Normal Thrombin Research Articles

Coagulopathy in a patient with nephrotic syndrome

A 55-year-old Caucasian woman presented with a 5week history of increasing lower limb edema associated with a two-pound daily weight gain. She was feeling well otherwise; she denied shortness of breath, chest discomfort, recurrent headaches, myalgia, arthlagia, or constitutional symptoms. She had respiratory infection 4 months before, but the infection has resolved. The patient presented with peripheral edema. The differential diagnosis includes right-sided heart failure, liver cirrhosis, nephrotic syndrome, drug-induced edema, and lymphoedema. Her preceding upper respiratory tract infection suggests postinfectious cardiac or renal pathology. The patient did not have any prior bleeding episode. The medical history was significant for Graves’ disease treated with radioactive iodine ablation. The only medication was daily thyroid supplementation. She was married and had two uncomplicated pregnancies. She was a lifelong nonsmoker. She denied any alcohol intake or illicit drug use. She was a pharmacist without any known exposure to toxic chemicals. On examination her vital signs were normal. She was not in any distress. Examination of her legs revealed bilateral pitting peripheral edema up to the knee level. There was no jaundice, lymphadenopathy, or bleeding sign. Her cardiac examination was normal and her jugular venous pressure (JVP) was not elevated. Respiratory examination was normal with no crackles on auscultation. Abdominal examination revealed no palpable hepatosplenomegaly or ascites. The fact that this patient was not on any medication other than long term thyroid supplement makes druginduced edema unlikely. The finding of normal JVP on clinical examination is against the diagnosis of heart failure. Lymphedema is typically nonpitting. Thus, the most likely differential diagnosis includes nephrotic syndrome and early liver cirrhosis. Patient’s hemoglobin level was 19.0 g/dL (normal 11.5– 16.5 g/L), platelet count 305,000/mm (normal 150,000– 400,000/mm). The blood urea nitrogen was 14.4 mg/dL (normal 7.0–18.0 mg/dL), creatinine 0.93 mg/dL (normal <1.5 mg/dL), albumin 2.0 g/dL (normal 3.5–5.5 g/dL), cholesterol 587 mg/dL (normal <200 mg/dL), and triglyceride 447.1 mg/dL (normal <160 mg/dL). The results of liver function tests were normal. Urinalysis revealed 11 blood, 31 protein, and granular casts. Twenty-four hour urine protein excretion was 15,000 mg/d. The combination of heavy proteinuria, minimal hematuria, hypoalbuminemia, and hypercholesterolemia suggests nephrotic syndrome. Nephrotic syndrome may be associated with primary kidney disease, such as minimal change disease, focal segmental glomerulosclerosis, and membranous nephropathy. Systemic diseases including diabetes mellitus, systemic lupus erythematosus, amyloidosis, or Fabry’s disease also cause nephrotic syndrome. International normalized ratio (INR) was increased to 2.6 and activated partial thromboplastin time (aPTT) was prolonged to 68 s but thrombin clotting time was normal. Clauss fibrinogen level was slightly elevated at 480 mg/dL (normal range 200–400 mg/dL). D-dimer level was not elevated. In general, patients with nephrotic syndrome are in a hypercoagulable state because of urinary loss of antithrombin III and enhanced platelet reactivity, but this patient’s coagulation tests were paradoxically prolonged although there was no bleeding symptom. Simultaneous prolongation of both INR and aPTT suggests common coagulation pathway abnormalities like liver dysfunction, vitamin K deficiency, disseminated intravascular coagulopathy (DIC), primary fibrinolysis or post-thrombolytic therapy. However, the normal thrombin clotting time and borderline high fibrinogen level make abnormalities in the conversion pathway of fibrinogen to fibrin less likely. Normal fibrinogen and D-dimer level are atypical for DIC. A deficiency of multiple coagulation factors in both intrinsic and extrinsic coagulation pathways commonly occurs in vitamin K deficiency or hepatic dysfunction. However, 48 hr after administration of 5 mg oral vitamin K, the coagulation indices remained prolonged with an INR at 2.6 and aPTT at 63 s. To further define the causes of coagulopathy, clotting factor levels were assayed individually. Factor II level was high at 166%, factor VII 179%, but factor VIII:C was low at 7%, factor X 3%, whereas factor XII level was borderline low at 47%. (Normal range of these clotting factors was 50–150%). Although acquired factor XII deficiency has been reported in patients with nephrotic syndrome [1], deficiency of multiple coagulation factors suggests acquired conditions, either due to insufficient production or excessive consumption. Coagulation factors II, V, VII, IX, X, XI, and XII are synthesized in the liver, and in addition factor VIII also

