Increase In Endogenous Thrombin Potential Research Articles

Fitusiran reduces bleeding in factor X–deficient mice

AbstractFactor X (FX) deficiency is a rare bleeding disorder manifesting a bleeding tendency caused by low FX activity levels. We aim to explore the use of fitusiran (an investigational small interfering RNA that silences antithrombin expression) to increase thrombin generation and the in vivo hemostatic potential under conditions of FX deficiency. We therefore developed a novel model of inducible FX deficiency, generating mice expressing <1% FX activity and antigen (f10low mice). Compared with control f10WT mice, f10low mice had sixfold and fourfold prolonged clotting times in prothrombin time and activated partial prothrombin time assays, respectively (P < .001). Thrombin generation was severely reduced, irrespective of whether tissue factor or factor XIa was used as an initiator. In vivo analysis revealed near-absent thrombus formation in a laser-induced vessel injury model. Furthermore, in 2 distinct bleeding models, f10low mice displayed an increased bleeding tendency compared with f10WT mice. In the tail-clip assay, blood loss was increased from 12 ± 16 μL to 590 ± 335 μL (P < .0001). In the saphenous vein puncture (SVP) model, the number of clots generated was reduced from 19 ± 5 clots every 30 minutes for f10WT mice to 2 ± 2 clots every 30 minutes (P < .0001) for f10low mice. In both models, bleeding was corrected upon infusion of purified FX. Treatment of f10low mice with fitusiran (2 × 10 mg/kg at 1 week interval) resulted in 17 ± 6% residual antithrombin activity and increased thrombin generation (fourfold and twofold to threefold increase in endogenous thrombin potential and thrombin peak, respectively). In the SVP model, the number of clots was increased to 8 ± 6 clots every 30 minutes (P = .0029). Altogether, we demonstrate that reduction in antithrombin levels is associated with improved hemostatic activity under conditions of FX deficiency.

Tissue Factor Pathway Inhibitor (TFPI) Beta and Gamma Isoforms Contribute to Bleeding in Hemophilia Mice

Key Points: Expression of any single TFPI isoform (α, β, or γ) is sufficient for mouse survival.Two-Kunitz domain TFPI isoforms, anchored β and circulating γ, each contribute to hemophilia bleeding.In vivo, TFPIβ is a more potent anticoagulant than TFPIα. Upon injury, tissue factor (TF) triggers coagulation by complexing with factor (F)VIIa to activate FIX and FX, leading to thrombin generation and a fibrin clot. Tissue factor pathway inhibitor limits TF-triggered coagulation. Humans and mice express transcripts encoding for 3-Kunitz domain TFPIα and membrane-anchored 2-Kunitz TFPIβ. Mice also express transcripts encoding untethered, 2-Kunitz TFPIγ. In humans, proteolysis of TFPIα and/or β produces “γ-like” 2-Kunitz TFPI. Hemophilia A and B, caused by deficiency in factors VIII and IX, respectively, are congenital X-linked recessive bleeding disorders that affect an estimated 1.1 million males worldwide. Hemophilia can cause excessive bleeding spontaneously or in response to trauma that can be life-threatening or significantly decrease the quality of life. Approved treatments for hemophilia include factor replacement, bypassing agents, the bispecific antibody Emicizumab, and recently approved gene therapies. Also in development are agents that reduce endogenous anticoagulant activity to counter the procoagulant deficiency of hemophiliacs, including monoclonal antibodies (Mab) to TFPI. These Mab target all TFPI isoforms to improve coagulation and correct bleeding in hemophilia patients, but with some demonstrated thrombosis risk. We investigated if TFPI-isoform-specific inhibition could provide equivalent efficacy which might reduce thrombotic risk. We generated TFPI-isoform-specific exon deletions in mice bred into hemophilia (FVIII-null) background. We find mice expressing any single TFPI isoform (α, β, or γ) appear healthy and reproduce. Our in vivo tail-vein re-bleeding assay is based on serial clot disruptions over 15 minutes, which we have shown is sensitive to anticoagulant and antiplatelet treatment, as well as factor replacement therapy. In this assay, we find C57Bl/6 WT mice clot 25.6 + 0.8 times (mean + SEM), whereas FVIII-null hemophilia mice clot 5.3 +0.4 times. In hemophilia mice, TFPIα-specific deletion is without impact, forming clots 5.1 + 0.8 times, whereas TFPIβ-specific or TFPIγ-specific deletion improves clot formation to 16.8 + 2.0 and 15.2 + 1.5 times, respectively (p&amp;lt;0001). Even hemophilia mice with heterozygotic deletion of the β exon form clots 11.8 + 0.8 times (p&amp;lt;0001). Thus, in vivo 2-Kunitz TFPI isoforms, anchored β and untethered γ each provides greater anticoagulant activity and impact on bleeding than 3-Kunitz TFPIα. Ex vivo plasma-based thrombin generation assays show that TFPIγ provides more anticoagulant activity than TFPIα. As shown in the figure, the deletion of TFPIγ increases thrombin generation to a greater extent than the deletion of TFPIα. The addition of an anti-TFPI antibody that effectively blocks TFPI activity shows a significant increase in endogenous thrombin potential in the plasma of TFPIαβ-deleted (γ-only) mice (370 nM.min), versus TFPIβγ-deleted (α-only) plasma (70 nM.min) and TFPIαγ-deleted (β-only) mice plasma (22 nM.min), (p&amp;lt;0.001). As expected, membrane-anchored TFPIβ is not detected in plasma. It is well established that 3-Kunitz TFPIα is a potent anticoagulant in plasma-based clotting time assays, while truncated 2-Kunitz TFPI, like mouse TFPIγ, appears inactive. Based on such assays, it was unexpected that TFPIγ-only expressing mice thrive, and in vivo, TFPIα is not the dominant endogenous anticoagulant. If mouse models are to be useful in providing insights into human hemostasis and TF-associated diseases, then it is critical to understand the similarities and differences between mice and humans. As humans do not express TFPIγ, the observed impact of depleting circulating 2-Kunitz TFPI on bleeding and thrombin generation in hemophilic mice is unlikely to translate to man. By contrast, our results demonstrating that, in vivo, TFPIβ is a more potent anticoagulant than TFPIα in mice has potential therapeutic implications in humans.

