Trousseau is considered to be the first to have recognised a relation between venous thromboembolism (VTE) and malignancy. Illtyd James and Matheson in 1935 were the first in the Anglo-Saxon literature, at least to our knowledge, to report the observation of a patient with occult cancer at time of his venous thrombosis. Meanwhile, cancer has become an established risk factor for VTE. The prevalence of concomitant cancer in patients presenting with VTE varies considerably between the studies due to differences in, for instance, threshold of suspicion, screening methods, and characteristics of the patients like age. A consistent observation is the low prevalence of concomitant cancer in patients with secondary thrombosis, comparable to the prevalence in the general population. A 3–19-fold increase in prevalence of concomitant cancer has been reported in patients presenting with an idiopathic VTE. The same applies for occult cancer in patients with secondary and idiopathic VTE. The prevalence of occult cancer in patients with secondary VTE is comparable with the prevalence of cancer in the general population, while the prevalence of occult cancer in patients with idiopathic VTE is 4–10%. It seems possible to detect a substantial number of additional cancer patients, at time of diagnosis of idiopathic VTE, by means of extensive screening. However, results of randomised trials, evaluating the effect on survival rate, are lacking.
Thrombin is a mammalian serine proteinase that plays a prominent role in the maintenance and regulation of hemostasis through its interaction with various substrates and/or ligands. The venoms of several snakes contain glycosylated serine proteinases that have been recognized to possess one or more of the essential activities of thrombin on fibrinogen (Fg) and/or platelets. These proteinases share about 60% sequence identity. One class of snake venom serine proteinases are those known as thrombin-like (TLE), named after their ability to directly clot Fg in order to preferentially produce fibrinopeptide A, fibrinopeptide B or both. To understand the molecular basis of this phenomenon, the corresponding amino acid sequences and molecular structures need to be analyzed. Given the absence of experimentally determined tertiary structures of snake venom, TLEs, three-dimensional molecular models should prove useful in this context. Towards this goal, we obtained models of snake venom TLEs that used TSV-PA as template, TSV-PA being the only snake venom serine proteinase whose crystal structure is known to date. Along with a comparative sequence analysis the models contribute to the identification and description of thrombin-homologous or alternative binding sites, helping thus to understand differences in specificity.
Surgery induces immediate hypercoagulability by direct alteration of the vascular bed, release of procoagulant substances from the extravascular spaces and blood flow decrease, and delayed hypercoagulation in response to tissue damage which triggers inflammatory responses. Thus, the postoperative period represents a high-risk time for thrombosis. Recognition of high-risk individuals would make it possible to improve thromboembolism prevention. We studied in women undergoing laparoscopic surgery a series of markers known to be related to the thrombotic risk and confronted their results with those of a global test, the thrombin generation test (TGT) described by Hemker’s group. Our results show that two groups of patients can be distinguished according to usual risk markers (PAI-1, TAT, body mass index): the higher risk group demonstrates higher initial TGT values, but also a postoperative decrease of the TGT values whose mechanisms remain to be defined.
Anticoagulant activities against the extrinsic and intrinsic coagulation pathways were identified in salivary gland extracts (SGE) prepared from four tabanids (Hybomitra muehlfeldi, Tabanus autumnalis, Haematopota pluvialis, Heptatoma pellucens). All extracts prolonged human plasma clotting time in a dose-dependent manner and inhibited thrombin activity in the chromogenic substrate assay. Horsefly SGE did not inhibit factor Xa. Partial purification of SGE proteins using reversed-phase high-performance liquid chromatography revealed species-specific differences in the elution profiles and range of fractions with anticoagulant activities.
