Plasma Coagulation Factors Research Articles

One case of hemophilia B combined with FIX inhibitor

Objective To explore the clinical characteristics and treatment of hemophilia B with coagulation factor FⅨ inhibitor. Methods On February 8, 2018, one case of child with hemophilia B and FⅨ inhibitor who was admitted to First Affiliated Hospital of Soochow University was selected as research subject into this study. The medical history of the children was collected, coagulation function indicators, plasma coagulation factors, coagulation factor inhibitors and other laboratory tests were conducted, And the diagnosis was made based on the results of above tests and gene sequencing. The child were treated with prednisone (15 mg/d) combined with cyclosporine (50 mg/time×2 times/d) and prednisone (15 mg/d) combined with rapamycin (1.5 mg/d). Treatment efficiency was evaluated by relevant laboratory test results of the child. Follow-up was conducted until September 30, 2019. Clinical characteristics, diagnosis, treatment and curative effect of this case were analyzed retrospectively. The procedure of this study was in accordance with the requirements of the revised World Medical Association Declaration of Helsinki in 2013. Results ① This child was a 8 old boy. On February 8, 2018, he was admitted to First Affiliated Hospital of Soochow University due to repeated joint pain and swelling for 7 years . The child was diagnosed as severe hemophilia B in May, 2010 at local hospital and received treatment with prothrombin complex and recombinant human FⅨ. The titers of FⅨ inhibitors were 3.2 and 6.2 BU/mL in March and April in 2012, respectively. The child suffered from frequent joint bleeding, and the pain was significantly worse than before. ② After admission, result of routine examination of coagulation function showed activated partial thromboplastin time (APTT) was 165.4 s, prothrombin time (PT) was 13.4 s, thrombin time (TT) was 16.6 s, fibrinogen (FIB) was 3.48 g/L, FⅨ∶C was 0.5%, FⅧ∶C was 20%. The results of revealed APTT were 152.7 and 129.6 s immediately and 2 h later, respectively. Antiphospholipid antibodies and lupus anticoagulant were negative. Titer of hemophilia B inhibitor>10 BU/ml. Genetic sequencing displayed a heterozygous mutation of FⅨ c. 7993C>T (p.Arg29X) in exon 2. ③ The child was diagnosed as severe hemophilia B with FⅨ inhibitor. The child did not achieve improvement after treating with prednisone combined with cyclosporine for 3 months. Then the therapy was adjusted to prednisone combined with rapamycin. Although the change of FⅨ inhibitor titer was not obvious, the frequency of joint hemorrhage was decreased after 6 months of treatment. Nine months later, the titer of FⅨ inhibitor was reduced to 50% of that at first diagnosis, with no recurrence of bleeding. The child achieved partial remission (PR). By the end of the follow-up, the child had no joint swelling or pain, and basically returned to normal life and study. Conclusions It should be alert if patients who diagnose as severe hemophilia B and appear situations such as the frequency of hemorrhage increases after multiple FⅨ infusions and the response to traditional therapy is poor. And titer of FⅨ inhibitor should be detected. Hemophilia B with FⅨ inhibitor is fairly rare, therapeutic efficacy is poor and prone to have adverse reactions. Therefore, correct diagnosis and proper treatment are of great significance to improve hemorrhage of hemophilia B. This conclusion is limited to the clinical analysis of one single case, and it requires to expand sample size for further study and verification. Key words: Hemophilia B; Blood coagulation factor inhibitors; Factor Ⅸ; Hemorrhage; Diagnosis; Drug therapy

Reduction of FXIII during myelosuppression in acute leukemia after chemotherapy and adverse relation with bleeding events

目的探讨急性白血病患者化疗后骨髓抑制期外周血凝血因子XIII（FXIII）浓度与出血事件的关系。方法纳入2017年8月至2018年3月期间55例PLT<50×109/L的化疗后骨髓抑制期急性白血病患者（非急性早幼粒细胞性急性髓系白血病35例，急性淋巴细胞白血病20例），以35例非血液疾病患者作为对照组。用ELISA方法检测外周血FXIII浓度，分析急性白血病患者化疗后骨髓抑制期外周血FXIII水平与出血事件的关系。结果急性白血病患者化疗后骨髓抑制期FXIII水平明显低于对照组（P<0.001），FXIII水平与出血评分呈负相关（Spearman相关系数为-0.761）。当FXIII浓度的临界值为103.9 µg/L时，诊断化疗后骨髓抑制期急性白血病患者出血的敏感性为0.939，特异性为0.909。结论化疗后骨髓抑制期急性白血病患者外周血FXIII水平减低，且与出血事件及严重程度呈负相关，是急性白血病化疗后骨髓抑制期患者出血的独立影响因素之一。

Clinical Applications, Pitfalls, and Uncertainties of Thrombin Generation in the Presence of Platelets.

