Hemostatic Defects Research Articles

Features of Blood Clotting on Thromboelastography in Hospitalized Patients With Cirrhosis

IntroductionThromboelastography (TEG) provides a global assessment of hemostasis and may have value for patients with cirrhosis who have multiple hemostatic defects. We sought to examine the characteristics of TEG in hospitalized patients with cirrhosis and its relationship with outcomes. MethodsWe performed a cohort study of all adults with cirrhosis hospitalized at Indiana University Hospital between November 2015 and October 2018 with a TEG. We examined the relationships among TEG, traditional measures of hemostasis, liver disease severity, and outcomes, including mortality, discharge to hospice, length of stay, and 30-day readmission. ResultsA total of 344 patients met inclusion and exclusion criteria. R-value was elevated (≥10 min) in 4.5%, alpha angle was low (<45°) in 9.3%, and maximum amplitude (maximum amplitude) was low (<55 mm) in 72.1%. K-value, alpha angle, and maximum amplitude were all correlated with both platelet count and fibrinogen (absolute rho range 0.52-0.67); R-value and international normalized ratio (INR) were not strongly correlated with traditional measures or TEG, respectively. Patients with bleeding had hypercoagulable profiles, and patients with infection had increased R-value and decreased alpha angle. A total of 35.8% died or were discharged to hospice, and these patients had a greater R-value and smaller alpha angle. However, after adjustment for model for end-stage liver disease (MELD), neither R-value nor alpha angle were associated with discharge outcomes. ConclusionsTEG provides insight into the hemostatic state of patients with cirrhosis beyond that of standard measures of hemostasis. It is associated with liver disease severity and outcomes and may play a role complementary to standard measures of hemostasis in this population.

Profiling of Inflammatory Biomarkers and Coagulation Factors in End‐Stage Renal Disease

BackgroundEarly diagnosis of chronic kidney disease (CKD) is necessary in preventing complications that develop into kidney failure and vascular complications. Currently, the detection of early stages of CKD is lacking predictive values, delaying the application of specific diagnostics and therapeutic interventions. End‐stage renal disease (ESRD) patients are at risk for cardiovascular complications and vascular thrombosis, exhibiting numerous coagulation and fibrinolytic abnormalities.ObjectivesProfiling inflammatory biomarkers and coagulation factors in ESRD patients may allow early onset detection and severity of kidney damage, furthering the understanding of the pathogenesis of this disease.Materials &amp; MethodsCitrated blood samples were collected from 95 confirmed ESRD patients undergoing maintenance hemodialysis under an IRB approved protocol. Biomarkers of inflammation and coagulation, including PAI‐1, TAFI, CRP, MMP‐9, MP, D‐Dimer, Factor VII, IX, X, and XIII were measured using commercially available ELISA methods. Normal controls were comprised of commercially available male and female citrated plasma (George King Biomedical). All results were compiled as mean ± SEM, and percent change in comparison to normal human plasma.ResultsBiomarkers including CRP (7ug/ml ± 0.6, 183%), MMP‐9 (88ng/ml ±8, 58%) D‐Dimer (1059ng/ml±138), Factor IX (124%±3, 23%), and Factor XIII (104%±1, 27%) showed significant increase when compared to normal plasma. Elevated CRP indicates severe inflammatory response and elevated D‐Dimer indicates endogenous fibrin formation. MP and PAI‐1 suggest endothelial dysfunction.DiscussionThese results indicate that the upregulation of inflammatory responses and hemostatic defects contribute to the multifactorial pathophysiology of ESRD. Profiling of these biomarkers may be helpful in the prognostic management of these patients.

Genetic Duplication of Tissue Factor Leads to Partial Specialization of Function in Zebrafish

