Thrombin generation, fibrin clot formation and hemostasis.

Hemostatic clot formation entails thrombin-mediated cleavage of fibrinogen to fibrin. Previous in vitro studies have shown that the thrombin concentration present during clot formation dictates the ultimate fibrin structure. In most prior studies of fibrin structure, clotting was initiated by adding thrombin to a solution of fibrinogen; however, clot formation in vivo occurs in an environment in which the concentration of free thrombin changes over the reaction course. These changes depend on local cellular properties and available concentrations of pro- and anti-coagulants. Recent studies suggest that abnormal thrombin generation patterns produce abnormally structured clots that are associated with an increased risk of bleeding or thrombosis. Further studies of fibrin formation during in situ thrombin generation are needed to understand fibrin clot formation in vivo.     

Thrombin generation and fibrin clot formation under hypothermic conditions: An in vitro evaluation of tissue factor initiated whole blood coagulation

Background

Despite trauma-induced hypothermic coagulopathy being familiar in the clinical setting, empirical experimentation concerning this phenomenon is lacking. In this study, we investigated the effects of hypothermia on thrombin generation, clot formation, and global hemostatic functions in an in vitro environment using a whole blood model and thromboelastography, which can recapitulate hypothermia.

Methods

Blood was collected from healthy individuals through venipuncture and treated with corn trypsin inhibitor, to block the contact pathway. Coagulation was initiated with 5pM tissue factor at temperatures 37°C, 32°C, and 27°C. Reactions were quenched over time, with soluble and insoluble components analyzed for thrombin generation, fibrinogen consumption, factor (f)XIII activation, and fibrin deposition. Global coagulation potential was evaluated through thromboelastography.

Results

Data showed that thrombin generation in samples at 37°C and 32°C had comparable rates, whereas 27°C had a much lower rate (39.2 ± 1.1 and 43 ± 2.4 nM/min vs 28.6 ± 4.4 nM/min, respectively). Fibrinogen consumption and fXIII activation were highest at 37°C, followed by 32°C and 27°C. Fibrin formation as seen through clot weights also followed this trend. Thromboelastography data showed that clot formation was fastest in samples at 37°C and lowest at 27°C. Maximum clot strength was similar for each temperature. Also, percent lysis of clots was highest at 37°C followed by 32°C and then 27°C.

Conclusions

Induced hypothermic conditions directly affect the rate of thrombin generation and clot formation, whereas global clot stability remains intact.     

Transfusing methylene blue-photoinactivated plasma instead of FFP is associated with an increased demand for plasma and cryoprecipitate

BACKGROUND: Photodynamic virus inactivation of plasma with methylene blue significantly decreases the recovery of fibrinogen and coagulation factors V and VIII. Because an adequate supply of fibrinogen is essential for the therapeutic efficacy of transfused plasma in many clinical settings, it was plausible that transfusing photoinactivated plasma (PIP) instead of FFP would result in an increased demand for plasma and cryoprecipitate. STUDY DESIGN AND METHODS: The study involved a retrospective analysis of the use of plasma at a university hospital (Barcelona, Spain) over three 1-year periods: one before the implementation of PIP therapy and two after. Blood components transfused to plasma recipients were listed by broad diagnostic categories based on the Diagnosis-Related Group classification. RESULTS: During the period under study, 2,967 patients were given plasma in this hospital. They received 27,434 units of plasma, 1,660 of cryoprecipitate, 10,079 of platelets, and 24,607 of packed RBCs. Patients undergoing surgical procedures accounted for 74 percent of all transfused plasma. In 71 percent of patient admissions, packed RBCs were transfused in addition to plasma. Diagnostic categories with the greatest requirement for plasma were cardiac valve surgery, liver transplant, wounds and traumatic injuries, and bowel surgery. The use of PIP was associated with a 56-percent increase in the aggregated demand for plasma, whereas the transfusion of non-virus-inactivated cryoprecipitate increased twofold the first year and threefold the second year. The growth in the use of plasma took place in all the diagnostic categories. In those categories that include patients with liver disease, a partial substitution of PIP for cryoprecipitate was observed during the second year after PIP therapy implementation. CONCLUSION: The use of PIP was associated with a marked increase in the demand for plasma and cryoprecipitate, which probably was due to the low hemostatic quality of the new component. It is possible that such an increase overrode the potential health benefits derived from transfusing virus-inactivated plasma.     