Intrinsic coagulation pathway-mediated thrombin generation in mouse whole blood.

Calibrated Automated Thrombography (CAT) is a versatile and sensitive method for analyzing coagulation reactions culminating in thrombin generation (TG). Here, we present a CAT method for analyzing TG in murine whole blood by adapting the CAT assay used for measuring TG in human plasma. The diagnostically used artificial and physiologic factor XII (FXII) contact activators kaolin, ellagic acid and polyphosphate (polyP) stimulated TG in murine blood in a dose-dependent manner resulting in a gradual increase in endogenous thrombin potential and peak thrombin, with shortened lag times and times to peak. The activated FXII inhibitor rHA-Infestin-4 and direct oral anticoagulants (DOACs) interfered with TG triggered by kaolin, ellagic acid and polyP and TG was completely attenuated in blood of FXII- (F12 -/-) and FXI-deficient (F11 -/-) mice. Moreover, reconstitution of blood from F12 -/- mice with human FXII restored impaired contact-stimulated TG. HEK293 cell-purified polyP also initiated FXII-driven TG in mouse whole blood and addition of the selective inhibitor PPX_Δ12 ablated natural polyP-stimulated TG. In conclusion, the data provide a method for analysis of contact activation-mediated TG in murine whole blood. As the FXII-driven intrinsic pathway of coagulation has emerged as novel target for antithrombotic agents that are validated in mouse thrombosis and bleeding models, our novel assay could expedite therapeutic drug development.

High Incidence of Intra-Cardiac Supra-Centimetric Thrombosis and Embolic Complications in Sickle Cell Disease Patients with Dual Lumen Ports

In Sickle Cell Disease (SCD) patients with cerebral vasculopathy and/or other severe complications, a transfusion program is indicated. A sustainable venous access is vital for patients engaged in these programs (5-10% of patients with SCD). Dual lumen ports (Vortextechnology) have emerged has a way to bypass the frequent local complications associated with arteriovenous fistulae. Between July 2015 and December 2020, a dual lumen port was implanted in 27 SCD patients in transfusion program at Necker Hospital. Over a 30 months period, we observed 20 venous thromboembolic events (VTE) in 12 patients (44%): 18 were voluminous intracardiac thrombosis (Fig 1), asymptomatic (n=15) or complicated of pulmonary embolism (n=3), one jugular thrombosis and one superior cellar syndrome, leading to long-term curative anticoagulation, except in cases of contraindication such as Moya-Moya. VTE were diagnosed on average 18 months after implantation (range 1 to 29 months). This observation has considerably limited the use of the device, even though no complications were observed in 56% of patients, suggesting that a subgroup of patients may be at lower risk of thrombosis and could still benefit from a Vortex. We conducted a clinical study aiming to identify potential biological markers related to Vortex thrombosis in SCD patients. Among the biological markers, alterations of thrombin generation and fibrino-formation, as well as potentially procoagulants items altered in SCD, such as microvesicules (MV), red blood cell (RBC) deformability and the intensity of splenic dysfunction were explored. Between January and June 2021, 11 healthy donors and 58 patients with SCD treated by erythrapheresis in the biotherapy department of Necker Hospital were included, 23 patients with a Vortex with (n=12) or without (n=11) intra-cardiac and/or other VTE and 36 patients without a Vortex (including 8 with a history of VTE). Thrombin generation Assay (TGA) in plasma and standard coagulation assays were performed on pre-transfusion sample, including Prothrombin Time (PT), activated partial thromboplastin time (APTT) and fibrinogen, dosage of FII, FV, FVII, FX and FXII, Protein C (PC), Protein C (PS), Antithrombin (AT) dosage. All samples were pretreated by activated carbon for removal of Direct Oral Anticoagulants from plasma. Quantification and characterization of plasma MV, determination of erythrocyte deformability and point of sickling (ektacytometry with oxygenscan) and assessment of splenic function (quantification of pocked RBC and Howell Jolly bodies) were also performed. We found a significant decrease in the mean levels of PC (69.7 vs 109.1%, p=0.0002) and PS (58.4 vs 99.4, p<0.0001) in SCD patients when compared to healthy donors while AT levels were only slightly decreased (91 vs 98,6%, p=0,04). We also observed a significant decrease in vitamin-K dependent coagulation factors: FII (77.5 vs 100.6%, p<0,0001), FVII (61.9 vs 84.5% p=0.005) and FX (71.8 vs 96.8%, p=0.0001) while FV (80.1 vs 93.8% p=0.02) was only slightly decreased and fibrinogen levels were comparable (2.62 vs 2.57g/L p=0.86). No significant differences were noted between SCD patient groups (with or without thrombosis). Thrombin generation Assay (TGA) in plasma showed an increase in endogenous thrombin potential (ETP; 714 vs 451nM.min, p=0.04) and Peak (161 vs 95 nM.min, p=0.03) in SCD patients compared to healthy donors but no differences were found between patient groups. Assessment of splenic function, point of sickling in oxygenscan and deformability of RBC measured by ektacytometry showed no differences between patient groups. Patients with SCD had a significant increase of circulating CD235+ MV (93268 vs 15 207/µL, p<0.0001), Annexin 5 MV (473553 vs 248404 /µL) and CD235+ erythrocyte ghosts (1757 vs 61/µL) compared to healthy donors. Again, no differences were seen between patient study groups. In conclusion, no biological predictive factors for intra cardiac Vortex thrombosis could be demonstrated by our study despite an extensive study of biological factors known to date to favor thrombosis. Given the high incidence of intracardiac thrombosis and the impossibility to date of predicting the risk of occurrence, we recommend that Vortex technology be reserved for patients who are already receiving effective anticoagulant therapy or for whom no other solution can be found, while maintaining a close surveillance of echocardiography.

