Disturbances of hemostasis in sepsis

Immune system and hemostasis are closely bound together. When one of these systems is activated, another is set in motion too. This is especially noticeable in polytraumas, inflammation, shocks etc. The most important activator of immune system and hemostasis is sepsis. In sepsis there is a vigorous stimulation of immune response because of a liberation of a lot of cytokines and proinflammatory molecules. This may lead to an extrem picture of systemic inflammatory response syndrome. In systemic inflammatory response syndrome a downregulation of thrombomodulin and endothelial protein C receptor on the surface of intact endothel may be detected and there is an upregulation of release of the tissue-type plasminogen activator with a switch to plasminogen activator inhibitor 1 release. There is lowering of activated protein C and fibrinolytic activation followed by fibrinolytic inhibition in septic patients. Consequently we can see consumption of coagulation factors, inhibitors (antithrombin, protein C, and tissue factor pathway inhibitor), microangiopatic hemolysis and thrombocytopenia with a picture of disseminated intravascular coagulation in these patients. The diagnosis of disseminated intravascular coagulation is not uniforme in the literature. Expression of tissue factor on monocytes and endothelium may aggravate this "circulus vitiosus" with serious microcirculatory failure in sense of MOF/MODS (mutliorgan failure/multiorgan dysfunction syndrome). The first steps in the therapy of sepsis represent the treatment of cause of sepsis, vigorous hydratation and maintenance of circulation and pulmonary function, glycemic control etc, the prevention and blocking of the undesirable activation of hemostasis and inflammation being equally important. The treatment with minidoses of heparin was implemented in the past and the question, if this therapy is indicated is not answered yet. The clinical studies of the suitability of the treatment with natural inhibitors of hemostasis (antithrombin, recombinant human activated PC or drotrecogin α activated, rhTFPI) were evaluated in the past and are still under way recently. The unequivocal contribution of these therapies was not proven and recombinant human activated PC was even pulled from the worldvide market.     

Endothelial cell function and thrombosis.

The endothelium is pivotal in the control of haemostasis and thrombosis because it is the primary source of many of the major haemostatic regulatory molecules. Healthy endothelial cells, unlike extravascular cells, are anticoagulant and antithrombotic. This is due to the regulated secretion of antiplatelet agents, including prostacyclin and nitric oxide. Following vessel injury, platelet adhesion to exposed matrix requires von Willebrand Factor, another endothelial cell product. Local generation of thrombin causes a series of receptor-mediated endothelial cell functional responses, while the surface of the endothelium is additionally the site for inactivation of thrombin by antithrombin, and its conversion to a coagulation inhibitor by interaction with thrombomodulin. Endothelial cells are also the source of circulating tissue-type plasminogen activator and its inhibitor, and Tissue Factor pathway inhibitor. In disease states, many of these endothelial cell properties are perturbed towards a more procoagulant and prothrombotic phenotype.     

A novel simultaneous clot-fibrinolysis waveform analysis for assessing fibrin formation and clot lysis in haemorrhagic disorders

Simultaneous evaluation of coagulation and fibrinolysis facilitates an overall understanding of normal and pathological haemostasis. We established an assay for assessing clot formation and fibrinolysis simultaneously using clot waveform analysis by the trigger of a mixture of activated partial thromboplastin time reagent and an optimized concentration of tissue-type plasminogen activator (0·63 μg/ml) to examine the temporal reactions in a short monitoring time (<500 s). The interplay between clot formation and fibrinolysis was confirmed by analysing the effects of argatroban, tranexamic acid and thrombomodulin. Fibrinogen levels positively correlated with coagulation and fibrinolytic potential and initial fibrin clot formation was independent of plasminogen concentration. Plasminogen activator inhibitor-1-deficient (-def) and α2-antiplasmin-def plasmas demonstrated different characteristic hyper-fibrinolytic patterns. For the specificity of individual clotting factor-def plasmas, factor (F)VIII-def and FIX-def plasmas in particular demonstrated shortened fibrinolysis lag-times (FLT) and enhanced endogenous fibrinolysis potential in addition to decreased maximum coagulation velocity, possibly reflecting the fragile formation of fibrin clots. Tranexamic acid depressed fibrinolysis to a similar extent in FVIII-def and FIX-def plasmas. We concluded that the clot-fibrinolysis waveform analysis technique could sensitively monitor both sides of fibrin clot formation and fibrinolysis, and could provide an easy-to-use assay to help clarify the underlying pathogenesis of bleeding disorders in routine clinical practice.     

