Restoration Of Hemostasis Research Articles

Changes in the levels of cytokines IL-1β, IL-4, IL-8, and IL-10 in dental orthopedic fixed prosthetics

Despite significant advances in materials science, introduction of new methods and principles of prosthetic treatment into the practice of dentists, complications after prosthetics with fixed structures are encountered. The result of dental prosthetics of patients with defects of hard tissues of teeth depends not only on the chosen construction and materials from which they are made, but is closely connected with the functional activity of the immune system. Cytokines are an important factor of mucosal immunity. The cytokine system represents one of the key components of mucosal immunity. It regulates normal physiological functions, restoration of haemostasis in the interaction of physical, chemical and biological factors on the body and participates in the pathogenesis of allergic, autoimmune, and autoinflammatory processes. The aim of the study is to compare the content of IL-1â, IL-8, IL-10, and IL-4 in unstimulated saliva and gingival fluid in patients during adaptation to metal-ceramic and oxide ceramic tooth-supported constructions. In the period from 2021-2024, we examined 3 groups of patients aged 46±2 years: group 1 – comparison group, patients without dental constructions in the oral cavity (25 people); group 2 – patients treated with tooth-supported metal-ceramic (MC) crowns (25 people); and group 3 – patients treated with tooth-supported zirconium dioxide (ZrO2) crowns (25 people). On the basis of IL-1â, IL-8, IL-10, and IL-4 we concluded that the mucosal immunity of oral mucous membranes is less affected by the structures made of oxide ceramics compared to the crowns made of metal-ceramics. Similar patterns of changes in the levels of IL-1â, IL-8, and IL-4 in saliva and gingival fluid in patients who underwent dental rehabilitation were found, which allows us to use only saliva in order to eliminate the complexity and specificity of gingival fluid sampling. Also, changes in cytokine indices in saliva and gingival fluid at different stages of prosthodontics reflect the level of reaction of oral mucosal immunity, which can serve as a criterion of the quality of the performed dental treatment.

Hemostasis restoring in postpartum hemorrhage — algorithm 2023

Postpartum hemorrhage (PPH) is still the leading cause of perinatal death in women of reproductive age. It is characterized by high dynamics, and most women die within the first day of puerperium and as many as 88% of them within the first 4 hours from the onset of hemorrhage. Management of each massive obstetric hemorrhage requires interdisciplinary, team-based and multidirectional action. The need for simultaneous application of uterotonic agents, surgical and/or endovascular methods as well as procedures aimed at restoring volume and hemostasis should be emphasized. The protocol for the management of severe PPH should be individualized and take into account hemorrhage etiology. Each time the volume of circulating blood as well as the degree of blood loss have to be determined, and the adequate procedures, the starting points for implementing aggressive treatment and the satisfactory end points must be defined. The most common cause of severe, life-threatening PPH is uterine atony. Restoration of hemostasis in massive PPH consists in transfusion of blood components, administration of antifibrinolytic drugs and coagulation factor concentrates, rFVIIa included. Patient survival is often determined by the very risk assessment for hemorrhage, early diagnosis and rapid implementation of PPH management algorithms.

Liver-directed lentiviral gene therapy corrects hemophilia A mice and achieves normal-range factor VIII activity in non-human primates

Liver gene therapy with adeno-associated viral (AAV) vectors delivering clotting factor transgenes into hepatocytes has shown multiyear therapeutic benefit in adults with hemophilia. However, the mostly episomal nature of AAV vectors challenges their application to young pediatric patients. We developed lentiviral vectors, which integrate in the host cell genome, that achieve efficient liver gene transfer in mice, dogs and non-human primates, by intravenous delivery. Here we first compare engineered coagulation factor VIII transgenes and show that codon-usage optimization improved expression 10-20-fold in hemophilia A mice and that inclusion of an unstructured XTEN peptide, known to increase the half-life of the payload protein, provided an additional >10-fold increase in overall factor VIII output in mice and non-human primates. Stable nearly life-long normal and above-normal factor VIII activity was achieved in hemophilia A mouse models. Overall, we show long-term factor VIII activity and restoration of hemostasis, by lentiviral gene therapy to hemophilia A mice and normal-range factor VIII activity in non-human primate, paving the way for potential clinical application.

