Showing papers in "Seminars in Thrombosis and Hemostasis in 2001"

The tissue factor pathway in disseminated intravascular coagulation.

Abstract: In most instances, tissue factor (TF) exposed to the circulation is the sole culprit underlying the initiation of disseminated intravascular coagulation (DIC), although notable exceptions because of a more direct activation of the coagulation system, by snake venoms, for example, do occur. Peripheral monocytes and subendothelial structures are the potential sources of such TF; in the former, TF emerges on the cell surface on synthesis induction and in the latter it becomes available subsequent to permeability changes or damage to the endothelium. Subendothelial TF is constitutively present in fibroblasts, pericytes, and macrophages and at a higher than normal level in tumor-associated macrophages. This scenario of coagulation activation probably describes the principal events underlying emerging acute DIC states under pathophysiological conditions such as abruptio placentae, septic abortion, amniotic fluid embolization, and pregnancy toxemia. Under disease conditions associated with DIC, the continuous exposure to excess TF typically exhausts the available tissue factor pathway inhibitor (TFPI), leading to rampant thrombin generation, persistent feedback activation of factor XI (FXI) by the generated thrombin, and hence virtually uncheckable ongoing fibrin generation (DIC). Recently, it was shown that patients subject to meningococcal sepsis had comparatively large amounts of mainly monocyte-derived circulating TF-containing microparticles. Because phosphatidylserine (PS) is exposed on such particles, in addition to TF, they probably contribute crucially to DIC during meningococcal sepsis. Although endothelial cells (EC) have been shown to express large amounts of TF in vitro, this observation hardly relates to the situation in vivo, where, in contrast, synthesis and exposure of EC TF is very limited and not likely to be of any significance in emerging and ongoing DIC.

Disseminated Intravascular Coagulation in Trauma Patients

Abstract: Disseminated intravascular coagulation (DIC) is characterized by the in vivo activation of the coagulation system, which results in the intravascular deposition of fibrin and consumption bleeding. DIC is a serious hemostatic complication of trauma. It can be clearly distinguished from physiological hemostatic response to trauma by using sensitive coagulofibrinolytic molecular markers. Physiological hemostasis to injuries is similar in all kinds of trauma without exception. There is an increase in circulating proinflammatory cytokines in DIC patients after trauma. Elevated cytokines induce tissue factor-mediated activation of coagulation, suppression of the anticoagulant pathway, and plasminogen activator inhibitor-1 (PAI-1)-mediated inhibition of fibrinolysis followed by disseminated fibrin deposition in the microvasculature. In addition to the occlusive microvascular thrombosis and hypoxia, sustained systemic inflammatory response characterized by neutrophil activation and endothelial damage plays a pivotal role in the development of multiple organ dysfunction syndrome (MODS) in posttrauma DIC patients. DIC associated with sustained systemic inflammatory response syndrome (SIRS) after trauma leads to the development of MODS, which is the main determinant of patients' outcome after trauma.

Regulatory role of cytokines in disseminated intravascular coagulation.

Abstract: Disseminated intravascular coagulation (DIC) can complicate a number of diseases. DIC in the setting of sepsis is considered to result from strong activation of the coagulation system and concurrent inhibition of fibrinolysis and other anticoagulant pathways. Cytokines have been implicated as important mediators in these hemostatic alterations. This article summarizes recent insights into which cytokines are likely to be involved in the procoagulant response to systemic infection.

Recombinant Activated Factor VII (rFVIIa): Characterization, Manufacturing, and Clinical Development

Abstract: Recombinant activated coagulation factor VII (rFVIIa) (NovoSeven) was developed for treatment of bleeding in hemophilia patients with inhibitors (antibodies) against factors VIII or IX. rFVIIa initiates the coagulation cascade by binding to tissue factor at the site of injury and causes the formation of sufficient amounts of thrombin to trigger coagulation. Patients with a variety of other coagulation deficiencies than hemophilia characterized by an impaired thrombin generation and life-threatening bleeding have been reported as successfully treated with rFVIIa. Data are now entered into clinical registries established to further monitor this experimental treatment with NovoSeven. rFVIIa is produced free of any added human protein. The amino acid sequence of rFVIIa is identical to plasma-derived FVIIa (pdFVIIa). Posttranslational modifications (i.e., gamma-carboxylations, N- and O-glycosylations) are qualitatively identical in pdFVIIa and rFVIIa although some quantitative differences exist. The activities of rFVIIa and pdFVIIa are indistinguishable. Manufacturing of rFVIIa involves expression in baby hamster kidney (BHK) cells followed by purification, including three ion-exchange and one immunoaffinity chromatography steps. The last anion-exchange chromatography step ensures completion of the autoactivation of recombinant factor VII (rFVII) to rFVIIa. This review describes the mechanism of action, characterization, manufacturing, and preclinical and current clinical evidence for the efficacy and safety of rFVIIa.

