Showing papers in "Seminars in Thrombosis and Hemostasis in 1996"

Quantitative determination of hirudin in blood and body fluids

Abstract: Recent clinical studies using hirudin as anticoagulant have demonstrated that an efficient method to determine the current blood level of hirudin is imperative for exact dose finding and adjustment Only the exact determination of the hirudin content in blood, performed within a few minutes, prevents overdosage involving side effects or, otherwise, a subtherapeutic dose regimen Therefore, a method for rapid, sensitive, and reproducible measurement of hirudin in blood, plasma, and other body fluids has been developed The method, which is based on coagulation measurement, is called ecarin clotting time (ECT) In this test, ecarin, a purified enzyme of the Echis carinatus snake venom, acts as a prothrombin activator In contrast to the "solid phase" prothrombin activation by prothrombinase, the ecarin-induced prothrombin activation proceeds in an alternative way, ie, without the need of cofactors, resulting in intermediates such as meizothrombin Compared to thrombin, meizothrombin has a lower procoagulant activity, but it still binds hirudin, which leads to the inhibition of meizothrombin Depending on the sample's concentration of hirudin, ecarin forms a residual, nonhirudin-bound amount of intermediates of the prothrombin-thrombin conversion that are able to concentration-dependently convert fibrinogen to fibrin There is an excellent linear correlation between ECT prolongation and the hirudin content of the sample in a range from 50 to 5,000 ng/mL blood or plasma This allows immediate measurement not only of the therapeutic blood level of hirudin, but also of its concentration in blood following under- or overdosage The ECT method is nearly independent of variations in the sample's content of fibrinogen (from 60% to 100%) and prothrombin (from 20% to 100%) Heparin is not able to catalyze the very low antithrombin inhibition of meizothrombin Therefore, it is also possible to determine hirudin in blood containing varying amounts of heparin Another advantage of the method is that it can be applied to different mechanical measuring systems used in coagulation diagnostics

Regulation of thrombin-mediated endothelial cell contraction and permeability.

Abstract: A variety of physical forces exist in a dynamic equilibrium in the vascular endothelium (EC) monolayer and serve to maintain EC responsiveness while preserving the integrity of the EC monolayer and barrier properties. Thrombin has potent effects on EC permeabilities disrupting the equilibrium between tethering forces (cadherins, focal adhesion plaque) and forces that increase centripetal tension primarily via myosin light chain (MLC) phosphorylation. Like other EC effects, thrombin-induced MLC kinase (MLCK) activation is dependent upon receptor proteolysis, Ca2+ mobilization, and activation of protein kinase C (PKC). While EC gap formation is central to barrier dysfunction and dependent upon activation of MLCK, (which phosphorylates MLC) an obligatory event in smooth muscle cell contraction, little is known regarding the events that reverse inflammatory responses, halt the contractile response, and initiate relaxation. However, as these events likely include MLC dephosphorylation, further examination of the processes that regulate MLC protein phosphatase activity, focal intercellular junctions, and extracellular matrix adhesions is needed. These investigations should yield new information as to how receptor occupancy is transduced into specific cellular responses, such as increased permeability, which promotes pathological vascular processes such as tissue edema formation and organ dysfunction.

Disseminated intravascular coagulation: objective clinical and laboratory diagnosis, treatment, and assessment of therapeutic response.

Abstract: Current concepts of the etiology, pathophysiology, clinical and laboratory diagnosis and management of fulminant and low-grade disseminated intravascular coagulation (DIC) have been presented Considerable attention has been devoted to interrelationships within the hemostasis system Only by clearly understanding the pathophysiological interrelationships can the clinician and laboratory scientist appreciate the divergent and wide spectrum of often confusing clinical and laboratory findings in patients with DIC Objective clinical and laboratory criteria for diagnosis of DIC have been delineated, thus avoiding needless confusion and empirical decisions regarding the diagnosis Many therapeutic decisions to be made are controversial and will remain so until more is published about specific therapeutic modalities and survival patterns Also, therapy must be highly individualized depending on the nature of DIC, age, etiology of DIC, site and severity of hemorrhage or thrombosis, and hemodynamic and other clinical parameters Also presented are clear criteria for severity of DIC and objective criteria for defining a response to therapy Also, since it is often difficult for the individual physician to decide when to stop expensive therapy, objective criteria by which therapy may be stopped when continuation is likely fruitless are presented as a guideline

Tissue transglutaminase and factor XIII in cartilage and bone remodeling.