Hemostasis in malignancy

Hemostatic abnormalities are present in a majority of patients with metastatic cancer. These abnormalities can be categorized as 1) increased platelet aggregation and activation, 2) abnormal activation of coagulation cascade, 3) release of plasminogen activator, and 4) decreased hepatic synthesis of anticoagulant proteins like Protein C and antithrombin III. The abnormal activation of coagulation cascade is mediated through release of Tissue Factor, Factor X activators, and other miscellaneous procoagulants from the plasma membrane vesicles of tumor cells. Macrophages of a tumor-bearing host also produce increased amounts of Tissue Factor. Production of Factor X activators and macrophage Tissue Factor is decreased by warfarin. The ability of the tumor cells to produce platelet-aggregating activity and plasminogen activator parallels their metastatic potential in animal and experimental systems. These studies also show that antiplatelet agents and antibodies against plasminogen activator can suppress the metastatic process. One or more laboratory abnormalities of hemostasis can be shown in up to 95% of patients with metastatic cancer. These abnormalities, however, are unable to predict subsequent development of thromboembolic or hemorrhagic complications. Clinical complications occur in 9-15% of the patients in the form of thrombotic or hemorrhagic disorders. The therapy of tumor-related coagulopathy should be guided by its clinical expression. Subclinical DIC should not be treated. Coumadin is generally ineffective for therapy of thrombosis in cancer patients. There is no consensus regarding the use of heparin in acute promyelocytic leukemia (APL). The defibrination in APL may be from disseminated intravascular coagulation as well as systemic fibrinolysis, as shown by decreased alpha 2 antiplasmin levels. In such cases, epsilon aminocaproic acid plus heparin therapy may be of benefit.     

Role of the plasminogen/plasmin system in thrombosis, hemostasis, restenosis and atherosclerosis evaluation in transgenic animals

Indirect evidence suggests a crucial role for the plasminogen/plasmin system, and its physiological triggers, tissue-type plasminogen activator, and urokinase-type plasminogen activator, in several proteolytic processes in blood vessels including blood clot dissolution (thrombolysis), hemostasis, aneurysm formation, neovascularization, restenosis, and atherosclerosis. The implied role of the fibrinolytic system in vivo is, however, deduced from correlations between fibrinolytic activity and (patho)physiological phenomena, which does not allow to establish a cause/consequence relationship. Recently, several transgenic mice, overexpressing or underexpressing fibrinolytic system components, have been generated. This article reviews briefly the physiological consequences of gain or loss of function of these fibrinolytic system components on thrombolysis/thrombosis, hemostasis, neointima formation, atherosclerosis, and associated effects on survival.     

Clot life span model analysis of clot growth and fibrinolysis in normal subjects: role of thrombin activatable fibrinolysis inhibitor.

Hypofibrinolysis plays a role in thrombophilic states, and a thrombelastography-based method incorporating tissue-type plasminogen activator to determine vulnerability to fibrinolytic stress has recently been developed. This study proposed to kinetically define fibrinolytic vulnerability and the contribution of thrombin activatable fibrinolysis inhibitor to fibrinolytic defenses in normal subjects. Plasma from 30 normal subjects was exposed to tissue factor/kaolin and tissue-type plasminogen activator (100 IU/ml). Prior to activation of coagulation, samples were either not exposed or exposed to potato carboxypeptidase inhibitor (25 microg/ml, a thrombin activatable fibrinolysis inhibitor). Data were collected until clot lysis time was observed. In plasma, time to onset of maximum rate of fibrinolysis was 200-1125 s (95% confidence interval), maximum rate of lysis was -2.0--0.8 dynes/cm2 per s, and clot lysis time was 555-1595 s. Thrombin activatable fibrinolysis inhibitor's inhibition decreased the time to onset of maximum fibrinolysis by 45%, increased the rate of maximum lysis by 50%, and decreased clot lysis time by 45%. The study established a range of fibrinolytic kinetic values and the contribution of thrombin activatable fibrinolysis inhibitor in normal subjects. Study of disease states involving potential hypofibrinolysis (e.g., in-situ ventricular assist device, cancer) could be conducted using this system to link fibrinolytic vulnerability and thrombophilia.     