Disseminated intravascular coagulation in acute leukemia.

Abstract: Malignancy is associated with a "hypercoagulable" state and a high risk for thrombohemorrhagic complications. Clinical complications may range from localized thrombosis to bleeding of varying degrees of severity because of disseminated intravascular coagulation (DIC). Life-threatening bleeding is frequent in acute leukemias, particularly in acute promyelocytic leukemia (APL). Laboratory assessments show profound hemostatic imbalance in this condition, with activation of coagulation, fibrinolysis, and nonspecific proteolysis systems. An important pathogenetic role is attributed to the leukemic cell properties interfering with the hemostatic mechanisms. However, chemotherapy and intercurrent infections also contribute to the bleeding risk in the patient with leukemia. In this article, we will attempt to describe what is currently known about the coagulopathy of acute leukemia, summarize the various aspects of the hemostatic abnormalities underlying this disorder, and revise the principal pathogenetic mechanisms. We will also try to provide information on the current therapeutic tools and recommendations for the management of life-threatening bleeding in this disease. Finally, a special focus will be devoted to the management of this complication in the era of all-trans retinoic acid (ATRA), a drug now indispensable in curing APL that has completely changed the natural history of APL and its coagulation/bleeding syndrome.

Fibrin degradation products, fibrin monomer and soluble fibrin in disseminated intravascular coagulation.

Abstract: Disseminated intravascular coagulation (DIC) is characterized by activation of hemostasis and fibrinolysis resulting in the formation of thrombin and plasmin, and the characteristic effects of these enzymes on plasma fibrinogen can be useful in diagnosis. Thrombin cleaves fibrinopeptides from fibrinogen, forming fibrin monomer that rapidly polymerizes to form a clot. Small amounts can circulate in plasma as "soluble fibrin," which may have a complex composition and include fibrinogen and a variable amount of cross-linking. Plasmic degradation of cross-linked fibrin forms a heterogeneous group of degradation products reactive in assays for D-dimer, and their levels provide a measure of the amount of fibrin formation and lysis. Caution should be exercised in comparing quantitative results using different assays because of problems with standardization and variable reactivity with different molecular forms. Marked elevations of fibrin(ogen) degradation products are a constant finding in experimental animal models of DIC. In human models of DIC resulting from endotoxin infusion, D-dimer is elevated early and high levels persist, reflecting lysis of microvascular fibrin deposits. Elevated levels of D-dimer and soluble fibrin are very sensitive for the diagnosis of DIC, and a normal level has a high negative predictive value. Serial monitoring of soluble fibrin or D-dimer assays may be of value in evaluating the response to therapy and possibly in identifying at-risk patients.

Complement factor H: physiology and pathophysiology

Abstract: The human plasma protein factor H, which is a multifunctional, multidomain protein, acts as a central regulator of the complement system. In addition to its complement regulatory activities, factor H has multiple physiological activities and 1) acts as an extracellular matrix component, 2) binds to cellular receptors of the integrin type, and 3) interacts with a wide selection of ligands, such as the C-reactive protein, thrombospondin, bone sialoprotein, osteopontin, and heparin. Recent genetic reports, which show involvement of factor H in the human disease hemolytic-uremic syndrome (HUS), have attracted the attention of both clinicians and basic complement researchers to the role of factor H in the pathophysiology of HUS.

How and when to monitor a patient treated with low molecular weight heparin.

Abstract: Low molecular weight heparins (LMWHs) are as efficient as unfractionated heparin (UFH) for prevention and treatment of thromboembolism. There is no evidence that monitoring the dose improves the clinical efficacy. In contrast, any overdosage increases the risk of hemorrhage. Because renal function plays a significant role in the elimination of LMWH, curative treatment should be monitored with an anti-factor Xa assay in patients presenting renal insufficiency, in the elderly, and in patients presenting an increased hemorrhagic risk. It is advisable to sample the patient at peak activity (3 to 5 hours after the subcutaneous [sc] administration) and to target the mean anti-factor Xa activity that was found efficient and safe in the clinical trial. This target is different for each LMWH and each dose regimen.

