Increase In Maximum Clot Firmness Research Articles

Fibrinogen contribution to clot strength in patients with sepsis and hematologic malignancies and thrombocytopenia—a prospective, single-center, analytical, cross-sectional study

BackgroundPatients with hematological malignancies (HM) frequently present thrombocytopenia and higher risk of bleeding. Although transfusion is associated with higher risk of adverse events and poor outcomes, prophylactic transfusion of platelets is a common practice to prevent hemorrhagic complications. Thromboelastometry has been considered a better predictor for bleeding than isolated platelet counts in different settings. In early stages of sepsis, hypercoagulability may occur due to higher fibrinogen levels. ObjectivesTo evaluate the behavior of coagulation in patients with HM who develop sepsis and to verify whether a higher concentration of fibrinogen is associated with a proportional increase in maximum clot firmness (MCF) even in the presence of severe thrombocytopenia. MethodsWe performed a unicentric analytical cross-sectional study with 60 adult patients with HM and severe thrombocytopenia, of whom 30 had sepsis (sepsis group) and 30 had no infections (control group). Coagulation conventional tests and specific coagulation tests, including thromboelastometry, were performed. The main outcome evaluated was MCF. ResultsHigher levels of fibrinogen and MCF were found in sepsis group. Both fibrinogen and platelets contributed to MCF. The relative contribution of fibrin was significantly higher (60.5 ± 12.8% vs 43.6 ± 9.7%; P < .001) and that of platelets was significantly lower (39.5 ± 12.8% vs 56.4 ± 9.7%; P < .001) in the sepsis group compared with the control group. ConclusionPatients with sepsis and HM presented higher concentrations of fibrinogen than uninfected patients, resulting in greater MCF amplitudes even in the presence of thrombocytopenia.

How Can Rotational Thromboelastometry as a Point-of-Care Method Be Useful for the Management of Secondary Thromboprophylaxis in High-Risk Pregnant Patients?

Thromboprophylaxis with low-molecular-weight heparin (LMWH) for patients with a history of venous thromboembolism (VTE) is suggested. Rotational thromboelastometry (ROTEM®) represents an innovative point-of-care method enabling the complex and quick evaluation of hemostasis. However, there are only episodic cases of its use for hemostasis assessment and guidance of LMWH in pregnancy. Therefore, we provide the results of unique prospective and longitudinal monitoring of hemostasis in high-risk pregnant women, which we used for the individualized optimalization of secondary thromboprophylaxis. According to the shortening of clot formation time (CFT) in EXTEM (p = 0.0007 from the 26th gestational week vs. controls) and INTEM (p = 0.002 from the 35th gestational week), increase in alpha angle (AA) in EXTEM, INTEM, and HEPTEM, and the persistence of increase in maximum clot firmness (MCF) in EXTEM, INTEM, and HEPTEM (p < 0.001 from the 26th and 35th gestational week vs. controls for EXTEM and INTEM, p = 0.0012 from the 26th gestational week in HEPTEM), LMWH dose was modified. Even after the postpartum period, AA in EXTEM was steeper than in controls (p = 0.0007), indicating that hemostasis is not fully normalized after 6–8 weeks following delivery. Therefore, ROTEM may be a useful tool for the individual evaluation of the termination of anticoagulant thromboprophylaxis.

Incidence of Antithrombin Deficiency and Anti-Cardiolipin Antibodies After Severe Traumatic Brain Injury: A Prospective Cohort Study.

Animal studies suggested that cerebral mitochondrial cardiolipin phospholipids were released after severe traumatic brain injury (TBI), contributing to the pathogenesis of thromboembolism. To determine the incidence of anti-cardiolipin antibodies after severe TBI and whether this was related to the severity of TBI and development of venous thromboembolism. Serial anti-cardiolipin antibodies, antithrombin levels, viscoelastic testing, and coagulation parameters were measured on admission, day-1, and between day-5 and day-7 in patients with severe TBI requiring intracranial pressure monitoring. Of the 40 patients included (85% male and median age 42years), 7 (18%) had a raised Ig-G or Ig-M anti-cardiolipin antibody titer after TBI. Antithrombin levels were below the normal level-especially on day-0 and day-1-in 15 patients (38%), and 14 patients (38%) developed an increase in maximum clot firmness on the viscoelastic test in conjunction with elevations in fibrinogen concentration and platelet count. Four patients (10%) developed deep vein thrombosis, and 10 patients (25%) died, both of which were not significantly related to the presence of anti-cardiolipin antibodies (P = 0.619 and P = 0.638, respectively). A reduction in antithrombin level and development of anti-cardiolipin antibodies were not rare immediately after severe TBI; these abnormalities were followed by an increase in in vitro clot strength due to elevations in fibrinogen concentration and platelet count. The quantitative relationships between the development of anti-cardiolipin antibodies and severity of TBI or clinical thromboembolic events deserve further investigation.