A central role of GPIb-IX in the procoagulant function of platelets that is independent of the 45-kDa GPIbα N-terminal extracellular domain

AbstractActivated platelets become procoagulant and efficiently promote the conversion of prothrombin to thrombin. A role of the GPIb-V-IX complex has long been postulated in view of the decreased prothrombin consumption in Bernard-Soulier patients. We evaluated the impact of GPIb-V-IX deficiency and the requirement for the GPIbα extracellular domain. In GPIbβ−/− mice, thrombin generation was profoundly decreased in tissue factor– or collagen-related peptide (CRP)–activated platelet-rich plasma and in washed platelets supplemented with normal plasma or with FVa, FXa, and prothrombin. Phosphatidylserine (PS) exposure was similarly decreased in response to thrombin, CRP, or CRP + PAR4 peptide despite a normal platelet phospholipid composition. The hypothesis that these defects originate from lack of the GPIbα N-terminal domain was evaluated after its removal from normal mouse and human platelets with Nk protease or O-sialoglycoprotein endopeptidase. Unexpectedly, the treated platelets exhibited normal thrombin generation and PS exposure, indicating that GPIb-V-IX regulates procoagulant activity independently of its GPIbα-binding region. These results suggested a more general structuring role through intracellular cytoskeleton-anchoring portions regulating responses leading to PS exposure. This hypothesis was supported by the decreased calcium mobilization observed in GPIbβ−/− platelets in response to several agonists, some acting independently of GPIb, in contrast to the normal calcium responses in Nk protease–treated platelets.

Impaired thrombin generation and fibrin clot formation in patients with dilutional coagulopathy during major surgery

Patients subjected to haemodilution during surgery are at increased risk of bleeding. We hypothesised that, in the acquired dilutional coagulopathy, insufficient haemostasis is due to either insufficient thrombin generation or insufficient fibrin clot formation. In tissue factor-activated plasmas from patients with coagulation deficiency, we measured time curves of thrombin generation and fibrin clot formation (thromboelastography). Investigated were in study A: 10 patients treated with vitamin K antagonist and five healthy subjects; in study B: 30 patients undergoing cardiopulmonary bypass (CPB) surgery and infused with on average 2,000 ml crystalloids and colloids (no major bleeding); in study C: 58 patients undergoing major general surgery, and transfused with >5,000 ml crystalloids, colloids and red cell concentrates, who experienced major bleeding and were post-transfused with fresh frozen plasma. The treatment with vitamin K antagonist led to a progressive reduction in thrombin generation but not fibrin clot formation. In CPB patients, plasma factor levels post-surgery were 53-60% of normal. This was accompanied by moderate reduction in both haemostatic processes. In plasmas from patients undergoing major surgery, factor levels were 38-41% of normal, and these levels increased after plasma transfusion. Taking preset thresholds for normal thrombin generation and fibrin clot formation, at least one of these processes was low in 88-93% of the patients with (persistent) bleeding, but only in 40-53% of the patients without bleeding. In conclusion, the ability of thrombin generation and fibrin clot formation is independently reduced in acquired dilutional coagulopathy, while minimal levels of both are required for adequate haemostasis.

Acquired coagulation disorders: revisited using global coagulation/anticoagulation testing

Acquired coagulation defects are characterized by a decrease of both pro- and anti-coagulants. Because of this, we hypothesise that global tests, such as the prothrombin and partial thromboplastin times (PT and APTT), might be unsuitable for their investigation. Indeed, these tests are not good predictors of bleeding in acquired coagulopathies as they are in the congenital ones. This article discusses the possible reasons for this, using cirrhosis and the neonatal period as epitomes of acquired coagulation defects. Both display normal thrombin generation in the presence of thrombomodulin, in spite of prolonged PT and APTT. We surmise that, because of their design, the PT and APTT are responsive to thrombin generated as a function of pro-coagulants, but much less to thrombin inhibited by the anti-coagulants, especially protein C, which is activated to a limited extent in the absence of thrombomodulin. In conclusion, the PT and APTT can tell us whether or not a patient is deficient in one or more pro-coagulants, but not whether this deficiency is counterbalanced by a parallel deficiency of anti-coagulants. Thrombin generation assays are more suitable than PT and APTT for use in acquired coagulation defects.

