A Genus-Wide Bioactivity Analysis of Daboia (Viperinae: Viperidae) Viper Venoms Reveals Widespread Variation in Haemotoxic Properties.

The snake genus Daboia (Viperidae: Viperinae; Oppel, 1811) contains five species: D. deserti, D. mauritanica, and D. palaestinae, found in Afro-Arabia, and the Russell's vipers D. russelii and D. siamensis, found in Asia. Russell's vipers are responsible for a major proportion of the medically important snakebites that occur in the regions they inhabit, and their venoms are notorious for their coagulopathic effects. While widely documented, the extent of venom variation within the Russell's vipers is poorly characterised, as is the venom activity of other species within the genus. In this study we investigated variation in the haemotoxic activity of Daboia using twelve venoms from all five species, including multiple variants of D. russelii, D. siamensis, and D. palaestinae. We tested the venoms on human plasma using thromboelastography, dose-response coagulometry analyses, and calibrated automated thrombography, and on human fibrinogen by thromboelastography and fibrinogen gels. We assessed activation of blood factors X and prothrombin by the venoms using fluorometry. Variation in venom activity was evident in all experiments. The Asian species D. russelii and D. siamensis and the African species D. mauritanica possessed procoagulant venom, while D. deserti and D. palaestinae were net-anticoagulant. Of the Russell's vipers, the venom of D. siamensis from Myanmar was most toxic and D. russelli of Sri Lanka the least. Activation of both factor X and prothrombin was evident by all venoms, though at differential levels. Fibrinogenolytic activity varied extensively throughout the genus and followed no phylogenetic trends. This venom variability underpins one of the many challenges facing treatment of Daboia snakebite envenoming. Comprehensive analyses of available antivenoms in neutralising these variable venom activities are therefore of utmost importance.

Vitamin K2 (Menaquinone-7) supplementation does not affect vitamin K-dependent coagulation factors activity in healthy individuals.

BACKGROUND: Vitamin K has long been regarded as a procoagulant drug by physicians, and concerns have been raised with regard to its effects on hemostasis. Although many studies have shown that vitamin K supplementation is safe for thrombotic events, the effect of vitamin K supplementation on the activities of vitamin K dependent procoagulation factors in healthy individuals is not available. OBJECTIVES: This study aimed to investigate whether vitamin K2 supplementation at recommended doses affects the activity of vitamin K dependent procoagulation factors in healthy individuals without any anticoagulation treatment. DESIGN: Forty healthy volunteers between 25 and 40 years of age were recruited. Menaquinone-7 (MK-7) was administrated at 90 µg for 30 days. Prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT), and blood coagulation factors II, VII, IX, and X activities and Protein induced by vitamin K absence or antagonist-II (PIVKA-II) were measured on days 0 and 30 after MK-7 administration. RESULTS: PT, APTT, and TT showed no significant differences on day 30 when compared with baseline. The activities of coagulation factors II, VII, IX, and X on day 30 showed no significant differences with those at baseline. PIVKA-II levels were unchanged after 30 days of MK-7 supplementation. CONCLUSIONS: MK-7 supplementation at recommended dosage does not affect vitamin K-dependent coagulation factors' coagulation activity, and does not enhance the carboxylation of prothrombin in healthy individuals. This indicated that MK-7 administration does not alter hemostatic balance in healthy populations without anticoagulation treatment.

Ignoring instead of chasing after coagulation factor VII during warfarin management: an interrupted time series study.

During warfarin management, variability in prothrombin time-based international normalized ratio (PT-INR) is caused, in part, by clinically inconsequential fluctuations in factor VII (FVII). The new factor II and X (Fiix)-prothrombin time (Fiix-PT) and Fiix-normalized ratio (Fiix-NR), unlike PT-INR, are only affected by reduced FII and FX. We assessed the incidence of thromboembolism (TE) and major bleeding (MB) in all 2667 patients on maintenance-phase warfarin managed at our anticoagulation management service during 30 months; 12 months prior to and 18 months after replacing PT-INR monitoring with Fiix-NR monitoring. Months 13 to 18 were predefined as transitional months. Using 2-segmented regression, a breakpoint in the monthly incidence of TE became evident 6 months after test replacement, that was followed by a 56% reduction in incidence (from 2.82% to 1.23% per patient-year; P = .019). Three-segmented regression did not find any significant trend in TE incidence (slope, +0.03) prior to test replacement; however, during months 13 to 18 and 19 to 30, the incidence of TE decreased gradually (slope, -0.12; R2 = 0.20; P = .007). The incidence of MB (2.79% per patient-year) did not differ. Incidence comparison during the 12-month Fiix and PT periods confirmed a statistically significant reduction (55-62%) in TE. Fiix monitoring reduced testing, dose adjustments, and normalized ratio variability and prolonged testing intervals and time in range. We conclude that ignoring FVII during Fiix-NR monitoring in real-world practice stabilizes the anticoagulant effect of warfarin and associates with a major reduction in TEs without increasing bleeding.