Fibrinogen concentrate for treatment of bleeding and surgical prophylaxis in congenital fibrinogen deficiency patients

BackgroundCongenital fibrinogen deficiency is an ultra‐rare disorder in which patients can experience severe and/or frequent bleeding episodes (BEs). Here, we present the largest prospective study to date on the treatment of this disorder. MethodsHemostatic efficacy of human fibrinogen concentrate (HFC; FIBRYGA®, Octapharma AG) for treatment of bleeding or surgical prophylaxis was assessed by investigators and adjudicated by an independent data monitoring and endpoint adjudication committee (IDMEAC) according to a four‐point scale, using objective criteria. Thromboelastometry maximum clot firmness (MCF) was also determined. ResultsTwenty‐five afibrinogenemia patients were treated with HFC: 24 for on‐demand treatment of 89 BEs, and nine as prophylaxis for 12 surgeries. For BEs, treatment success (rating of excellent or good) evaluated by investigators was 96.6% (90% confidence interval [CI], 0.92‐0.99; two missing ratings, classified as failures) and by the IDMEAC was 98.9% (90% CI, 0.95‐0.999). Mean ± standard deviation (SD) increase in MCF was 5.8 ± 2.5 mm one hour after the first HFC infusion (mean ± SD dose, 61.88 ± 11.73 mg/kg). For the 12 surgeries (median [range] HFC dose/surgery, 85.80 mg/kg [34.09‐225.36]), intraoperative and postoperative treatment success were both rated 100% (90% CI, 0.82‐1.00) by investigators and the IDMEAC. Three adverse events were possibly treatment related, including a moderate case of thrombosis. There were no deaths, no severe allergic or hypersensitivity reactions, and no clinical evidence of neutralizing antifibrinogen antibodies. ConclusionsHuman fibrinogen concentrate was efficacious for on‐demand treatment of bleeding and as surgical prophylaxis, with a favorable safety profile, in patients with congenital afibrinogenemia.

The Effect of Immunosuppression on Coagulation After Liver Transplantation.

Everolimus (EVR) is a mammalian target of rapamycin (mTOR) inhibitor commonly used for immunosuppression (IS) after liver transplantation (LT). However, there are concerns about whether mTOR inhibitors may move the hemostatic balance toward a higher likelihood of thrombosis. The present study aimed to investigate potential coagulation disorders after the administration of EVR. We evaluated 54 patients after conversion to an EVR-based IS regimen (n=26) and compared those patients with patients who were switched to extended-release tacrolimus (TAC) but had never received EVR (n=28). At baseline and again at 1 month and 6 months after conversion, we measured international normalized ratio, activated partial thromboplastin time, and anticoagulation and fibrinolysis factors, and we performed rotational thromboelastometry (ROTEM). Data were analyzed with a Mann-Whitney U test, a repeated-measure analysis of variance, and a Fisher's exact test. Statistical significance was set at the level of P ≤ 0.05. Plasma levels of von Willebrand factor, fibrinogen, and factor VIII were significantly higher than baseline levels at 1 month and 6 months after conversion of IS to EVR (P<0.001); plasma levels of protein C, protein S, and plasminogen also increased significantly (P<0.001). ROTEM confirmed a significant increase in maximum clot firmness in EXTEM, INTEM, and FIBTEM assays (P<0.001). In all assays, maximum lysis was significantly lower than baseline levels at 1 month and 6 months after conversion to EVR. Patients converted to IS with extended-release TAC exhibited no significant changes in coagulation variables. Retrospective analysis showed a significantly higher incidence of thromboembolic complications among patients treated with EVR-based IS than among those treated with extended-release TAC (P<0.01). In conclusion, the administration of EVR after LT seems to modify hemostasis to a procoagulant state. Thrombophilia screening before conversion may determine which patients will benefit from conversion to EVR-based IS.

Correlation Between ROTEM FIBTEM Maximum Clot Firmness and Fibrinogen Levels in Pediatric Cardiac Surgery Patients.

This study evaluated whether rotational thromboelastometry (ROTEM; Tem International GmbH, Munich, Germany) FIBTEM maximum clot firmness (MCF) can be used to predict plasma fibrinogen level in pediatric patients undergoing cardiac surgery. Linear regression was conducted to predict plasma fibrinogen level using FIBTEM MCF (0.05 level of significance). Scatter plot with the regression line for the model fit was created. Fifty charts were retrospectively reviewed, and 87 independent measurements of FIBTEM MCF paired with plasma fibrinogen levels were identified for analysis. Linear regression analysis suggested a significant positive linear relationship (P < .0001) between plasma fibrinogen levels and MCF. Both MCF intercept and slope were significantly correlated with fibrinogen level (P < .0001). The estimated regression equation (predicted fibrinogen = 78.6 + 12.4 × MCF) indicates that a 1-mm increase in MCF raises plasma fibrinogen level by an average of 12.4 mg/dL. The statistically significant positive linear relationship observed between MCF and fibrinogen levels (P < .001) suggests that MCF can be used as a surrogate for fibrinogen level. This relationship is of clinical relevance in the calculation of patient-specific dosing of fibrinogen supplementation in this setting.

Thromboelastometric assessment of hemostasis following hydroxyethyl starch (130/0.4) administration as a constant rate infusion in hypoalbuminemic dogs