Tissue factor (TF) is a critical factor for hemostasis in response to tissue injury. Among mouse knockouts of procoagulant factors, those lacking TF have the most severe phenotype, with complete lethality by midgestation. Furthermore, complete loss of TF has never been described in humans. Together, these suggest additional roles in embryonic development beyond coagulation. Zebrafish are a small freshwater teleost fish with a well described hemostatic system, including conservation of the coagulation cascade. Zebrafish are prolific breeders that reproduce through external fertilization, with subsequent rapid and transparent development, allowing studies not possible in mammals. Due to an ancient genomic event, 30-40% of the teleost genome is duplicated, resulting in two TF paralogs (TFa and TFb) with unknown functions. Here we use CRISPR/Cas9 to produce null alleles of TFa and TFb and uncover partial subspecialization of these duplicates. It has been shown previously that both TFs are expressed before the initiation of blood circulation, between 24 and 48 hours post fertilization, yet complete loss of TFa and TFb yielded no gross abnormalities. Embryos and larvae were able to develop normally through juvenile stages but succumbed to hemorrhage by early adulthood at 9 weeks of age. Surprisingly, a single allele of either TFa or TFb was able to rescue survival in the context of complete loss of the other gene. To evaluate for hemostatic effects of TF deficiency, laser-mediated endothelial injury was used in the venous and arterial systems at 3 and 5 days post fertilization (dpf), respectively. Loss of TFb alone at 3 dpf resulted in no observable hemostatic defects. Conversely, loss of TFa led to a 50% increase in the time to venous occlusion (TTO), which was exacerbated by concomitant loss of one allele of TFb. Total TF deficiency led to a complete inability to form occlusive venous thrombi, indicating that both TFs can trigger coagulation but TFa is able to completely compensate for the loss of TFb. Concordant with these data, loss of TFb resulted in transcriptional upregulation of TFa but not vice versa. Interestingly, the roles are reversed in the arterial vasculature. Loss of TFa had no effect, loss of TFb lead to a 60% reduction in the number of occlusive thrombi, and complete deficiency resulted in no arterial occlusion. Combined with the venous results, these data point to differentiated roles of TFa and TFb in the venous and arterial systems. In order to test whether these differences were functional, recombinant TF (rTF) molecules were expressed in E. coli, purified, and incorporated into 80% phosphatidylcholine/20% phosphatidylserine liposomes. Ex vivo tube-tilt clotting assays were performed by using each rTF to activate citrated plasma from lake trout. rTFa triggered stable clot formation within 1-2 minutes of recalcification. rTFb usually failed to induce clot formation, with occasional delayed fibrin thrombi that appeared to be grossly disorganized and were easily disrupted following agitation. Taken with the in vivo data, this hints at an altered kinetic profile, with TFa being a more potent cofactor for factor VIIa in low flow (venous) settings. The laboratory is an artificially safe environment, so a synthetic chemical stress test was performed on 3 dpf larvae. Prolonged treatment with cortisol and epinephrine led to the development of cardiac tamponade in larvae with complete TF deficiency (61%), but similar results were only found at low levels in wild type siblings (2-5%). The same assay in prothrombin mutants also revealed a high rate of tamponade (75%), but lower levels in fibrinogen-deficient larvae (20%). These data suggest an extrahemostatic risk factor for tamponade that is modified by prothrombin and tissue factor levels and is independent of fibrin formation. Our results intimate that TFa and TFb have overlapping procoagulant functions but differential kinetic profiles in venous vs arterial systems. We also find that the duplication provides a layer of quantitative regulation and creates a titratable level for regulation of hemostatic and extrahemostatic roles of TF. Overall, this novel model provides new structural and physiologic information about TF function in vivo, including potential previously unknown roles in perivascular development, cardiovascular stability, remodeling and/or regeneration. Disclosures Morrissey: PrevThro Pharmaceuticals: Equity Ownership; Cayuga Pharmaceuticals: Equity Ownership; Kerafast, Inc: Research Funding; Issued and pending patent applications relating to medical uses of polyphosphate and polyphosphate inhibitors: Patents &amp; Royalties. Shavit:Bayer: Consultancy; Sanofi: Consultancy; Shire/Takeda: Consultancy; Spark Therapeutics: Consultancy; CSL-Behring: Consultancy; Novo Nordisk: Consultancy.

Liver Diseases and the Hemostasis (Rewiew) Part 1. Non-Cholestatic Diseases of the Liver and Hemostasis

In liver diseases, most commonly in the terminal stage of liver failure, a variety of hemostatic defects affecting almost all parts of the blood coagulation system are developing. This leads to diminishing the capabilities of patients with liver diseases to correctly maintain the hemostatic balance.The severity of hemostatic disorders depends on the nosological form and degree of a liver damage. Depending on the imbalance of the hemostasis system and accumulated clinical/laboratory data, patients with liver diseases can be subdivided into three groups as exhibiting: 1. non-cholestatic liver damage; 2. cholestatic liver damage and 3. liver damage of vascular origin.The first part of the review discusses multiple alterations in the hemostasis system in patients with noncholestatic liver diseases, which are commonly accompanied by hypocoagulation.