Design and Implementation of an Anti-Factor Xa Heparin Monitoring Protocol.

BACKGROUND: The VA Northeast Ohio Healthcare System introduced a new nurse-driven anti-factor Xa (anti-Xa) protocol for monitoring unfractionated heparin to replace the previous activated partial thromboplastin time protocol. OBJECTIVE: To design, implement, and evaluate the efficacy of the anti-Xa monitoring protocol. METHODS: An interdisciplinary team of providers collaborated to develop and implement a nurse-driven, facility-wide anti-factor Xa protocol for monitoring unfractionated heparin therapy. The effectiveness of this protocol was evaluated by retrospective analysis. RESULTS: We reviewed 100 medical records for compliance with the new anti-Xa monitoring protocol. We then evaluated 178 patients whose anticoagulation was monitored with the anti-Xa assay to determine the time to therapeutic range. We found that 80% of patients receiving the anti-Xa protocol achieved therapeutic anticoagulation within 24 hours, as compared with 54% of patients receiving the activated partial thromboplastin time protocol (P < .001). Protocol conversion also yielded a decrease in blood draws, dose adjustments, and potential calculation errors. CONCLUSIONS: Monitoring intravenous heparin therapy with the anti-Xa assay rather than activated partial thromboplastin time resulted in a shorter time to therapeutic anticoagulation, longer maintenance of therapeutic levels, and fewer laboratory tests and heparin dosage changes. We believe the current practice of monitoring heparin treatment with activated partial thromboplastin time assays should be reexamined.

A hemophilia A mouse model for the in vivo assessment of emicizumab function.

The bispecific antibody emicizumab is increasingly used for hemophilia A treatment. However, its specificity for human factors IX and X (FIX and FX) has limited its in vivo functional analysis to primate models of acquired hemophilia. Here, we describe a novel mouse model that allows emicizumab function to be examined. Briefly, FVIII-deficient mice received IV emicizumab 24 hours before tail-clip bleeding was performed. A second infusion with human FIX and FX, administered 5 minutes before bleeding, generated consistent levels of emicizumab (0.7-19 mg/dL for 0.5-10 mg/kg doses) and of both FIX and FX (85 and 101 U/dL, respectively, after dosing at 100 U/kg). Plasma from these mice display FVIII-like activity in assays (diluted activated partial thromboplastin time and thrombin generation), similar to human samples containing emicizumab. Emicizumab doses of 1.5 mg/kg and higher significantly reduced blood loss in a tail-clip-bleeding model using FVIII-deficient mice. However, reduction was incomplete compared with mice treated with human FVIII concentrate, and no difference in efficacy between doses was observed. From this model, we deducted FVIII-like activity from emicizumab that corresponded to a dose of 4.5 U of FVIII per kilogram (ie, 9.0 U/dL). Interestingly, combined with a low FVIII dose (5 U/kg), emicizumab provided enough additive activity to allow complete bleeding arrest. This model could be useful for further in vivo analysis of emicizumab.

Simple Laboratory Test Utilization Interventions to Reduce Inappropriate Specialty Coagulation Testing.

OBJECTIVES: The naming convention in coagulation may cause confusion in electronic ordering systems, leading to inappropriate test orders. We implemented test utilization efforts and studied utilization before and after interventions for two specialty coagulation assays. METHODS: Two interventions were implemented: test names were changed from factor assay to activity, and residents reviewed all factor V and X requests. A retrospective review of factor V and X activity orders was performed for the period 1 year before and after interventions. RESULTS: After interventions, factor V and X activity orders decreased by approximately 40%. Resulted tests decreased by 53.8% and 47.8%, corresponding to reductions of $2,493.05 and $1,867.80 per year in laboratory charges for factor V and factor X activity, respectively. Abnormal factor V activity results increased from 45% to 59%. Factor V activity orders from outpatient clinics decreased by 21.6%. CONCLUSIONS: Simple interventions can reduce inappropriate specialty coagulation test orders and unnecessary costs.