BackgroundThe primary aim was to evaluate by means of thromboelastometry (ROTEM) the effects of hydroxyethyl starch (HES) 130/0.4 administered as a constant rate infusion (CRI) on hemostasis in hypoalbuminemic dogs. The second aim was to use ROTEM analysis to detect whether all hypoalbuminemic dogs of our population were hypercoagulable.ResultsThe study sample was 20 hypoalbuminemic dogs (albumin < 2 g/dl) with normal perfusion parameters and requiring intravenous fluid therapy. In order to support plasma colloid osmotic pressure, in addition to crystalloid, HES 130/0.4 was administered as a constant rate infusion at 1 ml/kg/h (group 1, n = 11) or 2 ml/kg/h for 24 h (group 2, n = 9). Blood samples were collected at baseline (T0) and 24 h postinfusion (T1); coagulation was assessed by standard coagulation profile (prothrombin time, activated partial thromboplastin time, and fibrinogen), and ROTEM analysis (in-TEM®, ex-TEM® and fib- TEM® profile).No statistically significant differences in ROTEM values in group 1 were observed (P > 0.05), whereas in group 2 statistically significant differences (P < 0.05) were found at T1 in the in-TEM® profile [decrease in clot formation time (P = 0.04) and increase in α angle (P = 0.02)] and in the ex-TEM® profile [increase in maximum clot firmness (P = 0.008) and α angle (P = 0.01)]; no changes were identified in the fib-TEM® profile. In both groups, a statistically significant decrease (P = 0.007) in hematocrit was noted, whereas no statistically significant differences in platelet count and standard coagulation profile were found. In group 2, a statistically significant increase in TS values (P = 0.03) was noted at T1. ROTEM tracings indicating a hypercoagulable state were observed in 7/20 dogs at T0 (5/11 in group 1 and 2/9 in the group 2).ConclusionOur findings suggest that HES 130/0.4 administered as CRI does not cause hypocoagulability in hypoalbuminemic dogs. A trend toward hypercoagulability, probably related to the underlying diseases, was observed in group 2 at T1.Although all dogs were hyoalbuminemic, only 7/20 were hypercoagulable at T0, confirming the lack of correlation between albumin level and prothrombotic state.

Thromboelastogram as a Tool to Predict Hypercoagulability in Children With Cystic Fibrosis.

Increased thrombophilic tendency in patients with cystic fibrosis (CF) has recently been reported. The determinants of thrombosis in children with CF remain largely unknown. Our aim in this study was to evaluate the thromboelastography (TEG) profile of children with CF through ROTEM (whole blood rotation thromboelastometry). Nineteen patients with CF and 20 controls were included in the study. Whole blood count, prothrombin time, activated prothrombin time, fibrinogen, d-dimer levels, and ROTEM assays (INTEM, EXTEM) were performed. Clotting time, clot formation time (CFT), and maximum clot firmness (MCF) were determined by INTEM and EXTEM analysis. In INTEM assay, MCF ( P = .001) value was significantly increased and CFT ( P = .031) value was decreased in patients with CF compared with those of the control group. In the EXTEM assay, there was a similar significant increase in MCF ( P = .023) value in patients with CF compared with that of the control group. There was a significant positive correlation between fibrinogen levels and MCF in EXTEM ( r = .72) and INTEM ( r = .76) assays, whereas there was a negative correlation with CFT in EXTEM ( r = -.61) and INTEM ( r = -.67). The results of our study indicated that TEG profiles in patients with CF were more hypercoagulable compared with those of the control group.

Infusion of Coagulation Factor VIII-Containing Induced Pluripotent Stem Cell (iPSC)-Derived Megakaryocytes (iMks) Shows Potential As Hemophilia a Treatment

Ectopically expressed factor VIII (FVIII) in megakaryocytes (MKs) and platelets (pFVIII) is stored in a-granules and released at sites of vascular injury by activated platelets (Plts), restoring hemostasis in FVIIInull mice, even in the presence of neutralizing inhibitors. These studies support the idea that unlike therapies that correct plasma levels of FVIII, pFVIII may be a useful therapy in patients with hemophilia A who have intractable inhibitors and significant bleeds. Expressing FVIII in Plts, however, has limitations that make pFVIII gene therapy through bone marrow transplantation (BMT) problematic: 1) pFVIII expressed during megakaryopoiesis can injure the Mks, potentially exacerbating post-BMT thrombocytopenia, and 2) pFVIII’s efficacy in joint and intracranial bleeds has yet to be shown, especially in the presence of inhibitors. Due to these limitations we propose an alternative strategy: infusing patient-specific iMks derived from personalized iPSCs and expressing either human B-domain-deleted (BDD) FVIII or variants of FVIII that have greater stability and longer half-lives. Our group has shown that infusing in vitro-grown Mks into mice releases functional Plts in the recipient animals. iPSCs are a renewable source of stem cells that can be pre-screened to select clones that both express high levels of pFVIII and also release high numbers of Plts after differentiation into iMks. As proof-of-principle, iMks were transfected with a self-inactivating lentivirus containing cDNA for 1 of 3 FVIII variants: wildtype BDD FVIII (WT FVIII), a PACE/furin cleavage site FVIII (FVIIIR1645H) variant, and an amino acid 1645 to 1648 deletion FVIII (FVIIIΔ) variant that removes the entire PACE/furin cleavage site. FVIIIR1645H and FVIIIΔ showed greater stability and consequently greater specific activity with no increase in injuring Mks. We previously published that hemophilia A mice expressing pFVIIIR1645H were more hemostatically corrected than comparable mice expressing WT pFVIII. All of the FVIII variant iMks expressed at least a 40-fold higher level of mRNA compared to the non-transduced control (N=6) and integration levels show the same number of viral copies between the groups (N=6). All variants expressed >550 pg FVIII/106 CD42b+ iMKs (N=6). Upon activation with thrombin, transduced Mks released the FVIII into the supernatant. To examine whether this pFVIII injured the developing Mks, baseline PAC-1 binding for Mk activation in culture (N=3), TUNEL staining and Annexin-5 binding for apoptosis (N=4) were analyzed with no differences observed with WT Mks not expressing pFVIII. To test the ability of FVIII-expressing iMks to correct the coagulopathy in hemophilia A, 5x105 iMks were added to FVIIInull murine whole blood (0.11 ml) and evaluated for clot formation using rotational thromboelastometry (ROTEM). Each pFVIII iMk variant showed a decrease in clotting time, clot formation time, and an increase in maximum clot firmness when compared to the non-transduced control (p<0.007 for each, N=4). These FVIII expressing iMks were also tested in vivo in a FeCl3 carotid artery injury murine model. 24 hours prior to infusion, recipient hemophilia A mice were treated with clodronate liposomes to eliminate circulating monocytes and to improve the survival of infused human iMks and their released Plts. Immediately post iMks (5x106)infusion, a 20% FeCl3 solution was applied to the carotid artery for 3 mins and flow rate through the injured vessel was measured for 30 mins. Both WT FVIII and FVIIIR1645H showed a significant decrease in blood flow through the injured vessel from 1.2 ml/min seen in FVIIInull mice receiving control iMks to 0.4 ml/min (p<0.05, N=10). Wild-type mice had a flow rate of 0.13 ml/min. These data indicate that pFVIII within iMKs or their derived Plts expressing FVIII can improve hemostasis in vitro and in vivo. These studies provide the groundwork to examine whether infused iMks pFVIII can improve hemostasis in the setting of inhibitors. DisclosuresArruda:Pfizer: Patents & Royalties, Research Funding. Sabatino:Spark Therapeutics: Research Funding. Camire:Bayer: Consultancy; Spark Therapeutics: Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties; Pfizer: Consultancy, Patents & Royalties, Research Funding; Novo Nordisk: Research Funding.