Combined quantitative (1)H and (13)C nuclear magnetic resonance spectroscopy for characterization of heparin preparations.

Abstract: The sulfation patterns of pig and bovine mucosal commercial heparin preparations can be characterized and distinguished from each other easily by analysis of their monodimensional proton and carbon nuclear magnetic resonance ((1)H and (13)C-NMR) spectra. NMR spectroscopy can detect and quantify signals associated with major sequences as well as with minor residues such as the typical ones associated with the antithrombin (AT) binding sequence and the "linkage region." Contaminants arising from industrial preparation processes are also detectable.

Acute immune thrombocytopenia (ITP) in childhood: retrospective and prospective survey in Germany.

Abstract: Treatment of acute childhood immune thrombocytopenia (ITP) is controversial For information on the present situation in Germany, a retrospective and a prospective survey were carried out In the retrospective survey, questionnaires were sent to all German pediatric hospitals asking about local policies for handling ITP and whether in the preceding year (starting on October 1995) death or ICH had occurred; 86% answered In the prospective survey, 94% responded to the mailings ("have you seen a case of ITP?") sent in monthly intervals between October 1, 1996 and September 30, 1997; 89% of the questionnaires were retrieved In the retrospective survey, no deaths and no ICH were reported If only mild bleedings, such as skin bleeds alone (or additional mild mucous membrane bleeding) were present, 205% (264%) preferred the "watchful waiting" regimen (supportive care), irrespective of the platelet count; 795% (736%) would treat if the platelet counts were 50 x 10(9)/L was considered as therapeutic success In the prospective survey, from the reported 323 children an annual incidence of 216 per 100,000 children was calculated The incidence depended on age and gender, being highest for boys younger than 2 years with 58 (girls 342) and low with 044 for boys (girls 089) older than l4 years About 60% of the patients had a preceding infection Although 83% had a platelet count <20 x 10(9)/L (56% <10 x 10(9)/L), almost all (975%) had only mild bleeding symptoms; 25% had serious bleeding symptoms requiring blood transfusion or nasal packing, none had ICH, and no death was reported The mean platelet count on admission was 11348 (lowest count 8253) x 10(9)/L Sixty-one percent received Ig, 19% GC (both either alone or as first choice), 6% Ig plus GC, and 14% no treatment Side effects were reported in 22% of the children treated with Ig The retrospective survey mirrored the uncertainty in regard to treatment The prospective survey provided new aspects on incidence, age, and gender distribution Although almost all patients had only mild bleeding symptoms, most received Ig and/or GC The decision to treat depended mainly on the platelet count From these surveys, conclusions about the effectiveness of treatment cannot be drawn Recommendations based primarily on platelet counts must be reconsidered

Production Processes of Licensed Recombinant Factor VIII Preparations

Abstract: The state-of-the-art treatment for hemophilia A is replacement therapy with recombinant factor VIII (rFVIII) made possible by genetic engineering advances. Currently, there are four different products licensed and available for hemophilia A patients. All are produced by recombinant mammalian cells in large-scale fermenter cultures, purified to high purity, formulated in stable formulations and freeze dried. The first-generation products Recombinate and Kogenate (also sold as Helixate by Aventis) are characterized as full-length human factor VIII molecules and formulated using human serum albumin as a stabilizer. The second-generation product ReFacto contains an improved albumin-free sucrose formulation and incorporates advanced antiviral safety procedures in the manufacturing process. It is a truncated B region-deleted form of factor VIII (FVIII) that makes use of a nonhuman peptide linker 14 amino acids in length to connect the 80 and 90 kD subunits. The most recently licensed rFVIII product is the second-generation Kogenate product called KOGENATE Bayer/Kogenate FS, which combines the advantages of the human full-length FVIII molecule with an albumin-free, sucrose-based synthetic formulation as well as an improved viral safety profile. In this article, the manufacturing processes for each of the four different products are discussed in detail, focusing on expression systems and cell lines, culture medium, technical culture systems, purification process (including viral removal potential), and final formulation.

Heparin-induced thrombocytopenia in pediatrics.