Protease-activated receptors are potential regulators in the development of arterial endofibrosis in high-performance athletes.

OBJECTIVE: High-performance athletes can develop symptomatic arterial flow restriction during exercise caused by endofibrosis. The pathogenesis is poorly understood; however, coagulation enzymes, such as tissue factor (TF) and coagulation factor Xa, might contribute to the fibrotic process, which is mainly regulated through activation of protease-activated receptors (PARs). Therefore, the aim of this explorative study was to evaluate the presence of coagulation factors and PARs in endofibrotic tissue, which might be indicative of their potential role in the natural development of endofibrosis. METHODS: External iliac arterial specimens with endofibrosis (n = 19) were collected during surgical interventions. As control, arterial segments of the external iliac artery (n = 20) were collected post mortem from individuals with no medical history of cardiovascular disease who donated their body to medical science. Arteries were paraffinized and cut in tissue sections for immunohistochemical analysis. Positive staining within lesions was determined with ImageJ software (National Institutes of Health, Bethesda, Md). RESULTS: Endofibrotic segments contained a neointima, causing intraluminal stenosis, which was highly positive for collagen (+150%; P < .01) and elastin (+148%; P < .01) in comparison with controls. Intriguingly, endofibrosis was not limited to the intima because collagen (+213%) and elastin (+215%) were also significantly elevated in the media layer of endofibrotic segments. These findings were accompanied by significantly increased α-smooth muscle actin-positive cells, morphologically compatible with the presence of myofibroblasts. In addition, PAR1 and PAR4 and the membrane receptor TF were increased as well as coagulation factor X. CONCLUSIONS: We showed that myofibroblasts and the accompanying collagen and elastin synthesis might be key factors in the development of endofibrosis. The special association with increased presence of PARs, factor X, and TF suggests that protease-mediated cell signaling could be a contributing component in the mechanisms leading to endofibrosis.

Procoagulant State in Current and Former Anabolic Androgenic Steroid Abusers.

BACKGROUND: Anabolic androgenic steroid (AAS) abusers are considered at increased risk of cardiovascular morbidity and mortality. We hypothesized that current and former AAS abuse would induce a procoagulant shift in the haemostatic balance. METHODS: Men 18 to 50 years of age were included as current AAS abusers, former AAS abusers or controls. Morning blood samples were collected after overnight fasting. Thrombin generation (lag time, time to peak, peak height, and endogenous thrombin potential [ETP]) and coagulation factor II (prothrombin), VII and X, antithrombin, protein C, free protein S and tissue factor pathway inhibitor (TFPI) were assessed. Groups were compared by ANOVA or Kruskal-Wallis test and probabilities were corrected for multiple comparisons. Associations were evaluated using linear regression models. RESULTS: ETP was increased around 15% in current (n = 37) and former (n = 33) AAS abusers compared with controls (n = 30; p < 0.001). Prothrombin and factor X were increased ≥10% in AAS abusers and prothrombin was a predictor of ETP (p < 0.0005). Lag time and time to peak were increased 10 to 30% in current AAS abusers (p < 0.001) and associated with higher concentrations of TFPI, antithrombin, protein C and protein S (p < 0.0005; = 0.005). Multivariate linear regression, with all coagulation inhibitors as covariates, identified TFPI to be independently associated with lag time and time to peak (p < 0.0005). CONCLUSION: Thrombin generation is augmented in current and former AAS abusers, reflecting a procoagulant state, with altered concentrations of coagulation proteins. Prospective studies are needed to clarify whether these findings translate into an increased thrombotic risk in AAS abusers potentially even after cessation.

Congenital FX Deficiency Rio Tercero: A New Heterozygous Missense Mutation (Cys241Gly) with a Potentiating Effect by a Polymorphism (c. 503-57C>T).