Efficacy of Human Fibrinogen Concentrate for on-Demand Treatment of Acute Bleeding and to Prevent Bleeding during and after Surgery in Subjects with Congenital Fibrinogen Deficiency

Patients with congenital afibrinogenaemia and hypofibrinogenaemia, frequently experience severe bleeding episodes starting already at birth or during early childhood. Bleeding may occur after a minor trauma or a surgical intervention into the skin, mucosa, muscles, gastrointestinal tract, or the brain. Therapeutic substitution with human fibrinogen concentrate (HFC) corrects the haemostatic defect and arrests bleeding in these patients. Octafibrin (Octapharma OPG, Vienna, Austria) is a plasma-derived, highly purified, lyophilized fibrinogen concentrate with two dedicated virus inactivation/removal steps was used in this study.This study was prospective, open-label and multinational, in adult and adolescent a- or hypofibrinogenemic patients, to investigate the safety and efficacy of Octafibrin a new HFC in the treatment of on-demand bleeding (BE) and surgical prophylaxis.A planned interim analysis comprises data of 11 adult and 2 adolescent patients. Efficacy was assessed using a 4 point objective scale completed by the investigator and adjudicated by an independent data monitoring and endpoint adjudication committee (IDMEAC) for each bleeding or surgical episode.Eleven of the 13 patients experienced a total of 23 minor BEs. Sixteen BEs (69.6%) were spontaneous and 7 (30.4%) were traumatic. A majority of BEs (21/23) required one HFC infusion (91.3%) and 2 (8.7%) BEs required two infusions. The median (range) dose of HFCadministered for the treatment of all BEs was 57.5 mg/kg (33.9-71.4 mg/kg) per infusion and 58.8 mg/kg (33.9-101.7 mg/kg) per BE.The success rate (efficacy rating of excellent or good) for all BEs was 100% (90% CI: 0.88, 1.00) as adjudicated by the IDMEAC (all excellent).Maximum clot firmness (MCF) using thromboelastometry (ROTEM®) performed in plasma was also determined for the first infusions administered for the treatment of all 23 BEs in the 11 BE patients. The mean (±SD) change in MCF from baseline (MCF 0) to 1 hour after the first infusion of HFCwas 6.5 mm (±2.0) (95% CI: 5.65, 7.40; p<0.0001). This significant overall increase in MCF from baseline coincided with the 100% haemostatic efficacy for all BEs.Four patients underwent 4 surgeries (3 major 1 minor, two of these patients were treated for both BE and surgery). The post-operative success rate (haemostatic efficacy excellent or good) was 100% (90% CI 0.5, 1.0), as assessed by the investigator and the IDMEAC.There have been no reports of serious adverse events related to the infusion of the HFC.In conclusion this study showed 100% hemostatic efficacy in on-demand treatment with HFC in bleeding patients with congenital fibrinogen deficiency. Also hemostatic efficacy in surgical prophylaxis in 4 patients was rated excellent/good. Maximum clot firmness values showed a statistically significant increase from baseline to 1 hour post-infusion in all patients after HFCadministrations reflecting the successful treatment of bleeding events. There were no related serious adverse events, no thromboembolic events, no allergic or severe hypersensitivity reactions, and no anti-fibrinogen antibodies that developed during the study. DisclosuresSchwartz:Octapharma: Employment. Knaub:Octapharma: Employment. Peyvandi:Bayer: Speakers Bureau; Biotest: Other: research funding paid to Luigi Villa Foundation, Research Funding, Speakers Bureau; Ablynx: Membership on an entity's Board of Directors or advisory committees, Other: research funding paid to Luigi Villa Foundation, Research Funding; CSL Behring: Speakers Bureau; Grifols: Speakers Bureau; Alexion: Other: research funding paid to Luigi Villa Foundation, Research Funding; Octapharma: Consultancy; Kedrion Biopharma: Consultancy, Other: research funding paid to Luigi Villa Foundation, Research Funding; LFB: Consultancy; Novo Nordisk: Other: research funding paid to Luigi Villa Foundation, Research Funding, Speakers Bureau; SOBI: Speakers Bureau.