Anticoagulation for Stroke Prevention after Restoration of Haemostasis with Emicizumab in Acquired Haemophilia A

Acquired haemophilia A (AHA) is a rare haemorrhagic disorder caused by the development of autoantibodies inhibiting factor VIII function. It predominantly affects the elderly, who are often burdened with a considerable number of comorbidities, and can result in life-threatening bleeding. The management of AHA consists of two aspects: inhibitor eradication with an immunomodulator and bleed control with a bypassing agent. Here we present a case of AHA with a high titre inhibitor in a patient with extensive comorbidities and atrial fibrillation in whom inhibitor eradication could not be achieved within a few weeks using corticosteroids alone. Due to coronavirus disease (COVID)-19 restrictions and complications of care, emicizumab offered an effective and convenient therapy, not only sparing the need for continued and intensified inhibitor eradication, but also allowing anticoagulation for stroke prophylaxis.

Anticoagulation for Stroke Prevention after Restoration of Haemostasis with Emicizumab in Acquired Haemophilia A.

Acquired haemophilia A (AHA) is a rare haemorrhagic disorder caused by the development of autoantibodies inhibiting factor VIII function. It predominantly affects the elderly, who are often burdened with a considerable number of comorbidities, and can result in life-threatening bleeding. The management of AHA consists of two aspects: inhibitor eradication with an immunomodulator and bleed control with a bypassing agent.Here we present a case of AHA with a high titre inhibitor in a patient with extensive comorbidities and atrial fibrillation in whom inhibitor eradication could not be achieved within a few weeks using corticosteroids alone. Due to coronavirus disease (COVID)-19 restrictions and complications of care, emicizumab offered an effective and convenient therapy, not only sparing the need for continued and intensified inhibitor eradication, but also allowing anticoagulation for stroke prophylaxis.LEARNING POINTSEmicizumab may offer a suitable option for bleeding prophylaxis when inhibitor eradication is not achievable with immunotolerance treatment, especially in the age of the COVID-19 pandemic when the consequences of immunosuppression can be detrimental.Bleeding prophylaxis with emicizumab may enable long-term anticoagulation in patients with acquired haemophilia A during inhibitor eradication.The prothrombotic risks of emicizumab are not yet sufficiently characterized.

Andexanet alfa for the treatment of hemorrhage

ABSTRACTIntroduction: While associated life-threatening and fatal bleeding events are less frequent with the direct factor Xa inhibitors compared to vitamin K antagonists, significant concern surrounding management of major bleeds and urgent periprocedural interruption of these agents exists among clinicians. Andexanet alfa is a recombinant human factor Xa decoy protein developed in response to this clinical gap in the care of patients receiving anticoagulation with factor Xa inhibitors.Areas covered: This paper reviews results from preclinical and healthy-volunteer studies demonstrating the ability of andexanet to rapidly and reliably normalize coagulation indices in patients treated with both direct and indirect factor Xa inhibitors. An interim analysis from an ongoing phase 3/4b clinical study assessing the efficacy and safety of andexanet in patients experiencing life-threatening hemorrhage in association with factor Xa inhibitors is discussed. It also provides an overview of the major safety concerns reported in these trials which include allergic and infusion reactions, development of anti-andexanet antibodies and, importantly, thrombosis.Expert commentary: While initial reports on restoration of hemostasis and safety are promising, further study of andexanet is required to gauge its efficacy and toxicity, including a potential prothrombotic effect. Further, its use in patients requiring urgent surgery should be studied.

Delayed double reading of whole blood clotting test (WBCT) results at 20 and 30 minutes enhances diagnosis and treatment of viper envenomation

BackgroundThe whole blood clotting test (WBCT) is a simple test of coagulation that is often used in the assessment, diagnosis, and therapeutic monitoring of snakebite patients in sub-Saharan Africa. WBCT requires only a clean glass tube and several milliliters of venous blood and is ideal for use in poorly equipped health centers throughout the rural areas where 95% of snakebites occur. However, questions surrounding the accuracy and reliability of the test remain unanswered due to variations in testing conditions and a lack of comparative research with which to validate them. This is the first study to evaluate WBCT results at both 20-min (WBCT20) and 30-min (WBCT30) reading times in the same group of snakebite patients.MethodsIn order to define the best reading time, the authors compared the results of serial WBCT evaluation at both 20 and 30 min after collection in 23 patients treated for snake envenomation in Bembèrèkè, northern Benin.ResultsWBCT results were identical at both reading times in patients without coagulopathy or when coagulation was restored permanently following a single dose of antivenom. Out of 17 patients with coagulopathy, 14 showed discrepancies between WBCT20 and WBCT30 results in at least one pair of serial evaluations. These could be completely contradictory results (e.g. normal clot at WBCT20 and no clot at WBCT30) or a marked difference in the quality of the clot (e.g. no clotting activity at WBCT20 and an unstable partial clot at WBCT30). WBCT discrepancies were encountered most frequently in three situations: initial normalization of hemostasis following antivenom therapy, detection of a secondary resumption of coagulopathy, or final restoration of hemostasis after a secondary resumption had occurred.ConclusionsThis study suggests that the WBCT is robust and that a sequential reading should improve the diagnosis and monitoring of venom-induced coagulopathies. It also indicates the possibility of discrepancies in the sensitivity of WBCT20 and WBCT30 for detecting the resolution or reoccurrence of coagulopathy and identifies how these findings, if confirmed, may be used to increase the efficacy and efficiency of antivenom treatment in the field.