Platelet-dependent thrombin generation is a helpful tool to assess ex vivo the interaction between platelets and plasma coagulation factors in the initiation, amplification, and inhibition of thrombin generation (TG). This review article discusses the most relevant available data on the clinical applications of fluorogenic TG, the most widely used TG assay, performed in the presence of platelets, i.e., in platelet-rich plasma. With respect to prothrombotic states, arterial hypertension and obesity were the most prominent cardiovascular conditions linked to increased platelet-dependent TG. In addition, platelet-associated hypercoagulability, assessed by the TG assay, has been shown in individuals with active cancer. In terms of bleeding, platelet-dependent TG has been applied to assess bleeding risk in individuals with hemophilia, von Willebrand disease, and Glanzmann thrombasthenia as well as in subjects with other congenital or acquired coagulation factor deficiencies. In addition to risk prediction, a role of the TG assay has been suggested in monitoring antiplatelet therapy in prothrombotic conditions and replacement therapy in bleeding diathesis. Finally, for the routine clinical use and as a biomarker of disease development and progression, better standardization and clinical validation of platelet-dependent TG are still needed.

Allergy and inhibitors in hemophilia - a rare complication with potential novel solutions

IntroductionHemophilia is a rare bleeding disorder caused by a deficiency of the plasma coagulation factors VIII and IX (hemophilia A [HA] and hemophilia B [HB], respectively). Replacement therapy with clotting factor concentrates is the mainstay of treatment. Unlike in patients with HB, anaphylaxis in patients with HA is extremely rare. MethodsA retrospective study of prospectively collected data on patients with hemophilia who experienced anaphylaxis was conducted in our center. Demographic and clinical data were collected, and laboratory workups that included thrombin generation were conducted. ResultsOur first patient underwent successful immune tolerance induction (ITI) following the administration of rituximab. The second patient was transitioned to emicizumab. The third patient receives recombinant activated VIIa (rFVIIa) on demand. Thrombin generation was performed following current medical management protocols for supporting hemostasis. DiscussionOur case series illustrates the difficulty in managing patients with anaphylaxis to replacement therapy. In the era of novel therapies, such as emicizumab, the management of HA patients who experience anaphylaxis to replacement therapy is becoming easier and may obviate the need for ITI. Current treatment strategies for HB patients with such anaphylaxis, however, are limited to rFVIIa, and it continues to pose a challenge.

Modern view on hemostasis system: cell theory

For many years, there has been no model capable of explaining the complex processes of interaction between various bloodclotting factors leading to a stop of bleeding. One of the most successful models able to partially reflect the mechanisms of hemostasis for a long time was the cascade theory. The cascade model perfectly explains the processes occurring during coagulation in vitro, but was completely inadequate in attempts to evaluate the processes occurring in vivo. A significant drawback of the cascade model is the impossibility to trace the interaction of cells carrying the tissue factor, platelets and plasma coagulation factors on their surface, since these conditions cannot be imitated. The cell theory, which has replaced the cascade theory, pays attention not only to the interaction of plasma coagulation factors, but also takes into account the role of platelets as important participants of coagulation processes. It is based on a four-stage reaction cascade that includes the following stages: initiation, amplification, propagation, and termination.The cell theory of hemostasis is able to reflect the complex process of interaction of all the links of hemostasis and answer questions related to the problems in patients with disorders of the coagulation system. The cell theory of hemostasis allows to reflect more precisely the processes of hemostasis in vivo and to interpret correctly the results of tests and pathophysiological mechanisms of disorders of the coagulation system. Global tests (thrombin generation assay, thromboelastography, thrombodynamics) used for hemostasis system evaluation are more complimentary with cell theory of hemostasis.

Toll‐like receptors 2 and 7 mediate coagulation activation and coagulopathy in murine sepsis