Abstract: As in adult patients, heparin is used for prophylaxis and treatment of thromboembolism in newborns, children, and adolescents. Patients receiving heparin are potentially at risk to develop heparin-induced thrombocytopenia (HIT). HIT type II has been extensively described in the adult population; only a few reports address HIT type II in pediatric patients (total of 15 neonates, 4 young children, 12 older children and adolescents). The available data are discussed, and the case of a patient with recurrent thrombosis and HIT type II without thrombocytopenia is presented. The review of the literature reveals that HIT type II occurs especially in neonates and adolescents, corresponding to the two age peaks of thrombosis in pediatric patients. Risk factors for thrombosis include hereditary factors, immobilization, and surgery. HIT complications are severe and partly lead to life-threatening thromboembolism. In three patients, an increasing heparin demand was found. In five cases, thrombocytopenia was absent. Heparin was replaced mostly by danaparoid sodium; in three patients hirudin was used as an alternative anticoagulant. HIT type II represents a potentially dangerous complication of heparin therapy in pediatric patients and should be taken into consideration whenever heparin is given for prophylactic or therapeutic use in newborns, children, or adolescents.

Production and characterization of saratin, an inhibitor of von Willebrand factor-dependent platelet adhesion to collagen.

Abstract: Platelets tether to collagen in both a von Willebrand factor (vWF)-dependent and a vWF-independent manner. We have recently characterized a recombinant protein, saratin, isolated from the saliva of the leech Hirudo medicinalis, expressed it in Hansenula polymorpha, and studied its effect on direct and indirect platelet-collagen interactions. Saratin dose dependently inhibited the binding of purified human vWF to human type I and III collagens (IC(50)= 0.23 +/- 0.004 and 0.81 +/- 0.04 microg mL(-1), respectively) and to calf skin collagen (IC(50)= 0.44 +/- 0.008 microg mL(-1)). Furthermore, saratin showed a similar inhibitory potency against the binding of human, rodent, and porcine plasma vWF to these collagens. In a flow chamber under conditions of elevated shear (2700 s(-1)), saratin dose dependently and potently inhibited platelet aggregate formation on a collagen-coated surface (IC(50)= 0.96 +/- 0.25 microg mL(-1)), but at reduced shear (1300 s(-1)) a rightward shift in the dose-response curve was noted (IC(50)= 5.2 +/- 1.4 microg mL(-1)). Surface plasmon resonance analysis revealed both high and low affinity binding sites for saratin on human collagen type III (K(d) 5 x 10(-8) M and 2 x 10(-6) M, respectively). Although low concentrations of saratin, which inhibited platelet adhesion under increased shear (i.e., saturation of high-affinity binding sites), had no effect on vWF-independent collagen-induced platelet aggregation, high concentrations (i.e., saturation of low-affinity binding sites) were found to inhibit platelet aggregation. These data demonstrate that saratin is a potent inhibitor of vWF-dependent platelet adhesion to collagen and hence may have therapeutic potential as an antithrombotic agent.

Toward a biotechnological heparin through combined chemical and enzymatic modification of the Escherichia coli K5 polysaccharide.

Abstract: A process to generate glycosaminoglycans with heparin- and heparan sulfate-like sequences from the Escherichia coli K5 capsular polysaccharide is described. This polymer has the same structure as N-acetylheparosan, the precursor in heparin/ heparan sulfate biosynthesis. The process involves chemical N-deacetylation and N-sulfation, enzymatic conversion of up to 60% of the D-glucuronic acid to L-iduronic acid residues, and chemical O-sulfation. Because direct sulfation afforded unwanted 3-O-sulfated (instead of 2-O-sulfated) iduronic acid residues, a strategy involving graded solvolytic desulfation of chemically oversulfated C5-epimerized sulfaminoheparosans was assessed using persulfated heparin and heparan sulfate as model compounds. The O-desulfation process was shown to increase the anti-factor Xa activity of oversulfated heparin.

Alloimmune thrombocytopenia in the fetus and newborn.

Abstract: Alloimmune thrombocytopenia is an interesting and challenging disease. Identification in the fetus and newborn by screening remains to be clarified. The primary clinical criterion for neonatal diagnosis appears to be a neonatal platelet count of <50 x 10(9)/L. Treatment of the neonate can be accomplished with intravenous immunoglobulin (IVIG) +/- steroids or with matched platelet transfusion. Cranial ultrasonography is important. Testing can be performed on the parents and requires a highly experienced laboratory. If an affected fetus is identified, based on a previous affected neonate and a homozygous father, antenatal management is needed. Studies have been completed that inform the still controversial decision. IVIG remains the basis of therapy but appears to require a higher dose (2 g/kg/week) and/or the addition of 1 mg/kg of prednisone in the highest risk cases, those with antenatal intracranial hemorrhage.

