Ivan Budnik

The in-vitro effect of fibrinogen, factor XIII and thrombin-activatable fibrinolysis inhibitor on clot formation and susceptibility to tissue plasminogen activator-induced fibrinolysis in hemodilution model.

Patients suffering major traumatic or surgical bleeding are often exposed to hemodilution resulting in dilutional coagulopathy. The aim of this study was to evaluate in vitro the effects of fibrinogen, factor XIII and thrombin-activatable fibrinolysis inhibitor (TAFI) on clot formation and resistance to fibrinolysis in hemodilution conditions. Citrated whole blood from 36 healthy volunteers was diluted to 30 and 60% with lactated Ringers solution. Blood samples were subsequently supplemented with fibrinogen, FXIII, TAFI or their combinations. Rotation thromboelastometry (ROTEM) in whole blood and thrombin generation in plasma were performed in the presence of CaCl2 and tissue factor/EXTEM reagent, and fibrinolysis was induced by tissue plasminogen activator (tPA). Hemodilution was expressed by decrease of peak height in thrombin generation and &agr;-angle and maximum clot firmness (MCF) in ROTEM. Fibrinogen, FXIII or TAFI did not correct the decrease in thrombin generation peak height. In ROTEM, spiking of diluted blood with fibrinogen stimulated clot propagation. In tPA-treated blood fibrinogen, FXIII and TAFI increased clot firmness and inhibited fibrinolysis. Stronger protection against fibrinolysis was achieved combining FXIII with TAFI. Hemodilution was associated with inhibition of thrombin generation; however, this effect was not sensitive to blood spiking with fibrinogen, FXIII and TAFI. In ROTEM, these hemostasis agents improved clot strength and decreased clot susceptibility to tPA in nondiluted and to more extent in diluted blood. The maximal protection against fibrinolysis was caused by TAFI. Combining FXIII with TAFI exerted synergistic inhibitory effect on fibrinolysis.

The impact of thrombin generation and rotation thromboelastometry on assessment of severity of factor XI deficiency

The phenotype of bleeding in patients with severe FXI deficiency is unpredictable and unlike other bleeding disorders, it is not directly correlated with levels of FXI. In this study we analyzed whether the global coagulation assays can serve as a clinical tool in predicting bleeding tendency in patients with severe FXI deficiency undergoing surgery, taking into account the large inter-individual variability of FXI levels and genotypes. Thrombin generation (TG) was measured in 39 platelet-poor plasma with or without tissue factor (TF) and in the presence or absence of corn trypsin inhibitor (CTI). Rotation thromboelastometry (ROTEM) was performed with fresh whole blood of 26 patients applying NATEM and INTEM tests. TG induced by recalcification can distinguish between bleeding and non-bleeding patients with severe FXI deficiency particularly among those with FXI activity of 2-20IU/dl. The addition of TF or TF and CTI to the TG assay masked the ability to differentiate between XI activity, genotype as well as bleeding and non-bleeding patients. ROTEM assays failed to distinguish bleeding from non-bleeding patients but could do so between different FXI activity levels and genotypes. In conclusion, in the current study we found a sensitive tool to distinguish between bleeding and non-bleeding patients. In order to recommend TG as a predictive tool for treatment tailoring, a larger patient group is required.

Differential roles of fibrinogen and von Willebrand factor on clot formation and platelet adhesion in reconstituted and immune thrombocytopenia.