Towards the Care of Hemophilia a Patients Using Induced Pluripotent Stem Cell (iPSC)-Derived Megakaryocytes (iMks) Expressing Coagulation Factor (F) VIII

We and others have shown that FVIII expressed ectopically in platelets (pFVIII) is stored in α-granules, released at sites of vascular injury and restores hemostasis in FVIIInull mice, even in the presence of neutralizing antibodies to FVIII. These studies support the concept that unlike therapeutic interventions that correct plasma FVIII, pFVIII may be a useful therapy in hemophilia A with intractable inhibitors and significant bleeds. We have also demonstrated this approach has several limitations that may make pFVIII gene therapy bone marrow transplantation (BMT) strategies problematic: 1) pFVIII is not equivalent to plasma FVIII and its efficacy in joint and intracranial bleeds has yet to be shown, especially in the presence of inhibitors, and 2) pFVIII expressed during megakaryopoiesis can cause injury to the Mks, potentially exacerbating post-BMT thrombocytopenia. We propose an alternative strategy: interval prophylactic infusions of FVIII-containing platelets generated from patient-specific iMks expressing either human B-domain-deleted (BDD) FVIII or variants of this FVIII that have greater stability and longer half-lives; making them especially efficacious as pFVIII as we previously demonstrated. iPSCs are a renewable source of cells that can be pre-screened prior to clinical usage for lines that express optimal levels of pFVIII and also release optimal numbers of platelets after differentiation into iMks. Such iPSCs were transfected with a self-inactivating lentivirus containing cDNA for one of three FVIII variants: wildtype BDD FVIII (WT FVIII), R1645H PACE/furin cleavage site FVIII (FVIIIR1645H), and amino acid 1645 to 1648 deletion FVIII (FVIIIΔ). FVIIIR1645H and FVIIIΔ show greater stability and consequently greater specific activity with no increase in injuring Mks. All FVIII variants were expressed using the MK-specific Cxcl4 promoter and were shown to be effective in several bleeding models in FVIIInull mice. Differentiated and transduced iMKs were analyzed for RNA and protein expression. All of the FVIII variant iMKs expressed at least forty-fold higher levels of mRNA compared to the non-transduced control (n=6) and protein was expressed at >550 pg/106 CD42b+ iMKs (n=6). Transduced MKs released FVIII into the supernatant when activated by thrombin showing the pFVIII was likely stored in α-granules. Annexin staining was the same between FVIII-expressing iMKs and control iMks suggesting that the level of pFVIII did not cause the iMks to become apoptotic. To test the ability of FVIII-expressing iMKs to correct the coagulopathy in hemophilia A, 5x105 iMKs were added to FVIIInull murine whole blood and evaluated for clot formation using rotational thromboelastometry (ROTEM). Each FVIII variant showed a decrease in clotting time, clot formation time, and an increase in maximum clot firmness when compared to the non-transduced control (n=4). These data show that iMKs expressing FVIII variants can improve hemostasis in a whole blood clotting assay. Our next goal is to generate sufficient platelets from these iMKs to test for correction of the bleeding diathesis in immunodeficient FVIIInull mice and to determine their efficacy in improving hemostasis in a number of clinically relevant hemostatic models. DisclosuresArruda:Pfizer: Consultancy, Patents & Royalties, Research Funding; Spark Therapeutics: Patents & Royalties. Camire:Pfizer: Consultancy, Patents & Royalties, Research Funding; NovoNordisk: Research Funding; Spark Therapeutics: Membership on an entity's Board of Directors or advisory committees.

Fibrinolysis Times As Outcome Measures Using ROTEM with Tpa-Induced Fibrinolysis in Chemotherapy Induced Thrombocytopenia

Background: Despite prophylactic platelet (PLT) transfusions, used to prevent bleeding in chemotherapy induced thrombocytopenia(CIT), some patients will still bleed, whereas others are spared even without PLT transfusion. Little research has been done on (hyper)fibrinolysis as a risk factor for bleeding in CIT. In this study a tPA-induced fibrinolysis in ROTEM was analysed to gain more insight in clot formation and clot lysis times in patients with CIT before and after PLT transfusions.Aim: to study clot vulnerability for fibrinolysis in CIT patients with tPA-ROTEM.Method: patients, older than 18 years, treated for a hematological malignancy and receiving a prophylactic PLT transfusion for thrombocytopenia were included. Blood was collected before PLT transfusion (baseline) as well as 1 hour and 24 hours after transfusion. A modified, EXTEM based,ROTEM in the presence of tPA was used to study the occurrence of fibrinolysis. The maximum clot firmness (MCF) and the time to maximum clot firmness (MCF-t) were determined. Also, 100-10% lysis times (A), 100-85% lysis times (B) and 85- 10% lysis times (C) were calculated. Reference values for the tPA-ROTEM were obtained from 40 healthy volunteers.Results: patient characteristics are shown in table 1. Baseline platelet counts and fibrinogen concentrations were median 6,000/mcL and 4,6 g/L, respectively (table 2). In the tPA-ROTEM a significant increase in MCF, MCF-t, 100-10% LT and 100-85% LT after transfusion was observed. The MCF remained significantly increased after 24 hrs (table 2). When baseline was compared to the healthy volunteers, the MCF, MCF-t were remarkably reduced in the CIT patients. The 100 -10% lysis time was significantly shorter in patients with CIT ( (27,4 versus 34,4 minutes, (P=0.020) (table 3), mainly due to a shorter 100 -85% LT (11.5 versus 17.9 (P<0,001). There were no differences in the 85 - 10% lysis times. A positive, moderate strength correlation was found between the 100-85% lysis times and fibrinogen concentrations (R=0,499 (P=0,035)).Conclusion: Patients with CIT have a shorter lysis times as measured with tPA-ROTEM than healthy volunteers, mainly occurring during the first phase (100-85%) of fibrinolysis. Higher fibrinogen concentrations were associated with a longer duration of the initial phase. Lysis times outcomes in tPA-ROTEM may be applicable in clinical trials assessing PLT transfusions.Table 1Patient characteristics:N=20SDMin- maxAge55 (mean)1323-72Men (%)10 (50%)Diagnosis9 Acute myeloid leukaemia4 Multiple myeloma4 Non-Hodgkin Lymphoma3 OtherTreatment7 Induction therapy (35%)13 Consolidation therapy and stem cell transplantation (65%)Table 2Results from baseline, 1h and 24h measurements (all as median)Baseline n=181hN=1924hN=18Healthy volunteersN=40Reference valuesPlatelet Count median*109/L6*#33*#21*#173150-400 x109/LFibrinogenmedian(g/L)4.58#4.40#4.64*#3.181.70-4.00Maximum Clot Firmnessmm23 #38*#34*#59Time to Maximum Clot FirmnessMin.11.5#15.7*15.3#18.2Lysis Onset TimeMin.23.0#29.7*#28.3#36.1Lysis TimeMin.38.9#44.4*#44.6#52.6* = P<0,05 versus baseline.# = P=<0,05 versus Healthy volunteers.Table 3Differences in lysis time speed between baseline measurement and healthy volunteersUnitPatient group baselineHealthy volunteersMean differenceP-value100 - 10% lysis timeMin.27.434.470.020100 - 85% lysis timeMin.11.517.96,4<0.00185 - 10% lysis timeMin.15.015.30,30.705 [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Tranexamic acid as adjunct therapy to bypassing agents in haemophiliaApatients with inhibitors