Noninvasive diagnosis of deep vein thrombosis in postoperative patients.

Abstract: The accuracy of noninvasive testing for the diagnosis of deep vein thrombosis (DVT) generally is less in asymptomatic patients than it is in those with symptoms suggestive of thrombosis. This is because asymptomatic DVT often is confined to the distal veins and, when it involves the proximal veins, the thrombi usually are smaller than in symptomatic patients with proximal thrombosis. Because the positive predictive value of noninvasive tests for asymptomatic DVT generally is 80% or less, abnormal results should be confirmed by venography. There are two main reasons why asymptomatic DVT is sought in the postoperative period: (1) to identify the need for full-dose anticoagulant therapy to prevent symptomatic episodes of venous thromboembolism (VTE), including fatal pulmonary embolism (this represents a form of secondary prophylaxis), and (2) to use this outcome as a surrogate for episodes of clinically important VTE in studies that are designed to evaluate methods of venous thrombosis prophylaxis. In relation to the first of these indications, evidence suggests that routine surveillance of high-risk patients to detect asymptomatic postoperative DVT does not result in improved clinical outcomes in patients who received appropriate VTE prophylaxis. In relation to the second indication, there is concern that asymptomatic VTE may not be a reliable surrogate for clinically important VTE, particularly if the effectiveness of different antithrombotic agents is being compared. Coupled with the comparatively low accuracy of noninvasive testing for asymptomatic DVT, this suggests that the results of such testing are unsuitable for the evaluation of new methods of prophylaxis in clinical trials.

Diagnosis and management of acute obstetrical DIC

Abstract: UNLABELLED Obstetrical disseminated intravascular coagulation (DIC) is usually a very acute, serious complication of pregnancy. The DIC diagnostic criteria in obstetrics (the obstetrical DIC score) help with making a prompt diagnosis and starting treatment early. These DIC diagnostic criteria, in which higher scores are given for clinical parameters than for laboratory parameters, have three components: (1) the underlying disease, (2) clinical symptoms, and (3) laboratory findings. It is justified that it is appropriate to initiate therapy for DIC when the obstetrical DIC score reaches 8 points or more before obtaining the results of coagulation tests. MANAGEMENT (1) Control of the underlying disease: because prolongation of exposure to the triggering factors worsens DIC, it is important to eliminate the etiologic factors as rapidly as possible. Elimination of the cause of DIC can be easily performed in obstetrics, for example, by cesarean section. (2) Antithrombin (AT) therapy: AT monotherapy (1,500 to 3,000 units/day, 2 days) is preferably employed instead of heparin monotherapy or heparin-AT therapy because of the hemorrhagic side effects of heparin. (3) Synthetic serine protease inhibitors: continuous infusion ofgabexate mesilate (FOY) or nafamostat mesilate (FUT) is effective for DIC. Controlled multicenter trials showed a significant improvement not only in clinical response but also in platelet counts and prothrombin time (PT) in the AT group compared with the FOY group. (4) Activated protein C (APC) can inhibit thrombin generation and accelerate fibrinolytic activity. APC (5,000 to 10,000 units) is administered for 2 days in patients with placental abruption complicated by DIC. APC is a very safe, effective, and useful agent for the treatment of DIC.

The role of virulence factors in enterohemorrhagic Escherichia coli (EHEC)--associated hemolytic-uremic syndrome.

Abstract: Enterohemorrhagic Escherichia coli (EHEC) are the most common cause of postdiarrheal hemolytic-uremic syndrome (HUS). The most important EHEC serotype implicated worldwide is O157:H7. However, several so-called non-O157 EHEC serotypes have emerged. After a mean incubation period of 3 days, patients develop watery diarrhea accompanied by cramping abdominal pain. During the next days, in most patients watery diarrhea changes to bloody diarrhea. One week after the onset of diarrhea, in about 15% of infected patients under 10 years of age EHEC infection results in a systemic complication, HUS. Shiga toxins (Stxs) are considered the major virulence factors of EHEC involved in the pathogenesis of HUS. It is generally believed that after intestinal infection with EHEC, Stxs cross the intestinal barrier and bind to endothelial cells. At this point they presumably injure the host cell by inhibition of protein synthesis, stimulation of prothrombotic messages, or induction of apoptosis. The B subunit of Stx binds to the membrane receptor globotriaosylceramide (Gb3). Gb3 facilitates the endocytosis and intracellular trafficking of the toxin. The Stx A subunit hydrolyzes a specific adenine residue of the 60S ribosomal subunit of mammalian cells. As a consequence, Stx shuts down the protein machinery of the susceptible cell. The HUS is the net effect of a variety of interacting factors, including background risk of acquisition, host factors (such as age), virulence characteristics of the infecting EHEC strain, and exogenous factors. All known EHEC virulence determinants are located on mobile genetic elements, and this has an important impact on the evolution of these pathogens. The evolution of EHEC has a dynamic component that includes different genetic mechanisms. The recent progress in understanding the pathogenesis and epidemiology of EHEC infections forms a basis for the development of future strategies to prevent EHEC infections in humans.