Thrombin generation and heparin-induced thrombocytopenia

Summary.  Background : Heparin-induced thrombocytopenia (HIT) is a severe complication of heparin therapy. IgG antibodies targeting the platelet factor 4-heparin complex activate platelets and generate microparticles with procoagulant activity. Objectives : To determine whether the thrombin generation assay is capable of detecting procoagulant activity induced by patient platelet-poor plasma (PPP) in donor platelet-rich plasma (PRP). Patients and methods : We explored two groups of patients; group 1 ( n  = 23): patients with a positive clinical and biological diagnosis of HIT; group 2 ( n  = 25): patients with a negative clinical and biological diagnosis of HIT. Mixtures of donor PRP and patient PPP (1:1) were incubated either with unfractionated heparin 0.2 U mL⁻¹ or with physiological saline. Thrombin generation was assessed by calibrated thrombinography. The effect of heparin on the mixtures was evaluated according to the ratio of the values with and without heparin (wH/woH) of the five thrombogram parameters. Results : With low heparin concentrations, plasma of group 1 activates donor platelets and generates procoagulant activity. A set of three ratios outside the cut-off values corresponds to the 'HIT thrombogram profile', characterized by a highly specific aspect of the thrombogram wH in relation to the thrombogram woH. None of the group 2 patients presented a HIT thrombogram profile. The results of thrombinography correlate well with the results of the platelet aggregation test. Conclusion : Our studies illustrate the central paradox of HIT, namely enhancement of thrombin generation in the presence of heparin. The HIT thrombogram profile as it is defined in this study can detect the procoagulant activity of HIT IgG antibodies.     

Thrombin generation in vascular tissue

BACKGROUND: Classically, it is thought that the vast majority of thrombin is generated on the surface of platelets, however, thrombotic events occur in patients despite treatment with potent antiplatelet agents. METHODS AND RESULTS: In freshly harvested left internal mammary artery (IMA) sections, addition of CaCl2 and platelet-poor plasma (PPP) were sufficient to stimulate a profound burst of thrombin and this effect was inhibited by antitissue factor antibodies. Ultracentrifugation of PPP to remove platelet microparticles had no effect on thrombin generation. Both the extrinsic and factor VIII-dependent pathways were necessary for IMA-supported thrombin generation as PPP derived from individuals deficient in factors V, VII, VIII or X did not support thrombin production. Small amounts of thrombin were generated utilizing factor IX (FIX)-deficient plasma, however, thrombin was not generated by aorta from FIX-deficient mice when FIX-deficient plasma was used. The addition of non-lipidated tissue factor (0.6 pM) and CaCl2 to actively proliferating cultured human aortic smooth muscle cells (SMC) resulted in a pronounced burst of thrombin generation occurring between 3 and 15 min after treatment. In the absence of tissue factor, thrombin was generated but at a slower rate and with a peak value 26% of that observed in the presence of tissue factor. CONCLUSION: Significant thrombin generation can occur on vascular tissue in the absence of platelets or platelet microparticles and on the surface of non-apoptotic SMC.     

Thrombin generation and venous thromboembolism

Venous thromboembolism is a chronic and potential fatal disease. Determination of recurrence risk is time-consuming and costly, and sometimes not feasible: many patients carry more than one risk factor, the relevance of some factors with regard to risk of recurrence is unknown, and existence of thus far unknown risk factors must be considered. A laboratory assay that measures multifactorial thrombophilia would be useful to identify patients at risk of thrombosis. The process of thrombin generation is the central event of the hemostatic process. Thrombin generation is increased in patients at risk of thrombosis including those with antithrombin deficiency or those who are taking hormonal contraceptives. Risk of first and recurrent venous thrombosis is higher in patients with increased thrombin generation. Thus, by use of a simple global marker of coagulation stratification of patients according to their risk of thrombosis is possible. Future studies are needed to improve the management of patients with VTE and increased thrombin generation.     

Antithrombin III in fresh frozen plasma, cryoprecipitate, and cryoprecipitate-depleted plasma

Antithrombin III (AT III) is a plasma protein that inhibits several activated procoagulants. Hereditary disease or acquired conditions such as severe hepatic dysfunction, nephrotic syndrome and intravascular coagulation may be associated with reduced levels of AT III. Its replacement may be essential in controlling thrombosis. In order to determine the most effective form of replacement, we compared AT III biological activity and antigen levels in conventionally prepared fresh frozen plasma, cryoprecipitate and cryoprecipitate depleted plasma (CDP). Both the activity and antigen levels were comparable in all three products (approximately 100%) and AT III was not concentrated in cryoprecipitate. These results indicate that conventionally prepared CDP, fresh frozen plasma and cryoprecipitate contain equal quantities volume for volume of AT III. On this basis, all products are equally effective as therapy for AT III deficiency, but CDP and fresh frozen plasma are recommended as convenient sources of this factor.     

Thrombin generation profiles in deep venous thrombosis

BACKGROUND: Reliable markers and methods to predict risk for thrombosis are essential to clinical management. OBJECTIVE: Using an integrated approach that defines an individual's comprehensive coagulation phenotype might prove valuable in identifying individuals at risk for experiencing a thrombotic event. METHODS: Using a numerical simulation model, we generated tissue factor (TF) initiated thrombin curves using coagulation factor levels from the Leiden Thrombophilia Study population and evaluated thrombotic risk, by sex, age, smoking, alcohol consumption, body mass index (BMI) and oral contraceptive (OC) use. We quantitated the initiation, propagation and termination phases of each individuals' comprehensive TF-initiated thrombin generation curve by the parameters: time to 10 nm of thrombin, maximum time, level and rate (MaxR) of thrombin generated and total thrombin. RESULTS: The greatest risk association was obtained using MaxR; with a 2.6-fold increased risk at MaxR exceeding the 90th percentile. The odds ratio (OR) for MaxR was 3.9 in men, 2.1 in women, and 2.9 in women on OCs. The association of risk with thrombin generation did not differ by age (OR:2.8 OR:2.5), BMI (OR:2.9 OR:2.3) or alcohol use. In both numerical simulations and empirical synthetic plasma, OC use created extreme shifts in thrombin generation in both control women and women with a prior thrombosis, with a larger shift in thrombin generation in control women. This suggests an interaction of OC use with underlying prothrombotic abnormalities. CONCLUSIONS: Thrombin generation based upon the individual's blood composition is associated with the risk for thrombosis and may be useful as a predictive marker for evaluating thrombosis on an individual basis.     