OBJECTIVE: The aim was to report a new family with congenital FX deficiency. PATIENTS AND METHODS: The proposita is a 41 year old female with a moderate bleeding tendency (easy bruising, menorrhagia). Parents were not consanguineous. Family history was positive for a mild bleeding tendency. RESULTS: Coagulation and genetics studies revealed that the proposita and two of her siblings were heterozygotes for a new mutation Cys241Gly in exon 6 but had different FX level (2-3% of normal in the proposita and about 50% in the two siblings. The same was true for one of her three children. The mother and the other two children of the proposita had also slightly decreased FX levels but no mutation. On the suspicion that the proposita was carrying another defect which had escaped the Sanger method, we carried out a whole exome analysis and discovered that the proposita and one of her siblings were also homozygous for a mutation of a known polymorphism (c.503-57C>T). The daughter of the proposita was instead, besides being a carrier of the missense new mutation Cys241Gly, heterozygous for the same polymorphism. The mother and two other daughters were also heterozygous for the polymorphism. There were no deletions or duplications. CONCLUSION: The polymorphism present in the family seems to be capable of potentiating the defect induced by the new mutation. This, safe for epigenetics phenomena, is the only possible explanation for the discrepancy found in the FX level between mother and daughter despite the fact that both carried the same new mutation.

Reduced Requirement for Prothrombin Complex Concentrate for the Restoration of Thrombin Generation in Plasma From Liver Transplant Recipients.

BACKGROUND: Plasma transfusion remains the mainstay hemostatic therapy during liver transplantation (LT) in most countries. However, a large volume is required for plasma to achieve clinically relevant factor increases. Prothrombin complex concentrate (PCC) is a low-volume alternative to plasma in warfarin reversal, but its efficacy has not been well studied in LT. METHODS: Blood samples were collected from 28 LT patients at baseline (T0) and 30 minutes after graft reperfusion (T1). Factor X and antithrombin levels were measured. Ex vivo effects of PCC (0.2 and 0.4 IU/mL) and 10% volume replacement with normal plasma were compared in LT and warfarin plasma by measuring lag time, thrombin peak, and endogenous thrombin potential (ETP) using thrombin generation (TG) assay. RESULTS: Coagulation status was worsened at T1 as international normalized ratio increased from 1.7 to 3.0, and factor X was decreased from 49% to 28%. TG measurements showed normal lag time and ETP at T0 and T1, but low-normal peak at T0, and below-normal peak at T1. Both doses of PCC increased peak and ETP, while 10% volume plasma had minimal effects on TG. Thrombin inhibition appears to be very slow after adding 0.4 IU/mL of PCC in LT plasma due to low antithrombin. The same doses of PCC and plasma were insufficient for warfarin reversal. CONCLUSIONS: Reduced TG in LT can be more effectively restored by using PCC rather than plasma. The required doses of PCC for LT patients seem to be lower than warfarin reversal due to slow thrombin inhibition.

Advances in the treatment of bleeding disorders.

Historically, the bleeding episodes in subjects with coagulation disorders were treated with substitution therapy, initially with whole blood and fresh frozen plasma, and more recently with specific factor concentrate. Currently, patients with hemophilia have the possibility of choosing different effective and safe treatments, including novel extended half-life and alternative hemostatic drugs. The availability of novel extended half-life products could probably overcome current prophylaxis limitations, particularly in hemophilia B patients, by reducing the frequency of injections, achieving a higher trough level, and improving the quality of life of the patients. In addition, subcutaneous administration of alternative therapeutics would simplify prophylaxis in patients with hemophilia A and B with and without inhibitors. Regarding von Willebrand disease, a recombinant von Willebrand factor was recently developed to control bleeding episodes in patients with this disease, in addition to available von Willebrand factor/factor VIII concentrates. The management of patients affected by rare bleeding disorders (RBDs) is still a challenge, owing to the limited number of specific products, which are mainly available only in countries with high resources. Some improvements have recently been achieved by the production of new recombinant factor (F) XIII A subunit-derived and FX plasma-derived products for the treatment of patients affected by FXIII and FX deficiency. In addition, the development of novel alternative therapeutics, such as anti-tissue factor pathway inhibitor, ALN-AT3, and ACE910, for patients with hemophilia might also have a role in the treatment of patients affected by RBDs.

Factor VIII-Mimetic Function of Humanized Bispecific Antibody in Hemophilia A.