Viper venom disintegrins have been used frequently to study the cellular receptors which characterize various types of cells, including platelets, endothelial cells and cancer cells. While the majority of such analyses have pointed to involvement of integrin receptors αvβ3, α5β1 or αIIbβ3, this may not always be so. Eristostatin, from Eristocophis macmahoni, is a potent inhibitor of ADP-induced platelet aggregation as well as of human and murine melanoma metastases in mouse model systems. This disintegrin requires an RGDW motif, as well as an intact C-terminus, in order to interact with both platelets and four different types of melanoma cells. Eristostatin causes nonmetastatic SBc12 melanoma cells to show higher susceptibility to specific killing by NK-like TALL-104 cells. While it is known that eristostatin binds to αIIbβ3 on platelets, the receptor with which eristostatin binds to the melanoma cells has not yet been identified.
Clinical laboratory investigation of hemostasis has considerably progressed during the last decade with the use of high-quality instruments and reagents. In order to transfer these improvements to the quality of patient care and to increase interand intra-laboratory reproducibility, more stringent requirements in the management of samples from blood collection to the validation of results are needed. The outcome of even the best clinical test in hemostasis is critically dependent upon the quality of the sample used. Therefore, the quality of blood sampling, the transport to the laboratory, sample preparation and storage are factors determining the test results. To minimize this significant source of variation, the implementation of good biological practice rules is compulsory. Both the National Committee for Clinical Laboratory Standards (NCCLS) and the European Concerted Action on Thrombosis have established and/ or reinforced such guidelines [1–5]. In addition, at least in France, the clinical pathologist is responsible for the whole preanalytical phase, including blood collection, even if it is carried out by a third party [6]. In this study, the criteria approved by the ‘Groupe d’Etude sur l’Hemostase et la Thrombose’ (GEHT) are summarized and updated [7].
Successful administration of thrombolytic agents is associated with vessel reocclusion in a high proportion of cases. We have previously established an animal model to investigate platelet-rich thrombolytic mechanisms in vivo and demonstrated that recombinant staphylokinase (rSAK)-induced thrombolysis was enhanced by the concomitant administration of the direct thrombin inhibitor argatroban. The present study expanded the use of this model by comparing arterial and venous thrombolysis using advanced image analysis software. Mural thrombi were formed by Helium-Neon laser irradiation in mesenteric arterioles and were shown to be lysed, dose-dependently, by smaller amounts of rSAK than those required in venules. Activated protein C (APC), as well as argatroban, enhanced the rSAK-induced thrombolysis. APC or argatroban also induced thrombolysis in the absence of rSAK, and the effect was inhibited by tranexamic acid. The enhanced thrombolysis induced by APC or argatroban in the presence or absence of rSAK may be due to increased endogenous thrombolysis mediated by the inhibition of thrombin activity or delayed thrombin generation.
Two G-to-A mutations at positions 1691 of the factor V (FV) gene and 20210 of the prothrombin (FII) gene have been associated with an increased risk of venous thromboembolism. We report a thrombosis-prone family in which one subject – the propositus who exhibited combined heterozygous FV G1691A and FII G20210A mutations – showed spontaneous and early clinical onset (at 23 years), recurrences of deep-vein thrombosis and pulmonary embolism. His asymptomatic father carried the FII G20210A substitution and his mother, characterized by an isolated thrombotic episode on occasion of surgery (at 48 years), carried the FV G1691A substitution. In the maternal lineage, one of the propositus’ uncles had thrombosis on occasion of a bone fracture (at 65 years) despite the absence of known prothrombotic defects. A sister of the propositus carried the FII G20210A and the brother the FV G1691A mutation. They have been asymptomatic until now. The propositus’ two children, 20 and 16 years old, both carry the FV G1691A substitution and have been asymptomatic until now. The plasma levels of FII were higher in carriers of the FII G20210A allele if compared with noncarriers, and the activated protein C resistance phenotype, associated with the FV Leiden mutation, showed a complete correlation with the FV G1691A mutation. Despite the very limited number of thrombotic cases involved in this survey, which does not allow statistically sound conclusions, the data obtained from this family suggest that the synergy of inherited factors and transient risk conditions could play a key role in the occurrence of thrombotic accidents.