The implementation of a ROTEM® sigma-based algorithm for the management of coagulopathic bleeding in a tertiary center

Introduction Viscoelastic assays are used for the management of perioperative or traumatic bleeding (1). Recently, the manufacturer of ROTEM® has introduced the automated sigma model. The change in technology results in new normal ranges and therefore new cutoff values triggering intervention compared to previous published ROTEM®delta cutoff values. Here, we describe the analysis performed to identify new cutoff values and the specific algorithm for the ROTEM®sigma. Methods 78 samples were obtained from 39 patients with various hemostatic defects and 12 healthy volunteers. Chosen standard laboratory test (SLT) cut-offs were fibrinogen (Fi) 37 sec or 1.5x the normal value. PLTEM was calculated as EXTEMA5 – FIBTEMA5. The clinically critical range (CCR) of SLT was defined as the range around the threshold that leads to treatment. Correlation was sought between SLT and ROTEM parameters. The best cut-off for the different ROTEM parameters to identify the chosen SLT thresholds (ROC analysis) were integrated in a step-by-step algorithm (Figure 1). Results Correlation between Fi and FIBTEMA5 was very strong (r = 0.94) but was lower in the CCR (0-2.5 g/l) (r = 0.65). The correlation between Fi and EXTEMA5 was weakly moderate (r = 0.42). PLTEMA5 showed very strong correlation (r = 0.96) with Pltc in the CCR ( Discussion In-house cut-off values of key ROTEM®sigma parameters differ from the published ones for ROTEM®delta. We report the first preliminary ROTEM®sigma-based algorithm for hemorrhage. Diagnostic and therapeutic performances shall be prospectively validated.

Roles of Factor XII in Innate Immunity.

Factor XII (FXII) is the zymogen of serine protease, factor XIIa (FXIIa). FXIIa enzymatic activities have been extensively studied and FXIIa inhibition is emerging as a promising target to treat or prevent thrombosis without creating a hemostatic defect. FXII and plasma prekallikrein reciprocally activate each other and result in liberation of bradykinin. Due to its unique structure among coagulation factors, FXII exerts mitogenic activity in endothelial and smooth muscle cells, indicating that zymogen FXII has activities independent of its protease function. A growing body of evidence has revealed that both FXII and FXIIa upregulate neutrophil functions, contribute to macrophage polarization and induce T-cell differentiation. In vivo, these signaling activities contribute to host defense against pathogens, mediate the development of neuroinflammation, influence wound repair and may facilitate cancer maintenance and progression. Here, we review the roles of FXII in innate immunity as they relate to non-sterile and sterile immune responses.

Angina bullosa hemorrhagica

Angina bullosa hemorrhagica (ABH) is a term used to describe acute, subepithelial oral mucosal blisters filled with blood without any systemic disorder or hemostatic defect. The diagnosis of ABH is largely clinical, and includes elimination of other diseases at histology. We present a 24-year-old patient suffering from ABH with some clinical peculiarities. Recognition of ABH is of great importance to dermatologists, to avoid misdiagnosis.

Tissue Factor-Negative Cell-Derived Microparticles Play a Distinctive Role in Hemostasis: A Viewpoint Review.

Circulating cell-derived microparticles (MPs) exhibit procoagulant activity and have been investigated for a possible role in some human pathologies. However, their potential role in hemostasis has been neglected and often denied. This review brings to attention a specific body of direct clinical evidence supporting an important but distinctive role of MPs in hemostasis. Evidence for a role of MPs in hemostasis includes: (1) two congenital bleeding disorders attributed to impaired release of MPs; (2) two recent studies of trauma patients relating naturally elevated endogenous MPs at admission to reduced transfusion requirements and better outcomes; (3) a study of coronary surgery patients showing that elevated MP before surgery reduces transfusion requirements during surgery; and (4) a clinical study of patients with immune thrombocytopenia demonstrating that those with high circulating MP have reduced bleeding compared to patients with similar platelet counts but lower MP levels. Mechanisms involving potentiating the contact factor pathway are thought to play a key role and are probably synergistic with polyphosphate released from activated platelets at sites of endothelial injury. Hemostatic defect of patients with deficient MP-mediated coagulation resembles deficiency of FXI (hemophilia C), distinct from hemophilia A or B, so can be termed type C hemostasis. A better understanding of this proposed hemostatic pathway may lead to improved methods for controlling excessive bleeding in surgery, trauma, and other clinical settings.

Analysis of factor V in zebrafish demonstrates minimal levels needed for early hemostasis

AbstractIn humans, coagulation factor V (FV) deficiency is a rare, clinically heterogeneous bleeding disorder, suggesting that genetic modifiers may contribute to disease expressivity. Zebrafish possess many distinct advantages including high fecundity, optical clarity, external development, and homology with the mammalian hemostatic system, features that make it ideal for genetic studies. Our aim was to study the role of FV in zebrafish through targeted mutagenesis and apply the model to the study of human F5 variants. CRISPR-mediated genome editing of the zebrafish f5 locus was performed, generating mutants homozygous for a 49 base pair deletion in exon 4. Thrombus formation secondary to vascular endothelial injury was absent in f5−/− mutant embryos and larvae. Despite this severe hemostatic defect, homozygous mutants survived before succumbing to severe hemorrhage in adulthood. Human F5 variants of uncertain significance from patients with FV deficiency were evaluated, and the causative mutations identified and stratified by their ability to restore thrombus formation in larvae. Analysis of these novel mutations demonstrates variable residual FV function, with minimal activity being required to restore hemostasis in response to laser-induced endothelial injury. This in vivo evaluation may be beneficial for patients whose factor activity levels lack correlation with bleeding symptomatology, although limitations exist. Furthermore, homozygous mutant embryos tolerate what is a severe and lethal defect in mammals, suggesting the possibility of species-specific factors enabling survival, and allowing further study not possible in the mouse. Identification of these factors or other genetic modifiers could lead to novel therapeutic modalities.