ВЛИЯНИЕ ИНТРАОПЕРАЦИОННОЙ НОРМОВОЛЕМИЧЕСКОЙ ГЕМОДИЛЮЦИИ НА ГЕМОСТАЗ, КРОВОПОТЕРЮ И ПОКАЗАНИЯ К ТРАНСФУЗИИ ДОНОРСКИХ ЭРИТРОЦИТОВ ПРИ ОПЕРАЦИЯХ НА СЕРДЦЕ В УСЛОВИЯХ ИСКУССТВЕННОГО КРОВООБРАЩЕНИЯ

Intr a-o perative acute normovolemic hemodilution or autologous blood transfusion, is one of the factors of the patient's blood conservation and reduction of the donor blood use. The objective: to assess the value of acute normovolemic hemodilution for the normalization of intr a-o perative homeostasis, reduction of blood loss and transfusion of donor erythrocytes during cardiac surgery with cardiopulmonary bypass. Methods. 270 patients undergoing surgery on heart valves and coronary vessels at the age from 18 to 79 patients were examined. 600−1,200 ml of autoblood were collected before cardiopulmonary bypass against the background of the patient's heparinization and installation of aortic cannula. The following parameters were studied: the frequency of normovolemic hemodilution use, volume of autohemoexfusion of blood, hemostasis rates in the early pos t-p erfusion period, the volume of intra- and pos t-o perative blood loss, frequency and volume of hemotransfusion. Results. Intr a-o perative autologous blood transfusion from right atrial appendage against full heparinization of the patient in the volume of 600–1,200 ml before cardiopulmonary bypass allowed reducing intr a-o perative blood loss by 1.3 times, the volume of transfused erythrocytes by 1.3–1.7 times and the frequency of the demand for it by 4–6 times in the patients having surgery with myocardial revascularization and cardiac defect management. The analysis of coagulation parameters in the pos t-p erfusion period after modified acute normovolemic hemodilution proved the reduction of hemostasis dysfunction mostly due to the platelet factor. Conclusion. Intr a-o perative acute normovolemic hemodilution is an effective and safe way to reduce intr a-o perative blood loss, use of donor blood and restoration of hemostasis in the patients undergoing cardiac surgery with cardiopulmonary bypass.

Clinical outcome of patients with a vitamin K antagonist‐associated bleeding treated with prothrombin complex concentrate

Unlabelled Box BackgroundVitamin K antagonists (VKA) are used for the treatment of thromboembolism. Patients with severe VKA‐associated bleeding require immediate restoration of haemostasis. Clinical studies on the effect of prothrombin complex concentrate (PCC) are heterogeneous with respect to outcome of bleeding. ObjectiveTo evaluate the clinical outcome of patients treated with PCC for VKA‐associated bleeding. MethodsWe performed a cohort study of consecutive patients who received PCC for VKA‐related bleeding in five Dutch hospitals. Data were collected by chart review on the bleeding event, international normalized ratio (INR), haemostatic efficacy, thromboembolic (TE) complications, and mortality. The primary outcome was effective haemostasis, assessed by an adaptation of the Sarode criteria with a surrogate outcome for patients with ICH without repeat CT. ResultsOne hundred patients were included. Mean age was 74 years, 54% were male and 79% received VKA for atrial fibrillation. Most patients presented with ICH (41%) or GI bleeding (36%). Effective haemostasis was achieved in 67/98 (68%) patients using the adapted classification. Surrogate outcomes were applied for 32 patients and data for two patients was missing. Median pre‐treatment INR was 3.9 (IQR 2.9‐5.8). One hour after PCC infusion, the INR was available for 50 patients and of these, 35 (70%) had an INR ≤1.4. TE complications occurred in five patients and 22 died (60% bleeding‐related) within 30 days. ConclusionPCC achieved effective haemostasis in 68% of evaluable patients with VKA‐associated bleeding. TE complication rates were low, but mortality rates were high, due to the large number of patients with ICH.