Abstract: While it is well established that factor XIII functions in crosslinking of the fibrin clot during blood coagulation and in wound healing, the physiological role of tissue transglutaminase is still unclear. Recent studies suggest that the expression of tissue transglutaminase correlates with (terminal) differentiation of cells and that the enzyme may play a role in extracellular matrix remodeling. In cartilage, tissue transglutaminase expression is restricted to hypertrophic chondrocytes and the enzyme is externalized at a distinct step in the chondrocyte maturation program. Upon activation by Ca2+, the transglutaminase modifies matrix constituents in a way that might predispose the matrix for the subsequent mineralization. Crosslinks of the structure gamma-glutamyl-epsilon-lysine are also abundant in bone matrix, but the transglutaminase expressed by osteoblasts and presumably involved in crosslinking of newly formed osteoid is likely to be distinct from both tissue transglutaminase and factor XIII. Matrix proteins thought to be crosslinked by transglutaminases in cartilage and bone matrix include glycoproteins such as osteonectin, osteopontin, fibronectin, fibrillin, and collagens II, III, V, and XI. Expression of the A subunit of factor XIII is restricted to megakaryocytes in the bone marrow cavity, and factor XIIIa is abundant in platelets that probably provide the major source for factor XIII in plasma.

Thrombin and vascular smooth muscle cell proliferation: implications for atherosclerosis and restenosis.

Abstract: Despite long-standing knowledge about the relationship between thrombosis and atherosclerosis, the specific role of thrombin in modulating atherosclerosis and the response to vascular injury is not well understood. Thrombin receptor stimulation in vitro signals many cellular events that are associated with the response to vascular injury (atherosclerosis) in vivo. Proliferation of smooth muscle cells (SMCs) is an important component of the response to vascular injury. We have previously shown that human alpha-thrombin and the 14-amino acid human thrombin receptor-activating peptide (huTRAP-14) stimulate proliferation of cultured rat aortic SMCs. However, thrombin-induced SMC proliferation demonstrates delayed kinetics relative to platelet-derived growth factor (PDGF-BB, another potent SMC mitogen). Several mechanisms may be responsible for these delayed kinetics in vitro, including production of necessary secondary growth factors and thrombin-induced upregulation of its receptor. In vivo studies have demonstrated that thrombin inhibition limits the response to vascular injury in a hypercholesterolemic rabbit model of focal femoral atherosclerosis. However, this effect does not appear to be mediated by effects on early SMC proliferation. In this discussion, we will address the mechanisms of thrombin-induced SMC proliferation in vitro and apply this knowledge to our understanding of the role of thrombin inhibition in limiting the response to vascular injury in vivo.

Hirudins: from leeches to man

Abstract: Hirudins are a group of highly homologous polypeptides from the medicinal leech, Hirudo medicinalis. They have an extremely high and specific affinity for thrombin and are consequently potent anticoagulants. They inhibit platelet aggregation induced by thrombin and efficiently inhibit thrombin when it is bound to a fibrin clot. Recombinant hirudins have now been produced and have been used to show a wide range of effects on thrombosis in animal models. They are particularly effective against venous-type thrombi and have greater effects than heparins on the platelet-rich thrombi in arteries. Recombinant hirudins are currently undergoing clinical trails in deep venous thrombosis and acute coronary syndrome and as an adjunct to thrombolysis in myocardial infarction. Results from pilot trials indicate promising advantages over the currently used antithrombotic agents. Larger clinical trails have been initiated and we await with great interest their imminent publication.

ETRO working party on factor XIII questionnaire on congenital factor XIII deficiency in Europe : Status and perspectives

Abstract: A questionnaire was sent out in 1993 to more than 350 European institutions caring for patients with hemorrhagic disorders with the request to provide data of patients with congenital factor XIII deficiency, to pursue the following aims: (1) establish a registry of congenital factor XIII deficiency patients, (2) promote exchange between clinicians and basic researchers, (3) improve diagnostic and therapeutic approaches, and (4) stimulate research on gene defects and their impact on factor XIII function. So far, 72 patient questionnaires from 60 families have been collected. Their bleeding pattern is typical, with frequent involvement of the umbilical cord and the central nervous system. Forty-nine patients receive regular factor XIII replacement, but obviously some patients with mild symptoms do not require prophylactic substitution, despite low factor XIII levels. On the other hand, 18 patients had factor XIII activities of > or = 5% of normal, but only 3 of those patients were reported to have no bleeding symptoms. Furthermore, 17 symptomatic, apparently heterozygous relatives in eight families were observed. Seven out of 30 females aged over 18 years had experienced spontaneous abortions; wound healing problems were seen in 26 patients. Currently, a second questionnaire is being distributed to obtain more detailed information on bleeding and other symptoms, diagnostic approaches, and exclusion of concurrent other bleeding diatheses. Future activities will be validation and standardization of assays, and study of gene defects and their impact on the structure of factor XIII and symptoms of patients. We intend to expand the survey to countries outside Europe.

Gene manipulation and transfer of the plasminogen and coagulation system in mice

Abstract: The blood coagulation and the fibrinolytic (or plasminogen/plasmin) systems determine the balance between the formation and dissolution of blood clots, but in addition contribute to the pathogenesis of various cardiovascular disorders such as thrombosis, atherosclerosis, and restenosis. Furthermore, they participate in a variety of other (patho)biological processes such as embryonic development, reproduction, wound healing, cancer, and brain function. Two recently developed technologies, gene targeting and gene transfer, that allow manipulation of the genetic balance of these proteinase systems in a controllable manner have allowed a more definitive elucidation of the biological role of these systems. This review summarizes the insights that have been obtained from the gene targeting studies and discusses the use of adenovirus-mediated transfer of fibrinolytic genes to study and possibly to develop novel strategies for the treatment of restenosis and thrombosis.