BACKGROUND: Bleeding tendencies in immune thrombocytopenia (ITP) do not always correlate with the number of platelets, suggesting platelet function variation. We used a model of normal whole blood thrombocytopenia to compare platelet function and other hemostatic variables with ITP patients. We further investigated the effect of in vitro spiking with von Willebrand factor (vWF) and fibrinogen on platelet function and hemostatic variables. METHODS: The Cone and Plate(let) Analyzer was used to measure platelet adhesion (surface coverage [SC], %) and aggregation (average size, &mgr;m2) under defined shear rate (1200 s−1). Rotational thromboelastometry was used to determine variables of clot formation triggered by CaCl2 and tissue factor. RESULTS: In both the model of thrombocytopenia as well as in ITP, the SC and to some extent the average size were correlated to the platelet number over a range of 5 to 80 × 106/mL. The results obtained for most ITP samples were within the boundaries of the lower and upper limits set by the whole blood model of thrombocytopenia. The addition of 2 U/mL vWF (Haemate-P) to whole blood (calculated to plasma volume) results in an increase in the SC and average size without affecting clot formation. Spiking with fibrinogen (100 and 300 mg/dL) did not affect platelet deposition but improved clot formation. CONCLUSIONS: Using a model of whole blood thrombocytopenia enables us to establish reference variables for the Cone and Plate(let) Analyzer and rotational thromboelastometry and to assess platelet function and clot formation in the presence of severe thrombocytopenia. We demonstrated that in most cases of ITP, platelet function is comparable to normal platelets. This work also suggests that vWF and fibrinogen differentially affect primary and secondary hemostasis and therefore both may perform a function in the bleeding phenotype and possibly may be considered for treatment in patients with ITP.

The in vivo effect of fibrinogen and factor XIII on clot formation and fibrinolysis in Glanzmann's thrombasthenia

Glanzmanns thrombasthenia (GT) is characterized by increased bleeding risk. The treatment options in GT are limited. The aim of this study was to test the effect of GT blood supplementation with fibrinogen and factor XIII on thrombin generation, blood clotting, and fibrinolysis. Whole blood samples of GT patients and normal donors treated with eptifibatide (GT model) were subjected to clotting by CaCl2 and tissue factor. Thrombin generation was measured in platelet-rich plasma. Clot formation and tPA-induced fibrinolysis were evaluated in whole blood by rotation thromboelastometry (ROTEM). Blood was supplemented with fibrinogen (3 g/L) and/or FXIII (2 IU/mL). Thrombin generation analysis of blood derived from GT model and GT patients revealed decreased endogenous thrombin potential and peak height and extended lag time compared to control. However, this method was not sensitive to blood spiking with fibrinogen and FXIII. ROTEM revealed lower maximum clot firmness (MCF) and area under curve (AUC) in the blood of GT model and GT patients. In the absence of exogenous tPA, blood spiking with fibrinogen markedly enhanced clot quality while FXIII had no effect. Combination of fibrinogen and FXIII did not add to the effect of fibrinogen. In contrast, by the addition of tPA, both fibrinogen and FXIII separately and, to more extent, in combination enhanced clot quality as well as resistance against tPA-induced fibrinolysis (increasing MCF, AUC, and lysis onset time). In conclusion, fibrinogen and FXIII exerted stimulation of blood clotting and inhibition of fibrinolysis. Treating normal blood with eptifibatide mimics the changes of coagulopathy in GT blood.

Model of trauma-induced coagulopathy including hemodilution, fibrinolysis, acidosis and hypothermia: Impact on blood coagulation and platelet function.

BACKGROUND Trauma-induced coagulopathy (TIC) is commonly seen among patients with severe injury. The dynamic process of TIC is characterized by variability of the features of the disease. METHODS A model of TIC was created. Hemodilution was produced by mixing the blood with 40% Tris/saline solution, fibrinolysis by treating the blood with 160 ng/mL tPA, acidosis by adding 1.2 mg/mL lactic acid achieving pH 7.0 to 7.1, and hypothermia by running the assay at 31°C. Intact blood tested at 37°C served as control. Clot formation was evaluated using rotation thromboelastometry. Platelet adhesion and aggregation were assayed at a shear rate of 1800 s−1 using Impact-R device. RESULTS Clotting time was not affected by any of the TIC constituents used. Clotting initiation was reduced by hemodilution and further reduced by additive hypothermia. The propagation phase of blood clotting was reduced by hemodilution, further reduced by additive hypothermia, and maximally reduced if additionally combined with fibrinolysis. No effect of fibrinolysis on clot propagation was observed at 37°C. Maximum clot firmness was reduced by hemodilution, further reduced by additive fibrinolysis, and maximally reduced if additionally combined with hypothermia. No effect of hypothermia on clot strength was observed in the absence of fibrinolysis. Platelet adhesion (percentage of surface coverage) and aggregation (aggregate size) under flow condition were reduced by hemodilution and further reduced by additive acidosis. Introduction of tPA to diluted blood had no effect on platelet function. CONCLUSION The study revealed a differential effect of TIC constituents—hemodilution, hypothermia, fibrinolysis, and acidosis—on clot formation and platelet function. The effect of one factor may influence that of another factor. These data may be helpful to better understand the pathogenesis of TIC and to elaborate an individually tailored treatment strategy. LEVEL Of EVIDENCE A new model of TIC is created. Contribution of various constituents to pathogenesis of TIC and their interactions are evaluated.