Andexanet Alfa Reverses Anticoagulation Effects of Enoxaparin and Associated Bleeding in a Rabbit Acute Hemorrhage Model

Introduction: Andexanet alfa (andexanet) is a modified recombinant human factor Xa (FXa), developed to reverse the anticoagulation effects of both direct and indirect FXa inhibitors. For antithrombin III (ATIII)-dependent FXa inhibitors, such as enoxaparin, andexanet binds to the ATIII-enoxaparin complex with high affinity and reverses the inhibition of coagulation factors Xa and IIa. This study evaluated the ability of andexanet to reverse anticoagulation effects and blood loss due to the indirect FXa inhibitor, enoxaparin, in a rabbit liver laceration model.Methods: In a rabbit model of surgically-induced, acute hemorrhage (liver laceration model), rabbits were randomized to receive enoxaparin (8 mg/kg; subcutaneous bolus) or enoxaparin vehicle. This route of administration and dose of enoxaparin was based on the pharmacokinetic profile of enoxaparin and pharmacodynamic activity in the rabbit (2-fold increase in blood loss relative to non-anticoagulated rabbits). Andexanet (15, 35, or 75 mg per rabbit) or andexanet vehicle was administered to anesthetized rabbits as a 5-minute bolus, 120 minutes after enoxaparin injection (from Time 120 to Time 125 minutes), prior to livery injury. The five treatment groups were enoxaparin vehicle + andexanet vehicle, enoxaparin + andexanet vehicle, or enoxaparin + andexanet (3 dose levels). For liver laceration, a standardized injury (10 1-cm-long and 2- to 3-mm-deep incisions) was made into two liver lobes with 5 incisions in each lobe, and bleeding was allowed to progress for 15 minutes (from Time 125 to Time 140 minutes). Study efficacy endpoints included evaluations of blood loss in rabbits anticoagulated with enoxaparin, and anti-FXa activity in plasma.Results: In rabbits anticoagulated with enoxaparin, andexanet significantly decreased blood loss 2-3 fold, relative to vehicle control (assessed at Time 140 minutes) at all doses of andexanet administered (15, 35, 75 mg per animal) (see Table 1). The mean blood loss in anticoagulated rabbits administered andexanet was similar to that in non-anticoagulated rabbits (enoxaparin vehicle + andexanet vehicle). In enoxaparin-anticoagulated rabbits, andexanet rapidly (5 minutes after the start of andexanet administration; Time 125 minutes) reduced mean anti-FXa activity, and the reduction was significant for the 35-mg and 75-mg doses of andexanet (see Table 1). Anti-FXa activity did not return to baseline levels for the duration of the study from Time 120 to Time 140 minutes. These results are consistent with previous studies in enoxaparin-anticoagulated rats where a similar decrease in anti-FXa activity was sufficient to significantly reduce blood loss. These findings with an indirect (ATIII-mediated) FXa inhibitor enoxaparin are distinct from those with a direct FXa inhibitor (e.g., rivaroxaban), which showed that larger reductions in anti-FXa activity were required for significant reductions in blood loss.Conclusions: Administration of andexanet resulted in reversal of the anticoagulant effects of enoxaparin as well as restoration of hemostasis in enoxaparin-anticoagulated rabbits, suggesting it could be clinically valuable for the management of major bleeding associated with indirect FXa inhibitors. This study suggests a potential difference in the extent of reduction in anti-FXa activity required for significant reduction in blood loss associated with indirect vs. direct FXa inhibitors. Further studies are ongoing to better understand correlations between anti-FXa activity and blood loss following andexanet administration in enoxaparin-anticoagulated animals. [Display omitted] DisclosuresPine:Portola Pharmaceuticals: Employment. Lu:Portola: Employment, Patents & Royalties. Canivel:Portola Pharmaceuticals: Employment. Pratikhya:Portola Pharmaceuticals: Employment. DeGuzman:Portola Pharmaceuticals: Employment. Karbarz:Portola Pharmaceuticals: Employment. Takeda:Portola Pharmaceuticals: Employment. Malinowski:Portola Pharmaceuticals: Employment. Leeds:Portola Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties, Research Funding. Curnutte:Portola Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties, Research Funding; 3-V Biosciences: Equity Ownership; Sea Lane Biotechnologies: Consultancy. Conley:Portola Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties, Research Funding.