Thrombin Generation Is Normal in Most Patients with Cirrhosis despite a Prolonged INR.

Introduction: Advanced liver disease is associated with prolongation of the prothrombin time (PT). In order to decrease the inter-laboratory variability of PT measurement, the international normalised ratio (INR) calculated as the ratio of patient's PT to a normal (control) sample, raised to the power of the international sensitivity index (ISI) of the particular thromboplastin used was developed. However, the ISI is derived from PT results of patients on warfarin and results cannot be extrapolated to liver patients. Despite this, the INR is still commonly performed to assess bleeding risk in patients with liver disease worldwide. Furthermore the INR is only affected by factors I, II, V, VII, and X and is not influenced by other factors such as factor VIII which is usually raised in hepatic cirrhosis. Recently it has been reportd that thrombomodulin addition (to take into account the protein C pathway) normalises thrombin generation (TG)1 despite these patients having a low TG if thrombomodulin is not used.Aim: We speculated that TG, which is a global assay of coagulation and sensitive to all coagulation factors, when triggered by a low tissue factor (TF) concentration might not correlate with the INR in patients with liver disease and that contact inhibition with corn trypsin inhibitor (CTI) might better reflect the coagulation potential in this patient group.Results: 73 unselected patients with liver cirrhosis due to various diseases and 25 normal subjects were studied. INR and TG using the calibrated automated thrombogram (CAT) at 1pM tissue factor (TF) with CTI, 5pM without CTI and with and without Protac (a Protein C activator) were performed using platelet poor plasma (PPP). The INR range was 0.8–4.0 (mean 1.6). At 5pM TF without Protac, the patient group had a significantly lower endogenous thrombin potential (ETP) than the controls (mean ETP difference 752nM/min; P <0.0001). With Protac, no significant differences could be detected between the 2 groups. However, if the ETP without Protac was divided by the ETP with Protac x 100, the liver group showed more resistance to PC activation (mean % difference 25.4; P 0.0002). At 1pM TF, the mean ETP in the cirrhosis cohort was slightly lower than the normal group (difference between means 216nM/min; P 0.03). However, only 7 (9.6%) patients had ETP values less than the normal range (mean±2SD). No correlation was found between the ETP at 1pM and the INR. The mean FVIII:C was raised at 185.6 (78–420U/dl).Conclusion: TG measured at low TF with CTI is normal in the majority of patients with cirrhosis. These patients are also more resistant to PC activation and have supranormal FVIII:C. Thus most patients have a normal or high thrombin potential despite an abnormal INR. These findings have important implications as in the absence of bleeding, “prophylactic” plasma and clotting factors are unnecessary.

First Experiences with the Measurement of Thrombin Generation (TGA) on the Ceveron® Alpha in the Routine Lab