The Fibrinolytic System in Neoplasia

Abstract: During activation of the fibrinolytic system plasminogen is converted to plasmin by tissue plasminogen activator (t-PA) or urokinase-type plasminogen activator (u-PA). t-PA is predominantly released from endothelial cells, u-PA primarily by renal parenchymal cells. The activation of plasminogen is regulated by plasminogen activator inhibitor-1 (PAI-1), plasmin is controlled by alpha 2-plasmin inhibitor. The fibrinolytic system is not only involved in the intravascular dissolution of fibrin (thrombi), it also plays a vital role in normal physiologic reproduction, wound repair, angiogenesis, and tissue remodeling. Fibrinolysis is also a vital component in the pathogenesis of neoplastic disease. It is essential in releasing cells from their primary site of origin, providing nutrition for neoplastic cell growth and promoting cell mobility and motility. In neoplastic cells the degradation of the extracellular matrix proteins is facilitated by excessive expression of u-PA, t-PA, and u-PAR. In many forms of carcinoma increased expression of u-PAR and u-PA is associated with significantly shorter survival. Greater expression of u-PA in breast cancer cells, for example, is associated with shorter survival and increased relapse rate. Progressively aggressive neoplastic cells evidence high expression of u-PA and u-PAR activities, variable expression of t-PA, and enhanced PAI-1 and PAI-2 activities. In acute nonlymphocytic leukemias, poor outcome correlates with high t-PA levels. In acute progranulocytic leukemia there is a high incidence of DIC. Neoplastic prostatic tissue also expresses high u-PA activity and the more aggressive the cell line, the greater the number of u-PAR and the higher the u-PA activity. In gynecologic malignancies, a greater expression of u-PA in combination with cathepsin D is associated with widespread disease and poor prognosis. High u-PA values were also seen in patients with brain, gastric, and hepatic malignancies. It is evident that the plasminogen-plasmin system is a vital component in the biology of neoplastic disease and that it is, in theses conditions, in no way beneficial to the host.

Factor XIII deficiency: pathogenic mechanisms and clinical significance.

Abstract: Congenital factor XIII deficiency is a rare disease, but has provided valuable information on the physiological role of factor XIII and the benefit of factor XIII replacement therapy. It could be shown that not only homozygous patients but also heterozygotes are at risk for bleeding complications. Acquired factor XIII deficiency, however, is much more common, and preliminary studies suggest a lack of factor XIII to be an important feature of various diseases. In acute states and severe hemorrhages, replacement therapy with factor XIII concentrates is recommended. Recent progress in assay methods and future clinical studies should help to evaluate the therapeutic potential of factor XIII.

Thrombin, its receptor and protease nexin I, its potent serpin, in the nervous system.

Abstract: The multifunctional serine protease, thrombin, the principal component of the blood coagulation cascade, is also active in nervous system growth and maintenance. In neural tissue culture, it prevents neurite outgrowth and modulates morphologic changes in both neurons and astrocytes. In recent studies, we found that it mediates polyneuronal synapse elimination, both in vivo and in vitro. Of relevance to neurologic disease, as well as to development, evidence also implicates thrombin in apoptosis of these cells. As with other serine proteases, thrombin is in "balance" with one or more endogenous protein inhibitors, members of the serpin superfamily of proteins. The most potent vertebrate inhibitor for thrombin is protease nexin I (PNI), which regulates thrombin's effect by forming post-translational, covalent complexes with the protease. We review some of the nervous system effects of the thrombin:PNI balance, and also present results of a recent study of this balance after peripheral nerve injury. We measured thrombin and prothrombin activity in extracts from adult mouse sciatic nerve using a specific chromogenic assay. We also performed reverse transcription polymerase chain reaction of RNA from nerve crush samples. We found a burst of activity at 3 days following injury distal to the crush site that was inhibited by thrombin specific inhibitors. It is possible that a significant fraction of the increased prothrombin in injured nerve was synthesized locally. Active PNI levels increased in these crush samples 6 to 9 days after the thrombin induction. These data suggest that nerve injury first induces the synthesis of prothrombin, which is subsequently converted to active thrombin. Nerve crush-induced thrombin is followed by the generation of functionally active PNI and may be directly responsible for its induction. These results suggest that the balance between serine proteases and their serpins is dysregulated during nerve injury and support a role for its reestablishment in nerve damage repair.

Are the available low-molecular-weight heparin preparations the same?