Clotting Factor VIII Overexpression Shows Signs of ER Stress but Does Not Cause Toxicity upon Gene Transfer

Hemophilia A is the X-linked bleeding disorder resulting from the loss of functional clotting factor VIII (FVIII). Hemophilia A patients with severe disease (<1% residual FVIII activity) experience spontaneous bleeds into the joints and closed spaces with severe morbidity. Restoration of hemostasis is managed by repeated infusions (2-3 times per week) of plasma derived or recombinant FVIII protein. While a standard treatment is available for patients, life-long infusions of FVIII protein is very expensive, has a negative impact on the patient's quality of life, and FVIII protein products are not available worldwide. Hence, there is a need to develop a more robust and cost effective treatment for hemophilia A patients. Liver-directed gene therapy using adeno-associated virus (AAV) represents a promising approach to treat hemophilia A. However, previous studies have shown that overexpression of human FVIII protein in the context of hydrodynamic delivery of plasmid vectors induces ER stress mediated through the unfolded protein response (UPR). Because human FVIII protein is inefficiently secreted into circulation, high AAV vector doses will be required to obtain therapeutic expression levels. Therefore, we sought to determine if AAV-FVIII gene delivery also triggers cellular UPR in hepatocytes in vivo. To this end, we selected to use a codon-optimized FVIII cDNA, which has been shown by our lab to significantly increase FVIII protein expression, and a high vector dose of AAV8-ApoE-hAAT-cohF8, containing a hepatocyte-specific enhancer/promoter combination. We evaluated this vector at doses of 1 x 1011 vg (4x1012 vg/kg) and 1 x 1012 vg (4x1013 vg/kg) in hemophilia A mice on a 129/BL6 background. Intravenous administration of the highest vector dose completely restored hemostasis, which was sustained and achieved super-physiological levels in some animals. Importantly, none of these mice developed inhibitors against FVIII. Next, we administered the vector at the same 2 doses to C67BL/6 mice, which show higher hepatic transduction efficiency than other strains. Experimental controls consisted of mice, with no vector or 1x1012 vg of AAV8-ApoE-hAAT-F9, expressing human factor IX (FIX) protein. Injection of tunicamycin, a potent inducer of the ER stress response, served as a positive control for all assays. Vector-treated mice were studied 2 and 4 weeks after gene transfer (n=3-4 per group). First, we evaluated the status of key molecular chaperones, known to be the mediators of the UPR: Bip, p-PERK, and p-eIF2a. Western blotting performed on the liver lysates indicated modest up-regulation of all three markers compared to normal control, but that effect was neither dose nor gene dependent. In addition, we tested the splicing of Xbp1 mRNA by PCR assay and observed low level of the 26 bp spliced fragment, indicative of the UPR, at the high vector dose. Immunohistochemistry on liver sections from each of our experimental groups including H&E staining, Tunnel staining for apoptotic cells, and reactive oxygen species staining. None of the stains yielded evidence for liver damage even in the 1x1012 AAV8-cohF8 treated mice compared to untreated controls. There was also no elevation of liver enzyme levels in plasma samples. Analysis of plasma from vector injected mice showed systemic levels of human FVIII and FIX proteins at ~30 ng/ml and ~6300 ng/ml, respectively (these ELISA-based measurements likely underestimate FVIII levels due to interference by von Willebrand factor). Overall our results suggest that over-expression of coagulation factors in hepatocytes from AAV vectors causes a mild cell stress response that is not strong enough to cause liver toxicity, is not specific for FVIII, and does impact expression or immunogenicity. DisclosuresNo relevant conflicts of interest to declare.

Four-Factor Prothrombin Complex Concentrate for Urgent Reversal of Vitamin K Antagonists in Patients With Major Bleeding