Thrombin generation potential is increased in patients with autoimmune inflammatory myopathies

PurposeDermatomyositis and polymyositis (DM/PM) are rare autoimmune inflammatory myopathies, characterized by an increased risk of cardiovascular and thromboembolic events, likely related to the prothrombotic plasma properties. The aim of this study was to assess the in vitro thrombin generation profile as a biomarker of plasma procoagulant properties in DM/PM patients. MethodsIn 58 clinically stable DM/PM patients and 67 controls matched for sex, age, body mass index, we measured plasma thrombin generation potential using the Calibrated Automated Thrombinography (CAT) and analyzed its relationship with clinical disease characteristics, including autoantibodies profile. ResultsPatients with DM/PM had a 21% increase in endogenous thrombin potential (ETP), 36% higher peak thrombin concentration, and 11% faster thrombin generation, compared to controls (p ​< ​0.001, all, also after adjustment for potential confounders). Interestingly, although both diseases did not differ in thrombin generation potential, heterogenous variables predicted elevated ETPs in both of them. In DM, that was higher fibrinogen, C-reactive protein, and total cholesterol, whereas in PM, presence of arthritis and increased blood platelet count. Surprisingly, thrombin formation capacity remained in a robust inverse relationship with serum troponin (r ​= ​−0.67, p ​< ​0.001) in the patient group. ConclusionsDM/PM patients are characterized by an increased thrombin generation potential, suggesting prothrombotic plasma properties in both diseases. However, more studies are needed to verify its rationale and role in DM/PM clinical course and unfavorable clinical outcomes.

Impact of Fibrinogen Infusion on Thrombin Generation and Fibrin Clot Structure in Patients with Inherited Afibrinogenemia.

Inherited afibrinogenemia is a very rare disease characterized by complete absence of fibrinogen in the circulation and an increased risk in both thrombosis and bleeding. Infusion of fibrinogen concentrate (FC) is the main approach for prevention and management of bleeding; however, it has been reported to carry a thrombotic risk. We investigated the impact of a standard dose (40-100 mg/kg) of FC infusion on the thrombin generation (TG) parameters and the fibrin clot structure formed in plasma samples of patients with afibrinogenemia. Blood samples were collected from 20 patients before (T0) and 1 hour after infusion of FC (T1). TG was studied with calibrated automated thrombography. Fibrin clot structure was assessed with turbidimetry and scanning electron microscopy. FC infusions (mean Clauss fibrinogen plasma level: 1.21 g/L at T1) led to a statistically significant increase in endogenous thrombin potential (ETP) (p < 0.0001) and thrombin peaks (p = 0.02). Nevertheless, when compared with healthy controls, patients' T1 lag times were longer (p = 0.002), ETP values were lower (p = 0.0003), and thrombin peaks were lower (p < 00001). All fibrin polymerization parameters (turbidimetry) obtained at T1 were comparable to those of patients with inherited hypofibrinogenemia matched for fibrinogen plasma levels. In summary, fibrinogen infusion with a standard dose of FC increased but did not correct TG and led to formation of fibrin clots similar to those of patients with hypofibrinogenemia. All in all, our results do not support the biological evidence of hypercoagulability induced by FC in patients with afibrinogenemia.

Donor and Storage Duration Impact Cold-Stored Platelet Function More Than Handling Protocols