Loss of fibrinogen in zebrafish results in an asymptomatic embryonic hemostatic defect and synthetic lethality with thrombocytopenia

Essentials Loss of fibrinogen in zebrafish has been previously shown to result in adult onset hemorrhage Hemostatic defects were discovered in early fga-/- embryos but well tolerated until adulthood Afibrinogenemia and thrombocytopenia results in synthetic lethality in zebrafish. Testing human FGA variants of uncertain significance in zebrafish identified causative mutations SUMMARY: Background Mutations in the alpha chain of fibrinogen (FGA), such as deficiencies in other fibrinogen subunits, lead to rare inherited autosomal recessive hemostatic disorders. These range from asymptomatic to catastrophic life-threatening bleeds and the molecular basis of inherited fibrinogen deficiencies is only partially understood. Zinc finger nucleases have been used to produce mutations in zebrafish fga, resulting in overt adult-onset hemorrhage and reduced survival. Objectives To determine the age of onset of hemostatic defects in afibrinogenemic zebrafish and model human fibrinogen deficiencies. Methods TALEN genome editing (transcription activator-like effector nucleases) was used to generate a zebrafish fga mutant. Hemostatic defects were assessed through survival, gross anatomical and histological observation and laser-induced endothelial injury. Human FGA variants with unknown pathologies were engineered into the orthologous positions in zebrafish fga. Results Loss of Fga decreased survival and resulted in synthetic lethality when combined with thrombocytopenia. Zebrafish fga mutants exhibit a severe hemostatic defect by 3 days of life, but without visible hemorrhage. Induced thrombus formation through venous endothelial injury was completely absent in mutant embryos and larvae. This hemostatic defect was restored by microinjection of wild-type fga cDNA plasmid or purified human fibrinogen. This system was used to determine whether unknown human variants were pathological by engineering them into fga. Conclusions These studies confirm that loss of fibrinogen in zebrafish results in the absence of hemostasis from the embryonic period through adulthood. When combined with thrombocytopenia, zebrafish exhibit synthetic lethality, demonstrating that thrombocytes are necessary for survival in response to hemorrhage.

Platelets compensate for poor thrombin generation in type 3 von Willebrand disease

In type 3 von Willebrand disease (VWD3), the most severe form with absent von Willebrand factor (VWF), the bleeding phenotype is variable. Platelet contribution to the hemostatic defect in VWD3 calls upon further studies. We investigated the contribution of platelets to in vitro thrombin generation (TG) and platelet procoagulant activity in VWD3. TG was assessed by calibrated automated thrombogram (CAT) in platelet-poor (PPP) and –rich plasma (PRP) from 9 patients before and in 6 patients also 30 min after receiving their regular VWF therapy. Responsiveness of PPP to FVIII and protein S was also investigated. TG data were compared with routine laboratory variables, rotational thromboelastometry (ROTEM) and platelet expression of P-selectin and phosphatidylserine in flow cytometry. Compared with healthy controls, TG was markedly decreased in VWD3 PPP (peak thrombin was 16% of normal median), but not in PRP (77% of normal median) (p = 0.002). Six out of nine patients (67%) were high responders in their platelet P-selectin, and 5/9 (56%) in phosphatidylserine expression. Replacement therapy improved TG in PPP, while in PRP TG only modestly increased or was unaffected. In PPP, FVIII levels associated with TG and in vitro FVIII-supplemented TG inclined up to threefold. Conversely, a FVIII inhibitory antibody reduced plasma TG in all, but especially in patients with remnant FVIII levels. Inhibition of protein S improved plasma TG, particularly at low FVIII levels. ROTEM failed to detect VWD3.In VWD3, TG is reduced in PPP and regulated by FVIII and protein S, but TG is close to normal in PRP. VWD3 platelets seem to compensate for the FVIII-associated reduction in TG by their exposure of P-selectin and phosphatidylserine.