Abstract: Low molecular weight heparins (LMWHs) are now universally accepted as drugs of choice for post-surgical prophylaxis of DVT. Currently these agents are also being developed for therapeutic and cardiovascular indications. Because of manufacturing differences, each of the LMWHs exhibit distinct pharmacologic and biochemical profiles. The specific activity of these agents in the anticoagulant assays ranges from 35 to 45 anti-IIa U/mg whereas the specific activity in terms of anti-Xa units is designated as 80 to 120 anti-Xa U/mg. These LMWHs are capable of producing product specific dose and time dependent antithrombotic effects in animal models of thrombosis. While the ex vivo effects are initially present at dosages that are antithrombotic, these agents have been found to produce sustained antithrombotic effects without any detectable ex vivo anticoagulant actions. In the experimental animal models and in various clinical trials, these agents have also been found to release tissue factor pathway inhibitor (TFPI) after both intravenous (i.v.) and subcutaneous (s.c.) administration. Repeated administration of LMWHs produces progressively stronger antithrombotic effects; however, the hemorrhagic responses vary and are largely dependent on products. The release of TFPI following i.v. and s.c. administration in a primate model also demonstrated the product individuality and the relevance of this inhibitor to the actions of LMWHs. The effect of repeated administration mimicking the post-surgical prophylaxis of DVT also exhibited product based augmentation of the antithrombotic or hemorrhagic effects. Antithrombotic and hemorrhagic studies are reported that compare the pharmacologic profile of some of the available LMWHs. Product individuality in terms of relative potency in different assays and the failure of standardization protocols to provide any guidelines for product substitution and prediction of the clinical effects is also addressed.

Thrombolytic therapy in acute myocardial infarction.

Abstract: Defibrotide is a polydeoxyribonucleotide that possesses profibrinolytic and cytoprotective activities. These properties have been associated with its capacity to induce the release of prostacyclin and tissue plasminogen activator (t-PA) from endothelial cells. In the present study, the bolus administration of defibrotide in humans was able to induce (100-800 mg) a dose-dependent decrease in plasminogen activator inhibitor (PAI) (from 19.4 +/- 7.11 to 7.20 +/- 6.41 AU/mL) and an increase in t-PA (from 3.70 +/- 0.96 to 4.50 +/- 1.20 IU/mL) and in the stable prostacyclin derivative 6-keto-PGF1 alpha (from 18.83 +/- 3.83 to 26.75 +/- 8.48 pg/0.1 mL) in the venous blood. In a second part of the study, defibrotide has been shown to inhibit dose-dependently (10-100 microns) neutrophil activation in vitro: it decreased lysosomal enzyme release and superoxide anion and chemiluminescence production induced by the oligopeptide fMLP and the ionophores A23187 and ionomycin. The increase in extracellular calcium concentration from 0.5 to 2 mm antagonized the inhibitory effect of the drug. Defibrotide was able to reduce the cytosolic free calcium increase induced by specific stimuli by blunting calcium entry. Such an inhibitory activity of defibrotide was antagonized by theophylline, an adenosine receptor antagonist. The study confirms some pharmacological activities of defibrotide (release of t-PA and prostacyclin in vivo), and it also suggests that the compound blocks Ca2+ entry into the cells, possibly by interfering with the adenosine receptors.

Molecular Interactions of Thrombin.

Abstract: Thrombin possesses at least three independent binding sites for substrate, inhibitor, and co-factor molecules, four counting the Na+ ion binding site. The S1 subsite of the active site is specific for an arginine side group, while S2 is a more extended apolar site. The highly electropositive S' subsites of the fibrinogen exosite circumnavigate about a third of the thrombin surface, although evidence suggests molecular recognition of a tetra- or pentapeptide sequence is sufficient for binding. Another highly electropositive region of thrombin that binds the second kringle of prothrombin is the heparin binding site. All three of these sites display distinct binding modes with different molecules. These can arise from tolerance of imprecision of binding and/or from reversal of ligand main chain direction in active site and fibrinogen exosite binding. Preliminary indications suggest similar principles may apply to the heparin site. Such varied behavior easily accounts for the diversity of thrombin functions at the molecular level. The complexity of the behavior is compounded even more by a Na+ ion binding site that produces a procoagulant fast form of thrombin. The slow form (in the absence of Na+ ion) is anticoagulant.

Physiopathological significance of catalytic phospholipids in the generation of thrombin.

Abstract: Thrombin generation is the culminating event of the coagulation cascade. It is initiated after the expression of tissue factor by endothelial cells and monocytes exposed to thrombogenic stimuli. Anionic phospholipids, chiefly phosphatidylserine, are necessary for the optimal activity of tissue factor and completion of the clotting process. They display a catalytic potential by allowing the formation of the characteristic enzyme complexes at the membrane surface. Platelets are viewed as the main source of procoagulant phospholipid referred to as platelet factor 3. The plasma membrane of resting cells presents an asymmetrical distribution of phospholipids, aminophospholipids being sequestered in the inner leaflet. Procoagulant phospholipids become available at the outer surface after cell stimulation. The collapse of the membrane asymmetry is thought to promote a phospholipid scrambling accompanied by the shedding of microparticles. The plasma membranes of such vesicles bear irreversibly externalized procoagulant phosphatidylserine and contain glycoproteins that testify to their tissue origin. Hence, microparticles could disseminate a dual procoagulant and adhesive potential. Thrombin autoamplification is exerted through feedback activation loops involving either coagulation factors or platelets. This article details the mechanisms by which procoagulant phospholipids promote the generation of an excess of thrombin. A new pharmacological approach of thrombosis is presented, based on the control of the exposure of procoagulant phospholipids and membrane microparticle shedding.