Cold storage of platelets has the potential to mitigate the logistical and financial burdens associated with platelet inventory management. In addition, cold-stored platelets (CS-PLT, stored at 2-6°C) have better preserved hemostatic function than room temperature (RT)-stored platelets (RT-PLT), suggesting that CS-PLT may provide improved hemostatic resuscitation in actively bleeding patients as compared to RT-PLT when transfused. With CS-PLT clinical use on the rise, questions regarding CS-PLT inventory management have been raised - specifically how should CS-PLT be stored and handled over the course of storage (after collection and prior to transfusion). RT-PLT are currently stored flat on specialized porous racks and agitated at 18-22°C, which allows for oxygenation. These products are stored out to 5 days, with 7 days allowed for large volume delayed sampling collections. In contrast, refrigerated blood products (i.e., packed red blood cells and whole blood) are stored vertically in pull out drawers, with the goal of reducing harmful oxidizing damage to red blood cells. Notably, refrigeration is known to induce aggregation of platelets, a feature historically associated with bacterial contamination in RT-PLT. In order to diminish concerns regarding aggregate formation, it may be beneficial to massage CS-PLT briefly each day to reduce aggregate formation, yet how this may impact hemostatic function is unknown. To this end, optimal storage and handling conditions for CS-PLT remain to be determined.The objective of our study was to measure CS-PLT hemostatic function in response to two major storage and handling variables: agitation and massage to reduce aggregation in the bag. Single donor apheresis platelet units (single Trima collection in plasma, split equally into two bags to control for donor variability; n=20 donors in 40 bags) were purchased from our regional blood center and delivered to our lab on day of collection. Upon arrival, units were spiked and sampled under sterile conditions for baseline (day 0) profiling, assigned to one of the study groups, and stored accordingly. Our study groups were as follows: “Agitated” (n=10 units), “Not Agitated” (n=10 units, paired with Agitated donors), “Massaged Daily”, (massaged 60s daily, n=10 units), and “Massaged at Sampling” (massaged only at sampling, n=10 units, paired with Massaged Daily donors). “Agitated” platelet units were stored in a custom refrigerated shaker (courtesy Helmer Scientific). For the other groups, platelet units were stored in a walk-in cold room. All units were stored on the same style perforated agitator rack, with agitation either powered on or off as assigned. Units from all groups were sampled under sterile conditions at days 2, 5, 7, 14, and 21 of storage, using 8 mL draws at each time point to ensure equal volume removal over the course of storage. Hemostatic function was assayed using light transmission aggregometry (LTA; ADP, collagen, epinephrine agonists), rotational thromboelastometry (ROTEM; ExTEM, InTEM agonists), and thrombin generation in response to 5 pM tissue factor. Platelet counts (x10 3/µL) were obtained using a hematology analyzer.During the first week of storage, there were no significant differences in the hemostatic profiles between study groups. While platelet counts, endogenous thrombin potential, and ROTEM clot formation time and maximum clot firmness were fairly stable over the first 7 days of storage across all groups, there was a > 50% reduction from baseline in aggregation responses to both ADP and collagen across all 4 groups, suggesting that platelet aggregation, as detected by LTA, may not be the best representative of CS-PLT function. By day 21 of storage, there was a robust increase in endogenous thrombin potential in all study groups when compared to baseline (Agitated, 22%; Not Agitated, 70%; Massaged Daily, 51%; Massaged at Sampling, 47%). Despite this increase in thrombin generation, after extrinsic activation day 21 CS-PLT in all groups had two-fold increased clot formation times compared to baseline (Agitated, 185%; Not Agitated, 223%; Massaged Daily, 236%; Massaged at Sampling, 190%), and reduced maximum clot firmness compared to baseline (Agitated, -44%; Not Agitated, -36%; Massaged Daily, -54%; Massaged at Sampling, -43%). These data suggest that storage duration, and not agitation nor massaging to reduce aggregates, has the most impact on CS-PLT hemostatic function. DisclosuresSpinella: Cerus Corporation: Consultancy, Research Funding; Secure Transfusion Services: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company.

Prophylactic fresh frozen plasma and platelet transfusion have a prothrombotic effect in patients with liver disease

Background and AimsPatients with liver disease acquire complex changes in their hemostatic system, resulting in prolongation of the international normalized ratio and thrombocytopenia. Abnormalities in these tests are commonly corrected with fresh frozen plasma (FFP) or platelet transfusions before invasive procedures. Whether these prophylactic transfusions are beneficial and truly indicated is increasingly debated. In this study, we studied ex vivo effects of FFP and platelet transfusions in patients with liver disease‐associated hemostatic changes in a real‐life clinical setting. MethodsWe included 19 patients who were deemed to require prophylactic FFP transfusion by their treating physician and 13 that were prescribed platelet transfusion before a procedure. Hemostatic status was assessed in blood samples taken before and after transfusion and compared with healthy controls (n = 20). ResultsEx vivo thrombin generation was preserved in patients with liver disease before FFP transfusion. Following FFP transfusion, both in and ex vivo thrombin generation significantly increased, as evidenced by a 92% and 38% increase in thrombin‐antithrombin and prothrombin fragment 1 + 2 levels, respectively, and a 20% increase in endogenous thrombin potential. Platelet counts increased from 28 [21‐41] × 109/L before to 43 [39‐64] × 109/L after platelet transfusion (P < .01), and was accompanied by increases in in vivo markers of hemostatic activation. ConclusionsFFP and platelet transfusion resulted in increased thrombin generation and platelet counts in patients with liver disease, indicating a prothrombotic effect. However, whether all transfusions were truly indicated and had a clinically relevant effect is questionable.

Reversal of hypercoagulability in patients with HCV-related cirrhosis after treatment with direct-acting antivirals.

The long-term impact of sustained virological response (SVR) after direct-acting antivirals (DAAs) on the hypercoagulability associated with HCV cirrhosis is unknown. We longitudinally evaluated the effect of DAAs treatment on cirrhotic coagulopathy. Pro- and anticoagulant factor levels and thrombin generation were assessed in patients with HCV-related cirrhosis at baseline, end of therapy (EOT), at 12, 24 and 48weeks (W) after EOT. Fifty-eight patients were enrolled (86% Child's A). SVR was 100%. Median factor VIII activity significantly decreased at EOT, 12weeks and 24weeks compared with baseline, whereas protein C significantly increased at 24weeks and 48weeks. Cirrhotic patients showed a slight but sustained increase in endogenous thrombin potential (ETP) with a statistically significant difference at EOT, 12weeks, 24weeks and 48weeks compared with baseline. Conversely, thrombomodulin-modified ETP was elevated before treatment and decreased over time to normal levels at 24weeks and 48weeks. The ETP ratio decreased slowly at EOT and 12weeks, and was significantly decreased at 24weeks and 48weeks compared with baseline (P<.001 for both comparisons), being not statistically different from ETP ratio measured in healthy controls. Child's B patients showed a significantly higher ETP ratio compared to Child's A at baseline and did not show any significant improvement in ETP ratio through 12weeks. Two Child's B patients developed PVT with an incidence rate of 1.1% p-yrs (95%CI, 0.18 to 3.58). DAAs therapy in HCV-related cirrhotic patients is associated with significant changes in thrombin generation suggesting a reversal of hypercoagulability particularly in Child's A patients.