Aims: Aim of the study was to show the suitability of the new coagulation analyzer Ceveron® alpha for determination of thrombin generation (TGA) in routine laboratories for discrimination of normal plasma samples from haemophilic and thrombophilic patient samples, from patients with a Lupus syndrome and patients with bleeding disorders.Methods: TGA was determined by a thrombin generation assay based on monitoring the formation of thrombin by means of a fluorigenic substrate upon activation of the coagulation cascade by tissue factor and phospholipids using 2 reagents with different phospholipid concentrations (RClow and RChigh) on Ceveron® alpha. Thrombin generation is measured with a specially adapted TGA fluorimetric module which is placed over the cuvette rotor. Readout parameters were lag time (tLag), peak thrombin (Peak), time to peak(tPeak), velocity index (VI) and area under the curve (AUC). In case of patients with Lupus Anticoagulants a ratio of peak thrombin values triggered with RClow and RChigh was formed. Four laboratory groups participated in the study. All patient samples used for thrombin generation measurement were characterized by routine parameters.Results: Group 1 analyzed samples with bleeding disorders. Peak thombin values showed the best discrimination between Hemophilia B patients (41.9, SD12.7nM), Hemophilia B patient after treatment (188.9nM) and normal controls (208.5nM, SD= 74.0). In Hemophilia A patients tLag was the best parameter to discriminate severe Hemophilia A (8.5min) from acquired hemophilia A and VWD (4.6 min, SD=2.1).Normal ranges for TGA established in group 1 (n=16) were tLag 2.7min (SD=0.8), tPeak 7.8min (SD=1.9), peak thrombin 208.5nM (SD= 74), AUC 1769.4 (SD=327.9).Group 2 analyzed samples of thrombophilic patients with pregnancy (n=30). Patient samples showed peak thrombin values (310.9nM, SD= 65.7) above normals (174.2nM, SD= 22.5). Even patients with heparin treatment had high peak thrombin values (340.5nM, SD=67.2). In heparin treated patients D-dimer values were pathologic as well.Group 3 determined thrombin generation in patients with Lupus Anticoagulants determined by dRVVT, APTT-LA, Lupus-Ceveron and anti-cardiolipin antibodies (ACA). Patients only with high ACA values showed normal peak thrombin ratios of RClow/RChigh 0.6 (SD= 0.07) whereas the ratio in patients with high lupus anticoagulants as measured in the coagulation tests was 0.98 (SD= 0.31) for high antibody titer and 0.83 (SD 0.01) for low antibody titer. Inter-assay CVs in the normal control determined by this laboratory were 2.67% for peak thrombin and 4.83% for AUC and in thrombophilic plasma 3.0% for peak thrombin and 1.41% for AUC.Group 4 determined TGA in orally anticoagulated patients. Normal range for peak thrombin established in this laboratory is 220nM (SD=52, n=12). Oral anticoagulant treatment lowered the AUC (from 1713.3nM, SD= 435.4 thrombin in samples with INR in the range 1 – 1.5 to 894.4nM, SD= 229.2 thrombin in samples with INR in the range 2 – 2.5) and peak thrombin formation (from 204.1nM, SD= 73.5 peak thrombin in samples with INR in the range 1 –1.5 to 79.0nM, SD= 35.2 peak thrombin in samples with INR in the range 2 – 2.5) and prolonged the tLag (from 5.5min, SD= 1.7 in samples with INR in the range 1 –1.5 to 7.6min, SD= 2.4 in samples with INR in the range 2 – 2.5.Conclusions: TGA can be measured reproducibly with low variation under routine laboratory conditions. Peak thrombin is the best parameter for discrimination of patient samples from normals in haemophiliacs, in thrombophilia, patients with a Lupus syndrome, pregnancy and bleeding disorders. With automated, standardised thrombin generation measurement on the Ceveron® alpha large prospective studies can be conducted to confirm these results.

Normal thrombin generation in neonates in spite of prolonged conventional coagulation tests

Conventional coagulation tests might be inadequate to explore mechanisms regulating thrombin generation in neonates, because they do not allow full activation of the reduced levels of protein C. Therefore, they do not reflect the action of pro- and anti-coagulants as does the endogenous thrombin potential assessed in the presence of thrombomodulin. Endogenous thrombin potential measured without thrombomodulin was greater than the lower-limit of the adult reference interval in 30% of 109 full-term and 49% of 55 pre-term neonates, a finding consistent with the reduced levels of procoagulants in this setting. When the test was modified adding thrombomodulin, endogenous thrombin potential reverted into the adult reference interval in 97% and 100% full-term and pre-term neonates. In conclusion, the coagulation balance in neonates is restored by the concomitant reduction of pro- and anticoagulants. The restored balance can be shown in vitro by the endogenous thrombin potential test that includes thrombomodulin, but not by conventional coagulation tests.

Establishment of an Acute Superior Mesenteric Artery Injury Model for Damage Control Surgery

Managements of superior mesenteric artery (SMA) injuries are difficult and often result in a disappointing outcome. Damage control surgery (DCS) has been approved to be an effective and reliable strategy for severe trauma victims. We aimed to build up a severe trauma-shock-hypothermia model of SMA injuries for DCS study and determine the optimal time to institute DCS. Pigs were anesthetized and instrumented with arterial and a thermodilution cardiac output catheter. SMA flow was interrupted while animals were hemorrhaged to 45% estimated blood volume. Pigs were maintained shock and intestine ischemia for three durations: intestine ischemia for 30 min (I-30; n = 6), 60 min (I-60; n = 6), and 90 min (I-90; n = 6). Cold lactated Ringer's (10 mL/kg) was infused to induce hypothermia. SMA was then declamped and kept in reperfusion for 6 h. Hemodynamic data and serum samples were collected during shock and resuscitation. Distal ileum was collected at the end of ischemia and reperfusion. All animals presented with disastrous conditions at the end of ischemia: low temperature, severe acidosis, decreased blood pressure, depressed cardiac output, and oxygen delivery. I-90 animals suffered the lowest temperature, the most severe acidosis, lowest blood pressure, and depressed cardiac output and oxygen delivery. Coagulopathy developed in I-90, whereas normal prothrombin time and thrombin time were detected in I-30 and I-60. Aspartate aminotransferase, lactate dehydrogenase, creatine kinase, and alkaline phosphatase were equally within groups (P > 0.05). All (6/6) of I-30, 83.3% (5/6) of I-60, and 16.7% (1/6) of I-90 pigs survived (P < 0.01). Base excess in I-90 was much lower than that in I-30 and I-60 animals. We first built up an acute SMA injury animal model for DCS investigations and determined that the optimal institution time of DCS was before 60 min after SMA injury in the trauma-shock-hypothermia swine model.