In vitro evaluation of clot quality and stability in a model of severe thrombocytopenia: effect of fibrinogen, factor XIII and thrombin-activatable fibrinolysis inhibitor.

BACKGROUND The treatment options in severe thrombocytopenia (platelet count ≤20×10(9)/L) are limited. The aim of this study was to investigate ways of improving blood clotting and stability in reconstituted thrombocytopenia. MATERIALS AND METHODS Thrombocytopenia (platelets [16±4]×10(9)/L) was created by differential centrifugation of normal blood followed by reconstitution of whole blood which was subjected to clotting in a rotation thromboelastometer by CaCl2 and tissue factor, and to fibrinolysis by tissue plasminogen activator (tPA). In separate experiments, blood was diluted by 40% with TRIS/saline solution. Blood was treated with fibrinogen (fib), factor XIII (FXIII), and thrombin-activatable fibrinolysis inhibitor (TAFI). RESULTS The maximum clot firmness of thrombocytopenic blood was approximately 2-fold less than that of intact blood. Supplementation of blood with fib and FXIII improved clot formation. In the presence of tPA, among fib, FXIII and TAFI, only fib stimulated clot propagation whereas each of these agents increased clot strength. There was a synergistic effect when fib was added together with FXIII or TAFI. Fibrinolysis was inhibited by TAFI and to a greater extent by TAFI + FXIII. Fourty percent dilution of blood reduced clot strength and increased susceptibility to tPA. Clot strength was increased by the treatments in the following order: fib/FXIII/TAFI > fib/TAFI > fib > TAFI > FXIII. In the presence of tPA, TAFI and FXIII lysed the clots significantly more slowly. This effect was stronger when blood was treated with the combination of fib/FXIII/TAFI. Doubling the fib concentration, alone or together with other agents, did not improve clot strength or stability. DISCUSSION Augmentation of clot formation and anti-fibrinolysis by combining fib, FXIII and TAFI may be beneficial for the treatment of patients with severe thrombocytopenia especially when complicated by haemodilution following introduction of fluids to compensate for massive blood loss.

Plasma tissue-type plasminogen activator increases fibrinolytic activity of exogenous urokinase-type plasminogen activator.

The relationship between tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) function is not fully understood. The aim of this study was to compare in vitro the fibrinolytic activity of tPA and uPA and evaluate their possible interaction. Blood coagulation and fibrinolysis were conducted by rotation thromboelastometry, whereas blood clotting was induced by CaCl2 and tissue factor and fibrinolysis additively by tPA and uPA. Effective concentration 50% of tPA and uPA fibrinolytic activity in blood was found to be 90 and 33 IU/ml relating to the units of activity established by manufacturers in the absence of blood. uPA-induced fibrinolysis in blood was faster compared with tPA used at the same units of activity. In contrast, in a blood-free system containing fibrinogen, plasminogen, and thrombin, fibrinolysis induced by uPA was weaker than by tPA. Treating of blood with tranexamic acid (60 mmol/l) was followed by decreased fibrinolytic potential of both exogenous tPA and uPA, despite uPA by itself is known to be not sensitive to aminocaproic acids. Thus, uPA exerted stronger activity in blood but weaker activity in blood-free system, compared with tPA. Taking into account the intermolecular binding of uPA to tPA, it could be suggested that interaction of exogenous uPA with plasma-containing tPA provided amplification of fibrinolysis due to formation of uPA/tPA complex possessing high affinity to fibrin.