Vitamin K antagonists (VKAs), such as warfarin, are widely used for prevention and treatment of arterial and venous thrombosis. Although oral or intravenous vitamin K and fresh frozen plasma are often used to reverse the anticoagulant effects of warfarin in patients who are bleeding, this approach has important limitations. Restoration of hemostasis with vitamin K relies on the hepatic synthesis of vitamin K–dependent procoagulant proteins, factors II (prothrombin), VII, IX, and X, a process that takes >6 hours. Fresh frozen plasma provides an immediate source of functional vitamin K–dependent clotting proteins, but large volumes are often required to normalize the international normalized ratio (INR). This can be problematic because it takes time to match blood type and thaw and infuse fresh frozen plasma, and the large volumes can lead to fluid overload, particularly in patients with compromised cardiac or renal function. Article see p 1234 Prothrombin complex concentrate (PCC) provides an alternative to fresh frozen plasma for reversal of VKA-induced coagulopathy (Table). Originally developed as a source of factor IX for treatment of patients with hemophilia B, 3-factor PCC contains factors II, IX, and X but little or no factor VII. In contrast to 3-factor PCC, 4-factor PCC also contains significant amounts of factor VII. Both 3- and 4-factor PCC contain protein C and protein S, and some may also contain small amounts of heparin, which is added to prevent activation of the clotting proteins. View this table: Table. Available Treatments for Reversal of Vitamin K Antagonist–Associated Coagulopathy PCC contains the vitamin K-dependent clotting proteins in a lyophilized form and can be stored at room temperature for several years. No thawing or blood-type matching is required, and after reconstitution with a small volume of sterile water (20–40 mL), PCC can be rapidly administered without risk of fluid overload. Additional advantages of PCC over fresh …

Hypodensity of extradural hematomas in children: an ominous sign

The aim of this study was to analyze the correlation of hypodensity in extradural hematomas on CT with the clinical profile in pediatric patients. This is the only study available in this age group. This was a prospective study conducted over a period of 3 years in which all children 18 years old or younger with a diagnosis of cranial extradural hematoma were included. The patients were allocated to 2 groups: those with mixed-density clots (17 cases) and those with classically hyperdense clots (52 cases). A comparative analysis between the 2 groups was conducted. Patients with mixed-density clots presented earlier to the hospital, had poor Glasgow Coma Scale scores at admission, exhibited large clot volumes, had a high incidence of active bleeding at surgery, and had increased morbidity and mortality as compared with the patients with hyperdense extradural hematomas. Early recognition and rapid evacuation of the mixed-density clot with restoration of hemostasis may result in a decline in morbidity and death in children with this entity.

Short‐term warfarin reversal for elective surgery – using low‐dose intravenous vitamin K: safe, reliable and convenient*

Peri-procedural management of warfarin reflects an intricate balance between the restoration of haemostasis and appropriate thromboprophylaxis. This prospective single-arm study assessed the safety and efficacy of a convenient schedule, incorporating low-dose intravenous vitamin K (vitK(IV) ) for short-term warfarin reversal prior to elective surgery, as well as vitK-dependent factor levels (vitK-Factors) and International Normalized Ratio (INR) pre- and post-vitK(IV) . One seventy eight patients on long-term warfarin received 3mg vitK(IV) 12-18 h pre-procedure with no adverse reactions. 167/178 (94%) achieved an INR≤1·5 post-vitK(IV) on the day of surgery, while all achieved INR≤1·7. Four patients had procedure-associated major bleeding, but importantly had achieved a pre-procedure INR<1·5 and vitK-Factors >0·30iu/ml. No patient suffered a symptomatic thromboembolism during the 6-week follow-up. Median days to re-establish a therapeutic INR were 4 (range 2-11). VitK(IV) near normalized all vitK-Factors, with a uniform pattern of depletion and repletion in association with an increase and decrease in INR, respectively; and from the data, INR<1·5 correlated with vitK-Factors >0·30iu/ml. Low-dose vitK(IV) for short-term warfarin reversal was reliable and safe, and successfully lowered the INR to an acceptable level for planned surgery, with no excess of bleeding, thromboembolism, delayed discharge, or resistance to warfarin. The protocol was simple and convenient for both the patients and the healthcare institution.