BackgroundSepsis is a life‐threatening condition often manifested as marked inflammation and severe coagulopathy. Toll‐like receptors (TLRs) play a pivotal role in inflammation, organ dysfunction and mortality in animal sepsis. ObjectivesTo investigate the role of TLR signaling in mediating sepsis‐induced coagulopathy (SIC) in a mouse model. MethodsPolymicrobial sepsis was created by cecal ligation and puncture (CLP) or fecal slurry peritoneal injection. To quantify global clotting function, two viscoelastic assays were performed with rotational thromboelastometry, and the results were presented as maximum clot firmness (MCF): (a) EXTEM to test tissue factor (TF)‐initiated clot formation; and (b) FIBTEM to test EXTEM in the presence of a platelet inhibitor, cytochalasin D. Plasma coagulation factors were quantified with ELISA. TF gene expression and protein expression were determined with real‐time quantitative reverse transcription PCR and flow cytometry, respectively. ResultsBetween 4 and 24 hours after CLP surgery, wild‐type mice showed significant MCF reduction in both EXTEM and FIBTEM tests. This was accompanied by marked thrombocytopenia and a significant increase in the levels of plasminogen activator inhibitor‐1, plasma TF, and D‐dimer. In comparison, TLR2−/− and TLR7−/− CLP mice showed preserved MCF and platelet counts, and near‐normal plasma TF levels. Bone marrow–derived macrophages treated with a TLR2 agonist Pam3cys‐Ser‐(Lys)4 (Pam3cys) or a TLR7 agonist (R837) showed marked increases in TF gene expression and protein expression. MicroRNA‐146a, a newly identified proinflammatory mediator that is upregulated during sepsis, induced TF production via a TLR7‐dependent mechanism. ConclusionsMurine sepsis leads to an increased procoagulant response, thrombocytopenia, and global coagulopathy. TLR2 and TLR7 play an important role in procoagulant production and in SIC.

HLA-DRB1*01:01, but not HLA-DRB1:1503 or HLA-DRB1*11, is associated with decreased inhibitor risk in Iranian hemophilia A patients

Background/objectiveHemophilia A is a genetic disorder through which patients suffer from recurrent bleeding. This can be caused by a defect in human plasma coagulation factor VIII. High incidence of FVIII inhibitors in some severe hemophilia A patients after FVIII therapy is a considerable complication. Determination of good predictive factors can improve the safety of this treatment. HLA-II have been shown as a predictive element for inhibitor development. The goal of this study is to determine the association between HLA-DRB1*15:03, HLA-DRB1*11 and HLA-DRB1*01:01 alleles and FVIII inhibitors in severe hemophilia A patients in Iran. Materials/methodsHLA-DRB1 genotyping was performed using Multiplex sequences Specific Primers (PCR-SSP) in two groups of severe hemophilia A patients comprising 51 and 50 individuals with and without FVIII inhibitors respectively. The levels of inhibitor were determined through Nijmegen-modified Bethesda assay. HLA-DRB1 allele frequencies were compared between groups by using multiple logistic regression models. ResultsHLA-DRB1*01:01 allele frequency was significantly higher in patients without inhibitor ORadj: 2.7 (95%CI: 1.08, 6.97; P = 0.034). There wasn’t any statistically significant difference in HLA-DRB1*11 allele frequency between groups ORadj: 0.7 (95%CI: 0.27, 1.82; P = 0.47). There was no connection between HLA-DRB1*15:03 and inhibitor development ORadj: 0.94 (95%CI: 0.38, 2.35; P = 0.94). ConclusionAn association between HLA-DRB1*01:01 and paucity of FVIII inhibitor showed that this allele has probably a protective effect in severe hemophilia A patients in Iran. Determination of the predictive and protective alleles are beneficial in pre-treatment activities and decrease the risk of unsuccessful therapy with FVIII in each population.

Anticoagulant Rodenticides Poisonings in Humans and Animals – Short Review

Anticoagulant rodenticides (AR) are among the most commonlyused rodent control pesticides. The current second-generationrodenticides in worldwide use are referred to as superwarfarins.These substances have relatively low toxicity to humans but significanttoxicity to animals, including pets.AR work at the level of hepatocytes by blocking the synthesisof plasma coagulation factors II, VII, IX, and X as well as proteinsC, S, and Z, resulting in severe coagulation disorders predominantin the clinical picture.Deaths associated with AR poisoning are the result of haemorrhagesinto the gastrointestinal tract, peritoneal cavity, or intracranialcavities.Medico-legal diagnosis of AR poisonings is based on the clinicalpicture, autopsy, and histopathological and toxicological examinations.

Effects of storage over a 36-month period on coagulation factors in a canine plasma product obtained by use of plasmapheresis.

To evaluate stability of coagulation factors in canine plasma obtained by use of plasmapheresis and stored over a 36-month period. Canine plasma obtained by use of plasmapheresis acquired from a commercial blood bank. Coagulation testing for fibrinogen concentration and activity of factors II, V, VII, VIII, and IX and von Willebrand factor was performed on canine plasma obtained by use of plasmapheresis. Samples were obtained for testing at 6-month intervals from plasma stored for up to 36 months. A simple mixed linear regression model was created for each analysis. Median value for the fibrinogen concentration was > 150 mg/dL for all time points, except at 467, 650, and 1,015 days of storage. Median value for factor VIII was > 70% only at 650 days. Median value for factor V was > 50% through 650 days. Median value for factors VII and X was > 50% through 833 days, and median value for factors II and VII was > 50% through 1,015 days. Median value for von Willebrand factor was > 50% for the entire study (1,198 days). Median value for factor X was always < 50%. Coagulation factors degraded over time at variable rates, and all labile factors remained at > 50% activity for longer than 1 year. Plasma collected by plasmapheresis potentially offers prolonged life span of some clotting factors. Plasmapheresis is an acceptable form of canine plasma collection for transfusion purposes, and further studies should be performed to determine all of its benefits.