Munc18-2, but not Munc18-1 or Munc18-3, regulates platelet exocytosis, hemostasis, and thrombosis

Platelet degranulation, a form of regulated exocytosis, is crucial for hemostasis and thrombosis. Exocytosis in platelets is mediated by SNARE proteins, and in most mammalian cells this process is controlled by Munc18 (mammalian homolog of Caenorhabditis elegans uncoordinated gene 18) proteins. Platelets express all Munc18 paralogs (Munc18-1, -2, and -3), but their roles in platelet secretion and function have not been fully characterized. Using Munc18-1, -2, and -3 conditional knockout mice, here we deleted expression of these proteins in platelets and assessed granule exocytosis. We measured products secreted by each type of platelet granule and analyzed EM platelet profiles by design-based stereology. We observed that the removal of Munc18-2 ablates the release of alpha, dense, and lysosomal granules from platelets, but we found no exocytic role for Munc18-1 or -3 in platelets. In vitro, Munc18-2-deficient platelets exhibited defective aggregation at low doses of collagen and impaired thrombus formation under shear stress. In vivo, megakaryocyte-specific Munc18-2 conditional knockout mice had a severe hemostatic defect and prolonged arterial and venous bleeding times. They were also protected against arterial thrombosis in a chemically induced model of arterial injury. Taken together, our results indicate that Munc18-2, but not Munc18-1 or Munc18-3, is essential for regulated exocytosis in platelets and platelet participation in thrombosis and hemostasis.

A case of mesenteric vein thrombosis developed in the first trimester of pregnancy after in vitro fertilization and embryo transfer in a patient with antiphospholipid syndrome

Combined thrombophilia is known as a probable cause of recurrent in vitro fertilization (IVF) failures. The present clinical case demonstrates the importance of considering the possibility of inherited or acquired thrombophilia in patients under the preparation for IVF. Thromboses of rare locations (hepatic vein, splenic vein, mesenteric vein, ovarian vein, retinal vein, cerebral vein, portal vein, axillary and subclavian vein) are life-threatening conditions. Most often thrombosis of atypical location develops in patients with inherited hemostatic defects, e.g. genetic thrombophilia. Such thromboses can also result from acquired abnormalities of hemostasis, which include antiphospholipid syndrome, pregnancy-associated hypercoagulability, IVF, oral contraception, or ovarian hyper-stimulation within IVF procedures.

PRIMARY AND SECONDARY THROMBOPHILIА: PATHOGENESIS, CLINICAL PRESENTATION, APPROACHES TO THROMBOTIC COMPLICATIONS PREVENTION AND TREATMENT

Introduction: Thrombophiliа is a predisposition to arterial or venous thrombotic complications as a result of congenital or acquired hemostatic system defects. Thrombophilia increases risk of fatal complications, disability of patients. The assessment of the risk of thrombotic complications makes it possible to prescribe adequate primary or secondary prophylaxis. However, there is no systematic information about estimation risk of thrombosis in various types of thrombophilia and conduction primary and secondary prophylaxis of thrombotic complications, choosing treatment. The aim: Analysis and arrangement information regarding pathogenesis, clinical features, approaches to diagnosis, risk assessment, primary and secondary prevention and peculiarities of thrombotic complications treatment in patients with thrombophilia. Materials and methods: There were used methods: content analysis, method of systemic approach. An analysis of the results of clinical trials, the review of articles in the field of hemostasis was conducted. Conclusions: Patients with deficiency of Pt C, S, antithrombin III deficiency and homozygous factor V Leiden mutation, malignancy, antiphospholipid syndrome, surgical interventions, pregnancy, usage of oral contraceptive pills (OCP s) and hormone replacement therapy (HRT) have the highest risk of thrombotic complications. The type of thrombophilia determinates the choice of anticoagulants, necessity for primary prophylaxis and the duration of secondary prophylaxis.

THE GENETIC SUSCEPTIBILITY TO ATHEROSCLEROSIS

Atherosclerosis is a complex multifocal arterial disease involving interactions of multiple genetic and environmental factors. Atherosclerosis is the main cause of death and disability in developed countries, while in developing countries the incidence of this pathology is growing rapidly. Advances in techniques of molecular genetics have revealed that genetic polymorphisms significantly influence susceptibility to atherosclerotic vascular diseases. A large number of candidate genes, genetic polymorphisms and susceptibility loci associated with atherosclerotic diseases have been identified in recent years and their number is rapidly increasing. In recent years, there is significant interest in identifying additional factors of genetic risk for atherosclerosis. In recent years, a large number of genetic studies have been carried out to prove the genetic effect on the atherosclerotic process. Rapid progress in the sequencing of the human genome and molecular genetic methods have helped in the definition of susceptibility loci and associated candidate genes with atherosclerosis and concomitant diseases. The association of a large number of susceptibility genes with atherosclerosis reflects the enormous complexity of the disease. Multiple factors, including endothelial dysfunction, lipid metabolism defects, inflammation and immune responses, oxidative stress, cell proliferation, tissue remodeling and hemostatic defects are involved in the pathogenesis of atherosclerosis. In this review we focus and discuss on some of the major candidate genes and genetic polymorphisms associated with human atherosclerotic vascular diseases.