The efficiency of anti-activated factor X and anti-activated factor II anticoagulants

Six thrombin-generation inhibitors or thrombin inhibitors were compared in the extrinsic coagulation activity assay (EXCA), where the normal thrombin generation is about 1 IU/ml within 1 min (37 degrees C). Unfrozen pooled normal citrated plasma was supplemented on flat-bottom wells (23 degrees C) with increasing concentrations of dalteparin, danaparoid, heparin, fondaparinux, hirudin, or argatroban. To 50 microl plasma, 5 microl of 1.5 ng/ml tissue factor, 6% bovine serum albumin, and 250 mmol/l CaCl2 were added. After 1 and 2 min coagulation reaction time at 37 degrees C (EXCA-1 and EXCA-2), 100 microl of 2.5 mol/l arginine and 0.16% Triton X 100, pH 8.6, were added. After 3 min (23 degrees C), 25 microl of 1 mmol/l CHG-Ala-Arg-pNA in 1.25 mol/l arginine, pH 8.7, were added, and the linear increase in absorbance with time was determined at 405 nm. The 50% inhibitory concentrations of plasmatic anticoagulants tested in the EXCA-1 (37 degrees C) were 0.025 IU/ml dalteparin, 0.13 U/ml danaparoid, 0.12 IU/ml heparin, 1.3 microg/ml fondaparinux, 2.4 ng/ml hirudin, and 1 microg/ml argatroban. From the 50% inhibitory concentration of hirudin it can be concluded that inhibition of about 30 mIU/ml thrombin halves the normal EXCA-1 value (i.e. if about 0.1 IU/ml thrombin are inactivated, then thrombin cannot self-amplify its generation 10-fold). The efficiency of any clinically used plasmatic anticoagulant can be monitored in the EXCA.

A neonatal presentation of factor V deficiency: A case report

BackgroundFactor V deficiency is a rare autosomal recessive coagulation disorder. Awareness of presenting features and management is important to avoid bleeding complications associated with mortality and neurodisability.Case presentationA 6-day-old Pakistani boy was admitted with bleeding from the left nipple. His parents were first cousins. A coagulation screen showed a prothrombin time of 41 s (control 14 s), a partial thromboplastin time of 132 s (control 33 s) and a normal thrombin time of 15 s (control 14 s). Factor V activity was <0.01 IU/ml. Oral tranexamic acid was started. At 5 weeks of age the child presented with irritability, lethargy and reduced feeding and a drop of hemoglobin to 5.6 g/dl. A cranial computed tomography scan showed a right intra-cerebral bleed extending from the frontal lobe to the parieto-occipital region with shift of the midline to the left. A regime of 20 ml/kg of fresh frozen plasma four times a week was instituted and has prevented further bleeds up to the present age of 21 months. Neurodevelopment remained normal.ConclusionThis case illustrates that in an unusually bleeding newborn of consanguineous parents rare severe homozygous bleeding disorders need to be considered. Nipple bleeding may be the first presentation of a congenital bleeding disorder. In cases of factor V deficiency where factor concentrates are not available long term use of fresh frozen plasma can prevent potentially life threatening bleeding.

