth for high specificity and shape for a finely tuned selection of substrates. Since new anticoagulant molecules, some with anti-thrombin activity, are emerging, it is important to understand the mechanisms allowing thrombin to be so specifically multifunctional.     

Thrombin generation and mortality during Staphylococcus aureus sepsis

Sepsis-induced abnormalities of coagulation may contribute to mortality during severe bacterial infection. The aim of this study was to examine changes in coagulation parameters and to assess the role of protein C supplementation during murine S. aureus sepsis. Gram-positive sepsis was characterized by a hypercoagulable state with predominant activation of the external coagulation pathway, registered as an early increase of tissue factor activity and concomitant reduction in protein C. The internal coagulation pathway was unaffected. No correlation between the changes of coagulation parameters and the intensity of inflammation, determined as serum IL-6 levels, was found. Supplementation with neither protein C or APC favoured survival in S. aureus sepsis. Reduction in thrombin generation in response to protein C supplementation was associated with significantly increased survival.     

Mechanical circulatory device thrombosis: a new paradigm linking hypercoagulation and hypofibrinolysis

This review considers the perhaps unappreciated role of contact pathway proteins in the pathogenesis of thrombotic/thromboembolic morbidity associated with mechanical circulatory support. Placement of ventricular assist devices (VADs) has been associated with consumption of circulating contact proteins and persistent generation of activated contact proteins such as Factor XII and high molecular weight kininogen. Importantly, activated contact proteins are absorbed to the surface of VADs via the Vroman effect. Further, hyperfibrinogenemia and persistent platelet activation exist in patients with VADs, likely contributing to speed of clot growth. Using thrombelastographic-based analyses, it has been determined that contact pathway protein activated coagulation results in a thrombus that develops strength at a significantly faster rate that tissue factor initiated coagulation. Further, thrombelastographic analyses that include the addition of tissue-type plasminogen activator have demonstrated that contact protein pathway activation results in thrombin activatable fibrinolysis inhibitor activation to a far greater extent than that observed with tissue factor initiated coagulation, resulting in a thrombus that takes significantly longer to lyse. These observations serve as the rational basis for clinical investigation to determine if regional suppression of thrombin generation with FXII/high molecular weight kininogen inhibition in concert with thrombin-activatable fibrinolysis inhibitor inhibition may decrease mechanical circulatory support-associated thrombotic morbidity.     

A new in vitro model to study interaction between whole blood and biomaterials. Studies of platelet and coagulation activation and the effect of aspirin

We have developed a versatile in vitro chamber model with a double purpose: first, to be able to study mechanisms of bio-incompatibility, and, second, to test biomaterials at all levels of interactions, in whole blood. The use of biomaterials in the form of microscope slides as walls in the chamber makes it possible to analyse both the biomaterial surface with regard to protein and cell binding, as well as the molecular events taking place in the fluid. Incubation of blood in the chamber, for 60 min at 37 degrees C resulted in the rapid binding of complement and coagulation proteins and of leukocytes and platelets to polyvinylchloride (PVC) slides. The cells formed a layer which more or less covered the underlying surface. Unlike complement activation, as reflected by soluble C3a and C5b-9, the thrombin-antithrombin formation was completely nullified in cell-depleted plasma. Despite the fact that thrombin-antithrombin generation was also negligible in platelet-rich plasma, inhibition of platelet aggregation on the material surface with aspirin resulted in suppressed generation of thrombin antithrombin complexes. Taken together, the coagulation activation in the chamber was dependent on the presence of blood cells which suggests that bound/aggregated platelets initiate a sequence of events involving leukocytes that results in coagulation activation.     

The roles of thrombin and protease-activated receptors in inflammation

Inflammation and coagulation constitute two host defence systems with complementary physiological roles in limiting tissue damage, restoring homeostasis and eliminating invading pathogens, functions reliant on effective regulation of both processes at a variety of levels. Dysfunctional activation or regulation of either pathway may lead to pathology and contribute to human diseases as diverse as myocardial infarction and septic shock. The serine protease thrombin, a key protein in the coagulation pathway, can activate cellular signalling directly via proteolytic cleavage of the N-terminal domain of a family of G protein-coupled receptors or indirectly through the generation of molecules such as activated protein C. These events transmit signals to many cell types and can elicit the production of various pro-inflammatory mediators such as cytokines, chemokines and growth factors, thereby influencing cell activation, differentiation, survival and migration. This review discusses recent progress in understanding how thrombin and protease-activated receptors influence biological processes, highlighting the detrimental and protective cellular effects of thrombin and its signalling pathways.     