Haemophilia patients with inhibitors require bypassing agents (BPA) like activated prothrombin complex concentrate (aPCC) and recombinant activated factor VII (rFVIIa) to control bleeds. Adjunct tranexamic acid (TXA) may improve haemostasis. The objective of this study was to investigate safety and haemostatic effect of TXA given in combination with BPA. Healthy volunteers (N=5) and haemophilia inhibitor patients (N=6) were enrolled in a prospective case crossover design. Controls were treated with TXA 20mgkg(-1) orally (O.R.) Patients were treated with aPCC 75IUkg(-1) intravenous (I.V.) on day 1 followed by TXA 20mgkg(-1) O.R. combined with aPCC 75IUkg(-1) I.V. on day 2. A 14-day washout occurred before crossover to rFVIIa 90μgkg(-1) I.V. ±TXA. Safety evaluation and blood sampling processes were performed at baseline, 15, 30, 60, 120, 180 and 240min post treatment. Primary outcome was maximum clot firmness (MCF) evaluated by whole blood thromboelastometry using a TF+tissue plasminogen activator-based assay. Healthy controls showed a 20-fold increase in MCF following TXA. Adjunct TXA to aPCC or rFVIIa induced a significant increase in MCF (P<0.0001) reaching levels indistinguishable from healthy controls treated with TXA (P>0.05). Infusion of aPCC or rFVIIa alone induced only 3-10 fold increase in MCF from baseline, with a decline in MCF starting after 60-120min. TXA did not increase the endogenous thrombin potential. No clinical or laboratory signs of thromboembolic events, disseminated intravascular coagulation, or hypercoagulability were observed. Combination of aPCC or rFVIIa with TXA normalizes clot stability in haemophilia patients with inhibitor as compared to healthy controls. No clinical or laboratory adverse events were observed.

Rotational thromboelastometry (ROTEM) in Behçet's disease

Dear Sirs, We very much appreciated the brief report by Bilge et al. recently published in Clinical Rheumatology [1] about the utility of rotational thromboelastometry (ROTEM) in Behcet's disease (BD). Bilge et al. reported a significant increase in maximum clot firmness (MCF) in a cohort of patients with BD, which may indicate a prothrombotic state in this disease. This report is in line with a study we recently published [2] highlighting the usefulness of ROTEM and of the calibrated automated thrombogram for studying the hypercoagulable state in BD patients. In our study, we found an increase in the MCF by the INTEM test in a group of BD patients with various degrees of disease activity. Interestingly, INTEM-MCF significantly correlates with disease activity and with plasma levels of Eselectin, a marker of endothelial damage/activation. We also observed a significant reduction in the CFT by the INTEM test in samples from BD. This last observation does not match with Bilge's report, and the difference might lie in the fact that none of their patients were in the active phase of the disease during the study. We recognize the high scientific value of the article by Bilge et al., and it strengthens our assumption that the ROTEM test may be a useful tool for monitoring the therapeutic response and disease progression in BD patients.