Structure and Function Studies of Factor XIIIa by X-Ray Crystallography

Abstract: The three-dimensional structures of several forms of the factor XIII A subunit have been determined using single crystal x-ray diffraction methods. Our crystallographic studies have provided the first detailed structural view of the factor XIII A subunit and information that is useful for understanding transglutaminase function. We have identified a conserved Cys314-His373-Asp396 catalytic triad of residues in the active site of the molecule and a number of other conserved residues that may play important roles as well. The calcium and strontium structures have revealed several conserved acidic residues (Asp438, Glu485, and Glu490) involved in ion binding. We have also been able to use our crystal structures as scaffolds to model the possible structural effects of missense mutations that have been identified in factor XIII-deficient patients.

The Macrophage and Fibrinolysis

Abstract: The monocyte/macrophage plays a central role in fibrinolysis. Cell-surface of components of the plasminogen activator system leads to the elaboration of plasmin, which facilitates degradation of fibrin in the pericellular environment, as well as activation of matrixins, which promote degradation of matrix components. Fibrin degradation also occurs by way of a proteolytic system within the macrophage lysosome that does not involve plasmin. This alternate pathway involves first the binding of fibrin(ogen) to the surface integrin Mac-1 (CD11b/CD18) followed by internalization of the complex into the lysosome where the aspartyl protease cathepsin D degrades the protein. These molecular events underlie the many physiologic and pathophysiologic processes in which the monocyte/macrophage is involved, including adhesion, migration, matrix degradation and remodeling, wound healing, fibrinolysis, and atherosclerosis.

Plasma levels of the molecular markers of coagulation and fibrinolysis in patients with peripheral arterial disease.

Abstract: In 103 patients with peripheral arterial disease (PAD) of the lower limbs, coagulation and fibrinolytic parameters were evaluated to identify hemostatic abnormalities characteristic of this patient population. PAD was defined as clinically stable Leriche stage 2 (based on clinical history, peripheral pulses, ankle-arm index, and treadmill test) for at least 3 months, walking distance > 100 m, and no other major illnesses, rest pain, or trophic lesions. Defibrotide, a polydeoxyribonucleotide derivative with vascular effects, was administered to the patients as part of a multicenter trial. The PAD patients exhibited a prothrombotic state as evidenced by high D-dimer in all but 24% of the patients (average 797 +/- 802 vs. 163 +/- 54 ng/mL normal population; p < 0.001) and high thrombin-antithrombin III complex (TAT) levels (10.2 +/- 8.9 vs. 2.5 + 1.5 ng/mL; p < 0.001) with low to normal levels of protein C (86 +/- 25 vs. 102 +/- 18%; p < 0.01) and plasminogen activator inhibitor-1 (PAI-1) antigen (5.9 +/- 4.5 vs. 1.3 + 0.7 ng/mL; p < 0.001) were elevated in 79% of the patients. These results suggest that there is ongoing thrombosis in the majority of PAD patients. Differences from normal controls were observed for t-PA, PAI-1, protein C, and protein S; however, it is not certain that the thrombosis in patients with PAD is due to these factors.

Platelet aggregation and coagulation inhibitors in leech saliva and their roles in leech therapy.

Abstract: Prolonged bleeding by the host after the leech ceases to feed and several reports that the use of leeches restores blood flow in the microcirculation after plastic surgery led us to search for inhibitors of platelet aggregation in Hirudo medicinalis saliva. Dilute leech saliva was collected by phagostimulating starved leeches with a solution of arginine in saline. The saliva is shown to inhibit human platelet aggregation induced by thrombin, collagen, adenosine diphosphate (ADP), epinephrine, platelet activating factor (1-O-alkyl-2-acetyl-sn-3-glycerophophoryl choline [PAF]), and arachidonic acid. We have isolated the PAF inhibitor and found it to be an amphipathic phosphoglyceride. We have also purified apyrase adenosine triphosphate ([ATP] diphosphohydrolase), which inhibits ADP-induced platelet aggregation, and have described collagenase. Besides well-known hirudin, Hirudo saliva contains a potent inhibitor of coagulation factor Xa. We also report antiaggregant and anticoagulant activities in the crop content of the closely related Nile leech, Limnatis nilotica. Anticoagulants of hematophagous species are surveyed. We have used medicinal leeches in plastic surgery for decompression of skin flaps and in patients with postphlebitic syndrome and peripheral arterial occlusions. Preliminary results indicate certain beneficial effects of leech therapy.