BACKGROUND: In patients with severe hemophilia A, standard treatment is regular prophylactic and episodic intravenous infusions of factor VIII. However, these treatments are burdensome, especially for children, and may lead to the formation of anti-factor VIII alloantibodies (factor VIII inhibitors). Emicizumab (ACE910), a humanized bispecific antibody mimicking the cofactor function of factor VIII, was developed to abate these problems. METHODS: We enrolled 18 Japanese patients with severe hemophilia A (with or without factor VIII inhibitors) in an open-label, nonrandomized, interindividual dose-escalation study of emicizumab. The patients received subcutaneous emicizumab weekly for 12 weeks at a dose of 0.3, 1.0, or 3.0 mg per kilogram of body weight (cohorts 1, 2, and 3, respectively). The end points were safety and pharmacokinetic and pharmacodynamic profiles. An additional, exploratory end point was the annualized bleeding rate, calculated as 365.25 times the number of bleeding episodes, divided by the number of days in the treatment period as compared with the 6 months before enrollment. RESULTS: Emicizumab was associated with neither serious adverse events nor clinically relevant coagulation abnormalities. Plasma concentrations of emicizumab increased in a dose-dependent manner. Activated partial-thromboplastin times remained short throughout the study. The median annualized bleeding rates in cohorts 1, 2, and 3 decreased from 32.5 to 4.4, 18.3 to 0.0, and 15.2 to 0.0, respectively. There was no bleeding in 8 of 11 patients with factor VIII inhibitors (73%) and in 5 of 7 patients without factor VIII inhibitors (71%). Episodic use of clotting factors to control bleeding was reduced. Antibodies to emicizumab did not develop. CONCLUSIONS: Once-weekly subcutaneous administration of emicizumab markedly decreased the bleeding rate in patients who had hemophilia A with or without factor VIII inhibitors. (Funded by Chugai Pharmaceutical; JapicCTI number, 121934.).

Experience of a new high-purity factor X concentrate in subjects with hereditary factor X deficiency undergoing surgery.

INTRODUCTION: Maintaining haemostasis in surgery is challenging for hereditary rare bleeding disorders in which multi-coagulation-factor concentrates are the only therapeutic option. Hereditary factor X (FX) deficiency affects 1:500 000 to 1:1 000 000 individuals, and no specific replacement FX concentrate has been available. A high-purity, plasma-derived FX concentrate (pdFX) has been developed for patients with hereditary FX deficiency. AIM: Our objective was to assess the safety and efficacy of pdFX in subjects with FX deficiency undergoing surgery. METHODS: Subjects with hereditary mild-to-severe FX deficiency (basal plasma FX activity [FX:C] <20 IU dL(-1) ) undergoing surgery received pdFX preoperatively to raise FX:C to 70-90 IU dL(-1) and postoperatively to maintain levels >50 IU dL(-1) until the subject was no longer at risk of bleeding due to surgery. Efficacy of pdFX was assessed by blood loss during surgery, requirement for blood transfusion, postoperative bleeding from the surgical or other sites, and changes in haemoglobin levels. Safety was assessed by adverse events (AEs), development of inhibitors, and clinically significant changes in laboratory parameters. RESULTS: Five subjects (aged 14-59 years) underwent seven surgical procedures (four major and three minor). Treatment duration was 1-15 days. For each procedure, pdFX treatment was assessed as "excellent" in preventing bleeding and achieving haemostasis. No blood transfusions were required, no AEs related to pdFX were observed, and no clinically significant trends were found in any laboratory parameters. CONCLUSION: These data demonstrate that pdFX is safe and effective as replacement therapy in five subjects with mild-to-severe FX deficiency undergoing surgery on seven occasions.

Ex vivo evaluation of 4 different viscoelastic assays for detecting moderate to severe coagulopathy during liver transplantation.