Changes in fibrinogen availability and utilization in an animal model of traumatic coagulopathy

BackgroundImpaired haemostasis following shock and tissue trauma is frequently detected in the trauma setting. These changes occur early, and are associated with increased mortality. The mechanism behind trauma-induced coagulopathy (TIC) is not clear. Several studies highlight the crucial role of fibrinogen in posttraumatic haemorrhage. This study explores the coagulation changes in a swine model of early TIC, with emphasis on fibrinogen levels and utilization of fibrinogen.MethodsA total of 18 landrace pigs were anaesthetized and divided into four groups. The Trauma-Shock group (TS) were inflicted bilateral blast femoral fractures with concomitant soft tissue injury by a high-energy rifle shot to both hind legs, followed by controlled exsanguination. The Shock group (S) was exposed to shock by exsanguination, whereas a third group was exposed to trauma only (T). A fourth group (C) served as control. Physiological data, haematological measurements, blood gas analyses and conventional coagulation assays were recorded at baseline and repeatedly over 60 minutes. Thrombelastometry were performed by means of the tissue factor activated ExTEM assay and the platelet inhibiting FibTEM assay. Data were statistically analysed by repeated measurements analyses method.ResultsA significant reduction of fibrinogen concentration was observed in both the TS and S groups. INR increased significantly in the S group and differed significantly from the TS group. Maximum clot firmness (MCF) of the ExTEM assay was significantly reduced over time in both TS and S groups. In the FibTEM assay a significant shortening of the clotting time and an increase in MCF was observed in the TS group compared to the S group.ConclusionDespite a reduction in clotting capability measured by ExTEM MCF and a reduced fibrinogen concentration, extensive tissue trauma may induce an increased fibrin based clotting activity that attenuates the hypocoagulable tendency in exsanguinated animals.

Effects of Fibrinogen Concentrate as First-line Therapy during Major Aortic Replacement Surgery

Fibrinogen is suggested to play an important role in managing major bleeding. However, clinical evidence regarding the effect of fibrinogen concentrate (derived from human plasma) on transfusion is limited. The authors assessed whether fibrinogen concentrate can reduce blood transfusion when given as intraoperative, targeted, first-line hemostatic therapy in bleeding patients undergoing aortic replacement surgery. In this single-center, prospective, placebo-controlled, double-blind study, patients aged 18 yr or older undergoing elective thoracic or thoracoabdominal aortic replacement surgery involving cardiopulmonary bypass were randomized to fibrinogen concentrate or placebo, administered intraoperatively. Study medication was given if patients had clinically relevant coagulopathic bleeding immediately after removal from cardiopulmonary bypass and completion of surgical hemostasis. Dosing was individualized using the fibrin-based thromboelastometry test. If bleeding continued, a standardized transfusion protocol was followed. Twenty-nine patients in the fibrinogen concentrate group and 32 patients in the placebo group were eligible for the efficacy analysis. During the first 24 h after the administration of study medication, patients in the fibrinogen concentrate group received fewer allogeneic blood components than did patients in the placebo group (median, 2 vs. 13 U; P < 0.001; primary endpoint). Total avoidance of transfusion was achieved in 13 (45%) of 29 patients in the fibrinogen concentrate group, whereas 32 (100%) of 32 patients in the placebo group received transfusion (P < 0.001). There was no observed safety concern with using fibrinogen concentrate during aortic surgery. Hemostatic therapy with fibrinogen concentrate in patients undergoing aortic surgery significantly reduced the transfusion of allogeneic blood products. Larger multicenter studies are necessary to confirm the role of fibrinogen concentrate in the management of perioperative bleeding in patients with life-threatening coagulopathy.

Whole blood rotation thromboelastometry (ROTEM®) in nine severe factor V deficient patients and evaluation of the role of intraplatelets factor V

Severe factor V (FV) deficiency (parahaemophilia) is a rare congenital hemorrhagic disorder characterized by very low or undetectable plasma FV levels and bleeding phenotype ranging from mild to severe. We evaluated whole blood (WB) rotation thromboelastometry (ROTEM) in parahaemophilia patients and the contribution of intraplatelets FV, if any, to clot formation. Standard ROTEM(®) assays were performed in WB from nine parahaemophilia patients and 50 healthy controls. In addition, platelets poor plasma from one parahaemophilia patient (PPP-Pt) or normal subjects (PPP-N) was reconstituted with washed platelets obtained either from one patient with parahaemophilia (Plts-Pt) or normal subjects (Plts-N) and ROTEM assays were performed in platelets rich plasma (PRP) samples. There was a prolongation of the WB clotting time (CT) in all assays in patients as compared with controls. However, maximum clot firmness (MCF) was similar in patients and controls. ROTEM in PPP-Pt showed both a prolongation of CT and a reduction of MCF as compared with PPP-N. The addition of either Plts-Pt or Plts-N to PPP-Pt resulted in similar increase in MCF and a decrease of CT which was more evident for PPP-Pt + Plts-N than PPP-Pt + Plts-Pt. In contrast, the addition of Plts-Pt or Plts-N to PPP-N had superimposable effects on both CT and MCF. In parahaemophilia patients, WB ROTEM(®) presents mainly with prolongation of CT and no relevant effect on MCF. Residual intraplatelets FV in parahaemophilia contributes significantly to thrombin generation as shown in artificially reconstituted PRP models.

Hemostatic Effects of Fresh Frozen Plasma May be Maximal at Red Cell Ratios of 1:2

Damage control resuscitation targets acute traumatic coagulopathy with the early administration of high-dose fresh frozen plasma (FFP). FFP is administered empirically and as a ratio with the number of packed red blood cells (PRBC). There is controversy over the optimal FFP:PRBC ratio with respect to outcomes, and their hemostatic effects have not been studied. We report preliminary findings on the effects of different FFP:PRBC ratios on coagulation. This is a prospective observational cohort study of trauma patients requiring >4 U of PRBCs. Blood was drawn before and after each 4-U PRBC interval for prothrombin time and analysis by rotational thromboelastometry. Interval change in coagulation parameters were compared with the FFP:PRBC ratio received during each interval. Sixty 4-U PRBC intervals from 50 patients were available for analysis. All measures of coagulation deteriorated with low FFP:PRBC ratios (<1:2). Maximal hemostatic effect was observed in the 1:2 to 3:4 group: 12% decrease in prothrombin time (p=0.006), 56% decrease in clotting time (p=0.047), and 38% increase in maximum clot firmness (p=0.024). Transfusion with ≥1:1 ratio did not confer any additional improvement. There was a marked variability in response to FFP, and hemostatic function deteriorated in some patients exposed to 1:1 ratios. The beneficial effects of plasma were confined to patients with coagulopathy. Interim results from this prospective study suggest that FFP:PRBC ratios of ≥1:1 do not confer any additional advantage over ratios of 1:2 to 3:4. Hemostatic benefits of plasma therapy are limited to patients with coagulopathy.