Fibrin degradation in the synovial fluid of rheumatoid arthritis patients: a model for extravascular fibrinolysis.

Abstract: The patterns of degradation and the influence of factor XIII polymerization on fibrin stability were examined in vitro following incubation with leukocyte elastase. In vivo experiments, various factor XIII-polymerized fibrin clots were implanted subcutaneously in mice to evaluate the stability of clots in the extravascular space. Both in vitro and in vivo lysis proceeded faster with nonpolymerized fibrin and was not influenced by the presence of cross-linked alpha 2-plasmin inhibitor. In vivo lysis of implanted clots was prevented by elastatinal, powerful elastase inhibitor, suggesting that granulocyte elastase is chiefly responsible for clot lysis in the extravascular space. To further extend investigations on the mechanisms of fibrinolysis in tissues, we evaluated fibrin and its degradation products in the synovial space. Expression of factor XIII in synovial cells and activities of coagulation factors, fibrinolytic enzymes, and inhibitors were investigated in the synovial fluid of rheumatoid arthritis patients. Immunohistochemical analysis showed deposits of insoluble fibrin on synovial membranes and pannus to an extent related to the progression of the disease. Factor XIII was expressed by fibroblasts and macrophages in the early stages of the disease, whereas in advanced stages factor XIII staining was associated with fibrin. The reduction of certain coagulation factors and high level of thrombin-antithrombin complexes in synovial fluid show a steady activation of the coagulation cascade. The evaluation of fibrinogen degradation products and the pattern of degradation of synovial fibrin(ogen) suggest the participation of leukocyte elastase in fibrin(ogen) lysis in synovial tissue of rheumatoid arthritis.

Determinants of substrate specificity for factor XIII.

Abstract: Plasma factor XIIIa (A*2) is a regulator in balancing the opposing coagulation and fibrinolytic processes. Its enzymatic activity is to catalyze epsilon-(gamma-glutamyl)lysyl bonds between certain substrate molecules to link them by strong bonds. The primary physiological substrates are crosslinks between the gamma and alpha chains of fibrin that produce gamma-gamma-dimer and alpha-polymer, between alpha 2-plasmin inhibitor (alpha 2-PI) and alpha chains of fibrin, and between fibronectin and fibrin. We have characterized a unique factor XIII antibody that is specific for the middle 54-kDa section of A*2. It does not react with the zymogen (A2) or the inactive intermediate (A'2), and it does not inhibit the active center, as do most patient antibodies to factor XIII. This antibody inhibits the formation of A*2-fibrin complexes. Because of this specificity, the antibody was used to study other substrate interactions. It inhibited formation of fibronectin-factor XIIIa complexes, similarly to fibrin, and there was very little crosslinking of fibronectin to a fibrin clot. However, the amount of alpha 2-PI crosslinked to a fibrin clot was normal. It was concluded that this antibody interferes with exosite binding of fibrin and fibronectin interferes with exosite binding of fibrin and fibronectin in a similar way, while at least one critical exosite binding domain for alpha 2-PI is different from those of the other two substrates. Furthermore, with this antibody, it was shown that both alpha 2-PI-alpha chain crosslinking and alpha-polymer formation are necessary to normalize the rate of fibrinolysis.

Intracellular factor XIII: cellular distribution of factor XIII subunit a in humans.

Abstract: In addition to the plasma where factor XIII subunit A is present as part of the heterodimers of the tetrameric fibrin-stabilizing factor, homodimers of factor XIII A can also be detected in cellular localization in different human tissues. Experimental findings verified that these cells belong to the megakaryocyte/ platelet and the monocyte/macrophage cell lines. Additionally, factor XIII A is present in hepatocytes localized mainly around central veins in the liver and occasionally expressed in certain malignantly transformed cells. It is reasonable to suppose that factor XIII A acts not only as a member of the clotting cascade, but also as a cellular enzyme that can catalyze molecular interactions intracellularly (during the assembly/reassembly of the cytoskeleton) or extracellularly (in the connective tissue matrix around the cells containing it).

Factor XIII in chronic inflammatory bowel diseases.

Abstract: Severe acute inflammation in chronic inflammatory bowel disease is associated with large wound areas and ulcerations that show spontaneous hemorrhage or marked friability. Therefore, an enormous potential of hemostasis and wound healing is required. Coagulation studies demonstrate a deficiency of factor XIII that is important for both clot formation and wound healing. Consequently, the substitution of factor XIII may be beneficial; the first case reports present favorable clinical results. In a prospective pilot study, we treated 12 patients with therapy-resistant ulcerative colitis. The colitis activity index (CAI) and the endoscopic score (ES) according to Rachmilewitz were elevated; all patients suffered from hematochezia. After substitution therapy with factor XIII concentrate (1,250 U/d) the stool frequency dropped and no further hematochezia was detected. The CAI and the ES declined highly significantly. Because of these encouraging results two placebo-controlled multicenter trials have been initiated. In the first study, patients with acute stage of ulcerative colitis associated with severe intestinal blood loss are treated with two different dosages of factor XIII concentrate (1,250 and 500 U/d, respectively) or placebo for 10 days. In the second trial, patients with therapy-resistant ulcerative colitis with a lack of remission in spite of a consequent therapy for 2 weeks are included; factor XIII concentrate or placebo is administered for 10 days. The aim of both trials is an end of intestinal bleeding and the fostering of a more effective wound-healing process.