Abstract 534: Aging is Associated with Peroxide Mediated Increased Thrombin Generation

Human aging is associated with increased incidence of deep vein thrombosis (DVT), but the mechanisms are not fully elucidated. Using experimental models of DVT, we and others have demonstrated that mice also display increased susceptibility to thrombosis during aging. Further, we established that aged mice overexpressing antioxidant glutathione peroxidase (Gpx1; that converts peroxides to water) are protected from age associated increased susceptibility to DVT. However, the mechanistic pathway for peroxide-dependent thrombosis remains elusive. Recent studies have suggested that hydrogen peroxide mediated activation of neutrophils causes release of neutrophil extracellular traps (NETs). NETs are known to activate coagulation and contribute to the development of DVT. We hypothesized that aging cause peroxide-dependent increased release of NETs and increased thrombin generation. We studied Gpx1 Tg mice and their wild type (WT) littermates at 4 (young) and 20 months (old) of age. Cell-free DNA (cfDNA), a circulating marker of NETs was measured in the plasma using Qubit assay. We observed a significant increase in cfDNA in old WT mice compared to young WT mice (28.2±2.2 vs. 11.4±0.8 ng/μl, P&lt;0.001), but there was no difference in cfDNA between old WT and old Gpx1 Tg mice. Next, we assessed thrombin generation using a Calibrated Automated Thrombogram (Daignostic Stago). The aged WT mice showed significant increase in endogenous thrombin potential (ETP, 430.2±26.7 vs. 277.9±42.1 nM.min, P&lt;0.05) as well as peak amplitude, compared to young WT mice (65.4±4.3 vs. 40.8±7.1 nM, P&lt;0.05). Interestingly, ETP was significantly decreased in aged Gpx1 Tg mice (P&lt;0.05 vs. aged WT), suggesting a peroxide mediated effect on thrombin generation with aging. To examine how the findings in aged mice translate to human aging, we examined plasma samples from healthy young (18-39 years, n=11) and older (50-79 years, n=17) human subjects. We observed a significant increase in cfDNA and ETP in older compared to younger humans (13.4±.9 vs. 12.3±.4 ng/μl, p&lt;0.05 for cfDNA and 1917.6±141.2 vs 1475.9±66.3 nM.min, p&lt;0.05 for ETP). These data suggest that both mouse and human aging is associated with increased release of cfDNA and higher ETP, and that in mice peroxide mediates effects of aging on thrombin generation.

Platelet aggregation impacts thrombin generation assessed by calibrated automated thrombography

A calibrated automated thrombogram (CAT) is performed usually with human platelet-free plasma (PFP) but may be more relevant with platelet-rich plasma (PRP). In this case, platelets are not stimulated by subendothelial molecules like collagen. Our aim was to assess the consequence of strong (collagen) or weak (ADP) induction of platelet release and aggregation on thrombin generation. Platelet aggregation in PRP was triggered with 10 µg/mL collagen or 10 µM ADP using a lumi-aggregometer. Thrombin generation curves were monitored by CAT in different conditions: PRP, PRP with activated platelets (actPRP), aggregated PRP (agPRP), aggregated platelets resuspended in autologous PFP (resPRP), PFP and PFP obtained after aggregation (agPFP). We found a 3-fold shortening of the lag time and time to peak and a marked increase in velocity and thrombin peak without changes in endogenous thrombin potential (ETP) in agPRP with both agonists compared with PRP. The same holds true in agPFP but with a marked increase in ETP compared with PFP. Similar changes in the kinetics of thrombin generation were observed with actPRP-collagen and to a lesser extent in resPRP-collagen compared with PRP. By contrast, there were no modifications of the thrombin generation curves in actPRP-ADP. Alpha-2-macroglobin-thrombin complexes were unchanged in the different PRP conditions but were increased in PFP prepared from agPFP compared to control PFP. Platelet aggregation during activation by agonists other than thrombin did not increase thrombin generation but accelerated its kinetics mainly via platelet content release and platelet-derived extracellular vesicules formation. In diseases characterized by altered platelet granule content or release as well as altered platelet activation, a platelet aggregation step prior to CAT analysis may be clinically relevant to improve laboratory estimation of the bleeding/thrombotic balance.

Effects of Fibrinogen Concentrate on Thrombin Generation, Thromboelastometry Parameters, and Laboratory Coagulation Testing in a 24-Hour Porcine Trauma Model.