Influence of coagulation factors on intrinsic thrombin generation

The intrinsic coagulation activity assay (INCA) is a new thrombin-generation test that imitates the intrinsic pathway of blood coagulation. The aim of the present study was to investigate the influence of the main coagulation factors on the INCA. The INCA was performed with citrated plasma samples supplemented with different amounts of fibrinogen. The INCA and activated partial thromboplastin time determination were performed with factor-depleted plasmas and with mixtures of depleted plasmas with normal plasma. Supplemented purified fibrinogen resulted in a decrease of intrinsic thrombin generation (50% inhibitory concentration = 0.8 g/l). The INCA depends on the intrinsic factors (factors VIII, IX, XI and XII) and on the factors of the common pathway (factors II, V and X): for normal thrombin generation, at least about 50% of normal factor II is necessary. For the majority of factors, the sensitivity of the INCA appears to be approximately one order of magnitude better than that of the activated partial thromboplastin time. The INCA allows one to diagnose defects in the intrinsic coagulation system and might be a useful test to support development and characterization of new drugs targeted at the intrinsic generation of thrombin.

Congenital Combined Defects of Factor VII: A Critical Review

Factor VII deficiency is the least rare among uncommon congenital coagulation disorders. The majority of cases are isolated deficiencies. In some cases, FVII deficiency has been found to be associated with the deficiency in another coagulation factor or with non-coagulation-related abnormalities or defects. The evaluation of all published studies on the subject has shown that the FVII defect has been reported in association with FV, FVIII, FIX, FX, FXI and protein C defects. Furthermore, FVII deficiency has been described in association with bilirubin metabolism disorders, mental retardation, microcephaly, epicanthus, cleft palate and persistence of ductus arteriosus. The most interesting association appears to be that with FX. This has been shown to be due to a deletion in part of the long arm of chromosome 13. This arm contains genes coding for both FVII and FX. Interestingly, this combined coagulation defect has been found to be associated with carotid body tumors and several other malformations. Combined defects in blood coagulation often create diagnostic difficulties since results cannot be explained if a single factor deficiency is assumed. For example the combined FVII and FX defect yields a rather peculiar laboratory picture (prolonged prothrombin time and partial thromboplastin time, but normal thrombin time) that could suggest FII or FV or FX single deficiency and not FVII deficiency, indicating the need for specific factor assays whenever data are confusing. Finally, the elevated incidence of mental and skeletal malformations present in these combined defects indicates the need for a careful evaluation of all these patients lest some aspects of the defect are missed.

Inhibition of extrinsic hemostasis activation by low-molecular-weight heparin

Low-molecular-weight heparins (LMWHs) are very important drugs; unfortunately, the routine global hemostasis assays activated partial thromboplastin time and prothrombin time are not sensitive to LMWHs. Here the 50% inhibitory concentration (IC(50)) values of heparin and LMWHs on extrinsic thrombin generation are determined. Pooled normal plasma was supplemented with 0-2 IU/ml unfractionated heparin, 0-2 IU/ml LMWH dalteparin, or 0-20 microg/ml pentosanpolysulfate in 5-ml polystyrole tubes (23 degrees C) and tested in the tissue-factor-triggered extrinsic coagulation activity assay (EXCA): 50 microl plasma + 5 microl tissue factor/CaCl(2), 1 and 2 min incubation time at 37 degrees C (coagulation reaction time for EXCA-1 and EXCA-2); + 100 microl of 2.5 mol/l arginine (pH 8.6), 20 min at room temperature; + 50 microl of 1 mmol/l CHG-Ala-Arg-pNA, 1.25 mol/l arginine; increase in absorbance/time at 23 degrees C; calibrator = 1 IU/ml bovine thrombin in 6.7% human albumin replacing the plasma sample; in EXCA-1, about 1 IU/ml thrombin is generated in pooled unfrozen normal citrated plasma. The IC(50) values in EXCA-1 are 0.1 IU/ml heparin, 0.02 IU/ml LMWH, and 4.7 microg/ml pentosanpolysulfate. In ECXA-2 the IC(50) values are 0.07 IU/ml, 0.01 IU/ml, and 4.6 microg/ml, respectively. The EXCA reflects the efficiency of anticoagulants on plasmatic coagulation. It is suggested to adjust the dosage of LMWH according to the EXCA value; about 30% of normal extrinsic thrombin generation might be the correct dose for prophylactic anticoagulation.