Recurrent pregnancy loss: etiology of thrombophilia.

Abstract: Congenital and acquired thrombophilia are associated with an increased risk of pregnancy-associated venous thrombosis and fetal loss. Two hundred eighty-nine patients with a history of recurrent spontaneous abortion were subjected to screening examinations for the etiology of these abortions. Endocrine abnormality (28.0%), uterine abnormality (10.4%), autoimmune diseases (1.4%), antiphospholipid antibody syndrome (4.5%), and balanced type chromosome translocation (4.2%) were found as underlying causes of recurrent abortions, and the remaining 55.0% of the 289 patients were classified as having an unexplained etiology. Congenital thrombophilia such as protein C (PC) deficiency, protein S (PS) deficiency, antithrombin deficiency, and factor V Leiden mutation was not frequently detected; only one patient had PS deficiency. A reduced factor XII activity was found at a frequency of 4.2%. The frequency of methylene tetrahydrofolate reductase gene C677T mutation in recurrent aborters (0.38) was the same as that found in a fertile control group. Although the prevalence of anti-beta2-glycoprotein I antibody (abeta2-GPI) syndrome was very low (1.7%), patients with a high titer of immunoglobulin G (IgG) class abeta2-GPI, despite anticoagulation therapy, experienced severe fetomaternal complications in subsequent pregnancies. The rate (13.8%) of positive tests for serum IgA class abeta2-GPI in patients with unexplained etiology was higher than that in the controls (0%) (P < .05). We conclude that congenital thrombophilia is rare in Japanese patients who had experienced consecutive spontaneous abortions.

Fibrinolysis in disseminated intravascular coagulation.

Abstract: Studies in experimental models for sepsis, the most common cause of disseminated intravascular coagulation (DIC), have put forward the concept of a procoagulant state that is characterized by thrombin generation exceeding that of plasmin. Convincing evidence indicates that this imbalance between coagulation and fibrinolysis is due to increased levels of plasminogen activator inhibitor type 1 (PAI-1). Levels of this fibrinolysis inhibitor indeed correlate with outcome and severity of multiple organ failure in patients with sepsis, as well as in patients with DIC from other causes. Hence we suggest that PAI-1 constitutes an important target for therapy in patients with DIC.

Clinical Monitoring of Hirudin and Direct Thrombin Inhibitors

Abstract: In addition to heparin, the standard medication for prophylaxis and therapy of thromboembolism, several other substances have been developed and tested for clinical use with the aim of decreasing or eliminating side effects. Most of all, hirudin, a direct antithrombin (AT), has proved to be effective. To define the therapeutic range and to avoid underdosage or overdosage, clinical monitoring is necessary. For monitoring of hirudin, thrombin time (TT) is not suited because of the missing linearity of the standard curve. Activated partial thromboplastin time (aPTT) can be used only in the lower hirudin level range, where the standard curve is quite linear. However, high and toxic hirudin levels cannot be determined using aPTT. Another drawback is a high variation in single measurements and in the normal value of patients. Methods using chromogenic substrates are suited for determination of hirudin in plasma but cannot be used at bedside. Especially for monitoring of hirudin, the ecarin clotting time (ECT) was developed. The standard curve is linear over the entire concentration range. There are no influences by other coagulation parameters or anticoagulants. For both acute clinical situations and long-term monitoring, this method capable of point-of-care therapy (POCT) will be the method of choice.

Recombinant antithrombin: production and role in cardiovascular disorder.