The role of the coagulation cascade in the continuum of sepsis and acute lung injury and acute respiratory distress syndrome

Sepsis is a common and life-threatening condition with a high mortality rate. Severe sepsis includes multiorgan dysfunction syndrome. The organ most often affected is the lung, with development of acute lung injury (ALI), which, in its most severe form, is referred to as acute respiratory distress syndrome (ARDS). Our understanding of inflammation in the pathogenesis of sepsis and ALI is continually growing. However, therapies aimed at the inflammatory cascade in sepsis have been unsuccessful. These failures have led investigators to consider other pathways that may be important in the development of sepsis and ALI, including the coagulation and fibrinolytic cascades. In fact, the first therapy to reduce mortality in sepsis modulates the coagulation cascade. With this clinical success, administration of drotecogin alfa (recombinant activated protein C), the importance of coagulation in the pathogenesis of human sepsis is becoming clearer. This review summarizes the current understanding of the role of coagulation and fibrinolytic abnormalities in sepsis and the development of ALI and ARDS. Both in vitro and in vivo studies of the role of the coagulation cascade in sepsis and lung injury will be discussed, including initiation of coagulation through modulation of tissue factor and tissue factor pathway inhibitor, propagation of coagulation via protein C and thrombomodulin, inhibition of thrombin generation and resolution through thrombolysis by plasminogen activator, and plasminogen activator inhibitor-1.     

Contribution of natural anticoagulant and fibrinolytic factors in modulating the clinical severity of haemophilia patients

The role of natural anticoagulants, fibrinolytic cascade factors and common prothrombotic gene polymorphisms in modulating disease severity were studied in 35 'clinically mild' and 37 'clinically severe' haemophilia patients with severe factor VIII or IX deficiency (<0.01 IU/ml). Strong association of deficiencies of proteins C and S, antithrombin III, tissue factor pathway inhibitor and tissue plasminogen activator, together with factor V Leiden and endothelial protein C receptor 23 bp insertion polymorphisms were observed in the 'clinically milder' group as compared with the 'clinically severe' group. These results indicate a synergistic modulation of bleeding tendency in haemophilia patients by factors in the anticoagulant and fibrinolytic systems.     

Evidence of endothelial dysfunction in patients with functionally univentricular physiology before completion of the Fontan operation

INTRODUCTION: Postoperative thrombosis after a cavopulmonary connection has been widely described. Abnormalities in coagulation seem to occur early in the course of patients with functionally univentricular physiology, and may precede surgery. Endothelial abnormalities due to chronic hypoxia, and hyperviscosity, may contribute to this scene. The purpose of our study was to investigate if patients with a superior cavopulmonary connection have altered levels of endothelial and coagulative markers in the plasma. METHODS: We compared findings in 10 patients, aged from 4 to 19 years, with 6 age-matched normal controls. We measured levels of von Willebrand factor antigen, thrombomodulin, tissue-type plasminogen activator, plasminogen activator inhibitor-1 and d-dimer in the plasma using enzyme-linked immunosorbent assay. RESULTS: We found increased levels of von Willebrand factor antigen (p = 0.01), tissue-type plasminogen activator (p = 0.01), and decreased levels of thrombomodulin (p = 0.008) in the patients when compared to controls, while levels of plasminogen activator inhibitor-1 were not different. Values of d-dimer were within the reference range. Levels of tissue-type plasminogen activator had a positive correlation with von Willebrand factor antigen (r = 0.66, p = 0.008). CONCLUSIONS: Altered levels of endothelial markers in the plasma, in the presence of normal levels of d-dimer, suggest that endothelial dysfunction may precede the occurrence of intravascular coagulation and thrombosis in patients with functionally univentricular physiology. These observations may have therapeutical implications.     