Sm22.6 antigen is an inhibitor to human thrombin

The physiological role of Schistosomiasis mansoni 22.6 antigen (sm22.6 Ag) and its pathogenic effect on the human host has never been reported. Recombinant sm22.6 Ag is a homogenous polymer under non-denaturing/non-reducing conditions, and an inhibitor to human thrombin. Kinetic and Western blot assays show that the recombinant protein interacts with human thrombin and inhibits proteolytic activity of the protease. Tests of whole blood revealed that coagulation time was significantly delayed (3-5 times longer) in the presence of the recombinant protein at a concentration similar to thrombin in normal blood samples. Kinetic studies revealed that the delayed coagulation time was due to the inhibition of alpha-thrombin proteolytic activity by the parasite protein in an irreversible pattern, and a reversible inhibition to gamma-thrombin. Also, Western blot analysis under non-denaturing/non-reducing conditions showed that sm22.6 Ag binds to both alpha- and gamma-thrombin. Our results strongly suggest that sm22.6 antigen plays a role in down-regulation of coagulation in humans.     

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.     

Crosstalk between inflammation and thrombosis

Inflammation shifts the hemostatic mechanisms in favor of thrombosis. Multiple mechanisms are at play including up regulation of tissue factor leading to the initiation of clotting, amplification of the clotting process by augmenting exposure of cellular coagulant phospholipids, inhibition of fibrinolysis by elevating plasminogen activator inhibitor 1 (PAI-1) and decreases in natural anticoagulant pathways, particularly targeted toward down regulation of the protein C anticoagulant pathway through multiple mechanisms. The decreased function of the natural anticoagulant pathways may be particularly problematic because these appear to play a role in dampening inflammatory responses. The protein C anticoagulant pathway provides a useful model for the impact of inflammation on coagulation. This pathway plays a major role in preventing microvascular thrombosis. The pathway is initiated when thrombin binds to thrombomodulin (TM) on the surface of the endothelium. An endothelial cell protein C receptor (EPCR) augments protein C activation by the thrombin–TM complex more than 10-fold in vivo. EPCR is shed from the endothelium by inflammatory mediators and thrombin. EPCR binds to activated neutrophils in a process that involves proteinase 3 and Mac-1 and appears to inhibit leukocyte extravisation. EPCR can undergo translocation from the plasma membrane to the nucleus where it redirects gene expression. During translocation it can carry activated protein C (APC) to the nucleus, possibly accounting for the ability of APC to modulate inflammatory mediator responses in the endothelium. TNF and other inflammatory mediators can down-regulate EPCR and TM and IL-6 can depress levels of protein S in experimental animals. Inhibition of protein C pathway function increases cytokine elaboration, endothelial cell injury and leukocyte extravisation in response to endotoxin, processes that are decreased by infusion of APC. In vitro, APC inhibits TNF elaboration from monocytes and to block leukocyte adhesion to selectins. Since thrombin can elicit many inflammatory responses in microvascular endothelium, loss of control of microvascular thrombin generation due to impaired protein C pathway function probably contributes to microvascular dysfunction in sepsis.     

Formation of the fibrin clot: the balance of procoagulant and inhibitory factors

Let us now briefly summarize some major known regulating mechanisms, most of which have already been discussed. A general regulating feature of the coagulation system is provided by the cofactors HMW-kininogen, tissue factor, factor V(a), factor VIII:C(a), protein S and thrombomodulin. Tissue factor and thrombomodulin, as cell membrane constituents, and the other cofactors, thanks to their affinity for certain surface sites, localize coagulation reactions and thus avoid generalized intravascular thrombosis when the clotting system is triggered. Thrombin activates factors V and VIII:C and activated protein C inactivates factors Va and VIII:Ca. Thrombin is regulated by AT III, alpha 2M and possibly heparin-cofactor II, whereby endothelial-cell-bound heparin-like molecules enhance thrombin neutralization. Moreover, binding of thrombin to thrombomodulin abolishes its clotting activity, at least in the case of rabbit thrombomodulin. Thrombin is able to cleave PT-fragment 1 from prothrombin, thus generating prethrombin 1, which lacks the gla-region and does not bind to phospholipids. The hypothesis that thrombin may regulate its own formation by this negative feedback, however, must probably be discarded, because no corresponding fragments are found after blood clotting in vitro (Aronson et al, 1977). Factor Xa and factor IXa are inhibited by AT III and endogenous heparin probably enhances their inactivation. However, phospholipid-bound factor Xa in the presence of factor Va (Marciniak, 1973) and phospholipid-bound factor IXa (Varadi and El?di, 1982) are relatively protected from inhibition. Platelet-bound factor Xa is completely protected from AT III, even in the presence of heparin (Miletich et al, 1978). Thus, specific cell surface sites modulate the inhibition of proteases in situ. Factor XIa is inhibited by several protease inhibitors, the most important being alpha 1-AT. beta-factor XIIa is inhibited mainly by C1-inhibitor and kallikrein by both C1-inhibitor and alpha 2M. No serine protease inhibitor for factor VIIa is as yet known. However, after initial rapid activation by factor Xa, factor VIIa is subsequently proteolytically inactivated by factor Xa, resulting in a transient burst of factor Xa generation by factor VIIa (Morrison and Jesty, 1984). This proteolytic regulation of factor VIIa by factor Xa dampens factor IX or factor X activation via tissue factor-factor VIIa by feedback proteolytic inhibition and this may constitute a major regulatory mechanism for factor VIIa.(ABSTRACT TRUNCATED AT 400 WORDS)     