Combination of hemostatic therapies for treatment of patients with hemophilia A and inhibitors

BACKGROUND Therapy application and monitoring of patients with hemophilia A (HA) and inhibitors are challenging. In the current study, combined FVIII - bypass therapy was implemented for a cohort of severe HA patients with inhibitors. METHODS Plasma of 15 HA patients with inhibitors was spiked ex vivo with FVIII, rFVIIa, FEIBA and their combinations and thrombin generation (TG) was studied. Some patients who experienced hemarthroses or required minor surgeries were treated by a combined concomitant administration of FVIII+FEIBA as IV bolus doses. RESULTS TG spiking studies showed individual responses not correlated to inhibitor titer. Combinations of agents augmented TG as compared to any single agent, while combined FVIII+FEIBA yielded the highest TG, supporting it as a potential treatment. Following emergent successful surgery of child treated by concomitant FVIII+FEIBA, a total of 396 episodes in 7/15 patients were treated with concomitant FVIII+FEIBA. Five patients were treated for bleeding episodes only, whereas 2 were children undergoing immune tolerance induction (ITI) with FEIBA prophylaxis. Four minor surgeries were performed on FVIII+FEIBA repeated infusions. Neither thrombosis nor any other adverse events were documented. CONCLUSION A combination of FVIII+FEIBA may be effective and safe as an alternative treatment option for some high-responding inhibitor patients.

Thrombin generation as a predictor of thromboembolic events in multiple myeloma patients.

BACKGROUND Multiple myeloma (MM) is characterized by an increased incidence of thromboembolic events, especially when immunomodulatory drugs are used. Currently, our ability to predict these thrombotic events is limited. We hypothesized that global coagulation tests may be predictive of thrombotic events in MM patients. METHODS Blood samples were taken from 36 MM patients before and during routine treatment. Thrombin generation (TG) tests including endogenous thrombin potential (ETP) and peak height were analyzed. RESULTS Patients were followed for a median of 2.5years. Those who developed thrombotic events were characterized by significantly higher ETP and peak height values compared to those who did not (P=0.001). In these patients, we identified a gradual increase in TG parameters that preceded the thrombotic event. Anticoagulation therapy was associated with a significant decrease in ETP and peak height values (P<0.001). There was no statistically significant difference in TG parameters between newly diagnosed MM patients and healthy subjects, as well as between MM patients prior to and during chemotherapy. CONCLUSIONS TG tests might predict thrombotic events in MM patients. Thus, TG tests may be incorporated into decision-making protocols of prophylactic anticoagulant therapy in MM patients.

Synergistic effect of signaling from receptors of soluble platelet agonists and outside-in signaling in formation of a stable fibrinogen-integrin αIIbβ3-actin cytoskeleton complex.

INTRODUCTION Thrombus formation in the injured vessel wall is a highly complex process involving various blood-born components that go through specific temporal and spatial changes as observed by intravital videomicroscopy. Platelets bind transiently to the developing thrombus and may either become stably incorporated into or disengage from the thrombus. The aim of the present study was to reveal the processes involved in the formation of a stable thrombus. METHODS Platelet-rich plasma and washed platelets were studied by the aggregometer. The aggregate stability was challenged by eptifibatide. Platelet Triton-insoluble fraction was prepared and the actin and αIIb content in the cytoskeleton was analyzed by western blot. RESULTS Maximal actin polymerization is achieved 1min after platelet activation while maximal αIIbβ3-actin cytoskeleton association requires 5 to 10min of activation and fibrinogen-mediated platelet-to-platelet bridging. Thus, actin polymerization is dependent on platelet activation and requires neither αIIbβ3 integrin occupation nor platelet aggregation. Formation of a stable aggregate requires platelet activation for more than 1min, complete increase in actin cytoskeleton fraction and partial association of αIIbβ3 with the actin cytoskeleton. However, direct αIIbβ3 activation is not sufficient for cytoskeleton complex formation. Thus, stable αIIbβ3-fibrinogen interaction, representing stable aggregate, is achieved after more than 1min agonist activation, involving inside-out and outside-in signaling but not after direct integrin activation, involving only outside-in signaling. CONCLUSIONS Formation of a stable fibrinogen-αIIbβ3-actin cytoskeleton complex is the result of the combined effect of platelet stimulation by soluble agonists, activation of αIIbβ3, fibrinogen binding and platelet-to-platelet bridging.