Distinct dose-dependent effects of plasmin and TPA on coagulation and hemorrhage

All thrombolytic agents in current clinical usage are plasminogen activators. Although effective, plasminogen activators uniformly increase the risk of bleeding complications, especially intracranial hemorrhage, and no laboratory test is applicable to avoid such bleeding. We report results of a randomized, blinded, dose-ranging comparison of tissue-type plasminogen activator (TPA) with a direct-acting thrombolytic agent, plasmin, in an animal model of fibrinolytic hemorrhage. This study focuses on the role of plasma coagulation factors in hemostatic competence. Plasmin at 4-fold, 6-fold, and 8-fold the thrombolytic dose (1 mg/kg) induced a dose-dependent effect on coagulation, depleting antiplasmin activity completely, then degrading fibrinogen and factor VIII. However, even with complete consumption of antiplasmin and decreases in fibrinogen and factor VIII to 20% of initial activity, excessive bleeding did not occur. Bleeding occurred only at 8-fold the thrombolytic dose, on complete depletion of fibrinogen and factor VIII, manifest as prolonged primary bleeding, but with minimal effect on stable hemostatic sites. Although TPA had minimal effect on coagulation, hemostasis was disrupted in a dose-dependent manner, even at 25% of the thrombolytic dose (1 mg/kg), manifest as rebleeding from hemostatically stable ear puncture sites. Plasmin degrades plasma fibrinogen and factor VIII in a dose-dependent manner, but it does not disrupt hemostasis until clotting factors are completely depleted, at an 8-fold higher dose than is needed for thrombolysis. Plasmin has a 6-fold margin of safety, in contrast with TPA, which causes hemorrhage at thrombolytic dosages.     

The overall hemostasis potential: a laboratory tool for the investigation of global hemostasis

A simple and rapid global haemostatic assay for determination of the overall hemostasis potential (OHP) in plasma represents a new approach in detecting alterations in the delicate balance between coagulation and fibrinolysis. The assay is based on repeated spectrophotometric registration of the fibrin-aggregation curve in platelet-poor plasma containing small amounts of exogenous thrombin, tissue-type plasminogen activator, and calcium. The overall coagulation potential and overall fibrinolytic potential are supplementary parameters of OHP, providing details of underlying changes in coagulation and/or fibrinolysis. The OHP assay was evaluated in connection with hypercoagulation in normal pregnancy, preeclampsia, some thrombophilias, coronary heart disease, diabetes, stroke, and vascular surgery as well as with hypocoagulability, especially in patients with hemophilia A or B. Preliminary results also indicate the possible usefulness of the assay in monitoring anticoagulant treatments. Large prospective clinical trials are needed before the method can be recommended for routine clinical application.     

Feedback activation of factor XI by thrombin in plasma results in additional formation of thrombin that protects fibrin clots from fibrinolysis

Recently, an alternative pathway for factor XI activation has been described in which factor XI is activated by thrombin. Patients with a factor XI deficiency bleed mostly from tissues with high local fibrinolytic activity. Therefore, the role of thrombin-mediated factor XI activation in both fibrin formation and fibrinolysis was studied in a plasma system. Clotting was induced by the addition of tissue factor or thrombin to recalcified plasma in the presence or absence of tissue- type plasminogen activator, after which clot formation and lysis were measured using turbidimetry. Thrombin-mediated activation of factor XI was found to take place in plasma under physiologic conditions in the absence of a dextran sulfate-like cofactor. At high tissue factor concentrations, no effect of factor XI was seen on the rate of fibrin formation. Decreasing amounts of tissue factor resulted in a gradually increasing contribution of factor XI to the rate of fibrin formation. In addition, thrombin-mediated factor XI activation resulted in an inhibition of tissue-type plasminogen activator-induced lysis of the clot. This inhibition occurred even at tissue factor concentrations at which no effect of factor XI was observed on fibrin formation. Trace amounts of activated factor XI (1.25 pmol/L, representing 0.01% activation) were capable of completely inhibiting fibrinolysis in our system. The inhibitory effect was found to be mediated by thrombin that is additionally generated in a factor XI-dependent manner via the intrinsic pathway and is capable of protecting the clot against lysis. We also observed that formation of additional thrombin continued after the clot had been formed. We conclude that thrombin-mediated factor XI activation can take place in plasma. The presence of factor XI during coagulation results in the formation of additional thrombin within the clot capable of protecting this clot from fibrinolytic attack. The large amounts of thrombin that are formed by the intrinsic pathway via factor XI may play an important role in the procoagulant and thrombogenic state of clots and may therefore have important clinical and therapeutic implications.     