Thrombomodulin: a new proteoglycan. Structure-function relation]

The endothelial cell surface receptor thrombomodulin (TM) displays various anticoagulant functions: it acts as a cofactor for the activation of protein C (PC) by thrombin, prevents the activation of fibrinogen, platelets and Factor V by thrombin. TM was also shown to accelerate the inhibition of thrombin by its physiological inhibitor antithrombin III (ATIII). The studies performed on rabbit lung TM were undertaken in order to provide better understanding, along with the identification and the characterization of functional domains, to the mechanism of action of TM. On the basis of the physical and chemical properties of TM, which were compatible with those of a proteoglycan, the presence of a sulfated polysaccharide chain covalently bound to TM, constituting an acidic domain independent of the protein C activation cofactor site, was suggested. Further enzymatic and chemical characterization showed that rabbit TM was in fact a chondroitin sulfate proteoglycan. Monoclonal antibodies raised against rabbit TM and proteins known for their ability to neutralize the activity of heparin, as well as TM submitted to chondroitinase digestion were used in order to identify the role of the different structural domains of TM. Binding of thrombin to TM at a primary site on the protein part is a prerequisite for all the biological activities of TM. However, while this binding is sufficient for TM to promote the activation of PC by thrombin, the inhibition by TM of thrombin-induced fibrinogen clotting and factor V activation requires the interaction of thrombin at a secondary site with the polysaccharide chain of TM. This interaction with the polysaccharide chain (which carries a highly sulfated trisaccharide at the non-reducing terminus) leads to the inhibition of the procoagulant functions of TM-bound thrombin towards fibrinogen and factor V as well as an increased reactivity of the enzyme with ATIII. These results were rationalized in the functional model proposed for the rabbit TM-proteoglycan. An original aspect of the TM-proteoglycan resides in the fact that the chondroitin sulfate side chain brings new anticoagulant activities, in addition to the PC activation cofactor activity, to the molecule. TM is a new type of proteoglycan with important regulatory function in hemostasis, which anticoagulant properties depend on both the protein core and the polysaccharide chain.     

New insight into the effects of heparinoids on complement inhibition by C1‐inhibitor

Complement activation is of major importance in numerous pathological conditions. Therefore, targeted complement inhibition is a promising therapeutic strategy. C1‐esterase inhibitor (C1‐INH) controls activation of the classical pathway (CP) and the lectin pathway (LP). However, conflicting data exist on inhibition of the alternative pathway (AP) by C1‐INH. The inhibitory capacity of C1‐INH for the CP is potentiated by heparin and other glycosaminoglycans, but no data exist for the LP and AP. The current study investigates the effects of C1‐INH in the presence or absence of different clinically used heparinoids on the CP, LP and AP. Furthermore, the combined effects of heparinoids and C1‐INH on coagulation were investigated. C1‐INH, heparinoids or combinations were analysed in a dose‐dependent fashion in the presence of pooled serum. Functional complement activities were measured simultaneously using the Wielisa^®‐kit. The activated partial thrombin time was determined using an automated coagulation analyser. The results showed that all three complement pathways were inhibited significantly by C1‐INH or heparinoids. Next to their individual effects on complement activation, heparinoids also enhanced the inhibitory capacity of C1‐INH significantly on the CP and LP. For the AP, significant potentiation of C1‐INH by heparinoids was found; however, this was restricted to certain concentration ranges. At low concentrations the effect on blood coagulation by combining heparinoids with C1‐INH was minimal. In conclusion, our study shows significant potentiating effects of heparinoids on the inhibition of all complement pathways by C1‐INH. Therefore, their combined use is a promising and a potentially cost‐effective treatment option for complement‐mediated diseases.     