Activators of blood coagulation factor X have been described in the venom of many snake species belonging to the genus Viperidae and Crotalidae as well as from a few Elapid species. Based on the structural and functional properties of purified activating principles, factor X activators are either metalloproteases or serine proteases. The best known activator is RVV-X from Russell's viper (Daboia russelli), a metalloprotease consisting of a heavy chain containing the catalytic domain and two light chains which share homology with C-type lectins and which are thought to exert a regulatory function in the Ca(2+)-dependent activation of factor X. This activator is also one of the best examples of the use of exogenous activators in coagulation research and in addition it is used in many diagnostic research kits. In this paper, an overview is given of the structural and functional properties of snake venom factor X activators thus far described in the literature.
We developed a simple assay using rabbit thrombomodulin (TM) based on an activated partial thromboplastin time method, which detected the response to TM in plasma coagulation. We call it thrombomodulin addition clotting time (TACT). The anticoagulant response to TM was calculated by dividing the clotting time with TM by the clotting time with buffer solution. Results were expressed as TACT ratio, which indicates the degree of inhibition of plasma clotting by TM. Using this assay, we measured the TACT ratio in 80 patients with deep-vein thrombosis (DVT) and in 126 controls matched to the patients according to age and sex. A significant difference in the TACT ratio was observed between patients with DVT (mean 1.874) and controls (mean 1.956) (p < 0.001). Twenty- three patients (29%) had TACT ratios below the 10th percentile (1.757) of distribution of control subjects (odds ratio: 3.5; 95% confidence interval (CI): 1.7–7.2). After excluding subjects with a deficiency of protein C, protein S and antithrombin III, we found an odds ratio for DVT of 3.4 (95% CI: 1.6–7.2). These data suggest that natural anticoagulant deficiencies do not influence the TACT ratio, and our case-control study may show that the plasma of patients with DVT has a low response to TM.
The aim of this study was to evaluate the accuracy of the portable coagulometer CoaguChek® (Roche Diagnostics) as a prothrombin time (PT) monitor, and to correlate capillary blood results with those of three different routine methods used for monitoring oral anticoagulant therapy (OAT): capillary, plasma and whole blood samples. Three hospitals participated in the study with a total of 235 patients on OAT. The international normalized ratio (INR) results obtained with CoaguChek were compared with those obtained using each of the routine methods. The study presents a good correlation between the PT monitor and the three methods studied: r = 0.9745 (hospital A), r = 0.9283 (hospital B), r = 0.9136 (hospital C). A simplified concordance test of the methods results in a nine-field comparison table showing concordances of 87.2, 85.7 and 68.4%, respectively. The absolute difference (mean ± SD) between laboratory A and CoaguChek INRs was 0.0571 ± 0.2042, with values of 0.04286 ± 0.3906 for laboratory B and 0.6986 ± 0.6170 for laboratory C. These results confirm that CoaguChek could be used as a new method for oral anticoagulant monitoring, and is in best agreement with the capillary blood PT system.
This work investigates relationships between platelet density and reactivity. 21 individuals subject to coronary angiography were studied. Peak platelet density was analyzed using a newly developed electronic device. The apparatus measures light transmission through test tubes containing density-separated platelets, thus allowing an estimation of the platelet distribution in the gradient. A flow cytometry technique was used for determining platelet reactivity after stimulating with ADP. Platelet counts, mean platelet volumes, peak platelet density and platelet reactivity were determined immediately before (day 1) and 24 h after cardiac catheterization (day 2). For all parameters changes during the day of angiography were compared with platelet density alterations. The subjects were divided into two groups according to density changes at angiography. Group 1 individuals showed density alterations (i.e. day 2 – day 1 value) ≧–8 × 10–5 kg/l. In contrast, group 2 subjects either displayed density changes <–8 × 10–5 kg/l or grossly disturbed platelet density patterns on day 2. Before angiography both groups had similar platelet counts and volumes. Then platelet reactivity when stimulating with ADP did not differ significantly between the two groups. After angiography, the number of fibrinogen-positive cells when stimulating with ADP rose by 6 ± 8% for group 2 patients. The corresponding figure for group 1 was –1 ± 6%. The difference was significant (p = 0.01). No such relationships were found when comparing density alterations and changes of platelet counts and volumes. We conclude that in this study platelet density alterations at coronary angiography are inversely related to variations of platelet reactivity.