Thromboembolic diseases: biochemical mechanisms and new possibilities of biological diagnosis

Abstract: The diagnosis of thromboembolic diseases is still difficult to establish before the occurrence of the pathological event, although it is now known that they are the result of a progressive alteration of the cardiovascular system. Introduction of new diagnostic tools for the evaluation of the thromboresistance capacity of the body or for the measurement of molecular markers allows the testing of the body defenses against thrombosis which is becoming a routine clinical diagnosis. Antithrombin III (AT III), protein C, protein S, and parameters of fibrinolysis have been recognized to be very important anticoagulant proteins and regulators of thrombin formation and thrombus extension. Furthermore, a normal factor V is necessary for the normal function of the protein C pathway. The presence of a factor V mutation leads to the activated protein C resistance syndrome. However, the major incidence of thrombotic events concerns the overall population. It has been epidemiologically related to the existence of risk factors producing blood activation, which progressively saturates the body's thromboresistance. This period is clinically silent for a long time. The new molecular markers recently introduced can show the existence of a preclinical state of blood activation at the plasma level (fibrinopeptide A, thrombin-antithrombin complexes, modified antithrombin III, fragments 1 + 2 of prothrombin, D-dimer) or at the platelet level (B-thromboglobulin, platelet factor 4, and thrombospondin), and promising developments concern the endothelial level (soluble thrombomodulin). The most universally used blood activation test is the D-dimer assay. This analyte has become very popular in past years for its high sensitivity, its long half-life, and its easy detection directly on citrated plasma. Its negative predictive value (in deep venous thrombosis or pulmonary embolism) as well as its use for monitoring of thrombotic risk in the post-operative period have been well documented clinically. New investigations are initiated to find analytes reflecting endothelial damage, an early platelet activation, or the involvement of blood cells (mainly monocytes and neutrophils) in abnormal processes. It also becomes possible to evaluate directly pathological causes inducing blood activation, such as the presence of antiphospholipid antibodies or other autoimmune antibodies.

The thrombin receptor in the nervous system.

Abstract: Neurite retraction and reversal of astrocyte stellation triggered by the serine protease thrombin are receptor-mediated events. This article summarizes the current knowledge about the cellular effects that are induced by thrombin and its receptor in neural cells. The data presented show that the thrombin receptor messenger RNA is expressed in cultured astrocytes and that the reversal of stellation caused by thrombin in these cells is prevented by the protein kinase inhibitor staurosporine. Peptides based in sequence on the tethered ligand domain of the thrombin receptor were shown to mimic the effect of thrombin in most systems investigated. Platelets of some species, however, aggregate only in response to thrombin but not to the peptides. This observation is confirmed here. Rodent receptor-activating peptides did not cause aggregation of rat or mouse platelets. In contrast, all peptides triggered reversal of stellation in rat astrocytes and neurite retraction in mouse neuroblastoma cells, supporting the proposed mechanism of cleavage-induced receptor activation in neural cells. Finally, evidence is presented that serum withdrawal causes a decrease in the amount of the thrombin receptor mRNA in different types of neuronal cells. The possible role played by the thrombin receptor in the nervous system is discussed.

Platelet-associated factor XIII in platelet activation, adhesion, and clot stabilization.

Abstract: Platelet-associated factor XIII provides a means by which to promote clot stabilization and platelet interaction with proteins of the coagulation and fibrinolytic pathways. In addition to its intracellular role within the platelet cytoplasm, activated factor XIII will bind to the surface of activated platelets. These platelets then participate in cell-cell or cell-clot interactions, thereby increasing the local concentration of factor XIIIa. The platelet-associated factor XIIIa may increase the amount of crosslinking in a fibrin clot, thereby contributing to the aging of the clot and the reduction in the degree of platelet binding. Clot resistance to fibrinolysis is enhanced by platelet factor XIIIa-mediated crosslinking of alpha 2-antiplasmin to fibrin. The binding of factor XIIIa to the platelet surface requires the activation of the platelet fibrinogen receptor, glycoprotein IIb-IIIa. Thus, platelet-associated factor XIIIa may be used as a marker of in vivo platelet activation. Since half of the factor XIII present in blood is provided by the platelets, it is not surprising that this form of factor XIII plays an important role in hemostasis.

An integrated view of the activities of defibrotide.