Evaluation and management of heavy menstrual bleeding in adolescents: the role of the hematologist.

Heavy menstrual bleeding (HMB) is frequently reported by adolescents. The role of the hematologist is threefold in evaluating such patients: (1) perform a clinical and laboratory evaluation for an underlying bleeding disorder on the basis of the degree of clinical suspicion, (2) identify and manage any concomitant iron deficiency, and (3) provide input to the referring provider regarding the management of HMB, particularly for patients with identified hemostatic defects. Several clues in the menstrual history should raise suspicion for an underlying bleeding disorder, such as menses lasting >7 days, menstrual flow which soaks >5 sanitary products per day or requires product change during the night, passage of large blood clots, or failure to respond to conventional therapies. A detailed personal and family history of other bleeding symptoms should also be obtained. Iron deficiency with and without anemia is commonly found in young women with HMB. Therefore, it is important to obtain measures of hemoglobin and ferritin levels when evaluating these patients. Iron supplementation is often a key component of management in the adolescent with heavy menses and is still needed in those who have received packed red cell transfusions as a result of severe anemia. Strategies for decreasing menstrual blood flow are similar for adults and adolescents with heavy menses, with combined hormonal contraceptives recommended as first-line therapy. However, there are adolescent-specific considerations for many of these agents, and they must be incorporated into shared decision-making when selecting the most appropriate treatment.

Efficacy and Safety of Fibrinogen Concentrate for on-Demand Treatment of Acute Bleeding and for Surgical Prophylaxis in Subjects with Congenital Fibrinogen Deficiency — a Phase 3 Study

Introduction: Congenital afibrinogenemia is a rare inherited disorder characterized by an absence of plasma fibrinogen. Affected individuals have a highly variable bleeding tendency, which can be severe and include life-threatening bleeding and spontaneous/trauma-related bleeds. Therapeutic substitution with human fibrinogen concentrate (HFC) can correct the hemostatic defect and arrest bleeding. The FORMA-02 study investigated the hemostatic efficacy of a new plasma-derived, double virus-inactivated HFC (Fibryga, Octapharma) for on-demand treatment of bleeding episodes (BEs) and for surgical prophylaxis in patients with afibrinogenemia.Methods: FORMA-02 was a prospective, open-label, uncontrolled, multicenter Phase 3 study. A total of 25 patients with congenital afibrinogenemia (≥12 years) received the new HFC for treatment of a BE and/or for surgical prophylaxis. HFC was individually dosed to achieve a recommended target fibrinogen plasma level dependent on the bleeding type or surgery type (minor or major). The primary endpoint was the efficacy of the new HFC for on-demand treatment of the first BE after signed consent was obtained. Secondary endpoints included hemostatic efficacy of the HFC for the treatment of all BEs during the study period and its efficacy in preventing bleeding during and after surgery. Hemostatic efficacy was assessed by investigators and an Independent Data Monitoring and Endpoint Adjudication Committee (IDMEAC) using objective four-point scales, with treatment success defined as a rating of excellent or good. Thromboelastometry maximum clot firmness (MCF) was also investigated as a surrogate marker of efficacy.Results: A total of 25 patients received the new HFC for treatment of a BE (N=24) and/or surgical prophylaxis (N=9). The mean±SD dose of HFC was 65.51±26.47 mg/kg per BE (89 events) and 40.45±30.78 mg/kg per surgery for surgical prophylaxis (12 events). Hemostatic efficacy for treatment of the first BE (primary endpoint) was rated as excellent or good for all patients by both the investigator and the IDMEAC (Success: 100%; 90% CI 0.89-1.00). When all BEs were evaluated (N=89 BEs in 24 patients), hemostatic efficacy was rated as excellent or good for 96.6% of events by the investigator and 98.9% by the IDMEAC. The first HFC infusion for treatment of the first BE for each patient led to a mean increase in blood fibrinogen concentration of 114.74±25.28 mg/dL 1 hour after administration, while the MCF increased by a mean of 6.48±3.07 mm.Intraoperative hemostatic efficacy for surgical prophylaxis was rated by the surgeon and the IDMEAC as excellent or good for 100% of the 12 surgeries that were performed (success: 100%; 90%CI 0.82-1.00). Postoperative efficacy was also rated as excellent or good in all cases. The first HFC infusion for each surgery led to a mean increase in blood fibrinogen concentration of 104.55±43.64 mg/dL at 1 hour after administration.A total of 15 serious adverse events (SAEs) occurred in 5 patients; only one was deemed to be related to the HFC by the investigator. This was a digital ischemia that resolved without sequelae. No inhibitory anti-fibrinogen antibodies were detected and there were no severe allergic or hypersensitivity reactions related to the HFC.Conclusions: These data indicate that the new HFC was efficacious for on-demand treatment of acute bleeding and for surgical prophylaxis in patients with congenital afibrinogenemia, with hemostatic efficacy rated as 100% in both settings. The HFC showed an acceptable safety profile in this study population. DisclosuresPeyvandi:Grifols: Speakers Bureau; Octapharma US: Honoraria; Roche: Speakers Bureau; Shire: Speakers Bureau; Octapharma US: Honoraria; Roche: Speakers Bureau; Shire: Speakers Bureau; Sobi: Speakers Bureau; Shire: Speakers Bureau; Novo Nordisk: Speakers Bureau; Sobi: Speakers Bureau; Novo Nordisk: Speakers Bureau; Octapharma US: Honoraria; Shire: Speakers Bureau; Novo Nordisk: Speakers Bureau; Octapharma US: Honoraria; Octapharma US: Honoraria; Novo Nordisk: Speakers Bureau; Ablynx: Other: Member of Advisory Board, Speakers Bureau; Ablynx: Other: Member of Advisory Board, Speakers Bureau; Ablynx: Other: Member of Advisory Board, Speakers Bureau; Ablynx: Other: Member of Advisory Board, Speakers Bureau; Ablynx: Other: Member of Advisory Board, Speakers Bureau; Kedrion: Consultancy; Kedrion: Consultancy; Kedrion: Consultancy; Kedrion: Consultancy; Kedrion: Consultancy; Grifols: Speakers Bureau; Grifols: Speakers Bureau; Grifols: Speakers Bureau; Novo Nordisk: Speakers Bureau; Grifols: Speakers Bureau; Roche: Speakers Bureau; Roche: Speakers Bureau; Roche: Speakers Bureau; Shire: Speakers Bureau; Sobi: Speakers Bureau; Sobi: Speakers Bureau; Sobi: Speakers Bureau. Schwartz:Octapharma US: Employment. Knaub:Octapharma AG: Employment.