Preparation of cryoprecipitate from riboflavin and UV light-treated plasma

Background and objectivesThe Mirasol® pathogen reduction technology system for plasma is based on a riboflavin and UV light treatment process resulting in pathogen inactivation due to irreversible, photochemically induced damage of nucleic acids. This study was undertaken to evaluate the possibility of making pathogen reduced cryoprecipitate from riboflavin and UV light- treated plasma that meets the quality requirements specified by UK and European guidelines for untreated cryoprecipitate.Materials and methodsCryoprecipitate was made from riboflavin and UV light-treated plasma. Plasma units were thawed over a 20 h period at 4 °C, and variable centrifugation settings (from 654 g for 2 min to 5316 g for 6 min) were applied to identify the optimal centrifugation condition. Plasma proteins in cryoprecipitate units were characterized on a STA Compact, Diagnostica STAGO and Siemens BCS analyzer.ResultsNeither the centrifugation speed or time appeared to have an effect on the quality of the final cryoprecipitate product; however the initial solubilization of the cryoprecipitate product was found to be easier at the lower centrifugation setting (654 g for 2 min). Cryoprecipitate units prepared from Mirasol-treated plasma demonstrated protein levels that were less than levels in untreated products, but were on average 93 IU/unit, 262 mg/unit and 250 IU/unit for FVIII, fibrinogen and von Willebrand ristocetin cofactor activity, respectively.ConclusionCryoprecipitate products prepared from Mirasol-treated plasma using a centrifugation method contain levels of fibrinogen, FVIII and von Willebrand ristocetin cofactor activity, that meet both the European and UK guidelines for untreated cryoprecipitate. Flexibility in centrifugation conditions should allow blood banks to use their established centrifugation settings to make cryoprecipitate from Mirasol-treated plasma.     

Thrombin generation capacity is impaired in methylene-blue treated plasma compared to normal levels in single-donor fresh-frozen plasma, a licensed solvent/detergent-treated plasma (Octaplas) and a development product (Uniplas).

Impaired capacity of thrombin generation (TG) was found in single-donor fresh-frozen plasma (FFP) units subjected to medical device treatment by a combination of methylene-blue dye and subsequent white-light exposure (MB plasma, MBP) compared to normal levels in non-MB-treated single-donor FFP, the licensed plasma product Octaplas, and a product under development (Uniplas; working title) applicable independently from the recipient's blood group. This result held true for MBP units obtained from two different European sources revealing a more significant TG impairment using the local inactivation system in blood banks ("in-house") than an industrial MB treatment. Supplementation of functional fibrinogen to physiological levels did not normalise the altered TG capacity in MBP, whereas addition of Octaplas did. No clear-cut correlation between coagulation factor levels in MBP and hampered TG capacity was found, suggesting a composite effect of impairment. A thorough elucidation and evaluation regarding the possible clinical impact of these findings seems prudent.     

Thrombin generation before and after multicomponent blood collection

BACKGROUND: Apheresis technology has made tremendous progress up to the development of automated blood component collection, which offers increased efficiency in donor blood use, but the concern about the contact of donor blood with artificial surfaces remains. Activation of the hemostatic system is a major issue in this context and is controversial. The aim of this study was to estimate the effect of apheresis on continuous thrombin generation (TG), representing a new tool to examine the overall function of the plasmatic clotting system. STUDY DESIGN AND METHODS: Twenty-six voluntary blood donors, fulfilling the law requirement for apheresis donation, participated in the study. Two units of platelets (6 x 10(11)) and 1 unit of red cells (250 mL; hematocrit level, 80%) were collected using two types of cell separators (Amicus, Fenwal, Inc.; and Trima Accel, Gambro BCT). Each donor underwent collection on both apheresis systems with at least 8 weeks in between. Samples of blood were collected before, immediately after, and 48 hours after apheresis. TG was measured using a slow fluorogenic substrate by means of calibrated automated thrombography (CAT). RESULTS: CAT data changed only slightly, and no significant changes were seen before, immediately after, and 48 hours after apheresis (p > 0.05). The variables did not differ significantly between the two different apheresis systems (p > 0.05). CONCLUSION: Using a CAT-based technique, no change in variables of continuous TG were observed, suggesting that multicomponent blood collection did not lead to severe alterations in the hemostatic system of the donors.