Abstract: Plasma-derived antithrombin (AT) concentrates have been used for the management of hereditary and acquired deficiencies since the early 1980s. Recombinant versions of other blood factors and their derivatives are increasingly becoming available, providing a safe and abundant supply of these important therapeutics. However, the complexity of the AT molecule and the large doses often required for supplementation treatments preclude the use of traditional cell culture bioreactors for recombinant production. The development of a very efficient expression system has been necessary for the cost-efficient recombinant production of AT. Transgenic production, with its ability to yield high levels of heterologous protein and its scale-up flexibility, is an attractive alternative to plasma fractionation. Purification of recombinant AT from the milk of transgenic dairy goats has been developed to provide a homogeneous, well-defined, and abundant supply of this factor. This article describes the production of recombinant AT and aspects of clinical applications of this molecule to cardiovascular disorders.

Advances in the Understanding of the Pathogenetic Pathways of Disseminated Intravascular Coagulation Result in More Insight in the Clinical Picture and Better Management Strategies

Abstract: Disseminated intravascular coagulation (DIC) is a syndrome characterized by systemic intravascular activation of coagulation leading to widespread deposition of fibrin in the circulation. There is ample experimental and pathological evidence that the fibrin deposition contributes to multiple organ failure. The massive and ongoing activation of coagulation may result in depletion of platelets and coagulation factors, which may cause bleeding (consumption coagulopathy). Recent knowledge on important pathogenetic mechanisms that may lead to DIC has resulted in novel preventive and therapeutic approaches to patients with DIC. DIC is not a disease in itself but is a complication of a variety of disorders. However, the pathogenesis of DIC follows similar pathways in almost all of these situations, with a pivotal role of proinflammatory cytokines. The cornerstone of the management of DIC is the specific and vigorous treatment of the underlying disorder. Strategies aimed at the inhibition of coagulation activation may theoretically be justified and have been found to be beneficial in experimental and initial clinical studies. These strategies comprise inhibition of tissue factor-mediated activation of coagulation and restoration of physiological anticoagulant pathways by means of the administration of (activated) protein C concentrate or antithrombin concentrate.

Thrombolytic therapy in children--clinical experiences with recombinant tissue-plasminogen activator.

Abstract: For the improvement of thrombolytic therapy with recombinant tissue-plasminogen activator (rt-PA) in children, more clinical data are needed. We retrospectively analyzed the clinical course of 20 patients (age ranging from 1 day to 16 years) with venous thrombosis (n = 16), arterial thrombosis (n = 2), and purpura fulminans by meningococcosis (n = 2). The venous thromboses were localized in the iliac-femoral veins (n = 9), brachiocephalic-jugular-subclavian veins (n = 6), and the superior caval vein (n = 1). The arterial occlusions were localized in the abdominal aorta and in the left pulmonary artery. Central venous catheters were of pathogenetic importance in seven cases. The patients were treated with rt-PA for 3 hours to 13 days. The dose ranged between 0.2 and 0.5 mg/kg for the initial bolus and 1.0 to 2.0 mg/kg/d for the continuous infusion. Nineteen patients received simultaneously low-dose unfractionated heparin. Complete clot lysis was detected in 11 cases, a partial lysis in 1, and in 8 patients thrombolytic therapy was not successful. An episode of hematemesis in one patient represented the only serious side effect observed in our study. A systemic decrease in fibrinogen concentration was also rare. In conclusion, thrombolysis with rt-PA represents an effective and safe therapy for children at the dosage used.

The nontreatment of childhood ITP (or "the art of medicine consists of amusing the patient until nature cures the disease").

Abstract: The management of childhood acute idiopathic thrombocytopenic purpura is controversial, with recent guidelines highlighting the lack of suitable evidence upon which to base management decisions. Three European centers have used an expectant policy and results over the past decade demonstrate that this is safe and convenient for the majority of children. Adequate parental education about the condition from an experienced specialist is essential, together with open access for children should they develop any problems. A clinical stratification of such patients must be incorporated into any future trials, together with quality of life assessment to discover the impact of restrictions on lifestyle, particularly in adolescents with chronic ITP who may need a different approach.

Vasospasms are characteristic in cases with eclampsia/preeclampsia and HELLP syndrome: proposal of an angiospastic syndrome of pregnancy.

Abstract: We attempted to prove the occurrence of generalized vasospasms in cases with eclampsia/preeclampsia and HELLP syndrome and to suggest the existence of an angiospastic syndrome of pregnancy. Sixteen cases with severe preeclampsia were evaluated. Among them, three cases were complicated by eclampsia and one case with HELLP syndrome. Cranial magnetic resonance angiography (MRA) was performed in 15 cases, and celiac angiography was performed in one case. Vasospasms were observed in 12 cases (75.0%), including all cases involving eclampsia and HELLP syndrome, and cerebral infarction was observed in one case. Typical vasospasms were found by MRA in the middle and posterior cerebral arteries with peripheral ischemic changes in cases with severe preeclampsia and eclampsia. Vasospasms of the hepatic arteries were observed by celiac angiography in a case with HELLP syndrome. Vasospasms are characteristic for patients with eclampsia/preeclampsia and HELLP syndrome, and the existence of an angiospastic syndrome of pregnancy is proposed.