Phospholipase D1 is specifically required for regulated secretion of von Willebrand factor from endothelial cells

Endothelial cells regulate thrombosis, hemostasis, and inflammatory responses by supplying the vasculature with several factors that include procoagulant von Willebrand factor (VWF) and fibrinolytic tissue-type plasminogen activator (tPA). Both proteins can be secreted in a Ca(2+)-regulated manner after endothelial activation but exhibit opposing physiologic effects. In search for factors that could modulate endothelial responses by selectively affecting the secretion of procoagulant or anticoagulant proteins, we identify here phospholipase D1 (PLD1) as a specific regulator of VWF secretion. PLD1 is translocated to the plasma membrane upon stimulation of endothelial secretion, and this process correlates with the generation of phosphatidic acid (PA) in the plasma membrane. Histamine-evoked secretion of VWF, but not tPA, is inhibited by blocking PLD-mediated production of PA, and this effect can be attributed to PLD1 and not PLD2. Thus, different mechanisms appear to control the agonist-induced secretion of VWF and tPA, with only the former requiring PLD1.     

Hemostasis and fibrinolysis in severe liver failure and their relation to hemorrhage

In a study of severe, decompensated liver failure, we tried to find a correlation between hemorrhage and parameters of hemostasis and fibrinolysis. Three groups of patients were studied: alcoholic cirrhosis; nonalcoholic cirrhosis, and acute liver failure without known prior liver disease. The two cirrhotic groups did not differ significantly from each other in coagulation or in fibrinolytic parameters, although liver function was more impaired in nonalcoholic cirrhosis. The levels of clotting factors, antithrombin III, prekallikrein, plasminogen and alpha 2-antiplasmin were significantly lower in the third group. Mean values of fibrinolytic activity (fibrin plate method) were slightly reduced as compared to normal in all three groups. Tissue plasminogen activator-related antigen tended to be elevated especially in alcoholic cirrhosis. The free fast-acting plasminogen activator inhibitor showed extremely high and extremely low levels in some patients among all three groups. Nonvariceal, capillary-type bleeding, including mucosal bleeding, hematomas and bleeding from puncture sites correlated with low thrombotest and normotest levels (p less than 0.01), low fibrinogen concentration (p less than 0.05) and with a high quotient of fibrinolytic activity (square root of lysis area) and normotest (p less than 0.001). The ratio between fibrin formation and dissolution appears to be an important parameter of hemorrhagic tendency in liver disease. Variceal bleeding appeared not to be related to impairment of hemostasis or fibrinolysis.     

Biochemical and physiologic principles of tissue-type plasminogen activator

After wound healing the protective fibrin clot is removed by the fibrinolytic system. In addition fibrinolysis is one of the most important counter-reactions of blood coagulation. Fibrinolysis is controlled by activation and inhibition processes. Tissue type plasminogen activator (t-PA) and Pro-urokinase (single chain urokinase; scu-PA) hold a key position in physiological plasminogen activation. Plasmin itself is a rather unspecific protease capable of degrading a great variety of proteins besides fibrin. In vivo however--except for certain pathological situations--the fibrinolytic process is restricted to its actual target the fibrin clot. This surprising situation in terms of structure function interrelation is physiologically managed by N-terminal modules in the protein structure of the essential factors providing fibrin affinity. Free plasmin will be immediately inactivated by alpha 2-antiplasmin. Therefore fibrin plays a central role as cofactor in the fibrinolytic system in determining initiation and localization of the fibrinolytic process. Because of the superior properties of t-PA and scu-PA with respect to fibrin specificity both activators must be regarded as the future thrombolytic agents for therapy.     