Efficacy and Pharmacokinetics of a New Fibrinogen Concentrate in Treating Acute Bleeding in Adolescent Patients with Congenital Fibrinogen Deficiency

Introduction: Individuals with congenital afibrinogenemia can experience frequent and/or severe bleeding episodes (BE). Human fibrinogen concentrate (HFC) can correct the hemostatic defect and arrest bleeding. We investigated the efficacy, safety and pharmacokinetics (PK) of a new highly purified, double virus-inactivated HFC (Fibryga, Octapharma) in adolescent patients.Methods: Data were analyzed from two multinational, prospective, open-label studies. FORMA-01 studied the PK of the new HFC vs. comparator (Haemocomplettan P), as well as surrogate efficacy and safety, after single infusion of 70 mg/kg. Surrogate efficacy was defined as thromboelastometric maximum clot firmness (MCF). FORMA-02 was a Phase 3 study in which the primary endpoint was the hemostatic efficacy of the new HFC for on-demand treatment of the first bleeding event. A 4-point objective scale, which was adjudicated by an Independent Data Monitoring and Endpoint Adjudication Committee (IDMEAC), was used for evaluation of efficacy. In addition, MCF and safety were evaluated for all BEs that occurred during the study period.Results: Data were available for 12 patients aged 12-17 years with afibrinogenemia. FORMA-01 included 5 patients aged 12-17 years (1 was excluded due to a protocol deviation); PK data for new HFC vs. comparator are shown in Table 1. For comparison, in adults from the same study AUCnorm was also larger and clearance slower for new HFC vs. comparator (both p=0.0027). At 1 h following infusion of new HFC or comparator, mean±SD MCF (n=6) increased from 0 mm to 9.0±2.0 and 8.8±2.6 mm, respectively (9.9±3.3 and 10.4±4.9 mm in adults).In FORMA-02, 6 adolescent patients received new HFC individually dosed for treatment of a BE. Hemostatic efficacy for treatment of the first BE was rated as excellent for all patients (success: 100%; 90% CI: 0.655-1.000). When taking all BEs into account (n=23), efficacy was again classed as excellent for all patients. Mean MCF increased by 4.83±0.98 mm from baseline to 1 h after first infusion (7.06±3.36 mm in adults). There were no related serious adverse events, severe allergic, hypersensitivity reactions, or thromboembolic events, and no inhibitory anti-fibrinogen antibodies were detected.Conclusions: This analysis of data from two prospective studies demonstrated favorable efficacy, safety, and PK parameters of the new HFC in adolescents with afibrinogenemia. PK parameters were broadly comparable with those of the comparator HFC. After infusion of the new HFC, MCF increased statistically significantly and hemostatic efficacy was rated excellent in all patients. No safety concerns relating to the new HFC were identified in these study patients. [Display omitted] DisclosuresPeyvandi:Ablynx: Other: Member of Advisory Board, Speakers Bureau; Ablynx: Other: Member of Advisory Board, Speakers Bureau; Octapharma US: Honoraria; Ablynx: Other: Member of Advisory Board, Speakers Bureau; Octapharma US: Honoraria; Ablynx: Other: Member of Advisory Board, Speakers Bureau; Ablynx: Other: Member of Advisory Board, Speakers Bureau; Kedrion: Consultancy; Kedrion: Consultancy; Kedrion: Consultancy; Kedrion: Consultancy; Grifols: Speakers Bureau; Kedrion: Consultancy; Grifols: Speakers Bureau; Grifols: Speakers Bureau; Novo Nordisk: Speakers Bureau; Novo Nordisk: Speakers Bureau; Grifols: Speakers Bureau; Grifols: Speakers Bureau; Roche: Speakers Bureau; Novo Nordisk: Speakers Bureau; Roche: Speakers Bureau; Novo Nordisk: Speakers Bureau; Novo Nordisk: Speakers Bureau; Roche: Speakers Bureau; Shire: Speakers Bureau; Shire: Speakers Bureau; Roche: Speakers Bureau; Roche: Speakers Bureau; Shire: Speakers Bureau; Sobi: Speakers Bureau; Shire: Speakers Bureau; Sobi: Speakers Bureau; Sobi: Speakers Bureau; Shire: Speakers Bureau; Octapharma US: Honoraria; Sobi: Speakers Bureau; Sobi: Speakers Bureau; Octapharma US: Honoraria; Octapharma US: Honoraria. Schwartz:Octapharma US: Employment. Knaub:Octapharma AG: Employment.