Response of Anticoagulant Pathways in Disseminated Intravascular Coagulation

Abstract: This article describes the microvascular endothelium as both a target and a regulator of events after hemostatic and inflammatory stress. The first section describes the four-quadrant hemostatic system as consisting of the two coagulant and anticoagulant domains that control clot formation and the two fibrinolytic and antifibrinolytic domains that control clot removal. This section concentrates on the anticoagulant domain that operates from the microvascular endothelium and protects it from the effects of hemostatic and inflammatory stress. The next two sections present examples of pure hemostatic and inflammatory stress and illustrate how the four functional domains of the hemostatic systems respond as a unit to these stresses. The following section correlates the response of molecular markers that reflect the activity of the hemostatic system with markers of microvascular endothelial injury to increasing sublethal concentrations of Escherichia coli in the baboon model of E. coli sepsis. The final section correlates the response of these same markers to endotoxin in the human model of endotoxemia. Both sections emphasize that the hemostatic and inflammatory stress and injury to the microvascular endothelium occur at surprisingly low concentrations of E. coli and endotoxin, long before there is evidence of fibrinogen consumption and before the other standard criteria for disseminated intravascular coagulation (DIC) are met. We conclude that this represents a compensated response to stress that can be measured and can be designated as nonovert DIC. We further conclude that as bedside assays of these molecular markers become available they should be helpful in diagnosing and staging early nonovert DIC.

Disseminated intravascular coagulation in sepsis.

Abstract: Disseminated intravascular coagulation (DIC) has been considered a rather rare syndrome characterized by severe bleeding. In fact, both of these beliefs are wrong. Bleeding is fairly rare in DIC. The clotting parameters are usually normal unless the DIC is fulminating. It is usually thought that fibrinogen may be low or absent in DIC. However, afibrinogenemia is rare. Fibrinogen is usually high in DIC because of the high rate of fibrinogen manufacture by the liver in response to stress. DIC is very common and most cases are never diagnosed. This is because it has been hard to find fibrin thrombi in autopsy cases and because acute severe bleeding is uncommon. The reason fibrin thrombi are rare may be because they have been lysed by endogenous fibrinolytic enzymes before the autopsy. The appearance of endogenous fibrinolytic response could be a defense mechanism to lyse the microclots of DIC. In fact, this response is often successful. This defense can be aided by the administration of plasminogen activators that will lyse the clots. Heparin has been used for the treatment of DIC but has proved useless and is, in fact, dangerous. This is because heparin will not dissolve clots and may actually promote platelet agglutination. Administration of plasminogen activators will actually prevent bleeding diathesis.

Hemolytic-uremic syndrome and complement factor H deficiency: clinical aspects.

Abstract: A subgroup of patients with hemolytic-uremic syndrome (HUS) has emerged with complement factor H deficiency. These have come to light because of persistent hypocomplementemia, although the molecular finding by Warwicker et al in one pedigree suggests that not all cases exhibit systemic hypocomplementemia. These patients have an atypical presentation in that they do not consistently have a prodrome of enterocolitis although precipitating factors may include infections of various kinds. Patients usually have severe hypertension early in the course of their illness, tend to relapse, and have a poor prognosis. The age of onset varies from neonates to adults. Plasma exchange and replacement with a plasma product containing factor H seem a reasonable option in the absence of better evidence. Kidney transplantation can meet with recurrence of HUS, but the risk of grafting has yet to be established, and a strategy to overcome graft recurrence is needed. Because of the great rarity of this disorder, a strong case can be made for international registries of atypical HUS cases so that clinical and laboratory investigation is promoted.

Production and clinical development of a Hansenula polymorpha-derived PEGylated hirudin.

Abstract: This article describes the expression of the hirudin gene heterologously in the methylotrophic yeast Hansenula polymorpha, the establishment of an industrial-scale production process and the subsequent clinical development of polyethylene glycol (PEG)-hirudin. PEGylation increases the molecular weight of hirudin, thereby reducing its kidney filtration rate and immunogenicity and increasing its half-life in the circulation.