Prolonged prothrombin time (PT) and its ratio are routinely used for the assessment of candidates for liver transplantation (LT), but intraoperative coagulation management of transfusion is hindered by its long turnaround time. Abnormal reaction time (R time) on thromboelastography (TEG) or clotting time (CT) of rotational thromboelastometry (ROTEM) are presumably an alternative, but there is a paucity of clinical data on abnormal R time/CT values compared to PT during LT. After receiving institutional review board approval and informed consent, we obtained blood samples from 36 LT patients for international normalized ratio (INR), factor (F) X level, and viscoelastic tests (EXTEM/INTEM and kaolin/rapid TEG) at baseline and 30 minutes after graft reperfusion. Receiver operating characteristic (ROC) curves were calculated for INR > 1.5 and viscoelastic R time/CT thresholds to assess the ability to diagnose FX deficiency at the moderate (<50%) or severe (<35%) level. The FX deficiency data were calculated using cutoff values of INR (>1.5) and abnormal R time/CT for TEG and ROTEM. Tissue factor (TF)-activated INR and EXTEM-CT performed well in diagnosing FX below 50%, but rapid TEG with combined TF and kaolin activators failed. Improved performance of INTEM-CT in diagnosing FX below 35% underlies multifactorial deficiency involving both intrinsic and common pathways. In conclusion, the differences among different viscoelastic tests and clinical situations should be carefully considered when they are used to guide transfusion during LT.

A novel Ala275Val mutation in factor X gene influences its structural compatibility and impairs intracellular trafficking and coagulant activity.

Factor X (FX) deficiency is an autosomal recessive severe bleeding disorder. Here, we identified a novel homozygous missense mutation (p.Ala275Val) in the F10 gene in a patient with severe FX deficiency. The novel mutation was analyzed by in vitro expression and modeling. Site-directed mutagenesis of FX cDNA was used to introduce the FX Ala275Val mutation, wild-type as well as mutant FX proteins were expressed in HEK293 cells, and subcellular localization experiments were performed. Expression experiments showed that the FX Ala275Val mutation led to a significant reduction in antigen and activity levels in the culture medium. Moreover, compared to the wild-type, mutant FX-Ala275Val was mainly distributed in the endoplasmic reticulum and rarely entered the Golgi apparatus, suggesting a transportation defect for FX from the endoplasmic reticulum to the Golgi apparatus. Molecular modeling analysis indicated that the Ala275 is spatially located to the catalytic triad of FXa, which is composed of His276, Asp322, and Ser419. The Ala to Val substitution may change the conformation of the catalytic pocket and alter protein folding and enzymatic activity. Our findings demonstrated that the Ala275Val substitution is a pathogenic mutation that causes the inherited FX deficiency.

Effects of storage and thawing conditions on coagulation testing.

INTRODUCTION: Current recommendations for coagulation testing storage and thawing are based on historical studies that were performed using unbuffered 3.8% sodium citrate. We sought to measure the effects of freezing and thawing conditions 3.2% buffered sodium citrate plasma samples that have been stored in vials with either snap or sealed screw tops, frozen in -70 °C freezer or dry ice and thawed either capped or uncapped. METHODS: Shed blood samples were pooled and then aliquoted into four snap top and four screw tops vials. Half the vials were stored in a -70 °C freezer, and half on dry ice for at least 16 h. Afterwards, half the frozen samples were thawed in 37 °C waterbath capped, and other half were thawed capped. After thawing cycles, samples were tested for PT, activated partial thromboplastin time (APTT), fibrinogen, D-dimer, factor assays, von Willebrand factor activity, plasminogen, antithrombin, protein C and lupus anticoagulant. RESULTS: Prothrombin time, APTT, factor X, and lupus anticoagulant testing were affected by all vials, freezing and thawing conditions, whereas fibrinogen, D-dimer, von Willebrand activity or protein C were not affected by any vial, freezing or storage condition. CONCLUSIONS: Storage vials, freezing and thawing condition affect coagulation testing, although these differences may not be clinically significant.

Consequences for the APTT due to direct action of factor XIa on factor X, resulting in bypassing factors VIII-IX.

BACK GROUND: It has recently been reported that factor XIa can activate factor X directly and can bypass factors VIII-IX. We evaluated the consequences for factor analysis with the one-stage APTT. METHODS: APTT was performed with the Actin FS reagent with ellagic acid as the standard. Silica, high lipid (PTT-A) or low lipid (PTT-LA) were also tested. Factor depleted and deficient plasma's were obtained from commercial sources. RESULTS: The APTT clotting times in factor XII, XI, High Molecular Weight Kininogen, factor X and factor V deficient plasma's were all significantly longer (>100s) than the clotting times of factor VIII- and IX-depleted or deficient plasma's (<100s). That the shorter times for factor VIII and IX deficient plasmas were due to contact activation was supported by biphasic inhibition of the clotting times with addition of Corn Trypsin Inhibitor and Trasylol. The role of factor XI and the by-passing of factor VIII/IX was shown by the use of quenching antibodies towards factor XI and VIII. Enriching factor VIII or IX depleted plasma with purified factor XI and addition of factor XIa showed a strong dependence on factor XI level. Calibration curves for factor analysis were steeper for factors FXII, HMWK, FX and FV, compared to those of both factors VIII and IX. Curves for VIII/IX were found steeper by the use of APTT-A/silica-based, 50% diluted substrate plasma and low factor XI in the substrate plasma. CONCLUSIONS: In factors VIII and IX deficient plasmas, the APTT shows an activity which can be attributed to contact activation of factor X by factor XIa. This direct activity is lower with silica reagent compared to ellagic acid, dilution of plasma and low factor XI in substrate plasma.