The intrinsic coagulation activity assay

A new assay for the contact-phase-mediated generation of thrombin activity has been developed - the intrinsic coagulation activity assay (INCA). Citrated plasma (50 microl) is incubated with 5 microl SiO2, 250 mmol/l CaCl2 in polystyrole flat-bottom wells. After exactly 4 and 5 min (37 degrees C) coagulation reaction times (INCA-4 and INCA-5), 100 microl of 2.5 mol/l arginine, pH 8.6, is added to inhibit hemostasis activation in the important ascending part of the thrombin generation curve and to depolymerize fibrin. After 20 min, 50 microl of 1 mmol/l (final concentration 0.24 mmol/l) chromogenic thrombin substrate CHG-Ala-Arg-pNA in 1.25 mol/l arginine, pH 8.7, is added. The increase in absorbance is determined at 405 nm using a microtiterplate photometer. The assay is calibrated against 1 IU/ml thrombin. The normal thrombin activity range of INCA-4 (main value) or INCA-5 (control value) is 100 +/- 30% of normal (mean value +/- 1 SD; 100% = 0.5 IU/ml for INCA-4 and 1.9 IU/ml for INCA-5). With the INCA the normal range of intrinsic hemostasis is reflected, low-molecular-weight heparins can be monitored, the plasma matrix is not changed significantly, and the assay results are a percentage of normal generated thrombin activity and not coagulation seconds.

Thalidomide increases thrombin generation in multiple myeloma patients

7602 Background: Numerous reports have demonstrated that the clinical use of thalidomide (THAL) carries a risk for deep venous thrombosis. Only limited investigations into the causative mechanisms for this complication have been conducted. Thrombin generation is a global measurement of hyper or hypocoagulability that has not been used to examine the hypercoagulable state caused by THAL. The aim of this study was to determine if THAL affects thrombin generation in patients with multiple myeloma (MM). Methods: MM patients on and off THAL were recruited. Patients were considered to be “on THAL” if they had been taking the medication for at least three months and “off THAL” if they had not been taking the medication for at least three months. Coagulation tubes of blood containing 3.2% sodium citrate were drawn from each patient. Platelet poor plasma was prepared from these specimens using standard technique. The samples were snap frozen at -70 C and stored until analysis. Thrombin generation was measured using the Calibrated Automated Thrombogram as described by Hemker(Pathophysiol Haemo Thromb 2003;33:4–15) using a microplate fluorometer, the Fluoroskan Ascent from Thermo Electron and the Thrombinoscope software package. The endogenous thrombin potential or ETP (area under the thrombin generation curve) and peak thrombin generation (the maximum release of thrombin per unit of time) were the variables of interest for this study. Results: Twenty seven patients were recruited. Five were excluded from analysis because they were on warfarin or enoxaparin. The normal ETP in females is 1803 ± 241 nM*min and in males is 1745 ± 259 nM *min. Normal peak thrombin generation in females is 318.5 ± 52.9 nM and in males is 293.4 ± 48.5 nM. The t-test was used to compare subjects to normal values.Peak thrombin generation was significantly increased in the 9 subjects taking THAL, 374 ± 10.6 nM (p&lt;0.001) and in the 13 subjects not taking THAL, 352 ± 14.7 nM (p=0.009) when compared to normal values. The ETP was not significantly increased 1818 ± 10.7 nM*min (p=0.69) in subjects not taking THAL. However, ETP was increased in subjects taking THAL, 1939± 90 nM*min and showed a trend toward statistical significance (p=0.10). Conclusions: Thalidomide therapy increases thrombin generation significantly in patients with MM. [Table: see text]

171 Evidence of normal thrombin generation in patients with liver cirrhosis using thromboelastography on native whole blood

171 Evidence of normal thrombin generation in patients with liver cirrhosis using thromboelastography on native whole blood

Pathogenic antibodies to coagulation factors. Part II. Fibrinogen, prothrombin, thrombin, factor V, factor XI, factor XII, factor XIII, the protein C system and von Willebrand factor.

Pathogenic antibodies to coagulation factors. Part II. Fibrinogen, prothrombin, thrombin, factor V, factor XI, factor XII, factor XIII, the protein C system and von Willebrand factor.