Protein C, protein S

Protein C is a potent inhibitor of blood coagulation, and, in addition, appears to be a profibrinolytic agent. In a first step, protein C must be converted to a serine protease. This activation is catalyzed by a complex formed between thrombin and thrombomodulin, an endothelial cell surface protein. Activated protein C exhibits its anticoagulant activity through the proteolytic inactivation of two blood coagulation cofactors, factors Va and VIIIa. This reaction requires phospholipids, originating from platelets or endothelial cells, and a cofactor protein, protein S. Protein S enhances the binding of activated protein C to phospholipids. In addition, activated protein C stimulates fibrinolysis, through the inactivation of the tissue plasminogen activator (tPA) inhibitor. An isolated constitutional, quantitative or qualitative, protein C or protein S deficiency increases the risk of thrombosis, the clinical features are different in the rare cases of homozygous protein C deficiency (neonatal purpura fulminans) or in the heterozygous patients (recurrent venous thrombosis in young adults). Acquired deficiency in protein C and S had been observed in liver disease, during vitamin K antagonists or L-Asparaginase treatment, and in disseminated intravascular coagulation.     

The second cofactor of heparin

Heparin cofactor II is a plasma protein that inhibits thrombin rapidity in the presence of heparin. It is definitely distinct from antithrombin III by immunological and physicochemical criteria, protease specificity and glycosaminoglycan specificity. HC II inhibits thrombin (but not the other proteases of the coagulation or fibrinolysis), chymotrypsin and chymotrypsin-like enzymes. Dermatan sulfate and pentosan sulfate but not heparin sulfate increase the rate of thrombin inhibition by HC II. The physiological role of HC II is presently unknown. II is likely that its antithrombin activity in vivo is restricted to the areas rich in dermatan sulfate. An additional role may be related to the inhibition of various chymotrypsin-like proteases involved in protein activation or degradation in the tissues. So far, there is no clinical evidence for a physiological role of HC II. Vascular thrombosis associated to constitutional deficiency in HC II have been reported in several instance but epidemiological data are lacking. The metabolism of HC II is very similar to that of AT III. In contrast, the anticoagulant effect of dermatan sulfate is strictly dependent on HC II. As this glycosaminoglycan is effective in an experimental model of thrombosis, HC II appears to be a potential target for new antithrombotic drugs.     

Endothelium—role in regulation of coagulation and inflammation

By its strategic position at the interface between blood and tissues, endothelial cells control blood fluidity and continued tissue perfusion while simultaneously they direct inflammatory cells to areas in need of defense or repair. The endothelial response depends on specific tissue needs and adapts to local stresses. Endothelial cells counteract coagulation by providing tissue factor and thrombin inhibitors and receptors for protein C activation. The receptor PAR-1 is differentially activated by thrombin and the activated protein C/EPCR complex, resulting in antithrombotic and anti-inflammatory effects. Thrombin and vasoactive agents release von Willebrand factor as ultra-large platelet-binding multimers, which are cleaved by ADAMTS13. Platelets can also facilitate leukocyte-endothelium interaction. Platelet activation is prevented by nitric oxide, prostacyclin, and exonucleotidases. Thrombin-cleaved ADAMTS18 induces disintegration of platelet aggregates while tissue-type plasminogen activator initiates fibrinolysis. Fibrin and products of platelets and inflammatory cells modulate the angiogenic response of endothelial cells and contribute to tissue repair.     

Relationship between the inflammation and coagulation pathways in patients with severe sepsis: implications for therapy with activated protein C

In patients with severe sepsis, thrombin has been implicated in the interrelationship between the coagulation and inflammation pathways. Thrombin is responsible for conversion of fibrinogen to fibrin (thrombus formation). Thrombin also activates endothelial cells, white blood cells and platelets. Regulation of both the coagulation and inflammation pathways is in part through the interaction of thrombin and activated protein C. Activated protein C has particular attributes that may inhibit microvascular thrombi, promote fibrinolysis and directly dampen the pro-inflammatory aspect of infection. In patients with severe sepsis, many investigators have demonstrated an active coagulopathic state, with low protein C levels. A phase III clinical trial has now demonstrated reduced mortality in patients with severe sepsis receiving activated protein C.