Healthy newborns have a very low risk of thrombosis. It has been suggested that this is partly due to the anticoagulant effect of alpha-2-macroglobulin (a2-M). This broad-spectrum protease binding glycoprotein is physiologically elevated in newborns over adult values and has been shown to complex generated alpha-thrombin. In our present study, we point out that a2-M also acts as a procoagulant by inhibiting activated protein C (APC). In all experiments performed in cord and adult plasma the anticoagulant action of APC was diminished in a dose-dependent manner when a2-M levels were successively elevated, reflected in increased thrombin potential (TP), and enhanced at low a2-M levels, reflected in decreased TP.
Blood coagulation factor V is a single-chain glycoprotein with Mr = 330,000 which plays an important role in the procoagulant and anticoagulant pathways. Thrombin activates factor V into factor Va, a two-chain molecule which is composed of a heavy (Mr = 105,000) and a light chain (Mr = 71,000/74,000). Factor Va accelerates factor Xa-catalysed prothrombin activation more than 1,000-fold and under physiological conditions the cofactor activity of factor Va in prothrombin activation is down-regulated by activated protein C. Factor V can also be activated by a wide variety of snake venoms (e.g. from Vipera species, Naja naja oxiana, Bothrops atrox) and by proteases present in the bristles of a South American caterpillar (Lonomia achelous). Some venoms, notably of Vipera lebetina turanica and Lonomia achelous, contain proteases that are able to inactivate factor V or factor Va. Venom factor V activators are excellent tools in studying the structure-function relationship of factor V(a) and they are also used in diagnostic tests for quantification of plasma factor V levels and for the screening of defects in the protein C pathway. In this review, the structural and functional properties of animal venom factor V activators and inactivators is described.
We report the arthroscopic treatment of pigmented villonodular synovitis (PVNS) in a 13-year-old Japanese boy with congenital partial deficiency of plasminogen activator inhibitor-1 (PAI-1). He was admitted to our hospital with recurrent haemarthrosis of his right knee. Characteristic abnormalities of fibrinolysis included shortened euglobulin lysis time, low PAI-1 activity and low PAI-1 antigen levels. In addition, levels of "active PAI" in the plasma, which is a measure of total PAI bound to exogenous plasminogen activator, were very low. These parameters remained low after venous occlusion. The diagnosis of PVNS was established by synovial membrane biopsy, and arthroscopic synovectomy was performed with adjuvant administration of intravenous tranexamic acid. Subsequent bleeding episodes have been well controlled by oral administration of tranexamic acid on demand.
The blood coagulation cascade is activated following vascular-wall injury. The serine protease thrombin is the final protease in this cascade that causes the formation of fibrin from fibrinogen. Thrombin also causes the activation of platelets, which are trapped in a fibrin net followed by hemostasis. Platelets gathered into fibrin clots release several growth factors such as platelet-derived growth factor and transforming growth factor β. In the present study, we demonstrated that the vascular endothelial growth factor (VEGF) could be bound to fibrin clots in the plasma, and that incubation of the endothelial cells with these VEGF-bound fibrin clots induced proliferation of endothelial cells. Thus, it suggests that clot-bound VEGF may play a role in wound healing through the proliferation of endothelial cells and vascular smooth-muscle cells. On the other hand, a noticeable migration of monocytes was observed when they were cultured on dishes in the presence of VEGF-bound fibrin clots. Moreover, peripheral blood monocytes incubated in the presence of VEGF-bound fibrin clots strikingly increased the production of IL-6 and IL-8, demonstrating that VEGF trapped in fibrin clots not only induces proliferation of human umbilical vein endothelial cells and migration of monocytes but also enhances secretion of IL-6 and IL-8. Thus, our data suggest that fibrin clots that contain several growth factors act as a bioactive reservoir and may play an important role in hemostasis as well as wound healing.