Evidence of normal thrombin generation in cirrhosis despite abnormal conventional coagulation tests†‡

The role played by coagulation defects in the occurrence of bleeding in cirrhosis is still unclear. This is partly due to the lack of tests that truly reflect the balance of procoagulant and anticoagulant factors in vivo. Conventional coagulation tests such as prothrombin time and activated partial thromboplastin time are inadequate to explore the physiological mechanism regulating thrombin, because they do not allow full activation of the main anticoagulant factor, protein C, whose levels are considerably reduced in cirrhosis. We used a thrombin generation test to investigate the coagulation function in patients with cirrhosis. Thrombin generation measured without thrombomodulin was impaired, which is consistent with the reduced levels of procoagulant factors typically found in cirrhosis. However, when the test was modified by adding thrombomodulin (i.e., the protein C activator operating in vivo), patients generated as much thrombin as controls. Hence, the reduction of procoagulant factors in patients with cirrhosis is compensated by the reduction of anticoagulant factors, thus leaving the coagulation balance unaltered. These findings help clarify the pathophysiology of hemostasis in cirrhosis, suggesting that bleeding is mainly due to the presence of hemodynamic alterations and that conventional coagulation tests are unlikely to reflect the coagulation status of these patients. In conclusion, generation of thrombin is normal in cirrhosis. For a clinical validation of these findings, a prospective clinical trial is warranted where the results of thrombin generation in the presence of thrombomodulin are related to the occurrence of bleeding.

Inherited bleeding disorder due to familial type 2 platelet cyclo-oxygenase deficiency

Inherited platelet cyclo-oxygenase (COX) deficiency is a rare bleeding disorder. We report here the first case of familial type 2 platelet COX deficiency responsible for a moderate bleeding phenotype. The propositus was admitted in the emergency department for major epistaxis following treatment with aspirin. Epinephrine closure time is very sensitive to drugs which inhibit COX but the test was normal in patients with inherited COX deficiency. This clinical and biological data suggest that the anti-platelet effect of aspirin may be dependent on mechanisms other than the inhibition of COX. Thrombin generation test confirmed mild bleeding phenotype in patients with COX deficiency as they had normal thrombin generating capacity.

Protease-Activator Receptor 4 Is Required for Maximal Thrombus Growth, but Not for Fibrin Generation in Thrombi after Laser Injury.

The serine protease, thrombin, is necessary for the conversion of fibrinogen to fibrin and is a potent activator of platelets. Thrombin-induced platelet activation, as measured by shape change, calcium mobilization, and ATP secretion, requires the protease-activator receptor 4 (PAR4). Platelets isolated from PAR4 knock-out mice are unresponsive to thrombin, and PAR4 null mice appear to be protected from thrombosis in a ferric chloride-induced injury model of thrombosis and a thromboplastin model of pulmonary embolism. To examine further the role of thrombin-induced platelet activation in developing thrombi, we have examined the in vivo kinetics of thrombus formation in living mice lacking PAR4 using high-speed widefield digital microscopy. In this study, platelets were labeled using anti-CD41 Fab fragments conjugated to Alexa-488. Thrombi were generated by laser-induced injury of the cremaster arteriolar vessel wall, and the total fluorescent antibody accumulation was monitored and quantitated for 5 minutes after injury. In PAR4 null mice, the thrombi generated were significantly smaller with an early arrest of thrombus growth when compared to thrombi generated in wild-type mice. The maximum thrombus platelet accumulation in PAR4 null mice (median of 30 thrombi in 3 mice) was 75% less than that seen in wild-type mice (median of 33 thrombi in 4 mice)(P<0.001). The time to half-maximal and the time to maximal thrombus formation in PAR4 null mice is approximately 5.5 seconds and 16 seconds, respectively, compared to 45 seconds and 74 seconds in wild-type mice (P<0.001). The shortened time to maximal platelet accumulation appears to be secondary to an early termination of thrombus growth. Fibrin generation was monitored using Alexa-647 conjugated to an anti-fibrin antibody that does not recognize fibrinogen in mice simultaneously infused with anti-CD41 Fab conjugated to Alexa-488. No difference in total fibrin accumulation was seen during the first 4 minutes of thrombus formation in PAR4 null mice (median of 23 thrombi in 3 mice) compared to thrombi generated in wild-type mice (median of 26 thrombi in 4 mice) despite a significant decrease in platelet accumulation in PAR4 null thrombi. Most of the fibrin deposition in both wild-type and PAR4 null thrombi was observed at the vascular wall/thrombus interface. In summary, thrombin-induced platelet activation via PAR4 is required for normal thrombus growth. However, in this model of thrombosis, neither PAR4 nor maximal thrombus growth appears to be necessary for fibrin deposition. This suggests that a platelet-independent mechanism of thrombin generation may exist. Alternatively, the amount of platelet accumulation and activation in PAR4 null mice may be sufficient for normal thrombin generation and subsequent fibrin deposition.