Introduction:In a 24-hour porcine model of liver injury, we showed that fibrinogen supplementation does not downregulate endogenous fibrinogen synthesis. Here we report data from the same study showing the impact of fibrinogen on coagulation variables.Materials and Methods:Coagulopathy was induced in 20 German land race pigs by hemodilution and blunt liver injury. Animals randomly received fibrinogen concentrate (100 mg/kg) or saline. Coagulation parameters were assessed and thromboelastometry (ROTEM) was performed.Results:Fibrinogen concentrate significantly reduced the prolongations of EXTEM clotting time, EXTEM clot formation time, and prothrombin time induced by hemodilution and liver injury. A decrease in clot strength was also ameliorated. Endogenous thrombin potential was significantly higher in the fibrinogen group than in the control group, 20 minutes (353 ± 24 vs 289 ± 22 nmol/L·min; P < .05) and 100 minutes (315 ± 40 vs 263 ± 38 nmol/L·min; P < .05) after the start of infusion. However, no significant between-group differences were seen in other thrombin generation parameters or in d-dimer or thrombin–antithrombin levels. Fibrinogen–platelet binding was reduced following liver injury, with no significant differences between groups. No significant between-group differences were observed in any parameter at ∼12 and ∼24 hours.Conclusion:This study suggests that, in trauma, fibrinogen supplementation may shorten some measurements of the speed of coagulation initiation and produce a short-lived increase in endogenous thrombin potential, potentially through increased clotting substrate availability. Approximately 12 and 24 hours after starting fibrinogen concentrate/saline infusion, all parameters measured in this study were comparable in the 2 study groups.

Enoxaparin has no significant anticoagulation activity in healthy Beagles at a dose of 0.8 mg/kg four times daily

Enoxaparin at 0.8 mg/kg every 6 h is used in dogs to prevent prothrombotic events, but has only been validated in healthy Greyhounds, a breed with documented hypocoagulable haemostatic profiles in vitro and bleeding tendencies in vivo. This study investigated the effects of enoxaparin at this dose rate on the coagulation parameters of eight healthy adult Beagles over a 48 h period. Anti-Xa activity was significantly increased 3 h after the second and third of nine injections, and target anti-Xa activity was only reached transiently in 3/8 dogs. Paradoxically, a transient increase in endogenous thrombin potential was observed 6 h after the third injection. Other haematologic, biochemical and coagulation parameters were not significantly altered from the baseline values during enoxaparin treatment (P > 0.05). This study suggests that assumptions regarding the pharmacodynamic effects of enoxaparin are not generalisable across breeds.

The assessment of thrombotic markers utilizing ionic versus non-ionic contrast during coronary angiography and intervention trial.

To determine how two different types of iodinated contrast media (CM), low-osmolar ionic dimer ioxaglate (Hexabrix) and iso-osmolar non-ionic dimer iodixanol (Visipaque), affect multiple indices of hemostasis. In vitro models demonstrate differential effects of ionic and non-ionic CM on markers of hemostasis. This blinded endpoint trial randomized 100 patients to ioxaglate or iodixanol. The primary endpoint was change in endogenous thrombin potential (ETP) following diagnostic angiography. Secondary endpoints included change in markers of fibrinolysis [tissue plasminogen activator (tPA) and plasminogen activator inhibitor 1 (PAI-1)] and platelet aggregation following diagnostic angiography and percutaneous coronary intervention (PCI) with bivalirudin. Data are presented as median [interquartile range]. ETP significantly decreased after diagnostic angiography in both ioxaglate (baseline 1810 nM*minute [1540-2089] to post-angiography 649 nM*minute [314-1347], p < 0.001) and iodixanol groups (baseline 1682 nM*minute [1534-2147] to post-angiography 681 nM*minute [229-1237], p < 0.001), but the decrease was not different between CM (p = 0.70). There was a significant increase in ETP during PCI (n = 45), despite the use of bivalirudin, suggesting a prothrombotic effect of PCI (post-angiography 764 nM*minute [286-1283] to post-PCI 1081 nM*minute [668-1552], p = 0.02). There were no significant differential effects on tPA, PAI-1, and markers of platelet activity. There were no significant differential effects between ioxaglate and iodixanol. Both CM led to significant reductions in thrombin generation and no significant effects on fibrinolytic activity or platelet activity, thereby contributing to a favorable antithrombotic milieu. © 2016 Wiley Periodicals, Inc.

Hypobaric Hypoxia Induces Hypercoagulability Due to an Increase in Microparticles

Studies investigating the role of hypoxia on coagulation, either by going to high altitude or after a long-haul flight, reveal contrasting results. Previous work from our group suggests that the cellular part of the blood is involved in causing a prothrombotic phenotype. Therefore this study investigated the effect of hypobaric hypoxia on blood coagulation, focusing on the role of cellular components.After approval from the local medical ethics committee, 16 healthy participants (aged 20 to 50 years old) were included in this study. Exclusion criteria were a history of cardiovascular disease or pulmonary disease, impaired mobility, and medication known to interfere with coagulation. Participants ascended by cable car to 3,883 meters above sea level, after acclimatizing to altitude for 6 days. At 50 meters and 3,883 meters above sea level, blood was drawn and vital signs (SpO2, heart rate) and the Lake Louise acute mountain sickness questionnaire (LLQ) were recorded. The following tests were performed on whole blood: hemoglobin (Hb), hematocrit (Ht), leucocyte count (L), platelet count (PC) and mean platelet volume (MPV); thrombin generation (TG) in whole blood (WB-TG) with 0.5 pM tissue factor (TF); platelet activation test (PAc-T) triggered by adenosine diphosphate (ADP), thrombin receptor activating peptide (TRAP) and collagen related peptide (CRP) in end concentrations of respectively 10 µM, 30 µM and 1 µg ml-1. The remainder of the blood was centrifuged to obtain platelet rich plasma (PRP) (once for 15 minutes at 230 g) and platelet poor plasma (PPP) (twice for 10 minutes at 2,821 g). PRP was used to test PRP-TG (1 pM TF) and plasma was used for PPP-TG (1 pM TF) and microparticles (MP)-TG (0 pM TF). The paired t-test with p <0.05 was used to determine statistical significance within participants.At 3,883 meters above sea level, oxygen saturation decreased and heart rate increased significantly. LLQ scores revealed mild acute mountain sickness (AMS) symptoms. One participant was withdrawn from the study at 3,030 meters due to moderate AMS. After applying TG in PPP, PRP and whole blood, we found that peak height and endogenous thrombin potential (ETP) were increased. Interestingly, we found a decrease in platelet activation and a decreased MPV. To find an explanation for the increased TG in the different media, we performed a TG assay specially designed to detect microparticles. As with the PPP-TG, PRP-TG and WB-TG, we found an increase in ETP and peak height, proving increased content of MPs.In conclusion, we found that exposure to hypobaric hypoxia increased TG in PPP, PRP and in whole blood. In contrast we found that platelet activation was decreased, indicating that platelets do not play a role in hypoxia-induced hypercoagulability. The increase in peak and ETP in the MP-sensitive TG assay indicates that MPs play a major role in hypoxia-induced hypercoagulability. DisclosuresDe Laat:Synapse bv: Employment.