Use of Viscoelastography in Malignancy-Associated Coagulopathy and Thrombosis: A Review.

The relationship between malignancy and coagulopathy is one that is well documented yet incompletely understood. Clinicians have attempted to quantify the hypercoagulable state produced in various malignancies using common coagulation tests such as prothrombin time, activated partial thromboplastin time, and platelet count; however, due to these tests' focus on individual aspects of coagulation during one specific time point, they have failed to provide clinicians the complete picture of malignancy-associated coagulopathy (MAC). Viscoelastic tests (VETs), such as thromboelastography (TEG) and rotational thromboelastometry (ROTEM), are whole blood analyses that have the advantage of providing information related to the cumulative effects of plasma clotting factors, platelets, leukocytes, and red cells during all stages of the coagulation and fibrinolytic processes. VETs have gained popularity in the care of trauma patients to objectively measure trauma-induced coagulopathy (TIC), but the utility of VETs remains yet unrealized in many other medical specialties. The authors discuss the similarities and differences between TIC and MAC, and propose a mechanism for the hypercoagulable state of MAC that revolves around the thrombomodulin-thrombin complex as it switches between activating the protein C anticoagulation pathway or the thrombin activatable fibrinolysis inhibitor coagulation pathway. Additionally, they review the current literature on the use of TEG and ROTEM in patients with various malignancies. Although limited research is currently available, early results demonstrate the utility of both TEG and ROTEM in the prediction of hypercoagulable states and thromboembolic complications in oncologic patients.

Фотогемотерапия плазменных факторов свертывания крови у спортсмена с генетической предрасположенностью к тромбозу глубоких вен

В статье рассматривается клинический случай развития острого венозного тромбоза у молодого спортсмена после выступления в сложных климатических условиях. Пациенту был поставлен диагноз «Окклюзионный тромбоз подключичной и подмышечной вен справа» и проведен курс консервативного лечения. Массивная антикоагулянтная терапия улучшила состояние больного, но не привела к нормализации тромботического потенциала крови. Генетическое исследование плазменных факторов системы свертывания крови выявило у пациента повышенную предрасположенность к возникновению тромбозов. Амбулаторное применение фотогемотерапии синим светом полностью нормализовадо гемостаз молодого спортсмена. The article discusses the clinical case of acute venous thrombosis in a young athlete after game in diffi cult climatic conditions. The diagnose was «Occlusive thrombosis of the subclavian and axillary veins on the right», and the patient received a course of medical treatment. Massive anticoagulant therapy improved patient’s state, but did not lead to normalization of blood thrombotic potential. Genetic testing of plasma factors of blood coagulation system revealed in patient an increased predisposition to thromboses occurrence. Outpatient use of photohemotherapy with blue light completely normalized the hemostasis of young athlete.

The impact of glucocorticoid on coagulation of smoke inhalation induced lung injury

Objective To explore the impact of glucocorticoid on coagulation through administrating on rats with smoke inhalation. Methods Totally 150 male S-D rats were randomly (random number) divided into 5 groups: control group (ambient air inhalation), smoke group (smoke inhalation for 30 min), smoke+high dosage methyl prednisolone group(MP 40 mg/kg, intraperitoneal injection, s+HMP group), smoke+medium dosage MP (4 mg/kg) group (s+MMP group), smoke+low dosage MP (0.4 mg/kg) group (s+LMP group) (all n=30). Survival rates were calculated 24 h after smoke inhalation. Lung tissues were collected for histopathology and wet to dry (W/D) ratio. Arterial blood was collected for blood gas test. Coagulation factors in lung and plasma were tested. Results Survival rates of three MP groups were markedly improved compared with the smoke group (all P<0.05), and was significantly higher in the medium dosage group(85.17%) than those in the low and high dosage groups (65.73% and 60.07%, all P<0.05). The W/D ratio and blood gas test were markedly improved in the high and medium groups (all P<0.05). Tissue factor (TF) and thrombin-antithrombin complex (TAT-c) in bronchoalveolar lavage fluid (BALF) increased dramatically after SI (P<0.01, P=0.005) with a remarkable drop of factor Ⅱ (FⅡ) (P=0.007), all of which were attenuated by MP with dosage dependence. The mRNA expression of TF increased dramatically after SI and recovered significantly with MP administration, while the expression of thrombomodulin (TM) recovered in the opposite direction with MP, all of which were in a dosage dependent manner. TF, fibrinogen (FIB), TAT-c increased significantly in plasma after smoke inhalation (P<0.01, P=0.027, P=0.005). FⅧ% increased with MP administration and TF was raised by high dosage MP compared with the smoke group. FIB and TAT-c were decreased in all MP groups, which were significant higher in the high and middle dosage groups. The change of TM and endothelial cell protein C receptor (EPCR) in circulation were similar with FIB or TAT-c with or without MP. Protein C (PC%) and antithrombin (ATⅢ%) dropped dramatically after SI, high and middle dosages of MP could restore the activity significantly, while low dosage would restore ATⅢ% but not PC%. Conclusions Glucocorticoid can significantly improve local and systemical coagulation disorder caused by smoke inhalation, and high- and medium-dosage hormones are effective. The regulation of hormones on the coagulation system is an important mechanism in the treatment of smoke inhalation induced lung injury. Key words: Smoke inhalation; Acute lung injury; Coagulation; Glucocorticoid; Thrombomodulin