Factor XI promotes hemostasis in factor IX‐deficient mice

Background In humans, deficiency of coagulation factorXI may be associated with a bleeding disorder, but, until recently, FXI-deficient mice did not appear to have a hemostatic abnormality. A recent study, however, indicated that FXI-deficient mice show a moderate hemostatic defect in a saphenous vein bleeding (SVB) model. Objectives To study the effect of FXI on bleeding in mice with normal levels of the FXI substrate FIX and in mice lacking FIX (a murine model of hemophiliaB). Methods Wild-type mice and mice lacking either FIX (F9- ) or FXI (F11-/- ) were tested in the SVB model. The plasma levels of FXI in F11-/- mice were manipulated by infusion of FXI or its active form FXIa, or by overexpressing FXI by the use of hydrodynamic tail vein injection. Results F9- mice showed a significant defect in the SVB model, whereas F11-/- mice and wild-type mice were indistinguishable. Intravenous infusion of FXI or FXIa into, or overexpression of FXI in, F9- mice improved hemostasis in the SVB model. Overexpression of a FXI variant lacking a FIX-binding site also improved hemostasis in F9- mice. Conclusions Although we were unable to demonstrate a hemostatic defect in F11-/- mice in the SVB model, our results support the premise that supraphysiological levels of FXI improve hemostasis in F9- mice through FIX-independent pathways.

Rap1 binding to the talin 1 F0 domain makes a minimal contribution to murine platelet GPIIb-IIIa activation

AbstractActivation of platelet glycoprotein IIb-IIIa (GPIIb-IIIa; integrin αIIbβ3) leads to high-affinity fibrinogen binding and platelet aggregation during hemostasis. Whereas GTP-bound Rap1 GTPase promotes talin 1 binding to the β3 cytoplasmic domain to activate platelet GPIIb-IIIa, the Rap1 effector that regulates talin association with β3 in platelets is unknown. Rap1 binding to the talin 1 F0 subdomain was proposed to forge the talin 1–Rap1 link in platelets. Here, we report a talin 1 point mutant (R35E) that significantly reduces Rap1 affinity without a significant effect on its structure or expression. Talin 1 head domain (THD) (R35E) was of similar potency to wild-type THD in activating αIIbβ3 in Chinese hamster ovary cells. Coexpression with activated Rap1b increased activation, and coexpression with Rap1GAP1 reduced activation caused by transfection of wild-type THD or THD(R35E). Furthermore, platelets from Tln1R35E/R35E mice showed similar GPIIb-IIIa activation to those from wild-type littermates in response to multiple agonists. Tln1R35E/R35E platelets exhibited slightly reduced platelet aggregation in response to low doses of agonists; however, there was not a significant hemostatic defect, as judged by tail bleeding times. Thus, the Rap1–talin 1 F0 interaction has little effect on platelet GPIIb-IIIa activation and hemostasis and cannot account for the dramatic effects of loss of Rap1 activity on these platelet functions.