Abstract: Defibrotide is a polydeoxyribonucleotide sodium salt extracted from mammalian organs. Its mean molecular weight is 15,000-30,000 daltons. Defibrotide contains aptamers, i.e., single-stranded polynucleotides with a well-defined base sequence and composition (5'-GGTTGG-ATT-GGTTGG-3' and 5'-GGTTGG-ATC-GGTTGG-3') that bind thrombin. For the time being, these aptamers are the only ones that have been identified in defibrotide, but there may also be other aptamers that bind proteins other than thrombin. Defibrotide has no anticoagulant activity, but in some other aspects it probably parallels heparin. Heparin is a sulfated polysaccharide sodium salt with a mean molecular weight ranging from 5,000 to 40,000 daltons extracted from mammalian organs. It contains disaccharide sequences of well-defined structure and frequency. Heparin binds an array of proteins, enzymes, and growth factors and shows inhibitory or stimulatory or protective activity. Defibrotide has a spectrum of interesting pharmacological properties that make this drug very useful for the treatment of arterial and venous thrombotic diseases. In fact, defibrotide has profibrinolytic, antithrombotic-thrombolytic, anti-ischemic, and antiatherosclerotic activity and protective activity in shock. What have all the above activities to do with a single drug? The explanation is that atherosclerosis, myocardial, renal, and liver ischemia, hemorrhagic and septic shock, and shock induced by occlusion of splanchnic artery and thrombosis are different facets of the same entity: the polyhedric inflammatory process.

The role of inflammatory cells and their proteases in extravascular fibrinolysis.

Abstract: Extravascular fibrin formation and dissolution is a pivotal event in numerous inflammatory and malignant diseases. In inflammatory cells such as monocytes/macrophages, neutrophil granulocytes appear to interact intimately with hemostasis and regulate the activity of the cascade systems of coagulation and fibrinolysis. Proteases such as neutrophil elastase are thought to influence components of hemostasis, and furthermore provide an alternative pathway of fibrinolysis. Histological, experimental, and clinical data suggest that extravascular fibrinolysis, mediated both by the plasmin system and by proteases like neutrophil elastase, is a prominent finding in various diseases such as lung cancer, chronic inflammatory bowel disease, vasculitis and connective tissue disease, bacterial sepsis, and septic shock.

Thrombin-mediated events implicated in mast cell activation.

Abstract: It has been suggested that mast cells contain receptors for thrombin because binding of thrombin to peritoneal mast cells (PMCs) results in heparin release. Peritoneal mast cell responsiveness to different thrombin forms was examined by measuring ion conductance, intracellular pH, the concentration of cyclic guanosine monophosphate (cGMP), and release of histamine. Several types of receptors for thrombin are suggested by the results, which demonstrate that: (1) PMCs responded to alpha-thrombin and diisopryopyl-phosphoryl-alpha-thrombin (DIP-alpha-thrombin), but not to gamma-thrombin, by activation of Na/H exchange in reactions involving protein kinase C and by a simultaneous elevation in cell conductance and capacitance; (2) the initial 1-nmol/L alpha-thrombin-induced acidification of PMC cytoplasm was absent in Ca-free medium, and higher doses of alpha-thrombin induced a biphasic reaction (acidification preceeded alkalinization); and (3) PMC stimulation by alpha-thrombin at low concentrations ( 1 mumol/L induced the acceleration of histamine release.

Management of acquired coagulation disorders in emergency and intensive care medicine

Abstract: Coagulation disorders usually confront the emergency physician as bleeding episodes or as abnormalities of laboratory tests. Bleeding has to be treated aggressively, while pathological coagulation tests should be related to a more differentiated diagnosis at first. The most common causes of acquired coagulation disorders are liver disease, vitamin K deficiency, and disseminated intravascular coagulation (DIC). More rarely, inhibitors, external factors such as drugs or extracorporeal circulation, or other diseases such as amyloidosis are present. Since localized hemorrhage is the most common bleeding source in liver disease, endoscopic and surgical therapeutic measures, respectively, are warranted. Careful and balanced substitution therapy according to laboratory findings should be initiated simultaneously and should consist of fresh frozen plasma (FFP), which contains all components of the coagulation system physiologically balanced. Prothrombin complex concentrates should be used in emergency situations only, keeping their potential hazards in mind. Adequate vitamin K substitution is indicated in liver disease as well as in coagulopathy due to vitamin K deficiency. Management of DIC primarily consists of aggressive treatment of the underlying disease. Substitution therapy is difficult and should be carefully monitored by the adequate laboratory tests. FFP is the adequate source of both procoagulants and inhibitors but may cause certain problems. Heparin therapy can be beneficial but is not recommended generally. Antithrombin III substitution cannot be assumed as established therapy so far. Inhibitors can lead to bleeding, but the most common inhibitor, lupus anticoagulant, rather predisposes to thrombosis. In bleeding patients with inhibitors against single clotting factors, treatment consists of adequate substitution before initiating the diagnostic workup.