Exploring the Heterogeneity of Nanoparticles in Their Interactions with Plasma Coagulation Factor XII.

Tuning the characteristics of nanoparticles (NPs) would be promising in improving their biocompatibilities, regarding biosafety and nanodrug considerations. Due to the high priority of the artificial NPs in contacting the circulatory system, understanding their interactions with plasma zymogens is of great importance. Four kinds of NPs, including 5 nm gold NPs (GNP-5), 5 and 20 nm silver NPs (SNP-5, SNP-20), and 20 nm silica NPs (SiNP-20), were investigated for their interactions with the coagulation factor XII (FXII). GNP-5 adsorbed FXII in a standing-up mode, and exhibited high binding affinity for the heavy chain of the protein without altering its secondary structure or inducing its activation. In contrast to GNP-5, FXII adsorption on the other tested NPs was in a lying-down mode, and their interactions with FXII induced its conformational changes, thus causing the evident zymogen cleavage. The structural alterations and activation of FXII induced by the NPs exhibited in specific surface area dependent manners, which were related with different NP cores and sizes. Additionally, the enzymatic activity of α-FXIIa was also influenced by NP incubation, and the alterations were dependent on the specific characters of the NPs as evidenced by the enzymatic inhibition effect of GNP-5 (noncompetitive) and SNP-5 (competitive), and enhanced enzymatic catalysis abilities of SNP-20 and SiNP-20. The interesting findings on the heterogeneity of NPs in their interactions with plasma FXII not only revealed the underlying mechanism for NP-triggered hematological responses, but also suggested the crucial role of tuning NP parameters in their potential bioapplication, like nanodrug design.

Value of Thromboelastography in Judging Abnormal Coagulation Function in Patients with Early Hemorrhagic Shock

Objective: To detect the changes of coagulation function in patients with early hemorrhagic shock by thromboelastography (TEG). Methods: TEG was performed in 50 patients with early hemorrhagic shock and surgical indications. The TEG parameters were compared with 50 healthy people. The coagulation and fibrinolysis in patients with early hemorrhagic shock were observed. Results: In terms of coagulation parameters, the R value decreased, the α angle increased, and the K value and MA value did not change significantly in patients with early hemorrhagic shock. Fibrinolytic aspects: EPL, LY30 observations have no significant changes compared to normal values. Conclusion: The plasma coagulation factor activity is increased in patients with early hemorrhagic shock; the fibrin level is increased; the blood is in a hypercoagulable state; and the fibrinolysis function is not changed. The timely detection of TEG can be used for coagulation function monitoring and blood transfusion therapy in patients with surgical hemorrhagic shock. It provides an important basis for preventing the formation of deep vein thrombosis.

Приобретенная гемофилия А: обзор литературы и собственное наблюдение

Резюме. Классическая гемофилия — наследственное заболевание крови, причиной развития которого является дефицит факторов свертывающей системы. Приобретенная же гемофилия относится к редким патологиям; в ее основе лежит продукция аутоантител, которые приводят к инактивации эндогенного фактора VIII. В статье представлен обзор литературы по этиологии, эпидемиологии, патогенезу, клинике, диагностике и лечению приобретенной гемофилии А. Приведено собственное клиническое наблюдение успешного лечения пациентки с приобретенной послеродовой гемофилией. Summary. Classical hemophilia is a hereditary blood disease caused by defi ciency of coagulation factors. Hemophilia A is a rare pathology caused by inactivation of plasma clotting factor VIII with autoantibodies. The review summarizes what is currently known about the epidemiology, pathogenesis, clinical peculiarities, diagnostics, treatment and prognosis of acquired hemophilia A. Case report of successful treatment of patient with acquired postpartum hemophilia is presented.

Whole blood mitigates the acute coagulopathy of trauma and avoids the coagulopathy of crystalloid resuscitation.