[Identification of Target Extracellular Proteases--Activators of Proteins of Haemostasis System Produced by Micromycetes Aspergillus ochraceus and Aspergillus terreus].

Effects of extracellular proteases of Aspergillus ochraceus and Aspergillus terreus on plasma hemostasis proteins, consist of initiating the activation of prothrombin complex proteins, was detected. Was discovered, that A. ochraceus proteases have a direct influence on protein C and coagulation factor X, and A. terreus proteases causes their activation indirectly through kallikrein system stimulation. The ability of extracellular proteases of micromycetes activate prekallikrein in human blood plasma on the example of A. terreus was first demonstrated.

Warfarin monitoring in antiphospholipid syndrome and lupus anticoagulant.

OBJECTIVE: To review the available literature on international normalized ratio (INR) and chromogenic factor X (CFX) monitoring in patients with antiphospholipid syndrome (APS), specifically lupus anticoagulant (LA), and furthermore, to identify benefits of one monitoring test compared with the other in the presence of LA. DATA SOURCES: A literature search was conducted through MEDLINE (1946-May 2014) utilizing the following MeSH terms chromogenic compounds, anticoagulants, and factor X. Further articles were identified from original literature citations. STUDY SELECTION: All English-language studies were included that involved INR and/or CFX monitoring in APS patients that focused on a therapeutic anticoagulation level with warfarin therapy. DATA SYNTHESIS: A total of 55 articles were identified, of which nine are referenced because of their relevance for this review: three articles focus on the efficacy of utilizing INR monitoring in patients with APS, five focus on CFX compared with INR for therapeutic warfarin dosing, and one compares different thromboplastins utilizing both INR and CFX monitoring. INR monitoring in patients with APS, specifically LA, was not found to be reliable because thromboplastin reagents are sensitive to LA. Furthermore, when INR was compared to CFX, patients with LA had supratherapeutic INRs despite having CFX within goal range. CONCLUSIONS: In a subgroup of APS patients, INR monitoring may not be safe for determining the dose of warfarin because their INR values can be falsely elevated. Although CFX monitoring is more accurate, it too comes with its own downsides. Managing warfarin therapy in the APS population needs to be individualized.

Coagulation factor and hemostatic protein content of canine plasma after storage of whole blood at ambient temperature.

BACKGROUND: Standard practice in canine blood banking is to produce fresh frozen plasma (FFP) by separating and freezing plasma produced from blood within 8 hours of collection. Within canine blood donation programs, this can limit the number of units collected. HYPOTHESIS/OBJECTIVES: The aim was to compare the coagulation factor and hemostatic protein content (CF&HPC) of plasma produced from blood stored at ambient temperature for 8, 12, and 24 hours. Another aim was to compare the CF&HPC between Greyhound types and other breeds. ANIMALS: None. METHODS: In vitro study. A convenience sample of 58 units of canine blood from a blood donor pool was processed to prepare and freeze plasma 8, 12, or 24 hours following collection. RESULTS: Regardless of time of processing, the units contained therapeutic CF&HPC. Frozen plasma prepared after 24 hours had significantly higher factor VIII (P = .014) and factor X (P = .03) when compared with the frozen plasma prepared at 8 hours. Factor X (P < .01), fibrinogen (P < .01), and vWF (P = .04) were significantly lower in plasma collected from Greyhound types than in plasma collected from other breeds. CONCLUSIONS AND CLINICAL IMPORTANCE: Storing whole blood for up to 24 hours is a suitable method for producing FFP. Lower values for some coagulation factors and hemostatic proteins in plasma produced from Greyhound types would not preclude these dogs as FFP donors.