Increased thrombin generation in women with polycystic ovary syndrome: A pilot study on the effect of metformin and oral contraceptives

ObjectivePolycystic ovary syndrome (PCOS) is associated with risk factors for cardiovascular disease (CVD) which may be modified by the use of metformin and oral contraceptives (OC). Thrombin generation (TG) measures are risk markers of CVD and address the composite of multiple factors that influence blood coagulation. This prospective, randomized, intervention study evaluated the potential influence of PCOS on TG measures and the effect of OC and/or metformin on TG measures in women with PCOS. Material and methodsNinety patients with PCOS and 35 controls were included. Patients were randomized to 12 months of treatment with metformin, metformin+OC or OC alone. C-reactive protein (CRP), fibrinogen, total cholesterol, trunk fat mass, body mass index, estradiol, testosterone, sex hormone binding globulin (SHBG) as well as TG measures, i.e. the lag time for formation of thrombin, the endogenous thrombin potential (ETP), peak thrombin concentration (peak) and time to peak were determined at baseline and after 12 months of treatment. ResultsCRP and total testosterone were significantly higher and SHBG significantly lower in PCOS women than in controls (P=0.012, P<0.001 and P=0.008, respectively). The TG measures ETP, peak and lag time were increased in women with PCOS compared to controls (P<0.01). Significant correlations were observed between TG measures and fibrinogen, CRP, SHBG and fat trunk mass (P>0.01). ETP (P=0.006), peak (P=0.003) and lag time (P=0.023) remained increased after adjustment for these potential confounders. Treatment with OC and metformin+OC further increased ETP (P<0.001) and peak (P<0.005) and reduced time to peak (P<0.04). The increase in ETP was significantly lower in the metformin+OC group than in the OC group (P<0.05). Metformin alone did not affect TG significantly. ConclusionsPCOS is associated with increase in TG measures independent of other risk factors of CVD. OC increase TG measures further and may thus add to the increased risk of CVD already present in women with PCOS.

Simultaneous Measurement of Thrombin Generation and Fibrin Formation in Whole Blood Applying Continuous Flow Is Predictive for the Amount of Blood Loss during/after Cardiothoracic Surgery

Background. Thrombin is the key enzyme converting fibrinogen into fibrin and assays based on the formation of thrombin and fibrin are currently often used separately both in research and clinical settings. Recently it was shown that thrombin generation and fibrin formation do not always go hand in hand as factor XIIa independently influences the crosslinking of fibrin and thereby the stability of the clot.Objective. The development and clinical validation of an assay that is able to simultaneously measure the generation of thrombin and formation of fibrin in whole blood.Methods and results. We have redesigned an air-bearing rheometer, based on the plate and cone viscositor, rendering it sensitive enough to measure fibrin formation based on the change in viscosity. A confocal fluorescent camera is placed into the rheometer, allowing the measurement of thrombin generation based on the conversion of a thrombin-sensitive substrate as in the original Calibrated Automated Thrombinography (CAT) method. Furthermore, the method allows the application of laminar flow conditions within the range of 100 s-1 until 1200 s-1. For all variables related to fibrin formation and thrombin generation, the intra- and inter-assay variation were below 10% in platelet-poor plasma (PPP). For both parameters endogenous thrombin potential (ETP) and peak we demonstrated an inverse relationship with increasing flow rates (100-1200 s-1) in PPP. This was in line with fibrin formation as maximum clot strength (based on viscosity) was inversely related to an increase in flow rate. Furthermore, as expected, the addition of increasing concentrations of fibrinogen to defibrinated plasma was accompanied with an increase in ETP, peak and maximum clot strength. Our method proved to be sensitive enough to detect an increase of 0.1 mg/ml fibrinogen. Furthermore, in the presence of tissue plasminogen activator, it allows to study fibrinolysis. The system was clinically validated in 70 patients undergoing cardiothoracic surgery. Blood samples were taken pre-bypass before heparinisation. Besides thrombin generation and fibrin formation measurements, clinical parameters together with the blood loss and the amount of transfusion products were recorded until 24 hours after surgery. Thrombin generation parameters obtained with the specially designed rheometer correlated well with the traditional whole blood CAT, whereas fibrin formation parameterscorrelated well with the fibrinogen content and the rotational thromboelastometry (ROTEM) parameters. Importantly, when patients were divided into two groups based on the median clot strength determined with the rheometer, a significant difference in perioperative and total blood loss was established.Conclusion. We have technically and clinically validated a new method capable of simultaneously measuring thrombin generation and fibrin formation in plasma and whole blood under continuous flow. Our method proved to be sensitive enough to detect differences in fibrinogen content and revealed an effect of different flow rates on both thrombin generation and clot strength. We hereby have not only combined thrombin generation and fibrin formation in whole blood, but by introducing continuous flow, our method is also one step closer to physiology.Importantly,in contrast to the traditionally used methods, our test applied pre-operatively was found to be predictive for the amount of blood loss during and after cardiothoracic surgery. DisclosuresNo relevant conflicts of interest to declare.