The contributions of type and timing of fluid resuscitation to coagulopathy in trauma remain controversial. As part of a multifunctional resuscitation fluid research effort, we sought to further characterize the coagulation responses to resuscitation, specifically as compared to whole blood. We hypothesized that early whole blood administration mitigates the acute coagulopathy of trauma by avoiding the coagulopathy of CR resuscitation. Anesthetized rhesus macaques underwent polytraumatic, hemorrhagic shock, then a crossover study design resuscitation (n = 6 each) with either whole blood first (WB-1st) followed by crystalloid (CR); or CR-1st followed by WB. Resuscitation strategies were the following: WB-1st received 50% shed blood in 30minutes, followed by twice the shed blood volume (SBV) of CR over 30minutes and one times the SBV CR over 60minutes, where CR-1st received twice the SBV of CR over 30minutes, followed by 50% of shed blood in 30minutes, and one times the SBV CR over 60minutes. Blood samples were collected at baseline, end-of-shock, end-of-first and end-of-second resuscitation stages, and end-of-resuscitation for assessment (thromboelastometry, platelet aggregation, and plasmatic coagulation factors). Statistical analyses were conducted using two-way analysis of variance ANOVA with Bonferroni correction and t-tests; significance was at p < 0.05. Survival, blood loss, hemodynamics, and shock duration were equivalent between the groups. Compared to baseline, parameters measured at first and second resuscitation stage time points directly following CR infusion revealed abnormalities in thromboelastometry (clot formation time, α angle, and maximum clot firmness), platelet aggregation response (to collagen, arachidonic acid, and adenosine diphosphate), and plasmatic coagulation (prothrombin time, anti-thrombin 3, and fibrinogen), while whole blood infusion resulted in stabilization or correction of these parameters following its administration. These data suggest that in the setting of trauma and hemorrhagic shock, the coagulation alterations begin before intervention/resuscitation; however, these are significantly aggravated by CR resuscitation and could perhaps be best termed acute coagulopathy of resuscitation. Translational animal model.

A Novel Humanized Hemophilia-a Mouse Model to Facilitate Preclinical In Vivo Studies of Human Specific Fviiia-Mimetic Bispecific Antibodies

Introduction: To improve treatment options for Hemophilia A patients with and without Factor VIII inhibitors, we and others are developing bispecific antibodies that replace the function of FVIII by bringing together activated FIX and FX to activate FX and restore normal hemostasis. Thus far, preclinical in vivo evaluation of these novel reagents has been limited to non-human primates, due to their lack of species cross-reactivity.To accommodate pharmacokinetic, pharmacodynamic and in vivo efficacy and safety studies of novel FVIIIa-mimetic bispecific antibodies we set out to generate two humanized FIX;FX double knock-in (DKI) mouse models: one with normal murine FVIII activity (hFIX;hFX-DKI) and one with hemophilia A (hFIX;hFX-DKI;mFVIII-KO).Methods and Results: Human FIX-knock-in (KI) and human FX-KI mice were generated, using classical C57/Bl6 embryonic stem cell targeting. Both hFIX and hFX encoding sequences replaced their murine counterparts with expression of the human genes driven by the intact murine promotor. hFIX;hFX-DKI mice were generated by intercrossing both lines to homozygosity. hFIX;hFX-DKI;mFVIII-KO mice were generated by CRISPR/Cas9 mediated knockout of murine FVIII in hFIX-KI mice, prior to intercrossing with hFX-KI mice. Both hFIX;hFX-DKI and hFIX;hFX-DKI;mFVIII-KO mice are fertile and viable.Plasma protein expression levels of both hFIX and hFX in single-KI, DKI, and hFIX;FX-DKI;mFVIII-KO mice were determined by human specific ELISAs and were approximately 1 and 2 µg/mL, respectively. These protein concentrations correspond to 20 % FIX and 10 % FX normal human plasma levels. Chromogenic assays showed matching plasma clotting factor activities compared to normal human plasma, i.e. 10-20% activity of hFIX and 5-10% of hFX. Similar data were obtained by one stage assay. Finally, functional FVIII deficiency in the hFIX;hFX-DKI;mFVIII-KO mice was confirmed by both chromogenic and one stage clotting assays.Importantly, hFIX;hFX-DKI mice showed only slightly reduced whole blood clotting activity compared to wild-type mice by rotational thromboelastometry (ROTEM; mean clotting times: 910 sec and 516 sec, respectively) whereas hemophilic mice showed significant prolonged clotting times (2500 sec). These data suggest a mild hemophilic phenotype due to reduced human clotting factor expression. Nonetheless the hFIX;hFX-DKI mice showed 100% survival in the tail vein transection model compared to nearly 0% survival for HemA and HemB mice. These findings indicate that hFIX;hFX-DKI;mFVIII-KO should be suitable for the tail vein transection bleeding efficacy evaluation of FVIIIa-mimetic antibodies. In contrast, the more severe tail clip bleeding model using hFIX;hFX-DKI mice showed only slightly less bleeding compared to hemophilia mice (median blood loss, 581 µl versus 766 µl), showing that 10% human clotting factor plasma levels compared to wild-type mice (62 µl) is not sufficient to stop bleeding in this efficacy model. The latter finding is in line with reported data showing that near normal FVIII or FIX clotting factor levels are needed in the severe tail clip bleeding model to stop bleeding in HemA or HemB mouse models, respectively (Dumont, Blood, 2012, Toby et al PLOS One 2016).Conclusions: We generated and characterized novel mouse models enabling pharmacokinetic, pharmacodynamic and in vivo efficacy and safety studies for novel FVIII-mimetic bispecific antibodies. Ongoing and future studies using these novel mouse models aim to elucidate the in vivo mode of action of this new class of non-replacement clotting factor drug candidates as well as their in vivo interactions with other bypass reagents DisclosuresLi:Bioverativ a Sanofi company: Employment, Equity Ownership, Honoraria. Hu:Biogen: Employment, Equity Ownership, Honoraria. Leksa:Bioverativ a Sanofi company: Employment. Aleman:Bioverativ, a Sanofi company: Employment. Peters:Bioverativ a Sanofi company: Employment, Equity Ownership. Salas:Bioverativ a Sanofi company: Employment, Equity Ownership. Van Der Flier:Bioverativ a Sanofi company: Employment, Equity Ownership, Honoraria.