In vitro factor XIII supplementation increases clot firmness in Rotation Thromboelastometry (ROTEM®)

Factor XIII (F XIII) is an essential parameter for final clot stability. The purpose of this study was to determine the impact of the addition of factor (F)XIII on clot stability as assessed by Rotation Thromboelastometry (ROTEM). In 90 intensive care patients ROTEM measurements were performed after in vitro addition of F XIII 0.32 IU, 0.63 IU, 1.25 IU and compared to diluent controls (DC; aqua injectabile) resulting in approximate F XIII concentrations of 150, 300 and 600%. Baseline measurements without any additions were also performed. The following ROTEM parameters were measured in FIBTEM and EXTEM tests: clotting time (CT), clot formation time (CFT), maximum clot firmness (MCF), maximum lysis (ML), maximum clot elasticity (MCE) and alpha-angle (alphaA). Additionally, laboratory values for FXIII, fibrinogen (FBG), platelets and haematocrit were contemporaneously determined. In the perioperative patient population mean FBG concentration was elevated at 5.2 g/l and mean FXIII concentration was low at 62%. The addition of FXIII led to a FBG concentration-dependent increase in MCF both in FIBTEM and EXTEM. Mean increases in MCF (FXIII vs. DC) of approximately 7 mm and 6 mm were observed in FIBTEM and EXTEM, respectively. F XIII addition also led to decreased CFT, increased alphaA, and reduced ML in FIBTEM and EXTEM. In vitro supplementation of FXIII to supraphysiologic levels increases maximum clot firmness, accelerates clot formation and increases clot stability in EXTEM and FIBTEM as assayed by ROTEM in perioperative patients with high fibrinogen and low FXIII levels.

Pharmacokinetic Evaluation of a Fibrinogen Concentrate in Patients with Congenital Fibrinogen Deficiency.

BACKGROUND: Congenital fibrinogen deficiency is a rare bleeding disorder and comprises fibrinogen abnormalities that result in either reductions in the quantity (hypofibrinogenemia and afibrinogenemia) or structure and functionality (dysfibrinogenemia) of fibrinogen. The pharmacokinetics (PK) and surrogate efficacy of a plasma-derived fibrinogen concentrate was studied in a prospective, open, non-controlled, multinational clinical study in patients with confirmed afibrinogenemia.METHODS: Patients had to have < 0.20 g/L plasma fibrinogen activity, be in a non-bleeding state and receive a single dose of 70 mg/kg body weight. Maximum clot firmness (MCF), determined via validated thrombelastography (TEG), was used as a surrogate endpoint for hemostatic efficacy. Standard PK parameters were analyzed in a central laboratory with a validated Clauss assay and an ELISA.RESULTS: A total of 15 patients were treated at 10 centers in the US and Italy. Of these, 5 (33.3%) were female. The mean age was 30 years, with 11 subjects (73.3%) in the age group of 16 to <65 years and the remaining between 8 and 14 years.PK RESULTS: Both median fibrinogen plasma antigen and activity levels reached a maximum within 30 minutes to 1 h post-infusion and decreased continuously afterwards. Median fibrinogen plasma activity levels were at or close to the limit of detection by Day 10 post-infusion. PK findings were similar for fibrinogen activity and antigen. Median Cmax and AUC were 1.3 g/L and 126.8 hours*mg/mL, with a median t1/2 of 77.1 hours for fibrinogen activity. For fibrinogen antigen, median Cmax and AUC were 1.3 g/L and 122.4 hours*mg/mL, with a median t1/2 of 88.0 hours. Whereas no statistically relevant effect of gender was seen on the PK parameters for fibrinogen activity, subjects <16 years old (N=4) had higher median Vss, and Cl values and lower median t1/2, AUC, and MRT values than older subjects (N=10). The median incremental IVR was 1.7 mg/dL increase per mg/kg body weight. for fibrinogen activity and antigen.EFFICACY: The mean change in MCF between pre-infusion and 1 hour post-infusion was (8.9 mm) (p<0.0001). A secondary analysis performed as a sensitivity analysis in the ITT population confirmed this result (mean change of 10.3 mm; p<0.0001). Mean change from pre-infusion to 1 hour post-infusion was similar for subjects <16 years of age (9.9 mm; N=4) and subjects ≥16 to <65 years of age (8.5 mm; N=10) as well as for males (9.0 mm; N=9) and females (8.8 mm; N=5).SAFETY: Four adverse events from two patients were reported. All were mild, non-serious and assessed by the investigators as not related to study medication.CONCLUSION: The PK results for this study of a plasma-derived fibrinogen concentrate showed a sufficient and rapid increase in fibrinogen plasma level and a long half-life. The study demonstrated a significant increase in MCF as a surrogate efficacy parameter and a good safety profile for fibrinogen concentrate in patients with afibrinogenemia.