Effect of prednisone administration on coagulation variables in healthyBeagle dogs

Long-term corticosteroid therapy has been associated with increased risk of thrombotic disease in dogs. The purpose of this prospective study was to use thrombelastography (TEG) and thrombin generation (TG) to detect development of a hypercoagulable state in healthy Beagle dogs receiving oral prednisone. We hypothesized that administration of corticosteroids would result in a hypercoagulable profile on TEG tracings and an increase in TG. Six healthy adult Beagles from the University of Montreal's research colony were used to conduct a prospective longitudinal study in which all dogs received 1 mg/kg of prednisone orally once daily for 2 weeks, followed by a 6-week washout period, and then 4 mg/kg of prednisone orally once daily for 2 weeks. TEG tracings on citrated whole blood and TG measurements on frozen-thawed platelet-poor plasma were obtained before prednisone administration (baseline), at the end of the washout period, and at the end of both corticosteroid trials. Significant differences compared with baseline values were obtained for K, α, and MA, with tracings compatible with a hypercoagulable profile following both corticosteroid trials. There was a significant increase in endogenous thrombin potential only after low-dose (1 mg/kg) prednisone. Administration of prednisone to healthy Beagles resulted in hypercoagulability as indicated by TEG tracings, whereas the effect on TG was more variable. Further studies are needed to determine the underlying mechanisms of hypercoagulability and its clinical impact.

Syncytiotrophoblast Microvesicles Released from Pre-Eclampsia Placentae Exhibit Increased Tissue Factor Activity

BackgroundPre-eclampsia is a complication of pregnancy associated with activation of coagulation. It is caused by the placenta, which sheds increased amounts of syncytiotrophoblast microvesicles (STBM) into the maternal circulation. We hypothesized that STBM could contribute to the haemostatic activation observed in pre-eclampsia.Methodology/Principal FindingsSTBM were collected by perfusion of the maternal side of placentae from healthy pregnant women and women with pre-eclampsia at caesarean section. Calibrated automated thrombography was used to assess thrombin generation triggered by STBM-borne tissue factor in platelet poor plasma (PPP). No thrombin was detected in PPP alone but the addition of STBM initiated thrombin generation in 14/16 cases. Pre-eclampsia STBM significantly shortened the lag time (LagT, P = 0.01) and time to peak thrombin generation (TTP, P = 0.005) when compared to normal STBM. Blockade of tissue factor eliminated thrombin generation, while inhibition of tissue factor pathway inhibitor significantly shortened LagT (p = 0.01) and TTP (P<0.0001), with a concomitant increase in endogenous thrombin potential.Conclusions/SignificanceSTBM triggered thrombin generation in normal plasma in a tissue factor dependent manner, indicating that TF activity is expressed by STBM. This is more pronounced in STBM shed from pre-eclampsia placentae. As more STBM are shed in pre-eclampsia these observations give insight into the disordered haemostasis observed in this condition.

Hypercoagulability in Patients With Chronic Noncirrhotic Portal Vein Thrombosis

Although they have normal liver histology and function, patients with chronic noncirrhotic nontumoral portal vein thrombosis (NC-PVT) frequently have abnormal results from coagulation tests. We investigated the significance of these results. We analyzed blood samples collected from 50 stable patients with NC-PVT secondary to a thrombophilic disorder (32%) or local factor (32%), or that was idiopathic (36%). We measured endogenous thrombin potential (ETP) with and without thrombomodulin, prothrombin time, activated partial thromboplastin time, coagulation factors (I, II, V, VII, VIII, IX, X, XI, and XII), antithrombin, proteins C and S, von Willebrand factor (vWF) antigen, vWF ristocetin cofactor, a disintegrin and metalloprotease with thrombospondin type 1 motifs 13 antigen, D-dimer, plasmin-antiplasmin complex, prothrombin fragment F1+2, activated factor VII, and clot lysis time. Samples from 50 age- and sex-matched healthy individuals were evaluated as controls. Compared with controls, patients with NC-PVT had significant increases in prothrombin time and activated partial thromboplastin time; they had significant reductions in levels of procoagulant factors II, V, VII, IX, X, XI, and XII, and the anticoagulants antithrombin, protein C, and protein S. The patients had increased levels of factor VIII and vWF antigen. Irrespective of etiology, patients with NC-PVT had a significant increase in ETP with thrombomodulin and higher levels of factor VIIa, prothrombin fragment F1+2, D-dimer, and plasmin-antiplasmin complex than controls, indicating in vivo activation of coagulation and fibrinolysis. Patients with NC-PVT have hypercoagulability that is independent of the underlying etiology, based on in vitro analyses of thrombin-generation capacity and increased levels of biomarkers in blood samples. Further studies are required to determine if activation of hemostasis increases the risk for thrombotic events.