Blood coagulation activation and fibrinolysis during a downhill marathon run.

Prolonged physical exercise is associated with multiple changes in blood hemostasis. Eccentric muscle activation induces microtrauma of skeletal muscles, inducing an inflammatory response. Since there is a link between inflammation and coagulation we speculated that downhill running strongly activates the coagulation system. Thirteen volunteers participated in the Tyrolean Speed Marathon (42,195 m downhill race, 795 m vertical distance). Venous blood was collected 3 days (T1) and 3 h (T2) before the run, within 30 min after finishing (T3) and 1 day thereafter (T4). We measured the following key parameters: creatine kinase, myoglobin, thrombin-antithrombin complex, prothrombin fragment F1 + 2, D-dimer, plasmin-alpha(2)-antiplasmin complexes, tissue-type plasminogen activator antigen, plasminogen-activator-inhibitor-1 antigen and thrombelastography with ROTEM [intrinsic pathway (InTEM) clotting time, clot formation time, maximum clot firmness, alpha angle]. Thrombin generation was evaluated by the Thrombin Dynamic Test and the Technothrombin TGA test. Creatine kinase and myoglobin were elevated at T3 and further increased at T4. Thrombin-antithrombin complex, prothrombin fragment F1 + 2, D-dimer, plasmin-alpha(2)-antiplasmin complexes, tissue-type plasminogen activator antigen and plasminogen-activator-inhibitor-1 antigen were significantly increased at T3. ROTEM analysis exhibited a shortening of InTEM clotting time and clot formation time after the marathon, and an increase in InTEM maximum clot firmness and alpha angle. Changes in TGA were indicative for thrombin generation after the marathon. We demonstrated that a downhill marathon induces an activation of coagulation, as measured by specific parameters for coagulation, ROTEM and thrombin generation assays. These changes were paralleled by an activation of fibrinolysis indicating a preserved hemostatic balance.     

Circadian mRNA expression of coagulation and fibrinolytic factors is organ-dependently disrupted in aged mice

To evaluate the effects of aging on the circadian gene expression of coagulation and fibrinolytic factors in the mouse tissues, we examined temporal mRNA expression profiles of plasminogen activator inhibitor-1 (PAI-1), tissue-type plasminogen activator (tPA), tissue factor (TF), and thrombomodulin (TM) genes together with circadian clock genes in the brains, hearts and livers of young (5weeks old) and aged (15months old) mice. Cardiac mRNA expression of β-myosin heavy chain (β-MHC), a molecular marker of cardiac hypertrophy, was obviously increased in the aged mice. Rhythmic expression of the clock genes mPer2 and BMAL1 in these organs was almost identical between young and aged mice, whereas that of PAI-1, TF and TM mRNAs and of clock-controlled genes such as DBP and Dec1 were damped to low levels in the livers of aged mice. Expression levels of tPA mRNA were significantly decreased and those of TF were significantly elevated throughout the day in the brain of aged mice. Expression levels of PAI-1 in the heart of aged mice were continuously elevated over 2-fold the peak levels of young mice throughout the day. However, day/night fluctuations in plasma PAI-1 levels were unaffected by aging. Aging tissue- and time-dependently affects the mRNA expression of coagulation and fibrinolytic factors. Aging-dependent constitutive PAI-1 induction in the heart might be a risk factor for cardiovascular diseases that is independent of plasma PAI-1 levels.     