Spatial Structure of Plasma Coagulation Factor XIII and Changes with Activation

IntroductionBlood coagulation factor XIII (FXIII) is a precursor of the active transglutaminase (FXIIIa) that catalyzes covalent cross-linking within polymeric fibrin to increase mechanical and proteolytic stability of blood clots and thrombi. Structurally, inactive plasma FXIII (pFXIII) with a molecular mass of 326 kDa is a heterotetramer (A2B2) with two catalytic A subunits (FXIII-A, 83 kDa) and two non-catalytic B subunits (FXIII-B, 80 kDa).The inactive pFXIII is converted into a catalytically active form by thrombin and Ca2+. Thrombin cleaves off an activation peptide (AP) from the N-terminus of each A subunit, and subsequently B subunits dissociate from thrombin-cleaved A subunits in presence of Ca2+. However, the oligomerization state of activated A and B subunits remain controversial.Both activated and inactive FXIII A subunits have been characterized crystallographically, while the atomic structure of the FXIII-A2B2 complex remains unknown. Therefore, our goal was to visualize and characterize the molecular structure of FXIII and its activated form FXIIIa using high-resolution atomic force microscopy (AFM).MethodsCommercially available pFXIII (Enzyme Research Laboratories, USA), non-activated or activated with thrombin and Ca2+, was adsorbed on the surface of graphite rendered hydrophilic with an amphiphilic hydrocarbon-glycine modifier followed by single-molecule AFM imaging, methodology developed by us to allow high-resolution imaging. Additionally, recombinant rFXIII-A2 (Zymogenetics, USA) and recombinant rFXIII-B2 (Zedira, Germany) were imaged to compare the structures and dimensions of FXIII and FXIIIa with individual dimeric subunits.ResultsIndividual molecules of pFXIII were visualized in AFM as globules with two (67% of the molecules), one (14%), or no (19%) thread-like extensions (Fig. 1A). The size (height) of the globular portions had a symmetrical distribution with a maximum at 3.5 nm. The strands had a peak contour length of 20 nm and a height of 0.4 nm. Based on the known polypeptide composition of pFXIII-A2B2, the observed molecular images could be interpreted as an A2 globule with one or two individual B subunits extending out from it. The structural diversity of FXIII forms may reflect an equilibrium between association and dissociation of B subunits from the A2 dimer. Alternatively, the B subunits can be partially or fully wrapped around the globular A2 dimer, making them invisible in AFM images.Although a crystallographic structure of FXIII-A2 is available, its AFM imaging was important to identify the A2 dimer within inactive and activated pFXIII molecules: rFXIII-A2 was a globule with the same height as the pFXIII globular core (Fig. 1B).Recombinant dimeric B subunits (rFXIII-B2) appear as thin flexible strands with an average contour length of 33 nm and a height of 0.6 nm (Fig. 1C). Thus, both the length and height of the free B2 dimers were significantly larger compared to the strands sticking out from the globular core of pFXIII-A2B2 molecules, we conclude that these strands in pFXIII-A2B2 comprise monomeric B subunits.In thrombin-activated pFXIIIa, the B-subunits dissociated from the A2 dimer and self-associated to form B2 dimers (Fig. 1D). The height distribution of globular particles remaining separated was bimodal with two peaks: one at 3.3 nm corresponded to the whole tetrameric pFXIII-A2B2 and, potentially, dimeric FXIII-A2, while the second major peak at 2.1 nm likely represented FXIII-A monomers.ConclusionsOur results provide qualitative and quantitative molecular structural characteristics of inactive and activated human pFXIII as well as dimeric A and B subunits. We have shown that inactive plasma FXIII (pFXIII-A2B2) consists of a globular A2 dimer core and individual B subunits extending out as flexible strands. Recombinant inactive FXIII-A subunits in the absence of FXIII-B subunits form a globular homodimer. Recombinant FXIII-B subunits in the absence of FXIII-A subunits form flexible homodimeric strands. Upon activation of pFXIII-A2B2 with thrombin and Ca2+, the initially monomeric B subunits dissociate from the globular core of the molecule and form B2 homodimers, while the initially dimeric activated A subunits dissociate into monomers.Fig. 1. AFM images of A: pFXIII-A2B2, B: rFXIII-A2, C: rFXIII-B2, D: thrombin-activated pFXIIIa. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Indoxyl Sulfate Promotes Arterial Thrombosis in Rat Model via Increased Levels of Complex TF/VII, PAI-1, Platelet Activation as Well as Decreased Contents of SIRT1 and SIRT3.