Lebetins from Macrovipera lebetina snake venom constitute a new class of inhibitors of platelet aggregation. There are two groups of peptides: lebetin 1 (L1; 11- to 13-mer) and lebetin 2 (L2; 37- to 38-mer). The short lebetins are identical to the N-terminal segments of the longer ones. They inhibit platelet aggregation induced by various agonists (e.g. thrombin, PAF-acether or collagen). The shortest lebetin (11-mer) shows potent inhibition of rabbit (IC(50) = 7 nM) and human (IC(50) = 5 nM) platelets. They prevent collagen-induced thrombocytopenia in rats. N- and C-terminal-truncated synthetic L1gamma (sL1gamma; 11-mer) is less active in inhibiting platelet aggregation than the native peptide. Results from Ala scan studies of the sL1gamma peptide indicated that replacement of the residues (P3, G7, P8, P9 or N10) resulted in a remarkable drop in the activity, whereas replacement of residues K2, P4 or K6 by Ala resulted in enhancement of the antiplatelet activity by at least 10-fold. To examine the activity of multimeric L1gamma, several multimeric peptides were synthesized using the multiple-antigen peptide system assembled on a branched lysine core and their antiplatelet activity was evaluated in vitro. The largest multimeric peptides showed a 1,000-fold increase in antiplatelet activity.
Response to: 'Glucocorticoid withdrawal in lupus: to do or not to do?' by AcharyaWe thank Acharya for her interest in our study showing that in patients with systemic lupus erythematosus (SLE) with a quiescent disease and a stable treatment regimen, for at least 1 year, withdrawal of 5 mg of prednisone was associated with a fourfold increase (ie, 27%), in the risk of flare onset, as defined by the SELENA-SLEDAI flare index and the British Isles Lupus Assessment Group index during a 1-year follow-up. 1 2Acharya states that these findings contrast with those from two previous published studies on the same subject. 3 4The latter studies are, however, not comparable to ours.As pointed out by Acharya, the Steroids In the Maintenance of remission of Proliferative Lupus nephritis (SIMPL) trial was a small pilot study, including only 15 patients, that was however not designed to assess the efficacy or safety of maintaining low-dose prednisone administration. 3With respect to the report of Moroni et al, in their study treatment withdrawal in patients with SLE with nephritis included not only glucocorticoids but also immunosuppressants. 4We would like to argue that the results of the CORTICOLUP trial are consistent with those of recently published observational studies, indicating that treatment with low-dose glucocorticoids prevents relapse in about one-fifth to one-third of patients with SLE with no or very low disease activity. 5 6 As suggested by Acharya, the results might have been different had the majority of patients been on immunosuppressant therapy.We, of course, do acknowledge that the results of the CORTI-COLUP study cannot be extrapolated to all patients in remission.The percentage of patients treated with an immunosuppressant in the CORTICOLUP study, amounting to 27%, reflects the clinical practice of our team.Moreover, our practice is comparable to that of other teams. 5 7Yet, the indication of an immunosuppressive treatment is not evidence based, especially in patients in remission, and depends to date on the decision of the physician and therefore varies according to his/her convictions.Finally, in the interaction analysis shown in figure 3 in our study, 2 there was no significant interaction between the effect of prednisone maintenance and immunosuppressants or hydroxychloroquine.To conclude, like Acharya, we believe that prescribing an immunosuppressant or a biologic might reduce the use of prolonged glucocorticoid therapy to prevent relapse of the disease.However, this belief has to be proved and balanced with the infectious and oncological risk possibly brought about by long-term exposure to this type of medication.