Targeted gene disruption of natural anticoagulant proteins in mice

The blood coagulation system is a complicated cascade of reactions and feedback regulations that executes a rapid response to vascular injury, yet avoids occlusion of the vessel. There are several key components of this system in the regulation of blood clot propagation, such as antithrombin (AT), tissue factor pathway inhibitor (TFPI), thrombomodulin (TM) and protein C (PC), of which defect causes thromboembolic diseases. In recent years, targeted gene disruption technique by homologous recombination has been introduced to investigate the physiological roles of those natural anticoagulant molecules, not only in thrombogenesis but also in embyrogenesis. We have studied the natural anticoagulantion system in a decade, and recently established AT knockout mice as well as ryudocan (syndecan-4) knockout mice. Ryudocan is a cell surface heparan sulfate proteoglycan, which bears heparin-like glycosaminoglycan (heparan sulfate) cahins, originally cloned from rat microvascular endothelial cells. We have demonstrated that ryudocan deficiency impairs the control of coagulation in fetal vessels of the placenta in mice. We have also reported that complete antithrombin deficiency in mice results in embryonic lethality, with severe fibrin deposition in the myocardium and the liver, accompanied with extensive subcutaneous hemorrhage. In this presentation, recent advances in understanding roles of natural anticoagulant molecules through the researches of targeted gene-knockout mice, including our experiences in antithrombin deficient mice and ryudocan deficient mice, will be discussed.     

Activation and inhibition of fibrinolysis in septic patients in an internal intensive care unit

Disseminated thrombotic processes in the microcirculation are considered to be an important cause of multiple organ failure in septic patients. Fibrinolysis is one endogenous mechanism protecting the circulation from overwhelming thrombosis. Therefore, we looked for alterations of fibrinolytic parameters (tissue plasminogen activator (t-PA), tissue plasminogen activator inhibitor (PAI), D-dimer, euglobulin-clot-lysis-time (ECLT), plasminogen, alpha 2-antiplasmin) and of some coagulation parameters (prothrombin time, fibrinogen, platelets, antithrombin III, protein C, factor XII) in clearly defined septic patients and for the relations of these values to the severity of the disease (APACHE II-score). An increase in D-dimer and t-PA-antigen was registered in all patients, while factor XII and plasminogen were decreased, indicating an activated fibrinolysis. In contrast the systemic fibrinolytic capacity of the blood was strongly inhibited: t-PA-activity was not detectable, PAI-function was elevated, the ECLT was prolonged and alpha 2-antiplasmin was normal. Coagulation was moderately activated: the platelets, antithrombin III and protein C were decreased, the prothrombin time was prolonged and fibrinogen was normal. The changes in t-PA-antigen, PAI-function, factor XII, prothrombin time and antithrombin III were significantly related to the APACHE II-score of the patients. We conclude that the activation of coagulation is accompanied by an activation of fibrinolysis in the microcirculation, but that systemically the increased inhibitors of fibrinolysis (PAI, alpha 2-antiplasmin) induce a decrease of the fibrinolytic capacity of the blood. The severity of the disease determines the extent of the alterations.     

Targeted gene disruption of natural anticoagulant proteins in mice

The blood coagulation system is a complicated cascade of reactions and feedback regulations that executes a rapid response to vascular injury, yet avoids occlusion of the vessel. There are several key components of this system in the regulation of blood clot propagation, such as antithrombin (AT), tissue factor pathway inhibitor (TFPI), thrombomodulin (TM) and protein C (PC), of which defect causes thromboembolic diseases. In recent years, targeted gene disruption technique by homologous recombination has been introduced to investigate the physiological roles of those natural anticoagulant molecules, not only in thrombogenesis but also in embyrogenesis. We have studied the natural anticoagulantion system in a decade, and recently established AT knockout mice as well as ryudocan (syndecan-4) knockout mice. Ryudocan is a cell surface heparan sulfate proteoglycan, which bears heparin-like glycosaminoglycan (heparan sulfate) cahins, orginally cloned from rat microvascular endothelial cells. We have demonstrated that ryudocan deficiency impairs the control of coagulation in fetal vessels of the placenta in mice. We have also reported that complete antithrombin deficiency in mice results in embryonic lethality, with severe fibrin deposition in the myocardium and the liver, accompanied with extensive subcutaneous hemorrhage. In this presentation, recent advances in understanding roles of natural anticoagulant molecules through the researches of targeted gene-knockout mice, including our experiences in antithrombin deficient mice and ryudocan deficient mice, will be discussed.