Role of anti‐idiotypic antibodies in immune tolerance induction

Replacement therapy using factor VIII (FVIII) elicits FVIII-specific antibodies (abs) in about 25% of the patients. A majority of such abs are directed towards specific FVIII regions in which major epitopes have been identified (C-terminal end of the C2 domain, the N-terminal end of the A2 domain and C1 domain in cases of mild/moderate haemophilia A). We derived five human monoclonal abs (mabs) that react with high affinity to the FVIII C1, C2 or A2 domains respectively and are representative of most of the specific inhibitors observed in haemophilia A patients. We generated mouse anti-idiotypic mabs (anti-Ids) against the paratope of each of the inhibitors. We demonstrated that a combination of these anti-Ids (anti-anti-A2, -C1, -C2) had the ability to neutralize the inhibitory properties of human polyclonal abs in plasma. In 16 of the 18 plasmas tested, the inhibiting FVIII activity was neutralized up to 100% by the anti-Ids mixture with restoration of full FVIII activity. These data allow us to conclude that polyclonal high-affinity FVIII inhibitors could be neutralized with an anti-Ids mixture and that only a limited number of anti-Ids were required for inhibitor neutralization in 90% of the patients. We also demonstrated that anti-Id Abs bound to anti-FVIII human B cell line produced the corresponding anti-FVIII Ab and that this binding was followed by surface capping of complexes. Data obtained in vitro at monoclonal and polyclonal level, confirmed by in vivo assays, and the preliminary results obtained at BCR level, indicate that anti-id mixture made of only a limited number of anti-Ids could be useful in the restoration of haemostasis in haemophilia patients with inhibitor.

Directed Engineering of a High-expression Chimeric Transgene as a Strategy for Gene Therapy of Hemophilia A

Human coagulation factor VIII (fVIII) is inefficiently biosynthesized in vitro and has proven difficult to express at therapeutic levels using available clinical gene-transfer technologies. Recently, we showed that a porcine and certain hybrid human/porcine fVIII transgenes demonstrate up to 100-fold greater expression than human fVIII. In this study, we extend these results to describe the use of a humanized, high-expression, hybrid human/porcine fVIII transgene that is 89% identical to human fVIII and was delivered by lentiviral vectors (LVs) to hematopoietic stem cells for gene therapy of hemophilia A. Recombinant human immunodeficiency virus-based vectors encoding the fVIII chimera efficiently transduced human embryonic kidney (HEK)-293T cells. Cells transduced with hybrid human/porcine fVIII encoding vectors expressed fVIII at levels 6- to 100-fold greater than cells transduced with vectors encoding human fVIII. Transplantation of transduced hematopoietic stem and progenitor cells into hemophilia A mice resulted in long-term fVIII expression at therapeutic levels despite <5% genetically modified blood mononuclear cells. Furthermore, the simian immunodeficiency virus (SIV) -derived vector effectively transduced the human hematopoietic cell lines K562, EU1, U.937, and Jurkat as well as the nonhematopoietic cell lines, HEK-293T and HeLa. All cell lines expressed hybrid human/porcine fVIII, albeit at varying levels with the K562 cells expressing the highest level of the hematopoietic cell lines. From these studies, we conclude that humanized high-expression hybrid fVIII transgenes can be utilized in gene therapy applications for hemophilia A to significantly increase fVIII expression levels compared to what has been previously achieved.

Nonsurgical bleeding diathesis in anemic thrombocytopenic patients: role of temperature, red blood cells, platelets, and plasma‐clotting proteins

Research at the Naval Blood Research Laboratory (Boston, MA) for the past four decades has focused on the preservation of red blood cells (RBCs), platelets (PLTs), and plasma-clotting proteins to treat wounded servicemen suffering blood loss. We have studied the survival and function of fresh and preserved RBCs and PLTs and the function of fresh and frozen plasma-clotting proteins. This report summarizes our peer-reviewed publications on the effects of temperature, RBCs, PLTs, and plasma-clotting proteins on the bleeding time (BT) and nonsurgical blood loss. The term nonsurgical blood loss refers to generalized, systemic bleeding that is not corrected by surgical interventions. We observed that the BT correlated with the volume of shed blood collected at the BT site and to the nonsurgical blood loss in anemic thrombocytopenic patients after cardiopulmonary bypass surgery. Many factors influence the BT, including temperature; hematocrit (Hct); PLT count; PLT size; PLT function; and the plasma-clotting proteins factor (F)VIII, von Willebrand factor, and fibrinogen level. Our laboratory has studied temperature, Hct, PLT count, PLT size, and PLT function in studies performed in non-aspirin-treated and aspirin-treated volunteers, in aspirin-treated baboons, and in anemic thrombocytopenic patients. This monograph discusses the role of RBCs and PLTs in the restoration of hemostasis, in the hope that a better understanding of the hemostatic mechanism might improve the treatment of anemic thrombocytopenic patients. Data from our studies have demonstrated that it is important to transfuse anemic thrombocytopenic patients with RBCs that have satisfactory viability and function to achieve a Hct level of 35 vol percent before transfusing viable and functional PLTs. The Biomedical Excellence for Safer Transfusion (BEST) Collaborative recommends that preserved PLTs have an in vivo recovery of 66 percent of that of fresh PLTs and a life span that is at least 50 percent that of fresh PLTs. Their recommendation does not include any indication that preserved PLTs must be able to function to reduce the BT and reduce or prevent nonsurgical blood loss. One of the hemostatic effects of RBC is to scavenge endothelial cell nitric oxide, a vasodilating agent that inhibits PLT function. In addition, endothelin may be released from endothelial cells, a potent vasoconstrictor substance,to reduce blood flow at the BT site. RBCs, like PLTs at the BT site, may provide arachidonic acid and adenosine diphosphate to stimulate the PLTs to make thromboxane, another potent vasoconstrictor substance and a PLT-aggregating substance. At the BT site, the PLTs and RBCs are activated and phosphatidyl serine is exposed on both the PLTs and the RBCs. FVa and FXa, which generate prothrombinase activity to produce thrombin, accumulate on the PLTs and RBCs. A Hct level of 35 vol percent at the BT site minimizes shear stress and reduces nitric oxide produced by endothelial cells. The transfusion trigger for prophylactic PLT transfusion should consider both the Hct and the PLT count. The transfusion of RBCs that are both viable and functional to anemic thrombocytopenic patients may reduce the need for prophylactic leukoreduced PLTs, the alloimmunization of the patients, and the associated adverse events related to transfusion-related acute lung injury. The cost for RBC transfusions will be significantly less than the cost for the prophylactic PLT transfusions.