Patients suffering from chronic kidney disease (CKD) are at a 20-fold higher risk of dying due to cardiovascular diseases (CVDs), primarily thrombosis following vascular injury. CKD is connected with retention of uremic toxins, especially indoxyl sulfate (IS), which are currently considered as a non-classical CKD-specific risk factor for CVDs. The present study aimed to examine the effect of chronic exposure to IS on the hemostatic system and arterial thrombosis in a model without greater interferences from the uremic milieu consisting of additional uremic toxins. Forty-eight male Wistar Crl:WI (cmdb) rats were divided into three groups: one control group and two experimental groups, which were exposed to 100 or 200 mg/kg of b.w./day of IS in drinking water for a period of 28 days. The control group received water without IS. At the end of the experiment, the induction of arterial thrombosis was performed. We investigated the impact of IS on thrombosis incidence, kinetics and strength of clot formation, platelet activity, aortic contents of sirtuin (SIRT) 1 and sirtuin 3 (SIRT3), hemostatic system, cardiorespiratory parameters, biochemistry of plasma and urine as well as histology of the thrombus, kidney, and liver. Obtained data revealed that chronic exposure to IS promotes arterial thrombosis via increased levels of complex tissue factor/factor VII, plasminogen activator inhibitor-1 (PAI-1), platelet activation, as well as decreased aortic levels of SIRT1 and SIRT3. Therefore, we hypothesize that IS enhances primary hemostasis leading to augmented formation of platelet plug with increased amounts of fibrin and affects secondary hemostasis through the influence on plasma coagulation and fibrinolysis factors, which results in the increased kinetics and strength of clot formation. The findings described may contribute to a better understanding of the mechanisms leading to increased thrombotic events in patients with CKD with elevated levels of IS.

Characteristics of hemostasis system and activity of trypsin-like blood enzymes in comorbid patients with chronic obstructive pulmonary disease and hypertension.

This work was aimed at studying the activity of the hemostasis system and trypsin-like blood enzymes (TLE) in patients with chronic obstructive pulmonary disease (COPD) and hypertension (H), depending on anthropometric, clinical features and smoking. The study included 87 patients. I group included 39 patients with COPD A – D, II-IV by GOLD and conco­mitant with H I-II stage, II group – 38 patients with H I - II stage, and K group – 10 apparently healthy persons. The study was conducted on the background of the use of standard basic therapy by patients from the 1st and 2nd groups in a stable period of disease. The degree of thrombocyte (tr) adhesion in I and II groups of patients was higher than in the K group. Both adhesive activity and the amount of tr in the I group was higher than in the II group. With positive status of smoking and increasing nicotine addiction in comorbid patients with COPD and H, the adhesive activity of tr increased. The level of the procoagulantive parameters of the plasma part of hemostasis in the two studied groups was not statistically different, but was higher than in the K group. Increased BMR and dyspnoea by the mMRS scale in group with COPD and H and increased age of patients with H led to the increase in the activity of plasma coagulation factors. In I and II groups, the increase in blood TLE was observed, which increased with the body weight, while in comorbid patients in the subgroup with FEV1≥50%, the incidence of COPD exacerbation additionaly effects the increase of TLE. In I group correlation between reduction of FEV1 with the decrease of TLE was revealed. In patients with COPD and AH activity of thrombocyte and plasma hemostasis, as well as trypsin-like blood enzymes, increases. Smoking, body weight gain and incidence of COPD exacerbation contribute to increased prothrombotic potential.