Mitigation of Coagulation by Removing Clotting Factors Part 2: Heparin-Free Extracorporeal Circulation in a Porcine Model

A subpopulation of patients would benefit from an anticoagulation strategy during extracorporeal circulation (ECC) that does not involve systemic administration of heparin and protamine. Inhibition of coagulation by adsorption of plasma clotting factors using protamine immobilized on a Sepharose matrix (PSM) has been explored. This investigation extends previous in vitro studies and demonstrates the feasibility of heparin-free ECC. In a porcine ex vivo circuit, plasma was separated from blood via plasmapheresis, passed through a column containing PSM beads, and returned to the animal. Hemodialyzers and stents were placed in the circuit before, during, and after ECC and examined for device thrombosis. After 90 minutes, prothrombin time (PT) was prolonged >10 times the baseline, and blood clotting Factors I, II, VIII, and X were decreased significantly (>90%); this state was maintained for 2.5 hours without detectable adverse consequences. After cessation of ECC, PT approached normal levels within 60 minutes. Examination of hemodialyzers and coronary stents placed in the circuit revealed that the removal of clotting factors significantly reduced device thrombosis and that transfusion of homologous blood ( approximately 10% V/V) resulted in immediate restoration of hemostasis. It is possible to remove clotting factors from circulating blood to allow extracorporeal circulation of blood without the use of heparin.

Delayed diagnosis of congenital factor IX deficiency (Christmas disease) in a girl with Turner's Syndrome

Patients with Turner's syndrome are at risk of X-linked recessive disorders. We report a case of a young girl with Turner's syndrome with persistent mildly abnormal coagulation studies associated with a mild to moderate bleeding diathesis. The abnormalities were initially attributed to intrahepatic cholestasis and were partially responsive to vitamin K. After an interval of several years an episode of unexplained iron deficiency anaemia prompted re-investigation of the mild coagulopathy. Disproportionate reduction in the factor IX concentration and restoration of haemostasis with factor IX concentrate lead to a revised provisional diagnosis of mild haemophilia B which was subsequently confirmed by sequencing the factor IX gene.

Abdominal trauma

Blunt abdominal trauma is much more frequent than penetrating abdominal trauma in Europe. As a consequence of improved quality of computed tomography, even complex liver injuries are increasingly being treated conservatively. However, missed hollow viscus injuries still remain a problem, as they considerably increase mortality in multiply injured patients. Laparoscopy decreases the rate of unnecessary laparotomies in perforating abdominal trauma and helps to diagnose injuries of solid organs and the diaphragm. However, the sensitivity in detecting hollow viscus injuries is low and the role of laparoscopy in blunt abdominal injury has not been defined. If intra-abdominal bleeding is difficult to control in hemodynamically unstable patients, damage control surgery with packing of the liver, total splenectomy, and provisional closure of hollow viscus injuries is of importance. Definitive surgical treatment follows hemodynamic stabilization and restoration of hemostasis. Injuries of the duodenum and pancreas after blunt abdominal trauma are often associated with other intra-abdominal injuries and the treatment depends on their location and severity.